Monsanto Company 12-AL-246U 1 of 407 

### Petition for the Determination of Nonregul ated Status for Reduced Lignin Alfalfa

KK179

The undersigned submits this petition under 7 CFR § 340.6 to request that the

Administrator make a determination that the article should not be regulated under

7 CFR Part 340

November 15, 2012

(Revised January 18, 2013)

OECD Unique Identifier: 

## MON-ØØ179-5

Monsanto Petition Number: 12-AL-246U

Submitted by:

Michael D. Hall, Ph.D.*

1 

### Monsanto Company 800 North Lindbergh Blvd. St. Louis, MO 63167 Phone: (314) 694-9134

Fax: (314) 694-3080 E-mail: mdhall@monsanto.com

*principal contact David J. Whalen

2 

### Forage Genetics International, LLC

N5292 S. Gills Coulee Rd.

West Salem, WI 54669 Phone: (608) 786-2121

Fax: (608) 786-2193 E-mail: dwhalen@foragegenetics.com

Prepared by:

1 

### A. E. Bernhard, B.S., 1 

### M. L. Breeze, M.S., 1 

### M. S. Giovannini, M.S., 1 

### M. D. Hall, Ph.D.,

1 

### T. H. Klusmeyer, Ph.D., 1 

### S. Paul, M.S., 1 

### K. A. Robinson, Ph.D., 1 

### R. W. Schneider, B.S.,

1 

### Y. Wang, Ph.D.

Contributors and/or Pr incipal Investigators:

1 

### L. D. Barberis, B.S., 1 

### E. L. Clawson, Ph.D., 1 

### S. R. Eskelsen, Ph.D.,

1 

### S. N. Fitzpatrick, M.S., 1 

### R. L. Girault, M.S., 1 

### J. R. Groat, M.S., 1 

### E. K. Hill, B.S.,

1 

### S. W. Hoi, M.S., 1 

### K. M. Huizinga, Ph.D., 1 

### K. R. Lawry, M.S., 1 

### T. P. Perez, M.S.,

1 

### S. L. Phillips, M.S., 2 

### M. S. S. Reddy, Ph.D., 1 

### S. Shah, M.S., 1 

### A. Skipwith, M.S.,

1 

### Z. Song, M.S., 2 

### S. J. Temple, Ph.D., 1 

### Q. Tian, M.D., Ph.D., 1 

### J. M. Ward, Ph.D.,

2 

### D. J. Whalen, B.S., 1 

### J. E. Whitsel, M.S., 1 

### T. K. Witten, Ph.D.Monsanto Company 12-AL-246U 2 of 407 

### RELEASE OF INFORMATION

Monsanto Company and Forage Genetics International are submitting the information in this petition for review by the USDA as part of the regulatory process. Monsanto Company and Forage Genetics International understa nd that the USDA complies with the provisions of the Freedom of Information Act (FOIA). In the event the USDA receives a FOIA request, pursuant to 5 U.S.C., § 552, and 7 CFR Part 1, covering all or some of the information in this petition, Monsanto Co mpany and Forage Genetics International expect that, in advance of the release of the document(s), USDA will provide Monsanto Company and Forage Genetics In ternational with a copy of th e material proposed to be released and the opportunity to object to the release of any information based on appropriate legal grounds, e.g ., responsiveness, confidentiality, and/or competitive concerns. Monsanto Company and Forage Genetics International understand that a CBIdeleted copy of this information may be made available to the public in a reading room and upon individual request as part of a pub lic comment period. Monsanto Company and Forage Genetics International also understand that when deemed complete, a copy of the petition may be posted to the USDA-APHIS BRS website or other U.S. government websites ( e.g. , www.regulations.gov ). Except in accordance with the foregoing, Monsanto Company and Forage Genetics Inte rnational do not authorize the release, publication or other distributi on of this information wit hout Monsanto Company's and Forage Genetics International’ s prior notice and consent.

© 2013 Monsanto Company and Forage Ge netics Internatio nal. All Rights

Reserved.

This document is protected under national and international copyright law and treaties. This document and any accompanying material are for use only by the regulatory authority to which it has been submitted by Monsanto Company and Forage Genetics International and only in suppor t of actions requested by Monsanto Company and Forage

Genetics International. A ny other use, copying, or tran smission, including internet posting, of this document and the materials desc ribed in or accompanying this document, without prior consent of Monsanto Company and Forage Genetics International, is strictly prohibited; except th at Monsanto Company and Forage Genetics International hereby grant such consent to the regulatory authority where require d under applicable law or regulation. The intellect ual property, information and materials described in or accompanying this document are owned by Monsanto Company and Forage Genetics International, which have filed for or b een granted patents on those materials. By submitting this document and any accompanying materials, Monsanto and Forage Genetics International do not grant any part y or entity any right or license to the information, material or intellectual property described or containe d in this submission.

### CERTIFICATION

The undersigned certify that, to the best knowledge and belief of the undersigned, this petition

includes all information and views on which to base a determination, and that it includes all

relevant data and information known to the petitioner that are unfavorable to the petition

.



## •

Michael D. Hall, Ph.D. Regulatory Affairs Manager

Address:

Monsanto Company

800 North Lindbergh Blvd., C3SD

St. Louis, MO 63167

Tel: (314)-694-9134

Fax: (314)-694-3080

David 

### J. Whalen, B.S.

Director of Regulatory Affairs and New Trait Development

Address: Forage Genetics International, LLC

N5292 S. Gills Coulee Rd.

West Salem, WI 54669

Phone: (608) 786-2121

Fax: (608) 786-2193

Monsanto Company 12-AL-246U 30f407Monsanto Company 12-AL-246U 4 of 407 

### EXECUTIVE SUMMARY

The Animal and Plant Health Inspection Serv ice (APHIS) of the United States (U.S.) Department of Agriculture (USDA) has resp onsibility under the Plant Protection Act (Title IV Pub. L. 106-224, 114 Stat. 438, 7 U.S.C. § 7701-7772) to prevent the introduction and dissemination of plant pest s into the U.S. USDA-APHIS regulation 7 CFR § 340.6 provides that an applicant may petition APHIS to evaluate submitted data to determine that a particular regulated arti cle does not present a pl ant pest risk and no longer should be regulated. If USDA-APHIS determines that the regulated article does not present a plant pest risk, the petiti on is granted, thereby allowing unrestricted

introduction of the article.

Monsanto Company (hereafter referred to as Monsanto) and Forage Genetics International (hereafter referred to as FG I) are submitting this request to USDA-APHIS for a determination of nonregulated status for the new biotechno logy-derived alfalfa product, KK179, any progeny derived from cr osses between KK179 and conventional alfalfa, and any progeny derived from cr osses of KK179 with biotechnology-derived alfalfa that have previously been granted nonregulated st atus under 7 CFR Part 340.

Product Description

Monsanto and FGI have developed biotechnology-derived alfalfa KK179 ( Medicago

sativa L.) which has reduced levels of guaiacyl lignin (G), a major subunit component of total lignin, compared to conventional alfalfa at the same stage of growth. This reduction in G lignin leads to reduced accumulation of total lignin in alfalfa forage, the principal feed product derived from alfalf a. Forage quality, as defined by market standards, is compromised by the presence of lignin which is sensitive to timing of harvest. KK179 is designed to provide alfalfa grow ers with greater flexibility in harvest timing in order to better manage forage quality and improve the ability to meet or exceed intended quality standards for alfalfa forage production.

KK179 reduces lignin in forage through the suppression of caffeoyl CoA

3- O -methyltransferase (CCOMT), a key enzyme in the lignin biosynthetic pathway. KK179 was produced by insertion of CCOMT gene segments, derived from alfalfa, assembled to form an inverted repeat DNA sequence. The invert ed repeat sequence produces double-stranded RNA (d sRNA) which suppresses endogenous CCOMT gene expression via the RNA interference (RNAi) pathway. Suppression of the CCOMT gene expression leads to lower CCOMT protein expr ession resulting in reduced synthesis of G lignin subunit compared to c onventional alfalfa at the sa me stage of growth. The reduction in G lignin subunit synthesis leads to reduced accumulation of total lignin, measured as acid detergent lignin (ADL).

Forage of KK179 and the conventional cont rol were analyzed for monomeric lignin subunits, the building blocks for lignin molecu les. These analyses confirm that the suppression of CCOMT acts to specifically reduce the level of one major lignin subunit, G lignin by approximately 19%, while not substa ntially affecting the levels of the other major lignin subunit, syringyl lignin (S lignin) , or minor lignin subuni ts, as predicted by the mode-of-action. The result is a lower pr oportion of G lignin a nd a greater proportionMonsanto Company 12-AL-246U 5 of 407 

### of S lignin, shown by an increase in the S to G lignin ratio. Analysis of the same forage for total lignin levels, measured as acid detergent lignin (ADL), by commercial forage testing methods shows a reduction of approxima tely 22% and verifies that the reduction in the G lignin leads to a concurrent reducti on in total lignin in KK179 forage compared

to the conventional control harveste d at the same stage of growth.

Alfalfa is grown to produce forage for direct use on farm or for sale of hay as animal feed. Growers consider both forage yield a nd forage quality as critical factors in determining the value of the crop. As the crop grows and forage biomass increases, the quality of conventional alfalfa forage begins to decrease rapidly due to increased lignin levels in the stems of maturing plants. Gr owers, therefore, must decide whether to harvest forage to obtain higher quality fora ge or higher yield (t onnage). There is a narrow interval of time, a matter of a few days, depending on the growing region, to harvest high quality forage prior to signi ficant lignin accumulati on at which point the quality of the forage declines rapidly. KK179 allows growers more flexibility to schedule harvests and thereby better manage the yield-quality relationship in forage production in order to meet market needs or intended on-farm uses as animal feed. This expanded harvest interval provides valuable fl exibility for growers to manage real-time production decisions. For example, over the life of an alfalfa field or stand, a grower using KK179 can, at each cutting, interchangeably : 1) maintain harvest schedule routines and obtain forage that is more likely to m eet or exceed the intended quality standard targeted by the grower; or 2) delay a harv est several days and obtain higher tonnage without sacrificing forage qua lity. At the same time, i ndependently of the production decision on when to harvest, a grower w ill benefit from flexibility to accommodate unexpected delays in harvesting forage cau sed by adverse weather conditions, equipment failure, or competing farming activities.

Data and Information Presented Confirm th e Lack of Plant Pest Potential and the Feed and Food Safety of KK179 Co mpared to Conventional Alfalfa

The data and information presented in this petition demonstrate that KK179 is agronomically, phenotypically, and compositiona lly comparable to conventional alfalfa, with the exception of the introduced trait fo r reduced G lignin and to tal lignin. Moreover, the data and information presented demonstr ate that KK179 is not expected to pose an increased plant pest risk, incl uding weediness, compared to conventional alfalfa. The feed, food and environmental safety of KK179 was confirmed based on multiple, well

established lines of evidence:

 Alfalfa is a familiar crop that has a history of safe consumption as animal feed, and serves as an appropriate ba sis of comparison for KK179.

 A detailed molecular characterization of the inserted DNA, which confirms the presence of a single, intact CCOMT suppression cassette stably integrated at a single locus within the alfalfa genome.

 The CCOMT suppression cassette in KK179 is extremely unlikely to produce a protein. The RNA-based suppression of the CCOMT alfalfa gene in KK179 is mediated by dsRNA molecules. Double- stranded RNAs are commonly used byMonsanto Company 12-AL-246U 6 of 407 

### eukaryotes, including plants, for endoge nous gene suppression and pose no novel risks from a feed/food and environment pe rspective. Nucleic acids, as the components of RNA, have a long history of safe consumption and are considered GRAS by the U.S. FDA.

 The compositional and nutritional assessmen t supports the conclusion that KK179 forage is compositionally equivalent, w ith the exception of the intended reduction in G lignin and total lignin (ADL), to that of conventional alfalfa at the same stage of growth.

 An extensive evaluation of KK179 phenotypi c and agronomic characteristics and

environmental interactions demonstrate that KK179 shows no increased plant pest risk potential compared to conventional alfalfa.

 An assessment of potential impacts to non-target organisms (NTOs) including organisms beneficial to agri culture indicates th at KK179 is not expected to have an effect on other organisms compared to conventional al falfa under normal agricultural practices.

 Evaluation of KK179 using cu rrent cultivation and ma nagement practices for alfalfa concluded that deregulation of KK179 is not expected to have an effect on alfalfa agronomic practices or land use.

Alfalfa is a Familiar Crop Lacking Weedy Characteristics

Alfalfa ( Medicago sativa L.) is grown for forage in all U.S. states and continues to be an important U.S. crop. It ranks fourth on the list of most widely grown crops by acreage and fourth in terms of value among agricu ltural crops. Approximately 20 to 24 million acres of alfalfa hay have been harvested annually over the past 10 years. Annual production has ranged from 68 to 82 million tons of hay. Average yields have remained fairly constant at 3.19 to 3.47 tons per acre over that same period. The annual value of production has ranged from $6.7 to $10.7 billion.

Because of its adaptability, alfalfa can survive outside of cultivation as feral populations. However, there is little evidence to suggest that alfalfa behave s as a weed, other than as a volunteer in agricultural setti ngs. Weed control experts from states where alfalfa is cultivated extensively and publicly available weed lists confirm that Medicago sativa is not considered or designated a weed or specie s with weediness potential. This aspect of alfalfa has been comprehensively reviewed by USDA-APHIS for Genuity ® 

### Roundup Ready ® 

### Alfalfa in its December 2010 Plant Pest Risk Asssessment and Determination and associated Environmental Impact Statement.

® Genuity and Roundup Ready are registered trademarks of Monsanto Technology LLCMonsanto Company 12-AL-246U 7 of 407 

### Conventional Alfalfa is an A ppropriate Comparator to KK179

A near-isogenic conventional alfalfa compar ator was developed using an identical breeding path used for the development of KK179. The same breeding process used to develop subsequent generati ons of KK179 was then follow ed to develop subsequent generations of near-isogenic conventional alfalfa comparators for each generation of KK179. All generations of KK179 and correspo nding generations of conventional alfalfa comparators used in safety assessments have closely related geneti c backgrounds with the exception of the introduced trait for reduced G lignin and total lignin absent in the conventional comparator. Conventional comme rcial alfalfa varieties (referred to as conventional commercial reference varieties), were used to establish ranges of natural variability or responses repres entative of commercial alfalfa varieties. The conventional

commercial reference varieties used were chosen based on their availability and agronomic fit for the geographic region.

Molecular Characterization Verified the Integrity and Stability of the Inserted DNA

in KK179

KK179 was developed through Agrobacterium tumefaciens -mediated transformation of conventional alfalfa, R2336, with the plas mid vector PV-MSPQ12633. The transformed plant was crossed with Ms208, an elite, male sterile, conventiona l alfalfa plant, to produce KK179. The PV-MSPQ12633 plasmid c ontains two separate T-DNAs, each delineated by Left and Right Border sequenc es to facilitate tran sformation. The first T-DNA, designated T-DNA I, contains the CCOMT suppression cassette, the Pal2 promoter and the nos 3 ′ UTR regulatory elements. The second T-DNA, designated T-DNA II, contains the nptII expression cassette under the regulation of the 35S promoter
and the nos 3 ′ UTR. During transformation, both T-DNAs were inserted into the alfalfa genome where T-DNA II, containing the nptII expression cassette, functioned as a marker gene for in vitro selection of transformed plantlets. Subsequent traditional alfalfa breeding methods and meiotic segregation, along with a combination of analytical techniques, were used to is olate a subset of transformed plants that contained the CCOMT suppression cassette (T-DNA I) but did not contain the nptII expression cassette (T-DNA II). This resulted in the subsequent identification of a single marker-free plant,

KK179.

Molecular characteriza tion of KK179 by Southern blot analyses confirmed that one copy

of the CCOMT suppression cassette (T-DNA I) was inte grated into the alfalfa genome at a single locus. No T-DNA II or bac kbone DNA sequences from plasmid vector PV-MSPQ12633 were detected in KK179. Th e complete DNA sequence of the insert and adjacent genomic DNA sequence in KK179 c onfirmed the integrity of the inserted

CCOMT suppression cassette within the inse rted sequences and identified the 5 ′ and 3 ′ insert-to-genomic DNA junctions. Additionally, Southern blot analysis and Mendelian segregation analysis of progeny from KK179 demonstrated that the inserted DNA has been maintained through four generations of breeding, thereby confirming the stability of the insert over multiple generations.Monsanto Company 12-AL-246U 8 of 407 

### RNA-Based Suppression Technology in KK179 Does not Pose Unique Safety Risks

The safety of KK179 expression products wa s assessed by taking into account multiple

factors. The KK179 insert contains a CCOMT suppression cassette. RNA-based suppression of CCOMT in KK179 is mediated by dsRNA molecules transcribed from the suppression cassette, which de crease the level of endogenous CCOMT RNA transcripts resulting in reduced levels of G lignin. Double-stranded RNAs are commonly found in eukaryotes, including plants, and functi on to suppress endogenous gene expression. RNA is composed of nucleic acids which have a long history of safe consumption and are generally recognized as safe (GRAS) by the U. S. FDA. There is no evidence to suggest dietary consumption of RNA is associated with mammalian toxicity, adverse health issues, or allergenicity. S everal biotechnology-derived pl ant products previously reviewed by the U.S. FDA, deregulat ed by USDA-APHIS, and approved by several international regula tory authorities were devel oped using RNA-based suppression mechanisms, including improved fatty acid profile soybean MON 87705, high-oleic soybean, plum pox virus-resistant plum trees , virus-resistant papaya, virus-resistant squash, and delayed-ripening tomatoes. The hairpin secondary structure of the dsRNA produced by the CCOMT suppression cassette precludes tr anslation initiation and protein synthesis, thus synthesis of the CCOMT pr otein or a putative polypeptide is highly unlikely. Based on this information, it is c oncluded that the inserted DNA and resulting dsRNA are safe and unlikely to produce a protein. As a result, the RNA-based suppression technology used in KK179 poses no novel risks from a feed, food or environment perspective.

KK179 is Compositionally Equivalent to Conventional Alfalfa

Detailed compositional analyses were conducted based on OECD guidelines for alfalfa to compare levels of key nutrients, anti-nut rients and secondary metabolites in KK179 forage to levels in the conven tional alfalfa control. Nutrient s analyzed in forage samples were proximates (ash, fat, moisture, and protein), carbohydrates by calculation, acid detergent fiber (ADF), neutral detergent fiber (NDF), acid detergent lignin (ADL), minerals (Ca, Cu, Fe, Mg, Mn, P, K, Na, and Zn), and amino acids (essential and nonessential). Anti-nutrients included daidzein, glycitein, genistein, coumesterol, formononetin, biochanin A, and saponins (t otal bayogenin, total hederagenin, total medicagenic acid, total soyasapogenol B, to tal soyasapogenol E, total zanhic acid and total saponins). Secondary metabolites an alyzed related to li gnin biosynthesis and phenylpropanoid metabolism in alfalfa included p- coumaric acid, ferulic acid, sinapic acid, total polyphenols, free phe nylalanine, and canavanine.

Compositional analyses on forage samples we re conducted to determine statistically significant differences ( α = 0.05) between KK179 and th e conventional control.

Statistical results from combined-site data we re evaluated using considerations relevant to the safety and nutritional quality of KK179 when compared to the conventional

control. Considerations to assess the releva nce of each statistically significant difference included: 1) the relative magnitude of the differe nces in the mean values of nutrient, antinutrient, and secondary metabolite com ponents between KK179 and the conventional control; 2) whether the KK179 component mean values we re within the range of variability of the component s as represented by the 99% tolerance interval of theMonsanto Company 12-AL-246U 9 of 407 

### conventional commercial referenc e varieties grown concurrently in the same field trial; and 3) an assessment of the differences with in the context of natural variability of available commercial alfalfa composition publis hed in the scientific literature.

Analysis of the observed signifi cant differences in nutrient, anti-nutrient, and secondary metabolite components with respect to magnitu de of differences, comparisons of mean analyte values to the 99% tolerance interval and to published values led to the conclusion that the differences were not biologically meaningful from a feed and food safety or nutritional perspective. Therefore, the genetic modification in KK179 does not meaningfully impact composition, other than the intended reduction in G lignin and total lignin (ADL). As a result, the feed and f ood safety and nutritional quality of this product are comparable to those of conventional alfa lfa, which has a history of safe use and consumption. When KK179 is grown on a comme rcial scale and used as a source of feed, alfalfa products derived from KK179 are not expected to be compositionally different from the equivalent feeds or iginating from conventional alfalfa.

KK179 Does Not Change Alfalfa Plant Pest Potential or Environmental Interactions

Plant pest potential of a biotechnology-de rived crop is assessed from the basis of familiarity that the USDA recognizes as an important underlying concept in risk assessment. The concept of familiarity is based on the fact that the biotechnologyderived plant is developed from a conventio nal plant whose biological properties and plant pest potential are well known. Familiar ity considers the biology of the plant, the introduced trait, the receiving environment, and the interactions am ong these factors. This provides a basis for comparative risk assessment between a biotechnology-derived plant and the conventional cont rol. Thus, the phenotypic, agronomic, and environmental

interaction assessment of KK179 included the near-isogenic conven tional control as a comparator. This evaluation used a weight of evidence approach and considered

statistical differences between KK179 and th e conventional control with respect to reproducibility, magnitude, and directionalit y. Characteristics asse ssed included seed dormancy and germination; pollen and flower morphologies; plant symbiont interactions in the laboratory or greenhouse; plant phenotypic observations ; and environmental

interaction evaluations conducted in the fi eld. Conventional commercial reference varieties grown concurrently were used to establish a range of natural variability for each

assessed characteristic in alfalfa. The phenotypic, agronomic, and environmental

interaction assessment demonstrated that KK179 is comparable to the conventional control. Thus, KK179 is not expected to have increased weediness or plant pest potential compared to conventional alfalfa.

Seed dormancy and germination characteri zation indicated that KK179 seed had no changes relative to the conven tional control in dormancy or germination characteristics

that could be indicative of increased plant weediness or pest potential of KK179 compared to the conventional c ontrol. Some st atistically significan t differences were

detected in which KK179 had higher percent germinated s eed and lower percent hard seed. These differences, however, were not c onsidered biologically meaningful in terms

of altered weediness of KK179 compared to the conventional control. No statistically significant differences were detected ( α = 0.05) between KK179 and the conventional control for percent viable pol len or pollen grain diameter . Furthermore, no visualMonsanto Company 12-AL-246U 10 of 407 

### differences in general pollen morphol ogy were observed between KK179 and the conventional control, demonstrating that th e introduced trait for reduced G lignin and total lignin did not alter the overall mo rphology or viability of pollen of KK179 compared to that of the conventional contro l. Similarly, no statistically significant

differences were detected between KK179 a nd the conventional control for morphology

of flowers.

The field evaluation of phenotypic, agronomic , and environmental characteristics of KK179 also supports the conclusion that KK 179 is not expected to have increased weediness or plant pest potential compared to conventional alfalfa. The evaluations were

conducted at nine field sites in the U.S. and one field site in Canada to provide a diverse range of environmental and agronomic conditi ons representative of commercial alfalfa production areas in North America. Trials were managed according to standard, local agronomic practices for forage production in or der to harvest forage at a growth stage of 1-10% bloom. For each growing season, assessments were made within each crop growth cycle and at each harvest. Inaddition to phenotypic and agronomic characteristics, observations were also made for plant responses to abiotic stressors, diseases, and arthropod inter actions. These studies were conducted over two complete growing seasons from 2010 to 2012.

The phenotypic characteristics observed durin g forage production from 2010 to 2012 across 10 field sites were comparable between KK179 and the conventio nal control. In a combined-site analysis of the phenotypic ch aracteristics in the first growing season (2010-2011), data showed no statistically signi ficant differences between KK179 and the conventional control for seedling emergen ce and early season vigor during stand establishment; no statis tically significant differences for lodging, crop growth stage, and regrowth after cutting for each of three harvests, and no statistically significant differences for fall plant height, total forage yield, spring vigor, spring stand recovery, and spring stand count. In a combined-site an alysis of the phenotypic characteristics in the second growing season (2011-2012), da ta showed no statistically significant

differences between KK179 and the conventiona l control for lodging, crop growth stage, forage yield, and regrowth after cutting for each of five harvests and no statistically significant differences for fall plant height, total forage yield, spring vigor, spring stand recovery, and spring stand count. In a comb ined-site and combined-year analysis, there were also no statistically significant differen ces for fall plant height, total forage yield, spring vigor, spring stand rec overy, and spring stand count.

The phenotypic characteristics observed during seed production at one field site in 2010 were also comparable between KK179 and th e conventional control. Data showed no statistically significant differences betw een KK179 and the conventional control for seedling establishment, seedling vigor, seed weight, seed per pod, or seed yield. Additionally, no statistically significant differences were detected between KK179 and the conventional control for lodging or split pods, both characteristics that could impact potential weediness.

Environmental interactions were assessed qualitatively within each growing season over two years and included plant response to abio tic stressors, disease damage and arthropod damage. In the first year, no differen ces were observed between KK179 and theMonsanto Company 12-AL-246U 11 of 407 

### conventional control for any of the 93 comparis ons of plant response to abiotic stressors, the 93 comparisons for plant damage caused by diseases, or the 96 comparisons for plant damage caused by arthropods. In the second year, no differences were observed between KK179 and the conventional control for any of the 129 comparisons of plant response to abiotic stressors, the 129 comparisons for plant damage caused by diseases, or the 129 comparisons for plant damage caused by arthropods.

Environmental interactions were assessed qua ntitatively within each growing season over two years and included assessments of al falfa weevil damage and potato leafhopper damage and pest- and benefici al-arthropod abundance. For alfalfa weevil or potato leafhopper damage, no statistically significant diff erences were detected in combined site analyses for either insect in 2010 and in 2011. For arthropod abundan ce, four differences out of 69 comparisons were detected at individua l sites in the first ye ar and one out of 89 comparisons in the second year. At the site s where statistical differences were observed, the mean abundance values for pests and ar thropods from KK179 were within the range

of the conventional commercial reference va rieties and/or the differences were not consistently detected across collection times or sites. Taken together, these data support the conclusion that compared to conventiona l alfalfa KK179 is no more susceptible to damage by alfalfa weevil or potato leafhopper and no more lik ely to promote increased abundance of these species.

In summary, phenotypic, agronomic, and enviro nmental interaction da ta were evaluated

to characterize KK179 and to as sess whether the introduced tr ait for reduced G lignin and total lignin in KK179 alters the plant pest po tential compared to c onventional alfalfa. The evaluation, using a weight of evidence approach, considered the reproducibility, magnitude, and direction of detected diffe rences between KK179 and the conventional control, and comparison to the range of the conventional commercial reference varieties. Results from the phenotypic, agronomic, an d environmental interactions assessment indicated that KK179 does not possess more or enhanced weediness characteristics, increased susceptibility or tolerance to specifi c abiotic stress, diseas es, or arthropods, or characteristics that would confer a plant pest risk compared to conventional alfalfa.

KK179 Will Not Have Effects On NTOs Incl uding Those Benefici al To Agriculture

Evaluation of the impacts of KK179 on non-targ et organisms (NTOs) is a component of the plant pest risk assessment. Since KK 179 does not possess pesticidal activity, all organisms that interact with KK179 are cons idered to be NTOs. The environmental

interactions assessment conducted over tw o growing seasons under field conditions

demonstrated that the introduction of th e CCOMT suppression cassette for reduced G lignin and total lignin in KK179 does not unexpe ctedly alter plant-ar thropod interactions, including beneficial arthropods, soil symbiont interactions, or alter disease su sceptibility compared to the conventiona l control. These results support the conclusion that cultivation of KK179 is not expected to effect non-target organisms and no altered

incidence of disease in KK179. The evaluati on also considered product characterization information including molecular characteri zation and composition, and the history of environmental exposure to lignin. Taken toge ther, these data support the conclusion that

KK179 has no reasonable mechanism for harm to NTOs, including organisms beneficial to agriculture, compared to commercial alfalfa.Monsanto Company 12-AL-246U 12 of 407 

### Potential for KK179 to outcross with sexually compatible species is not expected since no known related species capable of crossing with cultivated alfalfa are known to be present in North America. Assessments of phenotypic, agronomic, and environmental interactions, including reproducti ve characteristics indicated that KK179 is not expected to outcross with sexually compatible species compared with conventional alfalfa. Additionally, under forage production conditi ons, the potential for pollen production and outcrossing is very limited. Cross-pollination of KK179 or its progeny with feral alfalfa plants or with related species, would not be expected to have an effect on the environment, because evaluations have show n the introduced trait for reduced G lignin and total lignin in KK179 does not enhance weedin ess or plant-pest potential relative to conventional alfalfa. Therefore, the envi ronmental consequence of pollen transfer

between KK179 and feral alfalfa or a relate d species is considered negligible. Outcrossing to feral alfalfa populations a nd other species has been comprehensively reviewed by USDA-APHIS for Roundup Rea dy Alfalfa in its December 2010 Plant Pest

Risk Assessment and Determination and asso ciated Environmental Impact Statement.

USDA-APHIS has concluded that outcrossing to fera l alfalfa by deregulated biotechnology-derived alfalfa does not pose greater environmental risk compared with

conventional alfalfa.

Deregulation of KK179 Is Not Expected To Change Alfalfa Agronomic Practices or

Land Use

An assessment of current alfalfa agronom ic practices was conducted to determine

whether the cultivation of KK 179 has the potential to impact current alfalfa management practices. Alfalfa fields are typically highly managed agricult ural areas that are dedicated to forage production. KK179 is likely to be used in common rotations on land previously used for agricultural purposes. Cultivation of KK179 is not expected to differ from current alfalfa cultivation, with the exception of allowing growers greater flexibility to manage the yield-quality relationship in fo rage production and harvesting schedules to

meet market needs or for intended on-farm uses as animal feed. Certified seed production will not be impacted by the introduc tion of KK179 and germplasm developers and seed producers will continue to use well- established industry pr actices to deliver a diverse range of high quality alfalfa seed to growers.

KK179 is similar to conventional alfalfa in its agronomic, phenotypic, environmental, and compositional characteristics and has levels of resistance to insects and diseases comparable to conventional alfalfa. Based on this assessment, KK179 is not expected to result in changes to current U.S. alfalfa agronomic practices. Therefore, no impacts on current cultivation and management practices for alfalfa are expected following the

introduction of KK179.

Conclusion

Based on the data and information presented in this petition, Monsanto and FGI have concluded that KK179 is not expected to be a plant pest. Therefore, Monsanto and FGI request a determination from USDA-APHIS that KK179 and any progeny derived from

crosses between KK179 and conventional al falfa or deregulate d biotechnology-derived alfalfa be granted nonregulated status under 7 CFR Part 340.Monsanto Company 12-AL-246U 13 of 407 

### TABLE OF CONTENTS

RELEASE OF INFORMATION......................................................................................2

CERTIFICATION .............................................................................................................3

EXECUTIVE SUMMARY ...............................................................................................4

TABLE OF CONTENTS ................................................................................................13

LIST OF TABLES ...........................................................................................................17

LIST OF FIGURES .........................................................................................................22

ABBREVIATIONS AND DEFINITIONS .....................................................................24

I. RATIONALE FOR THE DEVELOPMENT OF KK179 ........................................28

I.A. Basis for the Request for a Determ ination of Nonregulated Status under 7 CFR § 340.6 ...............................................................................................................28

I.B. Rationale for the Developmen t of Reduced Lignin Alfalfa KK179 .....................28

I.B.1 Plant lignin biosynthesis ..............................................................................30

I.B.2 Mode-of-Action and Evidence of Suppression of the CCOMT Gene .........32

I.B.3. Intended Changes to Lignin Levels in KK179 Forage ...............................38

I.C. Submissions to Other Regulatory Agencies ..........................................................43

I.C.1. Submission to FDA .....................................................................................43

I.C.2. Submissions to Fore ign Government Agencies ..........................................43

II. THE BIOLOGY OF ALFALFA ..............................................................................44

II.A. Alfalfa as a Crop ..................................................................................................44

II.B. Characteristics of the Recipient Plant ..................................................................46

II.C. Alfalfa as a Test System in Product Safety Assessment ......................................46

III. DESCRIPTION OF TH E GENETIC MODIFICATION ....................................48

III.A. The Plasmid Vector PV-MSPQ12633 ................................................................48

III.B. Description of the Transformation System .........................................................49

III.C. The CCOMT Segment Sequence (T-DNA I) ......................................................52

III.D. The nptII Coding Sequence and nptII Protein (T-DNA II) ................................52

III.E. Regulatory Sequences .........................................................................................52

III.F. T-DNA Border Regions ......................................................................................52

III.G. Genetic Elements Outside the T-DNA Border Regions .....................................53

IV. CHARACTERIZATION OF THE GENETIC MODIFICATION .....................57

IV.A. Insert and Copy Number of T-DNA I in KK179 ...............................................63

IV.A.1. Probe 1 .....................................................................................................63

IV.A.2. Probe 2 and Probe 4 .................................................................................64Monsanto Company 12-AL-246U 14 of 407 

### IV.A.3. Probe 3 .....................................................................................................65

IV.B. Southern Blot Analysis to De termine the Presence or Absence of TDNA II Sequences in KK179 ........................................................................................71

IV.B.1. Probe 5 .....................................................................................................71

IV.C. Southern Blot Analysis to De termine the Presence or Absence of PV-MSPQ12633 Backbone Sequences in KK179 ........................................................73

IV.C.1. Backbone Probe 6 and Probe 8 ................................................................73

IV.C.2. Backbone Probe 7 and Probe 9 ................................................................73

IV.D. Organization and Sequence of th e Insert and Adjacent Genomic DNA in

KK179 ......................................................................................................................... ..77

IV.E. PCR and DNA Sequence Analyses to Examine the KK179 Insertion Site ........79

IV.F. Southern Blot Analysis to Examine Insert Stability in Multiple

Generations of KK179 ...................................................................................................81

IV.F.1. Probe 2 and Probe 4 ..................................................................................81

IV.G. Inheritance of the Genetic Insert in KK179 .......................................................84

IV.H. Characterization of the Genetic Modification Summary and Conclusion .........88

V. CHARACTERIZATION AND SA FETY ASSESSMENT OF KK179

EXPRESSED PRODUCTS .............................................................................................89

V.A. History of Safe Use of RNA-based Suppression of Endogenous CCOMT .........89

V.B. Safety Assessment of KK 179 Expressed Products Summary and

Conclusion .................................................................................................................... .90

VI. COMPOSITIONAL AS SESSMENT OF KK179 ..................................................91

VI.A. Compositional Equivalence of KK 179 Forage to Conventional Alfalfa ...........92

VI.B. Nutrients, Anti-Nutrients, and Secondary Metabolites in KK179 Forage .........94

VI.C. Compositional Assessment of KK179 Conclusion ..........................................108

VII. PHENOTYPIC, AGRONO MIC, AND ENVIRONMENTAL

INTERACTIONS ASSESSMENT ...............................................................................109

VII.A. Characteristics Measured for Assessment ......................................................109

VII.B. Interpretation of Phenotypic a nd Environmental Interactions Data ................114

VII.B.1. Interpretation of Detected Differences Criteria ....................................114

VII.B.2. Interpretation of Envi ronmental Interactions Data ...............................116

VII.C. Comparative Assessments of the Phenotypic, Agronomic, and

Environmental Interaction Characteristics of KK179 .................................................117

VII.C.1. Seed Germination and Dormancy Characteristics ................................117

VII.C.2. Field Phenotypic, Agronom ic, and Environmental Interaction

Characteristics ......................................................................................................122

VII.C.3. Reproductive Characteristics ................................................................142Monsanto Company 12-AL-246U 15 of 407 

### VII.C.4. Symbiont Interactions ...........................................................................146

VII.D. Conclusions for Phenotypi c, Agronomic, and Environmental

Interactions Evaluations ..............................................................................................148

VIII. U.S. AGRONOMIC PRACTICES.....................................................................149

VIII.A. Introduction ...................................................................................................149

VIII.B. Overview of U.S. Alfalfa Production ............................................................150

VIII.B.1. Alfalfa Forage Production ...................................................................150

VIII.B.2. Alfalfa Seed Production ......................................................................160

VIII.C. Alfalfa Forage Producti on Management Considerations ...............................168

VIII.C.1. Pre-Season ...........................................................................................168

VIII.C.2. Planting and Stand Establishment .......................................................171

VIII.C.3. Mid- to Late-Season ............................................................................173

VIII.C.4. Harvest.................................................................................................174

VIII.D. Management of Insect Pests ..........................................................................176

VIII.E. Management of Diseases and Other Pests .....................................................178

VIII.F. Weed Management .........................................................................................179

VIII.F.1 Weed Management in Roundup Ready Alfalfa ....................................186

VIII.G. Alfalfa Stand Termination and Volunteer Management ...............................186

VIII.H. Management of Feral Alfalfa ........................................................................189

VIII.I. Crop Rotation Practices in Alfalfa ..................................................................190

VIII.J. Impact of the Introduction of KK179 on Agricultural Practices ....................190

VIII.K. Stewardship of KK179 ..................................................................................192

IX. ENVIRONMENTAL ANALYSIS .......................................................................193

IX.A. Introduction ......................................................................................................193

IX.B. Plant Pest Assessment of KK179 .....................................................................193

IX.B.1. Characteristics of the Genetic Insert ......................................................194

IX.B.2. Compositional Char acteristics of KK179 ..............................................195

IX.B.3. Phenotypic and Agronomic and Environmental Interaction

Characteristics ......................................................................................................196

IX.C. Weediness Potential of KK179 ........................................................................198

IX.D. Potential for Gene Flow and Introgression ......................................................201

IX.D.1. Gene Flow Among Alfalfa ( Medicago sativa) Populations ..................201

IX.D.2. Hybridization with Annual Species of Subgenus Medicago .................206

IX.D.3. Hybridization with the Perennial Species of Subgenus Medicago ........206Monsanto Company 12-AL-246U 16 of 407 

### IX.D.4. Transfer of Genetic Informa tion to Species with which Alfalfa Cannot Interbreed (Horizontal Gene Flow) .........................................................206

IX.E. Potential Impact of KK179 on Alfalfa Agronomic Practices ...........................207

IX.F. Summary of Plant Pest Assessments ................................................................208

X. ADVERSE CONSEQUENC ES OF INTRODUCTION .......................................209

REFERENCES ...............................................................................................................210

APPENDICES ................................................................................................................229

Appendix A: USDA Permits ........................................................................................230

Appendix B: Materials and Methods Used for Molecular Analyses of KK179 ......236

Appendix C: Materials a nd Methods Used for Northern Blot Analyses of

CCOMT Suppression in KK179 ....................................................................................243

Appendix D: Materials, Methods, a nd Results for Intended Changes to Lignin Levels in KK179 Forage ....................................................................................247

Appendix E: Materials, Methods, and Re sults for Compositional Analysis of KK179 Forage ................................................................................................................26 3

Appendix F: Materials and Methods for Seed Germination and Dormancy

Assessment of KK179 ....................................................................................................328

Appendix G: Materials, Methods, and Individual Site Results from Phenotypic, Agronomic, and Environmental Interaction Assessment of KK179 under Field Conditions during Forage Production .......................................332

Appendix H: Materials and Methods from Phenotypic and Agronomic

Assessment of KK179 under Field Condi tions during Seed Production ..................386

Appendix I: Materials and Methods for Pollen Morphology and Viability

Assessment .................................................................................................................... ..391

Appendix J: Materials and Methods for Flower Morphology Assessment .............396

Appendix K: Materials and Metho ds for Symbiont Assessment..............................403Monsanto Company 12-AL-246U 17 of 407 

### LIST OF TABLES

Table I-1. Alfalfa Forage Lignin Subunit Levels and S:G Ratio for KK179

vs. Conventional Control .................................................................................................40

Table I-2. Alfalfa Forage Lignin Subunit Levels as Percent of Total HGS for

KK179 vs. Conventional Control....................................................................................41

Table I-3. Alfalfa Forage Total Lignin (ADL) Levels for KK179 vs.

Conventional Control ......................................................................................................42

Table III-1. Summary of Gene tic Elements in PV-MSPQ12633 ................................54

Table IV-1. Summary Chart of th e Expected DNA Segments Based on Hybridizing Probes and Restriction Enzymes Used in KK179 Analysis ....................61

Table IV-2. Summary of Ge netic Elements in KK179 ................................................62

Table IV-3. Segregation of the KK179 Insert During Mendelian Inheritance Testing ....................................................................................................................... ........87

Table VI-1. Summary of Differences Observed in the Combined-Site Analysis (p<0.05) of Alfalfa Forage Component Levels for KK179 vs.

Conventional Control ......................................................................................................96

Table VI-2. Statistical Summary of Al falfa Forage Nutrients for KK179 vs.

Conventional Control ......................................................................................................97

Table VI-3. Statistical Summary of Alfa lfa Forage Secondary Metabolites for

KK179 vs. Conventional Control..................................................................................103

Table VI-4. Statistical Summary of Al falfa Forage Anti-Nutrients for KK179

vs. Conventional Control ...............................................................................................104

Table VI-5. Literature and OECD Ranges for Compostional Components in Alfalfa Forage ................................................................................................................ .106

Table VII-1. Phenotypic, Agronomic, and Environmental Interaction

Characteristics Evaluated in North Ameri can Field Trials and Laboratory or

Greenhouse Studies ........................................................................................................111

Table VII-2. Comparison of Germinat ion Characteristics of Non-scarified

Seed of KK179 and the Conventional Control ............................................................120

Table VII-3. Comparison of Germinatio n Characteristics of Scarified Seed

of KK179 and the Conventional Control .....................................................................121

Table VII-4. Field Phenotypic Eval uation Sites for KK179 during 2010-2012 .......124

Table VII-5. Combined-Site Comparis on of KK179 to the Conventional Control for Phenotypic and Agronomic Characteristics in the First Year (2010-2011) of Forage Production ................................................................................126

Table VII-6. Combined-Site Comparis on of KK179 to the Conventional Control for Phenotypic and Agronomic Characteristics in the Second Year (2011-2012) of Forage Production ................................................................................128Monsanto Company 12-AL-246U 18 of 407 

### Table VII-7. Combined-Site and Comb ined-Year Comparison of KK179 to the Conventional Control for Phenotypic and Agronomic Characteristics during Forage Production .............................................................................................130

Table VII-8. Summary of Qualitat ive Environmental Interactions Assessments in the First Year (2010) ...........................................................................133

Table VII-9. Summary of Qualitat ive Environmental Interactions Assessments in the Second Year (2011) .......................................................................134

Table VII-10. Combined-Site Comparison of Potato Leafhopper and Alfalfa Weevil Damage to KK179 Compared to the Conventional Control in the First Year (2010) ............................................................................................................1 37

Table VII-11. Combined-Site Comparison of Potato Leafhopper and Alfalfa Weevil Damage to KK179 Compared to the Conventional Control in the Second Year (2011) ........................................................................................................138

Table VII-12. Summary of Arthropod Abundance Assessments and Detected

Differences in 2010 and 2011 ........................................................................................139

Table VII-13. Comparison of KK179 to the Conventional Control for Phenotypic and Agronomic Charact eristics during Seed Production ......................141

Table VII-14. Pollen Characteristics of KK179 Compared to the

Conventional Control ....................................................................................................143

Table VII-15. Flower Characterist ics of KK179 Compared to the

Conventional Control ....................................................................................................145

Table VII-16. Symbiont Interaction Assessment of KK179 and the

Conventional Control ....................................................................................................147

Table VIII-1. Alfalfa Hay Producti on in the U.S. from 2000 to 2010 1 

### .....................152

Table VIII-2. Alfalfa and Alfalfa Mixtures Forage (Hay and Haylage) Production by State in 2010 1 

### . ........................................................................................154

Table VIII-3. Costs and Returns for Establishment and Production of NonIrrigated Conventional Alfalfa in the North Central U.S. 1 

### ........................................158

Table VIII-4. Costs and Returns for Establishment and Production of Irrigated Conventional Alfalfa in the San Joaquin Valley, California. 1 

### ..................159

Table VIII-5. Alfalfa Seed Production in the U.S. 1 

### ....................................................162

Table VIII-6. Common Weeds Present in Alfalfa in the U.S 1 

### . ..................................181

Table VIII-7. Alfalfa Tolerance and Herbicide Effectiveness Ratings on Direct Seedlings of Alfalfa 1 

### ...........................................................................................184

Table VIII-8. Alfalfa Tolerance and Po stemergence Herbicide Effectiveness Ratings on Common Weeds in Established Alfalfa Stands 1 

### . .....................................185

Table VIII-9. Herbicides for Alfalf a Stand Termination and Control of

Volunteer Alfalfa 1 

### . .........................................................................................................188

Table IX-1. Potential Scenarios for Po llen-mediated Gene Flow in Alfalfa (adapted from Van Deynze et al., 2008) .......................................................................202Monsanto Company 12-AL-246U 19 of 407 

### Table A-1. USDA Notifications and Permits Approved for KK179 and

Status of Trials Conducted under These Permits .......................................................231

Table B-1. Hybridization Conditions of Utilized Probes ...........................................239

Table C-1. DNA Probes and Positive Hybridization Controls .................................246

Table D-1. Conventional Commercial Reference Varieties ......................................247

Table D-2. Re-expression Formulas for Statistical Analysis of Composition

Data .......................................................................................................................... .......250

Table D-3. Statistical Summary of Al falfa Forage Lignin Subunits for KK179 vs. Conventional Control (Conducted at Samuel Roberts Noble Foundation) ........251

Table D-4. Statistical Summary of Alfa lfa Forage Total Lignin (ADL) Levels for KK179 vs. Conventional Control (Co nducted at Dairy One Forage Lab) .........260

Table E-1. Conventional Commercial Reference Varieties ......................................263

Table E-2. Re-expression Formulas for Statistical Analysis of Composition

Data .......................................................................................................................... .......273

Table E-3. Statistical Summary of Si te CAPR Alfalfa Forage Nutrients for KK179 vs. Conventional Control (Conducted at Covance Laboratories) ................274

Table E-4. Statistical Summary of Site CAPR Alfalfa Forage Secondary

Metabolites for KK179 vs. Convention al Control (Conducted at Covance Laboratories) ................................................................................................................. .280

Table E-5. Statistical Summary of Site IARL Alfalfa Forage Nutrients for KK179 vs. Conventional Control (Conducted at Covance Laboratories) ...............281

Table E-6. Statistical Summary of Si te IARL Alfalfa Forage Secondary

Metabolites for KK179 vs. Convention al Control (Conducted at Covance Laboratories) ................................................................................................................. .287

Table E-7. Statistical Summary of Site ILCY Alfalfa Forage Nutrients for KK179 vs. Conventional Control (Conducted at Covance Laboratories) ...............288

Table E-8. Statistical Summary of Site ILCY Alfalfa Forage Secondary

Metabolites for KK179 vs. Convention al Control (Conducted at Covance Laboratories) ................................................................................................................. .294

Table E-9. Statistical Summary of Site KSLA Alfalfa Forage Nutrients for KK179 vs. Conventional Control (Conducted at Covance Laboratories) ................295

Table E-10. Statistical Summary of Si te KSLA Alfalfa Forage Secondary

Metabolites for KK179 vs. Convention al Control (Conducted at Covance Laboratories) ................................................................................................................. .301

Table E-11. Statistical Summary of Site TXCL Alfalfa Forage Nutrients for KK179 vs. Conventional Control (Conducted at Covance Laboratories) ................302

Table E-12. Statistical Summary of Si te TXCL Alfalfa Forage Secondary

Metabolites for KK179 vs. Convention al Control (Conducted at Covance Laboratories) ................................................................................................................. .308Monsanto Company 12-AL-246U 20 of 407 

### Table E-13. Statistical Summary of Site WIDL Alfalfa Forage Nutrients for KK179 vs. Conventional Control (Conducted at Covance Laboratories) ................309

Table E-14. Statistical Summary of Si te WIDL Alfalfa Forage Secondary

Metabolites for KK179 vs. Convention al Control (Conducted at Covance Laboratories) ................................................................................................................. .315

Table E -15. Statistical Summary of Alfalfa Forage Anti-Nutrients for KK179 vs. Conventional Control (Co nducted at Samuel Roberts Noble Foundation) ................................................................................................................... .316

Table F-1. Starting Seed of KK179, Control and Conventional Commercial Alfalfa Reference Varieties Used in Dormancy Assessment ......................................330

Table G-1. Starting Seed for Phenot ypic, Agronomic, and Environmental Interaction Assessment during Forage Production ....................................................334

Table G-2. Field and Pl anting Information ................................................................336

Table G-3. Study Site Management ............................................................................337

Table G-4. Definition of Morpholo gical Stages of Development for

Individual Alfalfa Stems Used to Calculate Crop Growth Stage ..............................339

Table G-5. Data Missing or Excluded from Analysis ................................................344

Table G-6. Individual-Site Phenot ypic Comparison of KK179 to the

Conventional Control in the First Ye ar (2010-2011) of Forage Production .............352

Table G-7. Individual-Site Phenot ypic Comparison of KK179 to the

Conventional Control in the Second Ye ar (2011-2012) of Forage Production.........357

Table G-8. Combined-Site Qualit ative Assessment: Abiotic Stressor Evaluations Using an Observationa l Severity Scale for KK179 and the

Conventional Control in the First Year .......................................................................366

Table G-9. Combined-Site Qualit ative Assessment: Disease Damage Evaluations Using an Observationa l Severity Scale for KK179 and the Conventional Control in the First Year (2010) ...........................................................367

Table G-10. Combined-Site Qualit ative Assessment: Insect Stressor Evaluations Using an Observationa l Severity Scale for KK179 and the Conventional Control in the First Year (2010) ...........................................................368

Table G-11. Individual-Site Analysis: Quantitative Assessment of Potato Leafhopper and Alfalfa Weevil Damage to KK179 Compared to the Conventional Control in the First Year (2010) ...........................................................369

Table G-12. Abundance of Pest Arthropods in Samples Collected from KK179, the Conventional Control, and the Conventional Commercial Reference Varieties in the First Year (2010) ...............................................................370

Table G-13. Abundance of Beneficial Ar thropods in Samples Collected from KK179, the Conventional Control, and the Conventional Commercial Reference Varieties in the First Year (2010) ...............................................................373Monsanto Company 12-AL-246U 21 of 407 

### Table G-14. Combined-Site Qualit ative Assessment: Abiotic Stressor Evaluations Using an Observationa l Severity Scale for KK179 and the Conventional Control in the Second Year (2011) .......................................................375

Table G-15. Combined-Site Qualit ative Assessment: Disease Damage Evaluations Using an Observationa l Severity Scale for KK179 and the Conventional Control in the Second Year (2011) .......................................................376

Table G-16. Combined-Site Qualit ative Assessment: Insect Stressor Evaluations Using an Observationa l Severity Scale for KK179 and the Conventional Control in the Second Year (2011) .......................................................377

Table G-17. Individual-Site Analysis: Quantitative Assessment of Potato Leafhopper and Alfalfa Weevil Damage to KK179 Compared to the Conventional Control in the Second Year (2011) .......................................................378

Table G-18. Abundance of Pest Arthropods in Samples Collected from KK179, the Conventional Control, and the Conventional Commercial Reference Varieties in the Second Year (2011) ...........................................................379

Table G-19. Abundance of Beneficial Ar thropods in Samples Collected from KK179, the Conventional Control, and the Conventional Commercial Reference Varieties in the Second Year (2011) ...........................................................383

Table H-1. Starting Seed for Phenot ypic and Agronomic Assessment during

Seed Production .............................................................................................................38 8

Table H-2. Field and Pl anting Information ................................................................389

Table I-1. Starting Seed for Pollen Morphology and Viability Assessment .............393

Table J-1. Starting Plants for Flower Morphology Assessment ................................399

Table K-1. Starting Seed of KK1 79, Conventional Control, and Conventional Commercial Reference Varieties Used in the Symbiont

Assessment .................................................................................................................... ..406Monsanto Company 12-AL-246U 22 of 407 

### LIST OF FIGURES

Figure I-1. Lignin biosynthetic pathway .....................................................................31

Figure I-2. Modified lignin biosynthetic pathway ......................................................33

Figure I-3. Northern Blot Analysis of CCOMT RNA Level in KK179 Forage

Tissue ........................................................................................................................ .........36

Figure I-4. Northern Blot Analysis of CCOMT RNA Level in KK179 Root

tissue ........................................................................................................................ ..........37

Figure III-1. Circular Map of PV -MSPQ12633 Showing Probe 1 through

Probe 9 ....................................................................................................................... .......50

Figure III-2. Schematic of the Development of KK179 ...............................................51

Figure IV-1. Schematic Representation of the Insert and Flanking DNA in

KK179 ......................................................................................................................... ......60

Figure IV-2. Southern Blot Analysis to Determine Insert and Copy Number

of T-DNA I in KK179: Probe 1 ......................................................................................67

Figure IV-3. Southern Blot Analysis to Determine Insert and Copy Number

of T-DNA I in KK179: Probe 2 and Probe 4 ................................................................68

Figure IV-4. Southern Blot Analysis to Determine Insert and Copy Number

of T-DNA I in KK179: Probe 3 .......................................................................................69

Figure IV-5. Southern Blot Analysis to Determine Insert and Copy Number

of T-DNA I in KK179: Probe 3 .......................................................................................70

Figure IV-6. Southern Blot Analysis to Detect the Presence or Absence of T-DNA II Sequences in KK179: Probe 5 .......................................................................72

Figure IV-7. Southern Blot Analysis to Determine the Presence or Absence of PV-MSPQ12633 Backbone Sequences in KK179: Probe 6 and Probe 8 ................75

Figure IV-8. Southern Blot Analysis to Determine the Presence or Absence of PV-MSPQ12633 Backbone Sequences in KK179: Probe 7 and Probe 9 ................76

Figure IV-9. Overlapping PCR Analys is across the Insert in KK179 .......................78

Figure IV-10. PCR Amplification of the KK179 Insertion Site in

Conventional Alfalfa ........................................................................................................80

Figure IV-11. Breeding History of KK179 ...................................................................82

Figure IV-12. Southern Blot Analysis to Examine Insert Stability in Multiple

Generations of KK179: Probe 2 and Probe 4 ................................................................83

Figure IV-13. Breeding Path for Gen erating Segregation Data for KK179 ..............86

Figure VII-1. Schematic Diagram of Agronomic and Phenotypic Data Interpretation Methods .................................................................................................115

Figure VIII-1. Geographic distribution of alfalfa hay harvested acres in the

U.S. .......................................................................................................................... ........152

Figure VIII-2. Commercial synthetic al falfa variety breeding schematic ...............164Monsanto Company 12-AL-246U 23 of 407 

### Figure I-1. General Morphology of Pollen from KK179, the Control, and Conventional Commerical Reference Varieties under 200X Magnification ............394

Figure J-1 Diagram of an Untri pped (Unopened) Alfalfa Flower ............................398

Figure J-2. General Morphology of Whole Racemes from KK179, the Conventional Control, and the Co nventional Commercial Reference

Varieties ..................................................................................................................... .....400

Figure J-3. General Morphology of Tripped Flowers from KK179, the Conventional Control, and the Co nventional Commercial Reference

Varieties ..................................................................................................................... .....401Monsanto Company 12-AL-246U 24 of 407 

### ABBREVIATIONS AND DEFINITIONS 1

Symbol or Abbreviation Definition 

### ~ Approximately
4CL 4-coumarate: CoA ligase

AACC American Association of Cereal Chemists

aadA Bacterial promoter, coding sequence, and 3 ′ UTR for an aminoglycoside-modifying enzyme, 3''(9)- O -nucleotidyltransferase from the transposon Tn 7 that confers spectinomycin and streptomycin resistance

ADF Acid detergent fiber

ADL Acid detergent lignin

APHIS Animal and Plant Health Inspection Service of the United States Department of Agriculture

AOAC Association of Analytical Chemists

AOCS American Oil Chemists Society

AOSA Association of Offi cial Seed Analysts

AOSCA Association of Official Seed Certification Agencies

AP Adventitious presence

ASSP Alfalfa Seed Stewardship Program

B-Left Border Region DNA region from Agrobacterium tumefaciens containing the Left Border sequence used for transfer of the T-DNA

B-Right Border Region DNA region from Agrobacterium tumefaciens containing the Right Border sequence used for transfer of the T-DNA

oC Degrees Celsius

C 0 

### The single progeny plant selected from the cross of alfalfa plants R2336 and Ms208 to develop the near-isogenic

conventional control

C 0 

### -Syn1 The near-isogenic conventional control for KK179 generation Syn1

C 0 

### -Syn1 Adv The near-isogenic co nventional control for KK179 generation Syn1 Adv

C3H p- coumarate-3-hydroxylase

C4H Cinnamate-4-hydroxylase
CAD Cinnamyl alcohol dehydrogenase

CCOMT Caffeoyl CoA 3- O -methyltransferase protein from Medicago

sativa

CCOMT Caffeoyl CoA 3- O -methyltransferase gene from Medicago

1 Alred, G.J., C.T. Brusaw, and W.E. Oliu. 2003. Handbook of Technical Writing, 7th edn., pp. 2-7. Bedford/St. Martin's, Boston, MA.Monsanto Company 12-AL-246U 25 of 407 

### sativa CCR Cinnamoyl-CoA reductase
CFR Code of Federal Regulations

COMT Caffeic acid 3- O -methyltransferase

CaMV Cauliflower mosaic virus

cpm Counts per minute

CRP Conservation Reserve Program

CS- rop Coding sequence for repressor of primer protein derived from the ColE1 plasmid for maintenance of plasmid copy

number in E. coli

CS- nptII Coding sequence of the neo gene from transposon Tn 5 of

E. coli encoding neomycin phospho transferase II (NPT II) that confers neomycin and kanamycin resistance
CTAB Cetyltrimethylammonium bromide

CWR Cell wall residue

d Day

DAP Days after planting

dATP Deoxyadenosine triphosphate

dCTP Deoxycytidine triphosphate

dNTP Deoxynucleotide triphosphate

dw Dry weight

E. coli Escherichia coli
DNA Deoxyribonucleic acid

EDTA Ethylenediamine tetraacetic acid

FASTA Algorithm used to find local high scoring alignments between a pair of protein or nucleotide sequences

FD4 Fall dormancy type 4
fw Fresh weight

FGI Forage Genetics International, LLC

F5H Ferulate 5-hydroxylase

g Gram(s)

G lignin Guaiacyl lignin subunits

G:F Gain:Feed

GRAS Generally Recognized As Safe

H lignin p- Hydroxyphenyl lignin subunits

HCT p- Hydroxycinnamoyl-CoA:shi kimate: hydroxycinnamoyltransferase HGS Combined H lignin, G lignin, and S lignin

ILSI International Life Sciences InstituteMonsanto Company 12-AL-246U 26 of 407 

### Kb Kilobase

kg Kilogram
LOQ Limit of quantitation

m Meter

MBC Modified backcross

Ms208 FGI proprietary conventiona l male sterile alfalfa plant

μg Microgram

μmol Micromole

mg Milligram

N Normal

NDF Neutral detergent fiber

NFTA National Forage Testing Association

NPTII Neomycin phosphotransferase II

NOS Nopaline synthase

NTO Non-target organism

OECD Organisation for Economic Co-operation and Development

ORF Open Reading Frame

OR- ori-pUC Origin of replication from plasmid pUC for maintenance of plasmid in E coli

OR- ori V Origin of replication from the broad host range plasmid RK2 for maintenance of plasmid in Agrobacterium

P 0 

### The single progeny plant selected from the cross of T 0 

### and

Ms208

P- 35S Promoter and leader from the 35S RNA of cauliflower mosaic virus (CaMV)

P- Pal2 Promoter of the Pal2 gene from Phaseolus vulgaris encoding the phenylalanine ammonia-lyase

PAL Phenylalanine ammonia-lyase

PCR Polymerase chain reaction

PEAQ Predictive Equations for Alfalfa Quality

polyA + 

### RNA PolyA enriched RNA

PPA Plant Protection Act

ppm Parts per million

PRESS Predicted residual sums of squares

PRT_2012 GenBank protein databa se, 187.0 (Released January 30, 2012) PV-MSPQ12633 Plasmid used to transf orm the alfalfa genome to produce

KK179
R2336 FGI proprietary conventiona l plant selected for ease ofMonsanto Company 12-AL-246U 27 of 407 

### transformation

RNA Ribonucleic acid

S lignin Syringyl lignin subunits

SAS Statistical Analysis System

S.E. Standard error

S:G lignin ratio Syringyl lignin s ubunit divided by guaiacyl lignin subunit

T-DNA Transfer DNA

Syn1 First generation KK179 synthetic population

Syn1 Adv Second generation KK 179 synthetic population

Tm Melting temperature

T 0 

### The transformed R2336 pl ant selected for KK179 development

T- nos 3 ′ UTR sequence of the nopaline synthase ( nos ) gene from Agrobacterium tumefaciens pTi encoding NOS that directs polyadenylation TUG Technology use guide

USD U.S. dollars

UTR Untranslated region

v/v Volume per volume

VNS Variety not statedMonsanto Company 12-AL-246U 28 of 407 

### I. RATIONALE FOR TH E DEVELOPMENT OF KK179

I.A. Basis for the Request for a Determ ination of Nonregulated Status under 7 CFR § 340.6

The Animal and Plant Health Inspection Serv ice (APHIS) of the United States (U.S.) Department of Agriculture (USDA) has res ponsibility, under the Plant Protection Act (Title IV Pub. L. 106-224, 114 Stat. 438, 7 U.S.C. § 7701-7772) to prevent the introduction and dissemination of plant pest s into the U.S. USDA-APHIS regulation 7 CFR § 340.6 provides that an applicant may petition USDA-APHIS to evaluate submitted data to determine that a particular regulated article does not present a plant pest risk and no longer should be regulated. If USDA-APHIS determines that the regulated article does not present a plant pest risk, the petition is granted, thereby allowing

unrestricted introduction of the article.

Monsanto Company (hereafter referred to as Monsanto) and Forage Genetics International (hereafter referred to as FG I) are submitting this request to USDA-APHIS for a determination of nonregulated status for the new biotechno logy-derived alfalfa product, KK179, any progeny derived from cr osses between KK179 and conventional alfalfa, and any progeny derived from cr osses of KK179 with biotechnology-derived alfalfa that have previously been granted nonregulated st atus under 7 CFR Part 340.

I.B. Rationale for the Developmen t of Reduced Lignin Alfalfa KK179

Alfalfa ( Medicago sativa L.) is the principal forage crop cultivated in the U.S. for animal feed. Approximately 20 to 24 million acres of alfalfa hay have been harvested annually in the U.S. in the majority of states over the past ten years to produce between 68 and 82 million tons of hay annually valued betw een approximately $6.7 and $10.7 billion USD (USDA-NASS, 2011b) Alfalfa is the fourth larges t agricultural crop in the U.S. in terms of acres harvested and fourth highest in value (USDA-NASS, 2012a; b). Approximately 40 percent of U.S. alfalfa acreage is planted as pure stands, while 30 percent is planted with cover or nurse crop and approximate ly 25 percent with grasses or another companion crop (USDA-APHIS, 2010).

Alfalfa forage products are va lued for their high protein content and highly digestible fiber for ruminants and horses (USDA-APHI S, 2010). The principal commercial product is hay, which is forage that has been dr ied and baled, or c ubed if mechanically compressed. Haylage, which is ensiled forage , is typically produced and used on-farm. The forage industry defines the quality of alfalfa hay by nutritional components, including crude protein, acid detergent fiber, neutral detergent fiber, total digestible nutrients, and relative feed value (Put nam et al., 2008b; USDA- APHIS, 2010). Other quality parameters evaluated include observati ons for the presence of weeds or molds, or anti-palatability factors such as poor text ure, evidence of heating, or unpleasant odors (Putnam et al., 2008b). USDA’s Agricultural Marketing Service uses the grades of supreme, premium, good, fair, and utility to re gularly report average pr ices for alfalfa hay in major producing areas (USDA-AMS, 2010). S upreme quality is defined as very early maturity, pre-bloom forage with no damage and with the highest nutritive value, while utility is forage harvested at very late ma turity, with heavy damage, and with minimalMonsanto Company 12-AL-246U 29 of 407 

### nutritive value. Price differences between supreme and fair quality can exceed 50% (Putnam et al., 2008b; USDA-AMS, 2012; USDA-APHIS, 2010).

Lignin deposition in maturing plants has a significant impact on the overall quality of alfalfa (Coors et al., 1986; Marten et al ., 1988; Schwab et al., 2005). Along with cellulose and hemicellulose constituents, lignin is a cell wall component that accumulates in the plant, particularly in the stem. At alfalfa crop maturity, lignin comprises 5-15% of dry matter (Putnam et al., 2008b), with this large range being dependent on many factors such as climate. After forage is harvested, re growth of stems and leav es initiates within a short time and the process of lignin accumulatio n is renewed. While a certain amount of lignin is essential for healthy alfalfa plants , lignin is indigestible and slows down the digestion of cellulose in the rumen of liv estock. Therefore, forage producers and commodity purchasers desire alfalfa with lower lignin levels but without loss of nutritional components, incl uding protein and fiber.

Alfalfa is grown as a perennial forage crop that is repeatedly harvested throughout the growing season. After each harvest, alfalf a plants go through a period of recovery followed by regrowth. Harvesting of alfalfa fo rage from an established stand can take place from two to eleven times in a growi ng season depending on the climate and region. Deciding when to harvest forage is a critical decision made by the gr ower that determines both forage yield and quality, and the ultimat e profitability and ut ility of the crop. Growers must compromise between obtaining high yield and high quality because the quality of forage declines rapidly as lignin accumulates in maturing plants. This inverse relationship means that crop maturity at the time of forage harvest affects yield and quality differently. Alfalfa forage yields can double from the pre-bud to full-bloom stages, but this rapid biomass increase is accompanied by a steep decline in quality due to lignin accumulation (Orloff and Putnam, 2008) . Conversely, alfalf a harvested at an immature or early maturity growth stage produc es the highest level of quality in forage due to lower lignin levels but at a lower yiel d (Orloff and Putnam, 2008). As a result, the interval of time during which forage quality and yield is optimized is relatively narrow and varies depending on which objective, quality or yield, is the priority for the grower. Planning the optimal schedule to harvest fora ge, therefore, is a difficult management decision involving market considerations, ag ronomic factors (Orlo ff and Putnam, 2008),

and weather.

Monsanto and FGI have developed biot echnology-derived alfalfa, KK179, which has reduced levels of guaiacyl (G) lignin subuni ts (hereafter referred to as G lignin), a primary subunit of total lignin, compared to conventional alfalfa at the same stage of growth. This reduction in G lignin subunits leads to reduced accumulation of total lignin in alfalfa forage. The levels of lignin in KK179 forage are generally similar to those found in conventional forage harvested seve ral days earlier under similar production conditions. As a result, grower s will have an expanded harv est interval and experience the benefit of greater flexibi lity to dynamically manage harv est strategies based on crop production priorities, such as:

 Maximizing forage quality: When aiming to maximize forage quality with KK179, the timing of harvest schedules would re main the same as with conventional varieties. KK179 harvested at a typical cr op cutting stage will produce alfalfa forageMonsanto Company 12-AL-246U 30 of 407 

### with lower levels of lignin compared to conventional alfalfa ha rvested at the same stage. As a result, the quality of the forage is more likely to meet or exceed the quality standard targeted by the grower. Th e forage yield will be maintained at the

same levels as with co nventional alfalfa. KK179 does not raise the maximum potential quality attainable for forage; rather, KK179 is more likely to meet or exceed the desired quality compared to c onventional alfalfa harvested at the same

stage.

 Maximizing forage yield: When aiming to maximize dry matte r yield of KK179, a grower can delay harvest for several da ys to accumulate more forage biomass without significantly forfeiting quality. During the reproductive growth stage, alfalfa dry matter can increase at th e rate of 200 pounds per acre per day (Undersander et al., 2009). Therefore, even a small delay in harvest timing can result in significant gains in forage yiel d. KK179 can be harvested several days later with quality comparable to that of conventional alfalfa harvested several days earlier, but with additional forage yield, a benefit not afforded by conventional systems. A similar delay with conventional alfalfa w ould provide a comparable yield, but the forage would have higher lignin content and, thus, lower quality. From a forageproduction perspective, the maximum potentia l yield of alfalfa attainable is not raised; rather, growers can more readily re ach the higher end of the potential yield range while maintaining a ta rgeted quality standard.

 Tolerating unexpected harves t delays: Unexpected delays in harvesting occur occasionally and are usually due to an untimely event such as rain, equipment failure, or the pressures of competing farming activities, e.g. , labor availability and dairy herd management. During the dela y period, forage quality often declines rapidly, leading to potential financial loss. A grower has more flexibility to withstand short delays in forage harves t with KK179, because there is less lignin accumulation during the delay period and, thus, less loss of quality by the time harvesting is resumed.

I.B.1 Plant lignin biosynthesis

An understanding of lignin bios ynthesis in alfalfa has pr ovided the means to reduce lignin levels and slow the accumulation of ligni n during the alfalfa growth cycle. Lignin is a high molecular weight, polymeric mol ecule composed princi pally of three lignin monomeric subunits: guaiacyl (G), syringyl (S), and p-hydroxyphenyl (H) lignin (Figure I-1) (Boerjan et al., 2003; Vanholme et al., 2010). The relative proportion of each lignin monomer can vary with plant species and tissue type (Boerjan et al., 2003). In alfalfa, G lignin and S lignin subunits co mprise up to 95% of the li gnin subunits. In the lignin biosynthetic pathway, the formation of the G an d S lignin subunits requires the activity of two O-methyltransferase enzymes for lignin biosynthesis, caffeoyl CoA 3-O-methyltransferase (CCOMT) and caffeic acid 3-O-methyltransferase (COMT). O-methyltransferases are a large family of enzymes that methylate the oxygen atom of secondary metabolites such as phenylpropanoid s, flavonoids, and alkaloids (Lam et al., 2007). CCOMT methylates caffeoyl CoA in the lignin biosynthetic pathway to produce feruloyl CoA acid, while COMT methylates caffeyl aldehyde to produce coniferyl aldehyde, and methylates 5-hydroxyconifery l aldehyde to produce sinapyl aldehydeMonsanto Company



### (Figure I- 1). Current literature on lignin production in alfalfa indicates that the COMT enzyme is specifically involved CCOMT enzyme acts in a parallel manner to form G lignin monomers (Figure I 

### et al., 2001; Zhou et al., 2010 principal enzyme to target suppression in order to lower the production of G lignin



### subunits.

Figure I-1. Lignin biosynthetic pathway

PAL: phenylalanine

C4H: cinnamate

4CL: 4- coumarate: CoA ligase;

HCT: hydroxycinnamoyl

C3H: p- coumarate

CCOMT: caffeoyl

COMT: caffeic acid
CCR1, CCR2: cinnamoyl

F5H: ferulate 5

CAD1: cinnamyl alcohol dehydrogenase; Reactions shown by dotted lines occur at very low rates in the wild type Shaded arrows indicate committed steps in the production of monolignin subunits.

12-AL-246U 

### 1). Current literature on lignin production in alfalfa indicates that the COMT enzyme is specifically involved in the formation of S lignin monomers while the CCOMT enzyme acts in a parallel manner to form G lignin monomers (Figure I

Zhou et al., 2010 

### ) . Of the two enzymes, CCOMT was identified as the principal enzyme to target suppression in order to lower the production of G lignin

Lignin biosynthetic pathway phenylalanine ammonia lyase;

cinnamate -4-hydroxylase; coumarate: CoA ligase; hydroxycinnamoyl

- CoA: shikimate hydroxycinnamoyl transferase;

coumarate -3-hydroxylase;

- CoA 3- O -methyltransferase;

caffeic acid 3- O -methyltransferase; cinnamoyl -CoA reductase;

ferulate 5 -hydroxylase; cinnamyl alcohol dehydrogenase; Reactions shown by dotted lines occur at very low rates in the wild type ( Zhou et Shaded arrows indicate committed steps in the production of monolignin subunits.

31 of 407 

### 1). Current literature on lignin production in alfalfa indicates that the COMT in the formation of S lignin monomers while the CCOMT enzyme acts in a parallel manner to form G lignin monomers (Figure I 

### -1) (Guo . Of the two enzymes, CCOMT was identified as the principal enzyme to target suppression in order to lower the production of G lignin

CoA: shikimate hydroxycinnamoyl transferase;

Zhou et al., 2010); Shaded arrows indicate committed steps in the production of monolignin subunits.Monsanto Company 12-AL-246U 32 of 407 

### I.B.2 Mode-of-Action and Evidence of Suppression of the CCOMT Gene

I.B.2.1 Mode-of-Action of KK179

The suppression cassette in KK179 functions by reducing the level of G lignin subunits, which are oxidatively coupled to other ligni n subunits to form complex lignin molecules (Boerjan et al., 2003). This specific reduction in G ligni n is achieved through use of endogenous alfalfa gene segments configured to suppress the CCOMT gene in order to lower CCOMT protein expression and thereby decrease the s ynthesis of G lignin (Figure I-2). KK179 contains CCOMT gene segments under the control of the Pal2 promoter from the phenylalanine ammonia-lyase gene in bean ( Phaseolus vulgaris ). PAL expression responds to endogenous cues for va scularisation and displays a pattern of expression that corresponds with sites of li gnin deposition in maturing plants (Guo et al., 2001; Leyva et al., 1992). Thus, KK179 transg ene expression correl ates with tissues where higher lignin deposition is observed. The assembled CCOMT gene segments produce a transcript with an inverted re peat sequence to form double-stranded RNA (dsRNA), which works via the RNA interferen ce mechanism to suppress the endogenous

CCOMT gene (Siomi and Siomi, 2009).

The RNAi mechanism is a natural process in eukaryotic organisms for regulation of gene expression (Dykxhoorn et al., 2003; Parrott et al., 2010). The dsRNA molecule that activates the mechanism is first processed by a class of RNAse III enzymes called Dicers into (~21-24 nucleotides) small interfering RNAs (siRNAs) (Hammond, 2005; Zamore et al., 2000). The resulting siRNA molecules are then incorporated into multiprotein RNAinduced silencing complexes (RISC) which facilitate target se quence recognition and mRNA cleavage (Hammond, 2005; Tomari and Zamore, 2005), in this case the degradation of CCOMT transcripts. The final outcome of this process is the suppression of the target CCOMT mRNA.

When CCOMT activity is reduced, an alternativ e path in the lignin biosynthetic pathway allows S lignin biosynthesis to continue through the conversion of caffeoyl-CoA to caffeyl aldehyde by the CCR2 enzyme (Zhou et al., 2010). As a result, the effect of CCOMT suppression is limited to lowering G lignin production. The decrease in actual amount of G lignin results in a lower proportion of G lignin re lative to all subunits. The decrease in G lignin also results in an increa se in the proportion of S lignin relative to all subunits, but not an increase in the actual amount of S lignin. These changes in subunit proportions result in an incr ease in the S:G lignin ratio, wh ich is charac teristic of CCOMT suppression (C hen et al., 2006).Monsanto Company



### Figure I-2. Modified lignin biosynth Enzymes in pathway are listed in indicates the suppression of CCOMT enzyme activity. Reactions shown by dotted lines occur at very low rates when CCOMT activity is reduced (Zhou et al., 2010).



### I.B. 2.2 Evidence of Suppression of the CCOMT Gene in Forage and Root

Northern blot analyses were used to compare the RNA levels of the endogenous gene in forage and root tissues of KK179 and conventional alfa 

### enriched RNA (polyA +


### samples of KK179 and the conventional control A CCOMT probe generated from a portion of the northern blots in order to compare the conventional control at an equivalent growth stage. Equivalent RNA loading and quality between the conventional control and KK179 was evaluated using an alfalfa actin p as an endogenous control. The northern blot data demonstrated a clear reduction in the



### level of CCOMT RNA in KK179 compared to the conventional control in both forage and root tissue. The details of the materials and methods are described in Appendi

12-AL-246U 

### Modified lignin biosynth 

### etic pathway Enzymes in pathway are listed in Figure I

- 1 legend: indicates the suppression of CCOMT enzyme activity. Reactions shown by dotted lines occur at very low rates when CCOMT activity is reduced



### 2.2 Evidence of Suppression of the CCOMT Gene in Forage and Root

Northern blot analyses were used to compare the RNA levels of the endogenous gene in forage and root tissues of KK179 and conventional alfa lfa. Polyadenylation 

### RNA), extracted from four replicate forage and root tissue samples of KK179 and the conventional control



### , was subjected to northern blot analyses. A CCOMT probe generated from a portion of the 

### CCOMT gene was hybr northern blots in order to compare the 

### CCOMT RNA levels in KK179 and the conventional control at an equivalent growth stage. Equivalent RNA loading and quality between the conventional control and KK179 was evaluated using an alfalfa actin p as an endogenous control. The northern blot data demonstrated a clear reduction in the

RNA in KK179 compared to the conventional control in both forage and root tissue. The details of the materials and methods are described in Appendi

33 of 407 Reactions shown by dotted lines occur at very low rates when CCOMT activity is reduced



### 2.2 Evidence of Suppression of the CCOMT Gene in Forage and Root

Northern blot analyses were used to compare the RNA levels of the endogenous 

### CCOMT lfa. Polyadenylation extracted from four replicate forage and root tissue

was subjected to northern blot analyses.

gene was hybr 

### idized to the

RNA levels in KK179 and the conventional control at an equivalent growth stage. Equivalent RNA loading and quality between the conventional control and KK179 was evaluated using an alfalfa actin p 

### robe as an endogenous control. The northern blot data demonstrated a clear reduction in the

RNA in KK179 compared to the conventional control in both forage and root tissue. The details of the materials and methods are described in Appendi 

### x C.Monsanto Company 12-AL-246U 34 of 407 

### I.B.2.2.1. Northern Blot Analysis of CCOMT RNA in Forage

PolyA + 

### RNA from each of four replicate sample s of forage tissue from the conventional control produced a strong hybr idization signal at ~1.1 kb, as expected, based on the predicted transcript size of CCOMT (Figure I-3, Panel A, Lanes 1, 3, 5, and 7), whereas no detectable hybridization signal was produced from the polyA + 

### RNA isolated from the forage tissue of KK179 (Figure I-3, Panel A, Lanes 2, 4, 6, and 8). These data

demonstrate a clear redu ction in the level of CCOMT RNA in KK179 compared to the

conventional control.

The CCOMT probe was stripped from the blot and the stripped blot was hybridized with the actin probe. The polyA + 

### RNA from the forage tissue of the conventional control (Figure I-3, Panel B, Lanes 1, 3, 5, and 7) and KK179 (Figure I-3, Panel B, Lanes 2, 4, 6, and 8) showed a strong hybr idization signal at ~1.5 kb , as expected for the actin transcript. The hybridization signals from th e forage tissue of the conventional control and KK179 of each replicate have similar in tensities, indicating that the RNA loading, RNA quality, and hybridization within each re plicate of the conventional control and KK179 were similar. When hybridized with th e actin probe template, in addition to the expected ~0.5 kb band, a very faint ~1.0 kb ba nd was detected (Figur e I-3, Panel B, Lane 11). This band most likely resulted from a hybridization of the actin probe to single stranded DNA formed during probe template purification (Qiage n, 2008) that has partially reannealed in various confirmations (Kasuga et al., 2001). Since the actin probe template loaded in this lane served as a pos itive hybridization contro l and showed that the probe hybridized to the target sequence, the presence of the faint ~1.0 kb band has no impact on the conclusions drawn from this an alysis. Therefore, the difference in the

CCOMT hybridization signals between the conv entional control and KK179 reflects the

difference in the CCOMT RNA levels (Figure I-3, Panel A).

I.B.2.2.2. Northern Blot Analysis of CCOMT RNA in Root

PolyA + 

### RNA from each of four replicate sample s of root tissue from the conventional control produced a strong hybrid ization signal at ~1.1 kb, as expected, based on the predicted transcript size of CCOMT (Figure I-4, Panel A, Lanes 1, 3, 5, and 7), whereas a greatly reduced signal wa s produced from the polyA + 

### RNA isolated from the root tissue of KK179 (Figure I-4, Panel A, Lanes 2, 4, 6, and 8). These data demonstrate a clear

reduction in the level of CCOMT RNA in KK179 compared to the conventional control.

The CCOMT probe was stripped from the blot and the stripped blot was hybridized with the actin probe. The polyA + 

### RNA from the root tissue of th e conventional control (Figure I-4, Panel B, Lanes 1, 3, 5, and 7) and KK179 (Figure I-4, Panel B, Lanes 2, 4, 6, and 8) showed a strong hybridization signal at ~1.5 kb, as expected for the actin transcript. The hybridization signals from the root tissue of the conventional control and KK179 of each replicate sample have similar intensities, indicating that the RNA loading, RNA quality, and hybridization within each replicate of the conventional control and KK179 are similar. As with the forage tissue analysis, a very faint ~1.0 kb ba nd detected (Figure I-4, Panel B, Lane 11) when hybridized with the actin probe template was not considered to have an impact on the conclusi ons drawn from this analysis. Therefore, the difference in

the CCOMT hybridization signal intensities between the conventional control and KK179Monsanto Company 12-AL-246U 35 of 407 

### reflect the difference in CCOMT RNA levels (Figure I-4, Panel A). In addition to the ~1.5 kb actin transcript, faint ~1.1 kb bands we re observed (Figure I-4, Panel B, Lanes 1, 3, 5, and 7). These faint bands likely resu lted from the incomplete removal of the CCOMT probe on the stripped blot prior to pr obing with the actin probe. The expected

~1.5 kb actin transcript is larg er than the ~1.1 kb CCOMT transcript. Therefore, the incomplete removal of the CCOMT probe had no impact on actin probe hybridization and no impact on the conclusions made from this analysis.Monsanto Company 12-AL-246U 36 of 407 

### Figure I-3. Northern Blot Analysis of CCOMT RNA Level in KK179 Forage Tissue

Panel A and Panel B are the same northern blot containing polyA + 

### RNA isolated from forage tissue of the conventional control and KK179. Panel A was hybridized with the CCOMT probe. Panel B was hybridized with the actin probe after stripping the CCOMT probe from the blot. Arrow heads indicate the CCOMT hybridization signal and stars

indicate the actin hybridization signal. Lane designations are as follows:

Lane 1 Conventional control (Replicate 1) 2 KK179 (Replicate 1) 3 Conventional control (Replicate 2) 4 KK179 (Replicate 2) 5 Conventional control (Replicate 3) 6 KK179 (Replicate 3) 7 Conventional control (Replicate 4) 8 KK179 (Replicate 4)
9 Empty 10 CCOMT probe template (5 pg) 11 Actin probe template (10 pg) Arrows denote the size of the RNA, in kilobase pairs, obtained from the RiboRuler High Range RNA Ladder on the ethidium stained gel. 

### 1 2 3 4 5 6 7 8 9 10 11

6.0
4.0 3.0 2.0 1.5 1.0

0.5

0.2



## A

* * 6.0

4.0 3.0 2.0 1.5 1.0

0.5
0.2 1 2 3 4 5 6 7 8 9 1011 

## B

*Monsanto Company 12-AL-246U 37 of 407 

### Figure I-4. Northern Blot Analysis of CCOMT RNA Level in KK179 Root tissue

Panel A and Panel B is the same northern blot containing polyA + 

### RNA isolated from root

tissue of the conventional control and KK179. Panel A was hybridized with the CCOMT probe. Panel B was hybridized with the ac tin probe after stripp ing the CCOMT probe

from the blot. Arrow heads indicate the CCOMT hybridization signal and stars indicate

the actin hybridization signal. Lane designations are as follows:

Lane 1 Conventional Control (Replicate 1) 2 KK179 (Replicate 1) 3 Conventional control (Replicate 2) 4 KK179 (Replicate 2) 5 Conventional control (Replicate 3) 6 KK179 (Replicate 3) 7 Conventional control (Replicate 4) 8 KK179 (Replicate 4)
9 Empty 10 CCOMT probe template (5 pg) 11 Actin probe template (10 pg) Arrows denote the size of the RNA, in kilobase pairs, obtained from the RiboRuler High Range RNA Ladder on the ethidium stained gel. 

### 6.0
4.0 3.0 2.0 1.5 1.0

0.5

0.2 1 2 3 4 5 6 7 8 9 10 11 

## A

6.0
4.0 3.0 2.0 1.5 1.0

0.5

0.2 1 2 3 4 5 6 7 8 9 10 11



## * * B

*Monsanto Company 12-AL-246U 38 of 407 

### I.B.3. Intended Changes to Lignin Levels in KK179 Forage

As described in Section I.B.2.1, KK179 reduces G lignin levels th rough the suppression of caffeoyl CoA 3- O -methyltransferase (CCOMT), a key enzyme in the lignin biosynthetic pathway. Suppre ssion of CCOMT acts to decr ease the amount of G lignin, resulting in a lower proportion of G lignin and a greater proportion of the other major lignin subunit, S lignin. The ch ange in lignin subunit proporti ons can be identified as a change in the ratio of S lignin levels to G lignin levels, or S:G ratio, which is characteristic of CCOMT suppression in alfa lfa (Chen et al., 2006). The reduction in G lignin in turn leads to reduced total lignin levels in forage compared to conventional alfalfa at the same stage of growth.

To demonstrate that the suppression of CCOMT in KK179 results in the intended reduction of the G lignin subunit, lignin subun it compositional analyses were conducted. Forage samples were collected from KK179 , a conventional alfalfa control, and conventional commercial alfalfa varieties gr own in the United States from the first cutting of a 2011 field producti on, described in Appendix D. The conventional control (C 0 

### -Syn1) used as a comparator was a near-i sogenic conventional al falfa population with a genetic background similar to that of KK179. Fourteen different conventional

commercial alfalfa varieties were included acr oss the field production to provide data on the natural variability of each compositiona l component analyzed. Field production of forage was conducted in typical alfalfa growing regions at six sites lo cated in California (CAPR); Iowa (IARL); Illinois (ILCY); Kans as (KSLA); Texas (TXCL); and Wisconsin (WIDL). KK179, conventional control and c onventional commercial varieties were planted in a randomized complete block design with four re plicated plots per site and grown under normal agronomic field conditions for their respective geographic regions. At the plant growth stage between 1 a nd 10% bloom, which is a normal stage for harvesting forage, samples of the whole alfalf a plant, 2-3 inches a bove the soil surface, were harvested at each site from the plan ts in the center of each individual plot.

The compositional analysis compared levels of the lignin subunits p- hydroxyphenyl lignin (H lignin), guaiacyl lignin (G lignin), syringyl lignin (S lignin), caffeyl lignin (derived from caffeyl aldehyde, Figure I-1), and 5-hydroxyguaiacyl lignin (derived from 5-hydroxy coniferyl aldehyde, Figure I-1). This analysis was performed by researchers at the Samuel Roberts Noble Foundation (Ardmore , OK). The method used to measure the lignin subunits, described in Appendix D, gene rated values expressed as μmol/g cell wall residue (CWR). Two lignin subunits, ca ffeyl lignin and 5-hydr oxyguaiacyl lignin, had

more than 50% of the observations below th e assay limit of quantit ation (LOQ) and, as a result, were excluded from the statistical an alyses. The S:G lignin ratio was calculated

from the values of the indivi dual components expre ssed as μmol/g CWR. The H, G, and S lignin values were expressed as proportions of each individual lignin subunit calculated as a percentage of the sum total of H, G, and S lignin (total HGS lignin).

To confirm that the reduction in G lignin leads to reduced tota l lignin in forage, levels of total lignin, as measured by ADL, were determined on the same samples. Forage samples of KK179 and the conventional control from the first cutting in 2011 at six sites, as described in Appendix D, were analyzed by the Forage Lab at Dairy One Cooperative, Inc., (hereafter referred to as Dairy One Fora ge Lab), a facility certified for analyticalMonsanto Company 12-AL-246U 39 of 407 

### assessments of forage quality by the National Forage Testing Associ ation (NFTA). Dairy One Forage Lab used a semi-automated ANKOM-based methodol ogy, described in Appendix D, as adopted by most commerc ial forage testing laboratories.

I.B.3.1. Intended Changes to Lignin Subunits H, G, and S in KK179 Forage

Assessment of KK179 lignin subunit composition compared to the conventional control showed a statistically significant (p<0.05) decrease in G li gnin when expressed as μmol/g CWR (Table I-1). The mean value of G lignin for KK179 was 68.10 μmol/g CWR, a decrease of 15.62 μmol/g CWR or 18.66% compar ed to the conventional control. As a result, the S:G ratio increased from 0.58 in the conventional control to 0.80 in KK179 (Table I-1), as predicted (Chen et al ., 2006). The proportion of G lignin for KK179, expressed as a percentage of total HGS lignin in KK179, was 53.69%, a relative decrease of 12.96% compared to the conventional contro l (Table I-2). These results support the conclusion that suppression of CCOMT in KK179 decreases the pr oduction of G lignin, resulting in a lower proportion of G lignin in total HGS and an increase in the S:G ratio compared to the conventional control.

I.B.3.2. Intended Changes to Total Lignin Levels in KK179 Forage

The NFTA-certified Dairy One Forage Lab ut ilizes standards and methods of analysis representative of those commonly adopted by the forage industry to measure forage quality-related parameters, including total li gnin (ADL). It is on the basis of these methods that quality of forage produced by growers and purchased by users is routinely determined; thus the commercia l value of the forage as f eed is determined. The ANKOM method, which has been adopt ed by most commercial fora ge testing laboratories, measures acid detergent lignin based on pro cedures developed by Goering and Van Soest (1970). These procedures involve a series of washes that expose the sample first to an acid detergent solution, then to acetone, foll owed by sulfuric acid, to gravimetrically

determine the amount of insoluble residue remaining. Compositional analysis of KK179 forage samples at the Dairy One Forage Lab confirmed the reduction in total lignin (ADL) levels. The mean value of to tal lignin (ADL) for KK179 was 5.39% dw, a decrease of 22.15% (p<0.05) from the mean value of 6.93% dw for the conventional control (Table I-3). These results confirm that commercial forage testing labs observe a measurable reduction in total lignin (ADL) in KK179, relative to conventional alfalfa harvested at the same stage of growth.Monsanto Company 12-AL-246U 40 of 407Table I-1. Alfalfa Forage LigninSubunit Levels and S:G Ratio for KK179 vs. Conventional ControlDifference (Test minus Control)Analytical Component (Units)1KK179Mean (S.E.)2(Range)Control3Mean (S.E.)(Range)Mean (S.E.)(Range)95%ConfidenceIntervalSignificance(p-Value)CommercialToleranceInterval4(Range)Lignin Subunits (μmol/g CWR)Guaiacyl (G) lignin68.10 (9.48)83.72 (9.40)-15.62 (6.12)-29.16, -2.070.0278.83, 176.39(21.17 - 134.96)(33.11 - 131.40)(-39.11 - 27.03)(25.34 - 153.11)Syringyl (S) lignin55.96 (8.83)50.41 (8.78)5.55 (5.11)-5.82, 16.920.3020, 120.96(9.82 - 87.67)(12.20 - 91.89)(-18.80 - 43.57)(5.64 - 110.93)p-Hydroxyphenyl (H) lignin5.05 (0.45)3.88 (0.43)1.17 (0.60)-0.16, 2.500.0771.59, 6.91(2.20 - 10.84)(0.58 - 5.49)(-1.76 - 7.24)(0.29 - 8.26)Syringyl to Guaiacyl Subunit ComparisonS:G Ratio0.80 (0.061)0.58 (0.060)0.22 (0.027)0.16, 0.29<0.0010.21, 0.96(0.43 - 1.16)(0.35 - 0.70)(-0.16 - 0.50)(0.22 - 0.92)1 CWR = Cell Wall Residue; S:G Ratio = Syringyl lignin subunit divided by Guaiacyl lignin subunit2Mean (S.E.) = least-square mean (standard error)3Control refers to the conventional alfalfa control, C0-Syn1.4With 95% confidence, interval contains 99% of the values expressed in the population of commercial alfalfa varieties. Negativelimits set to zero.Monsanto Company 12-AL-246U 41 of 407Table I-2. Alfalfa Forage Lignin SubunitLevels as Percent of Total HGS for KK179vs. Conventional ControlDifference (Test minus Control)Analytical Component(Units)1KK179Mean (S.E.)2(Range)Control3Mean (S.E.)(Range)Mean (S.E.)(Range)95%ConfidenceIntervalSignificance(p-Value)CommercialTolerance Interval4(Range)Lignin Subunits (% Total HGS)Guaiacyl (G) lignin53.69 (1.87)61.69 (1.87)-8.00 (0.71)-9.42, -6.58<0.00146.69, 76.44(44.92 - 63.78)(56.88 - 70.56)(-14.63 - 4.22)(50.02 - 76.69)Syringyl (S) lignin42.09 (2.35)35.24 (2.35)6.85 (0.75)5.34, 8.36<0.00117.39, 53.32(26.98 - 52.01)(24.60 - 40.26)(-6.77 - 13.61)(17.07 - 46.14)p-Hydroxyphenyl (H) lignin4.22 (0.54)3.07 (0.54)1.15 (0.28)0.53, 1.760.0010, 6.74(2.04 - 9.78)(0.34 - 5.18)(-0.85 - 4.60)(0.18 - 6.23)1Total HGS is the sum of Hydroxyphenyl (H), Guaiacyl (G) and Syringyl (S) lignin subunits (μmol/g CWR); CWR = Cell Wall Residue2Mean (S.E.) = least-square mean (standard error)3Control refers to the conventional alfalfa control, C0-Syn1.4With 95% confidence, interval contains 99% of the values expressed in the population of commercial alfalfa varieties. Negative limits set tozero.Monsanto Company 12-AL-246U 42 of 407Table I-3. Alfalfa Forage Total Lignin (ADL) Levels for KK179 vs. Conventional ControlDifference (Test minus Control)Analytical Component(Units)1KK179Mean (S.E.)2(Range)Control3Mean (S.E.)(Range)Mean (S.E.)(Range)Relative %DifferenceSignificance(p-Value)CommercialTolerance Interval4(Range)Acid Detergent Lignin5(% dw)5.39 (0.64)6.93 (0.64)-1.53 (0.42)-22.150.0041.39, 12.54(2.73 - 7.60)(2.23 - 10.10)(-4.33 - 1.30)(1.70 - 10.03)1dw = dry weight2Mean (S.E.) = least-square mean (standard error)3Control refers to the conventional alfalfa control (C0-Syn1).4With 95% confidence, interval contains 99% of the values expressed in the population of commercial alfalfa varieties. Negative limits set tozero.5ADL determined using the semi-automated ANKOM method (Weston et al., 2006).Monsanto Company 12-AL-246U 43 of 407 

### I.C. Submissions to Other Regulatory Agencies

Under the Coordinated Framework for Regul ation of Biotechnology, the responsibility for regulatory oversight of biotechnology-derived crops fa lls primarily on three U.S. agencies: the U.S. Food and Drug Administ ration (FDA), the United States Department of Agriculture (USDA), and in the case of plant-incorporated protectants, the Environmental Protection Agency (EPA). Deregulation of KK179 by USDA constitutes only one component of the overall regulatory overs ight and review of this product. As a practical matter, KK179 cannot be released and marketed until FDA has completed its

review and assessment under it s respective jurisdiction.

I.C.1. Submission to FDA

KK179 falls within the scope of the 1992 FDA policy statement concerning regulation of products derived from new plant varietie s, including those developed through biotechnology (U.S. FDA, 1992). In complia nce with this policy, Monsanto and FGI

have initiated a consultation with the FD A on KK179, identified under BNF No. 138. A feed/food safety and nutritional assessment summary document was submitted in August

2012.

I.C.2. Submissions to Forei gn Government Agencies

Consistent with their commitments to the Biotechnology Indus try Organization’s Excellence Through Stewardship Program, Mons anto and FGI intend to obtain import approvals from all key alfalfa import market s with functioning regu latory systems prior to commercial planting of KK179. As a ppropriate, notifications will be made to countries that import significan t quantities of alfalfa and al falfa products and do not have formal regulatory review processe s for biotechnology-derived crops.Monsanto Company 12-AL-246U 44 of 407 

### II. THE BIOLOGY OF ALFALFA

The Canadian Food Inspection Agency docum ent on the biology of alfalfa, Biology Document Bio 2005-02 (CFIA, 2005), provides key information on:

- general description of alfalfa biology

- use of alfalfa as a crop plant

- geographic centers of origin

- taxonomy and genetics

- reproductive biology

- breeding and seed production

- gene flow

- inter-species/genus introgression

- interactions with other organisms

- summary of the ecology of alfalfa

An extensive review of the history a nd biology of alfalf a can be found in Alfalfa and Alfalfa Improvement (Hanson et al., 1988) and in the USDA Final Environmental Impact Statement on Roundup Ready alfalfa (USDA-AP HIS, 2010). Taxonomic information for alfalfa is available in the U.S. Depart ment of Agriculture’s Natural Resources

Conservation Service PLANTS database (USDA-NRCS, 2012).

To support the evaluation of the plant pest potential of KK179 rela tive to conventional alfalfa, additional information regarding seve ral aspects of alfalfa biology can be found elsewhere in this petition. This includes: ag ronomic practices for alfalfa in Section VIII; volunteer management of alfalfa in Section VIII; and inter-species/genus introgression potential in Section IX.D.

II.A. Alfalfa as a Crop

Alfalfa ( Medicago sativa L.), including both cultivated alfalfa and closely related subspecies, originated in Asia Minor, Tran scaucasia, Turkmenista n, and Iran. With the spread of agriculture, alfalfa became e ndemic throughout the Mediterranean region, North Africa, the Middle East, most of Eu rope, Siberia, northern India, and China (Michaud et al., 1988; Quiros and Bauchan, 1988). Also known as lucerne, it has the longest history of any plant grown solely fo r forage (Michaud et al., 1988). Due to its importance as an animal feed, it has sp read globally and become acclimatized in Australia, New Zealand, North America, South America, and South Africa.

Alfalfa belongs to the order Fabales, family Fabaceae , tribe Trifolium , genus Medicago

. Medicago includes more than 80 described speci es, including perennials and annuals (Small and Jomphe, 1989; Steele et al., 2010). Alfalfa is a perennial legume species composed of several subspecies of the same ka ryotype with the abilit y to cross with each other, including subsp. sativa , subsp. falcata , subsp. coerulea , and subsp. glomerata (Chandra et al., 2011; Quiros and Bauchan, 1988; Small and Jomphe, 1989; USDA-ARS, 2007). In addition, the crossbreeds produ ced by crossing of the above mentioned subspecies, including subsp. varia , subsp. hemicycla, and subsp. tunetana are alsoMonsanto Company 12-AL-246U 45 of 407 

### classified into Medicago sativa L. (Quiros and Bauchan, 1988). The subspecies are classified according to ploidy level, flower color, and shape of seedpod. Most alfalfa cultivated worldwide is the tetraploid (2n=4x=32) M. sativa L. subsp. sativa, which produces predominantly violet flowers and coiled seedpods. The tetraploid M. sativa L. subsp. falcata L., which produces yellow flowers and straight or sickle-shaped seedpods, has been mostly used for selective breeding with M. sativa L. subsp. sativa to improve cold-tolerance, drought tolera nce, and disease resistance (Quiros and Bauchan, 1988). Modern alfalfa cultivars, especially those adapted to temperate growing zones, contain germplasm originally derived from both subspecies sativa and falcata

.

Early alfalfa breeding efforts were dedicated to collecting, evaluating, and comparing various sources of germplasm around the worl d. A key milestone in advancing alfalfa breeding and development was reached in the early 20 th 

### century with the development of a system for classifying alfalfa germplasm in to several distinct fall dormancy groups and

selection of more winterhar dy types within each group (Melt on et al., 1988). This system made it possible to separate fall dormancy fr om winter hardiness. In recent years, a better understanding of autotetr aploid genetics and its e ffects on breeding and variety synthesis has improved genetic gains for fo rage yield (Rumbaugh et al., 1988). Commercial alfalfa breeding programs focus on developing varieties with improved characteristics in several major areas, including: 1) greater resistance to insects, nematodes, and diseases; 2) gr eater yield potential; 3) improve d stand persistence, and 4) increased forage quality (Putnam et al., 2008a; Undersander et al., 2011).

Cultivated alfalfa is widely adapted, allowi ng production across varying climatic regions and geographies under both irriga ted and non-irrigated systems. It is typically planted to establish perennial stands that remain in th e field from three to seven years, depending upon geography and agronomic practice. Forage is harvested from tw o to eleven times per season, depending on the region and the sy stem of harvest management. In certain regions, alfalfa is cultivated as a mixture with perennial grasses, where it may be harvested as forage or used for grazing live stock. As a legume, it is also desired for rotational use to improve soil characteristics such as nitrogen conten t (Undersander et al., 2011).

Alfalfa is among the most important forage cr ops in the United States and ranks as the fourth most widely grown crop by acr eage, after corn, soybean, and wheat. Approximately 20 million acres of alfalfa and alfalfa-grass mixtures were grown for hay in 2010 across most states, with the highest ac reages harvested in California, Colorado, Idaho, Iowa, Michigan, Minnesota, Montana, Nebraska, North Da kota, South Dakota, and Wisconsin (USDA-NASS, 2012a). Alfalfa hay production in 2010 totaled 67,971,000 tons and was valued at approximately $8 billion USD, ranking fourth overall among agriculture crops in terms of total value (USDA-NASS, 2012b).

A small amount of conventional alfalfa is consumed by humans in the United States, predominately in the form of sprouts, but al so as dietary supplements and herbal teas. KK179 does not present any concerns with respect to human consumption; however, its intended commercial use will be forage pr oduction, which relies on treated seed and agronomic practices that are incompatible with non-forage purposes. Monsanto and FGIMonsanto Company 12-AL-246U 46 of 407 

### do not permit commercially sold Roundup Ready alfalfa seed to be used for sprout production (Monsanto Company, 2012), which is a restriction enforced through signed agreements between Monsanto/FGI and seed purch asers. This same restriction will apply to KK179 seed as a commercial product.

II.B. Characteristics of the Recipient Plant

The alfalfa plant used as the recipient of the DNA insertion to create KK179 was R2336, a conventional FGI proprietary plant (propagated vegetatively via stem cuttings), and

selected for ease of transformation from an elite, high-yielding breeding population. A single, transformed R2336 alfalfa plant (T 0 

### ) was crossed with Ms208, a conventional male-sterile alfalfa plant (propogated vege tatively via stem cuttings) to produce F 1 

### progeny plants. A single plant (P 0 

### ) was selected from these progeny plants (Figure IV-11) and used for mol ecular characterization of the F 1 

### generation, as described

in Section IV.

Due to inbreeding depression and self-incom patibility in an outcrossing species like alfalfa, it is not possible to breed pure isoge nic lines by self-pollination. Therefore, the subsequent generations were developed following traditional population breeding techniques for development of commercial alfalfa varieties (Figure IV-11). The P 0 

### plant, containing KK179, was hand-crossed with each of 10 elite alfalfa genotypes with a fall dormancy 4 phenotype (FD4) to produce the next generation; these FD4 plants were used as the female seed parents. This breedi ng step, known as a modified backcross (MBC), resulted in the first KK179 population with re lated individuals (MBC1). Repeating this step by hand crossing the MBC1 generation w ith the same 10 elite alfalfa genotypes with the FD4 phenotype resulted in the MBC2 ge neration, again using FD4 plants as the female seed parents. Fina lly, a population of MBC2 genera tion plants (N=80) was hand

crossed inter se (with itself) in a breeding step known as a polycross. The resulting progeny were the Syn1 generation, as they are the first synthetic population of KK179 and the preferred population for entry into comm ercial variety development. Analyses of the expressed products, described in S ection I.B.2.2., and composition of KK179, described in Section VI.B., were conducted with the Syn1 generation. Null plants, (individuals without KK179) as determined by event-specific PCR analysis, were removed at each generation prior to crossi ng in the KK179 synthetic populations. All

hand-crosses were cond ucted in a greenhouse.

II.C. Alfalfa as a Test System in Product Safety Assessment

The identical breeding process descri bed above was followed using the C 0 

### plant in order to produce a C 0 

### -Syn1 generation, which is a conventi onal synthetic population, to serve as the conventional comparat or for the Syn1 generation, re spectively. A single, nontransformed R2336 plant was crossed w ith Ms208 to produce conventional F 1 

### progeny plants, from which a conven tional alfalfa comparator C 0 

### was selected. The same breeding process used to develop subsequent generations of KK179 was then followed to develop subsequent generations of near-is ogenic conventional alfalfa comparators for each generation of KK179 as shown in Figure IV-11. As a result, all generations ofMonsanto Company 12-AL-246U 47 of 407 

### KK179 and the conventional controls used in the safety assessment studies have closely related genetic backgrounds, w ith the exception of the intended trait for reduced G lignin and total lignin. The C 0 

### and C 0 

### -Syn1 plants are both referre d to as the conventional

control. R2336 and Ms208 are referred to as conventional pa rental controls.

In addition, conventional commercial alfa lfa varieties (referred to as conventional commercial reference varieties), were used to establish ranges of natural variability representative of commercial alfalfa variet ies. The conventiona l commercial reference

varieties used at each location were chosen based on their availa bility and agronomic adaptation for the respective geographic region.Monsanto Company 12-AL-246U 48 of 407 

### III. DESCRIPTION OF TH E GENETIC MODIFICATION

Molecular analyses are an integral part of the characterization of crop products with new traits introduced by methods of biotechnol ogy. Vectors and methods are selected for transformation to achieve high probability of obt aining the trait of inte rest and integration of the introduced DNA into a single locus in the plant genome. This helps ensure that only the intended DNA encoding the desired tra it is integrated into the plant genome and

facilitates the molecular characterization of the product. Information provided here allows for the identification of the genetic ma terial present in the transformation vector delivered to the host plant and for an analysis of the data supporting the characterization of the DNA inserted in the pl ant found in Section IV.

KK179 was developed through Agrobacterium tumefaciens -mediated transformation of

conventional alfalfa R2336 l eaf tissue utilizing plasmi d vector PV-MSPQ12633. This section describes the plasmid vector, the donor genes, and the regulatory elements used in the development of KK179. In this section, tr ansfer DNA (T-DNA) refers to DNA that is transferred to the plant during transformati on. The suppression cassette refers to the sequences and regulatory elements necessa ry for the suppression of the endogenous

CCOMT RNA transcript. An expression cassette refers to the sequences and regulatory elements necessary for the expression of those sequences.

III.A. The Plasmid Vector PV-MSPQ12633

PV-MSPQ12633 was used for the transformati on of conventional alfalfa to produce KK179 and is shown in Figure III-1. PV -MSPQ12633 is approximately 10.6 kb in length and contains two T-DNAs, each delineated by Le ft and Right Border regions to facilitate transformation. The first T-DNA, de signated as T-DNA I, contains the CCOMT suppression cassette, whic h is regulated by the Pal2 promoter and the nos 3 ′ UTR. The second T-DNA, designated as T-DNA II, contains the nptII expression cassette, which is regulated by the 35S promoter and the nos 3 ′ UTR. During transformation, both T-DNAs

were inserted into the alfalfa genome (Section IV.B.) where T-DNA II, containing the nptII expression cassette, functioned as a marker gene for the selection of transformed plantlets. Subsequently, tr aditional breeding methods a nd segregation, along with a combination of analytical techniques, were used to isolate those plants that contained the

CCOMT suppression cassette (T-DNA I) but did not contain the nptII expression cassette (T-DNA II).

The backbone region of PV-MSPQ12633, which is located outside both of the T-DNAs contains two origins of replication ( oriV and ori-pUC ) for maintenance of the plasmid vector in bacteria, a bacteria l selectable marker gene ( aadA ), and a coding sequence for repressor of primer (ROP) protein for th e maintenance of the plasmid vector copy

number in E. coli . A description of the genetic elements and their prefixes ( e.g. , P-, OR-, B-, CS-, and T-) in PV-MSPQ1263 3 is provided in Table III-1.Monsanto Company 12-AL-246U 49 of 407 

### III.B. Description of the Transformation System

KK179 was developed through Agrobacterium- mediated transformation of alfalfa, based on the published method (Schenk and Hildeb randt, 1972; Walker and Sato, 1981) and allows for the generation of transformed plants . Briefly, alfalfa R2336 leaf cuttings were placed in a tissue culture media and co-cultured with Agrobacterium carrying the plasmid vector. R2336 is an FGI proprietary single alfa lfa plant, selected for regenerability from an elite, high yielding, fall dormant alfalf a breeding population. After three days, explants were placed on selection medium containing the antibiotics, kanamycin and timentin, to inhibit the growth of untransformed plant cells and excess Agrobacterium

, respectively. The kanamycin-resistant ca lli developed with somatic embryos. The somatic embryos were placed in media conducive to shoot and root development. Rooted plants (hereafter called T 0 

### plants) with normal phenotypic ch aracteristics were selected and transferred to soil for gr owth and further assessment.

The T 0 

### plants were crossed with Ms208, a conven tional male sterile plant selected from a population with a fall dormancy (FD4) phenotype, to produce F 1 

### plants, in which the

unlinked insertions of T-DNA I and T-DNA II we re segregated. Subsequently, plants that were positive for T-DNA I and negative for T-DNA II were identified by a polymerase chain reaction (PCR) based analysis. KK179 (P 0 

### ) was selected as the lead event based on superior phenotypic characte ristics and its molecular profile. P 0 

### is an

individual F 1 

### plant produced from crossing T 0 

### with Ms208. It has the reduced lignin phenotype without the T-DNA II. The major development steps of KK179 are depicted in Figure III-2. The result of this proce ss was the production of marker-free alfalfa

KK179.Monsanto Company 12-AL-246U 50 of 407 

### Figure III-1. Circular Map of PV-MSP Q12633 Showing Probe 1 through Probe 9 A circular map of PV-MSPQ12633 used to deve lop KK179 is shown. PV-MSPQ12633 contains two T-DNAs, designated as T-DNA I and T-DNA II. Ge netic elements and restriction sites (with positions relative to the size of the plasmid) used in Southern blot analyses are shown on the exterior of the map. The probes used in the S outhern analyses are shown on the interior of the map. The dashed arcs indicate that prob es were not generated for that region.

a This portion of the CCOMT sequence is contained in Probe 3 and not included in the T-DNA I probes.

b The Left Border sequences as well as some intervening sequences of T-DNA II share 100% identity to those of T-DNA I, which are covered by Probe 1 and, thus, not included in the T-DNA II probe.

c The Right Border sequences as well as some intervening sequences of T-DNA II share 100% identity to those of T-DNA I, which are covered by Probe 4 and, thus, not included in the T-DNA II probe. PV-MSPQ12633

10608 bp Eco RI 478

Eco RI 2674 Xba I 2105 Xba I 8998 B-Left Border Region

P- Pal2

T- nos

B-Right Border Region

aadA

OR- ori-pUC CS- rop OR- ori V B-Left Border Region CS- nptII T- nos B-Right Border Region

P- 35S Dra III 1284 Dra III 138

CCOMT CCOMT

Xmn I 7690

Dra III 7173

Xmn I 5696 Dra III 3913 Xmn I 3303 Dra III 3296 T-DNA I

T-DNA II

1

2
3

6

7 8 4 9

5 c

b a

Probe Probe Type Start Position (bp) End Position (bp) Total Length (~kb)

1 T-DNA I 1 853 0.9

2 T-DNA I 761 1568 0.8

3 T-DNA I 1507 2119 0.6

4 T-DNA I 2411 3084 0.7

5 T-DNA II 7510 9005 1.5

6 Backbone 3085 4219 1.1

7 Backbone 4126 5740 1.6

8 Backbone 5635 7035 1.4

9 Backbone 9406 10608 1.2Monsanto Company 12-AL-246U 51 of 407 

### Figure III-2. Schematic of the Development of KK179 Assembled Agrobacterium binary plasmid PV-MSPQ12633 and transferred to
Agrobacterium tumefaciens strain ABI

Transformed leaf tissue from R2336 with PV-MSPQ12633 in Agrobacterium
tumefaciens

Selected transformants and generated rooted shoots from the transformed leaf

tissues

Screening of transformed plants for the presence of T-DNA I ( CCOMT suppression cassette) and presence of the T-DNA II ( nptII expression cassette)

Identified KK179 as lead candidate based on analysis of the genomic insert and evaluation of progeny generations in laboratory and field assessments Selected T 0 

### plants were crossed to male sterile clone Ms208

Screening of F 1 

### plants for the presence of T-DNA I and the absence of

T-DNA IIMonsanto Company 12-AL-246U 52 of 407 

### III.C. The CCOMT Segment Sequence (T-DNA I)

The T-DNA I suppression cassette present in KK179 contains a partial gene segment

from CCOMT configured into an invert ed repeat sequence. The CCOMT partial gene segment is CCOMT sequence from Medicago sativa CCOMT gene that encodes the caffeoyl CoA 3- O -methyltransferase protein (Inoue et al., 1998). The suppression cassette is comprised of ~0.8 kb of sequence from the CCOMT coding sequence designed to express an RNA that contains an inverted repeat of the CCOMT gene segments. The gene transcript with the inve rted repeat produces dsRNA that, via an RNA interference (RNAi) pathway (Siomi and Siom i, 2009), suppresses endogenous CCOMT RNA levels,

which results in reduced biosynthesis of G lignin.

III.D. The nptII Coding Sequence and nptII Protein (T-DNA II)

The nptII expression cassette (T-DNA II) that is not present in KK179 encodes neomycin phosphotransferase II (NPT II). The nptII coding sequence is the neo gene from transposon Tn 5 of E. coli encoding the NPT II protein (B eck et al., 1982). The NPT II protein confers kanamycin resistance (Fraley et al., 1983) and was used as a selectable marker during the transformation selection pr ocess. Plants that did not contain the nptII expression cassette were isolated through tr aditional cross-pollinated breeding methods and segregation, along with a combina tion of analytical techniques.

III.E. Regulatory Sequences

T-DNA I contains an inverted repeat of a CCOMT gene segment under the regulation of

the Pal2 promoter, and the nos 3 ′ untranslated region. The Pal2 promoter is the promoter for phenylalanine ammonia-lyase gene from Phaseolus vulgaris (Cramer et al., 1989), which functions to direct transcription within vascular tissue and re sults in a pattern of expression that closely mirrors deposition of lignin as the plant matu res (Guo et al., 2001; Leyva et al., 1992). The nos 3 ′ untranslated region is the 3 ′ untranslated region of the nopaline synthase ( nos ) gene from Agrobacterium tumefaciens pTi encoding NOS, which functions to direct polyadenyl ation of the RNA transcripts (Bevan, 1984; Fraley et al., 1983). T-DNA II contains the nptII coding sequence under the regulation of the 35S promoter and the nos 3 ′ untranslated region. The 35S promoter is the promoter for 35S RNA of cauliflower mosaic virus (CaMV) (Ode ll et al., 1985), which functions to direct transcription in plant cells.

III.F. T-DNA Border Regions

PV-MSPQ12633 contains Left and Right Border regions (Figure II I-1 and Table III-1)

that were derived from Agrobacterium tumefaciens (Barker et al., 1983; Depicker et al., 1982; Zambryski et al., 1982). Th e border regions each contain a 24-25 bp nick site that is the site of DNA exchange during transformation. Left and Right Border regions separate the T-DNA from the plasmid backbone region and are involved in the efficient transfer into the alfalfa genome. B ecause PV-MSPQ12633 is a 2-T-DNA vector, it contains two Left Border regions and two Ri ght Border regions, where one border region

set flanks T-DNA I and the other bo rder region set flanks T-DNA II.Monsanto Company 12-AL-246U 53 of 407 

### III.G. Genetic Elements Outside the T-DNA Border Regions

Genetic elements that exis t outside of the T-DNA border regions are those that are

essential for the maintenance or selection of PV-MSPQ12633 in bacteria and are referred to as plasmid backbone. The origin of replication, oriV , is required for the maintenance of the plasmid in Agrobacterium and is derived from the broad host plasmid RK2 (Stalker et al., 1981). The origin of replication, ori-pUC , is required for the maintenance of the plasmid in E. coli and is derived from the plasmid vector pUC (Vieira and Messing, 1987). Coding sequence rop encodes the repressor of primer (ROP) protein, which is necessary for the maintenance of plasmid vector copy number in E. coli (Giza and Huang, 1989). The selectable marker aadA is a bacterial promoter and coding sequence for an enzyme from transposon Tn 7 that confers spectinom ycin and streptomycin resistance (Fling et al., 1985) in E. coli and Agrobacterium during molecular cloning.

Because these elements are outside the borde r regions, they are not expected to be transferred into the alfalfa genome.Monsanto Company 12-AL-246U 54 of 407 

### Table III-1. Summary of Gene tic Elements in PV-MSPQ12633

Genetic Element Location in Plasmid Function (Reference)

T-DNA I

B 1 -Left Border Region 1-442 DNA region from Agrobacterium tumefaciens containing the Left Border sequence used for transfer of the T-DNA (Barker et al., 1983) 

### Intervening Sequence 443-490 Sequence used in DNA cloning

P 2

- Pal2 491-1567 Promoter of the Pal2 gene from Phaseolus vulgaris encoding the phenylalanine ammonia-lyase that directs transcription in plant cells (Cramer et al., 1989) 

### Intervening Sequence 1568-1584 Sequence used in DNA cloning

CCOMT * 1585-2103 Partial coding sequence of the Medicago sativa CCOMT gene that encodes the caffeoyl CoA

3- O -methyltransferase protein (Inoue et al., 1998) that forms part of the suppression cassette 

### Intervening Sequence 2104-2110 Sequence used in DNA cloning

CCOMT * 2111-2410 Partial coding sequence of the Medicago sativa CCOMT gene that encodes the caffeoyl CoA

3- O -methyltransferase protein (Inoue et al., 1998) that forms part of the suppression cassette 

### Intervening Sequence 2411-2418 Sequence used in DNA cloning

T 3

- nos 2419-2671 3 ′ UTR sequence of the nopaline synthase ( nos ) gene

from Agrobacterium tumefaciens pTi encoding NOS that directs polyadenylation (Bevan, 1984; Fraley et al., 1983) 

### Intervening Sequence 2672-2727 Sequence used in DNA cloning



### B-Right Border Region 2728-3084 DNA region from Agrobacterium tumefaciens containing the Right Border sequence used for transfer of the T-DNA (Depicker et al., 1982; Zambryski et al., 1982)

Vector Backbone Intervening Sequence 3085-3199 Sequence used in DNA cloningMonsanto Company 12-AL-246U 55 of 407 

### Table III-1 A (continued). Summary of Genetic Elements in PV-MSPQ12633

Genetic

Element Location in

Plasmid Function (Reference)

aadA 3200-4088 Bacterial promoter, coding sequence, and 3 ′ UTR for an aminoglycoside-modifying enzyme, 3''(9)- O

nucleotidyltransferase from the transposon Tn 7 (Fling et al., 1985) that confers spectinomycin and streptomycin

resistance 

### Intervening Sequence 4089-4618 Sequence used in DNA cloning



### OR 4



### - ori-pUC 4619-5196 Origin of replication from plasmid pUC for maintenance of plasmid in E. coli (Vieira and Messing, 1987) 

### Intervening Sequence 5197-5623 Sequence used in DNA cloning



### CS 5



### - rop 5624-5815 Coding sequence for repressor of primer protein from the ColE1 plasmid for maintenance of plasmid copy

number in E. coli (Giza and Huang, 1989) 

### Intervening Sequence 5816-6552 Sequence used in DNA cloning



### OR- oriV 6553-6949 Origin of replication from the broad host range plasmid RK2 for maintenance of plasmid in Agrobacterium (Stalker et al., 1981)



### Intervening Sequence 6950-7035 Sequence used in DNA cloning



### T-DNA II

B-Left Border Region 7036-7477 DNA region from Agrobacterium tumefaciens containing the Left Border sequence used for transfer of the T-DNA (Barker et al., 1983) 

### Intervening Sequence 7478-7527 Sequence used in DNA cloning



### P- 35S 7528-7851 Promoter and leader from the 35S RNA of cauliflower mosaic virus (CaMV) (Odell et al., 1985) that directs transcription in plant cells 

### Intervening Sequence 7852-7884 Sequence used in DNA cloningMonsanto Company 12-AL-246U 56 of 407 

### Table III-1 (continued). Summary of Genetic Elements in PV-MSPQ12633

Genetic

Element Location in

Plasmid Function (Reference)

CS- nptII 7885-8679 Coding sequence of the neo gene from transposon Tn 5 of

E. coli encoding neomycin phos photransferase II (NPT II) (Beck et al., 1982) that confers neomycin and kanamycin resistance (Fraley et al., 1983) 

### Intervening Sequence 8680-8710 Sequence used in DNA cloning



### T- nos 8711-8963 3 ′ UTR sequence of the nopaline synthase ( nos ) gene

from Agrobacterium tumefaciens pTi encoding NOS that directs polyadenylation (Bevan, 1984; Fraley et al., 1983) 

### Intervening Sequence 8964-9048 Sequence used in DNA cloning



### B-Right Border Region 9049-9405 DNA region from Agrobacterium tumefaciens containingthe Right Border sequence used for transfer of the TDNA (Depicker et al., 1982; Zambryski et al., 1982)



### Vector Backbone Intervening Sequence 9406-10608 Sequence used in DNA cloning

1 B, Border

2 P, Promoter

3 T, Transcription Termination Sequence

4 OR, Origin of Replication

5 CS, Coding Sequence

* Within the CCOMT suppression cassette, bases 1654-1953 are reverse complement to bases

2410-2111.Monsanto Company 12-AL-246U 57 of 407 

### IV. CHARACTERIZATION OF THE GENETIC MODIFICATION

This section contains a comprehensive mo lecular characterization of the genetic modification present in KK179. It provides information on the DNA insertion into the plant genome of KK179, and additional inform ation relative to the arrangement and stability of the introduced genetic material. The information provided in this section

addresses the relevant factors in Codex Plan t Guidelines, Section 4, paragraphs 30, 31, 32, and 33 (Codex Alimentarius, 2009).

A multi-faceted approach was taken to characterize the genetic modification that produced KK179. The results confirmed that KK179 contains a single copy of the

CCOMT suppression cassette (T-DNA I) that is stab ly integrated at a single locus and is inherited according to Mendelian principles over multiple generations. Additionally, the results confirmed that T-DNA II and plasmid v ector backbone sequences are not detected

in KK179. These conclusions we re based on several lines of evidence: 1) Southern blot analyses assayed the entire alfalfa genom e and demonstrated the presence of T-DNA I sequences and the absence of T-DNA II and plasmid vector backbone sequences derived from PV-MSPQ12633, and confirmed that a si ngle copy of T-DNA I was inserted at a single locus; 2) DNA sequence analyses to de termine the exact sequence of the inserted DNA and the DNA sequence flanking the 5 ′ and 3 ′ end of the insert; 3) DNA sequence comparison of the inserted DNA sequence to the T-DNA I sequence in PV-MSPQ12633 confirmed that only the expected sequences were integrated; 4) sequence comparison of the DNA sequences flanking the 5 ′ and 3 ′ ends of the T-DNA I insert to the insertion site sequence in conventional alfalfa demonstrat ed the lack of any rearrangements that occurred at the insertion site during tran sformation; 5) Southern blot analysis demonstrated insert stability across multiple generations, and 6) segregation analysis

further confirmed that T-DNA I resides at a single locus and is inherited according to Mendelian principles of inher itance. Taken together, the ch aracterization of the genetic modification demonstrates that a single copy of T-DNA I was inserted at a single locus of the alfalfa genome and that no plasmid vector backbone sequences are present in KK179.

Southern blot analyses were used to determ ine the number of copies, to characterize the insertion site of T-DNA I, as well as to assess the presen ce or absence of T-DNA II and plasmid vector backbone sequences. The Sout hern blot strategy wa s designed to ensure that all potential inserted segments would be identified. The entire alfalfa genome was assayed with probes that spanned the comp lete plasmid vector PV-MSPQ12633 to detect the presence of T-DNA I, as well as the absence of T-DNA II and plasmid vector backbone sequences. This was accomplished by using probes that were less than 2 kb in length, ensuring a high level of sensitivity. This high level of sensitivity was demonstrated for each blot by detection of a positive control added at 0.1 copies per genome equivalent. Two sets of restriction enzymes were specifically chosen to fully characterize T-DNA I and detect any poten tial segments from the plasmid vector PV-MSPQ12633. The restriction enzyme sets we re chosen such that each enzyme set

cleaves once within the inserted T-DNA and at least once w ithin the known DNA sequence flanking the 5 ′ or 3 ′ end of the insert. As a result, the enzyme sets produce overlapping segments that contain the en tire insert sequence and adjacent 5 ′ or 3 ′ flanking DNA sequence. Therefore, at least one segm ent containing a portion of the insert withMonsanto Company 12-AL-246U 58 of 407 

### the adjacent 5 ′ flanking DNA generated by one set of the enzyme(s) is of a predictable size and overlaps with a nother predictable size segment cont aining a portion of the insert with the adjacent 3 ′ flanking DNA generated by another set of the enzyme(s). This twoset enzyme design ensures that the entire insert is identified in a predictable hybridization pattern. Additionally, this two-enzyme set design also maximizes the possibility of detecting an insertion elsewhere in the ge nome that could be overlooked if that band co-migrated with an expected band.

To determine the number of copies and the insertion sites of T-DNA I, and the presence or absence of T-DNA II and the plasmid vect or backbone sequences, duplicated samples that consisted of equal amount s of digested DNA were run on the agarose gel (Figures IV-2 through IV-8). One set of samples was run for a longer period of time (long run) than the second set (short run) . The long run allows for greater resolution of large molecular weight DNA, whereas the short run allows the detection of small molecular weight DNA. The molecular weight markers on the left of the figures were used to estimate the sizes of the bands present in the lo ng run lanes of the Southern blots, and the molecular weight markers on the right of the figures were used to estimate the sizes of bands present in the short ru n lanes of the Southern blot s. Southern blot results

demonstrated that KK179 contai ns a single copy of T-DNA I at a single insertion site in the alfalfa genome, and no T-DNA II or b ackbone sequences from PV-MSPQ12633 were

detected in KK179.

PCR and DNA sequence analyses of KK179, which complement the Southern blot analyses, determined the complete DNA sequence of the insert, confirmed the organization of the elements within the insert, and determined the 5 ′ and 3 ′ insert-to-plant junctions (Figure IV-9 and Figure IV-10). In addition, DNA sequencing analyses confirmed each genetic element in the insert and the sequence of the insert matches the corresponding sequence in PV-MSPQ12633. Furt hermore, genomic organization at the KK179 insertion site was determined by comparing the 5 ′ and 3 ′ flanking sequences of the insert to the sequence of the inse rtion site in conventional alfalfa.

The stability of the T-DNA I present in KK179 across multiple generations was demonstrated by Southern blot fingerpri nt analysis. Geno mic DNA from four generations (P 0 

### , MBC1, MBC2, and Syn1) of KK179 (F igure IV-11) was digested with one of the enzyme sets used for the insert and copy number analysis and was hybridized with a probe that detects re striction segments that enco mpass the entire T-DNA I insert (Figure IV-1). This fingerprint strategy cons ists of two border segments that assess not only the stability of T-DNA I, but also the stab ility of genomic DNA directly adjacent to T-DNA I. Generational stability analysis dem onstrated that the expected Southern blot fingerprint of KK179 was maintained through four generations of the breeding history, thereby confirming the stability of T-DNA I in KK179 (Figure IV-12).

Segregation analysis showed that heritability and stability of the insert occurred as expected across multiple generations (Figure IV-13, Table IV-3), which corroborates the molecular insert stability analysis and es tablishes that T-DNA I in KK179 is inherited according to Mendelian principles of inheritance.Monsanto Company 12-AL-246U 59 of 407 

### A circular map of PV-MSPQ12633 annotated with the probes us ed in the Southern blot analysis is presented in Figure III-1. A linear map depicting restriction sites within the insert, as well as the DNA flanking the insert in KK179 is shown in Figure IV-1. Based on the plasmid map and the linear map of th e insert, a table summarizing the expected DNA segments for Southern analyses is presen ted in Table IV-1. The genetic elements

within the KK179 insert are summarized in Ta ble IV-2. The results from the Southern blot analyses are presented in Figure IV-2 th rough Figure IV-8. PCR amplification of the

KK179 insert and the insertion site in conve ntional control for DNA sequence analysis is shown in Figure IV-9 and Fi gure IV-10, respectiv ely. The generations used in the generational stability an alysis are depicted in the breedi ng history shown in Figure IV-11 and the results from the generational stability an alysis are presented in Figure IV-12. The breeding path for the segregation data is s hown in Figure IV-13 and the results for the segregation analysis are presented in Table IV-3. Materials and methods used for the

characterization of the insert in KK179 are found in Appendix B.Monsanto Company 12-AL-246U 60 of 407Figure IV-1. Schematic Representation ofthe Insert and Flanking DNA in KK179DNA derived from T-DNA I of PV-MSPQ12633 integrated in KK179. Right-angled arrowsindicate the ends of the integratedT-DNA I and the beginning of the flanking sequence. Identified onthe map are genetic elements within the insert, as well asrestriction sites with positions relative to the size of the DNA sequence (flanks and insert).The relative sizes and locations of theT-DNA I probes and the expected sizes of restriction fragments are indicated. This schematic diagram is not drawn to scale.Locations of genetic elements andT-DNA I probes are approximate. Probes are described in Figure IV-1.SwaI 10175' Flank3' FlankDraIII 2165XbaI 2986SwaI 434514885Probe 1Probe 2Probe 3Probe 4

B-Left Border Region r1 P- Pal2 CCOMT T- nos B-Right Border Region r1

CCOMTXbaI andSwaI~ 2.0 kb ~ 1.4 kbXmnI andDraIII≥2.2 kb~ 2.0 kbXmnI 4186XmnI 4511Monsanto Company 12-AL-246U 61 of 407Table IV-1. Summary Chart of the Expected DNA Segments Based on Hybridizing Probes and Restriction Enzymes Used inKK179 AnalysisSouthern Blot Analysis T-DNA I T-DNA II BackboneFigureV-2 V-3 V-4 V-5 V-6 V-7 V-8Probe Used1 2, 4 3 3 5 6, 8 7, 9Probing TargetDigestionenzymeExpected Band Sizes on each Southern BlotPV-MSPQ12633XbaI~6.9 kb~3.7 kb~6.9 kb~3.7 kb ~~2~~2~6.9 kb~6.9 kb~3.7 kb~6.9 kb~3.7 kbEcoRI~~2~~2~2.2 kb ~2.2 kb ~~2~~2~~2Probe Templates1N/A--3~0.8 kb~0.7 kb--3--3--3~1.1 kb~1.4 kb~1.6 kb~1.2 kbKK179XbaI andSwaI~2.0 kb~2.0 kb~1.4 kb~2.0 kb~1.4 kb ~~2~1.4 kb NA4NAXmnI andDraIII≥2.2 kb≥2.2 kb~2.0 kb ~~2~2.0 kb ~2.0 kb NA NA1probe template spikes were used as positive hybridization controls in Southern blotanalyses when multiple probes were hybridized to the blotsimultaneously2‘~~’ indicates that this digest was not performed.3‘--’ indicates that probe templates were not used.4Not Applicable.Monsanto Company 12-AL-246U 62 of 407 

### Table IV-2. Summary of Ge netic Elements in KK179

Genetic Element Location in Sequence Function (Reference)

5 ′ flank 1-1047 Sequence flanking the 5 ′ end of the

insert

B 1 -Left Border Region r1 1048-1322 DNA region from Agrobacterium tumefaciens containing the Left Border sequence used for transfer of the T-DNA (Barker et al., 1983)



### Intervening Sequence 1323-1370 Sequence used in DNA cloning

P 2

- Pal2 1371-2447 Promoter of the Pal2 gene from Phaseolus vulgaris encoding the phenylalanine ammonia-lyase that directs transcription in plant cells (Cramer et al., 1989)



### Intervening Sequence 2448-2464 Sequence used in DNA cloning

CCOMT * 2465-2983 Partial coding sequence of the Medicago

sativa CCOMT gene that encodes the caffeoyl CoA 3- O -methyltransferase protein (Inoue et al., 1998) that forms part of the suppression cassette 

### Intervening Sequence 2984-2990 Sequence used in DNA cloning

CCOMT * 2991-3290 Partial coding sequence of the Medicago

sativa CCOMT gene that encodes the caffeoyl CoA 3- O -methyltransferase protein (Inoue et al., 1998) that forms part of the suppression cassette 

### Intervening Sequence 3291-3298 Sequence used in DNA cloning

T 3

- nos 3299-3551 3 ′ UTR sequence of the nopaline synthase ( nos ) gene from Agrobacterium tumefaciens pTi encoding NOS that directs polyadenylation (Bevan, 1984; Fraley et al., 1983) 

### Intervening Sequence 3552-3607 Sequence used in DNA cloning



### B-Right Border Region r1 3608-3629 DNA region from Agrobacterium tumefaciens containing the Right Border sequence used for transfer of the T-DNA (Depicker et al., 1982; Zambryski et al., 1982)



### 3 ′ flank 3630-4885 Sequence flanks the 3 ′ end of the insert

1 B, Border

2 P, Promoter

3 T, Transcription Termination Sequence

r1 Superscript in Left Border and Right Border re gions that indicates the sequences in KK179 were truncated compared to the sequences in PV-MSPQ12633.

* Within the CCOMT suppression cassette, bases 2534-2833 are reverse complement to bases

3290-2991.Monsanto Company 12-AL-246U 63 of 407 

### IV.A. Insert and Copy Number of T-DNA I in KK179

The copy number and insertion sites of T- DNA I sequences in the KK179 genome were evaluated by digesting the P 0 

### generation of KK179 and the appropriate control genomic DNA samples with two sets of restriction enzymes: a combination of Xmn I and Dra III,

a combination of Xba I and Swa I, and hybridized Southern bl ots with probes that span the T-DNA I (Figure III-1). Each restriction digest is exp ected to produ ce a specific banding pattern on the Southern blots (Table IV-1). Any additional copies and/or integration sites would be de tected as additional bands.

The combination of Xmn I and Dra III cleaves once within th e inserted DNA and at least

once within the known 3 ′ flanking sequence in KK179 (Fi gure IV-1). Therefore, if T-DNA I sequences were present as a single co py at a single integration site in KK179, the digestion with Xmn I and Dra III was expected to generate two border segments with expected sizes of ≥ 2.2 kb and ~2.0 kb (Figure IV-1 and Table IV-1). The combination of

Xba I and Swa I cleaves once w ithin the inserted DNA a nd once within the known 5 ′ and

3 ′ flanking sequences in KK179 (Figure IV-1). Therefore, if T-DNA I sequences were present as a single copy at a single integration site in KK179, the digestion with Xba I

and Swa I was expected to generate two border se gments with expected sizes of ~2.0 kb and ~1.4 kb (Figure IV-1 and Table IV-1).

The Southern blots were hybridized with probes spanning the entire T-DNA I sequence (Figure III-1, Probes 1, 2, 3, and 4). Each Sout hern blot contains at least one negative control and one or more positive controls. Conventional control genomic DNA digested with appropriate restriction enzymes was used as a negative control in all Southern blots. The conventional control, C 0 

### , is derived from a cross of the untransformed R2336 with

the elite conventional male sterile plant Ms208, resultin g in a near isogenic line comparator to KK179. Alfalfa is an autotetr aploid (Yang et al., 2009) and, therefore, contains multiple copies of each endogenous gene, which are randomly segregating. Southern blots hybridized with sequences specific to the CCOMT gene are expected to have different hybridization banding patt erns due to random segregation of the endogenous CCOMT gene. Therefore, for blots that were probed with CCOMT

containing sequences (Probe 3), the conven tional parental plants R2336 and Ms208 were also included as negative controls. Conve ntional control genomic DNA spiked with either digested PV-MSPQ12633 DNA and/or probe template(s) served as positive hybridization controls. The results of this analysis ar e shown in Figures IV-2 through

IV-8.

IV.A.1. Probe 1

Conventional control genomic DNA di gested with a combination of Xmn I and Dra III (Figure IV-2, Lane 8) or with a combination of Xba I and Swa I (Figure IV-2, Lane 3 and Lane 10) and hybridized with Probe 1 (Figur e III-1) showed no de tectable hybridization bands, as expected. Conventional control genomic DNA digested with Xmn I and Dra III and spiked with PV-MSPQ12633 DNA previously digested with Xba I produced two expected size bands at ~6.9 kb and ~3.7 kb (F igure IV-2, Lane 6 and Lane 7). The

~3.7 kb band and ~6.9 kb band were both de tected because the Left Border regionMonsanto Company 12-AL-246U 64 of 407 

### contained in Probe 1 sequence is pres ent in both the ~3.7 kb and the ~6.9 kb Xba I segments from PV-MSPQ12633. Detection of th e spiked controls indicates that the probe hybridized to its target sequences.

KK179 genomic DNA digested with a combination of Xmn I and Dra III and hybridized with Probe 1 (Figure III-1) pr oduced a band at ~4.5 kb (Figure IV-2, Lane 2 and Lane 9). The ~4.5 kb band is the expected band, which represents the 5 ′ end of the inserted DNA and the adjacent DNA flanking the 5 ′ end of the insert; this correlates with the expected border fragment size of ≥ 2.2 kb.

KK179 genomic DNA digested with a combination of Xba I and Swa I and hybridized with Probe 1 produced the expected band at ~2 .0 kb (Figure IV-2, Lane 4 and Lane 11). The ~2.0 kb band is the expected band, which represents the 5 ′ end of the inserted DNA and the adjacent DNA flanking the 5 ′ end of the insert. The results presented in Figure IV-2 indicate that the sequence covered by Probe 1 resides as one copy at a single detectable locus of integration in KK179.

IV.A.2. Probe 2 and Probe 4

Conventional control genomic DNA di gested with a combination of Xmn I and Dra III (Figure IV-3, Lane 1 and Lane 8) or with a combination of Xba I and Swa I (Figure IV-3, Lane 3 and Lane 10) and hybridized with Pr obe 2 and Probe 4 (Figure III-1) showed no detectable hybridization bands, as expect ed. Conventional control genomic DNA digested with a combination of Xmn I and Dra III and spiked with PV-MSPQ12633 DNA previously digested with Xba I produced two expected bands at ~6.9 kb and ~3.7 kb (Figure IV-3, Lane 5). Conventional contro l genomic DNA digested with a combination

of Xmn I and Dra III and spiked with probe templa tes generated from PV-MSPQ12633 (Figure IV-1, Probe 2 and Probe 4) produced the expected bands at ~0.8 kb and ~0.7 kb, respectively (Figure IV-3, Lane 6 and Lane 7). Detection of the spiked controls indicates that the probes hybridized to their target sequences.

KK179 genomic DNA digested with a combination of Xmn I and Dra III and hybridized with Probe 2 and Probe 4 (Figure III-1) produced two bands at ~4.5 kb and ~2.0 kb (Figure IV-3, Lane 2 and Lane 9). The ~ 4.5 kb band is the expe cted band representing

the 5 ′ end of the inserted DNA and the adjacent DNA flanking the 5 ′ end of the insert;

this correlates with the expected border fragment size of ≥ 2.2 kb. The ~2.0 kb band is the expected band representing the 3 ′ end of the inserted DNA and the adjacent DNA flanking the 3 ′ end of the insert.

KK179 genomic DNA digested with a combination of Xba I and Swa I and hybridized with Probe 2 and Probe 4 (Figure III-1) pr oduced two expected bands at ~2.0 kb and ~1.4 kb (Figure IV-3, Lane 4 and Lane 11) . The ~2.0 kb band is the expected band, which represents the 5 ′ end of the inserted DNA and the adjacent DNA flanking the 5 ′ end of the insert. The ~1.4 kb band is the expected band, which represents the 3 ′ end of the inserted DNA and the adjacent DNA flanking the 3 ′ end of the insert. The results presented in Figure IV-3 i ndicate that the sequence c overed by Probe 2 and Probe 4 resides as one copy at a single detect able locus of integration in KK179.Monsanto Company 12-AL-246U 65 of 407 

### IV.A.3. Probe 3

Alfalfa is an autotetraploid (Yang et al., 2009) and, therefore, contains multiple copies of each endogenous gene that are randomly segregating. Probe 3 covers the CCOMT region of PV-MSPQ12633, which contains seque nces specific to the endogenous CCOMT gene in the alfalfa genome. Therefore, the random segregation of the endogenous CCOMT in the alfalfa genome is expected to lead to different hybridization banding patterns with Probe 3. In order to show all endogenous CCOMT alleles, both parental plants R2336 and Ms208 were included as conventional pare ntal controls in addition to conventional

control C 0 

### when probed with Probe 3. A hybridiz ation band in KK179 that corresponds with a band detected in either one or both of the conventional parental plants R2336 and Ms208 would indicate that it is an endogenous hybridization signal and, therefore, not specific to the inserted DNA in KK179.

The conventional control, conventional pa rental controls R2336 and Ms208, and KK179 genomic DNA were digested with a combination of Xba I and Swa I (Figure IV-4) or

with a combination of Xmn I and Dra III (Figure IV-5) and probed with Probe 3 (Figure IV-1). As expected, different hybridization bands were present in the

conventional control and conve ntional parental c ontrols. All observed bands in the

conventional and conventional parental controls repres ent hybridization with the endogenous CCOMT gene in the alfalfa genome.

Conventional control genomic DNA digested with Xba I and Swa I (Figure IV-4, Lane 1 and Lane 8) and hybridized with Probe 3 (F igure IV-1) displayed hybridization bands at ~7.9 kb and ~11.0 kb. Conventional parental control R2336 genomic DNA digested with

Xba I and Swa I (Figure IV-4, Lane 2 and Lane 9) and hybridized with Probe 3 (Figure 1) displayed hybridization bands at ~7.9 kb a nd ~10.0 kb. Conventional parental control Ms208 genomic DNA digested with Xba I and Swa I (Figure IV-4, Lane 3 and Lane 10) and hybridized with Probe 3 (Figure III-1 ) displayed hybridization bands at ~7.9 kb, ~11.0 kb, and ~14.0 kb, ~16.0 kb, and ~20.0 kb. Since the conventional control is

derived from a cross between R2336 and Ms 208, as expected, the hybridization bands

detected in the conventional are present in either R2336 or Ms208.

The conventional control ge nomic DNA digested with Xba I and Swa I and spiked with PV-MSPQ12633 DNA previously digested with Eco RI produced the expected band at ~2.2 kb (Figure IV-4, Lane 6 and Lane 7) in addition to the endogenous hybridization

bands at ~7.9 kb and ~11.0 kb. Detection of th e positive control indi cates that the probe hybridized to its target sequences.

KK179 genomic DNA digested with Xba I and Swa I (Figure IV-4, Lane 4 and Lane 11) and hybridized with Probe 3 (Figure III-1 ) displayed bands at ~1.4 kb, ~2.0 kb, ~7.9 kb, ~10.0 kb, ~11.0 kb, ~14.0 kb, and ~16.0 kb. The ~7.9 kb, ~10.0 kb, ~11.0 kb, ~14.0 kb, and ~16.0 kb bands represent endogenous hybridiz ation as these bands have also been

observed in either the R2336 or Ms208 conventional parental controls (Figure IV-4, Lanes 2, 3, 9, and 10). The ~1.4 kb and ~2.0 kb bands are the expected hybridization bands (Table IV-1) from the inserted T-DNA. The ~2.0 kb band is the expected band representing the 5 ′ end of the inserted DNA and the adjacent DNA flanking the 5 ′ end ofMonsanto Company 12-AL-246U 66 of 407 

### the insert. The ~1.4 kb band is the expected band representing the 3 ′ end of the inserted DNA and the adjacent DNA flanking the 3 ′ end of the insert.

The conventional control ge nomic DNA digested with Xmn I and Dra III (Figure IV-5, Lane 1 and Lane 8) and hybridized with Pr obe 3 (Figure III-1) displayed hybridization bands at ~6.9 kb, ~7.9 kb, ~11.0 kb, ~14.0 kb, a nd ~16.0 kb. The conventional parental control R2336 genomic DNA digested with Xmn I and Dra III (Figure IV-5, Lane 2 and Lane 9) and hybridized with Probe 3 (Figur e III-1) displayed the hybridization bands at ~4.2 kb, ~5.9 kb ~6.2 kb, ~11.0 kb, ~14.0 kb, and~15.0 kb. The conventional parental control Ms208 genomic DNA digested with Xmn I and Dra III (Figure IV-5, Lane 3 and Lane 10) and hybridized with Probe 3 (Figure III-1) displayed the hybridization bands at ~6.9 kb, ~7.9 kb, ~11.0 kb, ~14.0 kb, ~16.0 kb, and ~20.0 kb. Since the conventional control is derived by a cro ss between R2336 and Ms208, as expected, the hybridization

bands detected in the conve ntional control are presen t in either R2336 or Ms208.

The conventional control genomic DNA digested with Xmn I and Dra III and spiked with PV-MSPQ12633 DNA previously digested with Eco RI produced an expected band at ~2.2 kb (Figure IV-5, Lane 6 and Lane 7) in addition to the endogenous hybridization bands at ~6.9 kb, ~7.9 kb, ~11.0 kb, ~14.0 kb, and ~16.0 kb. Detection of the positive control indicates that the probe hybr idized to its target sequences.

KK179 genomic DNA digested with Xmn I and Dra III (Figure IV-5, Lane 4 and Lane 11) and hybridized with Probe 3 (Figure III-1) displayed bands at ~2.0 kb, ~4.2 kb, ~6.2 kb, ~6.9 kb, ~11.0 kb, ~14.0 kb, and ~20.0 kb. The ~4.2 kb, ~6.2 kb, ~6.9 kb, ~11.0 kb, ~14.0 kb, and ~20.0 kb bands are endogenous, as these bands have also been

observed in either the R2336 or Ms208 conventional parental controls (Figure IV-5, Lanes 2, 3, 9, and 10). The ~2.0 kb band is th e expected band from the inserted DNA representing the 3 ′ end of the inserted DNA and the adjacent DNA flanking the 3 ′ end of

the insert.

The results presented in Figure IV-4 and Figure IV-5 indica tes that the sequence covered by Probe 3 resides as one copy at a single de tectable locus of integration in KK179.Monsanto Company 12-AL-246U 67 of 407 

### Figure IV-2. Southern Blot Analysis to Determine Insert and Copy Number of

T-DNA I in KK179: Probe 1 The blot was hybridized with one 32 

### P-labeled probe that spanned a portion of the T-DNA I sequence (Figure IV-1, Probe 1). E ach lane contains ~ 10 μg of digested genomic DNA isolated from leaf tissue. Lane designations are as follows:

Lane 1 Conventional Control ( Xmn I and Dra III)
2 KK179 ( Xmn I and Dra III)
3 Conventional Control ( Xba I and Swa I)
4 KK179 ( Xba I and Swa I)

5 Blank
6 Conventional Control ( Xmn I and Dra III) spiked with PV-MSPQ12633 ( Xba I) [~1.0 genome equivalent] 7 Conventional Control ( Xmn I and Dra III) spiked with PV-MSPQ12633 ( Xba I) [~0.1 genome equivalent] 8 Conventional Control ( Xmn I and Dra III)
9 KK179 ( Xmn I and Dra III)
10 Conventional Control ( Xba I and Swa I)
11 KK179 ( Xba I and Swa I)

Arrows denote the size of the DNA, in kilobase pairs, obt ained from the 1 kb DNA Extension Ladder (Invitrogen) on the ethidium bromide stained gel. 2 3 1 4 5 6 7 8 9 11 10

40 20 15 10

8.1 7.1 6.1

5.1

4.1

3.1

2.0

1.6

1.0

0.5 40 20 15 10 8.1

5.1

3.1

2.0 1.6

1.0

0.5 6.1

4.1 7.1 Long Run Short RunMonsanto Company 12-AL-246U 68 of 407 

### Figure IV-3. Southern Blot Analysis to Determine Insert and Copy Number of

T-DNA I in KK179: Probe 2 and Probe 4 The blot was hybridized with two 32 

### P-labeled probes that sp anned a portion of the T-DNA I sequence (Figure IV-1, Probe 2 and Probe 4). Each lane contains ~10 μg of digested genomic DNA isolated from leaf tis sue. Lane designations are as follows:

Lane
1 Conventional Control ( Xmn I and Dra III)
2 KK179 ( Xmn I and Dra III)
3 Conventional Control ( Xba I and Swa I)
4 KK179 ( Xba I and Swa I)
5 Conventional Control ( Xmn I and Dra III) spiked with PV-MSPQ12633 ( Xba I) [~1.0 genome equivalent] 6 Conventional Control ( Xmn I and Dra III) spiked with Probe 2 and Probe 4 [~1.0 genome equivalent] 7 Conventional Control ( Xmn I and Dra III) spiked with Probe 2 and Probe 4 [~0.1 genome equivalent] 8 Conventional Control ( Xmn I and Dra III)
9 KK179 ( Xmn I and Dra III)
10 Conventional Control ( Xba I and Swa I)
11 KK179 ( Xba I and Swa I)

Arrows denote the size of the DNA, in kilobase pairs, obt ained from the 1 kb DNA Extension Ladder (Invitrogen) on the ethidium bromide stained gel. 40 20 15 10

8.1 7.1 6.1

5.1

4.1

3.1

2.0

1.6

1.0 40 20 15 10 8.1

5.1

3.1

2.0

1.6

1.0

0.5 6.1

4.1 7.1 10 78 9 1 2 3 4 5 6 11 Long Run Short RunMonsanto Company 12-AL-246U 69 of 407 

### Figure IV-4. Southern Blot Analysis to Determine Insert and Copy Number of

T-DNA I in KK179: Probe 3 The blot was hybridized with one 32 

### P-labeled probe that spa nned portions of the T-DNA I sequence (Figure IV-1, Probe 3) . Each lane contains ~10 μg of digested genomic DNA

isolated from leaf tissue. La ne designations are as follows:

Lane
1 Conventional Control ( Xba I and Swa I)
2 Conventional Parental Control R2336 ( Xba I and Swa I)
3 Conventional Parental Control Ms208 ( Xba I and Swa I)
4 KK179 ( Xba I and Swa I)

5 Blank
6 Conventional Control ( Xba I and Swa I) spiked with PV-MSPQ12633 ( Eco RI) [~1.0 genome equivalent] 7 Conventional Control ( Xba I and Swa I) spiked with PV-MSPQ12633 ( Eco RI) [~0.1 genome equivalent] 8 Conventional Control ( Xba I and Swa I)
9 Conventional Parental Control R2336 ( Xba I and Swa I)
10 Conventional Parental Control Ms208 ( Xba I and Swa I)
11 KK179 ( Xba I and Swa I)

Arrows denote the size of the DNA, in kilobase pairs, obtained from the 1 kb DNA Ex tension Ladder (Invitrogen) on the ethidium bromide stained gel. 40

20
15

10

8.1

7.1

6.1

5.1

4.1

3.1

2.0

1.6

1.0 40 20 15

10 8.1

5.1

3.1

2.0
1.6

1.0

0.5 6.1

4.1 7.1 2 3 1 4 5 6 7 8 9 11 10 Long Run Short RunMonsanto Company 12-AL-246U 70 of 407 

### Figure IV-5. Southern Blot Analysis to Determine Insert and Copy Number of

T-DNA I in KK179: Probe 3 The blot was hybridized with one 32 

### P-labeled probe that spa nned portions of the T-DNA I sequence (Figure IV-1, Probe 3) . Each lane contains ~10 μg of digested genomic DNA

isolated from leaf tissue. La ne designations are as follows:

Lane
1 Conventional Control ( Xmn I and Dra III)
2 Conventional Parental Control R2336 ( Xmn I and Dra III)
3 Conventional Parental Control Ms208 ( Xmn I and Dra III)
4 KK179 ( Xmn I and Dra III)

5 Blank
6 Conventional Control ( Xmn I and Dra III) spiked with PV-MSPQ12633 ( Eco RI) [~1.0 genome equivalent] 7 Conventional Control ( Xmn I and Dra III) spiked with PV-MSPQ12633 ( Eco RI) [~0.1 genome equivalent] 8 Conventional Control ( Xmn I and Dra III)
9 Conventional Parental Control R2336 ( Xmn I and Dra III)
10 Conventional Parental Control Ms208 ( Xmn I and Dra III)
11 KK179 ( Xmn I and Dra III)

Arrows denote the size of the DNA, in kilobase pairs, obtained from the 1 kb DNA Ex tension Ladder (Invitrogen) on the ethidium bromide stained gel. 40 20 15 10 8.1

5.1

3.1

2.0

1.6

1.0

0.5 6.1

4.1 7.1 40

20
15

10

8.1 7.1

6.1

5.1

4.1

3.1

2.0

1.6

1.0

0.5 2 1 3 4 5 6 7 8 9 11 10 Long Run Short RunMonsanto Company 12-AL-246U 71 of 407 

### IV.B. Southern Blot Analysis to Det ermine the Presence or Absence of T-DNA II Sequences in KK179

The presence or absence of T-DNA II se quences in the KK179 alfalfa genome was evaluated by di gesting the P 0 

### generation of KK179 and the conventional control genomic DNA samples with two sets of restriction enzymes: a combination of Xmn I and Dra III

and a combination of Xba I and Swa I. The Southern blot was hybridized with a probe spanning the T-DNA II sequence, except for the border regions and some of the intervening sequences (Figur e III-1, Probe 5). Since th e border sequences and those intervening sequences of T-DNA II share 100% homology to the border and intervening sequences of T-DNA I, these border regions and intervening sequences were covered by T-DNA I Probes 1 and 4. A portion of Probe 5 contains sequences that are 100% homologous to the nos 3 ′ UTR sequence present in T-DNA I. Therefore, hybridization with Probe 5 is expected to result in de tection of the T-DNA I segment containing the nos 3 ′

UTR in KK179. If T-DNA II sequences are present in KK179, then hybridization with

Probe 5 would result in the de tection of unique hybridizati on bands in addition to the expected bands from the T-DNA I insert containing the nos 3 ′ UTR. The result of this analysis is shown in Figure IV-6.

IV.B.1. Probe 5

Conventional control genomic DNA digested with combination of Xmn I and Dra III (Figure IV-6, Lane 1 and Lane 8) or a combination of Xba I and Swa I (Figure IV-6, Lane 3 and Lane 10) and hybridized with Pr obe 5 (Figure III-1) s howed no detectable hybridization bands, as expect ed. Conventional control ge nomic DNA digested with

combination of Xmn I and Dra III and spiked with PV -MSPQ12633 DNA previously digested with Xba I produced an expected band at ~6.9 kb (Figure IV-6, Lane 6 and Lane 7). Detection of the spiked controls i ndicates that the probe hybridized to its target

sequences.

KK179 genomic DNA digested with Xmn I and Dra III (Figure IV-6, Lane 2 and Lane 9) and hybridized with Probe 5 (Figure IV-1) pr oduced the expected band at ~2.0 kb only visible in the longer expo sure (data not shown) due to the homology of the nos 3 ′ UTR in Probe 5 with T-DNA I. KK179 DNA digested with Xba I and Swa I (Figure IV-6, Lane 4 and Lane 11) and hybridized with Probe 5 (F igure III-1) produced an expected band at ~1.4 kb in a longer exposure (data not shown) due to the homology of the nos 3 ′ UTR in

Probe 5 with T-DNA I. This low level of intensity is ex pected because the nos 3 ′ UTR sequence is AT-rich and represents only a small portion of Probe 5. There are no additional hybridization bands other than the one expected from T-DNA I insert, indicating that KK179 contains no detect able T-DNA II elements from Probe 5 of PV-MSPQ12633.Monsanto Company 12-AL-246U 72 of 407 

### Figure IV-6. Southern Blot Analysis to De tect the Presence or Absence of T-DNA II Sequences in KK179: Probe 5 The blot was hybridized with one 32 

### P-labeled probe that spanned a portion of the T-DNA II sequence (Figure IV-1, Probe 5). E ach lane contains ~10 μg of digested genomic DNA isolated from leaf tissue. Lane designations are as follows:

Lane 1 Conventional Control ( Xmn I and Dra III)
2 KK179 ( Xmn I and Dra III)
3 Conventional Control ( Xba I and Swa I)
4 KK179 ( Xba I and Swa I)

5 Blank
6 Conventional Control ( Xmn I and Dra III) spiked with PV-MSPQ12633 ( Xba I) [~1.0 genome equivalent] 7 Conventional Control ( Xmn I and Dra III) spiked with PV-MSPQ12633 ( Xba I) [~0.1 genome equivalent] 8 Conventional Control ( Xmn I and Dra III)
9 KK179 ( Xmn I and Dra III)
10 Conventional Control ( Xba I and Swa I)
11 KK179 ( Xba I and Swa I)

Arrows denote the size of the DNA, in kilobase pairs, obtained from the 1 kb DNA Extension Ladder (Invitrogen) on the ethidium bromide stained gel. 40 20 15

10

8.1 7.1
6.1

5.1

4.1

3.1

2.0

1.6

1.0

0.5 40 20 15 10 8.1

5.1

3.1

2.0 1.6

1.0

0.5 6.1

4.1 7.1 10 78 9 1 2 3 4 5 6 11 Long Run Short RunMonsanto Company 12-AL-246U 73 of 407 

### IV.C. Southern Blot Analysis to Determine the Presence or Absence of PV-MSPQ12633 Backbone Sequences in KK179

The presence or absence of PV-MSPQ12633 b ackbone sequences in the alfalfa genome was evaluated by digesting the P 0 

### generation of KK179 and th e appropriate conventional control genomic DNA samples with two sets of restriction enzymes: a combination of

Xmn I and Dra III and with a combination of Xba I and Swa I. Digested genomic DNA was hybridized with overlapping probes spanning the backbone sequence of PV-MSPQ12633 (Figure III-1, Probes 6, 7, 8, and 9). If backbone DNA sequences were present in KK179, then hybridizing with overlapping probes corresponding to the backbone sequence should result in the det ection of hybridization bands on the Southern

blot. The results of th is analysis are shown in Figures IV-7 and IV-8.

IV.C.1. Backbone Probe 6 and Probe 8

Conventional control genomic DNA di gested with a combination of Xmn I and Dra III (Figure IV-7, Lane 1 and Lane 8) or with a combination of Xba I and Swa I (Figure IV-7, Lane 3 and Lane 10) and hybridized with Pr obe 6 and Probe 8 (Figure IV-1) showed no detectable hybridization bands, as expect ed. Conventional control genomic DNA digested with Xmn I and Dra III and spiked with PV-MSPQ12633 previously digested

with Xba I produced two expected bands at ~6.9 kb and ~3.7 kb (Figure IV-7, Lane 5).

The ~6.9 kb band was detected because Probe 6 and Probe 8 hybridized with the ~6.9 kb

Xba I segment from PV-MSPQ12633. The ~3.7 kb band was detected because a small region of the intervening sequence contained in Probe 8 is also present in the ~3.7 kb

Xba I segment from PV-MSPQ12633 in the region corresponding to the intervening sequence contained in Pr obe 9 (Figure III-1).

Conventional control genomic DNA di gested with a combination of Xmn I and Dra III and spiked with probe templates (Figure III -1, Probe 6 and Probe 8) generated from PV-MSPQ12633 produced the expected bands at ~1.1 kb and ~1.4 kb, respectively (Figure IV-7, Lane 6 and Lane 7). Detection of the spiked controls indicates that the probes hybridized to their target sequences.

KK179 genomic DNA digested with a combination of Xmn I and Dra III (Figure IV-7, Lane 2 and Lane 9) or with a combination of Xba I and Swa I (Figure IV-7, Lane 4 and Lane 11) and hybridized with Probe 6 and Pr obe 8 (Figure IV-1) produced no detectable bands, as expected. These data indicate that KK179 contains no detectable backbone elements from Probe 6 and Probe 8 of PV-MSPQ12633.

IV.C.2. Backbone Probe 7 and Probe 9

Conventional control genomic DNA di gested with a combination of Xmn I and Dra III (Figure IV-8, Lane 1 and Lane 8) or with a combination of Xba I and Swa I (Figure IV-8, Lane 3 and Lane 10) and hybridized with Pr obe 7 and Probe 9 (Figure III-1) showed no detectable hybridization bands, as expect ed. Conventional control genomic DNA digested with Xmn I and Dra III and spiked with PV-MSPQ12633 previously digested

with Xba I produced two expected bands at ~6.9 kb and ~3.7 kb (Figure IV-8, Lane 5).Monsanto Company 12-AL-246U 74 of 407 

### Conventional control genomic DNA di gested with a combination of Xmn I and Dra III and spiked with probe templates generated from PV-MSPQ12633 (Figure III-1, Probe 7 and Probe 9) produced the expected bands at ~1.6 kb and ~1.2 kb (Figure IV-8, Lane 6 and Lane 7). Detection of the spiked controls indicates that the probe s hybridized to their

target sequences.

KK179 genomic DNA digested with a combination of Xmn I and Dra III (Figure IV-8, Lane 2 and Lane 9) or with a combination of Xba I and Swa I (Figure IV-8, Lane 4 and Lane 11) and hybridized with Probe 7 and Probe 9 produced no detectable bands, as expected. These data indicate that KK179 contains no detectable backbone elements from Probe 7 and Probe 9 of PV-MSPQ12633

.Monsanto Company 12-AL-246U 75 of 407 

### Figure IV-7. Southern Blot Analysis to Determine the Presence or Absence of PV-MSPQ12633 Backbone Sequences in KK179: Probe 6 and Probe 8 The blot was hybridized with two 32 

### P-labeled probes that sp anned a portion of the PV-MSPQ12633 backbone sequence (Figure IV- 1, Probe 6 and Probe 8). Each lane contains ~10 μg of digested genomic DNA isol ated from leaf tissue. Lane designations

are as follows:

Lane
1 Conventional Control ( Xmn I and Dra III)
2 KK179 ( Xmn I and Dra III)
3 Conventional Control ( Xba I and Swa I)
4 KK179 ( Xba I and Swa I )
5 Conventional Control ( Xmn I and Dra III) spiked with PV-MSPQ12633 ( Xba I) [~1.0 genome equivalent] 6 Conventional Control ( Xmn I and Dra III) spiked with Probe 6 and Probe 8 [~1.0 genome equivalent] 7 Conventional Control ( Xmn I and Dra III) spiked with Probe 6 and Probe 8 [~0.1 genome equivalent] 8 Conventional Control ( Xmn I and Dra III)
9 KK179 ( Xmn I and Dra III)
10 Conventional Control ( Xba I and Swa I)
11 KK179 ( Xba I and Swa I)

Arrows denote the size of the DNA, in kilobase pairs, obtained from the λ DNA/ Hin d III Fragments (Invitrogen) on the ethidium bromide stained gel. 9.4 23.1

6.6

4.4

2.3

2.0 23.1

9.4

6.6

4.4

2.3

2.0

0.6 12 6 3 7 4 8 5 9 10 11 Long Run Short RunMonsanto Company 12-AL-246U 76 of 407 

### Figure IV-8. Southern Blot Analysis to Determine the Presen ce or Absence of PV-MSPQ12633 Backbone Sequences in KK179: Probe 7 and Probe 9 The blot was hybridized with two 32 

### P-labeled probes that spanned portions of PV-MSPQ12633 backbone sequence (Figure IV- 1, Probe 7 and Probe 9). Each lane contains ~10 μg of digested genomic DNA isol ated from leaf tissue. Lane designations

are as follows:

Lane 1 Conventional Control ( Xmn I and Dra III)
2 KK179 ( Xmn I and Dra III)
3 Conventional Control ( Xba I and Swa I)
4 KK179 ( Xba I and Swa I)
5 Conventional Control ( Xmn I and Dra III) spiked with PV-MSPQ12633 ( Xba I) [~1.0

g enome e q uivalent ] 6 Conventional Control ( Xmn I and Dra III) spiked with Probe 7 and Probe 9 [~1.0

g enome e q uivalent ] 7 Conventional Control ( Xmn I and Dra III) spiked with Probe 7 and Probe 9 [~1.0 genome equivalent] 8 Conventional Control ( Xmn I and Dra III) 9 KK179 ( Xmn I and Dra III) 10 Conventional Control ( Xba I and Swa I) 11 KK179 ( Xba I and Swa I)

Arrows denote the size of the DNA, in kilobase pairs, obtained from the λ DNA/ Hin d III Fragments (Invitrogen) on the ethidium bromide stained gel. 9.4 23.1

6.6

4.4

2.3

2.0 23.1

9.4

6.6

4.4

2.3
2.0

0.6 2 1 3 4 5 6 7 8 9 11 10 Long Run Short RunMonsanto Company 12-AL-246U 77 of 407 

### IV.D. Organization and Sequence of th e Insert and Adjacent Genomic DNA in

KK179

PCR and sequence analyses were performe d on genomic DNA extracted from KK179 and the conventional parental control R2336 to examine the organization and sequence of

the elements within the KK179 insert. PCR primers were designed with the intent to amplify five overlapping DNA regions that sp an the entire length of the T-DNA I insert and the associated DNA flanking the 5 ′ and 3 ′ ends of the insert (Figure IV-9). The amplified DNA segments were subjected to DNA sequence analyses. The analyses determined that the DNA sequence of the KK 179 insert is 2582 bp long (Table IV-2) and is identical to the corresponding T-DNA I se quence of PV-MSPQ12633 as described in Table III-1. From the sequence analys es, 1047 base pairs flanking the 5 ′ end of the KK179 insert and 1256 base pairs flanking the 3 ′ end of the KK179 insert (Table IV-2)

were also determined.Monsanto Company 12-AL-246U 78 of 407 

### Figure IV-9. Overlapping PCR Analys is across the Insert in KK179 PCR was performed on both parental cont rol genomic DNA and KK179 genomic DNA using five pairs of primer s to generate overlapping P CR fragments from KK179 for sequencing analysis. To verify synthesis of the PCR products, 2-5 μl of each of the PCR

reactions were loaded on the gel. The expected product size for each amplicon is provided in the illustrati on of the insert in KK179 that app ears at the bottom of the figure. This figure is a representative of the data generated in the st udy. Lane desi gnations are as

follows:

Lane Lane

1 1 Kb DNA Extension Ladder 11 KK179 2 R2336 12 No template DNA control

3 KK179 13 R2336 4 No template DNA control 14 KK179 5 R2336 15 No template DNA control 6 PV-MSPQ12633 16 R2336 7 KK179 17 PV-MSPQ12633 8 No template DNA control 18 KK179 9 R2336 19 No template DNA control 10 PV-MSPQ12633 20 1 Kb DNA Extension Ladder
Arrows on the agarose gel photograph denote the size of th e DNA, in kilobase pairs, obtained from the 1 kb DNA Extension Ladder (Invitrogen) on th e ethidium bromid e stained gel. 9 10 12 13 19 1 4 5 15 18 2 3 14 1617 8 7 6 11 20

3.1

0.5 1.0 1.6 2.0 4.1 5.1 40

0.4 3.1

0.5 1.0 1.6 2.0 40

5.1 4.1

0.4

5 ′ Flank 3 ′ FlankB-Left Border Regionr1P-Pal2CCOMTT-nosB-Right Border Regionr1CCOMT Product D: ~2.1 kb Product E: ~0.5 kb Product C: ~0.8 kb

Product B: ~0.8 kb Product A: ~2.1 kb Product

A Product

B Product

C Product

D Product

EMonsanto Company 12-AL-246U 79 of 407 

### IV.E. PCR and DNA Sequence Analyses to Examine the KK 179 Insertion Site

PCR and sequence analyses were performe d on genomic DNA extracted from KK179 and the conventional parental control R2336 to examine the integrity of the DNA insertion site in KK179. The PCR was perfor med with a forward prim er specific to the genomic DNA sequence flanking the 5 ′ end of the insert paired with a reverse primer specific to the genomic DNA sequence flanking the 3 ′ end of the insert (Figure IV-10). The amplified PCR product from the conventio nal parental control was subjected to DNA sequence analysis. Sequence alignments were performed between the conventional parental control sequence and the sequences flanking the 5 ′ and 3 ′ end of the KK179 T-DNA I insert. The alignment between the sequence flanking the 5 ′ end of the KK179 insert and the conventional parental control sequence showed that the 5 ′ flanking sequence of the KK179 insert is identical to the conventional parental control sequence, except for one base which is a G within the 5 ′ flanking sequence of the KK179 insert and is a G/T heterozygote. Th e alignment between the 3 ′ end of the KK179 insert and the conventional parental control sequence showed that the conventiona l parental control sequence is identical to the sequence flanking the 3 ′ end of the KK179 insert, except for

one base which is a G within the 3 ′ flanking sequence of th e KK179 insert and is a A/G heterozygote. These two heteroz ygotes were most likely caused by single nucleotide polymorphisms segregating in the autotetraploid alfalfa population (Yang et al., 2009). The alignment analyses also indicat ed a deletion of 102 base pairs from the conventional genomic DNA occurred upon T-DNA I insertion in KK179. This deletion presumably resulted from double-stranded brea k-repair mechanisms in the plant during

the Agrobacterium -mediated transformation process (Salomon and Puchta, 1998).Monsanto Company 12-AL-246U 80 of 407



### Figure IV-10. PCR Amplification of the KK179 Insertion Site in Conventional

Alfalfa PCR analysis was performed to evaluate the insertion site. PCR was performed on DNA from the conventional parental control R2336 and KK179 using Primer A, specific to the

5 ′ flanking sequence, and Primer B, specific to the 3 ′ flanking sequence of the insert in

K K179. The DNA generated from the parental control PCR was used for sequencing analysis. This illustration depicts the KK179 insertion site in the conventional parental control (upper panel) and the KK179 insert (lower panel). Approximately 5 μl aliquot of each PCR reaction was loaded on the gel. Lane designations are as follows:

Lane

1 1 Kb DNA Extension Ladder

2 Conventional Parental Control R2336

3 KK179

4 No template DNA control

5 1 Kb DNA Extension Ladder
Arrows on the agarose gel photograph denote the size of the DNA, in kilobase pairs, obtained from the 1 kb DNA Extension Ladder (Invitrogen) on the ethidium bromide stained gel. 1 2 3 4 5

3.1

0.5 1.0 1.6 2.0 40

5.1
3.1

0.5 1.0 1.6 2.0 4.1 5.1 40

4.1

5 ′ Flank 3 ′ FlankB-Left Border Regionr1P-Pal2CCOMTT-nosB-Right Border Regionr1CCOMT Primer A Primer B

Primer A Primer BMonsanto Company 12-AL-246U 81 of 407 

### IV.F. Southern Blot Analysis to Examine In sert Stability in Multiple Generations of

KK179

In order to demonstrate the stability of the DNA insert in KK179, Southern blot analysis was performed using genomic DNA obtained from four generati ons of KK179 (Figure IV-11). Genomic DNA isolated from each of the selected generations of KK179 was digested with the restriction enzymes Xmn I and Dra III (Figure IV-12) and hybridized with Probe 2 and Probe 4 (Figur e III-1). Probe 2 and Probe 4 are designed to detect both fragments generated by the Xmn I and Dra III digest at ≥ 2.2 kb and ~2.0 kb. Any instability associated with th e insert would be detected as novel bands on the Southern blot. The molecular weight markers were used to estimate the band sizes present. The results are shown in Figure IV-12.

IV.F.1. Probe 2 and Probe 4

Conventional control C 0 

### genomic DNA digested with Xmn I and Dra III (Figure IV-12, Lane 1) and hybridized with Probe 2 and Pr obe 4 (Figure III-1) showed no detectable hybridization bands, as expect ed. Conventional control ge nomic DNA digested with

Xmn I and Dra III and spiked with PV-MSPQ12633 previously digested with Xba I produced two bands at ~6.9 kb and ~3.7 kb (F igure IV-12, Lane 2) , as expected. Conventional control genomic DNA di gested with a combination of Xmn I and Dra III and spiked with probe templates generated from PV MSPQ12633 (Figure III-1, Probe 2 and Probe 4) produced the expected bands at ~0.8 kb and ~0.7 kb, respectively (Figure IV-12, Lane 3 and Lane 4). An additiona l faint ~1.4 kb band in the probe templates control lane (Figure IV-12, Lane 3) was obser ved and is most likely single stranded DNA formed during purification (Qiagen, 2008) th at has partially reannealed in various conformations (Kasuga et al., 2001). Since this ~1.4 kb band was de tected in only one conventional control (Lane 3) and not in any other lanes, the detection of this ~1.4 kb

band does not affect the conclusion of the an alysis on KK179. Detection of the spiked controls indicates that the probes hyb ridized to their target sequences.

KK179 DNA extracted from four generations (P 0 

### , MBC1, MBC2, and Syn1), digested

with Xmn I and Dra III (Figure IV-12, Lanes 5, 6, 7, and 8), and hybridized with Probe 2 and Probe 4 (Figure IV-1) produced two bands at ~4.5 kb and ~2.0 kb, as expected. The ~4.5 kb band is the expected band representing the 5 ′ end of the inserted DNA and the adjacent DNA flanking the 5 ′ end of the insert, which correla tes with the expected border fragment size of ≥ 2.2 kb. The ~2.0 kb band is the expected band representing the 3 ′ end of the inserted DNA and the adjacent genomic DNA flanking the 3 ′ end of the insert. The presence of ~4.5 kb and ~2.0 kb bands in the P 0 

### , MBC1, MBC2, and Syn1 generations demonstrates the stability of the T-DNA I insert across multiple generations of KK179.Monsanto Company



### Figure IV- 11. Breeding History of K



### The P 0 

### generation was used for the molecular analyses of KK179 reported in Figures V 

### through V- 8 and is referred to as KK179 in all Southern blot figures. The P 

### MBC2, and Syn1 generations were used for the insert stability of KK179 reporte 

### Figure IV-12. The Syn1



### described in Section I.B .3. and composition Alfalfa terminology

T 0 : the original transformed R2336 plant;

Ms208: conventio nal mal

P 0 : the single KK179 plant selected from the progeny of T

FD4: 10 elite alfalfa genotypes with fall dormancy rating 4 phenotype;

MBC1: KK179 generation produced from crossing P

breed ing step called modified backcross;

MBC2: subsequent KK179 generation produced from crossing MBC1 plants and FD4 elite genotypes through modified backcross; Syn1: a synthetic population of KK179 produced by crossing the MBC2 population of plants with eac Syn1 Adv: a subsequent synthetic population of KK179 produced by crossing the

population of plants with each other in a polycross.

12-AL-246U 

### 11. Breeding History of K 

### K179 generation was used for the molecular analyses of KK179 reported in Figures V 8 and is referred to as KK179 in all Southern blot figures. The P generations were used for the insert stability of KK179 reporte Syn1 generation was used for intended changes to lignin studies .3. and composition studies described in Section VI

the original transformed R2336 plant;

nal mal e sterile alfalfa plant;
the single KK179 plant selected from the progeny of T 0 × Ms208,
10 elite alfalfa genotypes with fall dormancy rating 4 phenotype;
KK179 generation produced from crossing P 0 and FD4 elite genotypes through a ing step called modified backcross; subsequent KK179 generation produced from crossing MBC1 plants and FD4 elite genotypes through modified backcross; a synthetic population of KK179 produced by crossing the MBC2 population of plants with eac h other in a breeding step called polycross; and a subsequent synthetic population of KK179 produced by crossing the population of plants with each other in a polycross.

82 of 407 

### generation was used for the molecular analyses of KK179 reported in Figures V 

### -2 8 and is referred to as KK179 in all Southern blot figures. The P 0 

### , MBC1, generations were used for the insert stability of KK179 reporte 

### d in generation was used for intended changes to lignin studies

studies described in Section VI 

### .B.

× Ms208,
10 elite alfalfa genotypes with fall dormancy rating 4 phenotype;
and FD4 elite genotypes through a

subsequent KK179 generation produced from crossing MBC1 plants and FD4

a synthetic population of KK179 produced by crossing the MBC2 population of

a subsequent synthetic population of KK179 produced by crossing the Syn1Monsanto Company 12-AL-246U 83 of 407 

### Figure IV-12. Southern Blot Analysis to Examine Insert Stability in Multiple

Generations of KK179: Probe 2 and Probe 4 The blot was hybridized with two 32 

### P-labeled probes that spanned portions of the T-DNA sequence (Figure IV-1, Probe 2 and Probe 4). Each lane contains ~10 μg of digested genomic DNA isolated from leaf tissue. Lane designations are as follows:

Lane
1 Conventional Control ( Xmn I and Dra III)
2 Conventional Control ( Xmn I and Dra III) spiked with PV-MSPQ12633 ( Xba I) [~1.0 genome equivalent] 3 Conventional Control ( Xmn I and Dra III) spiked with Probe 1 and Probe 2 [~1.0 genome equivalent] 4 Conventional Control ( Xmn I and Dra III) spiked with Probe 1and Probe 2 [~0.1 genome equivalent] 5 KK179 (P 0 ) ( Xmn I and Dra III)
6 KK179 (MBC1) ( Xmn I and Dra III)
7 KK179 (MBC2) ( Xmn I and Dra III)
8 KK179 (Syn1) ( Xmn I and Dra III)

Arrows denote the size of the DNA, in kilobase pairs, obt ained from the 1 kb DNA Exte nsion Ladder (Invitrogen) on the ethidium bromide stained gel. 40 20
15

10 8.1

5.1

3.1

2.0

1.6

1.0

0.5 6.1

4.1 7.1 2 3 1 4 56 78Monsanto Company 12-AL-246U 84 of 407 

### IV.G. Inheritance of th e Genetic Insert in KK179

During development of KK179, segregation data were generated to a ssess the heritability and stability of the T-DNA I pres ent in KK179 using Chi square ( χ 2) analysis over several generations. The Chi square analysis is based on comparing the observed segregation ratio to the expected segregation ra tio according to Mendelian principles.

The KK179 breeding path for generating segreg ation data is described in Figure IV-13.

The transformed T 0 

### plant was cross-pollinated to an elite male sterile alfalfa plant, Ms208, to produce F 1 

### seed. From the F 1 

### segregating populati on, an individual plant (designated as P 0 

### ) negative for T-DNA II and positive for the KK179 insert was identified via Southern blot analysis and construct-level gel-based PCR assay.

The selected P 0 

### plant was crossed with a population of 10 plants with conventional, elite genotypes with a fall dormancy 4 (FD4) rating to give rise to a modified backcrossed (MBC), designated as MBC1 plants. The pol len from 20 MBC1 plants that showed positive for the insert by Endpoint TaqMan PCR was used to pollinate the same conventional FD4 population to produce MBC2 seed. The pollen from 24 MBC2 plants that showed positive for the insert by Endpoint TaqMan PCR was used to pollinate the same conventional FD4 population to produce MBC3 seed. Finally, another 80 MBC2 plants shown to be positive for the insert by Endpoint TaqMan PCR were crossed to each other (polycross) to produ ce Syn1 seed (Figure IV-13).

The MBC2, MBC3, and Syn1 plants were tested for the expected segregation pattern for the insert using the Endpoint TaqMan PCR assay. Endpoint TaqMan PCR captures sample fluorescence reading following the completion of the PCR reaction. The Endpoint TaqMan PCR assay was designed to detect specific DNA sequences in flankinsert junction regions and is used to dete rmine the presence or absence of the KK179 insert in the generations evaluated. The M BC2 and MBC3 populations were predicted to segregate at a 1:1 (KK179 positive:KK179 negative) ratio; the Syn1 population was predicted to segregate at a ratio of 3:1 (KK179 positive:KK179 negative) according to Mendelian inheritance principles.

A Chi square ( χ 2) analysis was performed us ing the statistical program R Version 2.12.0 (2010-10-15) to compare th e observed segregation ratios to the expected ratios according to Mendeli an inheritance principles. The χ 2 was calculated as:

χ 2 

### = ∑ [( | o – e | ) 2 

### / e]

where o = observed frequency of the phenot ype and e = expected frequency of the phenotype. The level of statistical signi ficance was predetermined to be 5% ( α = 0.05).

The results of the χ 2 analysis of the segregating proge ny of KK179 are presented in Table

IV-3. The χ 2 values in the MBC2 and MBC3 popul ations indicated no statistically significant difference between the observed an d expected 1:1 segregation ratio (KK179 positive:KK179 negative) of the KK179 insert. Likewise, the χ 2 

### value in the Syn1 population indicated no statis tically significant differen ce between the observed and expected 3:1 segregation ratio (KK179 positiv e:KK179 negative) of the KK179 insert.Monsanto Company 12-AL-246U 85 of 407 

### These results support the conclu sion that the KK179 insert sequence in KK179 resides at a single locus within the alfalfa genome and is inherited according to Mendelian inheritance principles. These results ar e also consistent with the molecular

characterization data that indicate KK179 contains a single, intact copy of the CCOMT suppression cassette that was inserted into the alfalfa genome at a single locus.Monsanto Company



### Figure IV- 13. Breeding Path for Generating Segregation Data for KK179 The MBC2, MBC3, and



### insert in multiple generations.

Alfalfa terminology

T 0 : the original transformed R2336 plant;

Ms208: conventional male sterile alfalfa

F1: KK179 progeny produced from the cross of T

P 0 : the single KK179 plant selected from the progeny of T

FD4: 10 elite alfalfa genotypes with fall dormancy rating 4 phenotype;

MBC1: KK179 generation produced from crossing P modified backcross;

MBC2: subsequent KK179 generation produced from crossing MBC1 plants and FD4 genotypes through modified backcross;

MBC3: subsequent KK179 generation produced from crossing MBC2 plants and FD4 genotypes through modified backcross; and Syn1: a synthetic population of KK179 produced by self crossing the MBC2 population of plants in *Chi square analysis was conducted on segregation data from the MBC2, MBC3, and

12-AL-246U 

### 13. Breeding Path for Generating Segregation Data for KK179 The MBC2, MBC3, and 

### Syn1 generations were used for analyzing the inheritance of the multiple generations.

the original transformed R2336 plant;

conventional male sterile alfalfa plant;
KK179 progeny produced from the cross of T 0 and Ms208.
the single KK179 plant selected from the progeny of T 0 × Ms208;
10 elite alfalfa genotypes with fall dormancy rating 4 phenotype;
KK179 generation produced from crossing P 0 and FD4 genotypes through a modified backcross; subsequent KK179 generation produced from crossing MBC1 plants and FD4 genotypes through modified backcross; subsequent KK179 generation produced from crossing MBC2 plants and FD4 genotypes through modified backcross; and a synthetic population of KK179 produced by self crossing the MBC2 population

a breeding step called polycross. *Chi square analysis was conducted on segregation data from the MBC2, MBC3, and

86 of 407



### 13. Breeding Path for Generating Segregation Data for KK179

generations were used for analyzing the inheritance of the

Ms208;
10 elite alfalfa genotypes with fall dormancy rating 4 phenotype;
and FD4 genotypes through a

subsequent KK179 generation produced from crossing MBC1 plants and FD4

subsequent KK179 generation produced from crossing MBC2 plants and FD4

a synthetic population of KK179 produced by self crossing the MBC2 population

*Chi square analysis was conducted on segregation data from the MBC2, MBC3, and Syn1 populations.Monsanto Company 12-AL-246U 87 of 407Table IV-3. Segregation of the KK179 Insert During Mendelian Inheritance Testing1:1 SegregationGenerationTotalPlants1Observed #PositivesObserved #NegativesExpected #PositivesExpected #Negativesχ2 ProbabilityMBC2 261 119 142 130.5 130.5 2.027 0.1545MBC3 263 132 131 131.5 131.5 0.004 0.95083:1 SegregationGenerationTotalPlants1Observed #PositivesObserved #NegativesExpected #PositivesExpected #Negativesχ2ProbabilitySyn1 504 376 128 378 126 0.042 0.83701Plants were tested for the presence of theKK179 insert by Endpoint TaqMan analysis. “Total plants” refers to the total numberof plants in which the presenceor absence of the insert could be determined using the assay.Monsanto Company 12-AL-246U 88 of 407 

### IV.H. Characterization of the Genetic Modification Summary and Conclusion

Molecular characteriza tion of KK179 by Southern blot analyses confirmed that one copy

of the CCOMT suppression cassette was integrated in to the alfalfa genome at a single locus. No T-DNA II or backbone DNA seque nce from plasmid vector PV-MSPQ12633

was detected in KK179.

PCR and DNA sequence analyses performe d on KK179 and conventional parental control R2336 determined the following: th e complete DNA sequence of the insert and adjacent DNA sequences in KK179; the organiza tion of the genetic elements within the insert; the expected sequence of each element in the inserted DNA; and the 5 ′ and 3 ′ insert-to-genomic DNA juncti ons. The PCR and DNA sequence analysis identified a 5 ′ flanking G/T heterozygote and a 3 ′ flanking A/G heterozygote in the sequence. These two heterozygotes were most likely due to single nucleotide polymorphisms which are normal DNA sequence variations that are segregating in the autotetraploid alfalfa population (Yang et al., 2009). The PCR and DNA sequence an alysis also identified a 102 base pair deletion that occurre d at the insertion site in KK179.

Southern blot analysis of KK179 demonstrated that the inserted DNA has been maintained through multiple generations of br eeding, thereby confirming the stability of the insert. Results from segregation analyses show heritability and stability of the insert occurred as expected across multiple generati ons, which corroborates the molecular insert stability analysis and establishes the geneti c behavior of the T-DNA I inserted in KK179 at a single chromosomal locus.Monsanto Company 12-AL-246U 89 of 407 

### V. CHARACTERIZATION AND SA FETY ASSESSMENT OF KK179

EXPRESSED PRODUCTS

A multistep approach is used to assess the safe ty of gene products introduced into plants using biotechnology. As described in Section III.C., the KK179 insert contains a

CCOMT suppression cassette. The CCOMT suppression cassette encodes for dsRNA, which is highly unlikely to encode for a prot ein. Information pertaining to the mode-ofaction for RNA-based suppression of the expr ession of an endogenous plant gene can be

found in Secton I.A. Section V.A. describes th e history of safe use of products developed using RNA-based gene suppression.

V.A. History of Safe Use of RNA-based Suppression of Endogenous CCOMT

RNA-based suppression of the CCOMT gene, leading to the in tended reduction of G lignin and total lignin in KK179, is mediat ed by dsRNA molecules. These dsRNA molecules, which are produced from assemble d gene transcripts in KK179 composed of an inverted repeat sequence, suppress endogenous CCOMT gene via the naturally operating endogenous RNAi pathway. Double- stranded RNAs are commonly found in eukaryotes, including plants, for endogenous gene suppression and are composed of nucleic acids (Siomi and Siomi, 2009). Nu cleic acids have a long history of safe consumption and are considered GRAS (Gener ally Recognized as Safe) by the U.S. FDA (U.S. FDA, 1992) because there is no evidence of mammalian toxicity or allergenicity to RNA or DNA (Burnside et al., 2008; Heisel et al., 2008; Ivashuta et al., 2009; Jonas et al., 2001; Parrott et al., 2010; Reddy et al., 2009; U.S. FDA, 1992; Zhou et al., 2009).

S everal biotechnology-derived plant products previously reviewed by the U.S. FDA, deregulated by USDA-APHIS, and approved by several international regulatory authorities were developed using RNA- based suppression mechanisms, including improved fatty acid profile soybean MON 87705; high oleic soybean, virus-resistant squash, virus-resistant papaya , delayed-ripening tomatoes, and plum pox virus-resistant plum trees (ANZFA, 2000; CFIA, 2001; 2009; EFSA, 2012; HC, 1999a; b; 2000; 2002; MOE, 2007; U.S. FDA, 1994; 1995a; b; 1997; 2008; 2009b; a; 2011; USDA-APHIS, 2012).

Analysis of KK179 DNA segments encoding ds RNA indicate that production of a protein from the dsRNA encoded by the insert in KK179 is highly unlikely. This is supported by evidence that eukaryotic dsRNA molecules are refractory to translation due to the inability of 40s ribosomal subunits to melt double-stranded regions, even ones as short as 18 nucleotides (Kozak, 1989). As a consequen ce, it is highly unlikely for the dsRNA produced by the transgene in KK179 to yield a translation product. Bioinformatic analyses of the KK179 DNA insert and fl anking sequences provided no evidence for concern regarding safety implications of putative polypeptides. Based on this information, the inserted DNA and resulting dsRNA are considered safe and unlikely to produce a protein or polypeptide.

Based on the ubiquitous nature of the RNA-based suppression mechanism utilizing dsRNA, the history of safe consumption of RNA with no docum ented evidence for toxicity or allergenicity of dietary RNA, and the lack of evidence of any expressedMonsanto Company 12-AL-246U 90 of 407 

### protein from the DNA inserted into KK 179, the use of RNA-based suppression of endogenous CCOMT gene expression in KK179 poses no risks as a result of exposure to expressed products of the DNA insert.

V.B. Safety Assessment of KK179 Expressed Products Summary and Conclusion

The information provided in this section addr ess the history of sa fe use for RNA-based gene suppression. In Section I.B.2.2., KK179 nor thern blot data confirm the expected suppression of endogenous CCOMT RNA in alfalfa forage and root tissue. As summarized in this section, it is extremely unlikely a protein could be produced from the suppression cassette. Therefore, based on the ubiquitous nature of the RNA-based suppression mechanism utilizing dsRNA, de monstration of mode-of-action through

CCOMT RNA suppression, the history of safe c onsumption of RNA, and the apparent lack of toxicity or allergenicity of diet ary RNA, the RNA-base d suppression technology used in KK179 poses no novel risks as a result of expressed products.Monsanto Company 12-AL-246U 91 of 407 

### VI. COMPOSITIONAL ASSESSMENT OF KK179

Data collected on the feed and food safety of KK179 are consistent with recommendations contained in FDA’s proposed rule for Premarket Biotechnology Notice (PBN) Concerning Bioengineered Foods (66F R 4706). Additionally, the data collected and the assessment approach follow the comparative safety assessment process of the Codex Plant Guidelines (Codex Alimentarius , 2009) in which the composition of grain and/or other raw agricultural commoditie s of the biotechnology-derived crop are compared to the appropriate conventional count erpart that has a history of safe use. Compositional assessments are also performed using the principles and analytes outlined in the OECD consensus document for alfalfa composition (OECD, 2005).

A recent review of compositional assessments conducted according to OECD guidelines, which encompassed a total of seven biotechnol ogy-derived crop varietie s, nine countries, and eleven growing seasons, concluded that incorpora tion of biotechnology-derived agronomic traits has had little impact on na tural variation in crop composition. Most compositional variation is attributable to growing region, agronomic practices, and genetic background (Harrigan et al., 2010). Numerous scientific publications have further documented the extensive variability in the concentrations of crop nutrients, antinutrients, and secondary metabolites that re flect the influence of environmental and genetic factors as well as extensive conventional breeding efforts to improve nutrition, agronomics, and yield (H arrigan et al., 2010).

Compositional equivalence between biotech nology-derived and conventional crops supports an “equal or increased assurance of the safety of fo ods derived from genetically modified plants” (OECD, 2002). OECD consensus documents on compositional considerations for new crop varieties emphasi ze quantitative measurem ents of essential

nutrients and known anti-nutrien ts. These quantitative measur ements effectively discern any compositional changes that impl y potential nutritional or safety, e.g., anti-nutritional) concerns. Levels of the components in forage and/or other raw agricultural commodities of the biotechnology-derived crop product are co mpared to: 1) corresponding levels in a conventional comparator, a genetically sim ilar conventional line, grown concurrently under similar field conditions; and 2) rang es generated from an evaluation of

conventional commercial reference varietie s grown concurrently, and from data published in the scientific lite rature. The comparison to data published in the literature places any potential differences between the assessed crop and its comparator in the

context of the well-documente d variation in the concentrations of crop nutrients, antinutrients, and secondary metabolites.

This section provides analyses of concentrat ions of key nutrients , anti-nutrients, and secondary metabolites of KK179 forage compared to that of forage harvested from a conventional counterpart grown and harvested under similar conditions. In addition,

conventional commercial alfalfa reference varieties were include d in the composition analyses to establish a range of natural variability for each component. The production of forage for compositional analyses used fiel d designs (randomized complete block design with four blocks) and sensitive analytical methods that allow accurate assessments ofMonsanto Company 12-AL-246U 92 of 407 

### compositional characteristics over a range of environmental conditions under which KK179 is expected to be grown.

The information provided in this section addresses relevant factors in Codex Plant Guidelines, Section 4, paragraphs 44 and 45 for compositional analyses (Codex Alimentarius, 2009).

VI.A. Compositional Equivalence of KK 179 Forage to Conventional Alfalfa

Forage samples were collected from the fi rst cutting of KK179, a conventional alfalfa control, and conventional commercial alfalf a varieties grown in a 2011 field production. The conventional control (C 0 

### -Syn1) used as a comparator was a near-isogenic conventional alfalfa population with a geneti c background similar to that of KK179.

Fourteen different conventional commercial alfalfa reference varieties were included across the field production site s to provide data on the natural variability of each compositional component analyzed. The field production was conducted in typical alfalfa-growing regions at six U.S. sites: California (CAPR), Iowa (IARL), Illinois (ILCY), Kansas (KSLA), Texas (TXCL) , and Wisconsin (WIDL). KK179, the

conventional control and the conventional co mmercial varieties were planted in a randomized complete block design with four replicated plots per site and were grown under normal agronomic field conditions for th eir respective geographi c regions. At the plant growth stage between 1 and 10% bloo m, which is a normal stage for harvesting forage, samples of the whole alfalfa plant were harvested at each site from the plants in the center of each individual plot by cutting th e plant 2-3 inches above the soil surface.

Compositional analyses were based on OECD consensus document for alfalfa (OECD, 2005) to compare levels of key nutrients, anti-nutrients and secondary metabolites in KK179 to levels in the conventional control. Forage samples were analyzed for the following nutrients: proximates (ash, fat, moisture, and protein), carbohydrates (determined by calculation), acid detergent fi ber (ADF), neutral detergent fiber (NDF), acid detergent lignin (ADL), minerals (Ca, Cu, Fe, Mg, Mn, P, K, Na, and Zn), and amino acids (essential and non-essential). Anti-nutrient and secondary metabolites included daidzein, glycitein, genistein, coumeste rol, formononetin, biochanin A, saponins (total bayogenin, total hederagenin, total medi cagenic acid, total soyasapogenol B, total soyasapogenol E, total zanhic acid, and total saponins), and canavanine. In addition to the OECD-recommended analytes listed above, p -coumaric acid, ferulic acid, sinapic acid, total polyphenols, and free phenylalanine were also analyzed to evaluate the potential effect of CCOMT s uppression on the lignin biosynth etic pathway and cell wallassociated metabolites.

Methods used in the assessments of nutrients , anti-nutrients, and secondary metabolites are described in Appendix E. Prior to compositional analysis, levels of total lignin (ADL) in forage samples from the 2011 fi eld production were measured by Dairy One

Lab as described in Section I.B.3. Dair y One Forage Lab used a semi-automated ANKOM-based methodology, described in Appendi x D, as adopted by most commercial forage testing laboratories. Covance Laboratories, Inc., which conducted the compositional analyses of key nutrients, an ti-nutrients, and secondary metabolitesMonsanto Company 12-AL-246U 93 of 407 

### outlined above, also included an analysis of total lignin (ADL) using a manual method described in Appendix E.

In all, 54 different components of nutrients , anti-nutrients, and secondary metabolites were measured. Of those 54 components, six anti-nutrients (daidzein, glycitein, genistein, coumesterol, formononetin, and bi ochanin A) and one secondary metabolite (sinapic acid) had more than 50% of th e observations below the assay limit of quantitation (LOQ) and, as a result, were exclud ed from statistical analyses. Therefore, 47 components were statistically assessed using a mixed-model an alysis of variance method. Values for all components were expr essed on a dry weight (dw) basis with the exception of moisture, expressed as percent fresh weight (fw).

The statistical comparison was based on compositional data combined across all field sites. Statistically significant differences between KK179 and the conventional control were identified at the 5% level. Compos itional data from the conventional commercial

varieties were combined across all field sites to calculate a 99% tolerance interval for each compositional component to estimate the natural variability of each component in

alfalfa.

Statistical significance does not imply biolog ical relevance from a feed/food safety or nutritional perspective (EFSA, 201 1). Considerations used to assess the relevance of each statistically significant combined-site diffe rence included: 1) the relative magnitude

of the difference in the mean values of nutri ent, anti-nutrient, and secondary metabolite components of KK179 and the conventional co ntrol; 2) whether the KK179 component mean value is within the range of natural va riability of that component as represented by

the 99% tolerance interval of the conventional commercial reference varieties grown concurrently in the same trial; and 3) an a ssessment of the differences within the context of natural variability of available commercial alfalfa composition published in the scientific literature. Statistical summaries of nutrient, anti-nutrient, and secondary metabolites for individual sites are found in Appendix E.

The compositional analysis provided a comprehensive comparative assessment of the levels of key nutrients, anti- nutrients, and secondary metabo lites in forage of KK179 and

the conventional control. Assessment of the results demonstrated that, with the exception of three compositional constituents (ash, canavanine, and ferulic acid), there were no statisitically significant differences in 44 of the 47 constituents statistically compared. For the three constituents where significant differences were detected, an analysis, including the magnitudes of the differences a nd comparisons of mean values to the 99 % tolerance interval and literature values, i ndicated that they we re not biologically meaningful from a feed/food safety or nutr itional perspective. Further assessment of statistically significant differences obs erved between KK179 and the conventional control is provided in the following section. These results support the overall conclusion that, with the exception of the intended change in reduced G lignin and total lignin levels compared to conventional alfalfa at the same growth stage presented in Section I.B.3, forage of KK179 is compositionally equivalent to conventional alfalfa.Monsanto Company 12-AL-246U 94 of 407 

### VI.B. Nutrients, Anti-Nutrients, and Secondary Metabolites in KK179 Forage

The means and ranges for nutrients, anti- nutrients, and sec ondary metabolites from

KK179 were consistent with values establis hed from the conventional alfalfa control (Tables VI-2, VI-3, and VI-4). No signifi cant differences (p>0.05) were identified for protein (and total amino acids), minerals , fat, moisture, ADF, NDF, ADL, carbohydrates by calculation, saponins, total polyphenols, free phenylalanine, and p- coumaric acid, although statistically si gnificant differences for ash, canav anine, and ferulic acid were observed. A summary of differences observed in the combined-site analysis can be found

in Table VI-1.

The mean level of ash was significantly lo wer (p<0.05) in KK179 forage than the conventional control in the combined-site analys is (Table VI-1). The absolute difference in magnitude was 0.41% dw, which is a relativ e small difference of -3.8%. Furthermore,

the mean level of ash was within the 99% tolerance interval of the conventional

commercial references varieties and within the range of values found in the published literature (Table VI-5). Therefore, the diffe rence in ash in KK179 forage compared to the

conventional control is not c onsidered biologically meani ngful from a feed/food safety and nutritional perspective.

The mean level of canavanine, an anti-nutri ent, was significantly lower (p<0.05) in KK179 forage than the conventional control in the combined-site analysis (Table VI-1). The absolute difference in magnitude was 16.9 4 ppm, which is a relative difference of - 29.6%. However, the mean level of canavanine was within the 99% tolerance interval of

the conventional commercial reference varietie s and within the range of values found in the published literature (Table V I-5). Lower levels of cana vanine would not be adverse

as it is considered an anti-nu trient in leguminous plants. Therefore, the difference in canavanine in KK179 forage compared to th e conventional control is not considered biologically meaningful fr om a feed/food safety and nutritional perspective.

The mean level of ferulic acid was signifi cantly higher (p<0.05) in KK179 forage than

the conventional control in th e combined-site analysis (Tab le VI-1). The absolute difference in magnitude was 110.60 ppm, which is a relatively small difference of 7.4%.

The mean level of ferulic acid was within th e 99% tolerance interval of the conventional

commercial reference varieties and within the range of values found in the published literature (Table VI-5). Ferulic acid is an important component of the overall cell wall structure, and may serve as an ‘anchor site ’ for lignin deposition (Grabber et al., 2000), thus it is not unexpected that alterations in lignin content could result in alterations in ferulic acid levels. Therefore, the difference in ferulic acid in KK179 forage compared to

the conventional control is not considered biologically meaningf ul from a feed/food safety and nutritional perspective.

Although the mean level of total lignin (ADL ) in KK179 was not sign ificantly lower as reported in Section I.B.3., it was numerically lower in KK179 forage compared to the conventional control in the combined-site analys is (Table VI-2). The absolute difference in magnitude was 0.32% dw, which is a re lative difference of -4.89%. The use of different methods, semi-automated ANKOM-bas ed assay by Dairy One Forage Lab andMonsanto Company 12-AL-246U 95 of 407 

### the manual assay by Covance Lab, likely contri buted to the variability in total lignin (ADL) values determined by th e two laboratories. Both methods did confirm a decrease in total lignin in KK179, with Dairy One Fora ge Lab reporting a significant decrease of

22.15% and Covance a numeri cal decrease of 4.89%.

Assessment of these compositional results supp orts the overall conclusion that, with the exception of the intended reductio n in G lignin and total lignin as presented in Section I.B.3., forage from KK179 is compositionally equivalent to conventional alfalfa with regard to levels of nutrients, anti-nutrients, and secondary meta bolites. In the case of ash and ferulic acid, the relative magnitudes of the differences were under 10%. The mean levels of all three analytes with observed statistical differences were within the 99% tolerance interval established from the popul ation of conventional commercial reference varieties and within the range of values found in the published literature (T able VI-5). Therefore, these differences are not consider ed biologically meaningf ul from a feed/food safety and nutritional perspective.Monsanto Company 12-AL-246U 96 of 407Table VI-1. Summary of Differences Observed in the Combined-Site Analysis (p<0.05) of Alfalfa Forage Component Levelsfor KK179 vs. Conventional ControlMean Difference(Test minus Control)Analytical Component (Units)1KK179Mean2Control3MeanMean Difference(% of Control)Significance(p-Value)TestRangeCommercialTolerance Interval4Statistical Differences Observed in Combined-Site AnalysisForage Proximate (% dw)Ash10.3810.79-3.770.0348.43 - 13.266.70, 13.54Forage MetaboliteCanavanine (ppm dw)40.3057.24-29.600.01311.42 - 87.830, 137.35Ferulic Acid (ppm dw)1596.411485.817.440.0081389.38 - 1884.17854.88, 2061.101dw = dry weight2Mean = least-square mean3Control refers to the conventional alfalfa control (C0-Syn1).4With 95% confidence, interval contains 99% of the values expressed in the population of conventional commercial reference varieties. Negativelimits set to zero.Monsanto Company 12-AL-246U 97 of 407Table VI-2. Statistical Summary of Alfalfa Forage Nutrients for KK179 vs. Conventional ControlDifference (Test minus Control)Analytical Component (Units)1KK179Mean (S.E.)2(Range)Control3Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval4(Range)Proximate (% dw)Ash10.38 (0.53)10.79 (0.52)-0.41 (0.19)-0.78, -0.0300.0346.70, 13.54(8.43 - 13.26)(8.79 - 12.95)(-1.80 - 1.06)(7.54 - 13.23)Carbohydrates66.55 (1.71)65.97 (1.70)0.58 (0.49)-0.55, 1.710.27250.57, 81.80(57.73 - 73.90)(59.94 - 72.91)(-3.45 - 8.36)(54.35 - 74.91)Moisture (% fw)78.26 (1.54)78.15 (1.54)0.11 (0.33)-0.64, 0.860.74865.06, 90.61(73.70 - 84.60)(70.50 - 83.70)(-2.70 - 3.60)(66.10 - 85.30)Protein20.83 (1.36)21.02 (1.35)-0.19 (0.39)-0.98, 0.600.6369.26, 33.78(15.50 - 29.03)(15.98 - 27.30)(-6.67 - 3.19)(14.52 - 30.07)Total Fat2.28 (0.17)2.28 (0.17)0.0039 (0.16)-0.31, 0.320.9800.73, 3.59(0.84 - 3.98)(1.08 - 3.38)(-1.30 - 1.46)(0.53 - 4.21)Fiber (% dw)Acid Detergent Fiber27.03 (2.45)27.02 (2.44)0.015 (0.96)-2.12, 2.150.9876.16, 49.06(15.71 - 37.26)(10.96 - 36.11)(-10.08 - 9.57)(7.07 - 39.11)Monsanto Company 12-AL-246U 98 of 407Table VI-2 (continued). StatisticalSummary of Alfalfa Forage Nutrients for KK179 vs. Conventional ControlDifference (Test minus Control)Analytical Component (Units)1KK179Mean (S.E.)2(Range)Control3Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval4(Range)Fiber (% dw)Acid Detergent Lignin6.22 (0.60)6.54 (0.59)-0.32 (0.27)-0.91, 0.280.2652.13, 11.99(2.72 - 10.31)(3.58 - 8.26)(-2.08 - 3.39)(3.38 - 9.67)Neutral Detergent Fiber33.95 (2.64)34.46 (2.63)-0.52 (0.97)-2.67, 1.630.60512.04, 58.18(18.57 - 48.67)(18.94 - 43.32)(-9.74 - 11.84)(18.97 - 49.82)Amino Acid (% dw)Alanine1.11 (0.074)1.13 (0.074)-0.017 (0.020)-0.062, 0.0280.4170.49, 1.79(0.84 - 1.52)(0.87 - 1.39)(-0.19 - 0.13)(0.80 - 1.66)Arginine0.99 (0.065)1.01 (0.065)-0.020 (0.020)-0.065, 0.0240.3260.44, 1.59(0.73 - 1.35)(0.75 - 1.28)(-0.17 - 0.11)(0.70 - 1.44)Aspartic acid2.77 (0.28)2.74 (0.28)0.027 (0.071)-0.13, 0.190.7110.44, 5.63(1.97 - 4.65)(2.04 - 4.08)(-0.64 - 0.71)(1.96 - 5.15)Cystine0.21 (0.012)0.21 (0.011)0.00079 (0.0074)-0.016, 0.0170.9160.12, 0.32(0.15 - 0.30)(0.15 - 0.29)(-0.041 - 0.062)(0.16 - 0.31)Monsanto Company 12-AL-246U 99 of 407Table VI-2 (continued). StatisticalSummary of Alfalfa Forage Nutrients for KK179 vs. Conventional ControlDifference (Test minus Control)Analytical Component (Units)1KK179Mean (S.E.)2(Range)Control3Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval4(Range)Amino Acid (% dw)Glutamic acid1.85 (0.12)1.91 (0.12)-0.053 (0.035)-0.13, 0.0240.1560.81, 3.01(1.40 - 2.55)(1.39 - 2.36)(-0.31 - 0.22)(1.31 - 2.80)Glycine0.95 (0.055)0.97 (0.055)-0.018 (0.013)-0.047, 0.0110.1900.49, 1.44(0.75 - 1.21)(0.73 - 1.14)(-0.10 - 0.085)(0.70 - 1.33)Histidine0.43 (0.020)0.44 (0.020)-0.0064 (0.0058)-0.018, 0.00530.2760.26, 0.63(0.35 - 0.55)(0.36 - 0.51)(-0.053 - 0.059)(0.34 - 0.61)Isoleucine0.86 (0.053)0.88 (0.053)-0.016 (0.014)-0.048, 0.0160.2840.43, 1.36(0.67 - 1.15)(0.66 - 1.07)(-0.12 - 0.12)(0.63 - 1.27)Leucine1.43 (0.089)1.47 (0.089)-0.033 (0.023)-0.086, 0.0190.1870.70, 2.25(1.09 - 1.90)(1.09 - 1.78)(-0.22 - 0.16)(1.03 - 2.05)Lysine1.14 (0.067)1.17 (0.067)-0.024 (0.017)-0.058, 0.00940.1530.55, 1.82(0.93 - 1.55)(0.92 - 1.44)(-0.15 - 0.12)(0.82 - 1.73)Monsanto Company 12-AL-246U 100 of 407Table VI 2 (continued). StatisticalSummary of Alfalfa Forage Nutrients for KK179 vs. Conventional ControlDifference (Test minus Control)Analytical Component (Units)1KK179Mean (S.E.)2(Range)Control3Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval4(Range)Amino Acid (% dw)Methionine0.25 (0.024)0.24 (0.024)0.0083 (0.012)-0.017, 0.0330.5080.068, 0.42(0.15 - 0.39)(0.15 - 0.37)(-0.12 - 0.14)(0.14 - 0.45)Phenylalanine0.98 (0.061)1.00 (0.061)-0.025 (0.016)-0.060, 0.00970.1380.48, 1.53(0.75 - 1.27)(0.74 - 1.21)(-0.15 - 0.083)(0.71 - 1.39)Proline0.89 (0.054)0.92 (0.053)-0.028 (0.021)-0.075, 0.0180.1990.43, 1.41(0.71 - 1.18)(0.71 - 1.21)(-0.28 - 0.11)(0.65 - 1.24)Serine0.87 (0.044)0.88 (0.044)-0.0061 (0.017)-0.045, 0.0330.7330.45, 1.35(0.68 - 1.16)(0.68 - 1.05)(-0.16 - 0.13)(0.66 - 1.25)Threonine0.86 (0.050)0.88 (0.050)-0.018 (0.016)-0.053, 0.0180.2880.45, 1.33(0.66 - 1.12)(0.67 - 1.05)(-0.13 - 0.10)(0.63 - 1.23)Tryptophan0.37 (0.020)0.37 (0.020)0.0036 (0.0082)-0.013, 0.0200.6630.20, 0.56(0.30 - 0.48)(0.27 - 0.45)(-0.056 - 0.065)(0.25 - 0.50)Monsanto Company 12-AL-246U 101 of 407Table VI-2 (continued). StatisticalSummary of Alfalfa Forage Nutrients for KK179 vs. Conventional ControlDifference (Test minus Control)Analytical Component (Units)1KK179Mean (S.E.)2(Range)Control3Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval4(Range)Amino Acid (% dw)Tyrosine0.71 (0.042)0.71 (0.042)0.0012 (0.015)-0.033, 0.0350.9390.35, 1.09(0.55 - 0.94)(0.53 - 0.89)(-0.098 - 0.10)(0.52 - 1.01)Valine1.05 (0.061)1.07 (0.061)-0.017 (0.015)-0.048, 0.0140.2800.52, 1.64(0.79 - 1.38)(0.81 - 1.32)(-0.16 - 0.13)(0.79 - 1.55)MineralCalcium (% dw)1.68 (0.16)1.72 (0.16)-0.037 (0.037)-0.12, 0.0450.3360.55, 2.56(1.12 - 2.62)(1.09 - 2.53)(-0.41 - 0.28)(0.95 - 2.07)Copper (mg/kg dw)8.86 (0.85)8.34 (0.85)0.52 (0.34)-0.16, 1.200.1311.87, 14.98(5.14 - 13.16)(5.18 - 11.93)(-1.62 - 4.04)(4.54 - 19.67)Iron (mg/kg dw)272.00 (31.45)315.74 (30.93)-43.74 (24.69)-98.55, 11.070.10641.59, 446.31(123.38 - 473.91)(163.92 - 547.83)(-279.88 - 115.21)(105.45 - 691.43)Magnesium (% dw)0.22 (0.023)0.23 (0.023)-0.0082 (0.0050)-0.018, 0.00190.1080.027, 0.41(0.12 - 0.31)(0.15 - 0.32)(-0.048 - 0.037)(0.11 - 0.34)Monsanto Company 12-AL-246U 102 of 407Table VI-2 (continued). StatisticalSummary of Alfalfa Forage Nutrients for KK179 vs. Conventional ControlDifference (Test minus Control)Analytical Component (Units)1KK179Mean (S.E.)2(Range)Control3Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval4(Range)MineralManganese (mg/kg dw)52.56 (6.30)52.45 (6.27)0.11 (2.65)-5.86, 6.090.96617.53, 69.85(30.52 - 106.47)(30.92 - 77.32)(-15.31 - 37.75)(23.24 - 98.04)Phosphorus (% dw)0.29 (0.019)0.28 (0.019)0.0037 (0.0057)-0.0079, 0.0150.5230.14, 0.46(0.22 - 0.40)(0.20 - 0.38)(-0.040 - 0.071)(0.18 - 0.43)Potassium (% dw)2.35 (0.052)2.41 (0.051)-0.055 (0.051)-0.17, 0.0590.3071.82, 3.04(2.16 - 2.65)(2.18 - 2.71)(-0.45 - 0.21)(1.85 - 3.35)Sodium (% dw)0.089 (0.024)0.077 (0.024)0.013 (0.0076)-0.0026, 0.0280.1020, 0.24(0.020 - 0.22)(0.018 - 0.15)(-0.056 - 0.083)(0.016 - 0.20)Zinc (mg/kg dw)27.83 (2.11)26.81 (2.09)1.02 (1.42)-2.15, 4.190.4898.89, 47.44(18.40 - 39.22)(17.38 - 40.42)(-5.64 - 11.08)(17.08 - 47.48)1dw = dry weight; fw = fresh weight2Mean (S.E.) = least-square mean (standard error)3Control refers to the conventional alfalfa control (C0-Syn1).4With 95% confidence, interval contains 99% of the values expressed in the population of conventional commercial reference varieties. Negativelimits set to zero.Monsanto Company 12-AL-246U 103 of 407Table VI-3. Statistical Summary ofAlfalfa Forage Secondary Metabolites for KK179 vs. Conventional ControlDifference (Test minus Control)Analytical Component (Units)1KK179Mean (S.E.)2(Range)Control3Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval4(Range)MetaboliteCanavanine (ppm dw)40.30 (13.53)57.24 (13.51)-16.94 (5.69)-29.62, -4.270.0130, 137.35(11.42 - 87.83)(12.69 - 134.50)(-79.53 - 5.32)(11.47 - 151.33)Ferulic Acid (ppm dw)1596.41 (59.57)1485.81 (58.83)110.59 (40.34)29.36, 191.830.008854.88, 2061.10(1389.38 - 1884.17)(1103.96 - 2007.38)(-301.26 - 503.18)(1103.32 - 1906.86)Free Phenylalanine (ppm dw)266.99 (28.84)283.70 (28.69)-16.71 (12.62)-42.11, 8.690.1920, 627.23(111.86 - 409.20)(154.07 - 457.63)(-125.58 - 75.77)(133.05 - 579.05)Total Polyphenols (mg/g dw)8.19 (0.34)7.99 (0.34)0.20 (0.23)-0.30, 0.710.3904.86, 11.15(6.35 - 10.19)(6.57 - 10.21)(-2.13 - 1.72)(6.17 - 11.17)p-Coumaric Acid (ppm dw)639.50 (37.62)623.54 (37.34)15.97 (19.93)-28.25, 60.180.441188.81, 949.95(458.33 - 870.13)(442.08 - 819.59)(-112.64 - 226.29)(326.19 - 945.58)1dw = dry weight; fw = fresh weight2Mean (S.E.) = least-square mean (standard error)3Control refers to the conventional alfalfa control (C0-Syn1).4With 95% confidence, interval contains 99% of the values expressed in the population of conventional commercial reference varieties. Negativelimits set to zero.Monsanto Company 12-AL-246U 104 of 407Table VI-4. Statistical Summary ofAlfalfa Forage Anti-Nutrientsfor KK179 vs. Conventional ControlDifference (Test minus Control)Analytical Component (Units)1KK179Mean (S.E.)2(Range)Control3Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval4(Range)Saponins (response units/μg)Total Bayogenin5.10 (0.76)5.67 (0.76)-0.57 (0.47)-1.53, 0.380.2300.92, 8.86(2.54 - 13.97)(2.20 - 11.28)(-2.85 - 5.81)(1.46 - 11.28)Total Hederagenin2.94 (0.35)3.47 (0.35)-0.53 (0.32)-1.24, 0.190.1310.85, 7.20(1.70 - 5.80)(1.58 - 6.85)(-3.51 - 1.21)(0.90 - 10.31)Total Medicagenic Acid21.88 (2.44)23.39 (2.44)-1.51 (2.51)-6.57, 3.550.5510, 44.42(9.09 - 45.08)(9.43 - 51.04)(-22.95 - 15.92)(2.04 - 48.33)Total Soyasapogenol B22.17 (3.02)24.53 (3.02)-2.36 (1.44)-5.56, 0.840.1317.83, 44.92(9.68 - 40.48)(7.05 - 41.93)(-12.47 - 8.72)(9.22 - 43.87)Total Soyasapogenol E2.77 (0.54)3.08 (0.54)-0.31 (0.26)-0.84, 0.220.2480, 6.59(1.20 - 5.02)(0.84 - 8.89)(-4.99 - 1.87)(0.91 - 7.53)Monsanto Company 12-AL-246U 105 of 407Table VI-4 (continued). Statistical Summary of Alfalfa Forage Anti-Nutrients for KK179 vs. Conventional ControlDifference (Test minus Control)Analytical Component (Units)1KK179Mean (S.E.)2(Range)Control3Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialToleranceInterval4(Range)Saponins (response units/μg)Total Zanhic Acid4.59 (0.58)5.16 (0.58)-0.57 (0.45)-1.48, 0.330.2100.32, 12.06(2.25 - 12.08)(2.62 - 8.69)(-3.97 - 3.69)(1.75 - 13.20)Total Saponins59.30 (4.94)65.58 (4.94)-6.28 (4.35)-15.05, 2.490.15621.87, 108.47(36.00 - 122.44)(29.20 - 96.50)(-32.96 - 25.94)(17.38 - 103.19)1dw = dry weight; fw = fresh weight2Mean (S.E.) = least-square mean (standard error)3Control refers to the conventional alfalfa control (C0-Syn1).4With 95% confidence, interval contains 99% of the values expressed in the population of conventional commercial reference varieties. Negativelimits set to zero.Monsanto Company 12-AL-246U 106 of 407 

### Table VI-5. Literature and OECD Ranges for Compostional Components in Alfalfa Forage Components 1 

### Literature Range 2 

### OECD Range 3

Forage Nutrients
Proximates (% dw)

Ash 8.62 - 14.81 a ; 6.86 - 15.25 b ; 5.8 - 7.5 c 8.4 - 15.3 Carbohydrates by calculation 56.63 - 74.80 b NA Fat, total 1.80 - 3.24 a ; 1.33 - 4.49 b 2.8 - 3.1 c 1.3 - 3.2 Moisture (% fw) 7.74 - 18.10 a ; 70.90 - 83.50 b 9.0 - 82.1

Protein 14.91 - 25.35 a ; 15.29 - 28.34 b ; 17.0 – 21.3 c 15.3 - 25.8 Fiber (% dw) Acid detergent fiber 23.17 - 42.59 a ; 21.26 - 39.25 b 23.1 - 33.4 Neutral detergent fiber 29.08 - 53.56 a ; 26.53 - 51.09 b 26.5 - 40.0 Acid Detergent Lignin 5.69 - 9.37 a ; 2.31 - 13.71 b 3.9 - 9.7 Amino Acids (% dw)

Alanine 0.93 - 1.21 c 0.70 - 1.59 Arginine 0.86 - 1.08 c 0.62 - 1.54 Aspartic acid 1.97 - 2.15 c 1.40 - 3.52 Cystine NA 0.18 - 0.35

Glutamic acid 1.88 - 2.40 c 1.20 - 3.03 Glycine 0.82 - 1.1 c 0.60 - 1.47

Histidine 0.48 - 0.60 c 0.28 - 0.74

Isoleucine 0.77 - 0.95 c 0.50 - 1.26

Leucine 1.35 - 1.62 c 0.90 - 2.25 Lysine 1.06 - 1.16 c 0.59 - 1.81

Methionine 0.28 - 0.37 c 0.18 - 0.48 Phenylalanine 0.87 - 1.08 c 0.72 - 1.59

Proline 0.65 - 1.26 c 0.70 - 1.34

Serine 0.76 - 0.95 c 0.60 - 1.36

Threonine 0.78 - 1.11 c 0.60 - 1.15 Tryptophan NA 0.16 - 0.35 Tyrosine 0.66 - 0.83 c 0.50 - 1.16

Valine 0.91 - 1.18 c 0.60 - 1.55

Minerals Calcium (% dw) 1.03 - 1.93 a ; 0.90 - 1.86 b 0.90 - 1.96 Copper (mg/kg dw) 3.43 - 14.72 b 5.3 - 13.4 Iron (ppm dw) 1 - 4749 a ; 63.49 - 1538.46 b 0.2 - 15.4 Magnesium (% dw) 0.20 - 0.40 a ; 0.11 - 0.45 b 0.11 - 0.45 Manganese (ppm dw) 16 - 64 a ; 15.91 - 109.50 b 31.5 - 109.5 Phosphorus (% dw) 0.24 - 0.42 a ; 0.22 - 0.46 b 0.22 - 0.45 Potassium (% dw) 1.59 - 3.21 a ; 1.39 - 4.31 b 1.39 - 4.31 Sodium (ppm dw) 1 - 3826 a ; 170 - 5100 b 0.2 - 2.1 Zinc (mg/kg dw) 15.20 - 43.62 b 18.0 - 36.0Monsanto Company 12-AL-246U 107 of 407 

### Table VI-5 (continued). Literature and OECD Ranges for Components in Alfalfa
Forage

Components 1 Literature Range 2 

### OECD Range 3

Forage Metabolite Ferulic acid (ppm, dw) 627 d ; 680 e ; 770 - 2840 f NA

p -Coumaric acid (ppm, dw) 398 d ; 254 e ; 630 - 1860 f NA Canavanine (%) (seedling) 1.3 - 2.4 g NA Free Phenylalanine NA NA Total polyphenols NA NA

1 fw=fresh weight; dw=dry weight

2 Literature range references: a (Dairyland Laboratories, 2011); b (McCann et al., 2006); c (Smith, 1969); d (Bourquin et al., 1990); e (Cherney et al., 1989); f (Jung and Fahey, 1983); g (Rosenthal and Nkomo, 2000)

3 (OECD, 2005)

4 NA=not availableMonsanto Company 12-AL-246U 108 of 407 

### VI.C. Compositional Assessment of KK179 Conclusion

Analyses of nutrient, anti-nutrient, and s econdary metabolite levels in KK179 and the

conventional control were conducted to assess compositional equivalence. The analytes

evaluated are consistent with those identified by OECD as important to understanding the safety and nutrition of biotechnology-deriv ed alfalfa (OECD, 2005). The compositional comparisons were made by analyzing forage harvested from the first cutting during the

2011 field season from six field sites in th e U.S. that are representative of normal agricultural regions for alfalfa production. The compositional analysis, based on OECD consensus document for alfalfa, also include d measurement of nutri ents, anti-nutrients and secondary metabolites in conventional comme rcial reference varie ties to provide data on the natural variability of each co mpositional component analyzed.

Compositional analyses based on the OECD c onsensus document confirmed that, other than the intended reduction in G lignin and to tal lignin presented in Section I.B.3., there is no meaningful effect on key nutrient, anti-nutrient, and secondary metabolite components in KK179 compared to a conventiona l alfalfa control. Of the 47 components statistically assessed, only three (ash, canavan ine, and ferulic acid) showed a significant difference in combined-site analysis between KK179 and the conventional control. Two of the three observed differences (ash and fe rulic acid) are less than 10% in relative magnitude. The mean values for all three components with statis tically significant

differences between KK179 and the conventiona l control fall within the 99% tolerance

interval determined from the conventional commercial alfalfa varieties grown concurrently with KK179 and the control. In addition, the levels of these three components are also within the ranges publis hed in the scientific literature.

These analyses provide a comprehensive comp arative assessment of the levels of key nutrients, anti-nutrients, and secondary metabolites in fo rage of KK179 compared to the conventional control. These results support the overall conclusion that, with the exception of the intended re duction in G lignin and tota l lignin, forage of KK179 is compositionally equivalent to that of conventio nal alfalfa; therefore, the feed/food safety and nutritional quality of this product is comparable to conventional alfalfa.

The processing of KK179 forage into animal f eed is not expected to be different from

that of conventional alfalfa. As described in this section, detailed compositional analyses of key components of KK179 have demons trated that KK179 is compositionally equivalent except for the intended reduction in G lignin and total lignin compared to conventional alfalfa at the same stage of growth. Therefore, when KK179 and its progeny are used on a commercial scale as a source of feed, these products are not expected to be different from equivalent feeds originati ng from conventional alfalfa.Monsanto Company 12-AL-246U 109 of 407 

### VII. PHENOTYPIC, AGRO NOMIC, AND ENVIRONMENTAL

INTERACTIONS ASSESSMENT

This section provides a comparative asse ssment of the phenotypic, agronomic, and

environmental interaction char acteristics of KK179 compared to a conventional control. The data support a conclusion that KK179 is not meaningfully different from the conventional control with the exception of th e trait for reduced G li gnin and total lignin, and therefore is not expected to pose a plant pest risk compared with conventional alfalfa. These conclusions are based on the results of multiple evaluations from laboratory and

field assessments.

Phenotypic, agronomic, and environmental in teraction characteris tics of KK179 were evaluated in a comparative manner to assess plant pest potential. These assessments included evaluation of seed germination char acteristics, plant growth and development characteristics, observations of plant responses to abiotic st ress, plant-disease and plantarthropod interactions, pollen characteristics, flower charact eristics, and plant-symbiont

interactions. Results from these assessmen ts demonstrate that KK179 does not possess 1) increased weediness characteristics; 2) incr eased susceptibility or tolerance to specific abiotic stressors, diseases, or arthropods; or 3) characteristics that would confer a plant pest risk compared to conventional alfalfa.

VII.A. Characteristic s Measured for Assessment

In the phenotypic, agronomic, and environm ental interactions a ssessment of KK179, data were collected to evaluate altered plant pest potential. A detailed description of the regulated article phenotype is requested as part of the petition for determination of nonregulated status in 7 CFR § 340.6, incl uding differences from the unmodified recipient organism that would “s ubstantiate that the regulated article is unlikely to pose a greater plant pest risk than the unmodified or ganism from which it was derived.” As part of the characterization of KK179, data were coll ected to provide a detailed description of the phenotypic, agronomic, and environmental interaction characteristics of KK179. A

subset of these data were included as an evaluation of specific characteristics related to weediness, e.g. , seed dormancy, lodging, and split pods, which is an element of the USDA-APHIS plant pest determination.

The plant characterization of KK179 encompassed seven general data categories: 1) seed germination, dormancy, and emergence; 2) ve getative growth; 3) winter survival; 4) reproductive development, including pollen, flow er, and seed character istics; 5) lodging and seed retention on the plant; 6) plant respons e to abiotic stress and interactions with diseases and arthropods; and 7) plant-symbiont interact ions. An overview of the characteristics assessed is presented in Table VII-1.

The phenotypic, agronomic, and environmental interactions data were evaluated from a basis of familiarity (OECD, 1993) and were comprised of a combination of field, greenhouse, and laboratory studies conducted by scientists who are familiar with the production and evaluation of alfalfa. In each of these assessments, KK179 was compared to a conventional control, C 0 

### -Syn1, which is a near-isogeni c population produced via anMonsanto Company 12-AL-246U 110 of 407 

### identical breeding path as KK179, with the ex ception of the intended reduction in G and total lignin compared to conventional alfalfa at the same stage of growth. In addition, multiple conventional commercial reference varieties developed through conventional breeding and selection (see A ppendices F-K and Tables F-1, G-1, H-1, I-1, J-1, and K-1) were included to provide a range of comp arative values for each characteristic that represent the vari ability in existing conventional comm ercial alfalfa varieties. Data

collected for the various ch aracteristics from the conv entional commercial reference varieties provide context for in terpreting experi mental results.Monsanto Company 12-AL-246U 111 of 407 

### Table VII-1. Phenotypic, Agronomi c, and Environmental Interaction

Characteristics Evaluated in North Ameri can Field Trials and Laboratory or

Greenhouse Studies

Data Category Characteristics

measured (associated section where discussed) Evaluation timing 1
(setting of evaluation) Evaluation description (measurement endpoints)

Seed germination, dormancy, and

emergence Normal germinated (VII.C.1.) Day 4 and 7 (20 oC) (Laboratory) Percentage of seed producing seedlings exhibiting normal developmental characteristics Abnormal germinated (VII.C.1.) Day 7 (20 °C) (Laboratory) Percentage of seed producing seedlings that could not be classified as normal germinated Germinated (VII.C.1.) Day 4, 7 and 12 (10 °C and 30 °C) (Laboratory) Percentage of seed that had germinated (both normally or abnormally) Dead (VII.C.1.) Day 4 and 7 (20 °C) and Day 4, 7, and 12 (10 °C and 30 °C) (Laboratory) Percentage of seed that had visibly

deteriorated and become soft to the touch (also included non-viable hard and nonviable firm-swollen seed) Viable hard (VII.C.1.) Day 7 (20 °C) and Day 12 (10 °C and 30 °C) (Laboratory) Percentage of seed that did not

imbibe water and remained hard to the touch (viability determined by

tetrazolium test 2 )

Viable firm-swollen (VII.C.1.) Day 7 (20 °C) and Day 12 (10 °C and 30 °C) (Laboratory) Percentage of seed that imbibed

water and were firm to the touch but did not germinate (viability determined by tetrazolium test 2 ) Seedling emergence (VII.C.2.1.) 10-21 Days after planting (DAP) (Field forage production) Average number of emerged plants per foot

Seedling establishment (VII.C.2.3.) 51 DAP (Field seed production) Number of established plants in each plot

Vegetative growth Early season vigor (VII.C.2.1.) 10-40 DAP (Field forage production) Rated on a 1-10 scale, where: 1 = poor vigor and 10 = excellent vigor

Seedling vigor (VII.C.2.3.) 51 DAP (Field seed production) Rated on a 1-10 scale, where: 1 = poor vigor and 10 = excellent vigor

Crop growth stage (VII.C.2.1.) 0-2 days before forage harvest (Field forage production) Mean Stage by Count method, calculation based on growth stage of 35-45 stems per plot 1 (Table G-4) Forage yield (VII.C.2.1.) 1-10% bloom (Field forage production) Fresh weight of forage (ton/acre) for

an individual harvest

Regrowth after forage

harvest (VII.C.2.1.) Approximately 10-15 days after a harvest (Field forage production) Rated on a 1-10 scale, where 1 = dead and 10 = excellent vigor and growth

Fall plant height (VII.C.2.1.) Varied by region depending on decreasing photoperiod and temperature (Field forage production) Distance (in) from so il surface to the uppermost node on the main stem of five representative plantsMonsanto Company 12-AL-246U 112 of 407 Table VII-1 (continued). Phenotypic, Ag ronomic, and Environmental Interaction

Characteristics Evaluated in North American Field Trials and Laboratory or Greenhouse

Studies

Data Category Characteristics

measured (associated section where discussed) Evaluation timing (setting of evaluation) Evaluation description (measurement endpoints) Vegetative growth (cont) Total forage yield (VII.C.2.1.) Post-season (Field forage production) Sum of the fresh weight of forage (ton/acre) for all harvests at a site

over a season Winter

survival Spring vigor (VII.C.2.1.) 4-6 inches of spring regrowth (Field forage production) Rated on a 1-10 scale, where: 1 = poor vigor and 10 = excellent vigor

Spring stand recovery (VII.C.2.1.) 4-6 inches of spring regrowth (Field forage production) Rated on a 1-10 scale, where: 1 =

0-10% of stand survived winter and 10 = 100% of uniform stand present Spring stand count (VII.C.2.1.) 6-12 inches of spring regrowth (Field forage production) Average number of stems per foot for three 1-ft segments

Reproductive development Pollen viability (VII.C.3.) Flowering, (Laboratory) Percentage of viable pollen, which stain purple due to the presence of living cytoplasmic content Pollen morphology (VII.C.3.) Flowering, (Laboratory) Average diameter (μm) of 10 representative viable pollen grains

Flowers per raceme (VII.C.4.) Flowering, (Laboratory) Average number of opened and unopened flowers on a raceme

Standard petal length (VII.C.4.) Flowering, (Laboratory) Average length (mm) of the standard petal per 10 flowers Keel petal length (VII.C.4.) Flowering, (Laboratory) Average length (mm) of the fused keel petals per 10 flowers Calyx tube diameter (VII.C.4.) Flowering, (Laboratory) Average diameter (mm) of the calyx tube per 10 flowers Sexual column length (VII.C.4.) Flowering, (Laboratory) Average length (mm) of the sexual column, after tripping the flower, per 10 flowers Wing petal length (VII.C.4.) Flowering, (Laboratory) Average length (mm) of one wing petal per 10 flowers

Flower color class (VII.C.4.) Flowering, (Laboratory) Color of completely open nonsenesced flowers scored on a whole

raceme basis for 10 racemes

Gross raceme morphology (VII.C.4.) Flowering, (Laboratory) Classified as typical or atypical based on overall appearance for 10

racemes Gross flower morphology (VII.C.4.) Flowering, (Laboratory) Classified as typical or atypical based on overall appearance for 10

racemes Seed weight (VII.C.2.3.) Post harvest (Field seed production) Mean seed weight (mg) per 50 seed

Reproductive development (cont) Seed per pod (VII.C.2.3.) Post harvest (Field seed production) Mean number of seed per 20 pods

Seed yield (VII.C.2.3.) At harvest (Field seed production) Mean seed weight (g) harvested per plotMonsanto Company 12-AL-246U 113 of 407 Table VII-1 (continued). Phenotypic, Ag ronomic, and Environmental Interaction

Characteristics Evaluated in North American Field Trials and Laboratory or Greenhouse

Studies

Data Category Characteristics

measured (associated section where discussed) Evaluation timing (setting of evaluation) Evaluation description (measurement endpoints) Lodging and

seed retention Lodging (VII.C.2.1.) 0-2 days before forage harvest (Field forage production) Rated on a 0-9 scale, where 0 = 0-10% erect stems (lodging susceptible) and 9 = 91-100% erect stems (lodging resistant) Lodging (at seed maturity; VII.C.2.3.) At seed harvest (Field seed production) Rated on a 0-9 scale, where 0 = 0-10% erect stems (lodging susceptible) and 9 = 91-100% erect stems (lodging resistant) Split pods (VII.C.2.3.) Ripe seed pod stage, approximately five weeks after pollination (Field seed production) Percentage of split pods per 10

racemes

Plantenvironmental

interactions Plant response to

abiotic stress (VII.C.2.2.) 3-5 observations per season at 0-10 days prior to forage harvest (Field forage production) Qualitative assessment of each plot, rated on a 0-9 scale, where 0 = no symptoms and 9 = severe symptoms

Disease damage (VII.C.2.2.) 3-5 observations per season at 0-10 days prior to forage harvest (Field forage production) Qualitative assessment of each plot, rated on a 0-9 scale, where 0 = no symptoms and 9 = severe symptoms

Arthropod-related damage (VII.C.2.2.) 3-5 observations per season at 0-10 days prior to forage harvest (Field forage production) Qualitative assessment of each plot, rated on a 0-9 scale, where 0 = no symptoms and 9 = severe symptoms

Alfalfa weevil damage (VII.C.2.2.) 3-5 observations per season at 1-9 days prior to forage harvest (Field forage production) Specific quantitative assessment of alfalfa weevil damage from 10 areas in each plot using a 0-5 scale, where 0 = no damage and 5 = >90% of foliage with skeletonized appearance Potato leafhopper damage (VII.C.2.2.) 3-5 observations per season at1-9 days prior to forage harvest (Field forage production) Specific quantitative assessment of potato leafhopper damage from 10 areas in each plot using a 0-5 scale, where 0 = no damage and 5 => 90% foliage with yellowing and puckering Arthropod abundance (VII.C.2.2.) 3-5 collections per season at 0-10 days prior to forage harvest (Field forage production) Number of pest and beneficial arthropods collected per plot

Plantsymbiont

interactions Biomass (VII.C.4) 6 weeks after emergence (Greenhouse) Nodule, root, and shoot dry weight (g) Total nitrogen (VII.C.4) 6 weeks after emergence (Greenhouse) Shoot total nitrogen (%, g)

Plantsymbiont

interactions Biomass (VII.C.4) 6 weeks after emergence (Greenhouse) Nodule, root, and shoot dry weight (g) Total nitrogen (VII.C.4) 6 weeks after emergence (Greenhouse) Shoot total nitrogen (%, g)

1 Alfalfa developmental stages were determined according to definitions provided by (Kalu and Fick, 1981).

2 Viability of hard and firm-swollen seed were determined by a tetrazolium test (AOSA, 2007c; a).Monsanto Company 12-AL-246U 114 of 407 

### VII.B. Interpretation of Phenotypic and Environmental Interactions Data

Plant pest risk assessments for biotech nology-dervided crops are comparative assessments, and are considered from a basis of familiarity. The concept of familiarity is based on the fact that the biotechnology-deri ved plant is developed from a conventional crop whose biological properties and plant pe st potential are well-known. Familiarity considers the biology of the cr op, the introduced trait, the re ceiving environment, and the interaction of these factors, and provides a basis for comp arative environmental risk assessment between a biotechnology-derived pl ant and its conventi onal counterpart.

Expert knowledge and experience with conve ntionally bred alfalfa were the basis for selecting appropriate endpoints and estimatin g the range of responses that would be considered typical for alfalfa. As su ch, KK179 was compared to the conventional control, C 0 

### -Syn1, in the assessment of phenotypi c, agronomic, and environmental

interaction characteristics. An overview of the characteristics assessed is presented in

Table VII-1. A subset of the data relati ng to well-understood weedy characteristics, e.g. , seed dormancy, lodging, and split pods, was used to assess whether there was an increase in weediness of KK179 compar ed to conventional alfalfa. Evaluation of environmental interaction characteristics, e.g ., plant-abiotic stress, plant- disease, and plant-arthropod interactions, was also considered in the plant pest assessment.

Prior to analysis, the overall dataset was ev aluated for possible evid ence of biologicallyrelevant changes and unexpected plant respons es. No unexpected obs ervations or issues

were identified. Based on all of the data co llected, an assessment was made to determine if KK179 is expected to pose an increased plant pest risk compared to conventional

alfalfa.

VII.B.1. Interpretation of Detected Differences Criteria

Comparative plant characteriz ation data between a biotec hnology-derived crop and the conventional control are interp reted in the contex t of contributions to increased plant pest/weed potential as assessed by USDA-APH IS. Under the framework of familiarity,

characteristics for which no differences are de tected support a conclu sion of no increased plant pest/weed potential of the biotechnology- derived crop compared to the conventional crop. Characteristics for which differences ar e detected are considered in a step-wise method (Figure VII-1) or in a similar fashion. All de tected differences for a

characteristic are considered in the cont ext of whether or not the difference would increase the plant pest/weed potential of the biotechnology-derived crop. Ultimately, a weight-of-evidence approach considering all characteristics and data is used for the overall risk assessment of differences and their significance. In detail, Figure VII-1 illustrates the stepwise assessment process employed:Monsanto Company 12-AL-246U 115 of 407 

### Figure VII-1. Schematic Diagram of Agronomic and Phenotypic Data Interpretation Methods Note: A “no” answer at any step indicates that the characteristic does not contribute to a biological or environmental change for the crop in terms of plant pest potential and subsequent steps are not considered. If the an swer is “yes” or “uncertain,” the subsequent step is considered.

Steps 1 and 2 - Evaluate Detected St atistically Significant Differences

Data on each measured characteristic are sta tistically analyzed, as appropriate, within

each individual site and in a combined-site analysis, in which the data are pooled among sites. All statistically significant differences are evaluated and considered in the context of a change in plant pest/weed potential. Differences detected in individual-site analyses that are not detected when data across multip le environments are pooled in the combinedsite analysis are considered not biologically meaningful in terms of plant pest/weed potential and, therefore, are not further consid ered in subsequent st eps. Any difference detected in the combined-site analysis is further assessed.

Step 3 - Evaluate Differences in the Context of Conventional Commercial Reference Varieties Included in the Study

If a difference for a characteristic is dete cted in the combined-site analysis across multiple environments, then the mean value of the biotechnology-derived crop for the Hazard identification and

risk assessment on

difference Outside variation for crop? No

Yes

No

Yes 

### Adverse in terms of plant pest potential? Yes 

### Outside variation of study references? No 

### Not adverse; the direction or magnitude of the

detected difference in the

measured characteristic

does not contribute to a biological or environmental change for the crop in terms of plant pest/weed potential Yes 

### Statistical differences detected in combined-site analysis? No 

### Differences detected in the combined-site and individual site analyses are evaluated

Step 2

Step 3

Step 4 

### The measured

characteristic does not contribute to a biological or environmental change

for the crop in terms of plant pest/weed potential

Step 5

Step 6 Step 1Monsanto Company 12-AL-246U 116 of 407 

### characteristic is assessed rela tive to the range of variation of the conventional commercial reference varieties included in the study, e.g. , reference range.

Step 4 - Evaluate Differences in the Context of the Crop

If the mean value of the ch aracteristic for a biotechnology-d erived crop is outside the

variation of the conventional commercial re ference varieties, the mean value of the biotechnology-derived crop is assessed rela tive to known values common for the crop,

e.g. , published values.

Step 5 - Plant Pest Potential

If the mean value of the ch aracteristic for a biotechnology-d erived crop is outside the range of values common for the crop, the detect ed difference for the characteristic is then

assessed for whether or not it is adverse in terms of plant pest/weed potential.

Step 6 - Conduct Risk Assessment on Identified Hazard

If an adverse effect (hazard) is identified, risk assessment on the difference is conducted.

The risk assessment considers contributions to enhanced plant pest/weed potential of the crop itself, the impact of differences detect ed in other measured characteristics, and potential for and effects of trait introgressi on into any populations growing outside of cultivated environments or into a sexually-compatible species.

VII.B.2. Interpretation of En vironmental Interactions Data

Plant responses to abiotic stress, dis ease damage, and arthropod damage were qualitatively assessed using a continuous sc ale of increasing damage severity to determine a range of responses observed acro ss four replications. The biotechnologyderived crop and the conventiona l control were considered no t different in plant response if the range of damage symptoms obs erved for the biotechnology-derived crop overlapped with the range of symptoms observe d for the conventional control at a site. Any observed differences between the bi otechnology-derived crop and conventional

control were assessed for bi ological significance in the c ontext of the range of the

conventional commercial referen ce varieties at that site and for consistency with other

observation times and sites. Differences that were not consistently observed at other

observation times and sites were not considered to be biologically meaningful in terms of plant pest potential or of an effect on the environment.

Damage caused by specific arthropods (alfal fa weevil and potato leafhopper) was further assessed quantitatively, and data were analy zed within individual sites and pooled across sites in a combined-site analysis. Statistic ally significant differences detected between the biotechnology-derived crop and the conven tional control were evaluated using the method outlined in Figure VII-1.

Pest- and beneficial-arthropod abundance was also quantitatively assessed, and data were analyzed within individual collection times a nd sites. Statistically significant differences between the biotechnology-derived crop and th e conventional control were assessed for biological significance in the context of the range of the conventional commercial

reference varieties at that site and for consistency with othe r observation times and sites.Monsanto Company 12-AL-246U 117 of 407 

### Differences that were not consistently detect ed at other observation times and sites were not considered to be biologically meaningful in terms of plant pest potential or to have an

effect on the environment..

VII.C. Comparative Assessments of the Ph enotypic, Agronomic, and Environmental

Interaction Characteristics of KK179

This section provides the results of compar ative assessments condu cted in re plicated laboratory, greenhouse, and field studies to provide a detailed phenotypic, agronomic, and environmental interactions descrip tion of KK179. The KK179 characteristics

evaluated in these assessments included: s eed germination and dormancy characteristics (Section VII.C.1.); plant phenotypic and envi ronmental interaction observations under field conditions of plants managed for fo rage production (Section VII.C.2.1. and Section VII.C.2.2); plant phenotypic obs ervations under field conditions of plants managed for seed production (Section VII.C .2.3.); reproductive characterist ics (Section VII.C.3); and plant-symbiont interactions (Section VII.C.4.) . Additional details for each assessment are provided in Appendices F through K.

VII.C.1. Seed Germination and Dormancy Characteristics

USDA-APHIS considers the potential for weed iness to constitute a plant pest factor (7 CFR § 340.6). Seed dormancy is an important characteristic that is often associated with plants that are considered weeds, a nd several dormancy mechanisms can occur in seeds (Anderson, 1996; Copeland and McDona ld, 2001). Information on germination and dormancy characteristics is therefore us eful when assessing a plant for increased weediness potential. To assess germination characteristics, standardized germination assays are routinely used. The Associati on of Official Seed Analysts (AOSA), an internationally recognized s eed testing organization, recomme nds a temperature of 20 °C as optimal for testing the germination and dormancy characteristi cs of alfalfa seed (AOSA, 2007c; 2010b; a).

Seed germination and dormancy mechanisms vary among species and their genetic basis tends to be complex. While not considered a weed, alfalfa does exhibit physical dormancy, with a hard seed coat that is im permeable to water (Ba ss et al., 1988; Rolston, 1978). The production environment has a great imp act on the seed coat of alfalfa. The percentage of hard seed may be increased by cool temperatures, high humidity, and excess available water during seed development (Bass et al., 1988; Copeland and McDonald, 2001).

A hard seed coat can be manually scarified, i.e ., scratched or nicked, to facilitate imbibition of water (Anderson, 1996). In fact , commercial alfalfa seed is mechanically scarified during post-harvest handling and conditioning. Since commercial seed is frequently scarified, and germination rates for certified seed are usually targeted for greater than 80%, a high percentage of hard s eed and poor germination, i.e. , below 70%, is unlikely to be consistently obs erved in the field (AOSCA, 2009).

A comparative assessment of seed germination and dormancy characteristics was

conducted on KK179 and the conve ntional control. Four conventional commercial

reference varieties were also included to provide a range of germination and dormancyMonsanto Company 12-AL-246U 118 of 407 

### characteristic values representa tive of commercial alfalfa vari eties. Descriptions of the evaluated germination and dormancy characteri stics and the timing of the evaluations for all temperature regimes are listed in Table VII-1. Additional details on the materials and experimental methods used in this eval uation are presented in Appendix F.

All starting seed were produced in the artif icial environment of a greenhouse during the winter of 2010 under cool, humid conditions during seed development. Therefore, a relatively high level of hard seed would be expected among the starting seed. To account for different dormancy effects, both non-scarif ied and scarified seed were evaluated.

Non-scarified seed were used to assess the germination and dormancy characteristics of KK179, without regard to a spec ific mechanism of dormancy. Scarified seed were used to assess whether any other seed dormancy effects, besides a hard seed coat, wereintroduced with the trait for reduced G lignin and total lignin. In addition to the AOSArecommended temperature of 20 °C, non-scarifie d and scarified seed were tested at two additional temperature regimes of 10 °C and 30 °C to assess seed germination properties.

In the non-scarified treatment in the AOSA -recommended temperature regime (20 °C), no statistically significant differences ( α =0.05) were detected between KK179 and the conventional control for perc ent abnormal germinated, dead, and viable firm-swollen seed (Table VII-2). KK179 had a higher pe rcent normal germinated seed than the conventional control (86.0% vs. 72.5%), an d the mean for KK179 for percent normal germinated seed was slightly above the ra nge of the conventional commercial reference varieties (71.8% – 85.3%). The mean value observed for percent germinated seed is typical of alfalfa, which exhibits a wide range of germination rates across varieties (29.5% – 90.0% germination) (Weihing, 1940). Therefore, the difference in percent normal germinated seed at 20 °C is not expected to be biologically meaningful in terms of plant pest/weed potential (S ee Figure VII-1, Step 4, answer “no”). KK179 also had a lower percent viable hard seed than the c ontrol (13.0% vs. 25.3%) at 20 °C, although the mean for percent viable hard seed was w ithin the reference range (12.5% – 24.4%). Therefore, the difference in percent viable ha rd seed at 20 °C is not expected to be biologically meaningful in te rms of plant pest/weed poten tial (See Figure VII-1, Step 3, answer “no”).

In the non-scarified treatment in both the 10 °C and 30 °C temperature regimes, no statistically significant differences were detected between KK179 and the conventional control for percent dead a nd viable firm-swollen seed. At 10 °C, KK179 had higher percent germinated seed than the conventional co ntrol (86.5% vs. 74.1%) and concurrently lower percent viable hard s eed than the conventi onal control (12.8% vs. 24.7%). The means for KK179 for percent germinated seed and percent viable hard seed were within the range of conventional co mmercial reference varieties (75.8% – 88.8% and 11.2% – 23.0%, respectively). At 30 °C, KK 179 had higher percent germinated seed than the conventional control (87.3% vs. 74.9 %) and concurrently lower percent viable

hard seed than the conven tional control (12.5% vs. 24.8%). The means for KK179 for percent germinated seed and viable hard seed were within the reference ranges (77.5% – 89.5% and 10.5% – 21.8%, respectiv ely). Therefore, the differences in percent germinated and viable hard seed at 10 °C a nd 30 °C are not expected to be biologically meaningful in terms of plant pe st/weed potential (See Figure VII- 1, Step 3, answer “no”).Monsanto Company 12-AL-246U 119 of 407 

### Increased germination and reduced seed dor mancy, regardless of the specific mechanism of dormancy, i.e. , hard seed coat or other mech anism, would likely shorten the persistence of seed in the soil. Long-term pers istence of seed in the soil is a characteristic common to some weed species, and for some pe rennial weeds, has been associated with a high percentage of dormant seed (Conn a nd Werdin-Pfisterer, 2010). Short-term persistence, on the other hand, has been associat ed with a high initial rate of germination (Hesse et al., 2007). Increased germina tion and reduced dormancy would reduce longterm persistence and thereby reduce the potential for weediness. Moreover, increased germination and reduced seed dormanc y are common agronomic properties of domesticated crops. To this end, alfalfa seed treatments, such as varying temperature and moisture conditions, have been sought to improve germina tion (Bass et al., 1988; Klos and Brummer, 2000). Therefore, the differen ces detected for non-scarified seed are not considered to represent a meaningful increase in plant pest/weed potential.

In the scarified treatment, no statistically significant differences were detected between KK179 and the conventional control for any of the assessed characteristics in the 10 °C or 30 °C temperature regimes (Table VII- 3). In the AOSA-recommended temperature regime (20 °C), KK179 had sign ificantly higher per cent normal germinated seed than the conventional control (93.5% vs. 89.8%) and th is value was slightly lower than the reference range (93.8% – 98.0%). Howe ver, the mean for KK179 for percent germination of scarified seed is consistent with published germination rates of scarified alfalfa seed (93 – 98%) (Ha ll et al., 1993). Therefore, the difference in percent germinated seed at 20 °C is not expected to be biologically meaningf ul in terms of plant pest/weed potential (See Figure VII- 1, Step 4, answer “no”).

Scarification disrupts the hard seed coat and facilitates imbibition to allow an assessment of any other potential dormanc y effects introduced with the trait. KK179 and the

conventional control did not exhibit consis tent differences in any of the assessed characteristics across different temperature re gimes following scarification. Therefore, it is not expected that additio nal dormancy effects were introduced with the trait for reduced G lignin and total lignin.

While the exact nature of seed coat imperm eability is not known fo r alfalfa, lignin is known to be present in the seed coat (Rolston, 1978); and if altered, could be hypothesized to alter the germin ation and dormancy characteristics of alfalfa seed. For example, a study with a nother legume species, Vigna radiata L., observed higher lignin

levels in the seed coat of dormant beans compared to non-dormant beans (Rodriguez and Mendoza, 1990). Conversely, a reduction in th e lignin content of the seed coat could potentially be correlated with a reduction in percent hard seed and an increase in germination rate.

The germination and dormancy characteristic s evaluated were used to assess KK179 in the context of plant pest risk. The results of this assessment , particularly the observance of lower percentage hard seed and no other changes in other dormancy characteristics, support the conclusion that the introduction of the trait for reduced G lignin and total lignin is not expected to result in incr eased plant pest/weed potential compared to

conventional alfalfa.Monsanto Company 12-AL-246U 120 of 407 

### Table VII-2. Comparison of Germination Ch aracteristics of Non-scarified Seed of

KK179 and the Conventional Control

Temperature Germination Mean % 1 (SE)

Regime Category KK179 Control Reference Range 2

10 °C Germinated * 86.5 (0.6) 74.1 (1.7) 75.8 – 88.8

Dead 0.3 (0.3) 0.3 (0.3) 0.0 – 0.3

Viable firm-swollen 0.5 (0.3) 0.8 (0.5) 0.0 – 1.0

Viable hard * 12.8 (0.6) 24.7 (1.6) 11.2 – 23.0

20 °C Normal germinated * 86.0 (1.2) 72.5 (1.7) 71.8 – 85.3

(AOSA) Abnormal germinated 0.8 (0.5) 1.8 (0.5) 1.3 – 3.2

Dead 0.0 (0.0) 0.3 (0.3) 0.0 – 0.3

Viable firm-swollen 0.3 (0.3) 0.3 (0.3) 0.0 – 0.3

Viable hard * 13.0 (1.1) 25.3 (1.7) 12.5 – 24.4

30 °C Germinated * 87.3 (2.4) 74.9 (1.3) 77.5 – 89.5

Dead 0.0 (0.0) 0.0 (0.0) 0.0 – 0.5

Viable firm-swollen 0.2 (0.2) 0.3 (0.3) 0.0 – 1.0

Viable hard * 12.5 (2.3) 24.8 (1.5) 10.5 – 21.8

Note: The experimental design was a two-way factorial design with four replications (n=4). The two factors assessed were material (i.e

. , KK179, the control, or a conventional commercial reference variety) and treatment (i.e., scarifie d or non-scarified).

SE = Standard Error *Statistically significant differences detected ( α =0.05) between KK179 and the conventional control using

ANOVA.

1 In some instances, the total percentage for both KK179 and the conventional control did not equal 100% due to numerical rounding of the means.

2 Reference range is the minimum and maximum mean values observed among the four conventional

commercial reference varieties.Monsanto Company 12-AL-246U 121 of 407 

### Table VII-3. Comparison of Germination Characteristics of Scarified Seed of

KK179 and the Conventional Control

Temperature Germination Mean % 1 (SE)

Regime Category KK179 Control Reference Range 2

10 °C Germinated 99.5 (0.3) 99.8 (0.3) 99.0 – 100.0

Dead 0.0 (0.0) 0.0 (0.0) 0.0 – 1.0

Viable firm-swollen 0.0 (0.0) 0.0 (0.0) 0.0 – 0.0

Viable hard 0.5 (0.3) 0.3 (0.3) 0.0 – 0.0

20 °C Normal germinated * 93.5 (1.3) 89.8 (0.6) 93.8 – 98.0

(AOSA) Abnormal germinated 6.0 (1.3) 10.3 (0.6) 2.0 – 5.8

Dead 0.3 (0.3) 0.0 (0.0) 0.0 – 0.0

Viable firm-swollen 0.0 (0.0) 0.0 (0.0) 0.0 – 0.0

Viable hard 0.3 (0.3) 0.0 (0.0) 0.0 – 0.5

30 °C Germinated 98.8 (0.5) 99.5 (0.3) 97.3 – 99.8

Dead 0.5 (0.3) 0.5 (0.3) 0.0 – 0.3

Viable firm-swollen 0.5 (0.3) 0.0 (0.0) 0.0 – 2.3

Viable hard 0.3 (0.3) 0.0 (0.0) 0.0 – 0.3

Note: The experimental design was a two-way factorial design with four replications (n=4). The two factors assessed were material (i.e., KK179, the cont rol, or a conventional commercial reference variety) and treatment (i.e., scarifie d or non-scarified).

SE = Standard Error *Statistically significant differences detected ( α =0.05) between KK179 and the conventional control using

ANOVA.

1 In some instances, the total percentage for both KK179 and the conventional control did not equal 100% due to numerical rounding of the means.

2 Reference range is the minimum and maximum mean values observed among the four conventional

commercial reference varieties.Monsanto Company 12-AL-246U 122 of 407 

### VII.C.2. Field Phenotypic, Agronomi c, and Environmental Interaction

Characteristics

Phenotypic, agronomic, and environmental inter actions of plants managed for forage and seed production were evaluated under field co nditions of forage and seed production as part of the plant characteri zation assessment of KK179. Thes e data were developed to provide USDA-APHIS with a detailed descrip tion of KK179 relative to the conventional

control and the conventional commercial reference varieties. According to 7 CFR § 340.6, as part of the petition to se ek deregulation, a petitioner must submit “a detailed description of the phe notype of the regulated article.” This information is being provided to assess whether there are phenot ypic differences between KK179 and the conventional control that may impact it s plant pest/weed potential. Specific characteristics that are related to weediness, e.g. , lodging, and split pod s, were used to assess whether there is a pot ential increase in weedin ess of KK179 compared to

conventional alfalfa. Environmental inter actions including plant response to abiotic stress, disease damage, specific arthropod-re lated damage, and pest- and beneficialarthropod abundance were also assessed as an indirect indicator of phenotypic changes to

KK179 and are also considered in the plant pest assessment.

The results of the assessments of agronomic and phenotypic characteristics of KK179 managed under conditions for both forage a nd seed production demonstrated that the introduction of the trait for reduced G lignin and total lignin did not meaningfully alter the plant pest/weed pote ntial of KK179 compared to conven tional alfalfa. Furthermore, the lack of meaningful differences in e nvironmental interacti ons also support the

conclusion that the introduction of the trait is not expected to result in increased plant pest/weed potential or to have an effect on the environment for KK179 compared to

conventional alfalfa.

VII.C.2.1. Field Phenotypic and Agronomi c Characteristics of KK179 Managed for Forage Production

A field study was conducted, beginning in 2010 and continuing through 2012, to evaluate phenotypic and agronomic char acteristics of KK179 compar ed to the conventional control when managed under the agronomic prac tices of forage production. The selected sites reflected the diverse range of environmental and agronomic conditions representative of commercial alfalfa forage production areas in North America. At each site, all plots of KK179, the conventional control, and the conventional commercial reference varieties were uniformly managed for forage production, in order to assess

whether the introduction of the trait for reduced G lignin and total lignin altered phenotypic and agronomic char acteristics of KK179 compar ed to the conventional

control.

Phenotypic and agronomic data we re collected from field trials at 10 sites within U.S. and Canadian alfalfa production regions (Table VII- 4). The experimental design at each site was a randomized complete block with four repl ications. At the nine U.S. sites, KK179, the conventional control, and four conventional commercial reference varieties were

evaluated. At the Canadian site, KK179, the conventional cont rol, and five conventionalMonsanto Company 12-AL-246U 123 of 407 

### commercial reference varieties were evaluated. A total of twelve unique conventional

commercial reference varieties were eval uated among the 10 sites (Table G-1).

The trials at each site were managed accordi ng to standard, local agronomic practices for forage production in order to harvest forage at a growth stage of 1-10% bloom. The total number of cuttings harvested per growing seas on at a site depends on numerous factors including agro-ecological envir onment, choice of variety, harvest schedule, and weather. The number of cuttings per growing season at each site is listed in Table G-3.

This study was conducted over two years, from spring 2010 through spring 2012. The first year was considered to be from pl anting in 2010 through th e winter survival assessment in the spring of 2011. The second y ear was considered to be from the first cutting of 2011 through the winter survival assessment in the spring of 2012. For each year, assessments were made w ithin each crop growth cycle, i.e. , the period of forage growth between cuttings, and at each cutting.

Descriptions of the evaluated phenotypic char acteristics and the timi ng of the evaluations are listed in Table VII-1. The materials, methods, details concerning the timing of phenotypic assessments, and detailed results of the individual-site data comparisons are presented and discussed in Appendix G. The results of the combined-site and combined-year analyses are summarized be low. The results of the agronomic and phenotypic assessment demonstrate that th ere were no unexpected changes in the phenotype of KK179 compared to the conventional control when managed under the agronomic practices for forage production. Thus, the introduction of the trait is not expected to result in increased plant pe st/weed potential of KK179 compared to

conventional alfalfa.Monsanto Company 12-AL-246U 124 of 407 

### Table VII-4. Field Phenotypic Eval uation Sites for KK179 during 2010-2012

Site Code County, Stat e/Province, Country

CADV Yolo County, CA, U.S.A.

CANA Branchton, Ontario, Canada

IABN Boone County, IA, U.S.A.

IDNP Canyon County, ID, U.S.A.

ILCL Clinton County, IL, U.S.A.

KSLA Pawnee County, KS, U.S.A.

NYNR Wayne County, NY, U.S.A.

PACO Lancaster County, PA, U.S.A.

WATC Walla Walla County, WA, U.S.A.

WIWS La Crosse County, WI, U.S.A.Monsanto Company 12-AL-246U 125 of 407 

### In the combined-site analysis of the phenotypic characteristics assessed in the first year (2010-2011), no statistically sign ificant differences (5% leve l of significance) were

detected between KK179 and the conventio nal control for any of the assessed characteristics, including seedling emerge nce, early season vigor; lodging, crop growth stage, or regrowth after cutt ing for cutting #1; lodging, crop growth stage, forage yield or regrowth after cutting for cutting #2; lodgi ng, crop growth stage, or forage yield for cutting #3; fall plant height, to tal forage yield; spring vigo r, spring stand recovery, or spring stand count (Table VII-5). A fourth growth period and cutting occurred only at two sites (CADV, KSLA), therefore, a combin ed-site analysis could not be conducted for the following phenotypic characteristics: regrowth after cutting #3, lodging #4, crop growth stage #4, and forage yield for cutting #4.

In the combined-site analysis of the phenotyp ic characteristics assessed in the second year (2011-2012), no statistically significant differences were detected between KK179

and the conventional control fo r any of the assessed charac teristics, including lodging, crop growth stage, forage yield or regrowth after cutting for cuttings #1, #2, #3, or #4; lodging, crop growth stage, or forage yield for cutting #5; fall plant height, total forage yield; spring vigor, spring stand recover y, or spring stand count (Table VII-6).

An additional, combined-year analysis was conducted for the characteristics measured in both growing seasons, which were fall plant height, total forage yield, spring vigor, spring stand recovery, and spring stand count. No statistically si gnificant differences

were detected between KK179 and the conve ntional control for any of the assessed characteristics in the combined-year analysis (Table VII-7).

Lodging was a characteristic used to speci fically assess the potential weediness of

KK179. No differences were observed betw een KK179 and the conventional control for lodging within any crop growth cycl e assessed over the two years.

Based on the assessed phenotypic and agronomic characteristics within each individual year and across years, the re sults demonstrate that there were no unexpected changes in phenotype indicative of increased plant pes t/weed potential of KK179 compared to the conventional control (See Figure VII-1, Step 2, answer “no”).Monsanto Company 12-AL-246U 126 of 407 

### Table VII-5. Combined-Site Comparison of KK179 to the Conventional Control for Phenotypic and Agronomic Characteri stics in the First Year (2010-2011) of Forage Production

KK179 Control Reference Range 1

Phenotypic Characteristic (units) Mean (SE) Mean (SE) Minimum Maximum

Seedling emergence (# plants/foot) 19.5 (1.51) 18.5 (1.41) 12.0 27.1

Early season vigor (1-10 rating) 4.8 (0.33) 5.1 (0.38) 3.3 7.0

Cutting #1

Lodging (0-9 rating) 8.5 (0.19) 8.4 (0.24) 7.6 9.0

Crop growth stage (stage number 0-6) 2.8 (0.12) 2.7 (0.14) 1.9 3.2

Regrowth after cutting (1-10 rating) 9.1 (0.16) 8.7 (0.19) 6.8 8.9

Cutting #2

Lodging (0-9 rating) 8.2 (0.19) 8.3 (0.22) 7.5 9.0

Crop growth stage (stage number 0-6) 3.1 (0.14) 3.0 (0.15) 1.2 3.7

Forage yield (fresh weight ton/acre) 7.0 (0.46) 6.6 (0.42) 4.5 8.7

Regrowth after cutting (1-10 rating) 8.8 (0.22) 8.7 (0.22) 6.4 8.4

Cutting #3

Lodging (0-9 rating) 8.7 (0.10) 8.6 (0.11) 8.4 9.0

Crop growth stage (stage number 0-6) 2.1 (0.15) 2.2 (0.13) 1.5 2.8

Forage yield (fresh weight ton/acre) 4.7 (0.38) 4.8 (0.40) 2.8 6.9

Regrowth after cutting (1-10 rating) † 8.0 (0.42) 8.1 (0.23) 6.5 7.4

Cutting #4

Lodging (0-9 rating) † 7.4 (0.68) 7.0 (0.78) 5.8 9.0

Crop growth stage (stage number 0-6) † 2.1 (0.04) 2.2 (0.04) 2.0 2.2

Forage yield (fresh weight ton/acre) † 4.1 (0.21) 3.4 (0.36) 3.7 4.4Monsanto Company 12-AL-246U 127 of 407 

### Table VII-5. Combined-Site Comparison of KK179 to the Conventional Control for Phenotypic and Agronomic Characteri stics in the First Year (2010-2011) of Forage Production (continued)

KK179 Control Reference Range 1

Phenotypic Characteristic (units) Mean (SE) Mean (SE) Minimum Maximum

Fall plant height (in) 5.5 (0.42) 5.6 (0.44) 2.8 6.8

Total forage yield (fresh weight ton/acre) 12.6 (0.85) 12.0 (0.88) 8.0 17.8

Spring vigor (1-10 rating) 7.8 (0.16) 7.9 (0.25) 5.8 7.2

Spring stand recovery (1-10 rating) 8.4 (0.23) 8.6 (0.23) 7.2 10.0

Spring stand count (# stems/foot) 62.9 (3.53) 63.4 (3.85) 42.9 73.1

Note: The experimental design was a randomized complete block with four replications. Means based on n=40 for all characteristics except as follows: for earl y season vigor and lodging at cutting #1, n=36 for both KK179 and the conventional control; for crop growth stage at cutting #1, n=35 for both KK179 and the conventional control; for regrowth after cutting #1, n=36 for both KK179 and the conventional control; for lodging at cutting #2, n=36 for both KK179 and the conventional control; for crop growth stage at cutting #2, n=35 for both KK179 and the conventional control; for crop growth stage at cutting #3, n=39 for KK179 and n=37 for the conventional control; for regrowth after cutting #3 and lodging at cutting #4, n=8 for both KK179 and the conventional control; for crop growth stage at cutting #4, n=6 for both KK179 and the conventional control; for forage yield #4, n=8 for KK179 and n=7 for the conventional control; for total forage yield, n=39 for the conventional control; for spring vigor, spring stand recovery, and spring stand count, n=36 for both KK179 and the conventional control.

SE = Standard Error No statistically significant differences were det ected between KK179 and th e conventional control ( α =0.05) using ANOVA. † No statistical comparisons were made beca use data were collected at only two sites.

1 Reference range is the minimum and maximum mean values among the conventional commercial

reference varieties.Monsanto Company 12-AL-246U 128 of 407 

### Table VII-6. Combined-Site Comparison of KK179 to the Conventional Control for Phenotypic and Agronomic Characterist ics in the Second Year (2011-2012) of Forage Production

KK179 Control Reference Range 1

Phenotypic Characteristic (units) Mean (SE) Mean (SE) Minimum Maximum

Cutting #1

Lodging (0-9 rating) 7.1 (0.32) 7.0 (0.36) 6.2 8.9

Crop growth stage (stage number 0-6) 2.9 (0.13) 2.8 (0.14) 2.6 3.7

Forage yield (fresh weight ton/acre) 12.1 (0.88) 12.3 (0.87) 8.7 13.7

Regrowth after cutting (1-10 rating) 8.8 (0.13) 9.1 (0.13) 6.3 8.9

Cutting #2

Lodging (0-9 rating) 7.7 (0.29) 7.5 (0.33) 7.0 9.0

Crop growth stage (stage number 0-6) 3.3 (0.16) 3.4 (0.16) 2.7 3.9

Forage yield (fresh weight ton/acre) 9.7 (0.63) 9.8 (0.65) 6.1 12.6

Regrowth after cutting (1-10 rating) 9.0 (0.13) 9.1 (0.16) 6.8 9.4

Cutting #3

Lodging (0-9 rating) 7.2 (0.32) 7.5 (0.28) 7.2 8.8

Crop growth stage (stage number 0-6) 3.8 (0.13) 3.8 (0.14) 3.2 4.1

Forage yield (fresh weight ton/acre) 8.3 (0.61) 8.2 (0.58) 4.8 10.3

Regrowth after cutting (1-10 rating) 8.8 (0.22) 9.0 (0.22) 6.3 9.2

Cutting #4

Lodging (0-9 rating) 7.9 (0.29) 8.1 (0.26) 6.9 9.0

Crop growth stage (stage number 0-6) 2.8 (0.10) 3.0 (0.12) 2.2 3.4

Forage yield (fresh weight ton/acre) 5.4 (0.44) 5.5 (0.43) 2.5 6.8

Regrowth after cutting (1-10 rating) 8.4 (0.29) 8.5 (0.31) 6.3 9.0

Cutting #5

Lodging (0-9 rating) 8.3 (0.30) 8.3 (0.31) 7.5 9.0

Crop growth stage (stage number 0-6) 1.7 (0.24) 1.8 (0.25) 1.0 3.2

Forage yield (fresh weight ton/acre) 4.6 (0.49) 4.4 (0.41) 1.4 4.9Monsanto Company 12-AL-246U 129 of 407 

### Table VII-6 (continued). Combined-Site Comparison of KK179 to the Conventional Control for Phenotypic and Agronomic Ch aracteristics in the Second Year (2011- 2012) of Forage Production

KK179 Control Reference Range 1

Phenotypic Characteristic (units) Mean (SE) Mean (SE) Minimum Maximum

Fall plant height (in) 6.2 (0.47) 6.6 (0.51) 3.1 8.0

Total forage yield (fresh weight ton/acre) 36.6 (2.28) 37.2 (2.28) 23.1 44.5

Spring vigor (1-10 rating) 7.7 (0.28) 7.3 (0.32) 6.0 7.6

Spring stand recovery (1-10 rating) 8.1 (0.21) 8.5 (0.17) 7.2 9.3

Spring stand count (# stems/foot) 54.6 (4.46) 51.9 (4.31) 39.5 71.0

Note: The experimental design was a randomized complete block with four replications. Means based on n=40 for all characteristics except as follows: for lodging at cutting #1, n=36 for both KK179 and the conventional control; for crop growth stage at cutting #1, n=30 for KK179 and n=29 for the conventional control; for forage yield at cutting #1, n=39 for the conventional control; for crop growth stage at cutting #2, n=26 for KK179 and n=28 for the conventional control; for forage yield at cutting #2, n=36 for both KK179 and the conventional control; for crop growth stage at cutting #3, n=34 for both KK179 and the conventional control; for forage yield at cutting #3, n=39 for both KK179 and the conventional control; for crop growth stage at cutting #4, n=31 for KK179 and n=35 for the conventional control; for regrowth after cutting #4, n=20 for both KK179 and the conventional control; for lodging at cutting #5, n=20 for both KK179 and the conventional control; for crop growth stage at cutting #5, n=16 for both KK179 and the conventional control; for forage yield at cutting #5, n=20 for both KK179 and the conventional control; for fall plant height, n=32 for both KK179 and the conventional control; for total forage yield, n=39 for both KK179 and the conventional control; and for spring vigor, spring stand recovery, and spring stand count,

n=39 for KK179.

SE = Standard Error No statistically significant differences were det ected between KK179 and th e conventional control ( α =0.05) using ANOVA.

1 Reference range is the minimum and maximum mean values among the conventional commercial

reference varieties.Monsanto Company 12-AL-246U 130 of 407 

### Table VII-7. Combined-Site and Combin ed-Year Comparison of KK179 to the Conventional Control for Phenotypic and Agronomic Characteristics during Forage

Production

KK179 Control Reference Range 1

Phenotypic Characteristic (units) Mean (SE) Mean (SE) Minimum Maximum

Fall plant height (in) 5.8 (0.31) 6.0 (0.34) 3.3 6.6

Total forage yield (fresh weight ton/acre) 24.5 (1.81) 24.6 (1.88) 16.2 31.0

Spring vigor (1-10 rating) 7.8 (0.17) 7.6 (0.21) 6.0 7.4

Spring stand recovery(1-10 rating) 8.3 (0.15) 8.5 (0.14) 7.3 9.6

Spring stand count (# stems/foot) 58.6 (2.89) 57.4 (2.97) 44.2 71.9

Note: The experimental design was a randomized complete block with four replications. Means based on n=80 for all characteristics except as follows: for fall plant height, n=72 for both KK179 and the conventional control; for total forage yield, n=79 for KK179 and n=78 for the conventional control; for spring vigor, spring stand recovery, and spring stand count, n=75 for KK179 and n=76 for the conventional

control.

SE = Standard Error No statistically significant differences were det ected between KK179 and th e conventional control ( α =0.05) using ANOVA.

1 Reference range is the minimum and maximum mean values among the conventional commercial

reference varieties.Monsanto Company 12-AL-246U 131 of 407 

### VII.C.2.2. Environmental Interaction Ch aracteristics of KK179 Managed for Forage Production

USDA-APHIS considers the environmental interactions of the biotechnology-derived crop compared to its conventional control to determine the potential for increased plant pest characteristics. Evaluations of environm ental interactions were conducted as part of the plant characterization of KK179 managed under forage production conditions. In the

2010-2012 field trials conducted for eval uation of phenotypic and agronomic characteristics of KK179, data were also coll ected on plant response to abiotic stressors, ( e.g. , drought, wind, nutrient deficiencies), disease damage, arthropod-related damage, and arthropod abundance (Tables VII-8 through VII-12 and Tables G-8 through G-18). These data were used as part of the envir onmental analysis (Section IX) to assess plant pest potential and provide an indication of potential effects of KK179 on non-target organisms (NTOs) compared to the conve ntional control. In addition, multiple

conventional commercial referen ce varieties were included in the analysis to establish a range of natural variability for each assessed characteristic. The results of the field

evaluations showed that the trait for reduced G lignin and total lignin did not unexpectedly alter the assessed environmental interactions of KK179 compared to the conventional control. The lack of significant biological differences in plant responses to abiotic stress, disease damage, arthropod-rela ted damage, alfalfa weevil damage, potato leafhopper damage, and pest- and beneficial -arthropod abundance support the conclusion

that the introduction of the trai t for reduced G lignin and tota l lignin is not expected to result in increased plant pest potential or to have an effect on the environment for KK179 compared to conventional alfalfa.

VII.C.2.2.1. Qualitative Environmen tal Interactions Assessment

KK179 was compared to the conventional control for qualitative environmental

interactions in 2010 and 2011. Qualitative assessmen ts were conducted at 10 sites and included plant responses to abiotic stresso rs, disease damage, a nd arthropod damage. The number of observation times at a site depend ed on the number of cu ttings at that site.

Assessments were conducted th ree to four times during the 2010 growing season and four to five times during the 2011 growing season.

Plant responses to ab iotic stressors, disease damage, a nd arthropod damage were assessed at natural levels, i.e ., no artificial infestation or imposed abiotic stress; therefore these levels typically varied between observations at a site and among sites. Plant responses to abiotic stress, disease damage, and arthropod damage data were colle cted from each plot using a 0 – 9 scale of increasi ng severity of observed damage for each stressor. This

scale was utilized to allow for the evaluatio n of the wide variety of potential abiotic stressor, disease damage, and arthropod damage symptoms potentially occurring across the season and across sites. Due to the non-sp ecific nature of the scale used, the data were not statistically analyzed but rather were placed into one of the following categories: none (0), slight (1-3 ), moderate (4-6), or severe (7-9) and then expressed as a range of responses observed across the four repli cations at a site (e.g, no ne to slight). For a particular stressor, all comp arisons of the range of responses for KK179 to the range of responses for the conventional c ontrol across all observation times and sites are reported.Monsanto Company 12-AL-246U 132 of 407 

### Descriptions of the evaluated environmental interactions characteristics and the timing of

the evaluations are listed in Table VII-1. The materials, methods, additional details concerning the qualitative environmental intera ctions assessments, and detailed results of the qualitative data comparisons are presente d and discussed in Appendix G (Tables G-8 through G-10 and G-14 through G-18).

In 2010, no differences in the range of res ponses were observed between KK179 and the conventional control for any of the 93 comparis ons of plant response to abiotic stressors, including drought, flood, frost, hail, heat, nut rient deficiency, soil compaction, and wind (Table VII-8 and Table G-8). Additionally, no differences in the range of responses were

observed between KK179 and th e conventional control for an y of the 93 comparisons for plant damage caused by diseases, including An thracnose, bacterial wilt, black stem, damping-off, downy mildew, Fusarium wilt, leaf spots, root rot, Sclerotinia crown and stem rot, stem nematode, and Verticillium w ilt (Table VII-8 and Table G-9). Finally, no differences in the range of responses were observed between KK179 and the conventional control for any of the 96 co mparisons for plant damage caused by arthropods, including alfalfa caterpillar, alfalfa weevil, a phid, armyworm, blister beetle, cutworm, grasshopper, meadow spittlebug, pl ant bug, potato leafhopper, spider mite, and thrips (Table VII-8 and Table G-10).

In 2011, no differences in the range of res ponses were observed between KK179 and the conventional control for any of the 129 comparis ons of plant response to abiotic stressors, including drought, frost, hail, heat, heavi ng, nutrient deficiency, soil compaction, wet soil, wind, and winter injury kill (Table VII-9 and Table G-14). Additionally, no differences in the range of responses were observed between KK179 and the conventional control for any of the 129 comparisons for plant damage caused by diseases, including Anthracnose, bacterial wilt, black stem, crown rot, downy mildew, Fusarium wilt, leaf spots, root rot, Sclerotinia crown and stem rot, and Verticillium wilt (Tables VII-9 and G-15). Finally, no differen ces in the range of re sponses were observed

between KK179 and the conventional control for any of the 129 comparisons for plant damage caused by arthropods, including alfalfa caterpillar, alfalfa leafminer, alfalfa weevil, aphid, armyworm, bean leaf beetle, blister beetle, cutworm, grasshoppers, green cloverworm, Japanese beetle, Lygus bug, meadow spittlebug, plant bug, potato leafhopper, southern corn rootworm beetle, spider mite, and thri ps (Table VII-9 and Table G-16).

The lack of differences observed between KK179 and the conventiona l control for plant responses to abiotic stressors, disease damage , and arthropod-related damage in multiple environments across the U.S. and Canada suppor ts the conclusion that the introduction of the trait for reduced G lignin and total ligni n is not expected to cause a biologically meaningful change in terms of plant pest/weed potential or to have an effect on the environment for KK179 compared to the conven tional control (See Section VII.B.2.)

.Monsanto Company 12-AL-246U 133 of 407 

### Table VII-8. Summary of Qualitative Envi ronmental Interactions Assessments in the First Year (2010)

Note: The experimental design was a randomized complete block with four replications. Observations were made during each crop growth cycle, prior to harvest. No differences were observed between KK179 and the conventional control during any observation for damage caused by any of the assessed stressors.

1 KK179 and the conventional control were considered different in susceptibility or tolerance if the range of injury symptoms across four replications did not overlap between KK179 and the

conventional control. Stressor Number of

observations

across all sites in 2010 Number of observations with

no differences between

KK179 and the conventional

control across all sites in

2010 1

Abiotic stressors 93 93

Disease damage 93 93

Arthropod-related damage 96 96

Total 282 282Monsanto Company 12-AL-246U 134 of 407 

### Table VII-9. Summary of Qualitative Envi ronmental Interactions Assessments in the Second Year (2011)

Note: The experimental design was a randomized complete block with four replications. Observations were made during each crop growth cycle, prior to harvest. No differences were observed between KK179 and the conventional control during any observation for damage caused by any of the assessed stressors.

1 KK179 and the conventional control were considered different in susceptibility or tolerance if the range of injury symptoms across four replications did not overlap between KK179 and the

conventional control. Stressor Number of

observations

across all sites in 2011 Number of observations with

no differences between

KK179 and the conventional

control across all sites in

2011 1

Abiotic stressors 129 129

Disease damage 129 129

Arthropod-related damage 129 129

Total 387 387Monsanto Company 12-AL-246U 135 of 407 

### VII.C.2.2.2. Quantitative Environmen tal Interactions Assessment

Quantitative arthropod assessments were condu cted at three sites (ILCL, KSLA, NYNR) and included alfalfa weevil damage, potato leafhopper damage, and pest- and beneficialarthropod abundance. Each quantitative assessm ent occurred three to four times in 2010

and four to five times in 2011 based on the nu mber of cuttings at each site. Alfalfa weevil and potato leafhopper damage were assessed in each plot from 10 nonsystematically selected 6 × 6 inch areas us ing an arthropod-specifi c 0–5 rating scale of increasing severity. Areas were evaluated because individual alfalfa plants in direct seeded plots are very difficult to separate.

Damage data were collected for the same two arthropod species at the three sites in 2010 and in 2011; Both an individual-site anal ysis and a combined-site analysis were conducted, in which the data were pooled am ong the three sites for each single year separately. Descriptions of the evaluated environmental inte ractions characteristics and the timing of the evaluations ar e listed in Table VII-1. The materials, methods, additional details concerning the specific arthropod damage assessments, and detailed results of the individual-site data compar isons are presented and disc ussed in Appendix G (Tables G-11 and G-17). The results of the combined-s ite analysis are summarized below.

In the combined-site analyses in both 2010 and in 2011, no statistically significant

differences were detected between KK 179 and the conventional control for any observations of plant damage caused by alfalfa weevil or potato leafhopper (Tables VII-10 and VII-11). Considering the lack of differences, in both years, the results indicate there was no biological diffe rence in alfalfa weevil or potato leafhopper damage that would contribute to increased plant pest/weed of KK179 compared to the conventional control (See Figure VII-1, Step 2, answer “no”).

Pest- and beneficial-arthropod abundance data we re also collected at the three sites in 2010 and in 2011. Variations in temporal activity and geographi cal distribution of arthropod taxa occur between sites, theref ore, only individual-s ite analyses were conducted for arthropod abundance data in each year. Additional de tails of the arthropod

abundance assessments and detailed results of the individual-site da ta comparisons are provided in Appendix G (Tables G-12, G-13, G-18, and G-19). The results of these analyses are summarized belo w and in Table VII-12.

In 2010, a total of 69 comparisons were made between KK179 and the conventional control for arthropod abundance involving the following pest- and beneficial-arthropods: aphid, alfalfa weevil, alfalf a looper, false chinch bug, green cloverworm, garden webworm, thrips, damsel bug, ladybird bee tle, parasitic wasps, and lacewing. No statistically significant differences were detected between KK179 and the conventional control for 65 out of 69 comparisons, incl uding 39 pest-arthropod comparisons and 30 beneficial-arthropod comparisons (Tables G-12 and G-13). Of the four differences detected, there were two statis tically significant differences ( α =0.05) in pest-arthropod abundance and two statistically significant diffe rences in beneficial-arthropod abundance.Monsanto Company 12-AL-246U 136 of 407 

### In the assessment of pest arthropod-a bundance, KK179 had significantly lower abundance of potato leafhopper in collection #1 at ILCL (152.3 vs. 329.0) and KK179 had significantly higher abundance of L ygus bug than the conventional control in collection #2 at KSLA (1.3 vs. 0.0; Table G- 12). The mean abundance values of potato leafhopper in collection #1 at ILCL and of Lygus bug for collection #2 at KSLA were outside the respective range of the conventiona l commercial referen ce varieties for that site and collection time (178.8 – 256.8 and 0.0 – 0.3, respectively). However, the differences for these taxa were not consistently detected across collection times or sites. Thus, the detected differences in arthr opod abundance were not considered to be biologically meaningful in te rms of plant pest/weed potential or potential to have an effect on the environment for KK179 comp ared to the conventional control.

In the assessment of beneficial-arthropod abundance, KK179 had significantly lower abundance of spiders than the conventional c ontrol in collection #2 at ILCL (0.0 vs. 1.5) and at KSLA (0.0 vs.1.3; Table G-13). The mean abundance values of KK179 were within the respective range of the conventional commercial re ference varieties for that site and collection time. In addition, the difference in abundance of spiders was not consistently detected across collection times and sites. Thus, the detected differences

were not considered to be bi ologically meaningful in term s of plant pest/weed potential of KK179 compared to the conventi onal control (See Section VII.B.2.).

In 2011, a total of 83 comparisons were made between KK179 and the conventional control for arthropod abundance involving the following pest- and beneficial-arthropods: aphid, armyworm, false chinch bug, blis ter beetle, green cloverworm, Lygus bug, meadow spittlebug, potato leafhoppe r, thrips, soybean looper, spiders, ladybird beetle, lacewing, nabids, and chalcid wasps. No statistically significant differences were

detected between KK179 and the conventi onal control for 82 out of 83 comparisons, including 50 pest-arthropod comparisons and 33 beneficial-arthropod comparisons (Tables G-18 and G-19).

In the assessment of pest -arthropod abundance, KK179 had a significantly lower abundance of alfalfa weevils compared to th e conventional control for collection #1 at ILCL (0.3 vs. 7.0; Table G-18). The mean abundance value of KK179 was within the range of the conventional commercial reference varieties for that site and collection time. Thus, the detected difference was not consider ed to be biologically meaningful in terms of plant pest/weed potential or potential to have an effect on the environment for KK179 compared to the conventional co ntrol (See Section VII.B.2.).Monsanto Company 12-AL-246U 137 of 407 

### Table VII-10. Combined-Site Comparison of Potato Leafhopper and Alfalfa Weevil Damage to KK179 Compared to the Conventi onal Control in the First Year (2010)

Damage (0-10 rating scale)

KK179 Control Reference Range 1

Arthropod Observation Mean (SE) Mean (SE) Minimum Maximum

Alfalfa weevil ( Hypera postica ) 1 0.1 (0.04) 0.2 (0.07) 0.0 0.7

2 0.2 (0.07) 0.2 (0.11) 0.0 0.9

3 0.0 (0.02) 0.1 (0.03) 0.0 0.3

4 † 0.0 (0.00) 0.0 (0.00) 0.0 0.0

Potato leafhopper ( Empoasca fabae ) 1 0.4 (0.09) 0.5 (0.11) 0.0 0.9

2 0.2 (0.11) 0.3 (0.11) 0.0 0.5

3 0.0 (0.03) 0.0 (0.01) 0.0 0.1

4 † 0.0 (0.00) 0.0 (0.00) 0.0 0.0

Note: The experimental design was a randomized complete block with four replications. Means based on n=12 except for Observation #4, in which n=4 for both KK179 and the conventional control.

SE = Standard Error No statistically significant differences were det ected between KK179 and th e conventional control ( α =0.05) using ANOVA. † No statistical comparisons were made due to lack of variability in the data.

1 Reference range is the minimum and maximum mean values among the four conventional commercial

reference varieties.Monsanto Company 12-AL-246U 138 of 407 

### Table VII-11. Combined-Site Comparison of Potato Leafhopper and Alfalfa Weevil Damage to KK179 Compared to the Conventional Control in the Second Year (2011)

Damage (0-10 rating scale)

KK179 Control Reference Range 1

Arthropod Observation Mean (SE) Mean (SE) Minimum Maximum

Alfalfa weevil ( Hypera postica ) 1 1.2 (0.47) 0.9 (0.38) 0.0 2.3

2 0.2 (0.09) 0.2 (0.10) 0.0 0.6

3 0.2 (0.08) 0.2 (0.08) 0.0 0.6

4 0.0 (0.02) 0.0 (0.02) 0.0 0.2

5 † 0.0 (0.00) 0.0 (0.00) 0.0 0.0

Potato leafhopper ( Empoasca fabae ) 1 0.8 (0.31) 0.6 (0.23) 0.0 1.5

2 0.1 (0.06) 0.1 (0.05) 0.0 0.4

3 0.1 (0.05) 0.1 (0.06) 0.0 0.3

4 0.7 (0.27) 0.8 (0.27) 0.0 1.4

5 † 0.0 (0.00) 0.0 (0.00) 0.0 0.0

Note: The experimental design was a randomized complete block with four replications. Means based on n=12 except for Observation #5, in which n=4 for both KK179 and the conventional control.

SE = Standard Error No statistically significant differences were det ected between KK179 and th e conventional control ( α =0.05) using ANOVA. † No statistical comparisons were made due to lack of variability in the data.

1 Reference range is the minimum and maximum mean values observed among the four conventional

commercial reference varieties.Monsanto Company 12-AL-246U 139 of 407Table VII-12. Summary of Arthropod Abundance Assessments and Detected Differences in 2010 and 2011Summary of Statistical Comparisons1Summary of Detected Differences2ArthropodAbundanceAssessmentNumberof sitesNumber ofcomparisonsacross sitesNumber ofcomparisons whereno differences weredetectedArthropod SiteCollectionNumberWithinreferencerange?Consistentlydetected acrosscollections orsites?20102010Pest3 39 37Potato leafhopper ILCL 1 No NoLygus bug KSLA 2 No NoBeneficial 3 30 28Spiders ILCL 2 Yes NoKSLA 2 Yes No20112011Pest3 50 49Alfalfa weevil ILCL 1 Yes NoBeneficial 3 33 33– – – – –1Quantitative arthropod abundance assessments were statistically analyzed atα=0.05 using ANOVA.2Five statistically significant differences were detected. These differences arefurther assessed following Section VII.B.2.– Indicates no differences were detected.Monsanto Company 12-AL-246U 140 of 407 

### VII.C.2.3. Field Phenotypic and Agronomi c Characteristics of KK179 Managed for

Seed Production

Alfalfa seed production requires different environmental conditions and agronomic practices than alfalfa grown for forage. Al falfa seed production is limited globally to more arid regions, and more than 85% of a ll seed production occurs in the five U.S. Western states of California, Idaho, Washington, Nevada , and Oregon (Mueller, 2008; Rincker et al., 1988). Seed production fields ar e typically planted in rows and are seeded at a much lower density than forage producti on fields (Mueller, 2008). Thinner stands help maximize seed yield, while dense stands help maximize forage yield. As a result of this agronomic practice, plants in seed production fields tend to have an increased number of stems per plant and an increased number of pods per stem (Mueller, 2008). Additionally, agronomic and phenotypic charact eristics at reproductive growth stages may only be assessed in seed production fields , as forage is typically cut between late vegetative and early bloom stages.

A seed production study was establis hed in 2010 in Canyon County, Idaho, which represents an environment that is appropria te for commercial alfalfa seed production in North America. The experimental design was a randomized complete block with six replications. All plots of KK179, the conventional cont rol, and seven conventional

commercial reference varieties were uniform ly managed under the agronomic practices for seed production, in order to assess whet her the introducti on of the trait for reduced G lignin and total lignin altered the phenotypi c and agronomic characteristics of KK179 compared to the conventional control.

Descriptions of the evaluated phenotypic char acteristics and the timi ng of the evaluations during seed production are listed in Table VII-13. The details of the materials and experimental methods used in this ev aluation are presented in Appendix H.

No statistically signi ficant differences ( α =0.05) were detected between KK179 and the conventional control for seedling establishmen t, seedling vigor, seed weight, seed per pod, or seed yield (Table VII-13). Additionally, no statistically significant differences

were detected between KK179 and the conve ntional control for lodging or split pods,

both characteristics related to weediness. T hus, the results demonstrate that there were no unexpected changes in phenotype indicative of increased plant pe st/weed potential or potential to have an effect on the envir onment for KK179 compared to the conventional control (See Figure VII-1, Step 1, answer “no”).

The results of the agronomic and phenotypic as sessment demonstrate that there were no unexpected changes in the phenotype of KK179 compared to the conventional control when managed under the agronomic practices for seed production. Thus, the introduction of the trait for reduced G lignin and total lignin is not expected to re sult in increased plant pest/weed potential of KK179 compar ed to conventional alfalfa.Monsanto Company 12-AL-246U 141 of 407 

### Table VII-13. Comparison of KK179 to th e Conventional Control for Phenotypic and Agronomic Characteristics during Seed Production

Characteristic (units) Mean (SE) Reference Range 1

KK179 Control Minimum Maximum

Seedling establishment (# plants/plot) 282.3 (35.72) 307.3 (26.53) 206.7 328.5

Seedling vigor (1-10) 7.3 (0.49) 7.3 (0.49) 5.8 7.5

Lodging (0-9) 6.0 (0.37) 5.7 (0.49) 5.3 7.0

Seed weight (mg) 2.5 (0.05) 2.5 (0.07) 2.3 2.6

Seed per pod (# seed/pod) 4.9 (0.40) 4.7 (0.34) 4.7 6.0

Split pods (%) 0.1 (0.14) 0.1 (0.13) 0.0 0.5

Seed yield (g/plot) 622.8 (27.14) 772.7 (89.71) 627.0 867.7

Note: The experimental design was a randomized complete block design with six replications (n=6).

SE = Standard Error No significant differences were detected between KK179 and the conventional control ( α =0.05) using

ANOVA.

1 Reference range is the minimum and maximum mean values observed among the four conventional

commercial reference varieties.Monsanto Company 12-AL-246U 142 of 407 

### VII.C.3. Reproductive Characteristics

USDA-APHIS considers the potential for gene flow and introgression of the biotechnology-derived trait into other alfalfa varieties and wild relatives to assess the potential for increased weedy or invasive characteristics of th e receiving species. Information on reproductive characteristic s including pollen mor phology and viability and flower morphology are pertinent to this as sessment, and therefore were assessed for KK179. In addition, characteri zations of pollen and flower s produced by KK179 and the conventional control are relevant to the plant pe st risk assessment because they add to the detailed description of the phenotype of KK179 compared to the conventional control.

VII.C.3.1. Pollen

The viability and morphology of pollen collect ed from KK179 compared to that of the

conventional control was also assessed. KK179, the conventional control, and four

conventional commercial reference variet ies were grown under similar agronomic conditions in a greenhouse. The trial wa s arranged in a randomized complete block design with four replications, with 10 plants of each material per re plication. Once all plants across the replications reached the flowering stage, flowers were collected and pollen extracted and stained for assessment.

Descriptions of the evaluated viability a nd morphology characteristics and the timing of

the evaluations are listed in Table VII-1. Th e details of the materi als and experimental

methods used in this evaluati on are presented in Appendix I.

No statistically signi ficant differences ( α =0.05) were detected between KK179 and the conventional control for percent viable pollen or pollen grain diameter (Table VII-14). Furthermore, no visual differences in gene ral pollen morphology were observed between

KK179 and the conventional c ontrol (Figure I-1).

The pollen characterization data contribute to the detail ed phenotypic description of KK179 compared to the conventional control. Based on the assessed characteristics, the results support a conclusion that neither pol len viability nor morphology of KK179 were altered compared to co nventional alfalfa.Monsanto Company 12-AL-246U 143 of 407 

### Table VII-14. Pollen Characteristics of KK179 Compared to the Conventional

Control

KK179 Control Reference Range 1

Pollen Characteristic Mean (SE) Mean (SE) Minimum Maximum

Viability 2 (%) 89.2 (2.72) 88.4 (1.06) 86.0 89.9

Diameter 3 (μm) 32.6 (0.63) 33.2 (0.45) 32.5 33.7

Note: The experimental design was a randomized complete block design with four replications (n=4).

SE = Standard Error No significant differences were detected between KK179 and the conventional control ( α =0.05) using

ANOVA.

1 Reference range is the minimum and maximum mean values observed among the four conventional

commercial reference varieties.

2 Evaluated from five sub-samples per replication for four replications.

3 Recorded from ten representative viable pollen grains per replication.Monsanto Company 12-AL-246U 144 of 407 

### VII.C.3.2. Flowers

The purpose of this evaluation was to asse ss the morphology of fl owers collected from KK179 compared to those of th e conventional control. Fl owers were collected from KK179, the conventional control, and four c onventional commercial reference varieties grown under similar agronomic conditions in a Wi sconsin field trial. The field trial was arranged in a randomized complete block desi gn with four replicat ions. Flowers were collected from the field once all plots had reached approximately 10% bloom.

Descriptions of the evaluated phenotypic char acteristics and the timi ng of the evaluations

are listed in Table VII-1. The details of the materials and ex perimental methods used in this evaluation are presented in Appendix J.

No statistically signi ficant differences ( α =0.05) were detected between KK179 and the conventional control for any of the assessed flower characteristics including number of flowers per raceme, standard petal length, k eel petal length, calyx tube diameter, sexual column length, and wing petal length (Table VI I-15). Furthermore, no visual differences in flower color class, gross raceme morphology, or gross fl ower morphology were

observed between KK179 and the conventiona l control (Figures J-2 and J-3).

The flower characterization data contribute to the detailed phenotypic description of KK179 compared to the conventional control. Based on the assessed characteristics, the results support a conclusion that flower mo rphology of KK179 was not altered compared

to conventional alfalfa.Monsanto Company 12-AL-246U 145 of 407 

### Table VII-15. Flower Characteristics of KK179 Compared to the Conventional

Control

Mean (SE) 1 Reference Range 2

Characteristic (units) KK179 Control Minimum Maximum

Flowers per raceme (#) 15.28 (0.71) 15.45 (0.67) 10.35 17.03

Standard petal length (mm) 10.60 (0.07) 10.43 (0.10) 10.33 10.80

Keel petal length (mm) 7.63 (0.15) 7.33 (0.13) 7.28 7.43

Calyx tube diameter (mm) 1.91 (0.02) 1.93 (0.05) 1.83 1.95

Sexual column length (mm) 7.50 (0.09) 7.58 (0.11) 7.33 7.45

Wing petal length (mm) 8.60 (0.14) 8.53 (0.12) 8.20 8.45

Flower color class 3 Variegated dark purple Variegated dark purple Light purple Variegated dark purple

Gross raceme morphology Typical Typical Typical Typical

Gross flower morphology Typical Typical Typical Typical

Note: The experimental design was a randomized complete block design with four replications.

SE = Standard Error No significant differences in flowers per raceme, st andard petal length, keel petal length, calyx tube diameter, sexual column length, or wing petal length were detected between KK179 and the control ( α =0.05) using ANOVA. No visual di fferences in flower color class, gross raceme morphology, or gross flower morphology were observed between KK179 and the conventional control.

1 Means based on n=4. Flower color class, gross raceme morphology, and gross flower morphology data were not subjected to statistical analysis.

2 The reference range for flower characteristics statis tically analyzed is the minimum and maximum mean values from among the conventional commercial reference varieties. The reference range for those

characteristics that were not statisti cally analyzed consists of the mi nimum and maximum observed ratings from among the conventional commercial reference varieties.

3 Flower color classification (USDA-ARS, 1972).Monsanto Company 12-AL-246U 146 of 407 

### VII.C.4. Symbiont Interactions

As part of the plant pest risk assessment, USDA-APHIS considers the impact of the biotechnology-derived crop on pl ant pest potential and the e nvironment as well as on agricultural or cultiva tion practices compared to its conve ntional counterpart. Potential changes in the symbiotic relationship betw een alfalfa and members of the bacterial families, Rhizobiaceae and Bradyrhizobiaceae , which inhabit the rhizosphere, could effect plant pest potential or agronomic practices, i.e. , the need to add additional nitrogen to sustain alfalfa production. In alfalfa, atmospheric nitrogen is fixed into organic nitrogen through a symbiotic association with the bacterium Sinorhizobium meliloti, which can contribute up to 64% of alfalfa’s nitrogen requi rement (Lanyon and Griffith, 1988). Thus, the purpose of this evaluation wa s to assess whether the introduction of the trait for reduced G lignin and total lignin a ltered the symbiotic inte raction of KK179 with
Sinorhizobium meliloti compared to that of th e conventional control.

KK179, the conventional control, and six c onventional commercial reference varieties were grown under similar agronomic conditions in a greenhouse. Descriptions of the evaluated phenotypic characteristics are list ed in Table VII-16. The details of the materials and experimental methods used are presented in Appendix K.

No statistically signi ficant differences ( α =0.05) were detected between KK179 and the conventional control for each measured parame ter, including dry weight of nodules, root material, shoot material, shoot percent tota l nitrogen, and shoot to tal nitrogen (Table VII-16).

Based on the assessed characteristics, the lack of statistical differences between KK179

and the conventional control i ndicate that introduction of th e trait for reduced G lignin and total lignin did not alter th e symbiotic relationship between S. meliloti and KK179 compared to that of conventiona l alfalfa. Thus, the results demonstrate that there were no unexpected changes indicative of increased plan t pest/weed potential or potential to have

an effect on the environment for KK179 co mpared to the conve ntional control.Monsanto Company 12-AL-246U 147 of 407 

### Table VII-16. Symbiont Interaction Asse ssment of KK179 and the Conventional

Control

Mean (SE) Reference Range 1

Measurements KK179 Control Minimum Maximum

Nodule Dry Wt (g) 0.23 (0.02) 0.24 (0.02) 0.19 0.25

Root Dry Wt (g) 3.01 (0.41) 3.06 (0.46) 3.08 3.41

Shoot Dry Wt (g) 5.22 (0.58) 5.11 (0.61) 4.98 6.35

Shoot Percent Total Nitrogen (% dwt) 3.49 (0.03) 3.46 (0.08) 3.17 3.53

Shoot Total Nitrogen (g) 0.18 (0.02) 0.18 (0.02) 0.16 0.21

Note: The experimental design was a randomized complete block design with 10 replications (n=10 for KK179 and n=8 for the conventional control).

SE = Standard Error No statistically significant differences were det ected between KK179 and th e conventional control ( α =0.05) using ANOVA.

1 Reference range is the minimum and maximum mean values observed among the six conventional

commercial reference varieties.Monsanto Company 12-AL-246U 148 of 407 

### VII.D. Conclusions for Phenotypic, Agro nomic, and Environmental Interactions

Evaluations

Comparative plant characteriz ation data between a biotec hnology-derived crop and the conventional control are interp reted in the contex t of contributions to increased plant pest/weed potential as assessed by USDA-APH IS. Under the framework of familiarity,

characteristics for which no differences are de tected support a conclu sion of no increased plant pest/weed potential of the biotechnology- derived crop compared to the conventional crop. Ultimately, a weight-of-evidence approach that considers all characteristics and

data is used for the overall risk assessment of differences and th eir significance.

An extensive and robust set of agronomic, phenotypic, and environmental interactions data, including specific weedy characteri stics, were used to assess whether the introduction of the trait for reduced G ligni n and total lignin altered the plant pest potential of KK179 compared to the conventiona l control, considered within the context of the variation among the conventional commercial reference varieties. These assessments included seven general data cate gories: 1) seed germination, dormancy, and emergence; 2) vegetative growth; 3) wint er survival; 4) reproductive development including pollen, flower, and seed characteris tics; 5) lodging and seed retention on the plant; 6) plant response to abiotic stress and interactions with dise ases and arthropods; and 7) plant-symbiont interactions. Within these data categories, data relevant to understanding specific characteristics associated with weediness were also assessed to determine whether there was a potential incr ease in weediness of KK179 compared to

conventional alfalfa.

Results from the phenotypic, agronomic, an d environmental interactions assessments comparing KK179 and the conventional c ontrol demonstrate that KK179 does not possess: 1) increased weediness characteristics ; 2) increased suscepti bility or tolerance to specific abiotic stress, diseases, or arthropods; or 3) characteristic s that would confer a plant pest risk compared to conventional alfalfa. Therefore, based on the results of multiple assessments discussed above and pr esented in the appendices, the weight of evidence indicates that KK179 is not meaningf ully different from conventional alfalfa with the exception of the trait for reduced G lignin and total lignin and is not expected to pose a plant pest/weed risk compar ed to conventional alfalfa.Monsanto Company 12-AL-246U 149 of 407 

### VIII. U.S. AGRO NOMIC PRACTICES

VIII.A. Introduction

As part of the plant pest assessment re quired by 7 CFR § 340.6(c)(4), impacts of deregulation on agricultural and cultivation pr actices must be considered. This section provides a summary of current agronomic practices in the U.S. for producing alfalfa and is included in this petition as a baseline to assess possible impacts to agricultural practices

due to the cultivation of KK179. Discussions in clude the importance of alfalfa as a crop; alfalfa production; seed production; plant growth and development; practices for the establishment, management, and terminatio n of alfalfa stands; pest management practices; alfalfa rotational crops; and volunteer and feral alfalfa management. Information on U.S. alfalfa agronomic practi ces can be found in th e published literature and in USDA-APHIS Environmental ImpactStatement for Roundup Ready alfalfa (Section III and Appendices G, H, and J) (USDA-APHIS, 2010). The overview presented here in Section VIII collectively di scusses the agricultural practices from those

sources.

Information presented in Section VII demonstr ated that KK179 is no more susceptible to diseases or pests than conventional alfalfa. Additionally, data presented in Section VII show that KK179 is not expected to pose a plant pest risk compared to conventional alfalfa. Thus, there are no changes to the inputs needed for KK179, and no specific impacts to most of the agronomic practices employed for the production of alfalfa. Where there is potential impact on agronomic practices from the deregulation of KK179, discussion delineating the scope and magn itude of those impacts is provided.

Alfalfa ( Medicago sativa L.) is grown for forage, grazing, and seed production. Alfalfa is one of the most important forage crops in th e U.S. and ranks fourth on the list of most widely grown crops by acreage, behind corn, soybean, and wheat (USDA-APHIS, 2010). In terms of value, alfalfa is ranked fourth among agricultural crops. More than 20 million acres of alfalfa forage production have been grown annually since 1950 (USDA-NASS, 2011b). Alfalfa grows in a wide range of so ils and climates as evidenced by production in all continental states, Alaska and Hawaii (USDA-APHIS, 2010). Although alfalfa production practices vary by region due to climatic differences, some practices are similar among growing regions. Alfalfa produc tion has two distinct growing phases: the establishment phase and the pr oduction phase associated withestablished stands (USDAAPHIS, 2010). Proper seedbe d preparation, correct variety selection, optimum planting dates, optimum plant population, and good inte grated pest management practices are important for optimizing the yield potent ial and economic returns of alfalfa.

Established mechanical and chemical measures are available to control volunteer alfalfa,

i.e. , alfalfa plants that carry over from an esta blished stand or seed that have germinated and emerged unintentionally in a subsequent rotational crop. Mechanical and chemical methods are also effective in removing or taki ng out old, thinning stands when rotating to other crops. Due to the lack of differen ces between KK179 and conventional alfalfa in phenotypic, agronomic, and environmental inte raction assessments presented in Section VII, KK179 is not meaningfully different in its weediness potential. Therefore,Monsanto Company 12-AL-246U 150 of 407 

### introduction of KK179 in the alfalfa production system would have a negligible impact on managing KK179 volunteer plan ts in rotational crops such as corn, soybean, sorghum,

and wheat. The numerous control measures th at are effective on c onventional volunteer alfalfa plants will continue to be effective on volunteer KK179 plants should they arise.

As shown in Sections VI and VII, with the exception of the trait fo r reduced G lignin and total lignin, no biologically meaningful phenotypic, compositional, or environmental

interaction differences between KK179 and c onventional alfalfa have been observed. Therefore, it is not anticipated that commer cialization of KK179 in the U.S. would have a notable impact on current alfa lfa cultivation practices.

VIII.B. Overview of U.S. Alfalfa Production

VIII.B.1. Alfalfa Forage Production

Alfalfa is grown today on all continents (except Antarctica) in some capacity, but primarily in temperate climates. Alfalfa was introduced into the United States through a number of channels, first by Spanish missi onaries from Mexico into Texas and the Southwest region, and later into the British coloni es during the first half of the 18th century. The first known plantings of alfalfa in the U.S. were in Georgia in 1736. The use of alfalfa spread slowly until it was introduced into California during the Gold Rush of 1851, after which it dispersed rapidly throughout the nation (Janick et al., 1974).

Alfalfa is grown for forage, grazing, and s eed production. Alfalfa is generally regarded as the “queen of forages” because of its hi gh protein content and highly digestible fiber for ruminants and horses (USDA-APHIS, 2010) . The highest quality alfalfa hay is generally used for dairy cows. For instance, dairy farms consume between 75 to 85% of the alfalfa hay production in California (US DA-APHIS, 2010). Another 10 to 15% is consumed by horses. Alfalfa hay that is lower in protein and higher in fiber is fed to beef cattle, horses, heifers, and non-la ctating dairy cows. Alfalfa fora ge is stored as hay (bales at 18 to 20% moisture), hayl age (round bale silage, baled at 50 to 60% moisture and wrapped in plastic), and silage (chopped a nd stored in silos) (USDA-APHIS, 2010). Grazing alfalfa in the vegetative state is pr acticed sometimes for dormant-season alfalfa stubble, a substitute for early or late s eason cutting, and rotational grazing during the season. However, grazing can cause gastrointe stinal bloating in animals and result in stand maintenance problems with over-grazi ng. Humans consume a limited amount of alfalfa in the form of sprouts, dietary supplements, and herbal teas. Over 95% of alfalfa (by weight) used for human cons umption is in the form of alfalfa sprouts. An indirect

use of alfalfa is its use as a common nectar source for supporting the hives of honey bees. Alfalfa plants are also used for a vari ety of non-agricultural purposes, including rehabilitation of over-grazed rangelands, erosi on-control projects in interior forests, treatment of compacted soils, re-vegetation of areas damaged by wildfire, and erosion reduction in mined soils (USDA-APHIS, 2010).

Of the 79 million acres grown worldwide, approximately 70% is produced in the United States, Russia, and Argentina (Klonsky et al ., 2008). In 2009, when U.S. production was valued at approximately $8 billion, U.S. e xports of alfalfa hay we re valued (averageMonsanto Company 12-AL-246U 151 of 407 

### prices × volumes) at approximately $354 millio n and imports at $3.8 million, resulting in a U.S. consumption value of $7.6 billion (USDA-APHIS, 2010). Pacific Rim nations, including Japan, Korea, and Taiwan, are ma jor importers of alfalfa hay and cubes (compressed bales of hay) from th e western U.S. (Klonsky et al., 2008).

Alfalfa is a deep-rooted and short-lived pere nnial plant with a l ong growing season. Alfalfa grows from early spring until late fall or early winter. Growth begins when the average temperature reaches 50°F and continue s until a killing freeze occurs. It is adapted to a wide range of climatic and soil conditions. Deep, medium- to coarsetextured soils with adequate water are id eal (Kansas State University, 1998). Finetextured soils are usually difficult to mana ge. In deep, well-aerated soil, roots may extend 8 to 12 feet deep (Kansas State Univ ersity, 1998). Because of its deep taproot alfalfa can use up to 70% of available soil water without stress or loss of production under arid conditions, thus it is often consider ed naturally drought-toler ant. It has a high water requirement under normal conditions, usin g in excess of 40 inches of water during the season (Kansas State University, 1998). Du ring the summer months, the water use is 6 to 7 acre-inches per ton of forage. The wa ter requirements for alfalfa are greater than

the annual rainfall in the Great Plains and Western regions of the U.S. Approximately 31% of the total U.S. alfalfa hay acreage was irrigated in 2007 (USDA-NASS, 2007). Excess moisture is conducive to developmen t of root and crown diseases, and shallow water tables limit root growth (Kansas State University, 1998).

Alfalfa is grown for forage in all continenta l states, Alaska and Hawaii. The distribution of alfalfa grown as forage across the U.S. is presented in Figure VIII-1. Alfalfa hay acreage peaked in the mid-1950s and 60s at approximately 30 million acres, and has slowly declined during the past 40 years to the 2010 level of approximately 20 million acres (USDA-NASS, 2011d). Cu rrently, the harvested acres of alfalfa hay represent approximately 33% of the harvested acres for all types of hay (USDA-NASS, 2011d). Approximately 20 to 24 million acres of alfa lfa hay have been harvested annually over the past 10 years (Table VIII-1). Approxima tely 2.5 to 3.3 million acres (12-13% of the harvested acres) are seeded annually for ne w alfalfa stands (Table VIII-1). Annual production has ranged from 68 to 82 million tons of hay. Average yields have remained fairly constant at 3.19 to 3.47 tons per acre over that same period. The annual value of production has ranged from $6.7 to $10.7 billion (due to most alfalfa being fed to livestock on-farm, the value is an estimat e based on multiplying average prices with production volumes and does not correspond to act ual sales). Thus, alfalfa has been and continues to be an important U.S. crop.Monsanto Company 12-AL-246U 152 of 407 

### Figure VIII-1. Geographic distribution of al falfa hay harvested acres in the U.S.
Source: (USDA-NASS, 2010).



### Table VIII-1. Alfalfa Hay Producti on in the U.S. from 2000 to 2010 1

Year Seeded Alfalfa Acres (000) Harvested Acres (000) Production (000 tons) Yield (tons/acre) Value of

Production 2

($000)
2000 3,065 23,463 81,520 3.47 6,812,286 2001 3,260 23,952 80,354 3.35 7,533,401 2002 3,282 22,923 73,014 3.19 7,137,469 2003 3,119 23,527 76,098 3.23 6,707,172 2004 2,793 21,697 75,375 3.47 6,961,519 2005 3,290 22,359 75,610 3.38 7,290,854 2006 3,184 21,138 70,548 3.34 7,519,232 2007 2,828 21,126 69,880 3.31 8,855,044 2008 2,699 21,060 70,180 3.33 10,747,161 2009 2,665 21,247 71,072 3.35 7,941,539 2010 2,545 19,966 67,971 3.40 7,728,468

1 Source: (USDA-NASS, 2011c)

2 The value is an estimate based on multiplying average prices with production volumes and does

not correspond to actual sales.Monsanto Company 12-AL-246U 153 of 407 

### For purposes of this agronomic practices disc ussion, alfalfa production (alfalfa and alfalfa mixtures) is divided into six major alfalfa growing regions: Nort h Central region (IA, MN, ND, SD, WI), East Central region (AR, DE, IL, IN, KY, MD, MI, MO, New England states, NJ, NY, NC, OH, PA, TN, VI , WV), Plains region (KS, NE, OK, TX), Intermountain region (CO, MT, UT, WY), P acific Northwest region (ID, NV, OR, WA), and Southwest region (AZ, CA, NM) (Table VIII-2). Approximately 20 million acres of alfalfa hay and 2 million acres of alfalfa ha ylage were harvested in 2010. The largest acreage of alfalfa (hay and haylage) is gr own in the North Central region (8.2 million acres) followed by the East Central region (4.2 million acres). The North and East Central regions grow 56% of the total alfalfa acreage. The states with the largest acreage are South Dakota (2.2 million), Wisconsin ( 2.2 million), Montana (2.0 million), North Dakota (1.6 million), and Minnesota (1.3 million), which represent 42% of the total U.S. acreage. The highest yields were reporte d in the Pacific Nort hwest and Southwest regions with 4.6 and 6.8 tons per acre, respectively. The yields in the other regions range from 2.8 to 3.9 tons per acre. The U.S. produced 79 million tons of alfalfa hay and haylage in 2010. Over 50 percent was produced in the North and East Central regions

and 32% in the 5-state area of the North Ce ntral region alone. California produced the largest amount of alfalfa hay (6,549,000 tons ), but Wisconsin produced the largest amount of hay and haylage combined (8,846, 000 tons). The valu e of the alfalfa production in the U.S. totaled $7.7 billion in 2010 at an average price of $118 per ton.Monsanto Company 12-AL-246U 154 of 407 

### Table VIII-2. Alfalfa and Alfalfa Mixtures Forage (Hay and Haylage) Production by State in 2010 1



### .

Region/State Harvest Acres (000) Average

Yield (tons/acre) Production (000 tons) Value of Hay

Production 2

($000)

North Central Region Iowa 910 3.6 3,233 347,072 Minnesota 1,315 3.7 4,916 435,600 North Dakota 1,560 2.3 3,588 218,868 South Dakota 2,185 2.4 5,245 417,960 Wisconsin 2,200 4.0 8,846 399,620 Totals 8,170 3.2 4 25,828 1,819,120

East Central Region Arkansas 10 3.5 35 4,760 Delaware 5 3.4 17 3,247 Illinois 360 3.9 1,418 174,420 Indiana 300 3.6 1,080 150,120 Kentucky 230 2.8 644 88,228 Maryland 40 3.0 120 22,920 Michigan 950 3.4 3,249 226,800 Missouri 250 2.9 731 90,720 New England

States 3 96 3.5 339 17,966 New Jersey 20 2.9 58 8,352 New York 740 3.2 2,391 110,250 North Carolina 5 3.2 16 2,480 Ohio 420 3.6 1,508 205,920 Pennsylvania 650 3.2 2,089 191,100 Tennessee 15 3.4 51 9,129 Virginia 80 2.3 184 31,280 West Virginia 20 2.6 52 8,060 Region Totals 4,191 3.3 4 13,982 1,350,162Monsanto Company 12-AL-246U 155 of 407 

### Table VIII-2 (continued). Alfalfa a nd Alfalfa Mixtures Forage (Hay and Haylage) Production by State in 2010 1



### .

Region/State Harvest Acres (000) Average

Yield (tons/acre) Production (000 tons) Value of

Production 2

($000)

Plains Region Kansas 665 3.8 2,536 279,110 Nebraska 895 4.2 3,714 284,622 Oklahoma 310 3.3 1,023 142,197 Texas 130 4.8 625 109,800 Region Totals 2,000 3.9 4 7,898 815,729

Intermountain Region Colorado 820 3.5 2,870 367,360 Montana 1,950 2.3 4,485 354,315 Utah 540 4.0 2,160 228,960 Wyoming 620 2.6 1,612 149,916 Region Totals 3,930 2.8 4 11,127 1,100,551

Pacific Northwest Regio n Idaho 1,150 4.5 5,208 602,742 Nevada 280 4.3 1,204 151,704 Oregon 415 4.3 1,785 267,750 Washington 465 5.0 2,329 299,250 Region Totals 2,310 4.6 4 10,526 1,321,446

Southwest Region Arizona 280 8.2 2,296 296,184 California 960 6.8 6,549 841,092 New Mexico 229 5.2 1,182 184,184 Region Totals 1,469 6.8 4 10,027 1,321,460

U.S. Totals 22,070 3.6 4 79,388 7,728,468

1 Source: (USDA-NASS, 2011c)

2 The value is an estimate based on multiplying average prices with production volumes and does

not correspond to actual sales. Excludes the value of haylage.

3 New England States include Connecticut, Maine, Massachusetts, New Hampshire, Rhode Island,

and Vermont



### . 4 Average yield of Regional and U.S. Totals are calculated by dividing the corresponding

Production (000 tons) by Harvest Acres (000) values.Monsanto Company 12-AL-246U 156 of 407 

### In the past, alfalfa was viewed in some regi ons as a low-value rota tion or pasture crop and used primarily as a supplement to ot her higher value row and specialty crops (Klonsky et al., 2008). However, alfalfa has become a valuable and profitable crop that competes successfully with many higher-value specialty crops, espe cially in the high production areas of California. Dairy produc ers are the most important consumers or purchasers of alfalfa hay (Putnam et al ., 2008c; USDA-APHIS, 2010). While the dairy market utilizes alfalfa with quality cate gories ranging from low to supreme quality grades, high producing dairy cows require high-quality hay (supreme and premium grades) to maximize milk production. The horse industry is also an important market for high-quality alfalfa and alfalfa-grass hay mixt ures. The beef, sheep, and goat markets are another important market for alfalfa, but these producers typically put less emphasis on high-quality hay. Medium- and lower-qualityhay are frequently acceptable for nonlactating dairy cows and meat-producing livesto ck. Alfalfa is also used for processed feeds and alfalfa pellets for pets and rabbits . Unlike many other regions, the majority of the alfalfa hay produced in the North and East Central regions is produced and consumed on the same farm (USDA-APHIS, 2010). Wher eas in the western regions of the U.S., over 95 percent of the al falfa is sold as a hay product on the open market (Putnam et al., 2008c).

Alfalfa hay quality is defined by a number of nutritional traits in cluding acid detergent fiber (i.e., cellulose and lignin content), neut ral detergent fiber (i.e ., lignin, cellulose and hemi-cellulose content), crude protein, total digestible nutrients, a nd relative feed value (Putnam et al., 2008b; USDA-APHIS, 2010). Cert ain visual or sensory observations are also important to assess hay quality, such as the presence of weeds, molds, or antipalatability factors like poor texture, evidence of heati ng, or unpleasant odor. These factors can significantly affect nutritional va lue and overall forage quality. The USDA’s Agricultural Marketing Service (USDA-AMS) uses the grades of supreme, premium, good, fair, and utility to regular ly report average prices for alfalfa in various states (USDA-APHIS, 2010). Prices va ry significantly by quality grade and region. Prices paid for supreme grade alfalfa hay averaged $160.63 /ton, while fair grad e alfalfa averaged $116.48/ton in California (Hanford-Corcoran- Tulare-Visalia counties) in 2010 (USDAAMS, 2010).

Alfalfa producers can implement one of several different strategies or a combination of the following strategies to market alfalfa: 1) the low-cost production strategy, 2) productquality differentiation or niche-marketing strategy, and 3) provision of additional service strategy (Klonsky et al., 2008) . Producers utilizing the low-cost strategy produce maximum yields, control costs, and compete with other growers primarily on price. Product-quality differentiation or niche-marketin g strategy targets certa in markets such as the dairy industry to get top dollar for a consistently hi gh-quality alfalfa product. Differentiation can include designations such as certified weed-free hay, certified organic hay, special cubed alfalfa containing grasse s for horses, and low-potassium hay. The additional service strategy involves providing sp ecial service for the customer such as making hay available to the customer on de mand, meeting specific baling requirements, developing favorable payment or delivery co ntracts, offering quality warrantees, or allowing customers to harvest the alfalfa themselves for silage.Monsanto Company 12-AL-246U 157 of 407 

### Managing input costs is a major component to the profitability of producing an alfalfa crop. Since alfalfa is a perenni al crop and harvested over several years, the analysis of production costs for alfalfa are generally presente d in two parts: 1) co sts associated with stand establishment, and 2) the annual cost s during the production ye ars. Table VIII-3 provides a summary of the variable a nd fixed costs for producing non-irrigated,

conventional alfalfa in the North Centra l region based on the Ag Decision Maker spreadsheet by Iowa State University (Joha nns, 2012). Assuming tw o cuttings and two tons yield per acre from a spring seedi ng, variable and fixed costs were $268.70 and $199.00 per acre, respectivel y, during the establishment year , with a total cost of $467.70 per acre. There was a net retu rn of $31.30 per acre over variab le costs, but a negative net
return of $167.70 over total costs. Assuming three cuttings, f our tons yield per acre per year, and three years of producti on after establishment year, va riable and fixed costs were $225.30 and $189.50 per acre, respectively, duri ng the production years with a total annual cost of $414.80 per acre. Average net return over variable costs during the production years was $374.70 per acre per year. Average net return over total costs was $185.20 per acre per year. When assessing cost s and gross returns for both establishment and production (four years) net return per ye ar averaged $289.75 per acre above variable
costs and $97.88 above total costs.

Similar cost studies were conducted by the University of California on irrigated conventional alfalfa in the San Joaquin Va lley in 2008 (Mueller et al., 2008a). These data are presented in Table V III-4. In these studies, the fall-seeded alfalfa was assumed to produce a total of nine cutti ngs yielding eight tons of hay and three green tons (one-ton hay equivalent) of haylage per acre. Oper ating costs and total overhead costs were $385 and $375 per acre ($760 total), respectively, for establishing alfalfa. Annual operating

costs and total overhead co sts were $708 and $654 per acr e ($1,362 total), respectively, during the production years. Net return above operating costs was $912 per acre per year. Net return above total costs was $258 per acre per year which includes the total cash costs of stand establishment ($478 per acre) amortized over the three-year stand life. In contrast to non-irrigated alfalfa grown in the North Central region, the gross returns are much higher with irrigated alfalfa but the cost of produc tion is substantially higher, mainly due to the cost of irrigation.Monsanto Company 12-AL-246U 158 of 407 

### Table VIII-3. Costs and Returns for Establishment and Production of NonIrrigated Conventional Alfalfa in the North Central U.S. 1

Operations/Cost Item Establishment

Year Cost/Acre ($) Production Years Cost/Acre ($)

Gross Returns: Hay ($150/ton) 300 600

Total Gross Returns 300 600

Variable Costs: Preharvest Machinery 11.80 1.20

Fertilizer 121.50 192.00

Lime 29.00 -

Seed 72.00 -

Herbicide 13.10 -

Harvest 21.30 32.10 Total Operating Costs 268.70 225.30

Fixed Costs: Preharvest Machinery 15.80 1.60

Labor 55.00 44.00 Land (cash rent equi valent) 100. 00 100.00

Harvest 28.20 43.90

Total Fixed Costs 199.00 189.50

Total Costs 467.70 414.80

Net Return Above Variable Costs 31.30 374.70

Net Return Above Total Costs -167.70 185.20 Average Annual Net Return Above

Variable Costs Over 4 Years 289.75

Average Annual Net Return Above Total

Costs Over 4 Years 97.88

1 Source: (Johanns, 2012). Assumptions: 300 acres of alfalfa; three year s hay production after es tablishment year; two cuttings and 2 tons/acre in establishment year; th ree cuttings and 4 tons/acr e in production years; no irrigation.Monsanto Company 12-AL-246U 159 of 407 

### Table VIII-4. Costs and Returns for Establishment and Production of Irrigated Conventional Alfalfa in the San Joaquin Valley, California. 1

Operations or Cost Item Establishment Year Cost/Acre ($) Production Years Cost/Acre ($)

Gross Returns: Hay (8 tons) - 1,480 Silage (3 tons) 2 - 140

Total Gross Returns - 1,620

Operating Costs: Land preparation 123 -

Fertilizer 67 41 Seed and seeding 109 - Irrigation 26 182

Weed control 48 98

Insect control - 43

Harvest 322 Other cost items (truck & ATV) 6 12 Interest on capital @ 6.75% 8 -11 Total Operating Costs 385 708

Cash Overhead Costs:

Insurance 3 3

Taxes 60 76 Office Expenses 24 30 Investment Repairs 6 13

Total Cash Overhead Costs 93 122

Total Cash Costs 478 830

Non-Cash Overhead Costs (Capital Recovery):

Land 247 308 Irrigation System 19 38 Building, Fuel, and Equipment 16 13

Alfalfa Establishment 173

Total Non-Cash Overhead Costs 282 532

Total Cost/Acre 760 1362

Net Return Above Operating Costs 912

Net Return Above Total Costs - 258

1 Source: (Mueller et al., 2008a). Suppo rting information: 1200 acre farm with 300 acres of alfalfa; fall seeding; 3-year stand life for alfalfa; irrigation - 8 acr e-inches of water used for establishment and 4.5 acrefeet in 10 irrigations for production; harvest cost for two cuttings of haylage and seven cuttings of hay, buyer of haylage paid harvest costs.

2 Haylage price equals market price less harvest cost (assumes buyer harvests haylage) multiplied by percent haylage dry matter divided by percent hay dry matter ($185-$45) × (30% DM/90% DM) =
$46.67/ton.Monsanto Company 12-AL-246U 160 of 407 

### VIII.B.2. Alfalfa Seed Production

Standardized seed production practices are responsible for maintaining high-quality seed stocks, an essential basis for U.S. agricultu re. By the early 20th century, agronomists learned how to develop specific plant varieties with desirable traits. In the U.S., state agricultural experiment stations developed many seed varietie s that were distributed to growers for use. Seed was saved by grower s and later sold to ne ighbors; however, the

desirable traits of the varieties often we re lost through random genetic changes and contamination with other crop a nd weed seed (Sundstrom et al., 2002). The value of seed quality (including genetic purity, vigor, and pr esence of weed seed, seed-borne diseases, and inert materials, such as dirt) was quickly identified as a major factor impacting crop yields. States developed seed laws and certif ication agencies to ensure that purchasers

who received certified seed could be assured that the seed met established seed quality standards (Bradford, 2006). The federal governme nt passed the U.S. Federal Seed Act of 1939 to recognize seed certification and the esta blishment of official certifying agencies. Regulations first adopted in 1969 under the Fede ral Seed Act recognize land history, field isolation, and varietal purity standards for foundation, registered, an d certified seed. Under international agreements such as th e Organisation for Econom ic Co-operation and Development (OECD), the U.S. and other c ountries mutually recognize minimum seed quality standards (Bradford, 2006). The Asso ciation of Official Seed Certifying Agencies (AOSCA) represents state and priv ate seed certification organizations in the U.S., and includes international member countries in North and South America, Australia, and New Zealand.

Alfalfa seed is separated into four seed cl asses: 1) breeder, 2) foundation, 3) registered, and 4) certified (AOSCA, 2011). Breeder seed is seed that is direc tly controlled by the originating or sponsoring plan t breeding organization or firm . Foundation seed is firstgeneration seed increased from breeder seed and is handled in a manner designed to maintain specific levels of varietal purity and identity. Registered se ed is the progeny of

foundation seed that is handled to maintain satisfactory varietal purity and identity. Certified seed is the progeny of breeder, foundation or registered seed, and is typically two generations removed from foundation see d. While not all alfalfa seed sold to growers is officially certified, commercial alfa lfa seed sold and planted for typical alfalfa production is produced predominately to meet or exceed certified seed standards. This section of the petition will provide a broad ov erview of the practices used in producing

certified seed.

Alfalfa seed breeders and producers have put in place practical measures to assure the quality and genetic purity of al falfa varietal seed for commercial planting. The need for such systems arose from the recognition that the quality of impr oved varieties quickly deteriorated in the absence of monitoring fo r quality and genetic purity (CAST, 2007). Seed certification programs were initiated in th e early 1900s in the U.S. to preserve the genetic identity and variety purity of seed. There are special land requirements, seed stock eligibility requirements, field inspec tions, and seed labeli ng standards for seed

certification. Seed certification services are available through va rious state agencies affiliated with AOSCA. Large seed produc ers implement their own seed quality assurance programs that go beyond mandated qua lity standards. However, large seedMonsanto Company 12-AL-246U 161 of 407 

### producers often will utilize the se rvices of state certifying agencies as a third party source to perform certain field in spections and audits.

In contrast to the broad geographic distri bution for forage production, most commercial alfalfa seed production is highly concentrated in the irrigated regions of the western U.S. (Table VIII-5). The arid climate of the western U.S. provides a warm, dry production and harvest season to maximize seed yield and quality. Over 121,000 acres of alfalfa seed were harvested in 2007, producing approxi mately 62 million pounds of seed with an average yield of approximately 510 pounds per acr e. Historically, California has been and remains the largest producer of alfalfa seed, but acreage has declined in recent years due to economics, environmental constraint s, and regulatory i ssues (USDA-APHIS, 2010). Ninety-five percent of the seed produced in California is of non-dormant varieties (FD 7-10) while the Pacific Northwest produc es seed of semidormant (FD 5-6) and dormant (FD 2-4) varieties (Mueller, 2008) . Approximately 42 million and 8.5 million pounds of certified and non-certif ied alfalfa seed, respectivel y, were exported from the U.S. in 2011 (WASGA, 2011 ). The largest qu antities of seed went to Saudi Arabia, Mexico, Argentina, and Canada.Monsanto Company 12-AL-246U 162 of 407 

### Table VIII-5. Alfalfa Seed Production in the U.S. 1

State Farms Seed Harvested (Acres) Seed Harvested (lbs) California 114 36,625 19,083,458 Washington 82 17,127 10,860,608 Idaho 92 12,788 9,346,709 Wyoming 62 10,548 5,915,816 Nevada 19 6,498 4,237,101 Montana 80 10,338 3,729,635 Oregon 32 4,959 3,183,375 Utah 54 3,803 2,077,813 Arizona 53 5,206 1,902,669 South Dakota 47 6,014 428,447 Oklahoma 29 2,004 281,121 Texas 24 546 79,885 Minnesota 17 611 63,461 Missouri 19 699 40,540 North Dakota 6 (D) 34,784 New Mexico 15 310 29,907 Kansas 5 342 22,430 Nebraska 29 545 21,216 Michigan 10 (D) 15,610 New York 3 27 6,180 Iowa 5 (D) (D) Ohio 1 (D) (D) Colorado 8 1,815 (D)

Undisclosed total
- 962 587,405 U.S. Total 806 121,467 61,948,170 (D) = Data withheld to avoid disclosing data for individual farms.

1 Source: (USDA-APHIS, 2010).Monsanto Company 12-AL-246U 163 of 407 

### California is credited with 31% of the seed production, Washington with 17%, and Idaho with 15% (USDA-APHIS, 2010). Over 60% of the seed pr oduction is concentrated in those three states, and if Arizona, Nevada , Oregon, Wyoming, Montana, and Utah are included, the total seed production among these nine states collectively represents over 95% of alfalfa seed production. The U.S. alfalfa seed growers compete with seedproducers in Canada, Australia , and other countries where comparative pr oduction costs are significantly lower (USDA- APHIS, 2010). The demand for alfalfa seed is driven by

the demand for seed to establish new stands of alfalfa forage, with a minor amount used as field seed stock or for human consumption. It is estimated that 2.5 percent or less of the seed produced is used for human cons umption as alfalfa sprouts (USDA-APHIS, 2010). Alfalfa seed is not consumed as a grai n and, therefore, is not used directly as a feed or food product (USDA-APHIS, 2010).

Seed production is limited by the alfalfa plant’ s genetics and pollination characteristics. Most alfalfa plants exhibit various forms of genetic self-incompatibility or self-sterility and will not successfully self-pol linate (Viands et al., 1988). Al falfa is adversely affected by inbreeding, i.e. , self-fertilized plants commonly de monstrate a dramatic reduction in seed yield potential (Rumbaugh et al., 1988). Inbreeding depression may be because of

the loss of heterosis and/or accumulation and unmasking of deleteri ous recessive alleles

that occur as a result of se lf-pollination and/or pollinat ion among close relatives. Therefore, commercial alfalf a breeding programs are struct ured to avoid significant inbreeding and the resulting negative effects of inbreeding depression (Rumbaugh et al., 1988).

Alfalfa varieties are primarily bred for fo rage yield (vegetative production), forage quality, longevity, and adaptation to a broad geographic area. A typical alfalfa variety may have ten to 200 parent plants that were initially crossed in is olation to form the breeder generation seed (Figure VIII-2). The breeder seed of commerc ial alfalfa varieties is produced by the random intercrossing (open pollination) of all parent plants. An alfalfa variety is maintained through multip le seed generations beyond breeder seed via the open pollination of their progeny in isolat ion from other alfalfa varieties or pollen sources. Plant varieties bred in this way are called synthetic varieties (Rumbaugh et al., 1988).

Individual plants w ithin a synthetic variety are genotypically and phenotypically heterogeneous, i.e ., no two individuals within the vari ety are exactly alike. Synthetic alfalfa varieties are closed popul ations that segregate, within a defined range, for most morphological traits and naturally occurring ge netic markers. Because alfalfa varieties are segregating heterogeneous populations, al falfa varieties are ro utinely described in terms appropriate to populations (mean or % trait expression). For example, alfalfa variety registration agencies re quire that the pest resistance of a variety be described as the mean percent of plants that express the se gregating trait when th e population is tested

under standardized conditions.Monsanto Company 12-AL-246U 164 of 407 

### Figure VIII-2. Commercial synthetic al falfa variety breeding schematic

As illustrated in Figure VIII-2, commercial seed of alfalfa varieties is commonly produced according to the following sequence: 1) A set of superior al falfa plants (usually 10-200 genotypes, also known as Syn0 parents) are identified by an alfalfa breeder for use as parent plants for a new variety. Th e Syn0 parents are randomly intercrossed to produce the first synthetic generation of seed (Syn1 seed); 2) Syn2 generation seed is produced from a random, isolated intercro ss of Syn1 plants; and -3) Syn3 seed is produced from a random, isolated intercro ss of Syn2 plants. Breeder, foundation, registered, and certified seed classes are de fined at the discretion of the plant breeder during the variety registration process. Br eeder and foundation seed are typically Syn1 and Syn2 generations, respectively. Commerci al seed (registered and certified) is typically Syn3 generation. Syn 0 parent plants are isolated and randomly intercrossed to produce Syn 1 seed

Plant Syn 1 seed and allow random, isolated intercrossing Selection of superior parent-plant group (n=10-200 parent genotypes)

Plant Syn 2 seed and allow random, isolated intercrossing 

### Syn0

Syn1

Syn2

Syn3 “Foundation Seed” “Breeder Seed”

Random intercrossing occurs

Random intercrossing occurs

Random intercrossing occurs 

## Selection & Breeding of

Parents

Production of

Commercial

Seed Phenotype and/or

genotype
information Po p ulation 2 Po p ulation 1



### “Commercial Seed”

Package Syn 3 seed and distribute to forage growers



### Forage Fields Elite members from non-related populations are used to initiate the breeding processMonsanto Company 12-AL-246U 165 of 407 

### Commercial variety testing a nd registration by alfalfa bree ders typically use Syn1 and Syn2 generation seed to establish variety tes ting and evaluation experiments. The U.S. alfalfa variety registration and review process is based on data from tests initiated with Syn1 generation seed although other Syn genera tion test data are al so recognized.

Alfalfa breeding and development of varieties with new quality and agronomic characteristics are conducted by both public and private research programs. Alfalfa varieties are the result of germplasm developm ent, enhancement, and the use of standard breeding techniques typical fo r cross-pollinated crops.

Alfalfa seed is produced by a number of comp anies that produce and sell seed, such as Forage Genetics International, Pioneer Hi-B red, Dairyland Seed, and Cal/West Seeds. Seed companies in turn cont ract with growers to produce the needed amount of alfalfa seed on their acreage. To produce the seed, seed companies identify top growers then monitor and inspect seed fields throughout the growing season. S eed companies have processing facilities to clea n, condition, and bag the harveste d seed as well as monitor and inspect all the processes at the plant.

The entire seed production process at the majority of the seed companies operate using International Organization for Standardization (ISO) certificat ion standards, and therefore include internal and external audits (IS O, 2012). ISO standards ensure desirable characteristics of seeds and services, such as quality, safety, reliabi lity, and efficiency. The ISO standards represent an international consensus on good management practices with the aim of ensuring that the organizatio n can consistently deli ver excellent products or services. The standards not only must meet customers’ and applicable seed regulatory requirements, but must aim to enhance cu stomer satisfaction and achieve continual improvement of its performance in pur suit of these objectives (ISO, 2012).

While many of the field operations and manage ment practices for producing alfalfa seed are similar to normal alfalfa forage production, there are significant differences. Special

attention is needed in cert ain areas to optimize yields and to produce seed with high quality, high germination rates, and high gene tic purity. Fields selected must not have grown alfalfa on the land for a four-, thr ee-, and one-year period prior to stand establishment for foundation, registered, and certified seed, respectively (AOSCA, 2011). A two-year restriction applies to certified seed produced in northern and central regions following production of varieties adapted to the southern regi on. This restri ction is to limit the spontaneous germination and sprouting of alfalfa seeds left in the seed bank. In addition, the land must be free of volunteer alfa lfa plants the year pr ior to seed production (AOSCA, 2011).

Successful seed production begins with proper st and establishment. The majority of the dedicated alfalfa seed production fields, i.e. , not including common or catch-crop seed, are established according to the terms of a two- or three-year seed company contract, on a variety basis. The company supplies the seed stock, e.g. , foundation seed, to the seed producer and genetic source va riety of the seed is documented (USDA-APHIS, 2010). Depending upon the relative market prices fo r forage versus common or variety not stated (VNS) alfalfa seed, a minor proportion of the U.S. alfalfa seed crop is producedMonsanto Company 12-AL-246U 166 of 407 

### without contract as a catch-crop, where solid -seeded hay fields are allowed to set and ripen seed instead of being cut for forage. Th is practice occurs sporad ically in the Plains, Pacific Northwest, and Desert Southwest. “C atch-crop” seed is typically produced with lower management and inputs, the genetic purity of the seed stock is often unspecified or unknown, and the resultant seed quality is highly variable and cannot be certified as to cultivar or variety identity (USDA-APHIS, 2010).

Alfalfa grown for the dedicated purpose of commercial seed production is always established alone without a companion cr op (Hower et al., 1999). Recommended planting dates are similar to forage producti on. Fall establishment predominates (58%) because first year seed yields in fall-esta blished stands are greater than for spring seedings (Hower et al., 1999; Mueller, 20 08). Alfalfa stands dedicated to seed production are planted in rows with spacings of 30 to 40 inches and are usually on beds to facilitate furrow irrigati on and early cultivation for weed control (Mueller, 2008). The plant density required to optimize seed yiel ds depends on row spacing and soil type, which influence growth and final size of the alfalfa plant. Successful stands have been

established with seed ing rates as low as 0.5 to 0.75 pounds per acre (Mueller, 2008). Although, some seed producers may use slightly higher seeding rates and mechanically thin stands after emergence. Higher seed yi elds with thinner stands are attributed to improved water use efficiency, pe st control, and pollination.

After stand establishment, fields are clipped in the spring. The cli pback serves several purposes: 1) it encourages plants to come into bloom uniformly and to synchronize bloom with the period in the season when pollinators are most active, 2) it removes growth that has over-wintered and cleans up the field for herbicide applications, and 3) it also provide a cultural method for c ontrolling the alfalfa seed chalcid ( Bruchophagus

roddi ) insect pest (Mueller, 2008).

Field inspections are vital to ensure the alfa lfa seed meets seed cer tification requirements, ISO certification standards, re gulatory standards, and lice nsing agreement standards. Field inspections are conducted on seed production fields throughout the growing season to visually evaluate variet y purity, ensure alfalfa plants are developing properly, and fields are maintained free of weeds, insects, and diseases. The fiel ds are also mapped to

ensure the seed field has the minimum isol ation requirement as a physical barrier.

Foundation seed fields must be isolated from alfalfa of di fferent varieties by 900 feet, while certified fields must be isolated by 165 feet (USDA-APHIS, 2010). However, the ten percent rule is followed for certified seed fields, where if ten percent or less of the

certified field is in the 165 foot isolation zone , the entire field is considered certified, but if more than ten percent is in the isolation zone, then that part of the field must be separated and not harvested as certified seed. Some states and seed producers may have a stricter isolation requirement. Volunteer plants may be a cause for rejection or reclassification of a seed field (AOSCA, 2011).

Weed management is critical in alfalfa seed production because weed competition can directly reduce seed yields and weeds produce abundant seed which can impact the strict purity standards required for certi fied seed. Since alfalfa for seed is often grown in wide rows, there is greater potential for weed i nvasion. All available weed control methods,Monsanto Company 12-AL-246U 167 of 407 

### including herbicides, mechanical cultivati on, and hand weeding are used to eliminate weeds including volunteer alfalfa during the growing season (Muell er, 2008). Herbicide use on alfalfa seed acres averaged 78% fr om 1988 to 1992 (Hower et al., 1999) and, undoubtedly, has increased in rece nt years. The separation of weed seeds from alfalfa seeds after harvesting is costly, so controlling the weeds in the fields is a more desirable method of seed quality cont rol than post-harvest scr eening and separation (USDAAPHIS, 2010). Dodder ( Cuscuta spp

. ), a parasitic weed that lives off alfalfa plants, is a particularly troublesome weed because there is zero tolerance for dodder seed in certified alfalfa seed (Canevari et al., 2008). Registered herbicid es for control of dodder must be applied prior to emergence and attachment to a host plant to be effective.

Insect pests can have an impact on alfalfa s eed yields and quality. The major insect pests in alfalfa include lygus bugs ( Lygus spp

. ), spider mites ( Tetrancychus spp

. ), and alfalfa seed chalcid ( Bruchophagus roddi ) (Mueller, 2008). Lygu s bugs are among the most significant pest species in California because they occur throughout the season, moving between crops, and they are the most difficult ins ect pest to manage in alfalfa seed fields. They feed on the reproductive parts of alfalf a plants causing premature drop of buds and flowers, seed deformation, and reduced seed viability. Seed producers monitor insect pest and beneficial insect populations th roughout the growing season and implement appropriate insect management strategies. Multiple pesticide applications are usually required during the bloom and pollination period. Applicator s must exercise special precautions and follow special label instructions to protect bee colonies that are present to facilitate the pollination necessa ry for seed production.

Alfalfa flowers require trippi ng and cross-pollination for ma ximum seed yields. Three types of pollinators are used in seed production: honey bees ( Apis mellifera L.), alfalfa leafcutting bees ( Megachile rotundata ), and alkali bees ( Nomia melanderi ) (Mueller, 2008; USDA-APHIS, 2010). Alfa lfa leafcutting bees are utilized in the Pacific Northwest region and, to a limited extent, in California. Alfalfa leafcutting bees are more efficient than honey bees, but often are more expensive and require greater management. Alkali bees are used only in certain areas of Washington. B ee colonies are usually placed

around seed fields when the al falfa plants are between the one-third to one-half bloom stage. The pollination process generally takes 30 days and it takes an additional 20 to 25 days for the plants to produce mature seed (Mueller, 2008). Costs associated with pollination can be $160 per acre, or 25 percent of the total seed production costs (USDAAPHIS, 2010).

Once alfalfa plants reach maturity, they must be dried before harvest to efficiently separate the seed from the pod and other plan t material. This is accomplished by cutting and windrow curing or chemically desiccatin g the standing crop. Windrow curing can begin once the majority of the seed is mature (two-thirds or three-fourths of the pods are dark brown in color) and harvesting can begin when the plant reaches 12 to 18% moisture (Mueller, 2008). As an alternate curing method, desiccants can be applied to the fields 7 to 10 days prior to harvest; the standing crop is then directly harvested using a combine. To minimize seed losses and obtain high-quality seed, harvest machinery adjustments and operations must match the field and crop conditi ons. Seed is typically harvested once per

year.Monsanto Company 12-AL-246U 168 of 407 

### Harvested seed is conditioned to remove soil, weed seeds, and other debris. Separating machines utilizing the differences in physical characteristics of alfa lfa seeds and the nonseed fractions complete the cleaning proces s. Seed scarification (mechanical or chemical) is performed on seed lots that cont ain high percentages of hard seed to improve germination (Mueller, 2008). All seed lots ar e tested for purity, ge rmination, and noxious weed seed content before bagging and sale.

Commercial certified alfalfa s eed must meet state and federal seed standards and seed bag tag labeling requirements. AOSCA standards for certified al falfa seed are as follows: 99% pure seed (minimum), 1% inert matter (maximum), 0.5% weed seed (maximum), 1.0% other crop seeds (maximum) – 2.0% ot her varieties (maximum) and 0.05% other kinds (maximum), and 80% germination (minimum) (AOSCA, 2011). The alfalfa industry historically sets a higher minimum ge rmination rate for labeling purposes. State seed certification standards vary slightly from state to state and can be more restrictive

than the seed standa rds of AOSCA.

The phenotypic, agronomic, and environmenta l interaction assessments presented in

Section VII have demonstrat ed that KK179 is not mean ingfully different from conventional alfalfa. Therefore, if dere gulated, KK179 seed would be produced in the

same manner as certified alfalf a seed, such that it will m eet all state and federal seed standards and labeling requirements.

VIII.C. Alfalfa Forage Produc tion Management Considerations

VIII.C.1. Pre-Season

Production decisions regarding crop rotation, tillage system, soil fertility, and variety

selection need to be made well in advan ce of planting the alfalfa crop. Many of the decisions in this area are made prior to or im mediately after harvest of the previous crop. Rotations with other crops should be an integr al part of a farm management program. Alfalfa provides soil improvement and other be nefits to subsequent crops (Mueller and Teuber, 2008). Alfalfa is commonly rotated with wheat, corn, cotton, sugar beets, and vegetables. Alfalfa assimilates nitrogen th rough nitrogen fixation and can provide levels of nitrogen ranging from 40 to 60 pounds per acre to the crop th at follows alfalfa in the rotation (Orloff, 2008). Vegetable growers re alize the added value from alfalfa in the rotation by improving water in filtration, soil fertility, and reduced weed pressure following alfalfa. Planting alfalfa after alfalfa is not recommended because of autotoxicity (Mueller et al ., 2008b; Undersander et al., 2011). Autotoxicity occurs because alfalfa plants from the previous stand produce chemical inhibitors that can reduce germination and growth of new alfalf a seedlings. Production costs, relative rate of return, and current market conditions will di ctate which crops to rotate with alfalfa.

Profitable alfalfa production begins with the sele ction of the proper sites or fields to plant alfalfa. Sites that provide adequate rooti ng depth, nutrition, aerati on, and water, and have no salinity or alkalinity pr oblems, can produce good fo rage yields with good management. Alfalfa is adaptable to a wi de range of soil types, but requires a welldrained soil for optimum production (Orloff, 2008) . Therefore, fields should be selectedMonsanto Company 12-AL-246U 169 of 407 

### that have good surface drainage and have soils with good internal drainage and lack subsoil impediments (Orloff, 2008; Unders ander et al., 2011). Wet soils create conditions suitable for diseases that may k ill seedlings, reduce fora ge yield, and kill established plants. Of major importance in the western alfalfa growing regions, is selecting sites that have an adequate suppl y of quality water available for season-long irrigation. In the arid Southwest region, ir rigation supplies between 70 and 100% of the total crop water requirement (Hanson et al., 20 08). Site selection s hould also take into account the previous crop and an y soil residual herbicides th at may preclude planting

alfalfa.

Tillage has been an integral part of production agriculture and is synonymous with seedbed preparation. The purposes of primary preplant tillage are to incorporate residue from the previous crop, reduce wheel tra ffic compaction from the previous season, improve water filtration and soil aeration, c ontrol weeds, loosen the soil for root penetration, and provide a suitable environmen t for the planting and germination of the seed (Hake et al., 1996; Mueller et al., 2008b). Proper field preparati on before planting is

critical because the alfalfa st and will be intensively manage d and harvested for three to five years, or longer in some areas. Deep til lage with moldboard and disc plows, rippers, and chisels can increase infiltration rate, fractures stratified layers, mixes the soil profile, and reduces bulk density and soil strength (Mueller et al., 2008b). The cost of deep tillage must be weighed against the poten tial benefit to determine whether it is economically feasible. Land leve ling is an important step in the western irrigated regions because water must flow evenly over the ground surface in flood irrigation systems. Due to the high potential for eros ion on slopes using conventional and deep tillage systems, there is a great deal of interest in reduced-t illage alfalfa establishment in the North and East Central regions (Undersa nder et al., 2011). Reduced tillage may involve chisel plowing instead of moldboard plowing or a single pass with a secondary tillage tool. Crop residue management is an important consideration inpracticing reduced-tillage seeding. No-till seed ers are recommended in reduced tilla ge and no-tillage systems with residue levels above 35% (Undersander et al., 2011). Cultipacker seeders, which

broadcast the seed on the soil su rface and then press the seed into the soil with rollers, have been used extensively in conventi onal tillage systems b ecause they provide consistently even seed depth placemen t and good seed-soil contact. However, cultipacker seeders do not perform well in resi due levels above 35% (Undersander et al., 2011). Weed control is more difficult in redu ced-tillage systems because there is limited to no tillage to decrease weed populations. Perennial weeds may also be more of a problem with the lack of deep tillage. Still, th ere is some interest in alfalfa establishment with no tillage because of the high potential for soil erosion on slopes with conventional tillage. Special attention mu st be given to soil fertility and pH in no-till alfalfa establishment. Fertilizer and lime requireme nts should be incorporat ed into the soil well

in advance of the alfalfa establis hment year in the cropping system.

Maintaining optimum crop nutrition and proper li me management is critical in achieving high yields and quality in alfalfa. Proper fertilization allows for good stand establishment and promotes early growth, increases yield and quality, improves winter-hardiness and stand persistence, improves the ability of alfa lfa to compete with w eeds, and strengthens disease and insect resistance (U ndersander et al., 2011). Seve nteen elements are essentialMonsanto Company 12-AL-246U 170 of 407 

### in varying amounts for plant growth (Mey er et al., 2008). Carbon, hydrogen, and oxygen

come from the water or air. The other 14 elements are obtained from either the soil or fixation of atmospheric nitrogen by bacteria in root nodules of alfalfa. Coba lt is essential to legumes for nitrogen fixation. Soil tes ting is the most convenient and economical method of evaluating the fertility levels of a soil and accurately assessing nutrient and liming requirements. However, plant tissue te sting at the 10% bloom stage is the most precise method of determining nutrients needs, especially for sulfur, boron, and molybdenum. The nutrients that are most commonly in short supply for alfalfa production are phosphorus, followed by potas sium, sulfur, molybdenum, and boron. Adequate levels of nitrogen are almost al ways provided by symbiotic nitrogen-fixing bacteria ( e.g. , Rhizobium meliloti ). The most common causes of nitrogen deficiency are poor inoculation and nodule formation. Alfalf a seed should be inoculated when planting into fields without a history of alfalfa pr oduction. Small amounts of nitrogen fertilizer (25 to 30 pounds per acre) have been shown to enhance establishm ent and first year yields when direct-seeding on low organic ma tter, coarse-textured soils (Undersander et al., 2011).

Maximum nutrient availability for most cr ops occurs when pH values are between 6.0 and 7.0 (Orloff, 2008). Higher pH values of 6.7 and 6.9 are recommended for alfalfa for maximum yields and to favor activity of nitrogen-fixing Rhizobium bacteria (Undersander et al., 2011). Soils with pH values above 7.5 ar e not recommended for alfalfa (Orloff, 2008). Lime should be app lied during the production years of rotational crops following alfalfa in the rotation sequence to allow time for th e lime to react with

the soil and be mixed into the soil with tilla ge prior to replanting the next alfalfa crop.

Variety selection is an important preseason de cision in alfalfa production that can affect crop yield, crop quality, and pest management. Since alfalfa is a perennial crop, growers must stay with their choice of variety for several years. Variety selection is a real challenge since there are over 250 alfalfa vari eties to choose from and new varieties become available each year. Alfalfa varieties are diverse populations of plants having multiple genotypes rather than uniform genetic strains (Putnam et al., 2008a). Alfalfa is a polyploid having four complete sets of chromosomes which means that the offspring of

alfalfa crosses are much more diverse than most crop species. This genetic diversity

enables alfalfa varieties to be well adapted over a wide ra nge of environments, and to resist a wide range of insects, diseases, and nematodes to a greater degree than any other crop. Variety selections should weigh the importance of yield potential, stand persistence, fall dormancy, winter-hardiness, disease resistance, and forage quality (Undersander et al., 2011).

Yield potential is typically the most importa nt economic factor with regard to variety

selection. Growers are advise d to look for the top yielding varieties in university variety trials which are grown in a site with as similar a soil type and climate to their farm as possible (Undersander et al., 2011). They s hould look for top yields over several sites because soils vary on the farm and weather conditions change from year to year.

Persistence of stands in north ern locations depends primarily on the winter-hardiness of the variety, while persistence in southern locations depend s more on pest resistance. Winter-hardiness is a measure (1 to 6 scale, 1 being the most hardy) of the alfalfa plant’sMonsanto Company 12-AL-246U 171 of 407 

### ability to survive the winter without inju ry. Fall dormancy measures the degree of growth (plant height) of alfalfa in the fall. Varieties with fall dorma ncy (FD) ratings of 1 to 4 are considered dormant, 5 to 7 semi- dormant, and 8 to 11 non-dormant (Putnam et al., 2008a). Growers should choose less dormant va rieties that meet their winter survival requirement. These varieties wi ll green up earlier in the sp ring and recover more quickly between cuttings to give hi gher total yields for the se ason. Plant breeders have successfully incorporated resistance to cert ain diseases, nematode s, and insects into alfalfa varieties over the past 40 years (Putnam et al., 2008a). Growers need to determine the most significant diseases, nematodes, and in sect pests in their region to decide which variety has the best pest-resistance packag e. Although agronomic practices such as cutting schedule and weed control influence forage quality to a greater degree than variety selection, varieties should also be a ssessed on forage quality by evaluating ratings on digestibility, intake, and relative feed value (Undersan der et al., 2011). Since the

introduction of alfalfa varie ties with the biotech nology-enhanced glyphosate-tolerant trait (Roundup Ready), growers also need to weigh the weed management benefits and costs this trait will provide to thei r alfalfa production program.

VIII.C.2. Planting and Stand Establishment

Stand establishment is a critical step in alfa lfa production that can im pact profitability for many years. Good seedling establishment results in dense, vigorous stands that produce high-quality, high yielding alfalfa throughout the life of the stand. Alfalfa seed germinates best at soil temperatures from 65 to 85°F (Mueller et al., 2008b). Alfalfa will germinate in approximately six days when the soil temperature is 40°F, but will only take two days at 65°F. Alfalfa seed contains a porti on of hard seed which is highly resistant to water penetration and germination of hard seed can be delayed many weeks or months after the majority of the seed germinates (Mueller and Teuber, 2008). Seed lots containing more than 10% hard seed may need to be scarified to improve germination. Research at the University of California in dicates that optimum root growth during the first month of seedling growth occurs between 69 to 76°F, depending on dormancy class. Shoot growth is optimum at temperatures ranging from 72 to 76° F (Mueller et al., 2008b). Alfalfa stops growing when the air temperature drops below 42-34°F.

Alfalfa can be established successfully in either the spring or in the late summer and fall. In the U.S., 70% of alfalfa acres are spri ng-seeded and the remaining 30% are planted during late summer and early fall (Hower et al., 1999). Spring seeding is preferred in the

northern states of the North and East Centra l regions, while late summer and early fall seeding is preferred in all the remaining regions of the U.S. (Mueller et al., 2008b; Undersander et al., 2011). Temperature, soil moisture, and length of growing season are important factors that impact seed germina tion and stand establishment and ultimately determine which planting time is most successf ul and provide the highest alfalfa yields for a given area. Spring seedi ng begins as soon as the potential for da mage from spring frosts is over which is normally April and May in the northern areas of the North and East Central regions (Undersande r et al., 2011). Although alfalfa is very tolera nt of cold temperatures at emergence, seedlings at th e second trifolia te leaf stage become more susceptible to cold injury and may be killed by four or more hours at or below 26°F (Undersander et al., 2011). Fa ll seeding of alfalfa requires at least six weeks of growthMonsanto Company 12-AL-246U 172 of 407 

### after germination to survive the winter in the Central regions. In the Imperial Valley of California, fall seeding of alfalfa begins following the harvest of the summer crop and with adequate soil moisture which is Septem ber to early October (Mueller et al., 2008b). Pre-irrigation or overhead irrigation after s eeding is often needed for successful stand establishment in the arid West. Planting date research in Central Valley of California has shown fall seeding can result in 20 to 30% higher yields in the establishment year compared to spring seeding (Mueller et al., 2008b).

Seeding alfalfa with a compan ion crop (or nurse crop) such as annual ryegrass, oats, spring barley, and rye is often practiced with spring seeding to he lp control erosion on steep slopes, reduce seedling damage from wi nd erosion on sandy soils, and reduce weed competition during alfalfa establishment (Undersander et al ., 2011; USDA-APHIS, 2010). Companion crops also provide addition al forage or grain, and the straw can be harvested for livestock bedding. Fall seeding of alfalfa with a comp anion crop is seldom practiced because of limited soil moisture and competition with alfalfa. Small-grain companion crops grown to mature grain can damage alfalfa either by competition or by lodging, which smothers the alfalfa seedlings. Direct-seeding alfalf a (seeding without a companion crop) can produce up to two extr a cuttings of alfalfa and produce higher quality forage in the seeding year (Undersa nder et al., 2011). However, total forage production may be less than that of compan ion-crop seeding. An advanced seeding technique can be used with companion crops to obtain the benefits of direct seeding while controlling erosion (Undersander et al., 2011; USDA-APHIS, 2010). Alfalfa is seeded with a companion crop to obtain the benefits of early weed control and erosion control. However, the companion crop is killed with a postemergence herbic ide at between four and six inches in height to avoid competition with the alfalfa crop.

Alfalfa forage may be grown in pure stands or mixed with various other forage species ( e.g. , cool-season grass mixtures, with or wit hout other legumes, such as forage peas, birdsfoot trefoil [ Lotus corniculatus L.], or clover [ Trifolium L. spp.]). The use of mixed stands is widespread in the eastern and s outhern regions of the U.S., where pure-stand alfalfa production is challenged by climate and/ or soil-type. In general, species mixtures compete with alfalfa plants for available nut rients, light, space, and moisture, and also limit the market value and alfalfa yield ( i.e. , percent alfalfa composition or tonnage). Therefore, an increasing number of forage fiel ds are direct (solo) seeded to exploit the benefits of pure-stand alfalfa.

Alfalfa is a small-seeded crop (220,000 s eeds per pound) and is recommended to be seeded into a fine, firm seedbed that provide s good seed-to-soil contac t (Mueller et al., 2008b). Seeding rates should be between 12 and 15 pounds per acre in the North and East Central regions (Undersander et al., 2011). For irrigated alfalfa, slightly higher seeding rates of 15 to 20 pounds per acre are recommended for drilled seeding and 20 to 25 pounds per acre for broadcast seeding (M ueller et al., 2008b) . Poor seedbed conditions, poor seeding depth control, insuff icient or excessive moisture, poor seed germination, seedling diseases, and inclemen t weather are factors that reduce alfalfa emergence and stand density (Mueller et al., 2008b). Under normal conditions in Wisconsin, only about 60% of the seeds germinate and nearly 60 to 80% of the seedlings die the first year (Undersander et al., 2011). Seed should be inoculated with strains ofMonsanto Company 12-AL-246U 173 of 407 

### alfalfa Rhizobium bacteria when seeding into soils without a recent history (within the past 10 years) of alfalfa (Mueller et al., 2008b). Within four week s after germination,
Rhizobium bacteria create nodules on al falfa roots, allowing the bacteria to fix adequate nitrogen for the life of the alfalfa stand. S eed treatment with a fungicide (metalaxyl) is also recommended to protect the seed from seedling diseas es (Mueller et al., 2008b).

Following the emergence of the cotyledons, the alfalfa plant produces the first unifoliolate leaf and the seedling stem continues to produce alternately arranged

trifoliolate or mutifo liolate leaves as well as secondary stems until it develops into a mature plant (Mueller and Teuber, 2008). Unique to ear ly alfalfa development is contractile growth or the formation of the crown during stand establ ishment. The crown provides protection of the growing points from desiccation, cold, or mechanical damage. Harvesting and other farming practices that might disrupt the formation of the crown should be delayed until this process is complete. When the plant is 8 to 12 inches in height, the alfalfa plant has produced sufficient energy to maintain growth, and root reserves are replenished in preparation for the next forage harvest or for winter survival.

VIII.C.3. Mid- to Late-Season

Alfalfa development is divided into four cat egories and ten growth stages – vegetative stages, bud stages, flowering stages, and s eed development stages (Mueller and Teuber, 2008). There are three vegetative stages (Stage 0, 1, and 2), distinguished by stem length. Flowering stages are early fl ower (Stage 3) and late flower (Stage 4), and are distinguished by one or more nodes with open flowers. Seed development stages are early seed pod (Stage 7), late seed pod (Stage 8), and ripe seed pod (Stage 9), distinguished by the number of nodes with green pods or nodes with mostly brown mature seed pods. With the growth and deve lopment of the alfalfa plant, the proportion of leaves and stems changes. Stems lengthe n and become more fibrous, increasing their total proportion in the forage. Overall forage quality de clines because there is no concomitant increase in leaf pe rcentage, and the forage qualit y of leaves is higher than stems. The stems of alfalfa plants deve lop through vegetative, bud, flower, and seed stages. Numerous stems at various stages of development are typically found on one plant. Therefore, a “Mean Stage of Developm ent” method is utilized to more accurately

relate relative matur ity to forage quality in a fiel d. The Mean Stage method involves examining individual stems and classifying th em according to the staging system outlined in a detailed protocol defi ned by Kalu and Fick (1981).

Alfalfa fields should be monitored routin ely for insect pests and foliar diseases throughout the growing season. Integrated pest management programs involving chemical and cultural methods can significantly reduce insect losses in alfalfa. These programs include correct insect identification, use of economic threshold values, careful monitoring and sampling of insects, and impl ementing control strategies that minimize

effects to natural enemies and other non- target species (Summers et al., 2008). Accelerating or delaying cutting schedules is also an important cultural method of managing some insect pests. Despite the use of cultural cont rol methods, insect pests can reach economic threshold levels requiring th e use of insecticides. Unlike many fieldMonsanto Company 12-AL-246U 174 of 407 

### crops, economic threshold va lues with alfalfa forage production must take into consideration both yield and quality losses.

Management practices need to maximize fora ge yield while achieving a level of quality that meets the nutritional requi rement for the intended use or market. The maturity of alfalfa plants at harvest has the greatest im pact on forage quality. Prior to flowering, forage yield generally increases faster th an quality declines. However, during the flowering period, reduction in qua lity is very rapid due to increased fiber (cellulose and lignin) concentration in the stems.

VIII.C.4. Harvest

Harvest management decisions are critical to forage yield, forage quality, and to the profitability of an alfa lfa crop. Identifying the best harv esting or cutting schedule is one of the most difficult management decisions beca use it involves market considerations in addition to several agronomic factors (O rloff and Putnam, 2008) Several cutting strategies can be considered or implemen ted including harvestin g on a calendar basis, certain growth stage, growing-degree day models, numerical staging, and Predictive Equations for Alfalfa Quality (PEAQ) (Orl off and Putnam, 2008) Each strategy has advantages and disadvantages, as well as different degrees of flexibility, that growers must weigh to determine which strategy is mo st suitable for their farming operation. A combination of cutting strategies may be the best strategy for some farming operations that supply hay to more than one market.

Deciding when to cut alfalfa forage is chal lenging because plant maturity affects yield and quality differently and can affect the life and vigor of the stand. Alfalfa yield and forage quality are almost always inversely rela ted within a growth cycle. Alfalfa yield can double from the pre-bud to full-bloom stage and generally reaches maximum yield at about the 50% bloom stage and then levels off (Orloff and Putnam, 2008). Alfalfa harvested at an immature growth stage (sho rt interval between cuttings) results in relatively low yield but high fo rage quality (Orloff and Pu tnam, 2008). Conversely, the cutting of alfalfa at a mature growth stage (long interval betw een cuttings) results in high yield but low quality forage. The growth stag e to cut alfalfa should reflect the intended use of the hay. The value of alfalfa forage and hay varies considerably by the level of quality or quality grade. In addition to the visual appear ance of alfalfa hay, alfalfa hay quality is defined by a number of nutritiona l traits including acid detergent fiber (i.e., cellulose and lignin content), neutral detergent fiber (i.e., lignin, cellulose and hemicellulose content), crude protein, total dige stible nutrients, and relative feed value(Putnam et al., 2008b). Alfalfa hay intended for the dairy market must be cut early (latebud stage at the latest) (Putnam et al., 2008b) . Beef cows and hor ses usually are fed lower quality alfalfa that can be cut later, at 10 to 30% bloom, to maximize forage yields.

Repeatedly cutting alfalfa plants at immatu re growth stages (pre -bud to bud) shortens

stand life because it does not allow sufficient time for the alfa lfa plants to replenish root reserves (Orloff and Putnam, 2008). Stand loss can lead to the inva sion of weeds that compete for available resources ( e.g. , water, nutrients, light, and space) and negativelyMonsanto Company 12-AL-246U 175 of 407 

### impact forage quality. Additi onally, cutting schedules influe nce the number of harvests possible in a year and influence seasonal yield and costs.

More cuttings per year do not necessarily equate to higher total production per year. University of California studi es have shown the opposite to be true (Orloff and Putnam, 2008). nine to ten times at pre-bud (harvest interval of 21 days) re sulted in a 3-year average yield of 7.5 tons per acre in the Ce ntral Valley. In compar ison, harvesting at full bloom (harvest interval of 37 days) with a tota l of five to six cuttings per year yielded an average of 11.6 tons per acre. Most Califor nia growers harvest during early to late-bud stage to obtain forage with lower lignin cont ent, because most of their alfalfa production is targeted for the dairy industry (Orlo ff and Putnam, 2008). The most common cutting

schedule in the Low Desert a nd Mediterranean regions of Ca lifornia is 28 days and as often as 21-24 days where high quality is the objective (Orloff and Putnam, 2008). To produce high quality hay in the North Central region, a 28- to 33-day interval between the first and second cutting is recommended an d a 30- to 35-day ha rvest interval is recommended for the remaining cuttings (Undersander et al., 2011).

Pest management decisions may impact cut ting schedules. Harves ting early can provide effective management of alfalfa weevil in the spring and worm management in the summer (Summers et al., 2008). Also, longer ha rvest intervals can en able the alfalfa crop to compete more vigorously with weeds.

When cutting alfalfa, university alfalfa special ists recommend leaving no more than 2-4 inches of stubble height (Orloff and Putn am, 2008). Studies have shown that cutting above two inches results in a yield reducti on of 0.5 ton per acre pe r year per inch of additional cutting height. However, raising the cutting height does increase forage quality.

High alfalfa forage yields and stand life depe nd on minimizing winter injury, particularly

in the northern states of the North and East Central regions. Fall management of alfalfa involves assessing the risk of winter injury and the need for additional forage. Snow cover, temperature, and moisture are severa l uncontrollable environmental factors that impact the risk of winter injury (Undersa nder et al., 2011). Extended periods of cool temperatures are required in the fall for alfalfa to develop resistance to cold temperatures. Winter-hardy alfalfa varieties can be inju red or killed by two weeks or more of temperatures below 5 to 15°F. Snow cover of 6 inches or more can protect alfalfa plants from these low temperatures. Warm fall we ather (40°F or higher) and midwinter thaws cause alfalfa to break dormancy and have le ss resistance to freezing temperatures. However, growers are able to utilize severa l management options to reduce the risk of winter injury. Maintaining good soil fertility is very important to maintaining productive stands and good winter survival (Undersander et al., 2011). Cuttings should be avoided during the critical fall period 6 weeks before the first killing frost, especially when previous harvest intervals are 35 days or less. This allows plants to enter the winter with higher root carbohydrates. Late-fall cuttings (October) should be made at a height of 6

inches to catch snow and insulate the soil. The decision to make a late-fall cutting needs to weigh the risk of winter injury against the need for additional forage.Monsanto Company 12-AL-246U 176 of 407 

### Harvesting hay is a four step process: 1) cutting the forage , 2) raking the partially cured hay into windrows, 3) baling the dry hay, an d 4) storing the hay (Orloff and Mueller, 2008). The moisture content of alfalfa grow ing in the field is generally about 80% (Undersander et al., 2011). Solu ble sugars and proteins are disso lved in the forage liquid. When forage is dried to hay be fore being baled, water in th e forage evaporates, resulting in a higher concentration of nutrients in the remaining liquid where cell growth and enzyme activity are restricted. The objective during the harvesting process is to accelerate the drying rate and minimize losses in dry matter. The drying rate, mechanical handling of the forage, and the moisture content at baling all affect th e quality of the hay. Rapid drying is important to minimize qua lity losses caused by bleaching, respiration, leaf loss, and rain damage (Orloff and Mu eller, 2008). Weather conditions can make harvesting hay a real challenge. Rainy weather causes delays in harvest which increases the neutral detergent fiber (NDF) and aci d detergent fiber (ADF) and decreases digestibility and crude protein of the hay (U ndersander et al., 2011). In addition, rain on hay before baling leaches soluble nutrients (protein and carbohydrates ). The following management practices are re commended for harvesting ha y to accelerate drying and minimize losses: 1) cut the forage early in th e day, 2) rake into a wide swath or windrow, 3) rake at 40 to 50% moisture, 4) bale hay at 18 to 20% moisture content, and 5) store hay under cover (Undersander et al., 2011).

Mechanical conditioning or crimping the alfa lfa during the cutting op eration is a widely accepted practice to accelerate curing (Orl off and Mueller, 2008). The purpose of crimping is to facilitate water loss from the st ems, making the drying rate of stems more

similar with that of the leaves. This can hasten the drying proce ss by as much as 30% (Orloff and Mueller, 2008). Ch emical conditioning or use of drying agents can also be used to speed the drying process. Drying ag ents such as potassium carbonate or sodium carbonate sprayed on the forage at cutting can shorten drying time by 5 to 24 hours (Undersander et al., 2011). Drying agents are most effective when the weather is warm and sunny. They provide little to no benefit during poor curing conditions. If rain falls on treated hay, the hay reabsorbs water more readily than untreated hay. For these reasons and others, university alfalfa special ists do not believe drying agents are cost

effective.

Preservatives are used to a limited degree to a llow storage of alfalfa hay baled at moisture contents higher than would ordinarily be c onsidered safe. Hay ba led and stored at too high a moisture content without a preserva tive is subject to problems with mold, discoloration, and even spontaneous com bustion (Orloff and Mueller, 2008). Hay preservatives are organic acids, primarily pr opionic acid or propionic-acetic acid blends that prevent mold growth and heating losses by lowering the pH and retarding the growth of microorganisms that cause hay spoilage. Preservatives are used by some growers in the North Central region but are not considered cost effective in the western states (Orloff and Mueller, 2008).

VIII.D. Management of Insect Pests

Many insects are present in alfalfa, but fewer than 20 cause injury, and fewer insects are considered serious pests (Summers et al., 2008). However, those injury-causing insectMonsanto Company 12-AL-246U 177 of 407 

### pests can cause substantial yield and quality losses in alfalfa when present in high numbers. Alfalfa weevil ( Hypera postica ) and Egyptian alfalfa weevil ( Hypera brunneipennis ) routinely cause damage annually in established alfalfa throughout the U.S. Damage and yield losses are more spor adic and less frequent with other insect pests. Integrated pest management progr ams involving chemical and cultural methods can significantly reduce insect losses in alfa lfa. These programs include correct insect identification, use of economic threshold va lues, careful monitori ng and sampling of insects, and implementing control strategies that minimize effects to natural enemies and other nontarget species (Summe rs et al., 2008). Selection of resistant varieties is an effective element in the management of several alfalfa pests. However, even with highly resistant varieties only a mini mum of 50% of the plant popula tion are resistant (Summers et al., 2008). Accelerating or delaying cutti ng schedules is also an important cultural method of managing some insect pests. In irrigated alfalfa area s, border-strips are utilized to serve as refuge for natural enem ies and to retain lygus bugs in the alfalfa where they do little harm and keeps them out of neighboring crops (Summers et al., 2008). Despite the use of cultural control methods, insect pests can reach economic threshold levels requiring the use of insecticides. Unlike many field crops, economic threshold values with alfalfa forage producti on must take into cons ideration both yield and quality losses.

As indicated previously, alfalfa weevil is one of the most serious pests in alfalfa. The

larvae feed on the terminal buds and upper leaves and unde r severe infestations can completely defoliate the plant. Additionally , the adult weevils feed on alfalfa stems. Damage is usually greatest on the first cu tting, although second and third generations of weevil larvae can cause damage in subsequent harvests (Kansas State University, 1998; Summers et al., 2008). Both the larvae and adult forms can suppress yields by delaying regrowth. Insecticides and early harvest are the main control options for alfalfa weevil. Varieties with sufficient resistance to heavy infestations are currently not available. General biological control agents are not eff ective, although several parasitic wasps are effective in some growing regions to s uppress weevil population buildup (Kansas State University, 1998). However, an alfalfa weev il-specific fungus aids in biological control (Summers et al., 2008).

Among species of aphid, several are important insect pests that can damage alfalfa, including pea aphid ( Acyrthosiphon pisum ), blue alfalfa aphid ( A. kondoi ), spotted alfalfa aphid ( Therioaphis maculate ), and cowpea aphid ( Aphis craccivora ) (Summers et al., 2008). Some Lepidoptera insects are also important pests, including armyworm ( Spodoptera exigua

, S. praefica ), alfalfa caterpillar ( Colias eurytheme ), and leafhoppers ( Empoasca spp.) (Summers et al., 2008). Of occasional importance are cutworms ( Agrotic subterranea

, Peridroma sausia ), alfalfa webworm ( Loxostege cereralis ), alfalfa looper ( Autographa californica ), clover root curculio ( Sitona hispidulus ), ground mealybug ( Rhizoecus kondonis ), spider mites ( Tetranychus spp.), silverleaf whitefly ( Bermisia argentifolii ), grasshoppers ( Melanoplus spp. and Trimerotropis spp .), Threecornered alfalfa hopper ( Spissistilus festinus ), blister beetles ( Epicauta spp., Lytta spp., Tegrodera spp.), and thrips ( Frankliniella spp., Caliothrips phaseoli ) (Summers et al., 2008).Monsanto Company 12-AL-246U 178 of 407 

### The phenotypic, agronomic, and environmenta l interaction assessments presented in

Section VII have demonstrat ed that KK179 is not mean ingfully different from conventional alfalfa. Therefore, if de regulated, KK179 would require the same management practices for insect pests as conventional alfalfa.

VIII.E. Management of Diseases and Other Pests

Diseases can kill alfalfa seedlings, reduce stand life, cause yield reduction, and reduce the feeding value of the forage (Frate and Davis, 2008; Undersande r et al., 2011). The occurrence and severity of diseases depends on environmental conditions, soil type, and crop management (Undersander et al., 2011) . Temperature and moisture are the environmental factors affecting the occurrence and severity of diseases. Pathogens that

cause alfalfa diseases include fungi, bacteria, viruses, and ne matodes. Selecting diseaseresistant varieties is one of the most effective methods to managing many of the alfalfa diseases. Other integrated strategies and tec hniques utilized to manage alfalfa diseases include irrigation management, planting met hods, promotion of crop vigor, manipulation of cutting schedules, canopy management, and crop rotation (Frate and Davis, 2008).

The first threat to alfalfa comes from the seedling diseases or damping-off caused by soilborne fungi, including Pythium spp. Rhizoctonia spp., and Phytophora spp. (Frate and Davis, 2008). Their development is favored by excessive soil moisture, compacted or poorly drained soils, and adve rse temperatures for seedling growth. Seedling diseases can be devastating to the long-term producti vity and life of a new alfalfa seeding.

Cultural and chemical control measures ar e generally effective in managing seedling

diseases.

Foliar diseases reduce photosynthesis and defolia te alfalfa plants subsequently decreasing yield and forage quality (Frate and Davis, 2008). Some impor tant foliar diseases include common leaf spot ( Pseudopeziza medicaginis ), downy mildew ( Peronospora trifoliorum ), spring and summer blackstem ( Phoma medicaginis and Cercospora medicaginis ), Stagonospora leaf spot ( Stagonospora meliloti ), Stemphylium leaf spot ( Stemphylium botryosum ), and rust ( Uromyces striatus ) (Frate and Davis, 2008; Undersander et al., 2011). Various cultural te chniques are usually utilized to manage the

foliar diseases.

Root, stem, and crown diseases are less obviou s but can cause just as much damage to alfalfa stands and yields. Anthracnose ( Colletotrichum trifoii ), Aphanomyces root rot ( Aphanomyces euteiches ), Phytophthora root rot ( Phytophthora megasperma ), Rhizoctonia root canker and crown rot ( Rhizoctonia solani ), Sclerotinia stem and crown rot ( Sclerotinia tifoliorum

, S. sclerotiorum ), and Stagonospora crown and root rot ( Stagonospora meliloti ) are important diseases in this group with Phytophthora root rot being one of the most common diseases in al falfa (Frate and Davis, 2008; Undersander et al., 2011). the exception of Rhiz octinia root canker and crown rot, resistant varieties are

available and effective for the other diseases in this group.

Bacterial wilt ( Clavibacter michiganensis ), fusarium wilt ( Fusarium oxysporum ), and verticillium wilt ( Verticillium alfo-altrum ) are among the most im portant wilt diseasesMonsanto Company 12-AL-246U 179 of 407 

### (Frate and Davis, 2008). The pathogens for w ilt diseases invade the vascular system of plants which leads to the plant wilting and us ually dying. Resistant varieties are the first line of defense for the diseases in this group.

Parasitic nematodes are microscopic, unseg mented roundworms that reduce yields and

cause considerable economic losses in alfalf a (Westerdahl and Frate, 2008). The stem nematode ( Ditylenchus dipsaci ), root-knot nematode ( Trichodorus spp .), and the rootlesion nematode ( Pratylenchus penetrans ) are the most important nematodes consistently associated with damage to alfalfa. Nemat odes feed on root hairs, feeder roots, and nitrogen-fixing nodules of alfalfa and do not typically kill plants (Undersander et al., 2011; Westerdahl and Frate, 2008). They stre ss the plants and act in conjunction with

other stress factors in the fiel d to reduce growth and yield. The key practices to manage nematodes include site selection, crop rotati on, variety selection, ce rtified seed, fallow, chemical nematicides, and other management techniques that prevent nematodes from spreading, such as using clean equipment a nd irrigation water (Westerdahl and Frate, 2008).

The phenotypic, agronomic, and environmenta l interaction assessments presented in

Section VII have demonstrat ed that KK179 is not mean ingfully different from conventional alfalfa. Therefore, if de regulated, KK179 would require the same management practices for diseases and othe r pests as for conventional alfalfa.

VIII.F. Weed Management

Annual and perennial weeds reduce alfalfa yi eld and quality and cause serious economic losses in alfalfa because they compete for th e same resources required for alfalfa growth and development: water, nutrients, light, and space (Canevari et al., 2008). Weed competition in alfalfa occurs in two distinct time periods: seedling establishment and in established stands. Seedling alfalfa plants gr ow slowly and compete poorly with weeds. Forage yield losses due to the presence of w eeds in new stands of alfalfa often exceed 1000 pounds per acre (Caddel et al., 2011). In many incidences, the overall tonnage of forage may be the same or actually higher wh en weeds are left uncont rolled (Canevari et al., 2008). However, the feeding value or nut ritional value of the hay is drastically reduced. Weeds affect quality because most weeds are much lower in protein, higher in fiber, and are generally less palatable and less nutritious than alfalfa. In a California study, protein content was as lo w as 9% in hay that contai ned 80% weeds (Canevari et al., 2008). When weeds were controlled with herbicides, the protein content was over 20%. Reductions in forage quality also depend on the w eed species present. Annual grasses have a significant impact on quality because they have high fiber content and decrease livestock intake (Unde rsander et al., 2011). Annual broadleaf weeds, such as curly dock, hoary alyssum, and yellow rocket are unpalatable and decrea se animal intake. In addition, hay containing foxtail ( Setaria spp .) and wild barley ( Hordeum spp .) can cause livestock to develop serious mouth and throat ulcerations (Canevari et al., 2008).

Some weeds can contribute “off” flavors in milk, and other weeds contain alkaloids that are toxic to livestock. Certain weed species can have a more direct impact on alfalfa production. For example, quackgrass and Cana da thistle release toxins or growth-Monsanto Company 12-AL-246U 180 of 407 

### inhibiting compounds upon decomposition, which can affect alfalfa growth and yield (Kommedahl et al., 1959; Wilson, 1981).

Annual, biennial, and perennial weeds are problematic during seedling establishment and

in established stands. Common weeds found in alfalfa in the U.S. ar e presented in Table VIII-6. Tillage is an important part of a weed management program when establishing alfalfa. Tillage controls emerged annual w eeds and sets back perennial weeds prior to planting. The absence of tillage during the production years naturally favors invasion by perennial weeds (Undersander et al., 2011). Controlling certain perennial weeds ( e.g. , nutsedge) may be managed more effectively in other crops of the rotation sequence preceding alfalfa establishment. In the Midwest, alfalfa is often planted with a companion crop for erosion control, but this practice also helps suppress weeds while the alfalfa is becoming establishe d (Undersander et al., 2011). Practices that encourage alfalfa germination and vigorous seedling grow th are effective for weed management. A dense, vigorous alfalfa stand is an effectiv e weed management strategy for establishing alfalfa and extending the life of the stand. Additional details on weed control when applying Roundup ® 

### agricultural herbicides to Roundup Ready alfalfa can be found in

Section VIII.F.1. Details on weed contro l throughout this sec tion are focused on conventional weed control programs.

® Roundup is a registered tradem ark of Monsanto Technology LLC.Monsanto Company 12-AL-246U 181 of 407 

### Table VIII-6. Common Weeds Present in Alfalfa in the U.S 1



### . Annuals

Annual bluegrass

Annual sowthistle Barnyardgrass

Blessed milk thistle Bristly oxtongue

California burclover Cheeseweed ( Malva spp.)

Coast feddleneck

Cocklebur

Common chickweed Common groundsel Common lambsquarters Common purslane Common ragweed Creeping swinecress

Dodder Field pennycress

Fillarees Foxtail, yellow and

green Henbit Hoary alyssum Italian ryegrass

Kochia

London rocket

Miner’s lettuce

Mustards Night-flowering catchfly Nightshades Persian speedwell Pigweed spp. Prickly lettuce

Prostrate knotweed Roughseed buttercup Shepherds’s purse

Toad rush

Velvetleaf Virginia pepperweed

Volunteer cereals Wild celery

Wild oats

Yellow starthistle

Biennials

Buckhorn plantain Mexican tea Spotted knapweed

Perennials

Bermudagrass Curly dock Dallisgrass

Dandelion

Field bindweed Hemp dogbane Johnsongrass Nutsedge Orange hawkweed Quackgrass White cockle Wirestem muhly

Yellow rocket

1 Source:(Canevari et al., 2008 ; Undersander et al., 2011).Monsanto Company 12-AL-246U 182 of 407 

### There are currently 16 herbicides registered for use in conven tional alfalfa. According to an extensive survey conducted by the USDA from 1988 to 1992, herbicides are used on approximately 17% of all alfalfa acreage (s eedling establishment a nd established stands) grown for forage (USDA-APHIS, 2010). Herb icides are used more extensively in the West (50%) compared to the Northeast (22%) and North Central (8%) areas of the U.S. (Hower et al., 1999). This may be due to th e more arid environment in the West, in which weed competition has a greater economic impact. At the time of alfalfa establishment, total herbicide-treated acres were 1.1 million acres in spring-seeded alfalfa and 0.4 million acres in fall-seeded alfalfa (H ower et al., 1999). However, since more than one herbicide and more than one appli cation were made on some acres, it is not possible to accurately calculate the percentage of spring- or fall-seeded alfalfa acres

treated with herbicides. Mechanical and cultural weed control methods ( e.g. , tillage and companion crops) are used for approximately 80% of the spring-seeded alfalfa and 18% of the fall-seeded alfalfa (U SDA-APHIS, 2010). Companion cr ops were utilized on 49% and 3% of the spring- and fall -seeded al falfa, respectively (Hower et al., 1999).

There are several herbicides available fo r effective weed c ontrol during seedling establishment, including 2,4- DB, benefin, bromoxinil, clet hodim, hexazinone, imazamox, imazethapyr, sethoxydim, paraquat, and pronami de (USDA-APHIS, 2010). Table VIII-7 provides a summary of the crop tolerance an d effectiveness ratings of herbicides on

common weeds in direct-seeded alfalfa. The effectiveness and spectrum of weed species

controlled with each of these herbicides varies; where rarely does one herbicide control all the weed problems in alfalfa in a particular area. In addi tion, the restric tions on alfalfa stage of growth for application and interval between application and harvest varies with each herbicide, making the selection of herbicide(s) more complex.

Alfalfa stands generally remain in produc tion from three to six years or more. Eventually, weeds invade the alfalfa stand as the stand growth slows and stand density declines due to poor soil fe rtility, disease, insect pr oblems, and winter injury (Undersander et al., 2011). The decision to apply an herbicide to remove undesirable weeds in established alfalfa will depend on th e weed density, weed species, stand density, and intended use of the hay (U ndersander et al., 2011). It is important to note that alfalfa does not spread into the open areas previous ly occupied by weeds. Approximately 5.1 million herbicide-treated acres of established alfalfa were treated annually with an herbicide during 1988 to 1992 in the U.S. (Hower et al., 1999). This low level of treated acres may be the result of the existen ce of alfalfa-grass mixtures , the low economic return from current herbicides, the ineffectivene ss of past herbicide programs, the use of

alternate weed control methods such as growing a companion crop, or cultural practices.

Several herbicides can be used in establ ished stands of alfalfa including: 2,4-DB, clethodim, diuron, EPTC, hexazinone, metribuz in, norfluzaon, paraquat, pendimethalin, sethoxydim, terbacil, and trif luralin (USDA-APHIS, 2010). Table VIII-8 provides a summary of the crop tolerance and effectiven ess ratings of herbic ides on common weeds

in established stands of alfalfa. The spectrum of weed species controlled varies significantly among these herbicides. In additio n, some of the herbicides are applied for residual control, some control emerged weeds, and some have residual andMonsanto Company 12-AL-246U 183 of 407 

### postemergence activity. The rotation restrictions with certain herbicides are an important consideration in the selection process.

The phenotypic, agronomic, and environmenta l interaction assessments presented in

Section VII have demonstrat ed that KK179 is not mean ingfully different from

conventional alfalfa. Theref ore, if deregulated, KK179 w ould require the same weed management practices as conventional alfalfa.Monsanto Company 12-AL-246U 184 of 407Table VIII-7. Alfalfa Tolerance and Herbicide Effectiveness Ratings on Direct Seedlings of Alfalfa1PreplantIncorporatedHerbicidesPostemergence HerbicidesEPTC Trifluralin Bromoxynil2,4-DB Glyphosate Sethoxydim Pendimethalin Imazethapyr Imazamox ClethodimAlfalfa ToleranceF/G G F/G G E2E E G G EGrassesBarnyardgrassG/E G/E P N E G/E G/E G G G/EFoxtailsG/E G/E P N E E G/E G G/E EQuackgrassP/F P N N G/E F/G P P P/F GBroadleavesC. chickweed F P P P E N F/G G G NC. lambsquartersFF/GG/EG/EG/ENG/EF/GGNC. ragweedF P G/E G/E E N P F F/G NEastern Blacknightshade F P G/E F E N P E E NPennycressP P G/E F E N P G/E G/E NPigweed spp.F/G G/E F G/E E N G/E E E NShepherd’spurse P P E F/G E N G G/E G/E NSmartweed spp.P P G P G/E N P G G NVelvetleafF/G N G G/E G/E N N G/E G/E NWild mustardP/F P G F/G E N N G/E G/E NWild radishP/F N G F E N N G G NHerbicide effectiveness ratings: E = excellent;G = good; F = fair; P = poor; N = no control.1Source: (Cullen et al., 2012).2Crop tolerance rating is for postemergence applications of glyphosate inRoundup Ready alfalfa varieties.Monsanto Company 12-AL-246U 185 of 407Table VIII-8. Alfalfa Tolerance and Postemergence Herbicide Effectiveness Ratings on Common Weeds in Established AlfalfaStands1.2,4-DB FlumioxazinGlyphosate2Metribuzin Sethox-ydimPendi-methalinImazeth-apyrImazamox Clethodim Hexazi-doneAlfalfa ToleranceG G/E E2F/G E E G G E GAnnualsCommon chickweedP E E E N P G G N EField pennycress F/G E E G/E N P E E N G/EFoxtails spp. N G/E E F E G/E F/G G/E E FShepherd’s purse F/G E E E N F/G G/E G/E N EPerennialsBroadleaf plantainF/G - G/E P N P F F N F/GCanada thistleN P G P N P P P/F N NC. dandelionP P G G/E N P P P/F N G/ECurly dockP/F P - F N P P/F P/F N FHemp dogbaneN - E P N P P - N NHoary alyssumF P3- F/G N P F F N GPerennial sowthistleN - E P N - G G N PQuackgrassN P G/E F/G F/G P P P/F G F/GWhite cockleP P3F/G G N P P P N FWirestem muhlyN - E P F/G - P P F/G FYellow nutsedgeN P F F N P P/F F N FYellow rocketP P3G/E G N P F/G F/G N G/EHerbicide effectiveness ratings: E = excellent; G = good; F = fair; P = poor; N = no control; - = no data.1Source: (Cullen et al., 2012).2Crop tolerance rating is for postemergence applicationsof glyphosate in Roundup Ready alfalfa varieties.3Provides excellent control of seedlings.Monsanto Company 12-AL-246U 186 of 407 

### VIII.F.1 Weed Management in Roundup Ready Alfalfa

Roundup Ready alfalfa treated with postemergen ce applications of Roundup agricultural herbicide products provides alfalfa growers broad spectrum weed control and excellent crop safety for seedling establishment and established stands. Roundup Ready alfalfa growers can experience more effective control of problem weeds in alfalfa with greater application flexibility and simplicity compared to conventional weed management options. Tables VIII-7 and VIII-8 provi de a summary of the crop tolerance and effectiveness ratings of se veral herbicides, including glyphosate, on co mmon weeds in

direct-seeded and established alfalfa stands . Glyphosate is rated excellent for Roundup Ready alfalfa tolerance, while several other herbicides are on ly rated good or fair to good for alfalfa tolerance. Glyphos ate is rated good to excell ent on almost all the common

weeds listed in direct seedi ngs and established stands of alfalfa. Postemergence application of glyphosate provid es improved control of some of the most difficult to control perennial weeds (bermudagrass, nutsedge, johnsongrass, and dandelion) (Canevari et al., 2008). It is expected that adequate control of these tough perennials could add years to alfalfa stand life (Canevari et al., 2008).

Roundup agricultural herbicide pr oducts can be applied pre- plant, at planting, and preemergence during alfalfa seedling establishment and in-crop postemergence to established stands. In-crop postemergence applications can be made to Roundup Ready alfalfa from emergence of alfalfa until five days prior to cutting, which will not limit weed management decisions. Single in-crop applications must not exceed 1.54 lbs a.e. (acid equivalent) of glyphosate per acre and the combined total per year for in-crop applications on newly established and establishe d stands must not exceed 4.64 lbs a.e. per acre. The combined total per year for all applications, incl uding preplant during the year of establishment, must not exceed 6.0 lbs a.e. of glyphosate (Mons anto Company, 2012).

Since glyphosate controls only emerged weeds w ith no residual control, it is important to apply Roundup agricultural herbic ide products after most of the weeds have emerged or when there is enough crop canopy to outcompete late-emerging weeds. Generally, the best time for making applications in seedling alfalfa is when the alfalfa is between the 3- and 6-trifoliate leaf stages (Canevari et al., 2008). Ea rlier applications may allow subsequent weed emergence. With later applications, weeds may be too large for adequate control and can result in heavy weed infestations, such that alfalfa stand density or vigor may be affected before herbicide application. A diverse weed control system that incorporates cultural methods as well as rotations or tank mi xes of glyphosate with

other mode-of-action herbicides can minimize th e risk of weed species shifts or weeds developing resistance to glyphosat e (Canevari et al., 2008).

VIII.G. Alfalfa Stand Termin ation and Volunteer Management

The productivity of alfalfa fields declines over time due to loss of plants and weakening of crowns from diseases, insect pests, weed competition, equipment traffic, soil compaction, and other factors (Canevari and Pu tnam, 2008). Eventually, the forage yield and quality decline to the point that the sta nd must be removed and the field rotated to another crop. In some cases, it may be ec onomically feasible to extend the life of theMonsanto Company 12-AL-246U 187 of 407 

### stand by overseeding with grasses and other legu mes. However, there must be a potential market for the alfalfa-grass mixture. The pl ant density as well as the health, size, and regrowth potential of the alfalfa plants are factors determining stand viability (Canevari and Putnam, 2008). When the plant density falls below four to six plants per square foot, yields begin to decline. However, stem densities may be more important than plant density. Growers should consider stand removal when stem densities fall below approximately 39 stems per square foot, or when yields fall 25% below normal and weeds become a factor (Can evari and Putnam, 2008).

Alfalfa stand removal or termination is ach ieved in the fall through the use of deep tillage, herbicides, or both. Since normal tillage operations alone often do not provide complete termination, herbicides are commonly used to effectively control alfalfa. For most effective control with herbicides, term ination should be done in the fall when the alfalfa plants have sufficient vegetative growth and the herbicides are more effective. Fall applications should be made after the last cutting to at least 6 to 8 inches alfalfa regrowth and before the first killing frost (Coulter, 2010). To terminate seed production fields, multiple tillage operations are used, followed by irrigation to induce germination of dropped seed and the decomposition of the k illed plants. Herbicides in combination with shallow tillage may be used to kill the seed parent plants and recently germinated seedlings.

Volunteer alfalfa plants can arise from viable alfalfa crowns in terminated alfalfa forage production fields. In former seed productio n fields, volunteers may be either from surviving or newly germinated plants. Grow ers currently use a combination of tillage

and herbicides for volunteer control.

Several herbicides are labeled for control of alfalfa, including: 2,4-D, chlorpyralid, dicamba, carfentrazone, flumioxazin, and gluf osinate. Glphosate only provides partial

control of conventional alfa lfa and is not recommended for volunteer management. Research has demonstrated that 2,4-D, chlo rpyralid, dicamba, or a combination of the 2,4-D and dicamba provide excellent control of alfalfa (Coulter, 2010; Dillehay and Curran, 2006; Mayerle, 2002). Table VIII-9 lists the herbicides labeled for preplant and in-crop applications that can be used for st and termination and to control volunteer alfalfa in crops that immediately follow alfalfa in the rotation. In certain rotational crops ( i.e.

, potato, tomato), herbicides are available fo r stand termination or volunteer control preplant to the crop, but no herbicides are la beled for in-crop control of volunteers. In these rotational crops, tillage and hand weeding are used for in-crop control of volunteer

alfalfa.

The phenotypic, agronomic, and environmenta l interaction assessments presented in

Section VII have demonstrat ed that KK179 is not mean ingfully different from conventional alfalfa. Therefore, if deregul ated, stand terminati on and volunteer control

of KK179 would use the same mana gement practices as utilized for conventional alfalfa.Monsanto Company 12-AL-246U 188 of 407 

### Table VIII-9. Herbicides for Alfalfa Sta nd Termination and Control of Volunteer

Alfalfa 1



### .

Crop/Product Rate/Acre (lbs a.e or a.i.) Preplant/Pre In-Crop

Corn

2,4-D 1.0 

## √ √

Dicamba 0.5 

## √ √ 2,4-D/Dicamba 0.5/0.5 

## √ √ Chlorpyrali d 0.094-0.188 

## √ √

Cotton

Dicamba 0.5 fl oz 

## √

- Carfentrazone 0.004-0.008 

## √ √

Flumioxazin 0.06 

## √

- Trifloxysulfuron 0.01 - 

## √

Small Grains
2,4-D 1.0 

## √ √

Dicamba 0.5 

## √ √

2,4-D/Chlorpyralid 0.75 + 0.07 

## √ √

Potato
2,4-D 1.0 

## √

- Dicamba 0.5 

## √

- 2,4-D/Dicamba 0.5/0.5 

## √

-

Sugarbeets Chlorpyralid 0.094-0.188 

## √ √

Glufosinate 0.42 

## √

-

Tomato
2,4-D 1.0 

## √

- Dicamba 0.5 

## √

- 2,4-D/Dicamba 0.5/0.5 

## √

-

1 Sources: (CDMS, 2011; Coulter, 2010; Dillehay a nd Curran, 2006; Rogan and Fitzpatrick, 2004; USDA-APHIS, 2010; Van Deynze et al., 2004). 

## √ Indicates that herbicide provides control of volunteer alfalfa. - Indicates herbicide does not provide control or is not labeled for the identified application.Monsanto Company 12-AL-246U 189 of 407 

### VIII.H. Management of Feral Alfalfa

Alfalfa has occasionally become feral, or naturalized, by establishment outside of agricultural fields or intentionally planted in non-agricultural lo cations (USDA-APHIS, 2010). The Medicago sativa subspecies sativa (purple-flowered alfalfa used in cultivation) has naturalize d populations in all 50 states (USDA-APHIS, 2010). M. sativa subspecies falcata (yellow-flowered or Siberian alfalf a) is naturalized in the northern and western states and is being pr omoted as a rangeland enhancer for grazing. Like alfalfa under cultivation, feral al falfa originated from introduced va rieties. Feral alfalfa can be found in air fields, canals, cemeteries, d itch banks, fence rows, highways, irrigation ditches, pipelines, railroads, rangeland, right s-of-way, roadsides, wasteland, preserves, parks, and recovery areas. Fe ral populations exist near locati ons used for alfalfa seed and forage production (USD A-APHIS, 2010).

In the event of the trait for reduced G li gnin and total lignin be ing transferred from KK179 to feral populations by cross-pollina tion, no environmental consequences are expected to occur. Furthermore, evaluati ons have shown that the introduced trait for reduced G lignin and total lignin in KK179 do es not enhance weediness or plant pest potential relative to conventional alfalfa. Also, Monsanto and FGI are not aware of a conceivable mechanism by which the trait coul d confer a selective advantage or to be selected for in an unmanaged setting.

In general, alfalfa is not a species specifi cally targeted for weed control in unmanaged areas or in roadsides. In fact, alfalfa ma y be considered a desirable species along some

roadsides in South Dakota and Wisconsin and is encouraged to grow. In situations where control is desired, feral alfalfa can be c ontrolled or discouraged using physical or chemical methods. Herbicides, such as 2,4-D, dicamba, or a combination of these herbicides, effectively contro l feral alfalfa, including Roundup Ready and KK179 alfalfa, in most sites. Glyphosate is not generally the herbicide of choice to control feral alfalfa, as discussed in Section VIII.G, because it is not highly effect ive on conventional alfalfa.

The phenotypic, agronomic, and environmenta l interaction assessments presented in

Section VII have demonstrat ed that KK179 is not mean ingfully different from

conventional alfalfa. Theref ore, if deregulated, control of KK179 would use the same physical and herbicide management practices as utilized for conve ntional alfalfa.Monsanto Company 12-AL-246U 190 of 407 

### VIII.I. Crop Rotation Practices in Alfalfa

The rotation of alfalfa with other crops is an integral part of a farm management program to maintain soil productivity, reduce soil er osion, avoid pathogen and pest buildup, and adjust to market conditions (USDA-APHI S, 2010). Alfalfa provides greater water infiltration, improves soil tilth, and provides nitrogen for subsequent crops in rotation (Canevari and Putnam, 2008; Orloff and Putnam , 1997). The extensive root system of alfalfa improves soil tilth and soil structure by creating ch annels that encourage water penetration and biological activity in the root zone. Considerable organic matter is added

to the soil over the life of the stand whic h greatly benefits the growth and yield of subsequent crops, such as corn, tomato, wheat, or specialty cr ops. Alfalfa fixes atmospheric nitrogen through the symbiotic relationship with Rhizobium bacteria which can provide from 40 to 60 pounds of nitrogen pe r acre to crops that follow alfalfa (Orloff, 2008). In turn, rotations with other crops be nefit alfalfa by breaki ng disease and insect cycles and improving weed control and soil fertility (Orloff, 2008; Orloff and Putnam, 1997).

Alfalfa is rotated most often with corn, cotton, wheat, oats, barley, sugar beets, and tomatoes (Putnam et al., 2008c; USDA-APHIS, 2010). Alfalfa-to-alfalfa rotations are uncommon because of the potential for autotoxicity and the ine fficient use of residual soil nitrogen credits. Approximatel y 18% of existing alfa lfa stands are terminated and rotated to a different crop each year (Hower et al., 1999). Current crop rotation options or patterns in the U.S. where alfalfa is grown are expected to remain the same upon the

introduction of KK179.

VIII.J. Impact of the Introduction of KK179 on Agricultural Practices

Lignin plays an important role in terms of providing strength to plants (Undersander et al., 2009). and allowing water to move up th e plant stem without leakage. As a component of plant cell walls, lignin molecu les bind with hemicellulose and fill the spaces between cellulose, hemicellulose, and pec tins. Lignin increases as plants mature and makes up the major portion of fiber in alfalfa forage (Undersander et al., 2009). However, lignin is indigestible and as a result, has a significant impact on the overall quality of alfalfa. It reduces digestion of the cellulose in the rumen of livestock. Therefore, forage and livestock producers desi re alfalfa forage with lower lignin values as well as high protein content.

KK179 alfalfa is a biotechnology-derived alfalf a with an intended re duction in G lignin and total lignin compared to conventional alfalf a at the same stage of growth. Because of its lower lignin content, KK179 will provide gr owers with production benefits, including: 1) greater flexibility in harvesting schedules and 2) potentially lower production costs.

Growers will have the flexibility at each fo rage harvest to choose one of two production strategies to improve the value of alfalfa production on their farm. They can maximize forage quality while maintaining yield, or maximize yield while maintaining forage quality, depending on plant grow th stage at harvest. When implementing the first production strategy (maximizing forage qual ity while maintaining yield), the cuttingMonsanto Company 12-AL-246U 191 of 407 

### schedules and the timing of harvest remains the same as is used with conventional varieties. For example, if the grower wa s producing high quality alfalfa hay for dairy cows, the grower would typically harvest alfa lfa at the earl y to late bud stage. KK179 harvested at this crop stage will provide alfalfa forage with lower levels of lignin compared to conventional alfalfa. As a result, the quality of the forage is more likely to meet or exceed the intended quality standard targeted by the grower.

The second production strategy (maximizing yield while maintaining forage quality) involves delaying harvest to maximize yield wi thout forfeiting forage quality compared

to conventional alfalfa. KK179 can be harv ested later and still produce high quality alfalfa forage that is comparable to earlier harvest timings with conventional alfalfa. However, the forage tonnage or yield from the cutting will likely be higher with KK179. Alfalfa forage yields during th is plant growth stage can incr ease at the rate of 200 pounds per acre per day (Undersander et al., 2009). Therefore, delaying harvest can result in significant increases in forage yield. A dditionally, when rain or adverse weather conditions coincide with a planned harvest in terval, growers now will have the flexibility to delay harvest. This will allow growers to postpone harvest to when more favorable weather conditions occur, while still mainta ining acceptable forage quality at the later harvest timing.

A delayed harvest schedule, with its longer harvest intervals, will potentially lower production costs over the life of the KK179 sta nd. A delayed harvest schedule likely will lead to one less forage harvest per year in the North Central region ( i.e. , three compared to four cuttings). The elimination of one cutt ing will result in a reduction in harvesting costs. The overall forage yield for the year or season is expected to be the same or even higher with fewer cuttings because of the l onger harvest intervals and resulting higher forage yields with each cutting. Fewer harves ts also means fewer trips across the field, resulting in less labor, fuel consumption and soil compacti on, plus potentially less crown damage to alfalfa plants in established stands. In additi on, longer harvest intervals have

been shown to extend the life of the alfalfa stand because the plants have a longer period of time to replenish carbohydrate root reserves (Orloff and Putnam, 2008).

The phenotypic, agronomic, and environmenta l interaction assessments presented in

Section VII have demonstrat ed that KK179 is not mean ingfully different from conventional alfalfa. Therefore, KK179 will utilize the same agronomic practices as conventional alfalfa production, including: tillag e operations, seedbed preparation, pest management, and harvesting procedures. In addition, KK179 alfalfa growers will have increased flexibility in their forage harves t schedules, but the resulting hay quality will

continue to meet the established USDA Agricultural Marketing Services’ quality classifications of supreme, premium, good, fa ir, and utility. Thes e classifications are based on several factors, includ ing: the presence of weeds, fi ber content, protein content, general color, and presence of mold (USDA-APHIS, 2010).

KK179 will be combined with Roundup Rea dy alfalfa utilizing tr aditional breeding techniques. The combined traits will allow growers planting Roundup Ready × KK179 alfalfa to take advantage of the weed management benef its of the Roundup Ready weed control system: broad spectrum weed contro l and excellent crop safety with greaterMonsanto Company 12-AL-246U 192 of 407 

### application flexibility and simplicity. These growers will also have the flexibility to choose the production strategy th at improves forage quality or yield and maximizes the profitability of alfalf a production for their farming operatio n. Increased flexibility will allow growers to better manage the yield-qua lity relationship and ha rvesting schedules to

meet market needs and intended on-farm uses for their alfalfa forage production.

VIII.K. Stewardship of KK179

Monsanto and FGI develop eff ective alfalfa products and t echnologies and are committed to assuring that their products and technologies are safe and environmentally responsible.

Monsanto and FGI demonstrate this commit ment by implementing product stewardship processes throughout the lifecycle of a product a nd by participation in the Excellence Through Stewardship ® 

### (ETS) Program 1 

### and the USDA-APHIS Biotechnology Quality Management System (BQMS) 2 

### . These policies and practi ces include rigorous field compliance and quality management systems and verification through auditing. Stewardship Principles are also articulate d in Technology Use Guides (TUG) distributed annually to growers who util ize Monsanto branded traits (Monsanto Company, 2012).

As an integral action of fulfilling this commitment, Monsanto and FGI will seek biotechnology regulatory approvals for KK179 in all key alfalfa import countries with a functioning regulatory system to assure global compliance and support the flow of

international trade. These actions will be consistent with the Biotechnology Industry Organization (BIO) Policy on Product Launch Stewardship 3 

### . Monsanto and FGI will

continue to monitor other countr ies that are key importers of alfalfa from the U.S. for the development of formal biotechnology approval processes. If new functioning regulatory processes are developed, Monsanto and FGI will make appropriate and timely regulatory

submissions.

Monsanto and FGI also commit to industry best practices on seed quality assurance and control to ensure the purity and integrity of KK179 seed. As with Monsanto’s and FGI’s other alfalfa products, before commercializing KK179 in any country, a detection method

for KK179 will be made available to alfalf a producers, processors, and buyers.

® Excellence Through Stewardship is a registered servicemark of th e Excellence Through Stewardship Organisation, Washington, DC.

1 http://www.excellencethr oughstewardship.org/

. 2 http://www.aphis.usda.gov/biotechnology/bqms_main.shtml

3 http://www.excellencethroughstewardship.org/f acts/documents/Guide%20for%20Product%20Launch%20 Stewardship.pdf

.Monsanto Company 12-AL-246U 193 of 407 

### IX. ENVIRONMENTAL ANALYSIS

IX.A. Introduction

This section provides a brief review and assess ment of the plant pest potential of KK179 and its impact on agronomic practices and on the environment. USDA-APHIS has responsibility, under the Plant Protection Ac t (PPA) (7 U.S.C. § 7701-7772), to prevent the introduction and dissemination of plant pe sts into the U.S. Regulation 7 CFR § 340.6 provides that an applicant may petition US DA-APHIS to evaluate submitted data to determine that a particular regulated article does not present a plant pest risk and should no longer be regulated. If US DA-APHIS determines that th e regulated article does not present a plant pest risk, the petition is gr anted, thereby allowing unr estricted introduction of the article. According to the PPA, the de finition of “plant pe st” includes the living stage of any of the following, or a similar arti cle that can directly or indirectly injure, damage, or cause disease in any plant or plant product: (A) a pr otozoan; (B) a nonhuman animal; (C) a parasitic plant; (D) a bacterium; (E) a fungus; (F) a viru s or viroid; or (G) an infectious agent or other pathogens. (7 U.S.C. § 7702[14]).

The regulatory endpoint under the PPA for biotechnology-derived cr op products does not involve zero risk, but rather a determination th at the regulated articl e is not expected to pose a plant pest risk. Informa tion in this petition related to plant pest risk characteristics of KK179 include: 1) mode-of-action and cha nges to plant metabolism; 2) composition; 3) characteristics of the expr essed product; 4) pot ential for weediness of the regulated article; 5) impacts to non-target organisms (NTOs); 6) disease and pest susceptibilities; 7) impacts on agronomic practices; and 8) impact s on the weediness of any other plant with which it can interbreed, as well as the potential for gene flow.

The following lines of evidence form the basis for the plant pest risk assessment in this petition: 1) insertion of a single functional copy of the CCOMT suppression cassette; 2) safety and mode-of-action of the CCOMT suppression cassette; 3) compositional equivalence of KK179 forage to conventional alfalfa with the excep tion of the intended reduction in G lignin and total lignin; 4) phenotypic and agronomic characteristics demonstrating no increased plant pest potential including disease and pest susceptibilities; 5) negligible risk to NTOs including organisms benefi cial to agriculture; 6) familiarity with alfalfa as a cultivated crop; and 7) no greater likelihood to impact agronomic practices, cultivation practices, or the management of weeds, diseases, and insects compared to commercially grown alfalfa.

Using the assessment above, the data and analys is presented in this petition lead to a conclusion that KK179 is not expected to be a plant pest, and therefore should no longer be subject to regulation under 7 CFR § 340.

IX.B. Plant Pest Assessment of KK179

This section summarizes the details of the genetic insert, characteristics of the genetic modification, safety of expressed product, composition, and phenotypic, agronomic,Monsanto Company 12-AL-246U 194 of 407 

### environmental interaction char acteristics of KK179 used to evaluate the feed, food, and environmental safety of KK179.

IX.B.1. Characteristics of the Genetic Insert

IX.B.1.1. Genetic Insert

As described in Section IV , KK179 was developed by Agrobacterium tumefaciens- mediated transformation of conventiona l alfalfa R2336 leaf tissue using the plasmid vector PV-MSPQ12633. PV-M SPQ12633 contains two T-DNAs, each delineated by Left and Right Border regions to facilitate transformation. The first T-DNA is designated as T- DNA I and contains the CCOMT suppression cassette, which is regulated by the Pal2 promoter and the nos 3 ′ UTR. The second T-DNA, designated as T-DNA II, contains the nptII expression cassette, which is regulated by the 35S promoter
and the nos 3 ′ UTR. During transformation, both T-DNAs were inserted into the alfalfa genome where T-DNA II, containing the nptII expression cassette, functioned as a marker gene for the in vitro selection of transformed plantlets. Subsequently, conventional breeding methods and heritable segregation, along with a combination of analytical techniques, were used to isol ate those progeny plants that contained the

CCOMT suppression cassette (T-DNA I) but did not contain the nptII expression cassette (T-DNA II).

Molecular analyses demonstrated that KK179 contains a single copy of the CCOMT suppression cassette integrated in to the alfalfa genome at a single locus. No T-DNA II or backbone DNA sequences from PV-MSPQ12633 we re detected in KK179. Recorded data confirmed the organization and sequence of the in sert, in addition to the stability of the insert over several generations.

IX.B.1.2. Mode-of-Action

KK179 reduces G lignin and total lignin in forage through the suppression of caffeoyl

CoA 3- O -methyltransferase (CCOMT), a key enzyme in the lignin biosynthetic pathway. KK179 was produced by insertion of CCOMT gene segments, derived from alfalfa, assembled to form an inverted repeat DNA sequence. The invert ed repeat sequence produces double-stranded RNA (d sRNA) which suppresses endogenous CCOMT gene expression via the RNA interference (RNAi) pathway. Suppression of the CCOMT gene expression leads to lower CCOMT protein expr ession resulting in reduced production of G lignin subunit compared to conventional alfa lfa at the same stage of growth. The reduction in G lignin subunit synthesis leads to reduced accumulation of total lignin, measured as acid detergent lignin (ADL) described in Section I.B.3.

Analyses of KK179 RNA by Northern blot confirm the suppression of endogenous

CCOMT RNA in alfalfa forage. Additionally, analysis of monomeric lignin subunits, which constitute the building bl ocks of lignin molecules, conf irms that the suppression of

CCOMT acts to specifically reduce the level of one major lignin s ubunit, G lignin, while not substantially affecting the levels of th e other major lignin subunit, syringyl lignin (S lignin), or a minor lignin subunit, p -hydroxyphenyl lignin (H lignin), as predicted by theMonsanto Company 12-AL-246U 195 of 407 

### mode-of-action. The result is a lower proporti on of G lignin and a greater proportion of S lignin, shown by an increase in the S to G lignin ratio. Analys is of total lignin levels by a commercial forage testing lab, measured as ac id detergent lignin (ADL), verifies that the reduction in the G lignin leads to a concurrent reduction in total lig nin in KK179 forage compared to the conventional control harvested at the same stage of growth.

IX.B.1.3. Safety of KK179 Expressed Products

As described in Section V, the CCOMT suppression cassette encodes for dsRNA and is extremely unlikely to encode for a protein. Double stranded RNAs are composed of nucleic acids and are commonly found in e ukaryotes, including plants, for endogenous gene suppression. Nucleic acids have a l ong history of safe consumption and are considered GRAS by the U.S. FDA as th ere is no evidence to suggest dietary consumption of RNA is associated with mamma lian toxicity or allerg enicity. Therefore, based on the ubiquitous nature of the RNA-based suppression mechanism utilizing dsRNA, demonstration of mode-of-action through CCOMT RNA suppression, the history of safe consumption of RNA and the apparent l ack of toxicity or a llergenicity of dietary RNA, the RNA-based suppre ssion of endogenous CCOMT ge ne expression in KK179 poses no novel risks as a result of exposur e to expressed products of the DNA insert.

IX.B.2. Compositional Characteristics of KK179

Compositional comparisons based on OECD guida nce were presented in Section VI.B. to assess whether levels of nutrients, anti-nutri ents, and secondary metabolites in forage derived from KK179 are comparable to levels in the conventional control and several

conventional commercial referen ce varieties for which there is an established history of safe consumption. Nutrients assessed in this analysis in cluded proximates (ash, fat, moisture, and protein), carbohydr ates by calculation, acid dete rgent fiber (ADF), neutral detergent fiber (NDF), acid detergent lignin ( ADL), minerals (Ca, Cu, Fe, Mg, Mn, P, K, Na, and Zn), and amino acids (essential and non-e ssential). Anti-nu trients assessed included daidzein, glycitein, genistein, coum esterol, formononetin, biochanin A, and saponins (total bayogenin, tota l hederagenin, total medicagen ic acid, total soyasapogenol B, total soyasapogenol E, total zanhic acid, and total saponins). Secondary metabolites

assessed included p- coumaric acid, ferulic acid, sina pic acid, total polyphenols, free phenylalanine, and canavanine.

Compositional and nutritional comparisons were conducted to determine statistically significant differences at the 5% level of significan ce between KK179 and the

conventional control. The relevance of each statistically significant difference observed was assessed through evaluation of: 1) the magnit ude of the difference in the mean values of components; 2) whether the mean value is within the range of natural variability of that component as established by the 99% tolerance interval of the conventional commercial reference varieties; and 3) an asse ssment of the differen ces within the context of natural variability of available commercial alfalfa composition published in the

scientific literature.Monsanto Company 12-AL-246U 196 of 407 

### Assessment of the compositional analyses confirmed that the differences observed were not meaningful to feed and food safety or the nutritional quality of KK179 compared to the conventional control. With the excepti on of the intended changes in G lignin and total lignin (ADL) described in Section I.B.3., the levels of assessed components in

KK179 were determined to be within the 99 % tolerance interval established from the

conventional commercial reference varietie s grown concurrently. While compositional analysis confirmed that the intended reduction in G lignin and total lignin, this compositional change does not pose meaningf ul or novel feed/food sa fety or environment safety concerns, as similar levels of G lignin and total lignin are also found in conventional alfalfa forage harvested at earlier stages of growth. With the exception of the intended reduction in G lignin and tota l lignin, these results support the overall conclusion that KK179 forage is compositionall y equivalent to conventional alfalfa in accordance with OECD guidelines.

IX.B.3. Phenotypic and Agronomic and En vironmental Interaction Characteristics

Extensive information and data were used to assess whether the introduction of the trait for reduced G lignin and total lignin altered the plant pest potential of KK179 compared

to the conventional control. Phenotypic, agronomic, an d environmental interaction

characteristics of KK179 were evaluated a nd compared to those of the conventional

control and considered with in the variation among conventional commercial reference

varieties. These assessments included se ven general data cate gories: 1) seed germination, dormancy, and emergence; 2) ve getative growth; 3) winter survival; 4) reproductive growth including po llen, flower, and seed charac teristics; 5) lodging and seed retention on the plant; 6) plant respons e to abiotic stress a nd interactions with diseases and arthropods; and 7) plant-sy mbiont interactions. Results from the phenotypic, agronomic, and environmental interaction assessment demonstrate that KK179 does not possess: 1) increased weedy char acteristics; 2) incr eased susceptibility or tolerance to specific abiotic stresses, diseas es or arthropods; or 3) characteristics that would confer a plant pest risk compared to the conventional control. Taken together, the results of the analysis support a determination that KK179 is not expected to pose a plant pest risk compared to conventional alfalfa.

IX.B.3.1. Seed Dormancy and Germination

Seed germination and dormancy mechanisms vary with species and often have complex genetic origins. Seed dormancy is a characte ristic that is often a ssociated with plants considered weeds (Anderson, 1996; Lingenfe lter and Hartwig, 2007). Apart from the impervious (hard) seed coat, alfalfa has no physiological seed dormancy mechanism (Copeland and McDonald, 2001; Rolston, 1978). A decrease in the percent of hard seed

was observed in KK179 relative to the conventi onal control, however, this difference is not considered a characteristic associated with increased weediness or plant pest potential. No other changes in the seed dormancy or germ ination characteristics were

observed in either scarified or non-scarified se ed that would be indicative of increased plant weediness or plant pest potential of KK 179 compared to the conventional control as

described in Section VII.C.1.Monsanto Company 12-AL-246U 197 of 407 

### IX.B.3.2. Plant Growth and Development

Evaluations of plant growth and development in the field described in Section VII.C.2. were based on assessment of phenotypic char acteristics including emergence, early season vigor, lodging, crop growth stage, regrow th after forage harves t, fall plant height, total forage yield, spring vigor, spring sta nd recovery, and spring stand count. Also assessed were phenotypic char acteristics during seed produc tion such as seedling vigor, seed maturity, lodging at seed maturity, and split pods. Of the growth and development

characteristics assessed between KK179 and the conventional contro l, no statistically significant differences were detected at a 5% level of significance ( α = 0.05). The data support the conclusion that there is no biol ogically meaningful change in terms of weediness or plant pest pot ential of KK179 compared to conventional alfalfa.

IX.B.3.3. Response to Abiotic Stressors

No biologically meaningful differences were observed during comparative field

observations between KK179 and the conventiona l control in their response to abiotic stressors, such as cold, heat , drought, wind, as described in Section VII.C.2. The lack of significant biological differences in plant responses to abiotic stress supports the

conclusion that the introduction of the trait for reduced G lignin and total lignin is not expected to result in increased plant pest potential for KK179 comp ared to conventional

alfalfa.

IX.B.3.4. Reproductive Characteristics

Evaluations of pollen morphology and viab ility and flower mo rphology in KK179 and

the conventional control as described in Sec tion VII.C.3 provide information useful in a plant pest risk assessment as it relates to th e potential for gene flow and introgression of the biotechnology-derived trait in to other alfalfa varieties a nd sexually compatible related species. Pollen morphology and viabilit y and flower morphology evaluations demonstrated no statistically significant differences ( α = 0.05) between KK179 and the conventional control for any of the assessed characteristics. Taken together, these comparative assessments indicate that KK179 is not different than conventional alfalfa and is not expected to have increased weed iness or plant pest potential compared to

conventional alfalfa.

IX.B.3.5. Interactions with Non-target Or ganisms Including Those Beneficial to Agriculture

Evaluation of KK179 for potential effects on NTOs is a compone nt of the plant pest risk assessment and was evaluated using a comb ination of biochemical information and experimental data. The information and data included molecular characterization, safety of expressed products, the hist ory of environmental exposur e to reduced lignin, results

from the environmental interactions assessmen t described earlier, and the demonstration of compositional, agronomic, and phenotypic e quivalence to conventional alfalfa. The nature of KK179 as a product with no pestic idal activity means a ll exposed organisms

can be considered NTOs. Observational data on environmental interactions for KK179Monsanto Company 12-AL-246U 198 of 407 

### and a conventional alfalfa control were collec ted at select U.S. sites in 2010 and 2011. Multiple conventional commercial conventional reference varieties were included in the analysis to establish a range of natura l variability for each characteristic. The

environmental interactions ev aluation, described in Sectio n VII.C., included qualitative assessment of plant response to insects a nd disease and quantitative assessment of arthropod pests and abundance. The results of these asse ssments indicated that the presence of the trait for reduced G lignin and total lignin does not alter plant-insect interactions, including beneficial arthr opods and pests, nor does it alter disease susceptibility of KK179 compared to conventional alfalfa. The results also indicated that reduction of lignin to levels al ready present in the environmen t does not alter plant-insect

interactions or diseas e susceptibility.

In the field, alfalfa forms a complex symbiotic relationship with members of the bacterial family Rhizobiaceae and Bradyrhizobiaceae . No significant differences in parameters related to symbiont interactions were de tected between KK179 and the control for, indicating no impact on either the symbiotic relationship or the symbiotic nitrogen-fixing

bacteria. This assessment demonstrated th at the symbiosis between nitrogen fixing

bacteria and alfalfa was not alte red as a result of the introdu ction of the tr ait for reduced G lignin and total lignin (Section VII).

Taken together, these data combined w ith biochemical information support the conclusion that KK179 is not expected to have an effect on NTOs including those beneficial to agriculture.

IX.C. Weediness Potential of KK179

Alfalfa is a widely adapted crop that can be found in all parts of the continental United States, Alaska, and Hawaii, preferring fert ile, well-drained soils. Because of its adaptability, it also survives outside of cultivation. Little evidence exists to suggest that alfalfa behaves as a weed, other than as a volunteer in agricultural settings (USDAAPHIS, 2010). Weed control experts from stat es where alfalfa is cultivated extensively, including Arizona, California, Idaho, Ore gon, Pennsylvania, South Dakota, Washington, and Wisconsin, have communicated that they do not consider alfalfa a weed (Rogan and Fitzpatrick, 2004; USDA-APHIS, 2010). Out of 12 weed lis ts available in the USDA

PLANTS Database (USDA-NRCS, 2012), Medicago sativa is found in only one weed identification guide by the Southern Weed Sc ience Society (SWSS). The author of the SWSS entry for alfalfa has clarified that al falfa is not an invasive weed and does not displace native species (USDA-APHIS, 2010).

Though not considered a weed, alfalfa does exist in a feral state outs ide of agricultural settings. These plants orig inated from introduced varietie s and can be found in sparse populations throughout the U.S. (USDA- APHIS, 2010; USDA-NRCS, 2012). As described in Section VIII.H., alfalfa can be found in many non-agricultural areas. It has been intentionally used for numerous non-ag ricultural purposes incl uding: rehabilitation of overgrazed rangelands to improve wildlife habitat and for lives tock; erosion-control projects in forest interiors; improvement of compacted soils; use in seed mixes for USDA’s Conservation Reserve Program (C RP); revegetation of areas damaged byMonsanto Company 12-AL-246U 199 of 407 

### wildfire; and erosion reduction in mined soil s (USDA-APHIS, 2010). These uses have in

some cases led to establishment of feral alfa lfa populations. Survey s have confirmed that minor feral populations exist in six major alfa lfa-producing states, in areas where alfalfa seed or forage is produced (R ogan and Fitzpatrick, 2004). The falcata subspecies of alfalfa (yellow-flowered) is naturalized on ra ngelands in the northern and western States, where it has been promoted as a rangela nd enhancer for grazing (USDA-NRCS, 2012).

In situations where control of feral alfalfa is desired, it can be controlled like cultivated alfalfa using cultural or chemical methods as described in Secti ons VIII.G and VIII.H.

Feral alfalfa can be found in various soils and environments in coexistence with many other plant species. As expe cted, feral alfalfa populations are not managed and lack regular external inputs like irrigation, herbicides, insecticides, and fertilizers. Thus, they are exposed to numerous environmental fact ors limiting establishment of seedlings including low or excessive water conditions, low light intensity, extreme soil temperatures, and low pH soils (Bagavathiannan and van Acker, 2009). Because active pest control and genetic resistance to pest s are largely absent, feral alfalfa is more negatively impacted by endemic pests than al falfa under cultivation (Bagavathiannan et al., 2011b). As a result, the competitivenes s and numbers of viable, vigorous seeds produced by feral alfalfa are likely constrained compared to cultivated alfalfa with improved genetics under management (Van Deynze et al., 2008).

Seed dormancy is an important characteristic of ten associated with plants that are weeds (Anderson, 1996). Dormancy mechanisms, incl uding hard seed, vary with species and are usually based on complex processes. Fo r most crops, the number of hard seed is negligible or nonexistent. However, when al falfa seed is produced, a portion of the seed is "hard," that is, the seeds do not absorb wa ter after a prescribed period of time because of an impermeable seed coat (AOSA, 2007b). The percentage of ha rd seed in alfalfa varies widely, and depends largely on environmental cond itions during and after seed maturation, harvesting and seed conditioning methods and on genetic factors (Bass et al., 1988). Seed aging, weathering or mechanical sc arification make the seed coat permeable to water and allows rapid germination unde r favorable conditions. Apart from an impervious seed coat, alfalfa has no physio logical seed dormancy mechanism to delay germination.

The viability of most alfalfa seed in soil declines over time (Bass et al., 1988). A portion of the residual alfalfa seed can persist in the so il for several years, and if it remains viable may germinate as volunteers (Bass et al ., 1988; Mueller, 2008). Alfalfa that has germinated and emerged unintentionally in a subsequent crop, also known as volunteer alfalfa, may compete with the succeeding rota tional crop. However, problems controlling volunteer alfalfa are not common (Van Deynze et al., 2008). Volunteers, including ones with herbicide-tolerant traits, can be manage d with pre-plant or selective post-emergent herbicide applications or by mechanical means as described in Section VIII.G.

In comparative studies described in Secti on VII.C. between KK179 and the conventional control, phenotypic, agronomic, and environm ental interaction data were evaluated for changes that would impact the plant pest potential, in particular, weediness potential.

Results of these evaluations show that ther e are no biologically significant differencesMonsanto Company 12-AL-246U 200 of 407 

### between KK179 and the conventional control fo r traits potentially associated with weediness. Furthermore, comparative field observations between KK179 and the conventional control including their responses to abiotic stressors, such as cold, heat, drought, and wind, indicated no differences and, therefore, no increased weediness potential. Data on environmental interactio ns also indicate that KK179 does not confer any biologically meaningful increased susceptibility or tolerance to specific diseases or insect pests. Collectively, these findings support the conclusi on that KK179 has no increased weediness potential compared to alfa lfa and is not expected to become a weed.Monsanto Company 12-AL-246U 201 of 407 

### IX.D. Potential for Gene Flow and Introgression

Gene flow refers to all mechanisms that may result in the moveme nt of genes between populations. Pollen-mediated gene flow, al so referred to as cross-pollination or hybridization, occurs when pollen of one plant successfully fertilizes ovules of a second sexually compatible plant. Pollen-mediated gene flow is affected by both biotic and abiotic factors such as the degree of sexual compatibility of the pollen donor and recipient plants, proximity and frequency of pollen sources and sinks, pollen dispersal, flowering phenology, synchrony of flowering times, environmental conditions such as temperature, humidity and field architecture. Because pollen-mediated gene flow is a natural biological process, it does not constitute an environmental risk in and of itself. Seed-mediated gene flow is an additional mechanism whereby genes may be dispersed between locales and populations.

Gene introgression is a proces s whereby one or more genes successfully incorporate into the genome of a recipient plant. Gene introgr ession must be considered in the context of the transgenes inserted into the biotechnol ogy-derived plant and th e likelihood that the presence of the transgenes and their subseque nt transfer to recipient plants and plant populations will result in increa sed plant pest potential.

The baseline potential for gene flow and introgression in conve ntional alfalfa in agricultural and feral settings has been comprehensively reviewed by USDA-APHIS (USDA-APHIS, 2010) and is rele vant to the environmental analysis of KK179. The lack

of differences between the KK179 and conventi onal control with resp ect to plant pest potential, described in Section VII.C., incl udes aspects related to gene flow and introgression such as pollen, flower, and seed characteristics. The following section summarizes the previous USDA-APHIS review and more recently published information that can be used to address the potential for gene flow and introgression from the

introduction of KK179.

IX.D.1. Gene Flow Among Alfalfa ( Medicago sativa) Populations

Alfalfa is dependent on cross-po llination by insects; therefore, pollen-mediated gene flow between different alfalfa populations is possibl e. Many factors infl uence the probability of successful gene flow betw een alfalfa populations, includ ing: timing and degree of flowering; relative abundance of pollen sources ; the presence and activity of pollinators; proximity of alfalfa populations; physical ba rriers between populati ons; the relative scale of the alfalfa populations; probability of seed maturation and germinat ion; probability of seedling survival; starting gene frequency within the source and sink alfalfa populations; and cultural practices employed (Putnam, 2006; Van Deynze et al., 2008). How these factors interact to impact the gene flow potential in alfalfa has been extensively studied and reviewed by academic researchers and the al falfa industry with respect to potential impacts on commercial alfalfa cultivation a nd the environment (Bagavathiannan et al., 2011b; a; Fitzpatrick et al., 2003; Hammon et al., 2006; NAFA, 2008; Putnam, 2006; St. Amand et al., 2000; Teuber et al., 2011; Teube r et al., 2005; Teuber et al., 2007; Van Deynze et al., 2008; Van Deynze et al., 2005). The gene flow literature has also been reviewed by USDA-APHIS (USDA-APHIS, 2010).Monsanto Company 12-AL-246U 202 of 407 

### The development and introduction of Roundup Ready alfalfa containing the glyphosate tolerance trait has provided a marker system to examine in detail the dynamics of gene flow for alfalfa, both conventional and biotechnology-derived, under experimental and

commercial conditions. Studies conducted under commercial seed production conditions in areas of intended use for KK179 have provi ded information on the realistic scenarios for gene flow (Fitzpatrick et al., 2003; Hagler et al., 2011; Teuber et al., 2011; Teuber et al., 2007). These studies along with previ ous studies and reviews have provided a comprehensive assessment of gene flow as a commercial and envir onmental risk factor (USDA-APHIS, 2010; Van Deynze et al., 2008). More recent studies have confirmed previous findings (Hagler et al., 2011; Teube r et al., 2011).

For purposes of analyzing gene flow, alfalfa populations can be cat egorized into three major types as sources and recipients of pollen: 1) alfalfa fi elds intended for hay production; 2) alfalfa fields intended for s eed production; and 3) naturalized or feral alfalfa populations existing outside of mana ged conditions. These categories provide a comprehensive range of scenarios shown in Table IX-1 under which pollen flow can occur and provide a framework to evaluate pollen-mediated gene flow (Van Deynze et al., 2008).

Table IX-1. Potential Scenarios for Pollen-mediated Gene Flow in Alfalfa (adapted from Van Deynze et al., 2008)

↓ From To → Hay Seed Feral

Hay Hay-to-Hay Hay-to-Seed Hay-to-Feral

Seed Seed-to-Hay Seed-to-Seed Seed-to-Feral

Feral Feral-to-Hay Feral-to-Seed Feral-to-Feral



### Commercial alfalfa seed produc tion typically requires the intentional introduction of large numbers of bee colonies in or near fi elds during the peak of flower production in order to achieve high rates of pollination and uniform seed ripening. Forage production does not entail the use of bees by growers at an y stage. The primary pollinators used are leafcutter bees, honey bees, and to a lesser extent alka li bees. Leafcutter bees and alkali bees pollinate at rates of over 80% while honey bees pollinate at 22% (Beekman and Ratnieks, 2000; Cane, 2002; Pitts-Singer a nd Cane, 2011). Leafcutter bees have a foraging range under 1 mile while honey bees ra nge up to 3 miles an d alkali bees even further (Beekman and Ratnieks, 2000; Hagler et al., 2011; Pitts-Singer and Cane, 2011). The studies show that cross-pollination rate s decrease with increasing distance from the source of pollen and vary with the predomin ant pollinator species (USDA-APHIS, 2010; Van Deynze et al., 2008).

Gene Flow into Hay Production Fields Greater than 99.5 percent of alfalfa planted in the U.S. is cultivated exclusively for alfalfa hay (forage) production (USDA-NASS, 2011a). The most commonly occurring alfalfa field interface is hay field-to-hay field, how ever, pollen-mediated gene flow is highly improbable between adjacent hay fields (Putnam, 2006; Van Deynze et al., 2008). Several factors in forage production limit potential gene flow from and into hay production fields: 1) harvest takes place at vege tative and early flower stages when littleMonsanto Company 12-AL-246U 203 of 407 

### to no pollen is produced and few flowers are present; 2) few natural pollinators of the optimal type are present; 3) biomass with flowers is removed on a regular basis which prevents seed setting; and 4) the competitio n and natural autotoxicity of the alfalfa prevents new seedlings resulting from rare out crossing events to successfully grow within established stands (Canevari and Putnam, 2008) Thus, normal forage production practices significantly lower the risk of pollen-mediated gene flow between hay production fields and outside populations (Van Deynze et al., 2008).

The most important factor for avoiding s eed-mediated gene flow in a hay production setting is the use of certified seed (Putnam, 2006; Teuber et al., 2007). Forage growers routinely use certified seed of registered va rieties for sowing alfalfa stands. High quality seed decreases the risk of seed-mediated ge ne flow during stand establishment and the

introduction of varietal mi xtures and off-types.

It is also improbable that pollen from an adjacent seed field would result in gene flow into a hay field. Normal forage production pr actices, which include multiple harvests per year of the hay field, coupled with physical isolation distance requir ements of certified alfalfa seed production fields keep the potential for gene flow from seed production fields to hay production fields very low (US DA-APHIS, 2010; Van Deynze et al., 2008). Under the remote possibility that an outcrossed seed in a hay field were to mature, competition from the stand and alfalfa’s natura l autotoxicity would reduce the probability of successful germination and surv ival within an existing stand.

Gene Flow into Seed Production Fields Pollen-mediated gene flow between adjacent seed production fields (seed-to-seed), while a higher risk scenario, is considered a comm on, managable, and measurable occurrence for conventional cultivars of most outcrossing crops including alfalf a. This scenario applies to a much smaller ar ea of approximately 121,000 acres, or 0.5 percent of the total U.S. alfalfa acres, concentrated in areas optimal for alfalfa seed production (USDAAPHIS, 2010). Conventional alfalfa seed pr oduction occurs in approximately 20 U.S. states. In the case of Roundup Ready alfalfa, the alfalfa industry has, for purposes of coexistence and trait stewardship, opted to li mit seed production contracts to only ten states: Arizona, Colorado, Idaho, Montana, Nevada, Oregon, Texas, Utah, Washingon, and Wyoming. Professional seed growers in these states routinely utilize state seed certification services and, together, produce the majority of the U.S. alfalfa seed crop.

Certified seed growers have relied for ma ny decades on physical isolation to minimize and mitigate pollen-mediated gene flow in or der to manage genetic purity of commercial varieties (Brown et al., 1986; Dunkle, 2011; Kalaitzandonakes, 2011; Van Deynze, 2011). Gene flow studies of alfalfa seed pr oduction fields have c onfirmed that current AOSCA and OECD seed isolation and production standards can be used to meet Federal Seed Act standards (7 CFR § 201.76) (AOS CA, 2011; Fitzpatrick et al., 2003; Hammon et al., 2006; OECD, 2012; Teuber et al ., 2011; Teuber et al., 2005; Teuber and Fitzpatrick, 2007; Teuber et al., 2007; Teuber et al., 2004). For special situations such as identity preserved crops where even grea ter purity is desired, seed producers use

additional isolation or sanitation to ensure high seed purity.Monsanto Company 12-AL-246U 204 of 407 

### Current AOSCA isolation requirements for cer tified alfalfa seed pr oduction have also been shown to effectively mitigate gene flow from hay production fields into seed fields (Teuber et al., 2007). The normal forage produ ction practice of harv esting a hay field at or before 10 percent bloom during the seed production polli nation period is sufficient to ensure that pollen-mediated gene flow from hay production fields into seed production fields is low. Furthermore, this practi ce is a stewardship requirement for Roundup Ready alfalfa hay growers, under terms of the technology use ag reement (Monsanto Company, 2012). Research has shown that harvesting ha y at stages of 20 to 50% bloom does not significantly raise the po tential gene flow to neighboring seed production fields and risk remains very low (Teuber and Fitzpatrick, 2007; Van Deynze et al., 2008).

Further studies under actual co mmercial alfalfa seed grower conditions have shown thatthe use of minimum isolation distances specific to pollinator species and identitypreserved production protocols successfully m itigate gene flow to levels observed for other biotechnology-derived cr op species to produce seeds of high genetic purity (>99 %). Adventitious presence levels can be managed to le ss than 0.5 percent (Fitzpatrick, 2007; Fitzpatrick et al., 2007; Fitzpatrick and Lowry, 2010). Levels of actual gene flow under these conditions have also been shown to be several times less than those predicted by research models developed using smaller research plots (Fitzpatrick, 2007; Fitz patrick et al., 2007).

The information from these studies has allo wed the alfalfa industry in conjunction with AOSCA to develop coexistence strategies to provide additional measures for seed producers interested in further reducing the risk of pollen-me diated gene flow to their commercially-sensitive seed production crops from hay production fields. The strategies

include FGI Best Practices for Stewar dship in Roundup Ready Seed Production, NAFA Best Management Practices for Roundup R eady Seed Production, and NAFA Best Management Practices for Adventitious Presence-Sensitive Alfalfa Seed Production (Fitzpatrick, 2007; NAFA, 2011b; 2012b; c; a) with or without identity-preserved protocols such as the AOSCA Alfalfa S eed Stewardship Program (ASSP) (AOSCA, 2012). The protocols in these strategies requ ire pollinator-specific isolation distances to proactively mitigate adventitious presence in conventional seed and are considered applicable to other traits in alfalfa (Van Deynze et al., 2008). Other measures may include planting larger size seed production fields (>5 acres) and harvesting the seed field borders as a separate lot. The AOSCA AS SP was launched in 2010 by participating state seed certification agencies to offer a vol untary identity-preserved, process-based certificate to seed producers concerned about low level presence of biotechnologyderived traits (AOSCA, 2012) . The establishment of formal, voluntary grower opportunity zones in many seed productio n areas since 2011 has facilitated local

coordination of coexistence efforts of alfa lfa seed growers for both conventional and biotechnology-derived al falfa (NAFA, 2011a).

Gene Flow via Feral Alfalfa As described in Section IX.C, populations of fe ral alfalfa have existed in the U.S. since

its introduction due to natural dispersal from cultivated alfalfa and from intentional introductions for rangeland development a nd other purposes (Kendrick et al., 2005; USDA-APHIS, 2010). Gene flow to feral alfa lfa plants from large-scale seed or hayMonsanto Company 12-AL-246U 205 of 407 

### production fields of conventi onal alfalfa and bi otechnology-derived alfalfa has been shown to occur (Hammon et al., 2006; St . Amand et al., 2000). However, typical conditions and practices for hay and seed produ ction all but preclude the chance of gene flow into hay or seed production fields as previously described (USDA-APHIS, 2010; Van Deynze et al., 2008).

Certified alfalfa seed producti on requires minimum isolation fr om all sources of alfalfa including feral populations (AOSCA, 2011). Removing or mowing feral alfalfa plants near cultivated fields prevents synchronous bl oom and reduces the risk of gene flow to near zero (USDA-APHIS, 2010). Seed produ cers are known to control these populations as there are fewer feral populat ions in areas of intensive al falfa seed production compared to hay production areas (Kendric k et al., 2005). This practi ce also lowers the potential for gene flow into as well as from fera l populations (Van Deynze et al., 2008). As explained previously, normal forage production practices also restrict gene flow from feral populations to hay production fi elds to extremely low levels.

A number of factors relative to feral alfalfa itself further reduce the risk of pollenmediated gene flow from feral alfalfa populations to extremely low levels (USDAAPHIS, 2010; Van Deynze et al., 2008). Firs t, most feral populations are found in relatively small populations at low densities. Second, the asynchronous timing of flowering and the low density of pollinators in feral areas limit the effectiveness of feral alfalfa populations as sources, si nks or bridges for gene flow . Third, feral populations, as described in Section IX.C., are not manage d, and therefore are more susceptible to

environmental stresses. U nder these conditions, pollen and seed production on feral plants are expected to be considerably less pr olific compared to alfalfa plants in managed seed production fields. These same factors are also determinant in the relatively low potential for gene flow between feral alfalfa populations (USDA-APHIS, 2010; Van Deynze et al., 2008).

A final consideration is that any gene, were it to be intr ogressed into a feral population, including a biotechnology-derived gene, woul d be present at a limited frequency unless there were pressure from a selection mechanis m to raise the frequency (Ellstrand et al., 1999). Monsanto and FGI are not aware of a conceivable mechanism by which the introduced trait for reduced G lignin and total lignin could confer a selective advantage to feral alfalfa plants or be selected for in an unmanaged setting. The data presented in this petition strongly suggest that the trait in KK 179 would not confer a selective advantage to a feral alfalfa plant. These factors taken toge ther lead to the conclusion that gene flow from KK179 under scenarios involving feral popul ations is not expected to be greater

than it is for conventional alfalfa and is unlikely to affect seed production, hay production, or existing feral alfalfa populations.

As summarized in Section IX.C., comp arative studies between KK179 and the

conventional control to evalua te possible changes in plant pest potential, and weediness potential in particular, did not reveal biologically significant differences in characteristics

associated with weediness. Collectively, these findings also support the conc lusion that KK179 has no increased potential compared to conventional alfalfa to outcross or hybridize with cultivated alfalfa under ha y production or seed production conditions.Monsanto Company 12-AL-246U 206 of 407 

### IX.D.2. Hybridization with Annual Species of Subgenus Medicago

Medicago sativa is very distantly relate d to the annual members of Medicago (Lesins and Lesins, 1979). No annual speci es of this genus is native to North America. One annual species, M. lupulina (black medic), however, is found in the U.S. and is considered a weed in lawns and unmanaged areas, as well as in forage seed crops due to its seeds contaminating other small-seeded forage le gume seed crops. Crosses between the annual and perennial species do not occur naturally, and even artificial cross-fertilization is unsuccesful (Fridriksson and Bolton, 1963; Sangdue n et al., 1983). Significant biological

barriers exist between annual and perennial species, whic h prevent successful unassisted hybridization. Annual species are self-pollinating, while pe rennial species outcross and require bees to facilitate pol lination. Ploidy and karyotype differences between alfalfa

and the annual Medicago species also prevent successf ul hybridization. Additional reproductive barriers include bot h pre- and post-fertilizati on abnormalities, such as abnormal pollen tube growth (Sangduen et al., 1983) and post-ferti lization abortion of ovules (Fridriksson and Bolton, 1963). Successful hybridizations between M. sativa and M. lupulina were reported once several decades ago, but numerous attempts to repeat the crosses have failed and the ability of the tw o species to hybridize is disputed (USDAAPHIS, 2010). Expert opinion concludes that no annual speci es is known to naturally hybridize with M. sativa (McCoy and Bingham, 1988; Quiros and Bauchan, 1988).

USDA-APHIS has also conclude d, due to lack of confirmatory evidence, hybridization

between M. lupulina and M. sativa is very unlikely to oc cur (USDA-APHIS, 2010). Recent research further supports that M. lupulina and M. sativa hybridization is unlikely (Chandra et al., 2011; St eele et al., 2010).

IX.D.3. Hybridization with the Perennial Species of Subgenus Medicago

The M. sativa complex has been sexually hybridized with 12 other perennial Medicago species (McCoy and Bingham, 1988). Many of these interspecific hybrids have only been accomplished by using in vitro embryo culture of the hybr id in the laboratory (McCoy and Smith, 1986), making them highly unlikely to occur in nature. Three of these perennial species, M. prostrata, M. cancellata, and M. saxatilis are considered the most capable of successfully crossing with M. sativa (Lesins, 1961; 1962; 1970; Quiros and Bauchan, 1988). No perennial Medicago species are present naturally in the Americas, Australia, New Zealand, or Sout h Africa (Quiros and Bauchan, 1988; USDAAPHIS, 2010). therefore, no risk of inte rspecific hybridization exists in the United States. This was confirmed by a search of survey databases for Medicago populations in the U.S. which only produced matches for Medicago sativa itself and for species that are sexually incompatible with M. sativa complex members (USDA-APHIS, 2010).

IX.D.4. Transfer of Genetic Informatio n to Species with which Alfalfa Cannot Interbreed (Horizontal Gene Flow)

Monsanto and FGI are not aware of any re ports confirming the transfer of genetic material from alfalfa to other sexually inco mpatible plant species. The probability for horizontal gene flow to occur is judged to be exceedingly small. The ecological risk associated with gene flow and introgre ssion from KK179 derives fr om the presence ofMonsanto Company 12-AL-246U 207 of 407 

### the CCOMT gene segments. The consequen ces of the introg ression of the CCOMT gene segments from KK179 into a sexually compatible species are neglible since, as data presented in this petition conf irm, it confers no increased plant pest potential to alfalfa. If the extremely remote possibility of horizontal gene transfer were to occur, it is highly unlikely the consequences would be greater than gene flow to sexually compatible species.

IX.E. Potential Impact of KK179 on Alfalfa Agronomic Practices

Current agronomic practices for alfalfa were assessed to determine whether cultivation of KK179 has the potential to impact current al falfa cultivation practices and management as described in Section VIII. Typically, al falfa fields are managed agricultural areas dedicated to forage production. KK179 is likely to be used in common rotations on land previously used for agricultural purposes. Cultivation of KK179 is not expected to differ

from current cultivation of conventional or Roundup Ready alfalfa. Certified seed production will continue to use well-establishe d industry practices to deliver high-quality seed containing KK179 to growers.

KK179 is similar to conventional alfalfa in its agronomic, phenotypic, ecological, and compositional characteristics, and has levels of resistance to insects and diseases comparable to conventional alfalfa. KK179 w ill utilize the same agronomic practices as conventional alfalfa production, including till age operations, seedbed preparation, pest management, and harvesting procedures. Ther efore, no impacts on current cultivation and management practices for alfalfa are e xpected following the introduction of KK179. KK179 will allow growers the flexibility to a lter timing of harvest to a limited extent. The resulting hay quality will continue to meet the established USDA Agricultural Marketing Services’ quality classifications of supreme, premium, good, fair, and utility. Growers will have the flexibility at each harvest to choose one of two production strategies to improve the valu e of alfalfa production on their farm. Either they can 1) maximize forage quality while maintaining yi eld or 2) maximize yield while maintaining forage quality, depending on plant growth stag e at harvest. When implementing the first production strategy, the cutting schedules and th e timing of harvest remains the same as

those used with conventional alfalfa. KK 179 harvested at this crop stage will produce forage with lower levels of lignin compared to conventional alfalfa. As a result, the quality of the forage is mo re likely to meet or exceed the intended quality standard targeted by the grower. The second produc tion strategy involves delaying harvest to maximize yield without forfeiting forage qu ality compared to conventional alfalfa. KK179 can be harvested later and still produce hi gh quality forage that is comparable to earlier harvest timings with conventional alfalf a. This strategy is expected to result in increased forage tonnage or yield from the harv est. Growers will also have flexibility to delay harvest when rain or adverse weather conditions coincide with a planned harvest interval. This will allow them to postpone harvest to when more favorable weather conditions occur, while maintaining acceptable forage quality at a later harvest timing.

A delayed harvest schedule with its longer cutting intervals could potentially lower production costs over the life of the KK179 alfalfa stand. A delayed harvest schedule could lead to one less forage harvest per year in the North Central region ( i.e. , threeMonsanto Company 12-AL-246U 208 of 407 

### compared to four cuttings). The elimination of one cutting could result in a substantial reduction in harvesting costs. At the same time, the overall forage yield for the year or season is expected to be sim ilar or even higher because of the longer cuttin g intervals and resulting higher forage yields with each cuttin g. Fewer cuttings also means fewer trips across the field, which results in less labor , fuel consumption, and soil compaction, plus potentially less crown damage to alfalfa plants in established stands.

Based on this assessment, the introduction of KK179 is not expected to have an effect on current U.S. alfalfa cultivation practices and will provide growers increased flexibility to better manage the yield-quality relationship and harvesting schedules to meet market

needs and intended on-farm uses for their alfalfa forage production.

IX.F. Summary of Plant Pest Assessments

Plant pests, as defined in the Plant Protec tion Act, are the living stage of any of the following, or a similar article, that can directly or indirect ly injure, damage, or cause disease in any plant or plant product: (A ) a protozoan; (B) a nonhuman animal; (C) a parasitic plant; (D) a bacterium; (E) a fungus; (F) a virus or vi roid; or (G) an infectious agent or other pathogens (7 U.S.C. § 7702[14]) . Data presented in Sections V through VII of this petition confirm that KK179, with the exception of reduc ed G lignin and total lignin in forage compared to conventional alfalfa cut at the same stage of growth, is not significantly different from conventional alfalf a, in terms of plant pest potential. Monsanto and FGI are not aware of any study results or observati ons associated with KK179 that would suggest that an increased plant pest risk would result from its

introduction.

The plant pest assessment was based on multiple lines of evidence developed from a

detailed characterization of KK179 compared to conventional alfalfa, followed by a risk assessment on detected differences. The plant pest risk assessment in this petition was based on the following lines of evidence: 1) insertion of a single functional copy of the

CCOMT supppression cassette; 2) safety of the expressed product, dsRNA; 3) compositional equivalence of KK179 forage co mpared to a conventional control; 4) phenotypic and agronomic characteristics de monstrating no increased plant pest potential; 5) negligible risk to NTOs includi ng organisms beneficial to agriculture; 6) familiarity with alfalfa as a cultivated crop and 7) no greater likelihood to impact agronomic practices, including land use, cu ltivation practices, or the management of weeds, diseases and insects, than conventional alfalfa.

Based on the data and information presented in this petition, Monsanto and FGI have concluded that, like conven tional alfalfa and previous ly deregulated Roundup Ready alfalfa products, KK179 is not expected to be a plant pest. Results also support a conclusion of no increased weediness poten tial of KK179 compared to conventional alfalfa. Therefore, Monsanto and FGI re quest a determination from USDA-APHIS that KK179 and any progeny derived from crosse s between KK179 and other commercial alfalfa be granted non-regulated status under 7 CFR § 340.Monsanto Company 12-AL-246U 209 of 407 

### X. ADVERSE CONSEQUENCES OF INTRODUCTION

Monsanto and FGI are not aware of any study results or observati ons associated with KK179 indicating that there would be unexpect ed environmental consequences from the

introduction of KK179. KK179 contains an D NA insert with gene segments from the endogenous CCOMT enzyme gene under the contro l of a promoter to drive a pattern of expression similar to that of lignin (Guo et al., 2001; Leyva et al., 1992). The assembled gene segments produce a transcript with an inverted repeat se quence that forms double stranded RNA (dsRNA), which specifica lly results in the degradation of CCOMT transcripts via an RNA inte rference mechanism and leads to the suppression of the endogenous CCOMT gene (Siomi and Siomi, 2009). The DNA insert is not capable of expressing protein products. The suppression of the CCOMT gene in the lignin biosynthetic pathway significantly lowers G lignin synthesis, which leads to reduced accumulation of total lignin in forage tissue. As demonstrated by field results and laboratory tests, the only phe notypic difference between KK 179 and conventional alfalfa is reduction in G ligni n and total lignin.

The data and information presented in th is petition demonstrate that KK179 is not expected to pose an increased plant pest risk compared to conventional alfalfa. This conclusion is reached based on multiple lines of evidence developed from a detailed characterization of the product compared to conventional alfalfa, followed by risk

assessment on detected differences. The ch aracterization evaluati on included molecular analyses, which confirmed the insertion of a single functional copy of the CCOMT suppression cassette at a single locus within th e alfalfa genome. Additionally, there is a history of safe use of RNA as an e xpressed product and no evidence of protein production from the CCOMT suppression cassett e. Analyses of key nutrients, antinutrients, and toxicants of KK179 seed demonstrate that KK179 is compositionally equivalent to conventional alfalfa, with the exception of the intended reduction in G lignin and total lignin. The phenotypic evalua tions of KK179, including an assessment of seed germination and dormancy characteristics, plant growth and development characteristics, reproductive char acteristics, and environmental interactions also indicated KK179 is unchanged compared to conventional alfalfa. There is no indication that

KK179 would have an effect on non-target organisms in cluding ones beneficial to agriculture. Therefore, based on the lack of increased pest potential compared to conventional alfalfa, the risks for humans, animals, and other NTOs from KK179 are negligible under the anticipa ted conditions of use.

The introduction of KK179 will not result in changes to cultivation practices or the management of weeds, diseases and insect s in alfalfa producti on systems. Growers

familiar with cultivation of conventional or Roundup Ready alfalfa will continue to employ the same crop rotational practices, w eed control practices and/or volunteer control measures currently in place for conventional or Roundup Ready alfalfa products.Monsanto Company 12-AL-246U 210 of 407 

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### APPENDICESMonsanto Company 12-AL-246U 230 of 407 

### Appendix A: USDA Permits

Field trials of KK179 have been conducted in the U.S. since 2007. The protocols for these trials include field performance, breeding and observation, agronomics, and generation of field materials and data necessary for this petition. In addition to the KK179 phenotypic assessment data, observational data on pest and disease stressors were collected from these product development trials. The majority of the final reports have been submitted to the USDA. However, so me final reports, mainly from the 2010-12 seasons, are still in preparat ion. A list of trials conduc ted under USDA permits and the status of the final repo rts for these trials are provided in Table A-1.Monsanto Company 12-AL-246U 231 of 407 

### Table A-1. USDA Notifications and Perm its Approved for KK179 and Status of

Trials Conducted under These Permits

USDA No. Effective Date Release State (Sites) Trial # Trial Status

Year Field Trials – 2007

07-035-101 n

07-317-101 rm

10-328-101 rm 4/1/2007

1/9/2008

1/9/2011 WI 7.001 Submitted to USDA

USDA No. Effective Date Release State (Sites) Trial Status

Year Field Trials – 2008

08-034-101 rm 04/14/2008 CA 8.010 Submitted to USDA

08-011-102 rm 05/09/2008 WI (2) 8.032 Submitted to USDA

8.033 Submitted to USDA

07-317-101 rm 01/09/2008 WI (2), ID (2),

OK 8.054 Submitted to USDA

8.056 Submitted to USDA

8.103 Submitted to USDA

8.105 Submitted to USDA

8.151 Submitted to USDA

USDA No. Effective Date Release State (Sites) Trial Status

Year Field Trials – 2009

08-359-101 rm

11-353-103 rm 03/18/2009

03/15/2012 ID 9.018 Submitted to USDA

08-345-111 rm

11-353-102 rm 03/18/2009

03/15/2012 ID (4) 9.019 Submitted to USDA

9.024 Submitted to USDA

9.025 Submitted to USDA

9.028 Submitted to USDA

08-359-101 rm

11-353-103 rm 03/18/2009

03/15/2012 ID 9.0221-4 Submitted to USDA

9.0225-22 Submitted to USDA

09-037-101 rm

11-353-104 rm 04/15/2009

04/15/2012 PA 9.035 Submitted to USDA

08-345-108 rm

11-350-104 rm 03/18/2009

03/15/2012 WI (6) 9.049 Submitted to USDA

9.050 Submitted to USDA

9.051 Submitted to USDA

9.055 Submitted to USDAMonsanto Company 12-AL-246U 232 of 407 9.056 Submitted to USDA

9.062 Submitted to USDA

08-345-109 rm

11-353-101 rm 03/18/2009

03/15/2012 IA (2) 9.076 Submitted to USDA

9.078 Submitted to USDA

08-345-107 rm

11-350-103 rm 03/18/2009

03/15/2012 WA 9.092 Submitted to USDA

08-345-110 rm 03/18/2009 OK 9.162 Submitted to USDA

09-096-103 rm 8/1/2009 No plantings

USDA No. Effective Date Release State (Sites) Trial Status

Year Field Trials – 2010

09-149-103 rm

12-073-103 rm 08/01/2009

08/01/2012 CA (2) 10.001 Submitted to USDA

10.004 In Progress

08-034-101 rm

11-066-102 rm 04/14/2008

04/15/2011 CA 10.027 In Progress

09-037-101 rm 04/15/2009 PA 10.033 In Progress

08-345-107 rm

11-350-103 rm 03/18/2009

03/15/2012 WA (2) 10.041 Submitted to USDA

10.043 Submitted to USDA

08-345-108 rm

11-350-104 rm 03/18/2009

03/15/2012 WI (6) 10.101 Submitted to USDA

10.103 In Progress

10.106 In Progress

10.109 In Progress

10.110 In Progress

10.111 In Progress

09-345-102 rm 04/15/2010 WI (2) 10.107 In Progress

10.114 In Progress

08-011-102 rm

11-070-103 rm 05/09/2008

05/09/2011 WI (2) 10.151 Submitted to USDA

10.152 In Progress

09-308-108 rm 03/24/2010 ID 10.201 Submitted to USDA

08-345-111 rm

11-353-102 rm 03/18/2009

03/15/2012 ID (3) 10.204 Submitted to USDA

10.211 Submitted to USDA

10.212 Submitted to USDA

08-359-101 rm 03/18/2009 ID 10.213 Submitted to USDA

10-004-102 rm 04/15/2010 ID 10.215 In Progress

09-357-107 rm 04/01/2010 KS (2) 10.241 In ProgressMonsanto Company 12-AL-246U 233 of 407 10.242 Submitted to USDA

09-308-102 rm 04/01/2010 IL 10.251 Submitted to USDA

09-357-106 rm 04/01/2010 IL 10.252 Submitted to USDA

08-345-109 rm

11-353-101 rm 03/18/2009

03/15/2012 IA (5) 10.262 Submitted to USDA

10.267 Submitted to USDA

10.268 Submitted to USDA

10.269 Submitted to USDA

10.270 Submitted to USDA

09-308-107 rm 04/01/2010 CA 10.301 In Progress

09-308-104 rm 04/01/2010 IA 10.311 In Progress

09-357-103 rm 04/01/2010 TX 10.321 In Progress

09-308-103 rm 04/01/2010 WI 10.331 In Progress

09-357-104 rm 04/06/2010 NY 10.341 Submitted to USDA

09-345-103 rm 04/15/2010 No plantings

10-225-104 rm No plantings

USDA No. Effective Date Release State (Sites) Trial Status

Year Field Trials - 2011

10-364-115 rm 04/01/2011 ID (2) 11.001 In Progress

11.002 In Progress

11-066-102 rm 04/15/2011 CA 11.052 In Progress

10-341-101 rm 03/15/2011 CA (2) 11.053 In Progress

11.054 In Progress

10-341-103 rm 04/15/2011 WI (9) 11.101 In Progress

11.102 In Progress

11.103 In Progress

11.104 In Progress

11.105 In Progress

11.107 Submitted to USDA

11.108 Submitted to USDA

11.110 In Progress

11.111 In Progress

10-363-114 rm 04/15/2011 WI 11.151 Submitted to USDA

10-341-102 rm 04/15/2011 PA (5), WA (6), ID (7), OK (2) 11.004 In Progress

11.005 In Progress

11.006 In Progress

11.008 Submitted to USDA

11.009 In ProgressMonsanto Company 12-AL-246U 234 of 407 11.010 Submitted to USDA

11.011 Submitted to USDA

11.201 Submitted to USDA

11.202 Submitted to USDA

11.203 Submitted to USDA

11.204 In Progress

11.205 In Progress

11.261 Submitted to USDA

11.262 Submitted to USDA

11.263 In Progress

11.264 In Progress

11.267 In Progress

11.268 In Progress

11.351 In Progress

11.352 In Progress

10-341-104 rm 04/15/2011 IA (8) 11.241 In Progress

11.242 In Progress

11.243 In Progress

11.245 In Progress

11.246 In Progress

11.247 In Progress

11.248 In Progress

11.249 In Progress

11-070-103 rm 05/09/2011 WI (6) 11.281 In Progress

11.282 In Progress

11.283 Submitted to USDA

11.284 Submitted to USDA

11.285 In Progress

11.286 In Progress

USDA No. Effective Date Release State (Sites) Trial Status

Year Field Trials - 2012

10-341-101 rm 03/15/2011 CA 12.001 In Progress

11-342-102 rm-a1 03/15/2012 CA (2) 12.002 In Progress

12.003 In Progress

11-349-101 rm 04/15/2012 WI (4) 12.101 In Progress

12.104 In Progress

12.105 In Progress

12.109 In Progress

11-353-103 rm 03/18/2012 ID 12.201 In ProgressMonsanto Company 12-AL-246U 235 of 407 10-364-115 rm 04/01/2011 ID (2) 12.202 In Progress

12.203 In Progress

11-349-103 rm 04/15/2012 ID (3) 12.205 In Progress

12.206 In Progress

12.207 In Progress

11-349-104 rm 04/15/2012 IA (2) 12.401 In Progress

12.402 In Progress

10-364-116 rm 04/15/2011 WI 12.501 In Progress

11-203-101 rm 10/1/2011 No plantings

11-349-103 rm 04/15/2012 

### PA 12.302 In Progress

11-349-103 rm 04/15/2012 

### WA 12.703 In Progress

11-066-102 rm 04/15/2011 

### CA (2) 12.711 In Progress



### 12.715 In Progress

09-357-107 rm 04/01/2010 

### KS (2) 12.721 In Progress

12.722 In Progress

09-308-103 rm 04/01/2010 

### WI 12.731 In Progress

09-308-104 rm 04/01/2010 IA 12.741 In ProgressMonsanto Company 12-AL-246U 236 of 407 

### Appendix B: Materials and Methods Us ed for Molecular Analyses of KK179

B.1. Materials

The genomic DNA used in molecular analyses was isolated from leaf tissues of the P 0 

### generation of KK179, the conventional control C 0 

### , and conventional pa rental controls R2336 and Ms208. For generational stability an alysis, genomic DNA was extracted from

leaf tissue of the P 0 

### , MBC1, MBC2, and Syn1 genera tions of KK179. PV-MSPQ12633 (Figure III-1) was used as a positive hybridization control in Southern blot analyses. Probe templates generated from PV-MSPQ 12633 were used as additional positive hybridization controls. Additional referen ce standards, the 1 kb DNA Extension Ladder,

and λ DNA/Hind III Fragments from Invitrogen (Carlsbad, CA) were used for size

estimations on Southern bl ots and agarose gels.

B.2. Characterization of the Materials

The identity of the source materials was verified by methods used in molecular characterization to confirm the presence or absence of KK179. The stability of the genomic DNA was confirmed by observation of interpretable signals from digested DNA samples on ethidium bromide-stained agarose gels and /or specific PCR products, and the samples did not appear visibly degraded on the ethidium bromide-stained gels.

B.3. DNA Isolation for South ern Blot and PCR Analyses

KK179, conventional control C 0 

### , and conventional parental controls R2336 and Ms208 genomic DNA samples were isolated from alfalf a leaf tissue. Prior to extraction, leaf tissue was processed to a fine powder by mortar and pest le using liquid nitrogen. Genomic DNA was extracted using a hexadecyltrimethylammonium bromide (CTAB) based method. Briefly, 5 ml of CTAB buffer (1.5% w/v CTAB, 75 mM Tris HCl, 100 mM EDTA, 1.05 M NaCl, and 0.75% w/v PVP) and 50 μ g RNase A were added to approximately 1 ml of ground leaf tissue and incubated at 60-70°C for 40-50 minutes with intermittent mixing. The samples were cooled to room temperature. An equal volume of phenol:chloroform:isoamyl alcohol (25:24:1) was added to the samples, mixed by hand for two to three minutes, and then cen trifuged at ~10,300 × g fo r eight minutes at room temperature. The upper aqueous phase was transferred to a clean tube and the above purification step was repeated once. Approximately 5 ml of chloroform was added to the samples and mixed by hand for two to three minutes, then centrifuged at 10,300 × g for eight to 10 minutes. The upper aqueous phase was put into a clean tube and the chloroform step was repeated twice. After the last chloroform step, the aqueous phase was put into a clean tube and the DNA wa s precipitated with approximately 4 ml of 100% ethanol. The sample was centrifuged at 5,100 × g for five to seven minutes to pelletize the precipitated DNA. The DNA pellets were washed with 10-12 ml of 70% ethanol by centrifuging the samples at 5,100 × g for five to seven minutes. The DNA pellets were air dried, then resuspended in 500 μ l of TE buffer (10 mM Tris HCl, 1 mM EDTA, pH 8.0). All extracted DNA was stored in a 4°C refr igerator or a 20°C freezer.Monsanto Company 12-AL-246U 237 of 407 

### B.4. Quantification of Genomic DNA

PV-MSPQ12633 DNA, probe templates, and extracted genomic DNA were quantified using a Qubit TM 

### Fluorometer (Invitrogen, Carlsba d, CA) according to manufacturer’s

instruction.

B.5. Restriction Enzyme Digestion of Genomic DNA

Approximately ten micrograms (μg) of genomi c DNA extracted from the test and control substances were digested with the appropria te restriction enzymes. When digesting genomic DNA with the restriction enzyme combination Xmn I (New England Biolabs (NEB), Ipswich, MA) and Dra III (NEB), 10X Buffer 1 (N EB) was used. The digests were performed at 37°C in a total volume of ~500 μl using ~50 units of each restriction enzyme. When digesting genomic DNA with the restriction enzyme combination Xba I (NEB) and Swa I (NEB), 10X Buffer 2 (NEB) was used. In these digests, the DNA was first incubated overnight with ~50 units of Xba I at 37°C in a volume of ~500 μl. After

the Xba I digest, 23 μl of 1 M Tris HCl (Fisher Scientific, Pittsburgh, PA) and 5 μl of 5 M NaCl (Sigma-Aldrich Corp., Saint Louis, MO) were added to the digests the next day to optimize the buffer condition for Swa I. Approximately 50 units of Swa I was added to the digests and incubated for five hour s at room temperature. For the purpose of running positive hybridizat ion controls, ~10 μg of genomic DNA extracted from the conventional control was digested, and the appropriate positive hybridization control(s) were added to these digests prior to loading the gel.

B.6. Agarose Gel Electrophoresis

Digested DNA was resolved on 0.8% (w/v) agarose gels. For all Southern blot analyses, individual digests containing ~10 μg each of KK179 and conventional control genomic DNA were loaded on the same gel in a long ru n/short run format. The long run allows for greater resolution of large molecular we ight DNA, whereas the short run allowed the detection of small molecular weight DNA. The positive hybridization controls were only

run in the short run format. For the insert st ability analysis, individual digests of ~10 μg of genomic DNA extracted from leaf tissue across multiple genera tions of KK179 were loaded on the agarose gel in a single run format.

B.7. DNA Probe Preparation fo r Southern Blot Analyses

Probe templates were prepared by PCR amplification using PV-MSPQ12633 as the template. The PCR products were separate d on an agarose gel by electrophoresis and purified from the gel using QIAquick Ge l Extraction Kit (Qiagen, Valencia, CA) according to the manufacturer’s instruction. The probe temp lates were designed based on the nucleotide composition (% GC) of the sequen ce, in order to optimize the detection of DNA sequences during hybridization. When possible, probes possessing a similar melting temperature (Tm) were combined in the same Southern blot hybridization. Approximately 25 ng of each probe template were radiolabeled with

TM Qubit is a trademark of Invitrogen, Carlsbad, CA.Monsanto Company 12-AL-246U 238 of 407 

### [ α

32 

### P]-deoxycytidine triphosphate (dCTP) (6000 Ci/mmol) or [ α

32 

### P]-deoxyadenosine triphosphate (dATP) (6000 Ci/ mmol) using the random prim ing method (Invitrogen). Probe locations relative to the genetic elements in PV-MSPQ12633 are depicted in Figure IV-1.

B.8. Southern Blot Analyses of Genomic DNA

Genomic DNA isolated from the test and contro l substances were digested and evaluated using Southern blot analyses (Southern, 1975). In Southern blots hybridized with a single probe, ~0.1 and ~1.0 genome equiva lent of the PV-MSPQ12633 DNA previously digested with Xba I or Eco RI (NEB) was added to di gested conventional control genomic DNA to serve as a positive hybridizati on control. In Southern blots hybridized with multiple probes, ~1.0 genome equiva lent of the PV-MSPQ12633 DNA previously digested with Xba I or Eco RI (NEB), as well as ~0.1 a nd ~1.0 genome equivalent of the appropriate probe templates were added to digested conventional control genomic DNA to serve as positive hyb ridization controls. The DNA was then separated by agarose gel electrophoresis and transferred onto a nylon me mbrane. Southern blots were hybridized and washed at 55°C or 60°C, de pending on the calculated Tm of the probe(s) used. The table below lists the hybridization and radiolabeling conditions of the probes used in this study. Multiple exposures of each blot were then generated using Kodak Biomax MS film (Eastman Kodak, Rochester, NY) in c onjunction with at least one Kodak Biomax MS intensifying screen in a -80°C freezer.Monsanto Company 12-AL-246U 239 of 407 

### Table B-1. Hybridization Conditions of Utilized Probes

B.9. DNA Sequence Analysis of the Insert

Overlapping PCR products, denoted as Produc t A, Product B, Product C, Product D, and Product E were generated to sp an the insert and adjacent 5 ′ and 3 ′ flanking DNA sequences in KK179 (Figure V-9). These pr oducts were analyzed to determine the nucleotide sequence of the insert in KK179, as well as that of the DNA flanking the 5 ′

and 3 ′ ends of the insert.

The PCR analyses for Product A, Product B and Product D were each conducted using 100 ng of genomic DNA template in a 50 μl reaction volume. The reaction volume contains a final concentration of 2 mM MgCl2, 0.2 μM of each primer, 0.2 mM of each dNTP, and 0.025 units/μl of TaKaRa Ex TaqTM DNA Polymerase (Takara Bio Inc., Shiga, Japan). TM



### The PCR analyses for Product C and Product E were conducted using 80 ng of genomic DNA template in a 50 μl reaction volume. The reaction volume contains a final concentration of 1.5 mM MgCl 2 

### , 0.2 μM of each primer, 0.08 mM of each dNTP, and 0.02 units/μl of Phusion Hot Start II High Fi delity DNA Polymerase (Finnzymes, Espoo, Finland).

The amplification of Product A and Product D was performed under the following cycling conditions: one cycle at 98°C for 30 seconds; 25 cycles at 98 C for 10 seconds, 55°C for 20 seconds, 63°C for two minutes and 50 seconds; one cycle at 63°C for

five minutes.

TM TaKaRa Ex Taq DNA Polymerase is a tr ademark ofTakara Bio Inc., Shiga, Japan.

Probe Probe Type Labeling

Method Probe labeled

with dNTP ( 32 P) Hybridization/Wash Temperature (  C)

1 T-DNA I RadPrime dATP 55

2 T-DNA I RadPrime dATP 55

3 T-DNA I RadPrime dATP 55

4 T-DNA I RadPrime dATP 55

5 T-DNA II RadPrime dCTP 60

6 Backbone RadPrime dCTP 60

7 Backbone RadPrime dCTP 60

8 Backbone RadPrime dCTP 60

9 Backbone RadPrime dCTP 60Monsanto Company 12-AL-246U 240 of 407 

### The amplification of Product B was perfor med under the following cycling conditions: one cycle at 98°C for 30 seconds; 35 cycles at 98°C for 10 seconds, 55°C for 30°seconds, and 63°C for one minute; one cy cle at 63°C for five minutes.

The amplification of Product C was perfor med under the following cycling conditions: one cycle at 98°C for 30°seconds; 25°cycles at 98°C for 10 seconds, 63°C for one minute and 15 seconds; one cycle at 63°C for five minutes.

The amplification of Product E was perfor med under the following cycling conditions: one cycle at 98°C for 30 seconds; 25 cycles at 98°C for 10 seconds, 63°C for one minute

and 15 seconds.

Aliquots of each PCR product we re separated on a 1.0% (w/v) agarose gel and visualized by ethidium bromide staining to verify that the products were the expected size. Prior to sequencing, each verified PCR product was purified using the QIAquick PCR Purification Kit (Qiagen, Inc., Valencia, CA ) and quantified using a Qubit fluorometer according to manufacturer’s instruction. The purified PCR products were sequenced using multiple primers, including primers used for PCR amplification. All sequencing was performed by Monsanto’s TGAC (The Genome Analysis Center) using BigDye terminator chemistry (Applied Biosystems, Foster City, CA).

A consensus sequence was generated by compiling multiple sequencing reactions performed on the overlapping PCR products. Th is consensus sequence was aligned to the PV-MSPQ12633 sequence to determine the integr ity and organization of the integrated

DNA and the 5 ′ and 3 ′ insert-to-flank DNA junctions in KK179.

B.10. PCR and DNA Sequence Analyses to examine the Integrity of the DNA

Insertion site in KK179

To examine the KK179 insertion site in conve ntional alfalfa, PCR and sequence analyses were performed on genomic DNA from bot h KK179 and the conventional parental control alfalfa R2336 (Figure V-10 ). The primers used in th is analysis were designed from the DNA sequences flanking the insert in KK179. A forward primer specific to the DNA sequence flanking the 5 ′ end of the insert was paired with a reverse primer specific to the DNA sequence flanking the 3 ′ end of the insert.

The PCR reactions were conducted using 80 ng of genomic DNA template in a 50  l

reaction volume. The reaction volume cont ains a final concentration of 1.5 mM MgCl 2, 

### 0.2  M of each primer, 0.08 mM of each dNTP, and 0.02 units/μl of Phusion Hot Start II High Fidelity DNA Polymerase (F innzymes, Espoo, Finland).

The amplification was performed under the following cycling conditions: one cycle at

98  C for 30 seconds; 35 cycles at 98  C for 10 seconds, 55  C for 15 seconds, 72  C for 40 seconds; one cycle at 72  C for three minutes.

Aliquots of each PCR product were separated on a 1% (w/v) agarose gel and visualized by ethidium bromide staining to verify that th e PCR products were the expected size priorMonsanto Company 12-AL-246U 241 of 407 

### to sequencing. Only the verified PCR produc t from the conventional parental control R2336 was purified with the QIAquick PCR Pu rification Kit (Qiagen, Inc., Valencia, CA) and quantified using a Qubit fluorometer according to the manufacturer’s instruction. The purified PCR product was sequenced using primers used for PCR amplification. All sequencing was perfor med by TGAC using BigDye terminator chemistry (Applied Biosystems , Foster City, CA).

A consensus sequence was generated by compiling multiple sequencing reactions performed on the verified PCR product. Th is consensus sequence was aligned to the 5 ′

and 3 ′ sequences flanking the KK179 insert to determine the integrity and organization of

the insertion site.Monsanto Company 12-AL-246U 242 of 407 

### References for Appendix B

Southern, E.M. 1975. Detection of specifi c sequences among DNA fragments separated by gel electrophoresis. Jour nal of Molecular Biology 98:503-517.Monsanto Company 12-AL-246U 243 of 407 

### Appendix C: Materials and Methods Us ed for Northern Blot Analyses of

CCOMT Suppression in KK179

C.1. Materials

The mRNA used in the northern blot analysis was isolated from four replicate samples of root and forage tissue of the Syn1 genera tion of KK179 and the conventional control C 0



### .

Probe templates were used as positive hybridiz ation controls in northern blot analyses. Prior to probe generation, DNA fragme nts from the caffeoyl coenzyme A

3- O -methyltransferase ( CCOMT) (Monsanto Sequence Database) gene was generated from conventional alfalfa genomic DNA using a forward primer and a reverse primer specific to the 3 ′ UTR of the gene. The resulting PCR fragments of the CCOMT gene were cloned into pCR ® 

### -TOPO (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. The seque nce of the plasmid DNA was confirmed by sequencing and comparison to the expected se quences using pair-wise alignments. The plasmid DNA was then used as a template for PCR reactions to amplify the CCOMT probe templates. The actin probe templa te was generated by PCR from conventional alfalfa genomic DNA using a forward primer a nd a reverse primer specific to the partialcoding region of the alfalfa actin gene. RiboRuler TM 

### RNA ladders (high range) from Fermentas Co (Glen Burnie, MD) were used for size estimations on northern blots and formaldehyde/agarose gels. The unique iden tities of the probes, as well as molecular weight markers are documented in the raw data.

C.2. Characterization of the Materials

The forage and root tissues fo r test and control substances used in this study were obtained from Forage Genetics International (FGI) (West Salem, WI). The synthesis

records for these materials are retained with FGI. No certificate of analysis (COA) or verification of identity (VOI) certificates were generated for these materials. Prior to the start of the study, the identity of the test a nd control substances was confirmed by eventspecific PCR; the raw data were archived w ith the study. The study director reviewed the chain of custody (COC) documentation, which confirmed the identit y of the test and control substances prior to use of the materials in the study.

Prior to the start of the analysis, the CCOMT gene was sequenced by the Monsanto Genomics Sequencing Center and the sequences were aligned with the expected sequences to confirm the identiti es of the probes. The raw da ta are archived at Monsanto.

Extracted RNA and RNA markers were stor ed in a -80°C freezer. DNA probes were stored in a -20°C freezer. The test, control, and reference substances were deemed stable during storage because there were no signs of degradation on formaldehyde/agarose gels and probing with endogenous genes yielded in terpretable signals on northern blots.

® pCR is a registered trademar k of Invitrogen, Carlsbad, CA.

TM RiboRuler RNA ladders is a trademark of Fermentas Co., Glen Burnie, MD.Monsanto Company 12-AL-246U 244 of 407 

### C.3. Total RNA Extraction

KK179 and conventional control total RNA sample s were isolated from alfalfa root and forage tissues. Prior to extraction, forage and root tissues were processed to a fine powder by mortar and pestle using liquid ni trogen. Total RNA was extracted using a TRIzol-based method. Briefly, 20 ml of TRIzol reagent is added to approximately 2 g of root or forage tissue sample. The sample is homogenized using a vor texer for one to two minutes and incubated at room temperature for 40-50 minutes. The samples were centrifuged at ~12,000 × g for five minutes at 4°C to remove the debris. The supernatant is transferred to a new tube. Approximate ly 6 ml of chloroform was added to the samples, mixed by vortexing for one to two mi nutes, and incubated at room temperature for five to 10 minutes, followed by centrifugatio n at ~12,000 × g for 10 minutes at 4°C. The upper aqueous phase was transferred to a clean tube. Approximately 10 ml of phenol:chloroform:isoamyl alcohol (25:24:1) was added to the samples, mixed by hand for one to two minutes, and then centrifuge d at ~12,000 × g for five minutes at 4°C for additional purification of RNA. The upper aque ous phase was transferre d to a clean tube. Approximately 6 ml of chloroform was added to the samples, mixed by vortexing for one to two minutes, and incubated at room temper ature for five to 10 minutes, followed by centrifugation at ~12,000 × g for 10 minutes at 4°C. The upper aqueous phase was

transferred to a clean tube and the RNA wa s precipitated with a pproximately 10 ml of isopropyl alcohol. The samples were stored in a -80°C freezer for at least 30 minutes to 24 hours to precipitate the RNA. Samples we re then centrifuged at ~12,000 × g for 15-20 minutes at 4°C. The RNA pellets were wash ed with 5 ml of 75% ethanol (prepared in diethylpyrocarboonate (DEPC) treated water) and centrifuged at ~12,000 × g for 20 minutes at 4°C to precipitate the RNA. The RNA pellets were air dried, then resuspended in 2ml of water. All extr acted RNA was stored in a -80°C freezer.

C.4. Quantification of RNA

Total RNA was quantified using a B eckman Coulter DU-650 Spectrophotometer (Beckman Coulter, Inc. Atlanta, GA) according to ma nufacturer’s instruction. PolyA + 

### RNA was quantified using the Qubit Fluorometer (Invitrogen, Carlsbad, CA) according to manufacturer’s instruction.

C.5. PolyA + 

### RNA Isolation

PolyA + 

### RNA from the KK179 and conventional c ontrol total RNA samples was extracted using the Poly(A) Purist MAG Kit (Amb ion Co., Austin, TX) according to the

manufacturer's instruction with minor modifi cations. Briefly, ~420 μg of total RNA was placed in a 2 ml microcentrif uge tube and Rnase-free water was added to obtain a final RNA concentration of ~600 μg/ml. An e qual volume of 2X Binding Solution was added to the RNA samples and mixed thoroughly by brief vortexing. The RNA samples were transferred to the microcentrifuge tube containing the washed MagBeads and mixed thoroughly by inversion. The RNA/bead mixtur e was incubated in a 65°C water bath for

five minutes and then incubate d for ~ 1 hour at room temperature with gentle agitation by nutation. The tubes containing the RNA/bead mixture were placed onto the magnetic stand for ~ 2 minutes to pull the beads to th e side of the tube. The supernatant wasMonsanto Company 12-AL-246U 245 of 407 

### discarded and the beads washed twice with an equal volume (to the starting RNA solution) of Wash Solution 1 followed by tw o washes with an equal volume (to the starting RNA solution) of Wash Solution 2, di scarding all wash solution. Approximately 200 μl RNA Storage Solution, prewarmed in a 60-80°C water bath, was added to the captured MagBeads. The mixture was gently swirled by hand, and the tube was placed back onto the magnetic stand to capture the bead s. The supernatant containing the eluted RNA was placed in a clean microcentrifuge tube . The elution step was repeated once to obtain a final volume of 400 μl. The RNA was precipitated by adding 1/10 th 

### volume 5M ammonium acetate, 1 μl of 5 mg/ml glycogen and 2.5 volumes of 100% ethanol to the samples. The samples were incubated in a -80°C freezer for 30-40 minutes and then centrifuged at ~12,000 × g for 30 minutes at 4°C. The supernatant is discarded and the samples were washed with 70% ethanol. Th e pellets were briefly air dried, and then resuspended in an appropriate volume of pr ewarmed RNA Storage Solution and heated in a 65°C water bath until the RNA pellet dissolved. All extracted polyA + 

### RNA is stored in 10 μl aliquots in a -80°C freezer.

C.6. Formaldehyde/Agarose Gel Electrophoresis

PolyA + 

### RNA samples from KK179 and the conve ntional control were resolved in 0.8% formaldehyde/agarose gels.

C.7. Northern Blot Analyses

Northern blot analyses were performed by transferring polyA + 

### RNA from formaldehyde/agarose gels ont o nylon membranes and probing with radiolabeled DNA.

CCOMT hybridization signals were stripped from the blots and the stripped blots were hybridized to a 32 

### P-labeled actin probe to show the relative amount of RNA loaded in each lane. Probes were prepared by random prime labeling according to manufacturer’s instruction. Each probe was added at approximately 1-2 × 10 6 

### cpm/ml of the hybridization solution. Northern blots were hy bridized and washed at 55°C based on the calculated Tm of the probes used. Multiple exposures of each blot were then generated using Kodak Biomax MS film (Eastman Koda k, Rochester, NY) in conjunction with at

least one Kodak Biomax MS-intensi fying screen in a -80°C freezer.

C.8. Positive Hybridization Controls and Probe Template

DNA probe templates were run on the formal dehyde/agarose gels to serve as positive controls for hybridization. On northern blot s the probe templates migrated slightly differently than predicted by the RNA ladder. The difference in migration is most likely due to the inaccuracy of comparing a DNA pr obe to an RNA ladder. The following table lists the DNA probes, the positiv e hybridization controls, an d the hybridization and wash temperatures for each northern blot analysis.Monsanto Company 12-AL-246U 246 of 407 

### Table C-1. DNA Probes and Positive Hybridization Controls

Analysis Probe Positive hybridization

controls Hybridization

and Wash Temperature

Expression of CCOMT CCOMT CCOMT probe template 55oC

Expression of actin actin actin probe template 55oCMonsanto Company 12-AL-246U 247 of 407 

### Appendix D: Materials, Methods, and Results for Intended Changes to Lignin Levels in KK179 Forage

D.1. Materials

Forage was evaluated from KK179 (Seed Lot Number 11266289) and the conventional control C0-Syn1 (Seed Lot Number 112662 92). The conventional control has background genetics similar to that of KK179. The convent ional commercial reference

varieties were 14 conventional al falfa varieties (Table D-1).

Table D-1. Conventional Commercial Reference Varieties

Material Name Seed Lot

Number Field Site Pioneer 54V54 11266293 ILCY, WIDL Croplan LegenDairy 5.0 11266294 IARL, ILCY, WIDL Producer's Choice PGI 437 11266295 ILCY, KSLA
Pioneer 54H11 11266296 IARL, TXCL

Pioneer 54V46 11266297 KSLA Pioneer 54V09 11266298 CAPR, TXCL
DKA50-18 11266299 IARL, KSLA

America's Alfalfa Archer III 11266301 CAPR WL 319HQ 11266302 CAPR, KSLA, TXCL Dow/Dairyland Hybriforce 400 11266303 ILCY, WIDL AMPAC seed company Attention 11266304 CAPR

Vernal 11266305 WIDL Ranger 11266306 IARL Dow/Dairyland Hybriforce 2400 11266307 TXCL



### D.2. Characterization of the Materials

The identities of forage samples from KK179, the conventional control C0-Syn1, and

conventional commercial reference varieties were confirmed by verifying the chain of custody documentation prior to analysis. To fu rther confirm the iden tities of KK179, the conventional C0-Syn1, and conventional commercia l reference varieties, event-specific polymerase chain reaction (PCR) analyses were conducted on th e forage from each site to confirm the presence or absence of KK179.

D.3. Field Production of the Samples

Forage samples from KK179, the conventional control C0-Syn1, and conventional

commercial alfalfa varieties were collected fr om each of four replicated plots from six field sites [Tulare County, California (CAP R); Jefferson County, Iowa (IARL); Clinton County, Illinois (ILCY); Pawnee County, Ka nsas (KSLA); Armstrong County, Texas (TXCL); and Walworth County, Wisconsin (WIDL) ]. The field plots were established in 2010 from plants that were grown in a greenhouse. Prior to transplanting in the field, the presence or absence of KK179 wa s verified using PCR. The fi eld plots were planted in a randomized complete block design. All samp les at the field sites were grown under normal agronomic field conditions for their respective geographic re gions. Between 1Monsanto Company 12-AL-246U 248 of 407 

### and 10% bloom, all whole plants, two to three inches above the soil surface, were

harvested from the center of each individual plot. A subsample (about 680g) of plants was randomly collected from all harvested plants in each plot.

Forage samples were shipped frozen from th e field sites to Monsanto Company (Saint Louis, Missouri). All samples were ground to a powder and stored in a freezer set to maintain -20°C located at Monsanto Company (Saint Louis, Missouri). Subsamples were lyophilized and shipped at ambient temperat ure to Samuel Roberts Noble Foundation (Ardmore, Oklahoma) for analysis of ligni n monomer subunit composition. Subsamples were shipped on dry ice to Dairy One Forage Lab (Ithaca, New York) for lignin analysis.

D.4. Summary of Analytical Methods

Forage samples were analyzed for p- hydroxyphenyl lignin (H lig nin), caffeoyl lignin, guaiacyl lignin (G lignin), 5-hydroxyguaiacy l lignin, and syringyl lignin (S lignin) subunits by the Samuel Roberts Noble Foundation to demonstrat e that suppression of the caffeoyl coenzyme A 3- O -methyltransferase ( CCOMT ) gene by dsRNA produced by

KK179 resulted in the intended re duction of the G lignin subun it, and thereby total lignin. Forage samples were also analyzed by Dair y One Forage Lab for acid detergent lignin (ADL) by an industry standard method used to provide a standard forage quality

assessment.

D.4.1. Lignin Composition by Thioacidol ysis (H, G and S Lignin) (Conducted at Samuel Roberts Noble Foundation)

The lyophilized, ground samples were extracte d with chloroform/methanol (2:1, v/v), methanol (100%) and water three times each to obtain cell wall residue (CWR). The samples were then freeze-dried using a mani fold freeze-drier. About 18 mg of dried samples were weighed into 12 ml glass tube s; 3 ml of thioacidolysis reaction mixture were added, and the reaction was conducte d at 100°C for 4 hours. After solvent extraction, 3 ml of solvent were dried under nitrogen and derivatized with 150 μl pyridine and BSTFA mixture (1:1, v/v) before ga s chromatography/mass spectroscopy (GC/MS) analyses (Lapierre et al., 1985; Lapierre et al., 1995). The limit of quantitation was 0.1 μmol/g CWR.

Internal Standards:

 Sigma-Aldrich, Docosane (Lot Number 18999MJ) 3.03 mg/ml in chloroform

D.4.2. Lignin (Acid Detergent Lignin) (Conductedat Dairy One Forage Lab)

Forage samples were dried and analyzed using an ANKOM based methodology that is semi-automated, uses filter bags and is comp arable to the conventional crucible method (Weston et al., 2006). Briefly, samples we re boiled in acid detergent fiber (ADF) solution, rinsed with water a nd acetone, and hydroly zed in sulfuric acid. Following acid hydrolysis, samples were rinsed in water and soaked in acetone befo re being dried to a constant weight. The dried samples are weighe d and then ashed in individual crucibles atMonsanto Company 12-AL-246U 249 of 407 

### 525oC for 3 hours. After ashing, samples are cooled in a desi ccator and weighed a final time. The limit of quantitation was 0.1%.

D.5. Data Processing and Statistical Analysis

After compositional analyses were performed, data spreadsheets containing individual values for each analysis were sent to M onsanto Company (St. Louis, Missouri) for

review. Data were then transf erred to Certus International (Chesterfield, Missouri) where they were converted into the appropriate units and statistically analyzed. The formulas that were used for re-expression of composition data for statistical analysis are listed in

Table D-2.

In order to complete a statistical analysis for a compositional component in this study, at

least 50% of the values for a component had to be grea ter than the assay limit of quantitation (LOQ). Components with more than 50% of ob servations below the assay LOQ were excluded from summa ries and analysis. The fo llowing two components with

more than 50% of the observations below the assay LOQ were ex cluded: caffeoyl lignin and 5-hydroxyguaiacyl lignin.

The data were assessed for potential outli ers using a studentized PRESS residuals

calculation. A PRESS residual is the di fference between any value and its value predicted from a statistical model that excludes the data point. The studentized version

scales these residuals so that the values te nd to have a standard normal distribution when outliers are absent. Thus, most values are expected to be between ± 3. Extreme data points that are also outside of the ± 6 stude ntized PRESS residual range are considered for exclusion as outliers from the final analyses . In lignin units outlie rs as a proportion of HGS lignin were indentified in three conventio nal commercial reference varieties. All lignin unit component data from the three conventional commercial reference varieties were removed from the statistical analyses.

Alfalfa forage compositional components we re statistically analyzed using a mixed model analysis. The six replicated field site s were analyzed as a combined data set. These combined-site analyses used model (1).

(1) Y ijk 

### = U + T i 

### + L j 

### + B(L) jk



### + LT ij 

### + e ijk 

### ,

where Y ijk 

### = unique individual observa tion, U = overall mean, T i 

### = substance effect, L j 

### = random location effect, B(L) jk



### = random block within location effect, LT ij 

### = random location by substance inte raction effect, and e ijk 

### = residual error.

A supplementary individual site analysis used model (2).

(2) Y ij 

### = U + T i 

### + B j 

### + e ij 

### ,

where Y ij 

### = unique individua l observation, U = overall mean, T i 

### = substance effect, B j 

### = random block effect, and e ij 

### = residual error.Monsanto Company 12-AL-246U 250 of 407 

### For each compositional component, a range of observed values and a 99% tolerance interval were calculated. A tolerance interval is an interval that one can claim, with a specified degree of confidence, contains at least a specifi ed proportion, p, of an entire sampled population for the parameter measure d. The calculated tole rance intervals are expected to contain, with 95% confidence, 99% of the quantitie s expressed in the population of conventional alfalfa. Each to lerance interval estimate was based upon the average observation for each unique reference material. Because negative quantities are not possible, negative calculated lower tolerance bounds were set to zero.

SAS  

### (Version 9.2) software was used to genera te all summary statistics and perform all analyses.

Report tables present p-values from SAS as either <0.001 or the actual value truncated to three decimal places.

Table D-2. Re-expression Formulas for St atistical Analysis of Composition Data

Component From (X) To Formula 1

Lignin unit (HGS) μmol/g CWR % Total (HGS) (100)Xj/  X, for each HGSj

where  X is

over all the

HGS

1 ‘X’ is the individual sample value

.

 SAS is a registered trademark of the SAS Institute, Inc. Cary, North Carolina.Monsanto Company 12-AL-246U 251 of 407Table D-3. Statistical Summary of AlfalfaForage Lignin Subunits for KK179 vs. Conventional Control (Conducted at SamuelRoberts Noble Foundation)Difference (Test minus Control)Analytical Component (Units)1Test2Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%ConfidenceIntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Site CAPRLignin Subunits (μmol/g CWR)Guaiacyl lignin unit72.86 (11.69)92.84 (8.27)-19.99 (14.32)-53.85, 13.880.2058.83, 176.39(70.00 - 75.71)(66.63 - 113.14)(-35.42 - -10.46)(25.34 - 153.11)Hydroxyphenyl lignin unit5.41 (0.62)4.40 (0.42)1.01 (0.66)-0.78, 2.790.1961.59, 6.91(5.17 - 6.05)(2.78 - 5.26)(0.27 - 1.39)(0.29 - 8.26)Syringyl lignin unit73.27 (10.15)62.98 (6.72)10.29 (10.99)-19.01, 39.590.3960, 120.96(63.21 - 87.67)(46.77 - 76.24)(6.87 - 15.10)(5.64 - 110.93)Monsanto Company 12-AL-246U 252 of 407Table D-3 (continued). Statistical Summaryof Alfalfa Forage Lignin Subunits for KK179 vs. Conventional Control (ConductedatSamuel Roberts Noble Foundation)Difference (Test minus Control)Analytical Component (Units)1Test2Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%ConfidenceIntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Site CAPRLignin Subunits (% Total HGS)Guaiacyl lignin unit47.59 (1.64)57.82 (1.16)-10.23 (2.01)-14.97, -5.480.00146.69, 76.44(44.92 - 50.27)(56.88 - 58.92)(-14.01 - -6.61)(50.02 - 76.69)Hydroxyphenyl lignin unit3.70 (0.35)2.75 (0.22)0.96 (0.40)-0.038, 1.950.0560, 6.74(3.07 - 4.34)(2.39 - 3.29)(0.47 - 1.05)(0.18 - 6.23)Syringyl lignin unit48.70 (1.83)39.43 (1.30)9.27 (2.24)3.96, 14.570.00417.39, 53.32(45.39 - 52.01)(38.48 - 40.26)(5.56 - 13.54)(17.07 - 46.14)RatioS:G Ratio1.03 (0.065)0.68 (0.046)0.35 (0.080)0.16, 0.540.0030.21, 0.96(0.90 - 1.16)(0.65 - 0.70)(0.20 - 0.50)(0.22 - 0.92)Site IARLLignin Subunits (μmol/g CWR)Guaiacyl lignin unit37.57 (6.10)37.48 (6.10)0.088 (8.63)-19.44, 19.610.9928.83, 176.39(21.17 - 65.54)(33.11 - 43.31)(-13.83 - 27.03)(25.34 - 153.11)Monsanto Company 12-AL-246U 253 of 407Table D-3 (continued). Statistical Summaryof Alfalfa Forage Lignin Subunits for KK179 vs. Conventional Control (Conductedat Samuel Roberts Noble Foundation)Difference (Test minus Control)Analytical Component (Units)1Test2Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%ConfidenceIntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Site IARLLignin Subunits (μmol/g CWR)Hydroxyphenyl lignin unit4.88 (1.08)2.69 (1.08)2.19 (1.52)-1.25, 5.640.1831.59, 6.91(2.20 - 10.14)(2.40 - 3.06)(-0.20 - 7.24)(0.29 - 8.26)Syringyl lignin unit17.46 (2.63)13.74 (2.63)3.72 (3.72)-4.69, 12.130.3430, 120.96(9.82 - 27.96)(12.20 - 15.36)(-2.38 - 13.42)(5.64 - 110.93)Lignin Subunits (% Total HGS)Guaiacyl lignin unit62.80 (0.81)69.50 (0.81)-6.70 (1.06)-9.30, -4.11<0.00146.69, 76.44(60.80 - 63.78)(68.47 - 70.56)(-9.36 - -5.09)(50.02 - 76.69)Hydroxyphenyl lignin unit7.55 (0.45)4.99 (0.45)2.56 (0.61)1.07, 4.050.0050, 6.74(6.39 - 9.78)(4.84 - 5.18)(1.43 - 4.60)(0.18 - 6.23)Syringyl lignin unit29.65 (0.90)25.51 (0.90)4.14 (1.03)1.63, 6.650.00617.39, 53.32(26.98 - 31.80)(24.60 - 26.57)(0.99 - 6.92)(17.07 - 46.14)Monsanto Company 12-AL-246U 254 of 407Table D-3 (continued). Statistical Summaryof Alfalfa Forage Lignin Subunits for KK179 vs. Conventional Control (Conductedat Samuel Roberts Noble Foundation)Difference (Test minus Control)Analytical Component (Units)1Test2Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%ConfidenceIntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Site IARLRatioS:G Ratio0.47 (0.019)0.37 (0.019)0.11 (0.022)0.051, 0.160.0030.21, 0.96(0.43 - 0.52)(0.35 - 0.39)(0.049 - 0.17)(0.22 - 0.92)Site ILCYLignin Subunits (μmol/g CWR)Guaiacyl lignin unit87.60 (12.13)104.06 (12.13)-16.46 (13.04)-48.36, 15.440.2538.83, 176.39(52.96 - 134.96)(77.34 - 131.40)(-29.52 - 3.56)(25.34 - 153.11)Hydroxyphenyl lignin unit6.19 (1.03)4.14 (1.03)2.05 (1.38)-1.34, 5.440.1881.59, 6.91(3.16 - 10.84)(2.92 - 5.49)(0.24 - 5.35)(0.29 - 8.26)Syringyl lignin unit67.00 (7.82)63.39 (7.82)3.61 (9.39)-19.37, 26.590.7130, 120.96(49.44 - 85.83)(47.39 - 91.89)(-18.80 - 27.05)(5.64 - 110.93)Lignin Subunits (% Total HGS)Guaiacyl lignin unit53.58 (1.89)60.96 (1.89)-7.37 (2.68)-13.43, -1.310.02246.69, 76.44(49.12 - 61.66)(57.44 - 63.59)(-13.10 - 4.22)(50.02 - 76.69)Monsanto Company 12-AL-246U 255 of 407Table D-3 (continued). Statistical Summaryof Alfalfa Forage Lignin Subunits for KK179 vs. Conventional Control (Conductedat Samuel Roberts Noble Foundation)Difference (Test minus Control)Analytical Component (Units)1Test2Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%ConfidenceIntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Site ILCYLignin Subunits (% Total HGS)Hydroxyphenyl lignin unit3.69 (0.30)2.40 (0.30)1.29 (0.43)0.32, 2.250.0140, 6.74(2.99 - 4.95)(2.29 - 2.54)(0.58 - 2.55)(0.18 - 6.23)Syringyl lignin unit42.73 (2.13)36.64 (2.13)6.09 (3.02)-0.74, 12.910.07417.39, 53.32(33.39 - 47.77)(34.02 - 40.17)(-6.77 - 12.52)(17.07 - 46.14)RatioS:G Ratio0.81 (0.063)0.60 (0.063)0.21 (0.089)0.0083, 0.410.0420.21, 0.96(0.54 - 0.97)(0.54 - 0.70)(-0.16 - 0.41)(0.22 - 0.92)Site KSLALignin Subunits (μmol/g CWR)Guaiacyl lignin unit62.88 (3.72)82.54 (3.72)-19.66 (5.25)-31.54, -7.770.0048.83, 176.39(59.88 - 66.12)(65.03 - 91.43)(-31.46 - 1.09)(25.34 - 153.11)Hydroxyphenyl lignin unit4.38 (0.28)4.38 (0.28)0.0050 (0.39)-0.88, 0.890.9901.59, 6.91(4.14 - 4.60)(3.79 - 5.02)(-0.70 - 0.35)(0.29 - 8.26)Monsanto Company 12-AL-246U 256 of 407Table D-3 (continued). Statistical Summary of Alfalfa Forage LigninSubunits for KK179 vs. Conventional Control(Conducted at Samuel Roberts Noble Foundation)Difference (Test minus Control)Analytical Component (Units)1Test2Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%ConfidenceIntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Site KSLALignin Subunits (μmol/g CWR)Syringyl lignin unit51.49 (3.43)52.06 (3.43)-0.58 (4.85)-11.54, 10.390.9080, 120.96(44.58 - 54.81)(40.48 - 62.77)(-18.19 - 14.33)(5.64 - 110.93)Lignin Subunits (% Total HGS)Guaiacyl lignin unit53.00 (0.72)59.46 (0.72)-6.46 (1.02)-8.77, -4.15<0.00146.69, 76.44(52.15 - 54.91)(57.56 - 61.36)(-9.21 - -2.65)(50.02 - 76.69)Hydroxyphenyl lignin unit3.70 (0.25)3.19 (0.25)0.52 (0.36)-0.30, 1.330.1830, 6.74(3.49 - 4.22)(2.82 - 3.76)(-0.19 - 1.33)(0.18 - 6.23)Syringyl lignin unit43.30 (0.82)37.35 (0.82)5.94 (1.16)3.33, 8.56<0.00117.39, 53.32(40.88 - 44.36)(35.81 - 39.56)(1.32 - 8.54)(17.07 - 46.14)RatioS:G Ratio0.82 (0.024)0.63 (0.024)0.19 (0.034)0.11, 0.27<0.0010.21, 0.96(0.74 - 0.85)(0.58 - 0.69)(0.057 - 0.27)(0.22 - 0.92)Monsanto Company 12-AL-246U 257 of 407Table D-3 (continued). Statistical Summary of Intended Changes in Forage Lignin Subunits for KK179 vs. ConventionalControl (Conducted at Samuel Roberts Noble Foundation)Difference (Test minus Control)Analytical Component (Units)1Test2Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%ConfidenceIntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Site TXCLLignin Subunits (μmol/g CWR)Guaiacyl lignin unit64.68 (9.26)91.11 (9.26)-26.44 (12.16)-56.19, 3.320.0728.83, 176.39(54.72 - 74.33)(71.43 - 105.30)(-39.11 - -9.45)(25.34 - 153.11)Hydroxyphenyl lignin unit4.18 (0.91)4.55 (0.91)-0.37 (1.28)-3.27, 2.530.7771.59, 6.91(2.74 - 5.41)(4.24 - 4.94)(-1.76 - 1.17)(0.29 - 8.26)Syringyl lignin unit59.13 (9.52)55.21 (9.52)3.92 (12.79)-27.37, 35.220.7690, 120.96(45.79 - 70.15)(45.73 - 63.25)(-17.46 - 15.72)(5.64 - 110.93)Lignin Subunits (% Total HGS)Guaiacyl lignin unit50.67 (1.07)60.26 (1.07)-9.59 (1.51)-13.01, -6.16<0.00146.69, 76.44(49.59 - 52.63)(58.06 - 64.22)(-14.63 - -5.43)(50.02 - 76.69)Hydroxyphenyl lignin unit3.28 (0.53)3.08 (0.53)0.20 (0.75)-1.49, 1.880.7970, 6.74(2.04 - 4.13)(2.59 - 4.05)(-0.85 - 1.02)(0.18 - 6.23)Monsanto Company 12-AL-246U 258 of 407Table D-3 (continued). Statistical Summary of Intended Changes in Forage Lignin Subunits for KK179 vs. ConventionalControl (Conducted at Samuel Roberts Noble Foundation)Difference (Test minus Control)Analytical Component (Units)1Test2Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%ConfidenceIntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Site TXCLLignin Subunits (% Total HGS)Syringyl lignin unit46.05 (0.99)36.66 (0.99)9.39 (1.38)6.00, 12.78<0.00117.39, 53.32(44.04 - 47.67)(33.20 - 39.14)(4.90 - 13.61)(17.07 - 46.14)RatioS:G Ratio0.91 (0.031)0.61 (0.031)0.30 (0.043)0.20, 0.40<0.0010.21, 0.96(0.84 - 0.95)(0.52 - 0.67)(0.16 - 0.43)(0.22 - 0.92)Site WIDLLignin Subunits (μmol/g CWR)Guaiacyl lignin unit84.34 (5.42)94.31 (5.42)-9.97 (7.66)-27.30, 7.360.2258.83, 176.39(77.36 - 88.36)(69.76 - 111.31)(-24.67 - 18.60)(25.34 - 153.11)Hydroxyphenyl lignin unit5.24 (0.69)3.10 (0.69)2.15 (0.78)0.23, 4.070.0331.59, 6.91(4.34 - 6.22)(0.58 - 5.48)(-0.38 - 3.76)(0.29 - 8.26)Syringyl lignin unit69.11 (4.76)55.09 (4.76)14.02 (6.73)-1.20, 29.250.0660, 120.96(63.76 - 78.51)(34.94 - 64.85)(-0.86 - 43.57)(5.64 - 110.93)Monsanto Company 12-AL-246U 259 of 407Table D-3 (continued). Statistical Summary of Intended Changes in Forage Lignin Subunits for KK179 vs. ConventionalControl (Conducted at Samuel Roberts Noble Foundation)Difference (Test minus Control)Analytical Component (Units)1Test2Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%ConfidenceIntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Site WIDLLignin Subunits (% Total HGS)Guaiacyl lignin unit53.18 (1.25)62.12 (1.25)-8.94 (1.76)-12.92, -4.95<0.00146.69, 76.44(51.32 - 54.77)(57.24 - 65.32)(-14.01 - -2.47)(50.02 - 76.69)Hydroxyphenyl lignin unit3.33 (0.42)2.04 (0.42)1.29 (0.49)0.088, 2.490.0390, 6.74(2.67 - 4.22)(0.34 - 3.33)(0.0067 - 2.33)(0.18 - 6.23)Syringyl lignin unit43.49 (1.03)35.84 (1.03)7.65 (1.46)4.35, 10.94<0.00117.39, 53.32(41.89 - 45.60)(32.72 - 39.43)(2.46 - 12.88)(17.07 - 46.14)RatioS:G Ratio0.82 (0.032)0.58 (0.032)0.24 (0.045)0.14, 0.34<0.0010.21, 0.96(0.76 - 0.89)(0.50 - 0.69)(0.076 - 0.39)(0.22 - 0.92)1Total HGS is the sum of Hydroxyphenyl, Guaiacyl and Syringyl lignin subunits (μmol/g CWR); S:G Ratio = Syringyl lignin unit divided byGuaiacyl lignin unit. CWR = Cell Wall Residue2Test refers to KK179.3Mean (S.E.) = least-square mean (standard error)4Control refers to the non-biotechnology derived, conventional control, C0-Syn1.5With 95% confidence, interval contains 99% of the values expressed in the population of conventional commercial reference varieties. Negativelimits set to zero.Monsanto Company 12-AL-246U 260 of 407Table D-4. Statistical Summary of Alfalfa Forage Total Lignin (ADL) Levels forKK179 vs. Conventional Control (Conductedat Dairy One Forage Lab)Difference (Test minus Control)Analytical Component (Units)1Test2Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%ConfidenceIntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Site CAPRAcid Detergent Lignin (% dw)5.79 (0.44)8.38 (0.44)-2.59 (0.54)-3.91, -1.270.0021.39, 12.54(4.93 - 6.57)(7.30 - 10.10)(-3.53 - -1.23)(1.70 - 10.03)Site IARLAcid Detergent Lignin (% dw)2.79 (0.46)3.70 (0.46)-0.91 (0.65)-2.37, 0.550.1931.39, 12.54(2.73 - 2.87)(2.23 - 5.20)(-2.47 - 0.63)(1.70 - 10.03)Site ILCYAcid Detergent Lignin (% dw)6.87 (0.60)6.87 (0.60)0 (0.84)-1.91, 1.911.0001.39, 12.54(6.43 - 7.60)(5.67 - 8.63)(-2.20 - 1.30)(1.70 - 10.03)Site KSLAAcid Detergent Lignin (% dw)5.35 (0.49)8.35 (0.49)-3.00 (0.70)-4.58, -1.420.0021.39, 12.54(3.47 - 6.53)(7.60 - 9.77)(-4.33 - -1.07)(1.70 - 10.03)Monsanto Company 12-AL-246U 261 of 407Table D-4 (continued). Statistical Summaryof Alfalfa Forage Total Lignin (ADL)Levels for KK179 vs. Conventional Control(Conducted at Dairy One Forage Lab)Difference (Test minus Control)Analytical Component (Units)1Test2Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%ConfidenceIntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Site TXCLAcid Detergent Lignin (% dw)4.65 (0.26)7.32 (0.26)-2.67 (0.37)-3.58, -1.76<0.0011.39, 12.54(4.07 - 5.03)(6.40 - 8.10)(-3.17 - -1.40)(1.70 - 10.03)Site WIDLAcid Detergent Lignin (% dw)6.92 (0.29)6.96 (0.29)-0.042 (0.37)-0.94, 0.860.9131.39, 12.54(6.70 - 7.07)(5.97 - 7.87)(-1.17 - 0.87)(1.70 - 10.03)1dw = dry weight.2Test refers to KK179.3Mean (S.E.) = least-square mean (standard error)4Control refers to the non-biotechnology derived, conventional control, C0-Syn1.5With 95% confidence, interval contains 99% of the values expressed in the population of conventional commercial reference varieties. Negativelimits set to zero.Monsanto Company 12-AL-246U 262 of 407 

### References for Appendix D

Lapierre, C., B. Monties and C. Rolando. 1985. Thioacidolysis of Lignin Comparison with Acidolysis. Journal of Wood Chemistry and Technology 5: 277-292.

Lapierre, C., B. Pollet and C. Rola ndo. 1995. New Insights into the Molecular Architecture of Hardwood Lignins by Ch emical Degradative Methods. Research on

Chemical Intermediates 21: 397-412.

Weston, T.R., V. Nayigihugu and B.W. He ss. 2006. Comparison of techniques for quantitative analysis of acid detergent ligni n in roughages. Pages 242-244 in Proceedings, Western Section, American Society of Animal Science, American Society of Animal Science, Champaign, Illinois.Monsanto Company 12-AL-246U 263 of 407 

### Appendix E: Materials, Methods, and Results for Compositional Analysis of KK179 Forage

E.1. Materials

Forage from KK179 (Seed Lot Number 1126628 9) and the conventi onal control C0-Syn1 (Seed Lot Number 11266292) was evaluated. The conventional control C0-Syn1has background genetics similar to that of KK179. The convent ional commercial reference

varieties were 14 conventional al falfa varieties (Table E-1).

Table E-1. Conventional Commercial Reference Varieties

Material Name Seed Lot

Number Field Site Pioneer 54V54 11266293 ILCY, WIDL Croplan LegenDairy 5.0 11266294 IARL, ILCY, WIDL Producer's Choice PGI 437 11266295 ILCY, KSLA
Pioneer 54H11 11266296 IARL, TXCL

Pioneer 54V46 11266297 KSLA Pioneer 54V09 11266298 CAPR, TXCL
DKA50-18 11266299 IARL, KSLA

America's Alfalfa Archer III 11266301 CAPR WL 319HQ 11266302 CAPR, KSLA, TXCL Dow/Dairyland Hybriforce 400 11266303 ILCY, WIDL AMPAC seed company Attention 11266304 CAPR

Vernal 11266305 WIDL Ranger 11266306 IARL Dow/Dairyland Hybriforce 2400 11266307 TXCL



### E.2. Characterization of the Materials

The identities of forage samples from KK179, the conventional control C0-Syn1, and

conventional commercial reference varieties were confirmed by verifying the chain of custody documentation prior to analysis. To fu rther confirm the iden tities of KK179, the conventional control C0-Syn1, and conventiona l commercial reference varieties, eventspecific polymerase chain re action (PCR) analyses were conducted on the forage from each site to confirm the pres ence or absence of KK179.

E.3. Field Production of the Samples

Forage samples from KK179, the conventional control C0-Syn1, and conventional

commercial alfalfa varieties were collected fr om each of four replicated plots from six field sites [Tulare County, California (CAP R); Jefferson County, Iowa (IARL); Clinton County, Illinois (ILCY); Pawnee County, Ka nsas (KSLA); Armstrong County, Texas (TXCL); and Walworth County, Wisconsin (WIDL) ]. The field plots were established in 2010 from plants that were grown in a greenhouse. Prior to transplanting in the field, the presence or absence of KK179 wa s verified using PCR. The fi eld plots were planted in a randomized complete block design. All samp les at the field sites were grown under normal agronomic field conditions for their respective geographic re gions. Between 1Monsanto Company 12-AL-246U 264 of 407 

### and 10% bloom, all whole plants that were two to three inches above the soil surface

were harvested from the center of each indi vidual plot. A subsample (about 680g) of plants was randomly collected from a ll harvested plants in each plot.

Forage samples were shipped frozen from th e field sites to Monsanto Company (Saint Louis, Missouri). All samples were ground to a powder and stored in a freezer set to maintain -20°C located at Monsanto Company (Saint Louis, Missouri). Subsamples were shipped on dry ice to Covance Laboratories Inc. (Madison, Wisconsin) for compositional analysis. Subsamples were lyophilized and shipped on dry ice to Samuel Roberts Noble Foundation (Ardmore, Oklahoma) for analysis of saponins.

E.4. Summary of Analytical Methods

Compositional assessments by Covance and th e Samuel Roberts N oble Foundation were conducted to establish the compositional equivalence of KK179 compared to conventional alfalfa using the principles and analytes outli ned in the OECD consensus document for alflafa compositi on (OECD, 2005). Forage sa mples analyzed by Covance Laboratories Inc. included nutrients, proxi mates (ash, fat, moisture, and protein), carbohydrates by calculation, acid detergent fibe r (ADF), neutral detergent fiber (NDF), acid detergent lignin (lignin), minerals (Ca, Cu, Fe, Mg, Mn, P, K, Na, and Zn), and amino acids (18 components). Anti-nutri ents assessed by Covance included daidzein, glycitein, genistein, coumesterol, formononeti n, and biochanin A. Secondary metabolites assessed by Covance included p- coumaric acid, ferulic acid, sinapic acid, total polyphenols, free phenylalanine, and canavanine. Forage samples were analyzed by

Samuel Roberts Noble Foundation for total sa ponins, total bayogeni n, total hederagenin, total medicagenic acid, total soyasapogenol B, total soyasapogenol E, and total zanhic

acid.

E.4.1. Acid Detergent Fiber (Conducted at Covance Laboratories Inc.)

The ANKOM2000 Fiber Analyzer automated th e process of removal of proteins, carbohydrates, and ash. Fats and pigments were removed with an ace tone wash prior to analysis. The fibrous residue that was primarily cellulose and lignin and insoluble protein complexes remained in the ANKOM filter bag, and was determined gravimetrically (Goering and Van Soest, 1970; Komarek et al., 1993). The limit of quantitation was 0.100%.

E.4.2. Amino Acid Composition (Conducted at Covance Laboratories Inc.)

The following eighteen amino acids were analyzed:

 Total alanine

 Total arginine

 Total aspartic acid (i ncluding asparagine)

 Total cystine (including cysteine)

 Total glutamic acid (including glutamine)

 Total glycineMonsanto Company 12-AL-246U 265 of 407 

###  Total histidine

 Total isoleucine

 Total leucine

 Total lysine

 Total methionine

 Total phenylalanine

 Total proline

 Total serine

 Total threonine

 Total tryptophan

 Total tyrosine

 Total valine

The samples were hydrolyzed in 6N hydrochloric acid for approximately 24 hours at approximately 106-110oC. Phenol was added to the 6N hydrochloric acid to prevent halogenation of tyrosine. Cystine an d cysteine are converted to S-2- carboxyethylthiocysteine by the addition of dithiodipropionic acid. Tryptophan was hydrolyzed from proteins by heating at approximately 110o C in 4.2N sodium hydroxide

for 20 hours.

The samples were analyzed by HPLC after pre-injection derivatization. The primary

amino acids were derivatized with o -phthalaldehyde (OPA) and the secondary amino acids are derivatized with fluorenylmethyl chloroformate (FMOC) before injection (AOAC, 2011g; Barkholt and Jensen, 1989; Schus ter, 1988). The limit of quantitation was 0.100 mg/g.

Reference Standards:

 Sigma-Aldrich, L-Alanine, 99.9%, Lot Number 1440397

 Sigma-Aldrich, L-Arginine Monohydroc hloride, 100%, Lot Number 1361811

 Sigma-Aldrich, L-Aspartic Ac id, 100.6%, Lot Number BCBB9274

 Sigma-Aldrich, L-Cystine, 99.9%, Lot Number 1418036

 Sigma-Aldrich, L-Glut amic Acid, 100.2%, 1423805

 Sigma-Aldrich, Glycine, 100%, Lot Number 1119375

 Sigma-Aldrich, L-Histidine Monohydrochl oride Monohydrate, 99.9%, Lot Number

BCBB1348

 Sigma-Aldrich, L-Isoleuci ne, 100%, Lot Number 1423806

 Sigma-Aldrich, L-Leucine, 98.6%, Lot Number BCBB1733

 Sigma-Aldrich, L-Lysine Monohydrochloride, 100.2%, 1362380

 Sigma-Aldrich, L-Methionine, 99.9%, Lot Number 1423807

 Sigma-Aldrich, L-Phenylalanin e, 100%, Lot Number BCBB9200

 Sigma-Aldrich, L-Proline, 99.7%, Lot Number 1414414

 Sigma-Aldrich, L-Serine , 99.9%, Lot Number 1336081

 Sigma-Aldrich, L-Threonin e, 100%, Lot Number 1402329

 Sigma-Aldrich, L-Trypt ophan, 99.8%, BCBB1284Monsanto Company 12-AL-246U 266 of 407 

###  Sigma-Aldrich, L-Tyrosine, 99.5%, Lot Number BCBB5393

 Sigma-Aldrich, L-Valine, 100%, Lot Number 1352709

E.4.3. Ash (Conducted at Covance Laboratories Inc.)

The sample was placed in an electric fu rnace at 550°C and ignited. The nonvolatile matter remaining was quantitated gravimetrical ly and calculated to determine percent ash (AOAC, 2011e). The limit of quantitation was 0.100%.

E.4.4. Biochanin A (Conducted at Covance Laboratories Inc.)

Samples were extracted using an acidified acetonitrile/water solution combined with mixing. The extracts were then quantitat ed using isocratic reverse phase highperformance liquid chromatography (HPLC) with UV detection (Chen et al., 2010; Saloniemi et al., 1995). The lim it of quantitation was 10.0 ppm.

Reference Standards:

 Sigma-Aldrich, Biochanin A, 97.4%, Lot Number S39234-418

E.4.5. Canavanine, Free (Conducted at Covance Laboratories Inc.)

The samples were extracted with 0.02N HCL an d purified with ultrafiltration. Analysis was performed with an automated amino aci d analyzer (AOAC, 2011a). The limit of quantitation was 5.00 μg/g.

Reference Standards:

 Sigma-Aldrich, L-Canavanine Free Base from Jack Beans, 99%, Lot Number

116K7026

E.4.6. Carbohydrate (Conducted at Covance Laboratories Inc.)

The total carbohydrate level was calculated by difference using the fresh weight-derived data and the following equation (USDA, 1973):

% carbohydrates = 100 % - (% protein + % fat + % moisture + % ash)

The limit of quantitation was 0.100%.

E.4.7. Coumesterol and Formononetin (C onducted at Covance Laboratories Inc.)

The samples were extracted with 0.02N HCL an d purified with ultrafiltration. Analysis was performed with an automated amino acid analyzer (Pettersson and Kiessling, 1984; Saloniemi et al., 1995). The lim it of quantitation was 5.00 μg/g.Monsanto Company 12-AL-246U 267 of 407 

### Reference Standards:

 Sigma Aldrich, Formononetin, 99.1%, Lot Number BCBD9644V

 Indofine Chemical Co., Coumestrol, 99.24%, Lot Number 1103121

E.4.8. Fat by Acid Hydrolysis (Conducted at Covance Laboratories Inc.)

The sample was hydrolyzed with hydrochloric acid. The fat was extracted using ether and hexane. The extract was dried down and filtered through a sodium sulfate column. The remaining extract was then evaporate d, dried, and weighed (AOAC, 2010a; 2010b). The limit of quantitation was 0.100%.

E.4.9. Free Phenylalanine (Conducted at Covance Laboratories Inc.)

The sample was extracted in 0.1N HCl. Determination was by high-performance liquid chromatography (HPLC) with fluorescence cy stine only detection. The phenylalanine

was derivatized with o -phthalaldehyde before injection (Schuster, 1988). The limit of quantitation was 0.0200 mg/g.

Reference Standards:

 Sigma-Aldrich, Phenylalanine, 100%, Lot Number BCBB9200

E.4.10. Minerals/ICP Emission Spectrome try (Conducted at Covance Laboratories Inc.)

The following nine minerals were analyzed:

 Calcium

 Copper

 Iron

 Magnesium

 Manganese

 Phosphorus

 Potassium

 Sodium

 Zinc

The sample was dried, precharred, and ashed overnight in a muffle furnace set to maintain 500°C. The ashed sample was re-a shed with nitric acid, treated with hydrochloric acid, taken to dryne ss, and put into a solution of 5% hydrochloric acid. The amount of each element was determined at appropriate wavelengths by comparing the emission of the unknown sample, measur ed on the inductively coupled plasma spectrometer, with the emission of th e standard solutions (AOAC, 2011c; 2011h).Monsanto Company 12-AL-246U 268 of 407 

### Reference Standards:

Inorganic Ventures Reference Standa rds and Limits of Quantitation:

Mineral Lot No. Concentration (μg/mL) LOQ (ppm)

Calcium E2-MEB393070MCA,

E2-MEB393072 200,

1000 20.0

Copper E2-MEB393070MCA,

E2-MEB393071MCA 2.00,

10.0 0.500

Iron E2-MEB393070MCA,

E2-MEB393073 10.0,

50.0 2.00

Magnesium E2-MEB393070MCA,

E2-MEB393071MCA 50.0,

250 20.0

Manganese E2-MEB393070MCA,

E2-MEB393071MCA 2.00,

10.0 0.300

Phosphorus E2-MEB393070MCA,

E2-MEB393072 200,

1000 20.0

Potassium E2-MEB393070MCA,

E2-MEB393072 200,

1000 100

Sodium E2-MEB393070MCA,

E2-MEB393072 200,

1000 100

Zinc E2-MEB393070MCA,

E2-MEB393071MCA 10.0,

50.0 0.400Monsanto Company 12-AL-246U 269 of 407 

### E.4.11. Isoflavones (Conducted at Covance Laboratories Inc.)

The sample was extracted using a solution of hydrochloric acid and reagent alcohol heated on hot plates. The extract was brough t to volume, diluted, and centrifuged. An aliquot of the supernatant was placed ont o a C18 solid-phase extraction column. Unwanted components of the matrix were ri nsed off with 20% methanol and then the

isoflavones were eluted with 80% metha nol. The sample was analyzed on a highperformance liquid chromatography system with ultraviolet detection and was compared

to an external standard curve of known standards for quantitation (Pettersson and Kiessling, 1984; Seo and Morr, 1984). The limit of quantitation was 10.0 ppm.

Reference Standards:

 LC Labs, Daidzein, 99.7%, Lot Number DA-121

 LC Labs, Glycitein, 99.8%, Lot Number ARH-114

 LC Labs, Genistein, 99.7%, Lot Number CH-148

E.4.12. Acid Detergent Lignin (Conducted at Covance Laboratories Inc.)

The samples were analyzed using the conve ntional crucible method that is a manual method (Goering and Van Soest, 1970). Th e protein, carbohydrate, and ash contents were dissolved using a boiling detergent solution and filtered off. The fats and pigments were removed via an acetone wash leaving the lignocellulose fraction in a frit. The

cellulose was then dissolved with sulfuric acid leaving the lignin fraction, which was determined gravimetrically. The limit of qua ntitation was calculat ed and reported on fresh weight basis. The lim it of quantitation was 0.100%.

E.4.13. Moisture (Conducted at Covance Laboratories Inc.)

The sample was dried in a vacuum oven at approximately 100°C. The moisture weight loss was determined and converted to pe rcent moisture (AOAC, 2011b; 2011d). The limit of quantitation was 0.100%.

E.4.14. Neutral Detergent Fiber (Conducted at Covance Laboratories Inc.)

The ANKOM2000 Fiber Analyzer automated the process of the rem oval of protein, carbohydrate, and ash. Fats and pigments were removed with an ac etone wash prior to analysis. Hemicellulose, cellulose, lignin, and insoluble protein fraction were left in the filter bag and determined gravimetrically (AACC, 1998; Goering and Van Soest, 1970; Komarek et al., 1993). The lim it of quantitation was 0.100%.

E.4.15. p -Coumaric Acid, Ferulic Acid, Sina pic Acid (Conducted at Covance Laboratories Inc.)

The sample was extracted with methanol followed by alkaline hydrolysis and buffering prior to injection on an analytical high-performance liquid chromatography (HPLC) system for quantification of sinapic acid, p- coumaric acid, and ferulic acid by ultravioletMonsanto Company 12-AL-246U 270 of 407 

### (UV) detection (Hagerman and Nicholson, 1982). The limit of quantitation was 33.0

ppm.

Reference Standards:

 Sigma-Aldrich, p -Coumaric acid, 99.5%, Lot Number 060m1774v

 Sigma-Aldrich, 3, 5-Dimethoxy-4-Hydroxycin namic acid (Sinapic Acid), 99%, Lot

Number 040M1943

 ACROS Organics, 4-Hydroxy-3-methoxycinna mic Acid (Ferulic Acid), 99.4%, Lot

Number A0261354

E.4.16. Protein (Conducted at Covance Laboratories Inc.)

The protein and other organic nitrogen in the sample were converted to ammonia by digesting the sample with sulfur ic acid containing a catalyst mixture. The acid digest was

made alkaline. The ammonia was distille d and then titrated with a previously standardized acid. The per cent nitrogen was calculated a nd converted to equivalent protein using the factor 6.25 (AOAC, 2010c; 2011f; AOCS, 2009). The limit of quantitation was 0.100%.

E.4.17. Saponin (Conducted at Samuel Roberts Noble Foundation)

The following seven saponins were analyzed:

 Total Bayogenin

 Total Hederagenin

 Total Medicagenic Acid

 Total Soyasapogenol B

 Total Soyasapogenol E

 Total Zanhic Acid

 Total Saponins

The lyophilized ground samples were extrac ted with 80% methanol solution that contained the Internal Standard (Huhma n et al., 2005; Huhman and Sumner, 2002). Samples were analyzed with a Wate rs Acquity Ultra Performance Liquid Chromotography (UPLC) system (Waters Co rporation, Milford, Massachusetts) fitted with a Quadrupole Time of Flight (Q TOF) Premier mass spectrometer (Waters Corporation, Milford, Massachusetts). A reverse phase, 1.7-um UPLC BEH C18, 2.1 x 150 mm column was used for separations. BEH stands for Bridged Ethylene Hybrids (BEH Technolgy, Waters Corporation, Milfor d, Massachusetts). The mobile phase consisted of eluent A (0.1% [v/v] CH3C OOH/water and Eluent B (acetonitrile) and separations achieved using a linear gradient of 95% to 30% A over 30 min, 30% to 5% A over 3.0 min, 95% to 95% A over 3.0 min. The flow rate was 0.56 mls/min and the column temperature was maintained at 60°C. Compounds were detected using ESI in conventional negative ion mode from 50-2000 m/z. The QTOF Premier was operated under the following instrument parameters: desolvation temperature of 385°C; desolvation nitrogen gas flow of 850 L/h; capillary voltage of 2.9 kV; cone voltage of 48Monsanto Company 12-AL-246U 271 of 407 

### eV; and collision energy of 10 eV. The mass spectrometer system was calibrated using sodium formate, and raffinose was used for lockmass. Samples were normalized to the internal standard 7-OH coumarin by dividing individual peak area by the peak area of internal standard and multiplying by 1000. After normalization the saponins were separated by proposed aglycone class with peak areas of individual saponins summed. The units were response units/50 μg dry weight , where peak areas equals response units.

Internal Standard:

 Fluka, 7-OH Coumarin, 0.018 mg/ml, Lot Number 1256641

Reference Standards:

 Sigma, Esculin, 42.25 ng/inj ( 100 pmol), Lot Number 75H0706

 Fluka, 7-OH Coumarin, 48.6 ng/inj (300 pmol), Lot Number 1256641

 Sigma, Rutin, 76.3 ng/inj (125 pmol), Lot Number 10K0177

 Sigma, Naringin, 72.6 ng/inj (125 pmol), Lot Number 55H1139

 Sigma, Narigenin, 36.0 ng/inj ( 125 pmol), Lot Number 95H0996

 Sigma, Quercetin, 42.3 ng/inj ( 140 pmol) , Lot Number 85H0694

 Mark A. Berhow USDA Lab, Glc-Gal-Gl cUA- Soyasaponin B, 2.62 ng/inj (2.7 pmol)

 Mark A. Berhow USDA Lab, Rha-Gal- GlcUA- Soyasaponin B, 47.10 ng/inj (50.0 pmol)

 Mark A. Berhow USDA Lab, Rha-Ara- GlcUA- Soyasaponin B, 21.7 ng/inj (23.7 pmol)

 Mark A. Berhow USDA La b, Gal-GlcUA- Soyasaponin B, 10.5 ng/inj (13.1 pmol),

 Mark A. Berhow USDA La b, Ara-GlcUA- Soyasaponin B, 1.9 ng/inj (2.5 pmol)

E.4.18. Total Polyphenols (Conducted at Covance Laboratories Inc.)

Polyphenols were extracted from the samples with methanol. The samples were reacted with Folin-Ciocalteu Reagent to produ ce a color that can be measured spectrophotometrically at 760 nm. Results ar e reported in units of mg/g gallic acid equivalents. The limit of quantitation was 1.00 mg/g.

E.5. Data Processing and Statistical Analysis

After compositional analyses were performed, data spreadsheets containing individual values for each analysis were sent to M onsanto Company (St. Louis, Missouri) for

review. Data were then transf erred to Certus International (Chesterfield, Missouri) where they were converted into the appropriate units and statistically analyzed. The formulas that were used for re-expression of composition data for statistical analysis are listed in

Table D-2.

In order to complete a statistical analysis for a compositional component in this study, at

least 50% of the values for a component had to be grea ter than the assay limit of quantitation (LOQ). Components with more than 50% of ob servations below the assayMonsanto Company 12-AL-246U 272 of 407 

### LOQ were excluded from summaries and an alysis. The following seven components

with more than 50% of the observations be low the assay LOQ were excluded: biochanin A, coumesterol, daidzein, formononetin, ge nistein, glycitein, a nd sinapic acid.

If less than 50% of the observations for a component we re below the LOQ, individual analyses that were below the LOQ were assign ed a value equal to one-half the LOQ. In this study 56 values for sodium were assi gned a value of 50.0 ppm fw and 50 values for canavanine were assigned a value of 2.5 μg/g fw.

The data were assessed for potential outli ers using a studentized PRESS residuals

calculation. A PRESS residual is the di fference between any value and its value predicted from a statistical model that excludes the data point. The studentized version

scales these residuals so that the values te nd to have a standard normal distribution when outliers are absent. Thus, most values are expected to be between ± 3. Extreme data points that are also outside of the ± 6 stude ntized PRESS residual range are considered for exclusion as outliers from the final analyses. One copper value from one commercial

reference at the ILCY site was identified as an outlier, but the value was similar to other nearby data points and was not removed from the statistical analysis.

Alfalfa forage compositional components we re statistically analyzed using a mixed model analysis. The six replicated field site s were analyzed as a combined data set. These combined-site analyses used model (1).

(1) Y ijk 

### = U + T i 

### + L j 

### + B(L) jk



### + LT ij 

### + e ijk 

### ,

where Y ijk 

### = unique individual observa tion, U = overall mean, T i 

### = substance effect, L j 

### = random location effect, B(L) jk



### = random block within location effect, LT ij 

### = random location by substance inte raction effect, and e ijk 

### = residual error.

A supplementary individual site analysis used model (2).

(2) Y ij 

### = U + T i 

### + B j 

### + e ij 

### ,

where Y ij 

### = unique individua l observation, U = overall mean, T i 

### = substance effect, B j 

### = random block effect, and e ij 

### = residual error.

For each compositional component, a range of observed values and a 99% tolerance interval were calculated. A tolerance interval is an interval that one can claim, with a specified degree of confidence, contains at least a specifi ed proportion, p, of an entire sampled population for the parameter measure d. The calculated tole rance intervals are expected to contain, with 95% confidence, 99% of the quantitie s expressed in the population of conventional alfalfa. Each to lerance interval estimate was based upon the average observation for each unique reference material. Because negative quantities are not possible, negative calculated lower tolerance bounds were set to zero.

SAS (Version 9.2) software was used to generate all summary statistics and perform all analyses.Monsanto Company 12-AL-246U 273 of 407 

### Report tables present p-values from SAS as either <0.001 or the actual value truncated to three decimal places.

Table E-2. Re-expression Formulas for St atistical Analysis of Composition Data

Component From (X) 1 To Formula 2
Proximates (excluding Moisture),

Fiber % fw % dw X/d

Copper, Iron, Manganese, Zinc ppm fw mg/kg dw X/d Calcium, Magnesium, Phosphorus, Potassium, Sodium ppm fw % dw X/(10 4 d)

Ferulic Acid, p- Coumaric Acid ppm fw ppm dw X/d

Canavanine μg/g fw ppm dw X/d Total Polyphenols mg/g fw mg/g dw X/d Free Phenylalanine mg/g fw ppm dw 10 3 (X/d) Amino Acids (AA) mg/g fw % dw X/(10d)

Saponins response units/50 μg dw response units/ μg dw X/50

1 fw = fresh weight and dw = dry weight.

2 ‘X’ is the individual sample value; ‘d’ is the fraction of the sample that is dry matter

.Monsanto Company 12-AL-246U 274 of 407Table E-3. Statistical Summary of SiteCAPR Alfalfa Forage Nutrients for KK179 vs. Conventional Control (Conducted atCovance Laboratories)Difference (Test minus Control)Analytical Component (Units)1KK179Mean (S.E.)2(Range)Control3Mean (S.E.)(Range)Mean (S.E.)(Range)95%ConfidenceIntervalSignificance(p-Value)CommercialTolerance Interval4(Range)Proximate (% dw)Ash8.82 (0.54)9.85 (0.36)-1.04 (0.58)-2.59, 0.520.1426.70, 13.54(8.73 - 9.18)(8.79 - 10.59)(-1.80 - -0.32)(7.54 - 13.23)Carbohydrates69.61 (0.98)66.41 (0.69)3.20 (1.20)0.37, 6.030.03150.57, 81.80(68.88 - 70.34)(64.59 - 68.12)(1.55 - 5.75)(54.35 - 74.91)Moisture (% fw)78.40 (0.92)79.90 (0.65)-1.50 (1.13)-4.17, 1.170.22565.06, 90.61(76.40 - 80.40)(79.10 - 81.40)(-2.70 - 0.60)(66.10 - 85.30)Protein19.69 (0.55)21.86 (0.46)-2.17 (0.46)-3.42, -0.920.0089.26, 33.78(19.41 - 20.41)(20.72 - 22.47)(-2.75 - -1.67)(14.52 - 30.07)Total Fat1.54 (0.40)1.92 (0.28)-0.38 (0.49)-1.54, 0.780.4630.73, 3.59(1.47 - 1.62)(1.31 - 2.67)(-1.19 - 0.31)(0.53 - 4.21)Fiber (% dw)Acid Detergent Fiber29.98 (2.64)27.75 (1.58)2.23 (2.95)-5.69, 10.150.4896.16, 49.06(27.91 - 32.12)(24.31 - 30.69)(-2.78 - 7.81)(7.07 - 39.11)Monsanto Company 12-AL-246U 275 of 407Table E-3 (continued). Statistical Summary of Site CAPR Alfalfa Forage Nutrients for KK179 vs. Conventional Control(Conducted at Covance Laboratories)Difference (Test minus Control)Analytical Component (Units)1KK179Mean (S.E.)2(Range)Control3Mean (S.E.)(Range)Mean (S.E.)(Range)95%ConfidenceIntervalSignificance(p-Value)CommercialTolerance Interval4(Range)Fiber (% dw)Acid Detergent Lignin6.50 (0.43)6.99 (0.31)-0.50 (0.53)-1.75, 0.760.3802.13, 11.99(5.92 - 7.07)(6.31 - 7.54)(-1.63 - 0.012)(3.38 - 9.67)Neutral Detergent Fiber36.38 (1.98)36.43 (1.65)-0.043 (1.63)-4.55, 4.460.98012.04, 58.18(37.14 - 38.14)(30.91 - 40.74)(-3.60 - 1.72)(18.97 - 49.82)Amino Acid (% dw)Alanine1.06 (0.022)1.17 (0.015)-0.11 (0.026)-0.17, -0.0470.0040.49, 1.79(1.06 - 1.07)(1.12 - 1.21)(-0.095 - -0.063)(0.80 - 1.66)Arginine0.98 (0.027)1.10 (0.017)-0.12 (0.031)-0.20, -0.0470.0080.44, 1.59(0.95 - 0.99)(1.09 - 1.11)(-0.16 - -0.094)(0.70 - 1.44)Aspartic acid2.69 (0.13)3.06 (0.091)-0.36 (0.16)-0.74, 0.0100.0550.44, 5.63(2.44 - 2.94)(2.95 - 3.14)(-0.64 - -0.012)(1.96 - 5.15)Cystine0.22 (0.0097)0.23 (0.0069)-0.010 (0.012)-0.039, 0.0180.4110.12, 0.32(0.20 - 0.23)(0.21 - 0.24)(-0.023 - -0.014)(0.16 - 0.31)Monsanto Company 12-AL-246U 276 of 407Table E-3 (continued). Statistical Summary of Site CAPR Alfalfa Forage Nutrients for KK179 vs. Conventional Control(Conducted at Covance Laboratories)Difference (Test minus Control)Analytical Component (Units)1KK179Mean (S.E.)2(Range)Control3Mean (S.E.)(Range)Mean (S.E.)(Range)95%ConfidenceIntervalSignificance(p-Value)CommercialTolerance Interval4(Range)Amino Acid (% dw)Glutamic acid1.82 (0.044)2.05 (0.031)-0.23 (0.054)-0.36, -0.100.0030.81, 3.01(1.77 - 1.88)(2.01 - 2.11)(-0.31 - -0.14)(1.31 - 2.80)Glycine0.94 (0.020)1.01 (0.015)-0.076 (0.019)-0.13, -0.0250.0140.49, 1.44(0.90 - 0.94)(0.99 - 1.05)(-0.10 - -0.046)(0.70 - 1.33)Histidine0.43 (0.013)0.46 (0.0096)-0.026 (0.012)-0.059, 0.00740.0970.26, 0.63(0.42 - 0.43)(0.43 - 0.48)(-0.038 - -0.0067)(0.34 - 0.61)Isoleucine0.86 (0.025)0.94 (0.017)-0.080 (0.027)-0.15, -0.00840.0350.43, 1.36(0.83 - 0.87)(0.91 - 0.98)(-0.097 - -0.039)(0.63 - 1.27)Leucine1.42 (0.043)1.56 (0.027)-0.15 (0.048)-0.27, -0.0220.0290.70, 2.25(1.38 - 1.43)(1.51 - 1.63)(-0.16 - -0.076)(1.03 - 2.05)Lysine1.13 (0.054)1.24 (0.034)-0.11 (0.059)-0.27, 0.0490.1290.55, 1.82(1.10 - 1.12)(1.15 - 1.34)(-0.14 - -0.046)(0.82 - 1.73)Monsanto Company 12-AL-246U 277 of 407Table E-3 (continued). Statistical Summary of Site CAPR Alfalfa Forage Nutrients for KK179 vs. Conventional Control(Conducted at Covance Laboratories)Difference (Test minus Control)Analytical Component (Units)1KK179Mean (S.E.)2(Range)Control3Mean (S.E.)(Range)Mean (S.E.)(Range)95%ConfidenceIntervalSignificance(p-Value)CommercialTolerance Interval4(Range)Amino Acid (% dw)Methionine0.21 (0.018)0.24 (0.011)-0.034 (0.020)-0.086, 0.0180.1590.068, 0.42(0.20 - 0.21)(0.21 - 0.27)(-0.019 - -0.0094)(0.14 - 0.45)Phenylalanine0.96 (0.028)1.06 (0.020)-0.10 (0.035)-0.18, -0.0180.0230.48, 1.53(0.95 - 0.97)(1.01 - 1.11)(-0.11 - -0.045)(0.71 - 1.39)Proline0.85 (0.034)0.95 (0.021)-0.10 (0.037)-0.20, -0.00180.0470.43, 1.41(0.83 - 0.84)(0.91 - 1.01)(-0.13 - -0.064)(0.65 - 1.24)Serine0.84 (0.022)0.92 (0.015)-0.087 (0.027)-0.15, -0.0240.0140.45, 1.35(0.80 - 0.88)(0.90 - 0.96)(-0.16 - -0.023)(0.66 - 1.25)Threonine0.84 (0.024)0.94 (0.014)-0.10 (0.026)-0.17, -0.0290.0160.45, 1.33(0.82 - 0.85)(0.91 - 0.97)(-0.12 - -0.064)(0.63 - 1.23)Tryptophan0.34 (0.028)0.38 (0.018)-0.033 (0.032)-0.11, 0.0470.3370.20, 0.56(0.34 - 0.35)(0.35 - 0.39)(-0.056 - -0.00099)(0.25 - 0.50)Monsanto Company 12-AL-246U 278 of 407Table E-3 (continued). Statistical Summary of Site CAPR Alfalfa Forage Nutrients for KK179 vs. Conventional Control(Conducted at Covance Laboratories)Difference (Test minus Control)Analytical Component (Units)1KK179Mean (S.E.)2(Range)Control3Mean (S.E.)(Range)Mean (S.E.)(Range)95%ConfidenceIntervalSignificance(p-Value)CommercialTolerance Interval4(Range)Amino Acid (% dw)Tyrosine0.70 (0.017)0.78 (0.012)-0.081 (0.021)-0.13, -0.0330.0050.35, 1.09(0.69 - 0.70)(0.76 - 0.80)(-0.098 - -0.053)(0.52 - 1.01)Valine1.03 (0.027)1.13 (0.019)-0.098 (0.033)-0.18, -0.0190.0210.52, 1.64(1.00 - 1.06)(1.09 - 1.18)(-0.12 - -0.028)(0.79 - 1.55)MineralCalcium (% dw)1.42 (0.092)1.58 (0.065)-0.16 (0.11)-0.43, 0.110.1960.55, 2.56(1.29 - 1.55)(1.42 - 1.72)(-0.36 - 0.13)(0.95 - 2.07)Copper (mg/kg dw)9.98 (1.09)10.79 (0.77)-0.81 (1.33)-3.96, 2.340.5611.87, 14.98(9.19 - 10.77)(10.10 - 11.93)(-1.62 - 0.67)(4.54 - 19.67)Iron (mg/kg dw)256.68 (78.54)348.49 (51.22)-91.81 (85.46)-320.55, 136.920.33741.59, 446.31(236.86 - 316.33)(174.40 - 516.75)(-279.88 - 5.44)(105.45 - 691.43)Magnesium (% dw)0.14 (0.0085)0.15 (0.0060)-0.016 (0.0089)-0.040, 0.00700.1290.027, 0.41(0.12 - 0.15)(0.15 - 0.16)(-0.028 - -0.00009)(0.11 - 0.34)Monsanto Company 12-AL-246U 279 of 407Table E-3 (continued). Statistical Summary of Site CAPR Alfalfa Forage Nutrients for KK179 vs. Conventional Control(Conducted at Covance Laboratories)Difference (Test minus Control)Analytical Component (Units)1KK179Mean (S.E.)2(Range)Control3Mean (S.E.)(Range)Mean (S.E.)(Range)95%ConfidenceIntervalSignificance(p-Value)CommercialTolerance Interval4(Range)MineralManganese (mg/kg dw)35.15 (4.47)40.72 (3.17)-5.57 (4.58)-18.07, 6.940.28817.53, 69.85(36.36 - 37.65)(30.92 - 48.33)(-11.97 - -5.81)(23.24 - 98.04)Phosphorus (% dw)0.26 (0.010)0.30 (0.0091)-0.038 (0.0063)-0.055, -0.0210.0030.14, 0.46(0.27 - 0.28)(0.29 - 0.31)(-0.040 - -0.033)(0.18 - 0.43)Potassium (% dw)2.19 (0.078)2.36 (0.055)-0.17 (0.096)-0.40, 0.0540.1141.82, 3.04(2.18 - 2.20)(2.31 - 2.48)(-0.13 - -0.11)(1.85 - 3.35)Sodium (% dw)0.16 (0.013)0.14 (0.0093)0.025 (0.013)-0.0086, 0.0590.1080, 0.24(0.15 - 0.16)(0.11 - 0.15)(0.0075 - 0.039)(0.016 - 0.20)Zinc (mg/kg dw)27.03 (1.05)29.30 (0.94)-2.28 (0.71)-4.23, -0.320.0318.89, 47.44(25.38 - 26.99)(27.27 - 30.81)(-3.26 - -1.89)(17.08 - 47.48)1dw = dry weight; fw = fresh weight2Mean (S.E.) = least-square mean (standard error)3Control refers to the non-biotechnology derived, conventional control (C0-Syn1).4With 95% confidence, interval contains 99% of the values expressed in the population of conventional commercial reference varieties.Negative limits set to zero.Monsanto Company 12-AL-246U 280 of 407Table E-4. Statistical Summary of Site CAPR Alfalfa ForageSecondary Metabolites for KK179 vs. Conventional Control(Conducted at Covance Laboratories)Difference (Test minus Control)Analytical Component (Units)1KK179Mean (S.E.)2(Range)Control3Mean (S.E.)(Range)Mean (S.E.)(Range)95%ConfidenceIntervalSignificance(p-Value)CommercialTolerance Interval4(Range)MetaboliteCanavanine (ppm dw)44.03 (9.98)69.09 (7.06)-25.05 (12.22)-53.96, 3.850.0790, 137.35(32.97 - 55.10)(58.60 - 79.23)(-26.84 - -23.61)(11.47 - 151.33)Ferulic Acid (ppm dw)1509.08 (114.39)1350.71 (80.89)158.37 (140.10)-172.92, 489.650.295854.88, 2061.10(1411.02 - 1607.14)(1103.96 - 1596.77)(80.87 - 503.18)(1103.32 - 1906.86)Free Phenylalanine (ppm dw)165.37 (43.57)204.65 (30.81)-39.28 (53.36)-165.45, 86.890.4850, 627.23(111.86 - 218.88)(154.07 - 278.22)(-59.34 - -42.20)(133.05 - 579.05)Total Polyphenols (mg/g dw)8.51 (0.39)7.45 (0.25)1.06 (0.46)-0.070, 2.180.0614.86, 11.15(8.31 - 8.72)(7.20 - 7.66)(0.65 - 1.40)(6.17 - 11.17)p-Coumaric Acid (ppm dw)567.23 (57.51)514.33 (40.66)52.91 (70.43)-113.64, 219.450.477188.81, 949.95(466.10 - 668.37)(442.08 - 645.16)(-31.51 - 226.29)(326.19 - 945.58)1dw = dry weight; fw = fresh weight2Mean (S.E.) = least-square mean (standard error)3Control refers to the non-biotechnology derived, conventional control (C0-Syn1).4With 95% confidence, interval contains 99% of the values expressed in the population of conventional commercial reference varieties.Negative limits set to zero.Monsanto Company 12-AL-246U 281 of 407Table E-5. Statistical Summary of Site IARL Alfalfa Forage Nutrients for KK179 vs.Conventional Control (Conducted atCovance Laboratories)Difference (Test minus Control)Analytical Component (Units)1KK179Mean (S.E.)2(Range)Control3Mean (S.E.)(Range)Mean (S.E.)(Range)95%ConfidenceIntervalSignificance(p-Value)CommercialTolerance Interval4(Range)Proximate (% dw)Ash10.05 (0.36)10.63 (0.36)-0.58 (0.51)-1.74, 0.570.2816.70, 13.54(9.88 - 10.39)(10.29 - 11.39)(-1.52 - 0.095)(7.54 - 13.23)Carbohydrates59.32 (0.78)61.15 (0.78)-1.83 (0.89)-4.01, 0.340.08450.57, 81.80(57.73 - 62.20)(59.94 - 62.42)(-3.45 - 1.12)(54.35 - 74.91)Moisture (% fw)84.13 (0.24)83.38 (0.24)0.75 (0.34)-0.025, 1.530.05665.06, 90.61(83.60 - 84.60)(83.00 - 83.70)(0.20 - 1.60)(66.10 - 85.30)Protein27.34 (0.72)25.71 (0.72)1.64 (0.59)0.19, 3.080.0329.26, 33.78(24.51 - 29.03)(24.73 - 27.30)(-0.58 - 3.19)(14.52 - 30.07)Total Fat3.25 (0.30)2.59 (0.30)0.66 (0.28)-0.011, 1.340.0520.73, 3.59(2.58 - 3.98)(1.72 - 3.38)(-0.21 - 1.07)(0.53 - 4.21)Fiber (% dw)Acid Detergent Fiber16.65 (1.42)15.76 (1.42)0.89 (1.58)-2.96, 4.750.5916.16, 49.06(15.71 - 17.85)(10.96 - 19.18)(-3.20 - 6.12)(7.07 - 39.11)Monsanto Company 12-AL-246U 282 of 407Table E-5 (continued). Statistical Summaryof Site IARL Alfalfa Forage Nutrients for KK179 vs. Conventional Control(Conducted at Covance Laboratories)Difference (Test minus Control)Analytical Component (Units)1KK179Mean (S.E.)2(Range)Control3Mean (S.E.)(Range)Mean (S.E.)(Range)95%ConfidenceIntervalSignificance(p-Value)CommercialTolerance Interval4(Range)Fiber (% dw)Acid Detergent Lignin3.22 (0.17)3.85 (0.17)-0.62 (0.24)-1.21, -0.0410.0392.13, 11.99(2.72 - 3.69)(3.58 - 4.23)(-1.51 - -0.16)(3.38 - 9.67)Neutral Detergent Fiber22.65 (2.23)22.89 (2.23)-0.24 (3.16)-7.38, 6.900.94112.04, 58.18(18.57 - 30.78)(18.94 - 26.83)(-8.11 - 11.84)(18.97 - 49.82)Amino Acid (% dw)Alanine1.40 (0.034)1.36 (0.034)0.038 (0.032)-0.041, 0.120.2810.49, 1.79(1.31 - 1.52)(1.30 - 1.39)(-0.042 - 0.13)(0.80 - 1.66)Arginine1.28 (0.029)1.23 (0.029)0.052 (0.022)-0.0012, 0.100.0530.44, 1.59(1.23 - 1.35)(1.19 - 1.28)(0.0092 - 0.11)(0.70 - 1.44)Aspartic acid4.05 (0.17)3.85 (0.17)0.20 (0.16)-0.19, 0.590.2530.44, 5.63(3.74 - 4.65)(3.47 - 4.08)(-0.29 - 0.57)(1.96 - 5.15)Cystine0.26 (0.013)0.26 (0.013)0.0044 (0.010)-0.021, 0.0290.6850.12, 0.32(0.23 - 0.30)(0.24 - 0.29)(-0.018 - 0.019)(0.16 - 0.31)Monsanto Company 12-AL-246U 283 of 407Table E-5 (continued). Statistical Summaryof Site IARL Alfalfa Forage Nutrients for KK179 vs. Conventional Control(Conducted at Covance Laboratories)Difference (Test minus Control)Analytical Component (Units)1KK179Mean (S.E.)2(Range)Control3Mean (S.E.)(Range)Mean (S.E.)(Range)95%ConfidenceIntervalSignificance(p-Value)CommercialTolerance Interval4(Range)Amino Acid (% dw)Glutamic acid2.39 (0.045)2.31 (0.045)0.079 (0.038)-0.013, 0.170.0800.81, 3.01(2.24 - 2.55)(2.27 - 2.36)(-0.026 - 0.22)(1.31 - 2.80)Glycine1.15 (0.020)1.12 (0.020)0.026 (0.020)-0.022, 0.0750.2300.49, 1.44(1.10 - 1.21)(1.10 - 1.14)(-0.022 - 0.085)(0.70 - 1.33)Histidine0.52 (0.0099)0.50 (0.0099)0.015 (0.012)-0.015, 0.0440.2700.26, 0.63(0.49 - 0.55)(0.49 - 0.51)(-0.012 - 0.059)(0.34 - 0.61)Isoleucine1.08 (0.021)1.05 (0.021)0.032 (0.028)-0.037, 0.100.2930.43, 1.36(1.02 - 1.15)(1.03 - 1.07)(-0.048 - 0.12)(0.63 - 1.27)Leucine1.80 (0.031)1.74 (0.031)0.061 (0.030)-0.012, 0.130.0850.70, 2.25(1.73 - 1.90)(1.71 - 1.78)(-0.011 - 0.16)(1.03 - 2.05)Lysine1.44 (0.034)1.40 (0.034)0.038 (0.021)-0.014, 0.0910.1240.55, 1.82(1.37 - 1.55)(1.34 - 1.44)(-0.0052 - 0.12)(0.82 - 1.73)Monsanto Company 12-AL-246U 284 of 407Table E-5 (continued). Statistical Summaryof Site IARL Alfalfa Forage Nutrients for KK179 vs. Conventional Control(Conducted at Covance Laboratories)Difference (Test minus Control)Analytical Component (Units)1KK179Mean (S.E.)2(Range)Control3Mean (S.E.)(Range)Mean (S.E.)(Range)95%ConfidenceIntervalSignificance(p-Value)CommercialTolerance Interval4(Range)Amino Acid (% dw)Methionine0.37 (0.015)0.34 (0.015)0.031 (0.017)-0.010, 0.0720.1170.068, 0.42(0.34 - 0.39)(0.30 - 0.37)(0.0086 - 0.062)(0.14 - 0.45)Phenylalanine1.22 (0.020)1.18 (0.020)0.034 (0.019)-0.013, 0.0800.1270.48, 1.53(1.16 - 1.27)(1.15 - 1.21)(-0.027 - 0.083)(0.71 - 1.39)Proline1.09 (0.030)1.07 (0.030)0.020 (0.022)-0.033, 0.0720.4010.43, 1.41(1.02 - 1.18)(1.05 - 1.11)(-0.024 - 0.10)(0.65 - 1.24)Serine1.09 (0.023)1.01 (0.023)0.076 (0.018)0.030, 0.120.0060.45, 1.35(1.03 - 1.16)(0.95 - 1.05)(0.019 - 0.13)(0.66 - 1.25)Threonine1.07 (0.017)1.02 (0.017)0.048 (0.016)0.0075, 0.0880.0270.45, 1.33(1.02 - 1.12)(0.99 - 1.05)(0.0064 - 0.10)(0.63 - 1.23)Tryptophan0.45 (0.012)0.43 (0.012)0.015 (0.016)-0.025, 0.0550.3990.20, 0.56(0.41 - 0.48)(0.40 - 0.45)(-0.027 - 0.038)(0.25 - 0.50)Monsanto Company 12-AL-246U 285 of 407Table E-5 (continued). Statistical Summaryof Site IARL Alfalfa Forage Nutrients for KK179 vs. Conventional Control(Conducted at Covance Laboratories)Difference (Test minus Control)Analytical Component (Units)1KK179Mean (S.E.)2(Range)Control3Mean (S.E.)(Range)Mean (S.E.)(Range)95%ConfidenceIntervalSignificance(p-Value)CommercialTolerance Interval4(Range)Amino Acid (% dw)Tyrosine0.90 (0.023)0.86 (0.023)0.040 (0.028)-0.028, 0.110.2000.35, 1.09(0.86 - 0.94)(0.84 - 0.89)(-0.0025 - 0.10)(0.52 - 1.01)Valine1.31 (0.024)1.27 (0.024)0.040 (0.033)-0.041, 0.120.2740.52, 1.64(1.24 - 1.38)(1.24 - 1.32)(-0.073 - 0.13)(0.79 - 1.55)MineralCalcium (% dw)1.67 (0.041)1.76 (0.041)-0.093 (0.058)-0.22, 0.0380.1420.55, 2.56(1.63 - 1.71)(1.69 - 1.86)(-0.20 - -0.0096)(0.95 - 2.07)Copper (mg/kg dw)9.38 (0.86)8.57 (0.86)0.80 (0.98)-1.60, 3.210.4441.87, 14.98(8.54 - 11.23)(8.20 - 8.83)(0.073 - 2.40)(4.54 - 19.67)Iron (mg/kg dw)169.80 (36.93)310.65 (36.93)-140.85 (52.22)-258.99, -22.710.02441.59, 446.31(123.38 - 207.36)(202.40 - 386.50)(-263.13 - 1.26)(105.45 - 691.43)Magnesium (% dw)0.30 (0.0095)0.31 (0.0095)-0.011 (0.014)-0.041, 0.0200.4520.027, 0.41(0.29 - 0.31)(0.30 - 0.32)(-0.023 - -0.0023)(0.11 - 0.34)Monsanto Company 12-AL-246U 286 of 407Table E-5 (continued). Statistical Summaryof Site IARL Alfalfa Forage Nutrients for KK179 vs. Conventional Control(Conducted at Covance Laboratories)Difference (Test minus Control)Analytical Component (Units)1KK179Mean (S.E.)2(Range)Control3Mean (S.E.)(Range)Mean (S.E.)(Range)95%ConfidenceIntervalSignificance(p-Value)CommercialTolerance Interval4(Range)MineralManganese (mg/kg dw)31.84 (2.81)39.98 (2.81)-8.14 (3.97)-17.12, 0.840.07017.53, 69.85(30.52 - 34.57)(34.37 - 42.39)(-11.42 - 0.20)(23.24 - 98.04)Phosphorus (% dw)0.37 (0.018)0.35 (0.018)0.016 (0.025)-0.040, 0.0720.5350.14, 0.46(0.33 - 0.40)(0.33 - 0.38)(-0.035 - 0.071)(0.18 - 0.43)Potassium (% dw)2.45 (0.11)2.53 (0.11)-0.077 (0.15)-0.42, 0.270.6241.82, 3.04(2.26 - 2.62)(2.41 - 2.71)(-0.45 - 0.18)(1.85 - 3.35)Sodium (% dw)0.050 (0.010)0.041 (0.010)0.0087 (0.014)-0.023, 0.0410.5570, 0.24(0.030 - 0.069)(0.029 - 0.075)(-0.044 - 0.039)(0.016 - 0.20)Zinc (mg/kg dw)36.08 (1.99)37.26 (1.99)-1.18 (2.32)-6.86, 4.510.6308.89, 47.44(33.90 - 39.22)(34.12 - 40.42)(-5.64 - 2.31)(17.08 - 47.48)1dw = dry weight; fw = fresh weight2Mean (S.E.) = least-square mean (standard error)3Control refers to the non-biotechnologyderived, conventional control (C0-Syn1).4With 95% confidence, interval contains 99%of the values expressed in the populationof conventional commercial reference varieties.Negative limits set to zero.Monsanto Company 12-AL-246U 287 of 407Table E-6. Statistical Summary of SiteIARL Alfalfa Forage Secondary Metabolites for KK179 vs. Conventional Control(Conducted at Covance Laboratories)Difference (Test minus Control)Analytical Component (Units)1KK179Mean (S.E.)2(Range)Control3Mean (S.E.)(Range)Mean (S.E.)(Range)95%ConfidenceIntervalSignificance(p-Value)CommercialTolerance Interval4(Range)MetaboliteCanavanine (ppm dw)15.76 (0.24)15.04 (0.24)0.72 (0.33)-0.035, 1.480.0590, 137.35(15.24 - 16.23)(14.71 - 15.34)(0.19 - 1.53)(11.47 - 151.33)Ferulic Acid (ppm dw)1591.83 (78.25)1390.17 (78.25)201.66 (94.83)-30.37, 433.700.077854.88, 2061.10(1402.60 - 1779.22)(1181.82 - 1580.84)(22.23 - 361.57)(1103.32 - 1906.86)Free Phenylalanine (ppm dw)378.02 (21.12)386.96 (21.12)-8.94 (26.59)-74.01, 56.130.7480, 627.23(345.73 - 409.20)(370.30 - 422.94)(-29.72 - 38.90)(133.05 - 579.05)Total Polyphenols (mg/g dw)9.68 (0.22)8.86 (0.22)0.82 (0.31)0.11, 1.520.0274.86, 11.15(8.96 - 10.19)(8.47 - 9.22)(0.048 - 1.72)(6.17 - 11.17)p-Coumaric Acid (ppm dw)748.40 (40.83)732.14 (40.83)16.26 (57.74)-114.36, 146.890.784188.81, 949.95(625.77 - 870.13)(660.61 - 784.43)(-79.99 - 128.95)(326.19 - 945.58)1dw = dry weight2Mean (S.E.) = least-square mean (standard error)3Control refers to the non-biotechnology derived, conventional control (C0-Syn1).4With 95% confidence, interval contains 99% of the values expressed in the population of conventional commercial reference varietiesNegative limits set to zero.Monsanto Company 12-AL-246U 288 of 407Table E-7. Statistical Summaryof Site ILCY Alfalfa Forage Nutrients for KK179 vs.Conventional Control (Conducted atCovance Laboratories)Difference (Test minus Control)Analytical Component (Units)1KK179Mean (S.E.)2(Range)Control3Mean (S.E.)(Range)Mean (S.E.)(Range)95%ConfidenceIntervalSignificance(p-Value)CommercialTolerance Interval4(Range)Proximate (% dw)Ash9.85 (0.31)10.33 (0.31)-0.48 (0.28)-1.17, 0.210.1396.70, 13.54(8.81 - 10.83)(9.80 - 11.22)(-1.35 - -0.025)(7.54 - 13.23)Carbohydrates69.37 (1.34)67.53 (1.34)1.84 (1.89)-2.44, 6.130.35550.57, 81.80(66.84 - 71.14)(61.73 - 71.28)(-2.74 - 8.36)(54.35 - 74.91)Moisture (% fw)79.73 (0.43)80.45 (0.43)-0.73 (0.49)-1.93, 0.480.19265.06, 90.61(78.10 - 81.30)(80.30 - 80.60)(-2.20 - 0.70)(66.10 - 85.30)Protein18.87 (1.15)20.06 (1.15)-1.18 (1.62)-4.86, 2.490.4859.26, 33.78(17.41 - 21.12)(16.56 - 25.00)(-6.67 - 2.15)(14.52 - 30.07)Total Fat1.99 (0.36)2.22 (0.36)-0.23 (0.50)-1.45, 0.990.6620.73, 3.59(0.84 - 2.62)(1.31 - 3.23)(-1.30 - 0.93)(0.53 - 4.21)Fiber (% dw)Acid Detergent Fiber27.25 (1.40)32.57 (1.40)-5.32 (1.84)-9.82, -0.820.0276.16, 49.06(20.53 - 29.59)(30.62 - 33.67)(-10.08 - -2.75)(7.07 - 39.11)Monsanto Company 12-AL-246U 289 of 407Table E-7 (continued). Statistical Summaryof Site ILCY Alfalfa Forage Nutrients for KK179 vs. Conventional Control(Conducted at Covance Laboratories)Difference (Test minus Control)Analytical Component (Units)1KK179Mean (S.E.)2(Range)Control3Mean (S.E.)(Range)Mean (S.E.)(Range)95%ConfidenceIntervalSignificance(p-Value)CommercialTolerance Interval4(Range)Fiber (% dw)Acid Detergent Lignin5.88 (0.31)6.81 (0.31)-0.94 (0.41)-1.94, 0.0690.0622.13, 11.99(5.51 - 6.32)(5.89 - 7.50)(-1.99 - -0.36)(3.38 - 9.67)Neutral Detergent Fiber34.67 (1.47)39.92 (1.47)-5.25 (1.62)-9.21, -1.290.01712.04, 58.18(30.00 - 38.58)(38.26 - 43.32)(-9.74 - 0.21)(18.97 - 49.82)Amino Acid (% dw)Alanine0.99 (0.027)1.01 (0.027)-0.020 (0.038)-0.11, 0.0660.6100.49, 1.79(0.91 - 1.06)(0.94 - 1.07)(-0.096 - 0.074)(0.80 - 1.66)Arginine0.89 (0.024)0.90 (0.024)-0.0090 (0.033)-0.089, 0.0710.7920.44, 1.59(0.83 - 0.94)(0.87 - 0.96)(-0.043 - 0.039)(0.70 - 1.44)Aspartic acid2.68 (0.11)2.50 (0.11)0.18 (0.15)-0.16, 0.520.2580.44, 5.63(2.36 - 3.13)(2.42 - 2.61)(-0.25 - 0.71)(1.96 - 5.15)Cystine0.20 (0.0081)0.20 (0.0081)0.0071 (0.011)-0.019, 0.0330.5500.12, 0.32(0.19 - 0.23)(0.18 - 0.21)(-0.021 - 0.018)(0.16 - 0.31)Monsanto Company 12-AL-246U 290 of 407Table E-7 (continued). Statistical Summaryof Site ILCY Alfalfa Forage Nutrients for KK179 vs Conventional Control(Conducted at Covance Laboratories)Difference (Test minus Control)Analytical Component (Units)1KK179Mean (S.E.)2(Range)Control3Mean (S.E.)(Range)Mean (S.E.)(Range)95%ConfidenceIntervalSignificance(p-Value)CommercialTolerance Interval4(Range)Amino Acid (% dw)Glutamic acid1.66 (0.046)1.70 (0.046)-0.043 (0.065)-0.19, 0.100.5240.81, 3.01(1.59 - 1.78)(1.59 - 1.80)(-0.16 - 0.14)(1.31 - 2.80)Glycine0.84 (0.022)0.88 (0.022)-0.033 (0.032)-0.11, 0.0380.3200.49, 1.44(0.80 - 0.90)(0.82 - 0.93)(-0.096 - 0.053)(0.70 - 1.33)Histidine0.41 (0.0095)0.40 (0.0095)0.0035 (0.013)-0.027, 0.0340.8000.26, 0.63(0.38 - 0.43)(0.38 - 0.43)(-0.012 - 0.032)(0.34 - 0.61)Isoleucine0.76 (0.023)0.80 (0.023)-0.038 (0.032)-0.11, 0.0340.2600.43, 1.36(0.73 - 0.82)(0.74 - 0.85)(-0.094 - 0.050)(0.63 - 1.27)Leucine1.27 (0.037)1.33 (0.037)-0.061 (0.052)-0.18, 0.0570.2700.70, 2.25(1.19 - 1.36)(1.23 - 1.41)(-0.15 - 0.075)(1.03 - 2.05)Lysine1.04 (0.028)1.05 (0.028)-0.0074 (0.040)-0.097, 0.0820.8560.55, 1.82(0.99 - 1.11)(0.97 - 1.14)(-0.11 - 0.11)(0.82 - 1.73)Monsanto Company 12-AL-246U 291 of 407Table E-7 (continued). Statistical Summaryof Site ILCY Alfalfa Forage Nutrients for KK179 vs. Conventional Control(Conducted at Covance Laboratories)Difference (Test minus Control)Analytical Component (Units)1KK179Mean (S.E.)2(Range)Control3Mean (S.E.)(Range)Mean (S.E.)(Range)95%ConfidenceIntervalSignificance(p-Value)CommercialTolerance Interval4(Range)Amino Acid (% dw)Methionine0.26 (0.023)0.23 (0.023)0.032 (0.032)-0.041, 0.100.3500.068, 0.42(0.17 - 0.32)(0.22 - 0.24)(-0.053 - 0.10)(0.14 - 0.45)Phenylalanine0.86 (0.023)0.91 (0.023)-0.050 (0.032)-0.12, 0.0220.1530.48, 1.53(0.82 - 0.90)(0.84 - 0.96)(-0.10 - 0.022)(0.71 - 1.39)Proline0.84 (0.025)0.81 (0.025)0.031 (0.035)-0.047, 0.110.3900.43, 1.41(0.81 - 0.87)(0.75 - 0.88)(-0.064 - 0.11)(0.65 - 1.24)Serine0.83 (0.023)0.83 (0.023)0.0042 (0.032)-0.068, 0.0770.8980.45, 1.35(0.77 - 0.90)(0.79 - 0.87)(-0.055 - 0.099)(0.66 - 1.25)Threonine0.79 (0.022)0.80 (0.022)-0.013 (0.032)-0.085, 0.0590.6950.45, 1.33(0.74 - 0.84)(0.76 - 0.85)(-0.058 - 0.061)(0.63 - 1.23)Tryptophan0.35 (0.010)0.35 (0.010)0.0032 (0.015)-0.030, 0.0360.8310.20, 0.56(0.32 - 0.38)(0.32 - 0.37)(-0.047 - 0.035)(0.25 - 0.50)Monsanto Company 12-AL-246U 292 of 407Table E-7 (continued). Statistical Summary of Site ILCY Alfalfa Forage Nutrients for KK179 vs. Conventional Control(Conducted at Covance Laboratories)Difference (Test minus Control)Analytical Component (Units)1KK179Mean (S.E.)2(Range)Control3Mean (S.E.)(Range)Mean (S.E.)(Range)95%ConfidenceIntervalSignificance(p-Value)CommercialTolerance Interval4(Range)Amino Acid (% dw)Tyrosine0.64 (0.015)0.65 (0.015)-0.0087 (0.022)-0.058, 0.0400.6980.35, 1.09(0.60 - 0.68)(0.63 - 0.67)(-0.024 - 0.025)(0.52 - 1.01)Valine0.95 (0.023)0.99 (0.023)-0.040 (0.033)-0.11, 0.0350.2630.52, 1.64(0.89 - 0.99)(0.91 - 1.04)(-0.090 - -0.00058)(0.79 - 1.55)MineralCalcium (% dw)1.45 (0.085)1.46 (0.085)-0.013 (0.12)-0.30, 0.270.9120.55, 2.56(1.27 - 1.62)(1.25 - 1.66)(-0.12 - 0.062)(0.95 - 2.07)Copper (mg/kg dw)7.04 (0.57)6.05 (0.57)0.99 (0.77)-0.88, 2.860.2431.87, 14.98(5.21 - 8.50)(5.18 - 7.32)(0.028 - 1.51)(4.54 - 19.67)Iron (mg/kg dw)212.81 (25.68)212.29 (25.68)0.52 (35.24)-85.70, 86.740.98841.59, 446.31(173.97 - 230.88)(163.92 - 313.78)(-82.89 - 52.13)(105.45 - 691.43)Magnesium (% dw)0.17 (0.010)0.18 (0.010)-0.0085 (0.012)-0.038, 0.0210.5140.027, 0.41(0.13 - 0.20)(0.17 - 0.19)(-0.040 - 0.019)(0.11 - 0.34)Monsanto Company 12-AL-246U 293 of 407Table E-7 (continued). Statistical Summary of Site ILCY Alfalfa Forage Nutrients for KK179 vs. Conventional Control(Conducted at Covance Laboratories)Difference (Test minus Control)Analytical Component (Units)1KK179Mean (S.E.)2(Range)Control3Mean (S.E.)(Range)Mean (S.E.)(Range)95%ConfidenceIntervalSignificance(p-Value)CommercialTolerance Interval4(Range)MineralManganese (mg/kg dw)77.86 (7.33)69.83 (7.33)8.02 (10.37)-15.89, 31.940.46117.53, 69.85(59.82 - 106.47)(58.16 - 77.32)(-15.31 - 37.75)(23.24 - 98.04)Phosphorus (% dw)0.29 (0.0074)0.30 (0.0074)-0.0078 (0.010)-0.033, 0.0180.4800.14, 0.46(0.28 - 0.31)(0.29 - 0.31)(-0.032 - 0.0061)(0.18 - 0.43)Potassium (% dw)2.37 (0.055)2.51 (0.055)-0.14 (0.077)-0.32, 0.0310.0961.82, 3.04(2.25 - 2.58)(2.44 - 2.60)(-0.35 - 0.082)(1.85 - 3.35)Sodium (% dw)0.090 (0.013)0.091 (0.013)-0.0014 (0.019)-0.043, 0.0410.9420, 0.24(0.065 - 0.14)(0.074 - 0.13)(-0.056 - 0.068)(0.016 - 0.20)Zinc (mg/kg dw)25.77 (1.88)20.32 (1.88)5.45 (2.58)-0.87, 11.770.0798.89, 47.44(18.40 - 32.73)(17.38 - 22.50)(-1.34 - 11.08)(17.08 - 47.48)1dw = dry weight; fw = fresh weight2Mean (S.E.) = least-square mean (standard error).3Control refers to the non-biotechnology derived, conventional control (C0-Syn1).4With 95% confidence, interval contains 99% of the values expressed in the population of conventional commercial reference varieties.Negative limits set to zero.Monsanto Company 12-AL-246U 294 of 407Table E-8. Statistical Summary of SiteILCY Alfalfa Forage Secondary Metabolites for KK179 vs. Conventional Control(Conducted at Covance Laboratories)Difference (Test minus Control)Analytical Component (Units)1KK179Mean (S.E.)2(Range)Control3Mean (S.E.)(Range)Mean (S.E.)(Range)95%ConfidenceIntervalSignificance(p-Value)CommercialTolerance Interval4(Range)MetaboliteCanavanine (ppm dw)12.37 (0.26)12.79 (0.26)-0.42 (0.30)-1.15, 0.310.2100, 137.35(11.42 - 13.37)(12.69 - 12.89)(-1.27 - 0.48)(11.47 - 151.33)Ferulic Acid (ppm dw)1617.62 (75.96)1605.38 (75.96)12.24 (107.43)-230.77, 255.260.911854.88, 2061.10(1508.02 - 1840.80)(1515.31 - 1809.28)(-301.26 - 292.08)(1103.32 - 1906.86)Free Phenylalanine (ppm dw)301.46 (23.08)297.57 (23.08)3.89 (32.03)-74.49, 82.270.9070, 627.23(232.62 - 362.69)(245.36 - 317.44)(-41.81 - 45.25)(133.05 - 579.05)Total Polyphenols (mg/g dw)8.43 (0.36)8.74 (0.36)-0.31 (0.51)-1.46, 0.840.5554.86, 11.15(7.86 - 9.00)(8.11 - 10.21)(-2.13 - 0.87)(6.17 - 11.17)p-Coumaric Acid (ppm dw)696.28 (24.51)715.16 (24.51)-18.88 (31.57)-96.12, 58.370.571188.81, 949.95(661.76 - 736.32)(649.75 - 819.59)(-108.36 - 44.01)(326.19 - 945.58)1dw = dry weight2Mean (S.E.) = least-square mean (standard error)3Control refers to the non-biotechnology derived, conventional control (C0-Syn1).4With 95% confidence, interval contains 99% of the values expressed in the population of conventional commercial reference varietiesNegative limits set to zero.Monsanto Company 12-AL-246U 295 of 407Table E-9. Statistical Summary of SiteKSLA Alfalfa Forage Nutrients for KK179 vs. Conventional Control (Conducted atCovance Laboratories)Difference (Test minus Control)Analytical Component (Units)1KK179Mean (S.E.)2(Range)Control3Mean (S.E.)(Range)Mean (S.E.)(Range)95%ConfidenceIntervalSignificance(p-Value)CommercialTolerance Interval4(Range)Proximate (% dw)Ash11.97 (0.36)12.43 (0.36)-0.46 (0.50)-1.69, 0.770.3986.70, 13.54(11.27 - 13.26)(12.11 - 12.88)(-1.04 - 0.83)(7.54 - 13.23)Carbohydrates64.61 (0.56)64.29 (0.56)0.32 (0.80)-1.49, 2.120.69850.57, 81.80(63.04 - 65.40)(62.84 - 65.77)(-2.72 - 2.22)(54.35 - 74.91)Moisture (% fw)76.90 (0.28)77.53 (0.28)-0.63 (0.37)-1.53, 0.280.14165.06, 90.61(76.30 - 77.20)(77.00 - 78.20)(-1.10 - 0.10)(66.10 - 85.30)Protein21.08 (0.56)20.94 (0.56)0.14 (0.79)-1.64, 1.920.8609.26, 33.78(20.57 - 21.40)(19.91 - 22.16)(-1.59 - 1.31)(14.52 - 30.07)Total Fat2.35 (0.32)2.31 (0.32)0.043 (0.42)-0.98, 1.070.9210.73, 3.59(1.90 - 2.77)(1.08 - 2.92)(-0.71 - 1.46)(0.53 - 4.21)Fiber (% dw)Acid Detergent Fiber27.84 (1.43)28.97 (1.43)-1.14 (1.14)-3.92, 1.650.3566.16, 49.06(25.19 - 30.13)(23.70 - 31.42)(-4.13 - 1.49)(7.07 - 39.11)Monsanto Company 12-AL-246U 296 of 407Table E-9 (continued). Statistical Summary of Site KSLA Alfalfa Forage Nutrients for KK179 vs. Conventional Control(Conducted at Covance Laboratories)Difference (Test minus Control)Analytical Component (Units)1KK179Mean (S.E.)2(Range)Control3Mean (S.E.)(Range)Mean (S.E.)(Range)95%ConfidenceIntervalSignificance(p-Value)CommercialTolerance Interval4(Range)Fiber (% dw)Acid Detergent Lignin6.43 (0.35)7.44 (0.35)-1.02 (0.49)-2.13, 0.0970.0682.13, 11.99(5.47 - 7.27)(6.65 - 8.17)(-2.08 - 0.61)(3.38 - 9.67)Neutral Detergent Fiber34.37 (0.83)36.31 (0.83)-1.94 (1.17)-4.59, 0.710.13212.04, 58.18(31.79 - 37.37)(35.86 - 37.09)(-4.22 - 1.08)(18.97 - 49.82)Amino Acid (% dw)Alanine1.11 (0.032)1.09 (0.032)0.021 (0.045)-0.082, 0.120.6540.49, 1.79(1.06 - 1.17)(1.03 - 1.14)(-0.083 - 0.070)(0.80 - 1.66)Arginine0.98 (0.040)0.99 (0.040)-0.0086 (0.057)-0.14, 0.120.8830.44, 1.59(0.96 - 1.00)(0.90 - 1.06)(-0.10 - 0.079)(0.70 - 1.44)Aspartic acid2.72 (0.081)2.61 (0.081)0.10 (0.11)-0.16, 0.370.3690.44, 5.63(2.55 - 3.02)(2.41 - 2.72)(-0.14 - 0.31)(1.96 - 5.15)Cystine0.21 (0.013)0.21 (0.013)0.00044 (0.018)-0.040, 0.0410.9810.12, 0.32(0.18 - 0.25)(0.19 - 0.23)(-0.041 - 0.062)(0.16 - 0.31)Monsanto Company 12-AL-246U 297 of 407Table E-9 (continued). Statistical Summary of Site KSLA Alfalfa Forage Nutrients for KK179 vs. Conventional Control(Conducted at Covance Laboratories)Difference (Test minus Control)Analytical Component (Units)1KK179Mean (S.E.)2(Range)Control3Mean (S.E.)(Range)Mean (S.E.)(Range)95%ConfidenceIntervalSignificance(p-Value)CommercialTolerance Interval4(Range)Amino Acid (% dw)Glutamic acid1.89 (0.051)1.87 (0.051)0.020 (0.073)-0.14, 0.180.7890.81, 3.01(1.83 - 1.97)(1.75 - 1.95)(-0.12 - 0.13)(1.31 - 2.80)Glycine0.98 (0.025)0.98 (0.025)0.0034 (0.035)-0.076, 0.0830.9240.49, 1.44(0.97 - 1.02)(0.93 - 1.04)(-0.071 - 0.048)(0.70 - 1.33)Histidine0.43 (0.012)0.43 (0.012)-0.0062 (0.017)-0.046, 0.0330.7300.26, 0.63(0.41 - 0.45)(0.41 - 0.45)(-0.022 - 0.0075)(0.34 - 0.61)Isoleucine0.87 (0.023)0.86 (0.023)0.0069 (0.033)-0.068, 0.0810.8390.43, 1.36(0.83 - 0.91)(0.82 - 0.90)(-0.040 - 0.056)(0.63 - 1.27)Leucine1.45 (0.041)1.45 (0.041)0.0010 (0.057)-0.13, 0.130.9860.70, 2.25(1.42 - 1.53)(1.36 - 1.53)(-0.10 - 0.085)(1.03 - 2.05)Lysine1.12 (0.034)1.15 (0.034)-0.031 (0.048)-0.14, 0.0770.5360.55, 1.82(1.09 - 1.16)(1.10 - 1.19)(-0.062 - -0.0039)(0.82 - 1.73)Monsanto Company 12-AL-246U 298 of 407Table E-9 (continued). Statistical Summary of Site KSLA Alfalfa Forage Nutrients for KK179 vs. Conventional Control(Conducted at Covance Laboratories)Difference (Test minus Control)Analytical Component (Units)1KK179Mean (S.E.)2(Range)Control3Mean (S.E.)(Range)Mean (S.E.)(Range)95%ConfidenceIntervalSignificance(p-Value)CommercialTolerance Interval4(Range)Amino Acid (% dw)Methionine0.22 (0.020)0.23 (0.020)-0.0029 (0.028)-0.066, 0.0610.9180.068, 0.42(0.19 - 0.25)(0.18 - 0.30)(-0.089 - 0.064)(0.14 - 0.45)Phenylalanine1.00 (0.029)0.99 (0.029)0.0049 (0.041)-0.088, 0.0970.9070.48, 1.53(0.97 - 1.04)(0.93 - 1.05)(-0.063 - 0.057)(0.71 - 1.39)Proline0.88 (0.024)0.88 (0.024)-0.0029 (0.034)-0.081, 0.0750.9330.43, 1.41(0.84 - 0.92)(0.85 - 0.91)(-0.075 - 0.038)(0.65 - 1.24)Serine0.86 (0.021)0.86 (0.021)0.0036 (0.030)-0.064, 0.0710.9060.45, 1.35(0.84 - 0.90)(0.82 - 0.89)(-0.030 - 0.034)(0.66 - 1.25)Threonine0.87 (0.024)0.87 (0.024)0.0042 (0.034)-0.074, 0.0820.9050.45, 1.33(0.86 - 0.90)(0.81 - 0.91)(-0.049 - 0.052)(0.63 - 1.23)Tryptophan0.38 (0.017)0.38 (0.017)0.00096 (0.024)-0.054, 0.0560.9690.20, 0.56(0.34 - 0.42)(0.36 - 0.39)(-0.050 - 0.065)(0.25 - 0.50)Monsanto Company 12-AL-246U 299 of 407Table E-9 (continued). Statistical Summary of Site KSLA Alfalfa Forage Nutrients for KK179 vs. Conventional Control(Conducted at Covance Laboratories)Difference (Test minus Control)Analytical Component (Units)1KK179Mean (S.E.)2(Range)Control3Mean (S.E.)(Range)Mean (S.E.)(Range)95%ConfidenceIntervalSignificance(p-Value)CommercialTolerance Interval4(Range)Amino Acid (% dw)Tyrosine0.71 (0.034)0.70 (0.034)0.0084 (0.048)-0.10, 0.120.8640.35, 1.09(0.68 - 0.73)(0.63 - 0.77)(-0.053 - 0.060)(0.52 - 1.01)Valine1.05 (0.027)1.04 (0.027)0.0063 (0.039)-0.081, 0.0940.8740.52, 1.64(1.01 - 1.09)(0.98 - 1.09)(-0.051 - 0.070)(0.79 - 1.55)MineralCalcium (% dw)2.04 (0.048)1.95 (0.048)0.093 (0.037)0.0021, 0.180.0460.55, 2.56(1.98 - 2.09)(1.85 - 2.10)(-0.017 - 0.15)(0.95 - 2.07)Copper (mg/kg dw)9.13 (0.52)9.18 (0.52)-0.053 (0.74)-1.72, 1.610.9431.87, 14.98(8.31 - 9.96)(8.60 - 9.59)(-0.98 - 1.35)(4.54 - 19.67)Iron (mg/kg dw)362.07 (58.09)451.45 (58.09)-89.38 (82.16)-275.23, 96.470.30441.59, 446.31(269.62 - 473.91)(375.69 - 547.83)(-278.21 - 67.61)(105.45 - 691.43)Magnesium (% dw)0.26 (0.0067)0.26 (0.0067)-0.0077 (0.0078)-0.027, 0.0110.3590.027, 0.41(0.24 - 0.27)(0.25 - 0.27)(-0.0094 - -0.0063)(0.11 - 0.34)Monsanto Company 12-AL-246U 300 of 407Table E-9 (continued). Statistical Summary of Site KSLA Alfalfa Forage Nutrients for KK179 vs. Conventional Control(Conducted at Covance Laboratories)Difference (Test minus Control)Analytical Component (Units)1KK179Mean (S.E.)2(Range)Control3Mean (S.E.)(Range)Mean (S.E.)(Range)95%ConfidenceIntervalSignificance(p-Value)CommercialTolerance Interval4(Range)MineralManganese (mg/kg dw)50.02 (1.51)49.82 (1.51)0.20 (1.50)-3.48, 3.880.89917.53, 69.85(47.68 - 53.48)(46.79 - 53.15)(-4.49 - 3.28)(23.24 - 98.04)Phosphorus (% dw)0.25 (0.0060)0.25 (0.0060)0.0036 (0.0083)-0.017, 0.0240.6820.14, 0.46(0.24 - 0.26)(0.23 - 0.26)(-0.0061 - 0.018)(0.18 - 0.43)Potassium (% dw)2.21 (0.041)2.23 (0.041)-0.019 (0.054)-0.15, 0.110.7321.82, 3.04(2.16 - 2.28)(2.18 - 2.29)(-0.12 - 0.071)(1.85 - 3.35)Sodium (% dw)0.17 (0.019)0.14 (0.019)0.035 (0.026)-0.028, 0.0990.2230, 0.24(0.13 - 0.22)(0.12 - 0.15)(0.0023 - 0.083)(0.016 - 0.20)Zinc (mg/kg dw)24.75 (0.88)22.94 (0.88)1.81 (1.18)-1.08, 4.710.1768.89, 47.44(23.28 - 27.57)(21.91 - 24.13)(-0.85 - 3.92)(17.08 - 47.48)1dw = dry weight; fw = fresh weight2Mean (S.E.) = least-square mean (standard error)3Control refers to the non-biotechnology derived, conventional control (C0-Syn1).4With 95% confidence, interval contains 99% of the values expressed in the population of conventional commercial reference varieties.Negative limits set to zero.Monsanto Company 12-AL-246U 301 of 407Table E-10. Statistical Summaryof Site KSLA Alfalfa Forage Secondary Metabolites forKK179 vs. Conventional Control(Conducted at Covance Laboratories)Difference (Test minus Control)Analytical Component (Units)1KK179Mean (S.E.)2(Range)Control3Mean (S.E.)(Range)Mean (S.E.)(Range)95%ConfidenceIntervalSignificance(p-Value)CommercialTolerance Interval4(Range)MetaboliteCanavanine (ppm dw)80.13 (7.01)111.94 (7.01)-31.81 (7.01)-48.97, -14.650.0030, 137.35(64.19 - 87.83)(87.61 - 134.50)(-53.36 - -23.42)(11.47 - 151.33)Ferulic Acid (ppm dw)1584.79 (68.95)1438.26 (68.95)146.54 (97.51)-74.04, 367.110.167854.88, 2061.10(1392.41 - 1729.26)(1334.86 - 1582.61)(-190.20 - 394.40)(1103.32 - 1906.86)Free Phenylalanine (ppm dw)225.68 (24.61)252.06 (24.61)-26.39 (34.02)-109.64, 56.860.4670, 627.23(172.15 - 253.48)(174.67 - 327.98)(-101.34 - 75.77)(133.05 - 579.05)Total Polyphenols (mg/g dw)7.25 (0.28)7.27 (0.28)-0.020 (0.40)-0.93, 0.880.9604.86, 11.15(6.62 - 8.34)(6.93 - 7.55)(-0.93 - 1.41)(6.17 - 11.17)p-Coumaric Acid (ppm dw)630.59 (26.98)595.81 (26.98)34.78 (38.15)-51.53, 121.090.385188.81, 949.95(544.30 - 716.16)(545.87 - 626.13)(-81.78 - 170.29)(326.19 - 945.58)1dw = dry weight.2Mean (S.E.) = least-square mean (standard error)3Control refers to the non-biotechnology derived, conventional control (C0-Syn1).4With 95% confidence, interval contains 99% of the values expressed in the population of conventional commercial reference varietiesNegative limits set to zero.Monsanto Company 12-AL-246U 302 of 407Table E-11. Statistical Summary of Site TXCL Alfalfa ForageNutrients for KK179 vs. Conventional Control (Conducted atCovance Laboratories)Difference (Test minus Control)Analytical Component (Units)1KK179Mean (S.E.)2(Range)Control3Mean (S.E.)(Range)Mean (S.E.)(Range)95%ConfidenceIntervalSignificance(p-Value)CommercialTolerance Interval4(Range)Proximate (% dw)Ash12.12 (0.30)12.27 (0.30)-0.15 (0.42)-1.10, 0.790.7256.70, 13.54(11.33 - 12.93)(11.53 - 12.95)(-1.32 - 1.06)(7.54 - 13.23)Carbohydrates65.05 (0.68)64.63 (0.68)0.42 (0.90)-1.79, 2.620.65950.57, 81.80(62.89 - 66.54)(63.47 - 65.69)(-2.80 - 3.07)(54.35 - 74.91)Moisture (% fw)74.18 (0.50)72.60 (0.50)1.58 (0.71)-0.021, 3.170.05265.06, 90.61(73.70 - 74.50)(70.50 - 74.40)(0.10 - 3.60)(66.10 - 85.30)Protein20.42 (0.46)20.58 (0.46)-0.17 (0.65)-1.63, 1.300.8019.26, 33.78(18.86 - 21.17)(19.56 - 21.90)(-1.55 - 1.61)(14.52 - 30.07)Total Fat2.43 (0.29)2.54 (0.29)-0.11 (0.29)-0.82, 0.600.7130.73, 3.59(1.45 - 2.99)(1.63 - 3.15)(-0.54 - 0.49)(0.53 - 4.21)Fiber (% dw)Acid Detergent Fiber26.32 (1.02)26.16 (1.02)0.16 (1.32)-3.06, 3.380.9076.16, 49.06(24.36 - 29.88)(24.75 - 27.66)(-2.63 - 3.36)(7.07 - 39.11)Monsanto Company 12-AL-246U 303 of 407Table E-11 (continued). Statistical Summary of Site TXCL Alfalfa Forage Nutrients for KK179 vs. Conventional Control(Conducted at Covance Laboratories)Difference (Test minus Control)Analytical Component (Units)1KK179Mean (S.E.)2(Range)Control3Mean (S.E.)(Range)Mean (S.E.)(Range)95%ConfidenceIntervalSignificance(p-Value)CommercialTolerance Interval4(Range)Fiber (% dw)Acid Detergent Lignin7.43 (0.66)6.64 (0.66)0.80 (0.66)-0.82, 2.410.2732.13, 11.99(5.86 - 10.31)(5.88 - 7.28)(-0.11 - 3.39)(3.38 - 9.67)Neutral Detergent Fiber32.47 (1.21)31.92 (1.21)0.55 (0.97)-1.83, 2.930.59112.04, 58.18(28.34 - 36.00)(30.64 - 33.71)(-2.30 - 2.82)(18.97 - 49.82)Amino Acid (% dw)Alanine1.22 (0.049)1.22 (0.049)-0.0012 (0.070)-0.16, 0.160.9860.49, 1.79(1.19 - 1.28)(1.09 - 1.39)(-0.19 - 0.13)(0.80 - 1.66)Arginine1.02 (0.036)1.02 (0.036)0.0031 (0.051)-0.11, 0.120.9520.44, 1.59(0.97 - 1.05)(0.95 - 1.08)(-0.031 - 0.063)(0.70 - 1.44)Aspartic acid2.11 (0.060)2.27 (0.060)-0.16 (0.084)-0.35, 0.0310.0900.44, 5.63(1.97 - 2.23)(2.04 - 2.42)(-0.30 - 0.19)(1.96 - 5.15)Cystine0.21 (0.0065)0.20 (0.0065)0.0073 (0.0088)-0.014, 0.0290.4370.12, 0.32(0.19 - 0.22)(0.18 - 0.22)(-0.0093 - 0.024)(0.16 - 0.31)Monsanto Company 12-AL-246U 304 of 407Table E-11 (continued). Statistical Summary of Site TXCL Alfalfa Forage Nutrients for KK179 vs. Conventional Control(Conducted at Covance Laboratories)Difference (Test minus Control)Analytical Component (Units)1KK179Mean (S.E.)2(Range)Control3Mean (S.E.)(Range)Mean (S.E.)(Range)95%ConfidenceIntervalSignificance(p-Value)CommercialTolerance Interval4(Range)Amino Acid (% dw)Glutamic acid1.86 (0.065)1.96 (0.065)-0.10 (0.092)-0.31, 0.110.3020.81, 3.01(1.80 - 1.92)(1.83 - 2.06)(-0.17 - 0.011)(1.31 - 2.80)Glycine1.03 (0.026)1.05 (0.026)-0.021 (0.037)-0.10, 0.0620.5830.49, 1.44(1.00 - 1.05)(0.98 - 1.10)(-0.077 - 0.042)(0.70 - 1.33)Histidine0.44 (0.011)0.45 (0.011)-0.012 (0.014)-0.047, 0.0230.4210.26, 0.63(0.43 - 0.44)(0.43 - 0.48)(-0.053 - 0.0095)(0.34 - 0.61)Isoleucine0.90 (0.028)0.91 (0.028)-0.0062 (0.039)-0.095, 0.0820.8770.43, 1.36(0.87 - 0.95)(0.84 - 0.97)(-0.070 - 0.051)(0.63 - 1.27)Leucine1.51 (0.046)1.53 (0.046)-0.023 (0.065)-0.17, 0.120.7340.70, 2.25(1.47 - 1.57)(1.42 - 1.62)(-0.11 - 0.057)(1.03 - 2.05)Lysine1.19 (0.035)1.20 (0.035)-0.0061 (0.049)-0.12, 0.110.9040.55, 1.82(1.16 - 1.24)(1.12 - 1.30)(-0.14 - 0.11)(0.82 - 1.73)Monsanto Company 12-AL-246U 305 of 407Table E-11 (continued). Statistical Summary of Site TXCL Alfalfa Forage Nutrients for KK179 vs. Conventional Control(Conducted at Covance Laboratories)Difference (Test minus Control)Analytical Component (Units)1KK179Mean (S.E.)2(Range)Control3Mean (S.E.)(Range)Mean (S.E.)(Range)95%ConfidenceIntervalSignificance(p-Value)CommercialTolerance Interval4(Range)Amino Acid (% dw)Methionine0.24 (0.034)0.21 (0.034)0.025 (0.049)-0.085, 0.130.6230.068, 0.42(0.19 - 0.29)(0.15 - 0.31)(-0.12 - 0.14)(0.14 - 0.45)Phenylalanine1.04 (0.029)1.06 (0.029)-0.019 (0.041)-0.11, 0.0750.6630.48, 1.53(1.01 - 1.07)(0.99 - 1.12)(-0.057 - 0.034)(0.71 - 1.39)Proline0.92 (0.039)1.03 (0.039)-0.11 (0.055)-0.23, 0.0150.0780.43, 1.41(0.88 - 0.94)(0.93 - 1.21)(-0.28 - -0.0051)(0.65 - 1.24)Serine0.85 (0.031)0.89 (0.031)-0.037 (0.044)-0.14, 0.0630.4250.45, 1.35(0.80 - 0.89)(0.83 - 0.93)(-0.10 - 0.031)(0.66 - 1.25)Threonine0.90 (0.031)0.93 (0.031)-0.031 (0.044)-0.13, 0.0680.4950.45, 1.33(0.87 - 0.93)(0.86 - 0.99)(-0.055 - 0.041)(0.63 - 1.23)Tryptophan0.40 (0.016)0.40 (0.016)0.0054 (0.023)-0.047, 0.0580.8190.20, 0.56(0.38 - 0.43)(0.36 - 0.45)(-0.026 - 0.035)(0.25 - 0.50)Monsanto Company 12-AL-246U 306 of 407Table E-11 (continued). Statistical Summary of Site TXCL Alfalfa Forage Nutrients for KK179 vs. Conventional Control(Conducted at Covance Laboratories)Difference (Test minus Control)Analytical Component (Units)1KK179Mean (S.E.)2(Range)Control3Mean (S.E.)(Range)Mean (S.E.)(Range)95%ConfidenceIntervalSignificance(p-Value)CommercialTolerance Interval4(Range)Amino Acid (% dw)Tyrosine0.75 (0.025)0.71 (0.025)0.040 (0.035)-0.040, 0.120.2820.35, 1.09(0.71 - 0.78)(0.66 - 0.78)(-0.017 - 0.066)(0.52 - 1.01)Valine1.08 (0.033)1.09 (0.033)-0.0079 (0.047)-0.11, 0.0990.8700.52, 1.64(1.04 - 1.13)(1.01 - 1.14)(-0.065 - 0.044)(0.79 - 1.55)MineralCalcium (% dw)2.34 (0.091)2.35 (0.091)-0.012 (0.13)-0.30, 0.280.9270.55, 2.56(2.12 - 2.62)(2.05 - 2.53)(-0.41 - 0.28)(0.95 - 2.07)Copper (mg/kg dw)11.09 (0.76)9.67 (0.76)1.42 (1.01)-1.32, 4.160.2281.87, 14.98(8.52 - 13.16)(8.75 - 11.64)(-0.67 - 4.04)(4.54 - 19.67)Iron (mg/kg dw)338.53 (28.04)308.71 (28.04)29.82 (35.02)-55.86, 115.500.42741.59, 446.31(221.18 - 417.97)(270.51 - 356.46)(-60.07 - 115.21)(105.45 - 691.43)Magnesium (% dw)0.24 (0.015)0.25 (0.015)-0.013 (0.018)-0.056, 0.0300.4860.027, 0.41(0.21 - 0.28)(0.23 - 0.29)(-0.048 - 0.037)(0.11 - 0.34)Monsanto Company 12-AL-246U 307 of 407Table E-11 (continued). Statistical Summary of Site TXCL Alfalfa Forage Nutrients for KK179 vs. Conventional Control(Conducted at Covance Laboratories)Difference (Test minus Control)Analytical Component (Units)1KK179Mean (S.E.)2(Range)Control3Mean (S.E.)(Range)Mean (S.E.)(Range)95%ConfidenceIntervalSignificance(p-Value)CommercialTolerance Interval4(Range)MineralManganese (mg/kg dw)61.36 (2.91)59.33 (2.91)2.02 (4.05)-7.89, 11.940.63517.53, 69.85(49.41 - 71.48)(57.63 - 61.25)(-9.18 - 11.63)(23.24 - 98.04)Phosphorus (% dw)0.23 (0.0056)0.21 (0.0056)0.014 (0.0079)-0.0040, 0.0320.1130.14, 0.46(0.22 - 0.24)(0.20 - 0.23)(-0.00067 - 0.030)(0.18 - 0.43)Potassium (% dw)2.45 (0.067)2.47 (0.067)-0.017 (0.079)-0.21, 0.180.8371.82, 3.04(2.29 - 2.65)(2.37 - 2.54)(-0.085 - 0.10)(1.85 - 3.35)Sodium (% dw)0.052 (0.010)0.033 (0.010)0.019 (0.014)-0.013, 0.0510.2170, 0.24(0.040 - 0.069)(0.018 - 0.058)(-0.013 - 0.049)(0.016 - 0.20)Zinc (mg/kg dw)23.52 (3.82)22.92 (3.82)0.59 (5.40)-11.62, 12.800.9158.89, 47.44(19.89 - 27.30)(20.58 - 25.09)(-5.21 - 6.72)(17.08 - 47.48)1dw = dry weight; fw = fresh weight2Mean (S.E.) = least-square mean (standard error)3Control refers to the non-biotechnology derived, conventional control (C0-Syn1).4With 95% confidence, interval contains 99% of the values expressed in the population of conventional commercial reference varieties.Negative limits set to zero.Monsanto Company 12-AL-246U 308 of 407Table E-12. Statistical Summary of SiteTXCL Alfalfa Forage Secondary Metabolites for KK179 vs. Conventional Control(Conducted at Covance Laboratories)Difference (Test minus Control)Analytical Component (Units)1KK179Mean (S.E.)2(Range)Control3Mean (S.E.)(Range)Mean (S.E.)(Range)95%ConfidenceIntervalSignificance(p-Value)CommercialTolerance Interval4(Range)MetaboliteCanavanine (ppm dw)44.54 (7.37)84.96 (7.37)-40.42 (10.42)-63.99, -16.850.0030, 137.35(29.96 - 53.52)(70.11 - 109.49)(-79.53 - -22.46)(11.47 - 151.33)Ferulic Acid (ppm dw)1831.12 (68.79)1763.27 (68.79)67.86 (76.96)-120.45, 256.170.411854.88, 2061.10(1737.25 - 1884.17)(1594.89 - 2007.38)(-148.06 - 248.86)(1103.32 - 1906.86)Free Phenylalanine (ppm dw)290.05 (26.29)346.46 (26.29)-56.41 (20.60)-106.82, -6.010.0330, 627.23(244.11 - 332.05)(298.89 - 457.63)(-125.58 - -6.99)(133.05 - 579.05)Total Polyphenols (mg/g dw)8.61 (0.36)8.22 (0.36)0.39 (0.46)-0.74, 1.520.4294.86, 11.15(7.99 - 9.35)(7.41 - 9.12)(-1.13 - 1.65)(6.17 - 11.17)p-Coumaric Acid (ppm dw)676.49 (34.94)700.57 (34.94)-24.08 (43.71)-131.03, 82.870.601188.81, 949.95(600.00 - 726.56)(644.53 - 797.05)(-112.64 - 40.43)(326.19 - 945.58)1dw = dry weight2Mean (S.E.) = least-square mean (standard error)3Control refers to the non-biotechnology derived, conventional control (C0-Syn1).4With 95% confidence, interval contains 99% of the values expressed in the population of conventional commercial reference varietiesNegative limits set to zero.Monsanto Company 12-AL-246U 309 of 407Table E-13. Statistical Summary of Site WIDL Alfalfa ForageNutrients for KK179 vs. Conventional Control (Conducted atCovance Laboratories)Difference (Test minus Control)Analytical Component (Units)1KK179Mean (S.E.)2(Range)Control3Mean (S.E.)(Range)Mean (S.E.)(Range)95%ConfidenceIntervalSignificance(p-Value)CommercialTolerance Interval4(Range)Proximate (% dw)Ash9.05 (0.27)9.21 (0.27)-0.16 (0.38)-1.03, 0.710.6856.70, 13.54(8.43 - 10.13)(8.99 - 9.63)(-0.79 - 1.01)(7.54 - 13.23)Carbohydrates72.45 (0.51)71.82 (0.51)0.63 (0.73)-1.01, 2.270.40850.57, 81.80(70.80 - 73.90)(70.54 - 72.91)(-2.11 - 2.08)(54.35 - 74.91)Moisture (% fw)75.83 (0.42)75.08 (0.42)0.75 (0.56)-0.62, 2.120.23065.06, 90.61(74.80 - 77.40)(74.20 - 75.90)(-0.20 - 2.50)(66.10 - 85.30)Protein16.75 (0.41)16.97 (0.41)-0.23 (0.58)-1.53, 1.080.7059.26, 33.78(15.50 - 17.65)(15.98 - 18.18)(-1.51 - 1.68)(14.52 - 30.07)Total Fat1.86 (0.18)2.07 (0.18)-0.22 (0.26)-0.80, 0.360.4210.73, 3.59(1.60 - 2.14)(2.01 - 2.17)(-0.47 - 0.13)(0.53 - 4.21)Fiber (% dw)Acid Detergent Fiber34.76 (1.41)30.88 (1.41)3.88 (1.99)-0.63, 8.390.0836.16, 49.06(31.41 - 37.26)(27.69 - 36.11)(-4.71 - 9.57)(7.07 - 39.11)Monsanto Company 12-AL-246U 310 of 407Table E-13 (continued). Statistical Summary of Site WIDL Alfalfa Forage Nutrients for KK179 vs. Conventional Control(Conducted at Covance Laboratories)Difference (Test minus Control)Analytical Component (Units)1KK179Mean (S.E.)2(Range)Control3Mean (S.E.)(Range)Mean (S.E.)(Range)95%ConfidenceIntervalSignificance(p-Value)CommercialTolerance Interval4(Range)Fiber (% dw)Acid Detergent Lignin7.88 (0.39)7.51 (0.39)0.37 (0.53)-0.93, 1.660.5162.13, 11.99(6.83 - 8.58)(6.15 - 8.26)(-0.77 - 2.24)(3.38 - 9.67)Neutral Detergent Fiber42.99 (1.53)39.31 (1.53)3.68 (1.61)-0.26, 7.630.06212.04, 58.18(37.47 - 48.67)(36.67 - 40.64)(-1.88 - 8.04)(18.97 - 49.82)Amino Acid (% dw)Alanine0.87 (0.024)0.93 (0.024)-0.055 (0.033)-0.13, 0.0210.1330.49, 1.79(0.84 - 0.90)(0.87 - 1.03)(-0.19 - 0.014)(0.80 - 1.66)Arginine0.76 (0.020)0.82 (0.020)-0.060 (0.029)-0.12, 0.00460.0650.44, 1.59(0.73 - 0.78)(0.75 - 0.90)(-0.17 - 0.014)(0.70 - 1.44)Aspartic acid2.25 (0.070)2.15 (0.070)0.10 (0.099)-0.13, 0.320.3410.44, 5.63(2.12 - 2.51)(2.09 - 2.23)(-0.035 - 0.28)(1.96 - 5.15)Cystine0.17 (0.0084)0.18 (0.0084)-0.0082 (0.010)-0.034, 0.0170.4610.12, 0.32(0.15 - 0.19)(0.15 - 0.20)(-0.030 - 0.017)(0.16 - 0.31)Monsanto Company 12-AL-246U 311 of 407Table E-13 (continued). Statistical Summary of Site WIDL Alfalfa Forage Nutrients for KK179 vs. Conventional Control(Conducted at Covance Laboratories)Difference (Test minus Control)Analytical Component (Units)1KK179Mean (S.E.)2(Range)Control3Mean (S.E.)(Range)Mean (S.E.)(Range)95%ConfidenceIntervalSignificance(p-Value)CommercialTolerance Interval4(Range)Amino Acid (% dw)Glutamic acid1.47 (0.040)1.54 (0.040)-0.075 (0.057)-0.20, 0.0540.2220.81, 3.01(1.40 - 1.52)(1.39 - 1.70)(-0.30 - 0.065)(1.31 - 2.80)Glycine0.77 (0.016)0.78 (0.016)-0.015 (0.023)-0.067, 0.0380.5450.49, 1.44(0.75 - 0.79)(0.73 - 0.85)(-0.099 - 0.022)(0.70 - 1.33)Histidine0.36 (0.0056)0.38 (0.0056)-0.013 (0.0068)-0.029, 0.00400.1130.26, 0.63(0.35 - 0.38)(0.36 - 0.40)(-0.033 - 0.00032)(0.34 - 0.61)Isoleucine0.70 (0.018)0.72 (0.018)-0.018 (0.026)-0.078, 0.0410.4970.43, 1.36(0.67 - 0.73)(0.66 - 0.78)(-0.12 - 0.024)(0.63 - 1.27)Leucine1.14 (0.031)1.19 (0.031)-0.051 (0.043)-0.15, 0.0470.2720.70, 2.25(1.09 - 1.18)(1.09 - 1.31)(-0.22 - 0.023)(1.03 - 2.05)Lysine0.93 (0.021)0.98 (0.021)-0.053 (0.027)-0.12, 0.0140.1000.55, 1.82(0.93 - 0.93)(0.92 - 1.08)(-0.15 - 0.0086)(0.82 - 1.73)Monsanto Company 12-AL-246U 312 of 407Table E-13 (continued). Statistical Summary of Site WIDL Alfalfa ForageNutrients for KK179 vs. ConventionalControl(Conducted at Covance Laboratories)Difference (Test minus Control)Analytical Component (Units)1KK179Mean (S.E.)2(Range)Control3Mean (S.E.)(Range)Mean (S.E.)(Range)95%ConfidenceIntervalSignificance(p-Value)CommercialTolerance Interval4(Range)Amino Acid (% dw)Methionine0.17 (0.017)0.18 (0.017)-0.015 (0.021)-0.066, 0.0360.5060.068, 0.42(0.15 - 0.21)(0.15 - 0.22)(-0.062 - 0.011)(0.14 - 0.45)Phenylalanine0.77 (0.020)0.81 (0.020)-0.038 (0.029)-0.10, 0.0270.2230.48, 1.53(0.75 - 0.80)(0.74 - 0.89)(-0.15 - 0.018)(0.71 - 1.39)Proline0.73 (0.015)0.75 (0.015)-0.022 (0.021)-0.069, 0.0240.3080.43, 1.41(0.71 - 0.76)(0.71 - 0.80)(-0.092 - 0.051)(0.65 - 1.24)Serine0.73 (0.022)0.75 (0.022)-0.012 (0.032)-0.083, 0.0600.7210.45, 1.35(0.68 - 0.80)(0.68 - 0.81)(-0.13 - 0.055)(0.66 - 1.25)Threonine0.69 (0.017)0.72 (0.017)-0.029 (0.024)-0.083, 0.0260.2660.45, 1.33(0.66 - 0.73)(0.67 - 0.79)(-0.13 - 0.019)(0.63 - 1.23)Tryptophan0.31 (0.0090)0.29 (0.0090)0.015 (0.013)-0.014, 0.0440.2640.20, 0.56(0.30 - 0.33)(0.27 - 0.31)(-0.011 - 0.062)(0.25 - 0.50)Monsanto Company 12-AL-246U 313 of 407Table E-13 (continued). Statistical Summary of Site WIDL Alfalfa Forage Nutrients for KK179 vs. Conventional Control(Conducted at Covance Laboratories)Difference (Test minus Control)Analytical Component (Units)1KK179Mean (S.E.)2(Range)Control3Mean (S.E.)(Range)Mean (S.E.)(Range)95%ConfidenceIntervalSignificance(p-Value)CommercialTolerance Interval4(Range)Amino Acid (% dw)Tyrosine0.57 (0.013)0.58 (0.013)-0.013 (0.019)-0.056, 0.0300.5040.35, 1.09(0.55 - 0.58)(0.53 - 0.63)(-0.053 - 0.024)(0.52 - 1.01)Valine0.85 (0.026)0.87 (0.026)-0.023 (0.036)-0.11, 0.0590.5430.52, 1.64(0.79 - 0.90)(0.81 - 0.96)(-0.16 - 0.046)(0.79 - 1.55)MineralCalcium (% dw)1.14 (0.031)1.22 (0.031)-0.082 (0.044)-0.18, 0.0180.0960.55, 2.56(1.12 - 1.15)(1.09 - 1.31)(-0.17 - 0.035)(0.95 - 2.07)Copper (mg/kg dw)5.78 (0.18)5.74 (0.18)0.047 (0.25)-0.52, 0.610.8541.87, 14.98(5.14 - 6.33)(5.42 - 5.93)(-0.65 - 0.91)(4.54 - 19.67)Iron (mg/kg dw)258.80 (29.54)262.86 (29.54)-4.05 (37.93)-96.85, 88.740.91841.59, 446.31(200.00 - 357.52)(210.08 - 366.93)(-46.89 - 41.91)(105.45 - 691.43)Magnesium (% dw)0.23 (0.0061)0.22 (0.0061)0.0043 (0.0086)-0.015, 0.0240.6270.027, 0.41(0.22 - 0.25)(0.21 - 0.24)(-0.025 - 0.022)(0.11 - 0.34)Monsanto Company 12-AL-246U 314 of 407Table E-13 (continued). Statistical Summary of Site WIDL Alfalfa Forage Nutrients for KK179 vs. Conventional Control(Conducted at Covance Laboratories)Difference (Test minus Control)Analytical Component (Units)1KK179Mean (S.E.)2(Range)Control3Mean (S.E.)(Range)Mean (S.E.)(Range)95%ConfidenceIntervalSignificance(p-Value)CommercialTolerance Interval4(Range)MineralManganese (mg/kg dw)56.24 (3.32)55.01 (3.32)1.24 (4.65)-10.13, 12.600.79917.53, 69.85(49.40 - 65.93)(44.94 - 62.15)(-8.50 - 5.20)(23.24 - 98.04)Phosphorus (% dw)0.30 (0.0075)0.29 (0.0075)0.011 (0.0098)-0.013, 0.0350.2870.14, 0.46(0.28 - 0.32)(0.27 - 0.31)(-0.025 - 0.035)(0.18 - 0.43)Potassium (% dw)2.39 (0.063)2.34 (0.063)0.055 (0.060)-0.092, 0.200.3931.82, 3.04(2.30 - 2.44)(2.23 - 2.56)(-0.13 - 0.21)(1.85 - 3.35)Sodium (% dw)0.021 (0.012)0.020 (0.012)0.00065 (0.017)-0.040, 0.0420.9700, 0.24(0.020 - 0.022)(0.019 - 0.021)(-0.00016 - 0.0022)(0.016 - 0.20)Zinc (mg/kg dw)29.54 (0.95)28.13 (0.95)1.42 (1.34)-1.61, 4.440.3168.89, 47.44(28.89 - 30.27)(25.82 - 32.16)(-2.53 - 4.45)(17.08 - 47.48)1dw = dry weight; fw = fresh weight2Mean (S.E.) = least-square mean (standard error)3Control refers to the non-biotechnology derived, conventional control (C0-Syn1).4With 95% confidence, interval contains 99% of the values expressed in the population of conventional commercial reference varieties.Negative limits set to zero.Monsanto Company 12-AL-246U 315 of 407Table E-14. Statistical Summary of SiteWIDL Alfalfa Forage Secondary Metabolites for KK179 vs. Conventional Control(Conducted at Covance Laboratories)Difference (Test minus Control)Analytical Component (Units)1KK179Mean (S.E.)2(Range)Control3Mean (S.E.)(Range)Mean (S.E.)(Range)95%ConfidenceIntervalSignificance(p-Value)CommercialTolerance Interval4(Range)MetaboliteCanavanine (ppm dw)43.03 (3.39)49.63 (3.39)-6.60 (4.79)-17.44, 4.240.2010, 137.35(38.76 - 46.25)(39.92 - 59.92)(-21.16 - 5.32)(11.47 - 151.33)Ferulic Acid (ppm dw)1443.73 (38.85)1367.10 (38.85)76.63 (54.94)-47.65, 200.920.196854.88, 2061.10(1389.38 - 1479.17)(1314.74 - 1403.10)(33.41 - 117.65)(1103.32 - 1906.86)Free Phenylalanine (ppm dw)224.65 (15.68)214.48 (15.68)10.17 (20.04)-38.87, 59.210.6290, 627.23(190.36 - 267.26)(155.04 - 246.15)(-55.79 - 58.45)(133.05 - 579.05)Total Polyphenols (mg/g dw)6.90 (0.43)7.38 (0.43)-0.48 (0.60)-1.84, 0.880.4474.86, 11.15(6.35 - 7.35)(6.57 - 8.22)(-1.87 - 0.77)(6.17 - 11.17)p-Coumaric Acid (ppm dw)526.67 (25.69)483.23 (25.69)43.44 (35.63)-43.74, 130.630.268188.81, 949.95(458.33 - 566.37)(469.64 - 503.88)(-22.99 - 88.28)(326.19 - 945.58)1dw = dry weight2Mean (S.E.) = least-square mean (standard error)3Control refers to the non-biotechnology derived, conventional control (C0-Syn1).4With 95% confidence, interval contains 99% of the values expressed in the population of conventional commercial reference varietiesNegative limits set to zero.Monsanto Company 12-AL-246U 316 of 407Table E -15. Statistical Summary of Alfalfa Forage Anti-Nutrients for KK179 vs. Conventional Control (Conducted at SamuelRoberts Noble Foundation)Difference (Test minus Control)Analytical Component (Units)1KK179Mean (S.E.)2(Range)Control3Mean (S.E.)(Range)Mean (S.E.)(Range)95%ConfidenceIntervalSignificance(p-Value)CommercialTolerance Interval4(Range)Saponins (response units/μg)Site CAPRTotal Bayogenin8.30 (2.52)4.98 (1.66)3.33 (2.85)-3.84, 10.490.2920.92, 8.86(3.00 - 13.97)(3.58 - 8.16)(-1.44 - 5.81)(1.46 - 11.28)Total Hederagenin4.16 (0.64)3.25 (0.53)0.91 (0.54)-0.55, 2.370.1630.85, 7.20(3.01 - 5.80)(2.14 - 4.59)(-0.11 - 1.21)(0.90 - 10.31)Total Medicagenic Acid38.13 (8.20)26.51 (5.15)11.63 (9.45)-12.14, 35.390.2690, 44.42(31.41 - 45.08)(9.43 - 40.45)(4.64 - 11.57)(2.04 - 48.33)Monsanto Company 12-AL-246U 317 of 407Table E-15 (continued). Statistical Summary of Alfalfa ForageAnti-Nutrients for KK179 vs. C0-Syn1 (Conducted at SamuelRoberts Noble Foundation)Difference (Test minus Control)Analytical Component (Units)1KK179Mean (S.E.)2(Range)Control3Mean (S.E.)(Range)Mean (S.E.)(Range)95%ConfidenceIntervalSignificance(p-Value)CommercialTolerance Interval4(Range)Saponins (response units/μg)Site CAPRTotal Soyasapogenol B26.47 (5.31)24.88 (3.97)1.59 (5.27)-12.28, 15.460.7757.83, 44.92(15.68 - 40.48)(17.41 - 31.77)(-10.25 - 8.72)(9.22 - 43.87)Total Soyasapogenol E3.42 (0.77)2.74 (0.49)0.68 (0.90)-1.56, 2.910.4810, 6.59(1.84 - 5.02)(2.12 - 3.16)(-1.30 - 1.87)(0.91 - 7.53)Total Zanhic Acid7.14 (2.29)5.03 (1.52)2.11 (2.56)-4.36, 8.570.4460.32, 12.06(2.58 - 12.08)(3.66 - 8.39)(-1.45 - 3.69)(1.75 - 13.20)Total Saponins87.15 (16.16)67.38 (11.60)19.76 (16.73)-23.88, 63.400.29321.87, 108.47(57.51 - 122.44)(38.88 - 96.50)(-2.99 - 25.94)(17.38 - 103.19)Site IARLTotal Bayogenin3.55 (0.41)2.80 (0.57)0.74 (0.70)-0.92, 2.410.3260.92, 8.86(3.06 - 4.44)(2.20 - 3.40)(-0.034 - 1.11)(1.46 - 11.28)Monsanto Company 12-AL-246U 318 of 407Table E-15 (continued). Statistical Summary of Alfalfa ForageAnti-Nutrients for KK179 vs. C0-Syn1 (Conducted at SamuelRoberts Noble Foundation)Difference (Test minus Control)Analytical Component (Units)1KK179Mean (S.E.)2(Range)Control3Mean (S.E.)(Range)Mean (S.E.)(Range)95%ConfidenceIntervalSignificance(p-Value)CommercialTolerance Interval4(Range)Saponins (response units/μg)Site IARLTotal Hederagenin2.37 (0.25)1.91 (0.35)0.47 (0.43)-0.54, 1.480.3090.85, 7.20(1.83 - 3.09)(1.58 - 2.23)(-0.40 - 0.73)(0.90 - 10.31)Total Medicagenic Acid18.75 (1.98)20.09 (2.98)-1.33 (3.28)-9.78, 7.120.7010, 44.42(15.66 - 21.15)(14.90 - 24.08)(-8.41 - 2.34)(2.04 - 48.33)Total Soyasapogenol B12.72 (1.53)9.61 (2.16)3.11 (2.65)-3.16, 9.380.2787.83, 44.92(9.68 - 18.28)(7.05 - 12.16)(-0.61 - 4.31)(9.22 - 43.87)Total Soyasapogenol E1.40 (0.12)1.10 (0.17)0.30 (0.21)-0.19, 0.790.1870, 6.59(1.20 - 1.73)(0.84 - 1.35)(-0.15 - 0.38)(0.91 - 7.53)Total Zanhic Acid2.80 (0.41)3.15 (0.59)-0.35 (0.72)-2.05, 1.350.6420.32, 12.06(2.25 - 4.21)(2.62 - 3.67)(-1.29 - -0.27)(1.75 - 13.20)Monsanto Company 12-AL-246U 319 of 407Table E-15 (continued). Statistical Summary of Alfalfa ForageAnti-Nutrients for KK179 vs. C0-Syn1 (Conducted at SamuelRoberts Noble Foundation)Difference (Test minus Control)Analytical Component (Units)1KK179Mean (S.E.)2(Range)Control3Mean (S.E.)(Range)Mean (S.E.)(Range)95%ConfidenceIntervalSignificance(p-Value)CommercialTolerance Interval4(Range)Saponins (response units/μg)Site IARLTotal Saponins41.59 (4.25)38.05 (6.01)3.54 (7.36)-13.86, 20.930.64521.87, 108.47(36.00 - 52.91)(29.20 - 46.90)(-10.90 - 8.59)(17.38 - 103.19)Site ILCYTotal Bayogenin3.79 (0.55)5.23 (0.55)-1.45 (0.59)-2.89, -0.00430.0490.92, 8.86(2.81 - 4.52)(2.87 - 6.77)(-2.25 - -0.061)(1.46 - 11.28)Total Hederagenin2.59 (0.40)3.10 (0.40)-0.50 (0.48)-1.68, 0.670.3330.85, 7.20(1.89 - 3.66)(2.37 - 4.68)(-1.82 - 0.83)(0.90 - 10.31)Total Medicagenic Acid27.83 (4.68)30.73 (4.68)-2.90 (5.40)-16.12, 10.310.6100, 44.42(19.21 - 36.75)(19.96 - 51.04)(-14.28 - 9.18)(2.04 - 48.33)Total Soyasapogenol B16.69 (2.17)21.58 (2.17)-4.89 (2.34)-10.61, 0.830.0817.83, 44.92(14.30 - 22.25)(13.56 - 26.38)(-11.46 - 0.75)(9.22 - 43.87)Monsanto Company 12-AL-246U 320 of 407Table E-15 (continued). Statistical Summary of Alfalfa ForageAnti-Nutrients for KK179 vs. C0-Syn1 (Conducted at SamuelRoberts Noble Foundation)Difference (Test minus Control)Analytical Component (Units)1KK179Mean (S.E.)2(Range)Control3Mean (S.E.)(Range)Mean (S.E.)(Range)95%ConfidenceIntervalSignificance(p-Value)CommercialTolerance Interval4(Range)Saponins (response units/μg)Site ILCYTotal Soyasapogenol E1.94 (0.26)2.58 (0.26)-0.64 (0.23)-1.21, -0.0730.0320, 6.59(1.47 - 2.72)(1.82 - 3.18)(-1.12 - -0.28)(0.91 - 7.53)Total Zanhic Acid3.49 (0.42)4.35 (0.42)-0.86 (0.52)-2.14, 0.420.1500.32, 12.06(2.80 - 3.88)(2.67 - 5.75)(-1.87 - 0.13)(1.75 - 13.20)Total Saponins56.32 (7.09)67.57 (7.09)-11.25 (7.52)-29.65, 7.160.18521.87, 108.47(42.54 - 68.38)(43.25 - 94.51)(-30.05 - 1.39)(17.38 - 103.19)Site KSLATotal Bayogenin4.83 (0.49)6.30 (0.49)-1.48 (0.59)-2.91, -0.0390.0450.92, 8.86(3.83 - 6.60)(5.45 - 6.71)(-2.39 - -0.12)(1.46 - 11.28)Total Hederagenin2.44 (0.53)4.04 (0.53)-1.59 (0.68)-3.26, 0.0730.0570.85, 7.20(2.09 - 3.13)(2.97 - 4.62)(-2.10 - -0.88)(0.90 - 10.31)Monsanto Company 12-AL-246U 321 of 407Table E-15 (continued). Statistical Summary of Alfalfa ForageAnti-Nutrients for KK179 vs. C0-Syn1 (Conducted at SamuelRoberts Noble Foundation)Difference (Test minus Control)Analytical Component (Units)1KK179Mean (S.E.)2(Range)Control3Mean (S.E.)(Range)Mean (S.E.)(Range)95%ConfidenceIntervalSignificance(p-Value)CommercialTolerance Interval4(Range)Saponins (response units/μg)Site KSLATotal Medicagenic Acid12.60 (3.67)18.16 (3.67)-5.57 (5.15)-18.16, 7.030.3210, 44.42(9.73 - 19.65)(15.48 - 20.96)(-8.70 - -1.31)(2.04 - 48.33)Total Soyasapogenol B24.34 (2.47)30.71 (2.47)-6.38 (2.62)-12.79, 0.0370.0507.83, 44.92(18.83 - 28.51)(23.91 - 34.73)(-10.72 - -0.70)(9.22 - 43.87)Total Soyasapogenol E4.05 (0.30)4.01 (0.30)0.045 (0.35)-0.82, 0.910.9020, 6.59(3.30 - 4.90)(3.43 - 4.31)(-1.01 - 0.70)(0.91 - 7.53)Total Zanhic Acid3.66 (0.42)5.49 (0.42)-1.83 (0.41)-2.83, -0.820.0040.32, 12.06(2.60 - 4.67)(4.88 - 6.55)(-2.62 - -0.21)(1.75 - 13.20)Total Saponins51.92 (7.16)68.72 (7.16)-16.79 (8.43)-37.41, 3.830.09321.87, 108.47(40.39 - 67.46)(62.00 - 76.11)(-22.46 - -8.65)(17.38 - 103.19)Monsanto Company 12-AL-246U 322 of 407Table E-15 (continued). Statistical Summary of Alfalfa ForageAnti-Nutrients for KK179 vs. C0-Syn1 (Conducted at SamuelRoberts Noble Foundation)Difference (Test minus Control)Analytical Component (Units)1KK179Mean (S.E.)2(Range)Control3Mean (S.E.)(Range)Mean (S.E.)(Range)95%ConfidenceIntervalSignificance(p-Value)CommercialTolerance Interval4(Range)Saponins (response units/μg)Site TXCLTotal Bayogenin7.45 (0.76)9.09 (0.76)-1.64 (1.08)-4.07, 0.790.1610.92, 8.86(5.90 - 8.43)(7.46 - 11.28)(-2.85 - 0.18)(1.46 - 11.28)Total Hederagenin3.66 (0.64)4.71 (0.64)-1.05 (0.90)-3.10, 0.990.2730.85, 7.20(2.51 - 4.92)(3.28 - 6.85)(-3.51 - 0.55)(0.90 - 10.31)Total Medicagenic Acid17.77 (3.55)17.59 (3.55)0.19 (5.02)-11.17, 11.540.9710, 44.42(9.09 - 35.00)(14.06 - 20.31)(-7.33 - 15.92)(2.04 - 48.33)Total Soyasapogenol B27.51 (3.31)31.83 (3.31)-4.32 (3.87)-13.80, 5.150.3067.83, 44.92(21.98 - 31.25)(25.02 - 41.93)(-12.47 - 3.21)(9.22 - 43.87)Total Soyasapogenol E3.80 (1.10)5.00 (1.10)-1.20 (1.21)-4.16, 1.750.3580, 6.59(2.42 - 4.49)(3.29 - 8.89)(-4.99 - 1.20)(0.91 - 7.53)Monsanto Company 12-AL-246U 323 of 407Table E-15 (continued). Statistical Summary of Alfalfa ForageAnti-Nutrients for KK179 vs. C0-Syn1 (Conducted at SamuelRoberts Noble Foundation)Difference (Test minus Control)Analytical Component (Units)1KK179Mean (S.E.)2(Range)Control3Mean (S.E.)(Range)Mean (S.E.)(Range)95%ConfidenceIntervalSignificance(p-Value)CommercialTolerance Interval4(Range)Saponins (response units/μg)Site TXCLTotal Zanhic Acid6.73 (0.92)6.77 (0.92)-0.037 (1.18)-2.93, 2.860.9760.32, 12.06(3.08 - 9.33)(6.16 - 7.89)(-3.39 - 2.76)(1.75 - 13.20)Total Saponins66.92 (8.31)74.99 (8.31)-8.07 (11.75)-34.65, 18.510.50921.87, 108.47(44.97 - 91.65)(59.72 - 95.91)(-31.55 - 19.44)(17.38 - 103.19)Site WIDLTotal Bayogenin4.16 (0.56)5.09 (0.56)-0.93 (0.79)-2.73, 0.860.2700.92, 8.86(2.54 - 5.52)(3.70 - 6.54)(-2.10 - -0.035)(1.46 - 11.28)Total Hederagenin2.79 (0.40)3.42 (0.40)-0.63 (0.56)-1.90, 0.650.2940.85, 7.20(1.70 - 3.67)(1.99 - 4.22)(-1.42 - 0.29)(0.90 - 10.31)Total Medicagenic Acid23.23 (5.18)26.30 (5.18)-3.07 (7.32)-19.63, 13.490.6840, 44.42(14.06 - 34.94)(15.02 - 42.64)(-22.95 - 12.43)(2.04 - 48.33)Monsanto Company 12-AL-246U 324 of 407Table E-15 (continued). Statistical Summary of Alfalfa ForageAnti-Nutrients for KK179 vs. C0-Syn1 (Conducted at SamuelRoberts Noble Foundation)Difference (Test minus Control)Analytical Component (Units)1KK179Mean (S.E.)2(Range)Control3Mean (S.E.)(Range)Mean (S.E.)(Range)95%ConfidenceIntervalSignificance(p-Value)CommercialTolerance Interval4(Range)Saponins (response units/μg)Site WIDLTotal Soyasapogenol B26.38 (2.10)26.31 (2.10)0.073 (2.97)-6.65, 6.800.9807.83, 44.92(21.42 - 29.70)(19.10 - 31.46)(-1.95 - 2.31)(9.22 - 43.87)Total Soyasapogenol E2.40 (0.23)2.80 (0.23)-0.39 (0.31)-1.15, 0.360.2490, 6.59(2.08 - 3.10)(2.00 - 3.45)(-0.73 - 0.081)(0.91 - 7.53)Total Zanhic Acid4.96 (0.81)5.90 (0.81)-0.93 (1.14)-3.52, 1.650.4340.32, 12.06(2.79 - 6.54)(3.77 - 8.69)(-3.97 - 1.86)(1.75 - 13.20)Total Saponins63.92 (7.99)69.81 (7.99)-5.89 (11.29)-31.43, 19.660.61421.87, 108.47(44.58 - 81.55)(45.57 - 91.36)(-32.96 - 10.64)(17.38 - 103.19)1 Response units equals peak area counts2Mean (S.E.) = least-square mean (standard error)3Control refers to the non-biotechnology derived, conventional control, C0-Syn1.4With 95% confidence, interval contains 99% of the values expressed in the population of conventional commercial reference varieties.Negative limits set to zero.Monsanto Company 12-AL-246U 325 of 407 

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AACC. 1998. Approved methods of the Ameri can Association of Cereal Chemists.

American Association of Cereal Ch emists, Inc., St. Paul, Minnesota.

AOAC. 2010a. AOAC official method 922.06: Fat in flour. Association of Analytical Communities International, Gaithersburg, Maryland.

AOAC. 2010b. AOAC offici al method 954.02: Fat (crude) or ether extract in pet food. Association of Analytical Communities International, Gaithersburg, Maryland.

AOAC. 2010c. AOAC official method 955.04: Nitroge n (total) in fertilizers. Association of Analytical Communities International, Gaithersburg, Maryland.

AOAC. 2011a. AOAC official method 982.30: Prot ein efficiency ratio. Association of Analytical Communities International, Gaithersburg, Maryland.

AOAC. 2011b. AOAC offi cial method 926.08: Loss on drying (moisture) in cheese. Association of Analytical Communities International, Gaithersburg, Maryland.

AOAC. 2011c. AOAC official method 985.01: Meta ls and other elements in plants and pet foods. Association of Analytical Comm unities International, Gaithersburg, Maryland.

AOAC. 2011d. AOAC official method 925.09: Solids (total) and loss on drying (moisture) in flour. Association of Analy tical Communities International, Gaithersburg, Maryland.

AOAC. 2011e. AOAC official method 923.03: Ash of flour. Association of Analytical Communities International, Gaithersburg, Maryland.

AOAC. 2011f. AOAC official method 979.09: Protein in grains. Association of Analytical Communities International, Gaithersburg, Maryland.

AOAC. 2011g. AOAC offi cial method 988.15: Tryptophan in foods and food and feed ingredients. Association of Analytical Communities International, Gaithersburg, Maryland.

AOAC. 2011h. AOAC official method 984.27: Calcium, copperm iron, magnesium, manganese, phosphorus, potassium, sodium, and zi nc in infant formula. Association of Analytical Communities International, Gaithersburg, Maryland.

AOCS. 2009. Nitrogen-ammonia- protein modified Kjeldahl method titanium dioxide + copper sulfate catalyst. Official Methods and Recommended Practices of the American Oil Chemists' Society, 5th Edition. Method Ac 4-91, American Oil Chemists' Society, Champaign, Illinois.Monsanto Company 12-AL-246U 326 of 407 

### Barkholt, V. and A.L. Jensen. 1989. Amino acid analysis determination of cysteine plus half-cystine in proteins af ter hydrochloric acid hydrolysis with a disulfide compound as additive. Analytical Biochemistry 177: 318-322.

Chen, Q.H., P. Li, B. Li, X.L. Li, J. Zhu and F.C. Chen. 2010. Simultaneous Determination of Formononetin, Biochanin A Daidzen and Genistein in Trifolium pratense (Red Clover) by HPLC. Lc Gc Europe 23: 406-411

Goering, H.K. and P.J. Van Soest. 1970. Forage and fiber analyses (apparatus, reagents, procedures, and some applications). Agri culture Handbook No. 379. U.S. Department of Agriculture, Agricultural Res earch Service, Washington, D.C.

Hagerman, A.E. and R.L. Nicholson. 1982. High-performance liquid chromatographic determination of hydroxycinnamic acids in the maize mesocotyl. Journal of Agricultural and Food Chemistry 30: 1098-1102.

Huhman, D.V. and L.W. Sumner. 2002. Metabolic profiling of saponins in Medicago

sativa and Medicago truncatula using HPLC coupled to an electrospray ion-trap mass spectrometer. Phytochemistry 59: 347-360.

Huhman, D.V., M.A. Berhow and L.W. Su mner. 2005. Quantification of saponins in

aerial and subterranean tissues of Medicago truncatula . Journal of Agricultural and Food Chemistry 53: 1914-1920.

Komarek, A.R., J.B. Robertson and P.J. Va n Soest. 1993. A comparison of methods for determining ADF using the Filter Bag Techni que versus conventional filtration. Journal

of Animal Science 72 114.

Komarek, A.R., J.B. Robertson and P.J. Va n Soest. 1994. Comparison of the filter bag technique to conventional filtration in the Van Soest NDF analysis of 21 foods. National Conference on Forage Quality, Evaluation, and Utilization, American Society of Agronomy, Inc., Crop Science Society of Ameri ca, Inc., Soil Society of America, Inc., Madison, Wisconsin.

OECD. 2005. Consensus document on compositiona l considerations fo r new varieties of alfalfa and other temperate forage legumes: Key feed nutrients, anti-nutrients and secondary plant metabolites. Organisation fo r Economic Co-operation and Development, Paris, France.

Pettersson, H. and K.H. Kiessling. 1984. Liqui d Chromatographic Determination of the Plant Estrogens Coumestrol and Iso Flavones in Animal Feed. Journal of the Association of Official Analytical Chemists 67: 503-506.

Schuster, R. 1988. Determination of amino aci ds in biological pharmaceutical plant and food samples by automated precolumn deri vatization and high-performance liquid chromatography. Journal of Chromatography 431: 271-284.Monsanto Company 12-AL-246U 327 of 407 

### Saloniemi, H., K. Wahala, P. Nykanenkur ki, K. Kallela and I. Saastamoinen. 1995. Phytoestrogen Content and Estrogenic Effect of Legume Fodder. Proceedings of the Society for Experiment al Biology and Medicine 208: 13-17.

Seo, A. and C.V. Morr. 1984. Improved high performance liquid chromatographic analysis of phenolic acids and isoflavonoids from soybean protein products. Journal of Agricultural and Food Chemistry 32: 530-533.

USDA. 1973. Energy value of foods: Basis and derivation. Agricultural Handbook No. 74. US Department of Agriculture, Agricultu ral Research Service, Washington, D.C.Monsanto Company 12-AL-246U 328 of 407 

### Appendix F: Materials and Methods for Seed Germination and Dormancy

Assessment of KK179

F.1. Materials

Seed germination and dormancy characteris tics were assessed on seed from KK179, the conventional control, and four conventional commercial refe rence varieties produced in a greenhouse at a single location in West Sale m, WI in 2010. Both non-scarified and scarified seed were assessed. Uniform, me chanical scarification of the seed was performed using a Forsberg manual seed scarifier.

F.2. Characterization of the Materials

The presence or absence of KK179 in th e starting seed from KK179, the conventional control ( C 0 -Syn1) 

### , and the conventional commercial re ference varieties was verified by event-specific polymerase chain reaction (PCR) analyses.

F.3. Germination Testing Facili ty and Experimental Methods

Seed germination and dormancy evaluations we re conducted at BioDiagnostics, Inc. in River Falls, WI. The princi pal investigator was qualified to conduct seed germination and dormancy testing consistent with the st andards established by the Association of Official Seed Analysts (AOSA), a seed trade association (AOSA, 2007, 2010a; b).

Seed of KK179, the conventiona l control, and four conven tional commercial reference

varieties was tested under th ree different temperature regimes. Three germination

chambers were maintained under dark conditio ns with one of the following temperature regimes: constant temperature of appr oximately 10 °C, 20 °C (AOSA recommended temperature for testing alfalfa), or 30 °C. The temperature inside each germination chamber was monitored and recorded every 15 minutes throughout the duration of the study.

Approximately 100 seeds of each treatment of KK179, the conventional control, and the

conventional commercial reference varieties were placed on pre-moistened germination towels using a vacuum planting system. A dditional pre-moistened germination towels were placed on top of the seed. The bottom edge of the towels was folded to prevent loss of seeds, and the towels were then rolled up in a wax cover. The labeled, rolled germination towels were then placed into an appropriately labeled bucket. Each replication consisted of 12 labeled germina tion towels, placed into a single, labeled bucket, for a total of four buckets per temper ature regime. Buckets were then placed in the appropriate germination chambers.

Each temperature regime constituted a separate experiment. The experiments were established in a two-way factorial design with four replications per temperature regime. The two factors assessed were material ( i.e. , KK179, conventional control, or a

conventional commercial refere nce variety) and treatment ( i.e. , scarified or nonscarified).Monsanto Company 12-AL-246U 329 of 407 

### A description of each germination characterist ic evaluated and the timing of evaluations are presented in Table VII-1. Seed pl aced in the AOSA-recommended temperature regime ( i.e. , 20 °C) was evaluated according to AOSA standards as normal germinated, abnormal germinated, hard, dead, or fi rm-swollen (AOSA, 2010a; b). AOSA only provides guidelines (AOSA, 2010a) for testing seed under optimal temperatures, whereas additional temperature regimes were included to test diverse environmental conditions. Therefore, seed placed in temperature regi mes of 10 °C and 30 °C were evaluated as germinated, hard, dead, or firm-swollen. Becau se temperature extremes could affect the development of seedlings, AOSA standards we re not applied and no distinction was made between normal or abnormal germinated seed. Therefore, any seedling with a

radical of 1 mm or more was cl assified as germinated.

The calculation of percent se ed in each assessment category was based on the actual number of seeds evaluated ( e.g. , 99 or 101). Across temperature regimes, the total number of seeds evaluated from each germination towel was approximately 100.

Within both AOSA and the additional temperat ure regimes, hard and firm-swollen seeds remaining at the final evaluation date were subjected to a tetrazolium (Tz) test for evaluation of viability according to AOSA standards (AOSA, 2007). The number of

nonviable hard and nonviable firm-swollen seed was added to the nu mber of dead seed

counted on both collection dates to determine th e total percent dead seed. Total counts for percent viable hard and viable firm-swolle n seed were determined from the Tz test.

F.4. Statistical Analysis

Analysis of variance (ANOVA) was conducted using SAS  

### (SAS, 2008). Within each temperature regime, the experiment was establ ished in a two-way factorial design with four replications. The two f actors assessed were material ( i.e. , KK179, conventional control, or a conventional commercial reference variety) and treatment ( i.e. , scarified or non-scarified). KK179 was compared to the conventional control for germination characteristics. The seed germination ch aracteristics analyzed included percent of germinated seed, percent of viab le hard seed, percent of dead seed, and percent of viable firm-swollen seed. The percent of germinated seed were categorized as either normal germinated or abnormal germinated for th e AOSA temperature regime. The level of statistical significance was pr edetermined to be 5% ( α =0.05). KK179 was not statistically compared to th e conventional commercial refe rence varieties, nor were comparisons made across temperature regimes. The minimum and maximum mean values (reference range) were determined from the conventional commercial reference varieties. Results from the analyses are presented in Table VII-2 and Table VII-3.

 

### SAS is a registered trademark of the SAS Institute, Inc., Cary North Carolina.Monsanto Company 12-AL-246U 330 of 407 

### Table F-1. Starting Seed of KK179, Cont rol and Conventional Commercial Alfalfa Reference Varieties Used in Dormancy Assessment

Material Name Genotype / Phenotype Treatment 1 Monsanto Lot

Number

KK179 Reduced Lignin; Syn1 population Non-scarified 11287366

C 0 -Syn1 Conventional; Syn1 population Non-scarified 11287367

Producer's Choice PGI 437 Conventional Non-scarified 11287370

Pioneer 54V54 Conventional Non-scarified 11287368

Croplan LegenDairy 5.0 Conventional Non-scarified 11287369

Vernal Conventional Non-scarified 11287371

KK179 Reduced Lignin; Syn1 population Scarified 11287372

C 0 -Syn1 Conventional; Syn1 population Scarified 11287373

Producer's Choice PGI 437 Conventional Scarified 11287376

Pioneer 54V54 Conventional Scarified 11287374

Croplan LegenDairy 5.0 Conventional Scarified 11287375

Vernal Conventional Scarified 11287377

1 Scarification scratches the seed coat to facilitate im bibition of water. Scarification was performed using a Forsberg manual seed scarifier. Each 7 g seed lo t was scarified with 25 revolutions of the handle.Monsanto Company 12-AL-246U 331 of 407 

### References for Appendix F

AOSA. 2007. Tetrazolium testing handbook. Contri bution No. 29. Association of Official Seed Analysts, Lincoln, Nebraska.

AOSA. 2010a. AOSA Rules for testing seeds Vol. 1: Principles and procedures.

Association of Official Seed Analysts, Ithaca, New York.

AOSA. 2010b. AOSA Rules for testing seeds Vol. 4: Seedling evaluation. Association of Official Seed Analys ts, Ithaca, New York.

SAS. 2008. SAS/STAT software version 9.2. SAS Institute, Inc., Cary, North Carolina.Monsanto Company 12-AL-246U 332 of 407 

### Appendix G: Materials, Methods, and I ndividual Site Results from Phenotypic, Agronomic, and Environmental Intera ction Assessment of KK179 under Field Conditions during Forage Production

G.1. Materials

Agronomic, phenotypic, and environmental in teraction characteristics were assessed for KK179, the conventional control, and 12 conv entional commercial re ference varieties grown under similar agronomic conditions of forage production (Table G-1).

G.2. Characterization of the Materials

The presence or absence of KK179 in th e starting seed of KK179, the conventional control ( C 0 -Syn1) 

### , and three of the conventional comm ercial reference varieties, which were produced in the same production with KK179 and the conventional control, was verified by event-specific PCR analyses.

G.3. Field Sites and Plot Design

Field trials were established in 2010 at 10 sites that provi ded a range of environmental and agronomic conditions representative of U.S. and Canadian alfalfa growing regions (Table VII-4). The Principal Investigator at each site was familiar with the growth, production, and evaluation of th e alfalfa characteristics.

At the nine U.S. sites, s eed of KK179, the conventional control, four conventional commercial reference varieties, and one othe r material were planted in a randomized complete block design with four replications. The plan ted plot dimensions varied between sites, due to variability in availa ble planting equipment (Table G-2). Each planted plot at CADV consisted of six rows spaced seven inches apart and 10 feet in length. Each planted plot at IABN consisted of five planted rows spaced six inches apart and 19 feet in length. Rows we re then thinned to 16 feet in length. Each planted plot at

IDNP consisted of five rows spaced six inches apart and 13 feet in length. Each planted plot at PACO consisted of five rows spaced si x inches apart and 17 feet in length. Rows were then thinned to 16 feet in length. Each planted plot at WATC and WIWS consisted of five rows spaced six inches apart and 16 feet in length. At these sites, all rows were

used for data collection.

Planted plots at ILCL, NYNR, and KSLA cons isted of 10 rows. At ILCL, rows were spaced six inches apart, with an additional si x inches between planter passes, and 12 feet in length. At KSLA, rows were spaced six inches apart, with an additional 6 inches between planter passes, and 19 feet in length. Row ends were then culled to 18 feet in length. At NYNR, rows were spaced seven inches apart, with approximately an additional 8.5 inches between planter passes, an d 12 feet in length. At these sites, rows #1-6 were used for phenotypic and qualitative environmental interactions data collection and rows #7-9 were used for quantitative envi ronmental interactions data collection.Monsanto Company 12-AL-246U 333 of 407 

### At the Canadian site, CANA, seed of KK1 79, the conventional control, and five

conventional commercial reference varieties were planted in a randomized complete block design with four replica tions. Each plot at CANA c onsisted of seven planted rows spaced approximately 6.7 inches and approximately 19.7 feet in length. In 2010, data were collected out of rows # 2-6. In 2011, all rows were used for data collection.

At some sites, the harvested plot area was di fferent from the planted plot area. Harvested plot areas used to calculate forage yield are presented in Table G-2.

At each site, the entire trial area ( i.e. , all replicates) was surrounded by a border approximately 3-10 feet of a conventional commercial alfalf a variety. Alleys between replicates were planted with approximately 3-8 feet of a conventional commercial alfalfa variety. The purpose of the pl anted border and alleys was to create a continuous alfalfa

stand across the entire trial area to ensure collection of more r obust arthropod abundance data within each plot.

G.4. Planting and Field Operations

Planting information, soil description, and crop ping history of the tr ial area are listed in Table G-2. Prior to planting, the Principal In vestigator at each site prepared a proper seedbed according to local agronomic practic es, including tillage, fertilization and pH adjustment, and pest management. During each growing season, the trial area was scouted for agronomic conditions and pest populations, including pest arthropods, diseases, and weeds. Fertilizer, irrigation, agricultural chemicals and other management treatments were applied as necessary. All maintenance operations were performed uniformly across the entire trial area.

G.5. Study Duration and Management

This study was conducted over two years (Spring 2010 through Spring 2012). Cuttings at each site were managed according to standard local agronomic practices for forage production of alfalfa and were timed to ha rvest forage at 1-10% bloom throughout both the 2010 and 2011 growing seasons. The number of cuttings per s ite for each growing

season is listed in Table G-3.Monsanto Company 12-AL-246U 334 of 407 

### Table G-1. Starting Seed for Phenotypic, Agronomic, and Environmental Interaction Assessment during Forage Production

Site Material Name Genotype / Phenotype Monsanto Lot Number

All C 0 -Syn 1 Conventional; Syn 1 population 11265474

All KK179 Reduced Lignin; Syn 1 population 11265471

CADV WL 319HQ Conventional 11265484

CADV Pioneer 54V09 Conventional 11265480

CADV Dow/Dairyland Hybriforce 2400 Conventional 11265770

CADV Producer's Choice PGI 437 Conventional 11265477

CANA Croplan LegenDairy 5.0 Conventional 11265476

CANA Pioneer 54V46 Conventional 11265479

CANA Dow/Dairyland Hybriforce 400 Conventional 11265485

CANA Pioneer 54H11 Conventional 11265478

CANA Pioneer 54V09 Conventional 11265480

IABN Croplan LegenDairy 5.0 Conventional 11265476

IABN Pioneer 54V54 Conventional 11265475

IABN Dow/Dairyland Hybriforce 400 Conventional 11265485

IABN Pioneer 54H11 Conventional 11265478

IDNP WL 319HQ Conventional 11265484

IDNP DKA50-18 Conventional 11265481

IDNP Dow/Dairyland Hybriforce 2400 Conventional 11265770

IDNP Producer's Choice PGI 437 Conventional 11265477

ILCL Croplan LegenDairy 5.0 Conventional 11265476

ILCL DKA50-18 Conventional 11265481

ILCL Pioneer 54V54 Conventional 11265475

ILCL Vernal Conventional 11265487

KSLA WL 319HQ Conventional 11265484

KSLA Pioneer 54V09 Conventional 11265480

KSLA Ranger Conventional 11265488

KSLA Producer's Choice PGI 437 Conventional 11265477Monsanto Company 12-AL-246U 335 of 407 

### Table G-1. Starting Seed for Phen otypic, Agronomic, and Environmental Interaction Assessment during Forage Production (continued)

Site Material Name Genotype / Phenotype Monsanto Lot Number

NYNR WL 319HQ Conventional 11265484

NYNR DKA50-18 Conventional 11265481

NYNR Dow/Dairyland Hybriforce 2400 Conventional 11265770

NYNR Vernal Conventional 11265487

PACO Croplan LegenDairy 5.0 Conventional 11265476

PACO Pioneer 54V46 Conventional 11265479

PACO Ranger Conventional 11265488

PACO Producer's Choice PGI 437 Conventional 11265477

WATC WL 319HQ Conventional 11265484

WATC Pioneer 54V54 Conventional 11265475

WATC Ranger Conventional 11265488

WATC Pioneer 54H11 Conventional 11265478

WIWS Croplan LegenDairy 5.0 Conventional 11265476

WIWS DKA50-18 Conventional 11265481

WIWS Dow/Dairyland Hybriforce 400 Conventional 11265485

WIWS Vernal Conventional 11265487

Note: The study also included one additional experimental material that was outside of the scope of the objectives of this evaluation of KK179.Monsanto Company 12-AL-246U 336 of 407Table G-2. Field andPlanting Information1A broad range of seeding rates is acceptable to produce adequate stands of alfalfa. Stands naturally self-thin over time, primarily due to competition betweenseedlings, and seedling survival further decreases with increasingseeding rate (Tesar and Marble, 1988). Plants can also compensate for low germination byincreasing the number of stems per plant. Stand and yield are ultimately influenced by the number of stems, not the number ofplants, per unit area.2OM = organic matter3CANA harvested a smaller area in2010 and cutting #1 of 2011 due to potential impacts to outer rows from heavy rain immediatelyafter planting. By 2011, itwas apparent that the impact of the heavy rain on the stand was minor and the entire plot area was harvested.4NYNR harvested six of 10 rows in 2010 due to a spotty stand.In 2011, the stand had filled in and all rows were harvested.Site1Harvested plot area (ft2)Rows (#/plot) Planting rate (lb/A)1Soil series OM2(%) 2009 CropCADV 35 6 18.0 Clay loam 1.8 WheatCANA354.9 / 76.9 7 9.6 Brant silt loam 1.3 SoybeansIABN 40 5 18.0 Nicollet loam 4.0 OatsIDNP 32.5 5 15.0 Power silt loam 1.0 FallowILCL 66 10 19.2 Cisne silt loam 2.7 FallowKSLA 99 10 16.4 Loam silt loam 2.6 Wheat/soybeansNYNR450.4 / 78.4 10 13.8 Elnora sand 2.5 WheatPACO 40 5 20.1 Hagerstown silt loam 1.2 Forage grassesWATC 40 5 14.6 Touchet silt loam 1.3 FallowWIWS 40 5 21.4 Toddville silt loam 3.7 OatsMonsanto Company 12-AL-246U 337 of 407Table G-3. Study Site ManagementNA Indicates activity not applicable for this site.1Month/day/year.2Cutting #3 of 2011 at ILCL occurred over two dates- reps 1 and 2 were cut on 7/21/11 and reps 3 and 4 were cut on 7/22/11.Dates1by SiteActivityCADVCANAIABNIDNPILCLKSLANYNRPACOWATCWIWSFirst YearPlanting3/29/106/2/104/20/104/26/104/19/104/21/104/23/104/12/104/10/104/20/10Cutting #16/8/107/19/106/30/107/7/106/25/106/21/107/1/106/18/107/14/107/14/10Cutting #27/16/108/10/107/29/108/13/108/6/107/20/108/3/107/28/108/16/108/27/10Cutting #39/3/109/23/109/8/1010/1/109/20/108/19/109/22/108/31/109/15/1010/22/10Cutting #411/6/10NANANANA9/20/10NANANANASecond YearCutting #14/22/116/10/116/6/116/6/115/17/115/23/116/3/115/24/115/28/116/2/11Cutting #25/27/117/8/117/7/117/12/116/20/116/22/117/6/116/24/117/1/117/6/11Cutting #37/5/117/28/118/9/118/16/117/21-22/1127/22/118/18/117/27/118/5/118/5/11Cutting #48/3/118/25/119/16/119/21/119/1/118/22/1110/14/119/1/119/9/119/14/11Cutting #59/9/119/29/11NANANA9/21/11NA10/5/1110/25/11NAMonsanto Company 12-AL-246U 338 of 407 

### G.6. Phenotypic Observations

The descriptions of the phenotypi c characteristics assessed ar e listed in Table VII-1. After planting, KK179 was compared to the co nventional control for seedling emergence and early season vigor. For each crop growth cycle and cutting of each year, KK179 was compared to the conventional control for lodging, crop growth stage (using Mean Stage by Count method; see Table G-4) , forage yield, and regrowth after cutting (except for the final cut of each season). At the end of each growing season, KK179 was compared to the conventional control for fall plant height, as an indicator of fall dormancy, and total forage yield. KK179 was also compared to the conventional control for winter survival, through the assessment of spring vigor, spring st and recovery, and spring stand count.

Phenotypic characteristics were assessed over each complete year. The first year was considered to be from planting in 2010 thr ough the winter survival assessment in the spring of 2011. The second year was consider ed to be from the fi rst cutting of 2011 through the winter survival as sessment in the spring of 2012. For each year, assessments were made within each crop growth cycle ( i.e. , the period of forage growth between cuttings) and at each cutting.Monsanto Company 12-AL-246U 339 of 407 

### Table G-4. Definition of Morphologi cal Stages of Development for Individual

Alfalfa Stems Used to Calc ulate Crop Growth Stage

Stage number Stage name Stage definition

0 Early vegetative Stem length ≤ 15 cm; no buds, flowers, or seed pods

1 Mid-vegetative Stem length 16 to 30 cm; no buds, flowers, or seed pod

2 Late vegetative Stem length  31 cm; no buds, flowers, or seed pods

3 Early bud 1 to 2 nodes with buds; no flowers or seed pods

4 Late bud  3 nodes with buds; no flowers or seed pods

5 Early flower One node with one open flower (standard open); no seed pods

6 Late flower  2 nodes with open flowers; no seed pods

Crop growth stage was determined using the Mean Stage by Count (MSC) method (Kalu and Fick 1981). Crop growth stage data were collect ed at each site during each crop gr owth cycle, prior to harvest. Approximately 35-45 stems from each plot were non-syste matically selected and ranked by growth stage. The MSC was calculated for each plot us ing the following formula: MSC = [ Σ (stage number * number of stems in stage)]/total number of stems evaluated.Monsanto Company 12-AL-246U 340 of 407 

### G.7. Environmental Observations

Environmental interactions ( i.e. , interactions between the cr op plants and their receiving environment) were used to characterize KK179 by evaluating plant response to abiotic stressors, disease damage, and arthropod -related damage using qualitative methods described in section G.8. In addition, specifi c arthropod damage and pest- and beneficialarthropod abundance were evaluated using the quantitative methods described in Section

G.9.

G.8. Plant Response to Abiotic Stress, Disease Damage, and Arthropod-Related Damage

KK179 and the conventional control were eval uated at all sites for plant response to abiotic stressors, disease damage, and arthr opod damage. Three abiotic stressors, three diseases, and three arthropod pests were evalua ted three to four times throughout the first growing season (2010) and four to five times throughout the second growing season (2011). The timing of each observation ranged from 10 days before the cut to the day of

the cut.

The Principal Investigator at each site chose abiotic st ressors, diseases, and arthropod pests that were either actively causing plant injury in the study area or were likely to occur during a given observation period. The t ype of abiotic stressors, diseases, and arthropod pests assessed vary between observa tion times at a site, between sites, and between years.

Abiotic stressors, disease damage, and arthr opod damage data were collected from all rows (or rows #1-6 at sites with arthropod collections) of each plot using a continuous 0- 9 scale of increasing severity. Data were collected numerically using the rating scale below. Due to the non-specific nature of the s cale used, the data were placed into one of the following qualitative categories of plant damage severity: none (0), slight (1-3), moderate (4-6), or severe (7-9) and then expressed as a range of responses observed across the four replic ations at a site.

Rating Severity of plant damage 0 none (no symptoms observed) 1 – 3 slight (symptoms not damaging to plant development) 4 – 6 moderate (intermediate between slight and severe) 7 – 9 severe (symptoms damaging to plant development)

For each stressor evaluated, the range of responses for KK179 was then compared to the range of responses for the conventional control. If the range of in jury symptoms across all four replications overlapped between KK 179 and the conventional control, the KK179

and the conventional control were considered not different in plant response. The stressors evaluated, the number of times KK179 and the conventional control were compared for each stressor, and the number of times those comparisons showed differences are reported.Monsanto Company 12-AL-246U 341 of 407 

### G.9. Specific Arthropod Dam age and Arthropod Abundance

Alfalfa weevil and potato leafhopper damage were assessed quantitatively at three sites, ILCL, KSLA, and NYNR, three to four times throughout the first growing season (2010) and four to five times throughout the sec ond growing season (2011). Alfalfa weevil damage was assessed at each site one to nine days before each cutting. At each assessment time, 10 non-systematically selected 6 × 6 inch areas within rows #8 and 9 of each plot were chosen for examination. Th e following rating scale was used for each evaluation of alfalfa weevil damage:

Rating Severity of plant damage 0 No damage
1 < 50% foliage have pin holes ( usually terminal leaves)

2 51-90 % foliage have pinholes with minor feeding damage

between the veins 3 < 50 % foliage with major feed ing damage between the veins
4 51-90% foliage with skeletonized appearance
5 >90 % foliage with skeletonized appearance

For potato leafhopper damage, 10 non-systematica lly selected 6 × 6 inch areas within rows #8 and 9 of each plot were chosen for examination from one to nine days before each cutting. The following rating scale was used for each evaluation of potato leafhopper damage:

Rating Severity of plant damage
0 No damage
1 < 50% foliage with yellowing
2 51-90% foliage with yellowing
3 < 50% foliage with yellowing and puckering
4 51-90% foliage with yellowing and puc kering (stunting of plant is evident)
5 >90% foliage with yellowing and puckering (severe st unting of plant)

Pest- and beneficial-arthropods were collect ed at ILCL, KSLA, and NYNR zero to 10 days prior to each cutting, which occurred th ree to four times during the first growing season (2010) and four to five times during the second growing season (2011). Arthropods were collected usi ng a vertical beat sheet samp ling method (Drees and Rice, 1985). The beat sheet was approximately a 36 × 36 inch sheet cons tructed of a stiff material with a collecting trough at the bottom. A total of six sub-samples were collected from rows #7-9 from each plot. The sheet was placed between rows #6 and 7, and the collecting trough was positioned near the base of the plants in row #7. Plants were shaken vigorously along the length of the beat sheet to dislodge arthropods from the plants. Another sub-sample was collected fr om the same row, approximately two feet from the first sub-sample. Two sub-samples were collected from each rows #8 and 9 inMonsanto Company 12-AL-246U 342 of 407 

### the same manner. The six sub-samples were combined into one pre-labeled container and placed on freezer ice packs. The samples were first sent to Monsanto Company, St. Louis, MO and then sent to Kansas State University, Manhattan, KS for arthropod

identification and enumeration.

From each collection in each year, a maximu m of five pest- and five beneficialarthropods were enumerated. For each individual collection ( e.g. , collection #1 at KSLA), four non-systematically selected samp les were examined to determine presence and relative abundance of up to five pest- and beneficial-art hropods to be enumerated for that particular collection and site. Thus, the suite of pe st- and beneficial-arthropods

assessed often varied between collections from a site and between sites due to differences in temporal activity and geographica l distribution of arthropod taxa.

G.10. Data Assessment

Experienced scientists familiar with the expe rimental design and evaluation criteria were involved in all components of data collection, summariza tion, and analysis. Study personnel assessed that measurements were ta ken properly, data were consistent with expectations based on experi ence with the crop, and the experiment was carefully monitored. Prior to analysis, the overall dataset was evaluated for evidence of biologically relevant changes and for possible evidence of an unexpected plant response. Any unexpected observations or issues that would impact the objectives were noted. Data were then subjected to statistical analysis.

G.11. Environmental Interactions Evalua tion Criteria for Qualitative Data

The qualitative environmental interactions data, including plant responses to abiotic stressors, disease damage, and arthropod damage were categorical an d, therefore, were not subjected to statis tical analysis. KK179 a nd the conventional cont rol were c onsidered different in plant responses if the range of injury symptoms across all four replications did not overlap between KK179 and the convent ional control. Any observed differences

between KK179 and the conventional control we re assessed for biologi cal significance in the context of the range of th e conventional commercial refere nce varieties at a site and for consistency with other observation times and sites. Differences that were not consistently observed at othe r observation times and sites we re not considered to be biologically meaningful in terms of plant pest potential or an effect on the environment.

G.12. Statistical Analyses

G.12.1 Statistical Analysis of Agronomic and Phenotypic Data

An analysis of variance was conducted accord ing to a randomized complete block design using SAS ® 

### to compare KK179 and the conventional control for the phenotypic

characteristics listed in Table VII-1. Fo r each year, comparisons of KK179 and the

conventional control were condu cted within sites (individual-site an alysis) and in a combined-site analysis in which the data we re pooled among the s ites. KK179 was also compared to the conventional control for a subset of phenotypic ch aracteristics in a combined-year analysis in which data were pool ed across years. The level of statisticalMonsanto Company 12-AL-246U 343 of 407 

### significance was predetermined to be 5% ( α =0.05). KK179 and the conventional control were not statistically compar ed to the conventional commer cial reference materials.

Minimum and maximum mean values were dete rmined for each characteristic from the 12 unique conventional commercial reference varie ties that were included at all sites.

G.12.2 Statistical Analysis for Specific Arthropod Damage

An analysis of variance was conducted accord ing to a randomized complete block design using SAS  

### to compare KK179 and the conventi onal control for potato leafhopper damage and alfalfa weevil damage assessed at three sites (ILCL, KSLA, and NYNR). For each year, comparisons of KK179 and the conventional control were conducted within sites (individual-site analysis). Since specific arthropod damage data were collected for the same two arthropods at the th ree sites, a combined-site analysis in which the data were pooled among the sites was al so conducted for each year. The level of statistical significance was pr edetermined to be 5% ( α =0.05). KK179 and the

conventional control were no t statistically compared to the conventional commercial

reference varieties. Minimum and maximum mean values were determined for each characteristic from the nine unique conventi onal commercial reference varieties that were

included at the three sites.

G.12.3 Statistical Analysis for Arthropod Abundance

An analysis of variance was conducted accord ing to a randomized complete block design using SAS  

### to compare KK179 and the conventional control for pest- and beneficialarthropod abundance assessed at three sites (ILCL, KSLA, and NYNR). Variations in temporal activity and geographi cal distribution of arthropod taxa occur between sites. Therefore, comparisons of KK179 and the conventional control were conducted only within sites (individual-site anal ysis) for both years. The leve l of statistical significance was predetermined to be 5% ( α =0.05). KK179 and the conventional control were not statistically compared to the conventional co mmercial reference materials. Minimum and

maximum mean values were determined fo r arthropod abundance from the conventional

commercial reference varieties at each site and collection time.

Data excluded from analysis for 2010 th rough 2012 and the reasons for exclusion are

listed in Table G-5.

 SAS is a registered trademark of the SAS Institute, Inc. Cary, North Carolina.Monsanto Company 12-AL-246U 344 of 407Table G-5. Data Missing orExcluded from AnalysisSite Plots Entry type DataReasonFirst YearAll All All Forage yield #1No yield data were collected from the first cutting at eachsite due to variability in establishment year.CADV, IABN,IDNP, WATC,WIWS, CADVAll All Forage quality sample weightsNot all sites weighed the quality sample that was cut priorto harvest; therefore, this weight was excluded from theplot weight of forage yield per cut.CANA, IABN,IDNP, ILCL,NYNR, PACO,WATCAll All Regrowth after cutting #3Rating was not required following the last harvest of theseason.CADV, IDNP,WATCAll All Fall plant height-Yr 1: 1stratingFall plant height data were collected twice at westernsites, as is regionally appropriate. Plants had not yetstarted going dormant at the first rating.CADV All All Percent stand Data collected byPI were not requested in the protocol.CADV All All Regrowth after cutting #1 and #4Regrowth after cutting #1 was not collected at theappropriate time and regrowth after cutting #4 was notrequired following the last harvest of the season.Monsanto Company 12-AL-246U 345 of 407Table G-5 (continued). Data Missing or Excluded from AnalysisSite Plots Entry type DataReasonCADV 107 Control Forage yield #4The forage weight for plot 107 was lost due to a harvestermalfunction.CANA All All Spring stand count- Yr 2Plants per foot were counted instead of stems per foot, asrequested.IABN All AllAbiotic Stressor Observation #1, ratingfor stressor #3Plots were evaluated for winter injury when plants hadyet to be exposed to winter.IABN All AllAbiotic Stressor, Disease Damage, andArthropod Damage Observation #3Ratings were not made atthe appropriate time asspecified in the protocol.KSLA 305 Reference Arthropod collection #1 Collection jar broke and the sample was lost.NYNR All AllSpring stand rating and spring vigorrating- Yr 2Data reporting was inconsistent.NYNR 102 Reference Spring stand count- subsample 2 Entry on paper was not readable.PACO All All Mean stage by count #2 Data were not collected at this site.PACO All All Disease Damage Observation #1PI did not complete evaluation or follow protocolinstructions.WATC All All2ndSeedling emergence rating; Earlyseason vigor; Mean stage by count #1;Lodging #1Seedling emergence was collected twice and the secondrating was unnecessary. Timing of data collection forEarly season vigor and Mean stage by count #1 did notfollow the protocol and Lodging #1data were notcollected.WATC101,102, 103References,additionalmaterialAllFrost event in April 2010 killed most plants in theseplots.Monsanto Company 12-AL-246U 346 of 407Table G-5 (continued). Data Missing or Excluded from AnalysisSite Plots Entry type DataReasonWIWS All All Lodging #2Heavy rains immediately preceding the rating causedforage in all plots to be weighed down. The rating wastherefore not an accurate assessment of lodging.All All All All mean stage by count evaluationsAny plots at any site for which fewer than 35 stems wereevaluated per plot.Second YearCADV, CANA,KSLA, PACOAll All Regrowth after cutting #5Rating was not required following the last harvest of theseason.IABN, IDNP,ILCL, NYNR,WIWSAll All Regrowth after cutting #4Rating was not required following the last harvest of theseason.CADV All All Forage yield #21Plot weight data could not bereconstructed. Additionally,the data collector is no longer employed by FGI and isthus unavailable to provide clarification.CADV 201 Control Forage yield #1 and #31There was gopher damage to a significant number ofplants in the plot.CADV 207 Reference Forage yield #51Plot weight was not collected due to an error inharvesting equipment operation.CADV All All Forage yield #6 Plot weight data were collected that were not requested.CADV301, 302,304, 305,305, 306All Arthropod Damage Observation #4Entries on paper were not readable.CADV All AllAbiotic Stressor, Disease Damage, andArthropod Damage Observation #2PI did not complete data evaluation or follow protocolinstructions.Monsanto Company 12-AL-246U 347 of 407Table G-5 (continued). Data Missing or Excluded from AnalysisSite Plots Entry type DataReasonCADV 301 TestSpring vigor, Spring stand recovery,and Spring stand countThere was significant gopher damage to the plot.CANA All All Fall plant height Yr 2 Data were not collected at the appropriate time.CANA 101 TestArthropod Damage Observation #5,stressor 1 and 2Raw data were not available.IDNP 307 Reference Mean stage by count#4 Raw data were not available.KSLA All All Mean stage by count #5The data were lost due to an electronic notebookmalfunction.KSLA 403, 407Control,referenceFall plant height-Yr 2, plant number 9 An extra plant was measured.KSLA203,304, 405References,additionalmaterialArthropod collection #3Collection samples were of poor quality and could not beenumerated.NYNR All All Mean stage by count # 1-4 PI did not take representative stem samples.NYNR All All Fall plant height-Yr 2 Data werenot collected at the appropriate time.PACO All All Lodging #1 Data were notcollected at the appropriate time.PACO All All Mean stage by count # 1 and 2Data were not collected at this site.PACO 303 Test Forage yield #31There was groundhog damage to a significant number ofplants in the plot.PACO All AllFall plant height- Yr 2, plant numbers6-10The protocol only required five plants to be measured.Monsanto Company 12-AL-246U 348 of 407Table G-5 (continued). Data Missing or Excluded from AnalysisSite Plots Entry type DataReasonPACO All AllAbiotic Stressor, Disease Damage, andArthropod Damage Observation #1PI did not complete data evaluation or follow protocolinstructions.WATC All All Mean stage by count #2Insect (Lygus) stripping of buds interfered with cropgrowth stage evaluation.WATC 402 Reference Forage yield #31Gopher mound caused excessive soil to enter sample,affecting plot weight.All All All All mean stage by count evaluationsAny plots at any site for which fewer than 35 stems wereevaluated per plot.1Forage yield in the second year was determined by adding a plot weight with the sample weight. For all instances of excluded plot weights, the correspondingsample weight was also excluded.Monsanto Company 12-AL-246U 349 of 407 

### G.13. Individual Field Site Plant Growth and Developmen t Results and Discussion

G.13.1. Individual Field Site Plant Gr owth and Development Results and Discussion- First Year (2010-2011)

In the individual-site analysis for the first year, 12 statistically significant differences

were detected between KK179 and the conventi onal control (Table G-6). No statistical comparisons could be made in some instances due to lack of variab ility in the data.

At the beginning of the 2010 season, one statis tically significant di fference was detected

between KK179 and the conventional contro l for seedling emergence. Seedling emergence was lower for KK179 than the co nventional control at ILCL (20.0 vs. 25.5 plants/ft).

At cutting #1, three statistically significant di fferences were detected between KK179 and

the conventional control. KK179 had a lo wer lodging rating than the conventional control at ILCL (8.5 vs. 9.0 rating). KK179 was at a later growth stage than the conventional control at IABN (2.7 vs. 2.3). KK179 showed greater regrowth after cutting

than the conventional control at WATC (9.3 vs. 8.0 rating).

At cutting #2, three statistically significant di fferences were detected between KK179 and

the conventional control. KK179 had a lo wer lodging rating than the conventional control at IABN (7.8 vs. 8.5 rati ng). KK179 was at a slightly earlier growth stage than the conventional control at NYNR (3.58 vs. 3.64; means are rounded in Table G-6). KK179 had significantly greater forage yield than the conventional control at WATC (11.0 vs. 8.2 fresh weight ton/acre).

At cutting #3, two statistically significant di fferences were detected between KK179 and

the conventional control. KK179 was at a later growth stage than the conventional control at WATC (3.0 vs. 2.8) and was at an earlier growth stage than the conventional control at WIWS (0.7 vs. 1.1).

At cutting #4, one statistica lly significant difference wa s detected between KK179 and

the conventional control. KK179 was at an earlier growth stage than the conventional control at KSLA (2.0 vs. 2.1).

At the end of the 2010 season, one statisti cally significant difference was detected

between KK179 and the conventional control for total forage yield, which is the sum of all cuttings over the season. KK179 had signifi cantly greater total forage yield than the conventional control at WATC (18.1 vs. 14.9 fresh weight ton/acre).

In the assessment of winter survival conducte d in spring 2011, one stat istically significant

difference was detected between KK179 and th e conventional control. KK179 had lower spring vigor than the conventional co ntrol at WATC (8.5 vs. 10.0 rating).

None of the statistical differen ces detected in the individual-s ite analysis were detected in the combined-site analysis, suggesting these differences were not indicative of a consistent plant response associated with th e trait and were not c onsidered biologicallyMonsanto Company 12-AL-246U 350 of 407 

### meaningful in terms of plant pest/weed poten tial of KK179 compared to the conventional control (Figure VII-1, St ep 2, answer “no”).

G.13.2. Individual Field Site Plant Gr owth and Development Results and Discussion- Second Year (2011-2012)

In the individual-site analysis for the second year, 17 statistically significant differences

were detected between KK179 and the conventi onal control (Table G-7). No statistical comparisons could be made in some instances due to lack of variab ility in the data.

At cutting #1, two statistically significant di fferences were detected between KK179 and

the conventional control. KK179 was at an earlier growth stage than the conventional control at ILCL (2.9 vs. 3.2). KK179 showed reduced regrowth after cutting than the conventional control at WATC (9.0 vs. 10.0 rating).

At cutting #2, three statistically significant di fferences were detected between KK179 and

the conventional control. KK179 had a hi gher lodging rating than the conventional control at IABN (5.8 vs. 4.3 rating). KK179 was at an earlier growth stage than the conventional control at CADV (2.0 vs. 2.5) and at KSLA (3.6 vs. 4.2).

At cutting #3, four statistically significant differences were detected between KK179 and

the conventional control. KK179 was at an earlier growth stage than the conventional control at WIWS (3.2 vs. 3.7). KK179 had a greater forage yi eld for cutting #3 than the conventional control at PACO (9.1 vs. 8.4 fresh weight ton/acre). KK179 showed reduced regrowth after cutting than the conve ntional control at IABN (7.0 vs. 8.3 rating) and greater regrowth after cu tting than the conventional control at PACO (10.0 vs. 9.5 rating).

At cutting #4, one statistica lly significant difference wa s detected between KK179 and

the conventional control. KK179 had a lowe r forage yield for cutting #4 than the conventional control at IABN (5.4 vs. 6.0 fresh weight ton/acre).

At cutting #5, one statistica lly significant difference wa s detected between KK179 and

the conventional control. KK179 had a lowe r forage yield for cutting #5 than the conventional control at WATC (4.8 vs. 5.4 fresh weight ton/acre).

At the end of the 2011 season, one statisti cally significant difference was detected

between KK179 and the conventional control. Plants of KK179 were shorter than the conventional control at WATC (8.8 vs. 9.7 in).

In the assessment of winter survival conducte d in spring 2012, five si gnificant differences

were detected between KK179 and the conventional control. KK179 had greater spring vigor than the conventional co ntrol at PACO (6.5 vs. 5.5 ratin g) and lower spring vigor

than the conventional cont rol at WATC (9.3 vs. 10.0 ra ting). KK179 had a reduced spring stand recovery than the conventional control at IABN (7.8 vs. 9.5 rating), showing greater gaps in the stand across the plot. KK179 had a higher spring stand count than the conventional control at IDNP (54.8 vs. 33.8 stem s/ft) and a lower spring stand count than the conventional control at NYNR (59.3 vs. 69.7 stems/ft).Monsanto Company 12-AL-246U 351 of 407 

### None of the statistical differen ces detected in the individual-s ite analysis were detected in the combined-site analysis, suggesting thes e differences were not indicative of a consistent plant response associated with th e trait and were not c onsidered biologically meaningful in terms of plant pest/weed poten tial of KK179 compared to the conventional control (Figure VII-1, St ep 2, answer “no”).Monsanto Company 12-AL-246U 352 of 407Table G-6. Individual-Site Phenotypic Comparison of KK179 tothe Conventional Control in the First Year (2010-2011) ofForage ProductionCutting #1Seedling emergence(# plants/foot)Early season vigor(1-10 rating)Lodging (0-9 rating)Crop growth stage(stage number 0-6)Regrowth after cutting1(1-10 rating)SiteKK179Mean (SE)ControlMean (SE)KK179Mean (SE)ControlMean (SE)KK179Mean (SE)ControlMean (SE)KK179Mean (SE)ControlMean (SE)KK179Mean (SE)ControlMean (SE)CADV5.4 (0.63)7.3 (1.71)† 1.0 (0.00)1.0 (0.00)† 9.0 (0.00)9.0 (0.00)2.6 (0.08)2.4 (0.04)––CANA28.8 (3.88)24.0 (1.83)5.3 (0.25)6.0 (0.41)† 9.0 (0.00)9.0 (0.00)1.8 (0.21)1.4 (0.13)9.0 (0.00)9.0 (0.00)IABN24.7 (1.60)21.5 (2.93)5.8 (0.48)6.3 (0.48)9.0 (0.00)8.5 (0.50)* 2.7 (0.04)2.3 (0.12)7.5 (0.50)7.3 (0.48)IDNP13.4 (2.20)10.4 (2.50)4.8 (0.48)4.5 (0.65)8.5 (0.50)8.5 (0.50)3.2 (0.06)3.3 (0.11)8.8 (0.25)8.8 (0.75)ILCL* 20.0 (0.53)25.5 (0.59)5.3 (0.48)5.8 (0.25)* 8.5 (0.29)9.0 (0.00)2.0 (0.27)1.9 (0.21)9.0 (0.58)8.0 (0.00)KSLA12.5 (0.22)12.3 (0.49)† 5.0 (0.00)5.0 (0.00)† 9.0 (0.00)9.0 (0.00)3.7 (0.05)3.5 (0.03)9.3 (0.48)8.5 (0.65)NYNR15.8 (0.37)12.5 (3.66)3.0 (0.71)4.3 (2.02)† 9.0 (0.00)9.0 (0.00)3.4 (0.02)3.5 (0.03)† 10.0 (0.00)10.0 (0.00)PACO28.0 (3.62)30.7 (3.36)5.0 (0.00)4.8 (0.25)† 9.0 (0.00)9.0 (0.00)2.4 (0.16)2.1 (0.17)10.0 (0.00)9.3 (0.48)WATC12.9 (1.82)13.5 (2.33)––––––* 9.3 (0.48)8.0 (0.41)WIWS33.0 (4.40)26.8 (2.66)8.3 (0.25)8.3 (0.48)5.8 (0.75)4.8 (0.48)3.5 (0.04)3.9 (0.11)9.3 (0.48)9.3 (0.48)Monsanto Company 12-AL-246U 353 of 407Table G-6 (continued). Individual-Site Phenotypic Comparisonof KK179 to the Conventional Control in the First Year (2010-2011) of Forage ProductionCutting #2Lodging (0-9 rating)Crop growth stage(stage number 0-6)Forage yield(fresh weight ton/acre)Regrowth after cutting1(1-10 rating)SiteKK179Mean (SE)ControlMean (SE)KK179Mean (SE)ControlMean (SE)KK179Mean (SE)ControlMean (SE)KK179Mean (SE)ControlMean (SE)CADV† 9.0 (0.00)9.0 (0.00)3.3 (0.51)3.3 (0.35)6.6 (0.66)6.9 (0.14)8.8 (0.25)9.0 (0.00)CANA† 9.0 (0.00)9.0 (0.00)1.2 (0.11)1.1 (0.05)1.1 (0.26)1.2 (0.10)† 8.0 (0.00)8.0 (0.00)IABN* 7.8 (0.48)8.5 (0.50)2.5 (0.12)2.4 (0.15)5.9 (0.51)5.2 (0.45)7.3 (0.85)7.3 (1.03)IDNP7.5 (0.65)7.3 (1.44)3.4 (0.28)3.2 (0.36)10.2 (0.81)10.5 (0.86)8.3 (0.48)8.3 (0.63)ILCL8.5 (0.50)9.0 (0.00)3.2 (0.25)3.2 (0.26)5.3 (0.49)4.7 (0.28)7.3 (1.31)6.8 (0.85)KSLA† 9.0 (0.00)9.0 (0.00)3.6 (0.03)3.5 (0.04)7.5 (0.25)8.0 (0.36)10.0 (0.00)9.8 (0.25)NYNR† 9.0 (0.00)9.0 (0.00)* 3.6 (0.01)3.6 (0.02)5.6 (0.41)5.5 (0.44)† 10.0 (0.00)10.0 (0.00)PACO6.0 (0.00)6.3 (0.25)––8.9 (0.37)8.2 (0.49)9.8 (0.25)9.0 (0.41)WATC8.3 (0.25)8.0 (0.00)3.9 (0.10)3.9 (0.08)* 11.0 (1.10)8.2 (1.03)8.8 (0.25)9.0 (0.41)WIWS––3.0 (0.17)3.1 (0.06)8.1 (0.38)8.0 (0.26)9.5 (0.29)10.0 (0.00)Monsanto Company 12-AL-246U 354 of 407Table G-6 (continued). Individual-Site Phenotypic Comparisonof KK179 to the Conventional Control in the First Year(2010-2011) of Forage ProductionCutting #3Lodging (0-9 rating)Crop growth stage(stage number 0-6)Forage yield(fresh weight ton/acre)Regrowth after cutting1(1-10 rating)SiteKK179Mean (SE)ControlMean (SE)KK179Mean (SE)ControlMean (SE)KK179Mean (SE)ControlMean (SE)KK179Mean (SE)ControlMean (SE)CADV8.0 (0.58)7.8 (0.48)2.1 (0.12)2.2 (0.05)4.7 (0.23)4.9 (0.38)7.0 (0.41)7.8 (0.25)CANA† 9.0 (0.00)9.0 (0.00)1.2 (0.39)1.9 (0.12)1.4 (0.28)1.7 (0.37)––IABN8.3 (0.48)8.5 (0.29)1.7 (0.27)1.6 (0.25)3.7 (0.76)3.2 (0.70)––IDNP8.5 (0.50)7.8 (0.48)2.0 (0.08)1.7 (0.19)7.9 (0.18)8.5 (0.38)––ILCL† 9.0 (0.00)9.0 (0.00)2.2 (0.12)2.2 (0.06)2.9 (0.21)2.8 (0.23)––KSLA† 9.0 (0.00)9.0 (0.00)3.5 (0.03)3.5 (0.04)8.7 (0.14)9.1 (0.64)9.0 (0.00)8.5 (0.29)NYNR† 9.0 (0.00)9.0 (0.00)3.4 (0.02)3.5 (0.03)4.5 (0.15)4.3 (0.20)––PACO† 9.0 (0.00)9.0 (0.00)1.5 (0.09)1.5 (0.07)3.6 (0.19)3.6 (0.18)––WATC8.5 (0.29)8.0 (0.00)* 3.0 (0.07)2.8 (0.15)7.1 (0.33)6.7 (0.56)––WIWS† 9.0 (0.00)9.0 (0.00)* 0.7 (0.10)1.1 (0.08)2.7 (0.26)2.8 (0.12)––Monsanto Company 12-AL-246U 355 of 407Table G-6 (continued). Individual-Site Phenotypic Comparisonof KK179 to the Conventional Control in the First Year (2010-2011) of Forage ProductionCutting #4Lodging (0-9 rating)Crop growth stage(stage number 0-6)Forage yield(fresh weight ton/acre)Fall plant height (in)SiteKK179Mean (SE)ControlMean (SE)KK179Mean (SE)ControlMean (SE)KK179Mean (SE)ControlMean (SE)KK179Mean (SE)ControlMean (SE)CADV5.8 (0.63)5.0 (0.41)2.2 (0.04)2.3 (0.02)4.3 (0.37)3.1 (0.85)1.8 (0.13)1.8 (0.12)CANANANANANANANA3.1 (0.15)3.1 (0.18)IABNNANANANANANA7.5 (0.47)7.6 (0.56)IDNPNANANANANANA4.1 (0.25)4.3 (0.30)ILCLNANANANANANA4.1 (0.33)4.1 (0.38)KSLA† 9.0 (0.00)9.0 (0.00)* 2.0 (0.02)2.1 (0.03)4.0 (0.22)3.6 (0.26)4.1 (0.24)4.2 (0.39)NYNRNANANANANANA4.2 (0.34)4.1 (0.63)PACONANANANANANA9.8 (0.85)10.2 (0.75)WATCNANANANANANA7.5 (0.29)8.3 (0.28)WIWSNANANANANANA8.8 (0.35)8.5 (0.41)Monsanto Company 12-AL-246U 356 of 407Table G-6 (continued). Individual-Site Phenotypic Comparisonof KK179 to the Conventional Control in the First Year(2010-2011) of Forage ProductionTotal forage yield(fresh weight ton/acre)Spring vigor (1-10 rating)Spring stand recovery(1-10 rating)Spring stand count(# stems/foot)SiteKK179Mean (SE)ControlMean (SE)KK179Mean (SE)ControlMean (SE)KK179Mean (SE)ControlMean (SE)KK179Mean (SE)ControlMean (SE)CADV15.6 (0.78)14.7 (0.81)7.8 (0.25)7.5 (0.50)7.8 (0.75)7.3 (0.48)39.8 (6.30)30.8 (4.82)CANA2.5 (0.52)2.9 (0.47)5.8 (0.25)5.3 (0.48)† 10.0 (0.00)10.0 (0.00)––IABN9.7 (1.23)8.4 (1.14)8.5 (0.50)9.0 (0.41)8.8 (0.25)9.5 (0.29)71.9 (3.38)73.8 (3.52)IDNP18.1 (0.72)19.1 (1.20)8.0 (0.41)8.0 (0.00)7.8 (0.63)8.5 (0.65)86.7 (12.05)89.8 (12.45)ILCL8.2 (0.70)7.5 (0.50)7.5 (0.29)7.3 (0.85)7.0 (0.71)7.5 (0.87)45.6 (1.85)45.7 (8.22)KSLA20.1 (0.35)20.7 (0.85)8.0 (0.00)7.8 (0.25)7.5 (0.29)8.0 (0.41)88.3 (2.32)84.6 (3.51)NYNR10.2 (0.56)9.8 (0.61)––––50.5 (7.23)57.0 (4.43)PACO12.5 (0.52)11.8 (0.67)7.8 (0.25)7.3 (0.48)9.8 (0.25)10.0 (0.00)45.3 (1.64)44.3 (2.50)WATC* 18.1 (1.02)14.9 (1.49)* 8.5 (0.29)10.0 (0.00)7.5 (0.29)7.3 (0.48)83.6 (3.02)87.3 (5.94)WIWS10.8 (0.60)10.8 (0.37)8.5 (0.29)9.0 (0.00)9.8 (0.25)9.8 (0.25)54.8 (3.60)57.8 (2.71)Note: The experimental design was a randomized complete block with four replications. Means based on n=4 for all characteristics except as follows: for cropgrowth stage #1 and crop growth stage #2, n=3 for both KK179 and the conventional control at CADV; for crop growth stage #3, n=2 for the conventionalcontrol at CANA, n=3 for KK179 at CADV, and n=3 for the conventional control at KSLA; for crop growth stage #4, n=2 for both KK179 and the conventionalcontrol at CADV; for forage yield #4, n=3 for the conventional control at CADV; and for total forage yield, n=3 for the conventional control at CADV.SE = Standard Error* Indicates a statistically significant difference was detected between KK179 and the conventional control (α=0.05) using ANOVA.† No statistical comparisons were made due to lack of variability in the data.– Indicates data were excluded from analysis.NA Indicates cutting is not applicable for this site.1Regrowth after cutting was not assessed following the last harvest of the season.Monsanto Company 12-AL-246U 357 of 407Table G-7. Individual-Site Phenotypic Comparison of KK179 tothe Conventional Control in the Second Year (2011-2012) ofForage ProductionCutting #1Lodging (0-9 rating)Crop growth stage(stage number 0-6)Forage yield(fresh weight ton/acre)Regrowth after cutting1(1-10 rating)SiteKK179Mean (SE)ControlMean (SE)KK179Mean (SE)ControlMean (SE)KK179Mean (SE)ControlMean (SE)KK179Mean (SE)ControlMean (SE)CADV† 9.0 (0.00)9.0 (0.00)2.9 (0.04)2.9 (0.03)9.5 (1.18)10.2 (0.39)9.0 (0.00)8.8 (0.25)CANA7.0 (0.58)8.0 (0.41)2.9 (0.19)2.9 (0.24)9.4 (0.89)9.7 (0.30)9.0 (0.00)9.0 (0.00)IABN6.5 (0.29)6.0 (0.71)3.5 (0.06)3.6 (0.27)12.1 (0.20)12.1 (0.27)8.5 (0.29)9.3 (0.48)IDNP4.5 (0.50)3.5 (0.29)2.3 (0.14)2.3 (0.07)23.3 (0.92)23.7 (0.79)9.0 (0.41)9.3 (0.25)ILCL8.0 (0.00)8.3 (0.25)* 2.9 (0.19)3.2 (0.05)9.3 (0.23)9.8 (0.85)10.0 (0.00)10.0 (0.00)KSLA† 9.0 (0.00)9.0 (0.00)1.4 (0.43)1.5 (0.32)4.5 (0.30)5.0 (0.30)8.0 (0.00)8.0 (0.00)NYNR† 9.0 (0.00)9.0 (0.00)––5.8 (0.39)6.4 (0.28)8.8 (0.95)9.5 (0.29)PACO––––13.6 (0.56)12.5 (0.46)8.0 (0.00)8.0 (0.41)WATC7.0 (0.71)6.0 (0.71)3.9 (0.05)3.9 (0.06)17.0 (0.66)17.5 (0.42)* 9.0 (0.00)10.0 (0.00)WIWS4.0 (0.00)4.0 (0.00)2.5 (0.11)2.3 (0.15)16.3 (0.52)15.7 (0.44)9.0 (0.00)9.0 (0.00)Monsanto Company 12-AL-246U 358 of 407Table G-7 (continued). Individual-Site Phenotypic Comparison of KK179 to the Conventional Control in the Second Year(2011-2012)of Forage ProductionCutting #2Lodging (0-9 rating)Crop growth stage(stage number 0-6)Forage yield(fresh weight ton/acre)Regrowth after cutting1(1-10 rating)SiteKK179Mean (SE)ControlMean (SE)KK179Mean (SE)ControlMean (SE)KK179Mean (SE)ControlMean (SE)KK179Mean (SE)ControlMean (SE)CADV† 9.0 (0.00)9.0 (0.00)* 2.0 (0.20)2.5 (0.04)––8.5 (0.29)8.3 (0.48)CANA† 9.0 (0.00)9.0 (0.00)2.7 (0.34)2.8 (0.42)3.8 (0.28)4.2 (0.46)8.0 (0.00)7.3 (0.75)IABN* 5.8 (0.25)4.3 (0.48)3.7 (0.23)3.6 (0.19)10.9 (0.51)11.4 (0.45)8.0 (0.00)9.0 (0.41)IDNP6.0 (1.22)6.0 (1.41)3.2 (0.18)3.1 (0.24)14.5 (0.41)15.3 (0.43)9.0 (0.41)9.5 (0.29)ILCL† 9.0 (0.00)9.0 (0.00)4.5 (0.16)4.7 (0.19)8.2 (0.14)7.5 (0.11)10.0 (0.00)10.0 (0.00)KSLA† 9.0 (0.00)9.0 (0.00)* 3.6 (0.34)4.2 (0.35)10.6 (0.25)10.9 (0.57)8.8 (0.25)9.0 (0.00)NYNR† 9.0 (0.00)9.0 (0.00)––3.4 (0.24)3.5 (0.22)† 10.0 (0.00)10.0 (0.00)PACO5.8 (0.25)5.5 (0.29)––12.4 (0.50)12.1 (0.41)9.0 (0.00)9.0 (0.00)WATC9.0 (0.00)9.0 (0.00)––13.1 (0.28)13.2 (0.66)9.3 (0.25)9.5 (0.29)WIWS5.0 (0.00)5.3 (0.25)3.0 (0.09)3.0 (0.09)10.5 (0.27)10.2 (0.23)9.8 (0.25)9.0 (0.41)Monsanto Company 12-AL-246U 359 of 407Table G-7 (continued). Individual-Site Phenotypic Comparison of KK179 to the Conventional Control in the Second Year(2011-2012) of Forage ProductionCutting #3Lodging (0-9 rating)Crop growth stage(stage number 0-6)Forage yield(fresh weight ton/acre)Regrowth after cutting1(1-10 rating)SiteKK179Mean (SE)ControlMean (SE)KK179Mean (SE)ControlMean (SE)KK179Mean (SE)ControlMean (SE)KK179Mean (SE)ControlMean (SE)CADV† 9.0 (0.00)9.0 (0.00)3.9 (0.05)4.0 (0.01)13.4 (0.96)12.6 (0.45)9.3 (0.48)9.5 (0.50)CANA† 9.0 (0.00)9.0 (0.00)3.0 (0.33)2.1 (0.52)1.6 (0.10)1.8 (0.43)8.5 (0.50)8.5 (0.50)IABN3.3 (0.25)4.5 (0.87)4.0 (0.22)4.1 (0.19)7.9 (0.49)8.5 (0.34)* 7.0 (0.00)8.3 (0.48)IDNP6.5 (0.87)7.8 (0.63)2.6 (0.12)2.8 (0.19)10.9 (0.72)10.9 (0.82)8.8 (0.48)9.0 (0.41)ILCL† 9.0 (0.00)9.0 (0.00)4.5 (0.23)4.3 (0.16)5.2 (0.15)5.0 (0.17)10.0 (0.00)10.0 (0.00)KSLA5.8 (0.75)5.3 (0.25)4.7 (0.15)4.3 (0.31)10.9 (0.53)11.4 (0.14)6.0 (0.00)5.8 (0.25)NYNR† 9.0 (0.00)9.0 (0.00)––3.7 (0.02)3.6 (0.23)† 10.0 (0.00)10.0 (0.00)PACO7.3 (0.25)7.5 (0.29)4.2 (0.20)4.0 (0.07)* 9.1 (0.35)8.4 (0.12)* 10.0 (0.00)9.5 (0.29)WATC7.8 (0.63)7.5 (0.96)4.0 (0.06)4.1 (0.04)12.1 (0.25)12.1 (0.65)9.0 (0.00)9.5 (0.50)WIWS5.5 (0.29)6.3 (0.25)* 3.2 (0.15)3.7 (0.33)8.6 (0.17)8.4 (0.20)9.3 (0.25)9.5 (0.29)Monsanto Company 12-AL-246U 360 of 407Table G-7 (continued). Individual-Site Phenotypic Comparison of KK179 to the Conventional Control in the Second Year(2011-2012) of Forage ProductionCutting #4Lodging (0-9 rating)Crop growth stage(stage number 0-6)Forage yield(fresh weight ton/acre)Regrowth after cutting1(1-10 rating)SiteKK179Mean (SE)ControlMean (SE)KK179Mean (SE)ControlMean (SE)KK179Mean (SE)ControlMean (SE)KK179Mean (SE)ControlMean (SE)CADV† 9.0 (0.00)9.0 (0.00)4.0 (0.17)4.0 (0.05)6.9 (0.25)7.4 (0.53)8.5 (0.29)9.0 (0.00)CANA† 9.0 (0.00)9.0 (0.00)2.3 (0.41)2.8 (0.39)1.9 (0.23)1.9 (0.39)† 9.0 (0.00)9.0 (0.00)IABN8.5 (0.29)8.5 (0.29)2.4 (0.07)2.4 (0.09)* 5.4 (0.07)6.0 (0.16)––IDNP6.8 (0.85)8.0 (0.71)3.0 (0.13)3.1 (0.18)9.2 (0.66)8.6 (0.64)––ILCL† 9.0 (0.00)9.0 (0.00)3.2 (0.20)3.6 (0.48)0.9 (0.08)0.8 (0.06)––KSLA† 9.0 (0.00)9.0 (0.00)2.6 (0.22)2.4 (0.29)6.7 (0.34)6.7 (0.14)6.0 (0.00)6.0 (0.00)NYNR† 9.0 (0.00)9.0 (0.00)––3.2 (0.21)3.3 (0.34)––PACO4.3 (0.25)4.5 (0.29)2.6 (0.13)2.6 (0.19)6.0 (0.19)6.1 (0.46)9.0 (0.00)8.5 (0.29)WATC† 9.0 (0.00)9.0 (0.00)3.4 (0.03)3.5 (0.03)9.3 (0.19)9.2 (0.26)9.5 (0.29)10.0 (0.00)WIWS5.3 (0.25)5.8 (0.25)2.4 (0.13)2.5 (0.09)4.8 (0.16)5.3 (0.13)––Monsanto Company 12-AL-246U 361 of 407Table G-7 (continued). Individual-Site Phenotypic Comparison of KK179 to the ConventionalControl in the Second Year(2011-2012) of Forage ProductionCutting #5Lodging (0-9 rating)Crop growth stage(stage number 0-6)Forage yield(fresh weight ton/acre)Fall plant height (in)SiteKK179Mean (SE)ControlMean (SE)KK179Mean (SE)ControlMean (SE)KK179Mean (SE)ControlMean (SE)KK179Mean (SE)ControlMean (SE)CADV8.5 (0.29)8.8 (0.25)3.3 (0.16)3.5 (0.09)6.9 (1.02)4.9 (0.78)4.7 (0.80)4.8 (0.62)CANA† 9.0 (0.00)9.0 (0.00)0.9 (0.07)1.0 (0.10)1.1 (0.13)1.2 (0.16)––IABN––––––3.4 (0.14)3.7 (0.28)IDNP––––––8.7 (0.13)9.3 (0.31)ILCL––––––10.6 (0.61)11.1 (0.93)KSLA† 9.0 (0.00)9.0 (0.00)––5.0 (0.21)5.4 (0.06)4.6 (0.39)5.1 (0.25)NYNR––––––––PACO5.8 (0.25)5.8 (0.48)1.6 (0.03)1.7 (0.06)5.3 (0.26)5.3 (0.31)4.9 (0.10)4.7 (0.13)WATC† 9.0 (0.00)9.0 (0.00)1.2 (0.06)1.2 (0.05)* 4.8 (0.40)5.4 (0.17)* 8.8 (0.31)9.7 (0.13)WIWS––––––4.3 (0.10)4.2 (0.14)Monsanto Company 12-AL-246U 362 of 407Table G-7 (continued). Individual-Site Phenotypic Comparison of KK179 to the Conventional Control in the Second Year(2011-2012) of Forage ProductionTotal forage yield(fresh weight ton/acre)Spring vigor (1-10 rating)Spring stand recovery(1-10 rating)Spring stand count(# stems/foot)SiteKK179Mean (SE)ControlMean (SE)KK179Mean (SE)ControlMean (SE)KK179Mean (SE)ControlMean (SE)KK179Mean (SE)ControlMean (SE)CADV236.7 (1.95)35.8 (0.91)7.3 (1.20)6.5 (0.29)8.0 (1.15)8.8 (0.25)11.4 (1.66)11.1 (0.28)CANA17.7 (1.55)18.7 (1.47)5.8 (0.25)5.8 (0.25)† 10.0 (0.00)10.0 (0.00)72.4 (3.92)67.8 (1.87)IABN36.3 (1.09)38.0 (1.00)7.5 (0.65)8.0 (1.08)* 7.8 (0.25)9.5 (0.29)50.2 (2.50)51.4 (3.27)IDNP57.9 (2.42)58.5 (1.12)7.5 (0.65)6.8 (0.75)7.3 (0.25)6.5 (0.65)* 54.8 (4.70)33.8 (8.22)ILCL23.6 (0.35)23.1 (0.96)9.5 (0.29)9.0 (0.00)8.3 (0.25)8.0 (0.00)31.0 (3.23)32.0 (1.62)KSLA37.7 (0.81)39.4 (0.69)9.0 (0.00)7.5 (1.50)8.3 (0.25)8.0 (0.00)105.8 (4.82)93.8 (6.00)NYNR16.0 (0.16)16.8 (0.68)5.0 (0.00)5.0 (0.00)7.0 (1.35)8.3 (0.25)* 59.3 (6.51)69.7 (1.86)PACO46.4 (1.27)44.5 (1.59)* 6.5 (0.29)5.5 (0.29)8.3 (0.25)8.3 (0.25)32.3 (4.00)38.1 (2.28)WATC56.3 (0.85)57.5 (1.71)* 9.3 (0.25)10.0 (0.00)7.8 (0.48)8.3 (0.48)86.2 (3.15)90.3 (4.18)WIWS40.1 (0.47)39.6 (0.79)9.8 (0.25)9.3 (0.25)8.8 (0.63)9.0 (0.41)32.3 (1.27)31.3 (2.35)Note: The experimental design was a randomized complete block with four replications. Means based on n=4 for all characteristics except as follows: for cropgrowth stage #1, n=3 for the conventional control at CANA, IABN, and KSLA and n=2 for KK179 at KSLA; for forage yield #1, n=3 for the conventionalcontrol at CADV; for crop growth stage #2, n=3 for KK179 at CADVand at KSLA; for crop growth stage #3, n=2 for KK179 at CADV and n=3 for theconventional control at CADV and CANA; for forage yield #3, n=3 for KK179 at PACO and n=3 for the conventional control at CADV;for crop growth stage#4, n=2 for KK179 at CADV, n=3 for KK179 at CANA, IDNP and KSLA, and n=3 for the conventional control at KSLA; for total forageyield , n=3 for KK179at PACO and n=3 for the conventional control at CADV; for spring vigor, spring stand recovery, and spring stand count, n=3 forKK179 at CADV.SE = Standard Error* Indicates a statistically significant difference was detected between KK179 and the conventional control (α=0.05) using ANOVA.† No statistical comparisons were made due to lack of variability in the data.NA Indicates the particular cutting is not applicable for this site.– Indicates data were excluded from analysis.1Regrowth after cutting was not assessed following the last harvest of the season.2Total forage yield at CADV was calculated from four cuttings, since forage yield for cutting #2 was excluded.Monsanto Company 12-AL-246U 363 of 407 

### G.14 Combined and Individual Field Site Environmental Interactions Results and

Discussion

G.14.1 Combined and Individual Field Site Environmental Interactions Results and Discussion - First Year (2010)

In the assessment for the first year (2010), no differences in the range of plant damage responses to abiotic stressors were observed for 93 comparisons between KK179 and the

conventional control. Abio tic stressors evalua ted included drought, flood, frost, hail, heat, nutrient deficiency, soil compaction, and wind (Table G-8).

No differences in the range of plant damage responses to disease stressors were observed for 93 comparisons between KK179 and the c onventional control. Diseases evaluated included Anthracnose, bacterial wilt, black stem, damping-off, dow ny mildew, Fusarium wilt, leaf spots, root rot, Sclerotinia crown and stem rot, stem nematode, and Verticillium wilt (Table G-9).

No differences in the range of plant dama ge responses to arthropod stressors were observed for 96 comparisons between KK179 an d the conventional control. Arthropods evaluated included alfalfa caterpillar, alfalf a weevil, aphid, armyworm, blister beetle, cutworm, grasshopper, meadow spittlebug, pl ant bug, potato leafhopper, spider mite, and thrips (Table G-10).

In an individual-site analysis for the firs t year (2010), no sta tistically significant

differences were detected between KK179 and the conventional contro l for alfalfa weevil and potato leafhopper damage among all observa tions (Table G-11). The lack of differences in both the individual-site analys is and the combined-site -analysis supports

the conclusion that the introducti on of the trait for reduced G lignin and total lignin is not expected to have an effect on the envir onment compared to the conventional control (Figure VII-1, Step 2, answer “no”).

In the individual-site analysis of arthropod abundance for the first year, no statistically significant differences were detected ( α =0.05) between KK179 and the conventional control for 65 of the 69 comparisons of pe st- and beneficial-art hropod abundance (Tables G-12 and G-13). No st atistically significant differenc es in abundance were detected between KK179 and the conventional control for the following pest and beneficial arthropods: aphid, alfalfa w eevil, alfalfa looper, false chinch bug, green cloverworm, garden webworm, thrips, damsel bug, ladybird beetle, parasitic wasps, and lacewing.

Two statistically significant differences in abundance were detected out of 39 pestarthropod comparisons (Table G-12). KK 179 had significantly higher abundance of Lygus bug than the conventional control in collection #2 at KSLA (1.3 vs. 0.0). KK179 had significantly lower abundance of potato leafhopper in collection #1 at ILCL (152.3 vs. 329.0). The mean abundance values of pot ato leafhopper in collect ion #1 at ILCL and of Lygus bug for collection #2 at KSLA were outside the reference ranges (178.8 – 256.8 and 0.0 – 0.3, respectively) However, the diffe rences detected for these taxa were not consistently detected across collection times or sites. Thus, the detected differences wereMonsanto Company 12-AL-246U 364 of 407 

### not indicative of a consistent response associated with the trait and were not considered biologically meaningful in te rms of effects on the environment from KK179 compared to the conventional control (S ee Section VII.B.2.).

Two statistically significant differences in a bundance were detected out of 30 beneficialarthropod comparisons (Table G-13). KK179 had significantly lower abundance of spiders than the conventional control in collection #2 at IL CL and at KSLA (0.0 vs. 1.5 and 0.0 vs. 1.3, respectively). The mean abund ance values of KK179 were within the respective reference ranges. Thus, the de tected differences were not considered biologically meaningful in te rms of effects on the environment from KK179 compared to

the conventional control (See Section VII.B.2.).

G.14.2. Combined and Individual Field Site Environmental Interactions Results and Discussion - Second Year (2011)

In an assessment for the second year, no differences in the range of plant damage responses to abiotic stresso rs were observed for 129 comp arisons between KK179 and

the conventional control. Abiotic stressors evaluated included drought, frost, hail, heat, heaving, nutrient deficiency, soil compac tion, wet soil, wind, and winter injury kill (Table G-14).

No differences in the range of plant damage responses to disease stressors were observed for 129 comparisons between KK179 and the c onventional control. Diseases evaluated included Anthracnose, bacterial wilt, black stem, crown rot, downy mildew, Fusarium wilt, leaf spots, root rot, Sclerotinia crow n and stem rot, and Verticillium wilt (Table G-15).

No differences in the range of plant dama ge responses to arthropod stressors were observed for 129 comparisons between KK179 a nd the conventional control. Arthropods evaluated included alfalfa caterpillar, alfalfa leafminer, alfalfa weevil, aphid, armyworm, bean leaf beetle, blister be etle, cutworm, grasshoppers, green cloverworm, Japanese beetle, Lygus bug, meadow spittlebug, plan t bug, potato leafhopper, southern corn rootworm beetle, spider mite , and thrips (Table G-16).

In the individual-site analysis for the s econd year, three stat istically significant

differences were detected between KK179 and the conventional contro l for alfalfa weevil and potato leafhopper damage among all observa tion times (Table G-17). Alfalfa weevil damage and potato leafhopper damage were higher for KK179 than the conventional control during the observation #1 at KSLA (damage rating of 3.4 vs. 2.7 and 2.2 vs. 1.7, respectively). Potato leaf hopper damage was lower for KK179 than the conventional control for observation #4 at KSLA (damage rating of 0.1 vs. 0.7). The differences

detected in the individual-site analysis were not detected when data were pooled in a combined-site analysis. Thus, the detected differences were not c onsidered biologically meaningful in terms of effects on the environment from KK179 compared to the conventional control (Figure V II-1, Step 2, answer “no”).Monsanto Company 12-AL-246U 365 of 407 

### In an individual-site analysis of arthropod abundance for the second year, no significant

differences were detected between KK179 a nd the conventional control for arthropod abundance for 82 of 83 comparisons (Tables G- 18 and G-19). No statistically significant

differences were detected between KK179 and the conventional control for the following pest- and beneficial-arthropods: aphid, armywo rm, false chinch bug, blister beetle, green cloverworm, Lygus bug, meadow spittlebug, po tato leafhopper, thrips, soybean looper, spiders, ladybird beetle, lacewi ng, nabids, and chalcid wasps.

One significant difference was detected out of 50 pest-arthropod comparisons between

KK179 and the conventional c ontrol (Table G-18). KK179 had a lower abundance of alfalfa weevils compared to the conventional control for collection #1 at ILCL (0.3 vs. 7.0). The mean abundance value of KK179 was within the reference range. Thus, the

detected difference was not c onsidered biologically meaningf ul in terms of effects on the environment from KK179 compared to the c onventional control (See Section VII.B.2.).

No statistically significant differences in abundance were detected between KK179 and

the conventional control out of 33 benefici al-arthropod comparisons (Table G-19). The lack of differences supports the conclusion th at the introduction of the trait for reduced G lignin and total lignin is not expected to affect the environment compared to the

conventional control.Monsanto Company 12-AL-246U 366 of 407 

### Table G-8. Combined-Site Qualitative A ssessment: Abiotic Stressor Evaluations Using an Observational Severity Scale fo r KK179 and the Conventional Control in

the First Year

Note: The experimental design was a randomized complete block with four replications. Observations were made during each crop growth cycle, prior to harvest. Data were not subjected to statistical analysis.

No differences were observe d between KK179 and the conv entional control during any observation for damage caused by an y of the assessed abiotic stressors.

1 Includes wet soil and excess moisture. Abiotic Stressor Number of observations

across all sites in 2010 Number of observations with no

differences between KK179 and

the conventional control across all

sites in 2010

Total 93 93

Drought 16 16

Flood 1 15 15

Frost damage 1 1

Hail damage 7 7

Heat 22 22

Nutrient deficiency 11 11

Soil compaction 5 5

Wind damage 16 16Monsanto Company 12-AL-246U 367 of 407 

### Table G-9. Combined-Site Qualitative Assessment: Disease Damage Evaluations Using an Observational Severity Scale fo r KK179 and the Conventional Control in the First Year (2010)

Note: The experimental design was a randomized complete block with four replications. Observations were made during each crop growth cycle, prior to harvest. Data were not subjected to statistical analysis.

No differences were observed between KK179 and the conventional control during any observation for damage caused by any of the assessed disease stressors.

1 Includes Phytophthora. Disease Stressor Number of observations

across all sites in 2010 Number of observations with

no differences between

KK179 and the conventional

control across all sites in

2010

Total 93 93

Anthracnose 6 6

Bacterial wilt 7 7

Black stem 8 8

Damping-off 3 3

Downy mildew 13 13

Fusarium wilt 6 6

Leaf spots 26 26

Root rot 1 6 6

Sclerotinia crown and stem rot 4 4

Stem nematode 4 4

Verticillium wilt 10 10Monsanto Company 12-AL-246U 368 of 407 

### Table G-10. Combined-Site Qualitative Assessment: Insect Stressor Evaluations Using an Observational Severity Scale fo r KK179 and the Conventional Control in the First Year (2010)

Note: The experimental design was a randomized complete block with four replications. Observations were made during each crop growth cycle, prior to harvest. Data were not subjected to statistical analysis.

No differences were observed between KK179 and the conventional control during any observation for damage caused by any of the assessed insect stressors. Insect Stressor Number of observations

across all sites in 2010 Number of observations with no

differences between KK179 and

the conventional control across all

sites in 2010

Total 96 96

Alfalfa caterpillar 1 1

Alfalfa weevil 5 5

Aphid 29 29

Armyworm 3 3

Blister beetle 4 4

Cutworm 7 7

Grasshopper 10 10

Meadow spittlebug 4 4

Plant bug 8 8

Potato leafhopper 10 10

Spider mite 2 2

Thrips 13 13Monsanto Company 12-AL-246U 369 of 407 

### Table G-11. Individual-Site Analysis: Quantitative Assessment of Potato Leafhopper and Alfalfa Weevil Damage to KK179 Compared to the Conventional Control in the First Year (2010)

Damage (0-10 rating scale)

Alfalfa weevil ( Hypera postica ) Potato leafhopper ( Empoasca fabae )

Site KK179 Mean (S.E.) Control Mean (S.E.) KK179 Mean (S.E.) Control Mean (S.E.)

Observation #1

ILCL 0.3 (0.03) 0.5 (0.16) 0.7 (0.04) 0.7 (0.14)

KSLA 0.1 (0.06) 0.2 (0.05) 0.5 (0.02) 0.7 (0.08)

NYNR † 0.0 (0.00) 0.0 (0.00) † 0.0 (0.00) 0.0 (0.00)

Observation #2

ILCL 0.5 (0.04) 0.7 (0.13) 0.7 (0.15) 0.8 (0.10)

KSLA 0.0 (0.03) 0.0 (0.00) † 0.0 (0.00) 0.0 (0.00)

NYNR † 0.0 (0.00) 0.0 (0.00) † 0.0 (0.00) 0.0 (0.00)

Observation #3

ILCL 0.1 (0.05) 0.2 (0.06) 0.1 (0.08) 0.1 (0.03)

KSLA † 0.0 (0.00) 0.0 (0.00) † 0.0 (0.00) 0.0 (0.00)

NYNR † 0.0 (0.00) 0.0 (0.00) † 0.0 (0.00) 0.0 (0.00)

Observation #4

KSLA † 0.0 (0.00) 0.0 (0.00) † 0.0 (0.00) 0.0 (0.00)

Note: The experimental design was a randomized complete block with four replications. Means based on

n=4.

S.E. = Standard Error No statistically significant differences were det ected between KK179 and th e conventional control ( α =0.05) using ANOVA. † No statistical comparisons were made due to lack of variability in the data.Monsanto Company 12-AL-246U 370 of 407Table G-12. Abundance of Pest Arthropods in SamplesCollected from KK179, the Conventional Control, and theConventional Commercial Reference Varieties in the First Year (2010)Pest ArthropodAphid (Aphididae)Alfalfa weevil (Hypera postica)Alfalfa looper (Autographa californica)SiteKK179Mean (SE)ControlMean (SE)ReferenceRange1KK179Mean (SE)ControlMean (SE)ReferenceRange1KK179Mean (SE)ControlMean (SE)ReferenceRange1Collection #1ILCL–––––––––KSLA22.3 (3.20)26.0 (4.56)17.5 – 27.30.8 (0.48)0.0 (0.00)0.0 – 0.5–––NYNR22.8 (4.77)15.8 (3.99)17.0 – 24.5––––––Collection #2ILCL12.8 (3.77)13.8 (3.71)13.8 – 22.8––––––KSLA167.8 (35.73)181.3 (25.38)114.0 – 294.5––––––NYNR156.5 (19.75)118.3 (6.16)116.8 – 159.5––––––Collection #3ILCL44.5 (17.33)41.5 (8.21)28.0 – 44.3––––––KSLA510.3 (62.90)509.0 (172.02)404.0 – 813.5–––0.8 (0.25)0.5 (0.29)0.5 – 0.8NYNR8.3 (0.85)12.3 (2.66)7.0 – 11.5––––––Collection #4KSLA5.8 (1.89)9.0 (1.78)5.8 – 9.0––––––Monsanto Company 12-AL-246U 371 of 407Table G-12 (continued). Abundance of Pest Arthropods in Samples Collected from KK179, the Conventional Control, and theConventionalCommercial Reference Varieties in the First Year (2010)Pest ArthropodFalse chinch bug (Nysius raphanus)Green cloverworm (Hypena scabra)Garden webworm (Achyra rantalis)SiteKK179Mean (SE)ControlMean (SE)ReferenceRangeKK179Mean (SE)ControlMean (SE)ReferenceRangeKK179Mean (SE)ControlMean (SE)ReferenceRangeCollection #1ILCL4.5 (1.32)10.0 (2.86)7.0 – 11.31.3 (0.25)1.8 (1.11)1.3 – 4.3–––KSLA9.0 (1.96)7.5 (0.50)6.3 – 13.8––––––NYNR–––––––––Collection #2ILCL7.3 (1.11)9.5 (1.94)10.0 – 13.3–––2.3 (0.63)2.8 (1.03)1.5 – 3.8KSLA1.0 (0.41)1.5 (1.50)0.8 – 1.3––––––NYNR2.0 (0.71)1.8 (0.75)0.8 – 1.5––––––Collection #3ILCL6.8 (1.93)7.5 (2.02)3.8 – 10.3––––––KSLA10.5 (3.01)10.3 (0.85)9.0 – 10.5–––3.3 (0.75)3.8 (0.75)1.8 – 3.0NYNR–––––––––Collection #4KSLA6.0 (1.58)3.0 (1.08)3.8 – 6.8––––––Monsanto Company 12-AL-246U 372 of 407Table G-12 (continued). Abundance of Pest Arthropods in Samples Collected from KK179, the Conventional Control, and theConventional Commercial Reference Varieties in the First Year (2010)Pest ArthropodLygus bug (Miridae)Potato leafhopper (Empoasca fabae)Thrips (Thysanoptera)SiteKK179Mean (SE)ControlMean (SE)ReferenceRangeKK179Mean (SE)ControlMean (SE)ReferenceRangeKK179Mean (SE)ControlMean (SE)ReferenceRangeCollection #1ILCL7.8 (1.49)8.8 (3.07)6.8 – 11.0*152.3 (30.90)329.0 (90.25)178.8 – 256.8–––KSLA0.3 (0.25)1.5 (0.96)0.7 – 1.36.5 (1.66)11.0 (1.78)6.7 – 13.3–––NYNR1.5 (0.50)1.0 (0.71)1.5 – 2.312.0 (3.24)18.5 (3.07)12.3 – 14.3–––Collection #2ILCL–––35.8 (7.79)29.0 (11.71)19.5 – 27.8–––KSLA* 1.3 (0.48)0.0 (0.00)0.0 – 0.3–––8.5 (2.72)15.8 (5.85)6.8 – 12.5NYNR11.8 (0.95)11.8 (3.71)5.3 – 11.0––––––Collection #3ILCL–––9.0 (1.68)10.3 (3.97)6.5 – 8.8KSLA0.3 (0.25)0.8 (0.48)0.0 – 0.5––––––NYNR1.8 (0.63)4.3 (2.36)1.8 – 4.825.5 (1.04)19.8 (1.49)14.8 – 21.812.0 (3.56)16.3 (2.50)11.5 – 26.0Collection #4KSLA–––1.5 (0.29)1.5 (0.29)0.3 – 1.327.0 (3.76)24.8 (2.78)16.3 – 26.8Note: The experimental design was a randomized complete block with four replications. Means based on n=4.SE = Standard Error* Indicates a statistically significant difference was detected between KK179 and the conventional control (α=0.05) using ANOVA.– Indicates arthropod was not evaluated.1Reference range is the minimum and maximum mean values among the conventional commercialreference varieties.Monsanto Company 12-AL-246U 373 of 407Table G-13. Abundance of Beneficial Arthropods in Samples Collected from KK179, the Conventional Control, and theConventional Commercial Reference Varieties in the First Year (2010)Beneficial ArthropodSpiders (Araneae)Damsel bug (Nabisspp.)Ladybird beetle (Coccinellidae)SiteKK179Mean (SE)ControlMean (SE)ReferenceRange1KK179Mean (SE)ControlMean (SE)ReferenceRange1KK179Mean (SE)ControlMean (SE)ReferenceRange1Collection #1ILCL1.5 (0.29)1.5 (0.50)0.8 – 1.51.8 (0.85)1.0 (0.41)0.8 – 2.02.0 (0.91)2.3 (0.85)2.3 – 3.0KSLA–––1.0 (0.58)0.8 (0.25)0.5 – 2.01.0 (0.71)0.3 (0.25)0.3 – 2.8NYNR–––0.0 (0.00)0.0 (0.00)0.0 – 0.30.0 (0.00)0.3 (0.25)0.0 – 0.5Collection #2ILCL* 0.0 (0.00)1.5 (0.65)0.0 – 1.80.5 (0.29)0.5 (0.29)0.3 – 2.01.5 (0.65)1.8 (0.75)0.5 – 3.0KSLA* 0.0 (0.00)1.3 (0.48)0.0 – 0.5–––0.3 (0.25)0.0 (0.00)0.0 – 0.3NYNR–––1.5 (0.65)0.5 (0.50)0.3 – 1.51.8 (0.85)1.3 (0.48)0.5 – 3.0Collection #3ILCL0.0 (0.00)0.5 (0.29)0.0 – 0.50.0 (0.00)0.5 (0.29)0.0 – 0.5–––KSLA2.5 (0.65)1.5 (0.87)1.3 – 2.5–––5.5 (1.04)2.8 (0.63)2.5 – 4.8NYNR–––0.5 (0.29)0.8 (0.48)0.3 – 1.00.3 (0.25)0.5 (0.29)0.8 – 1.8Collection #4KSLA1.3 (0.48)1.0 (0.41)0.8 – 2.30.5 (0.29)0.3 (0.25)0.0 – 0.5–––Monsanto Company 12-AL-246U 374 of 407Table G-13 (continued). Abundance of Beneficial Arthropods in Samples Collected from KK179, the Conventional Control,and the Conventional Commercial ReferenceVarieties in the First Year (2010)Beneficial ArthropodParasitic wasps (Hymenoptera)2Lacewing (Chrysopidae)SiteKK179Mean (SE)ControlMean (SE)Reference RangeKK179Mean (SE)ControlMean (SE)Reference RangeCollection #1ILCL––––––KSLA5.0 (1.47)9.8 (1.18)5.0 – 12.3–––NYNR0.0 (0.00)0.5 (0.50)0.0 – 0.5–––Collection #2ILCL––––––KSLA0.8 (0.25)2.0 (0.91)0.8 – 1.5–––NYNR1.0 (1.00)1.0 (0.71)0.5 – 1.5–––Collection #3ILCL–––0.0 (0.00)0.0 (0.00)0.0 – 0.5KSLA3.0 (1.08)1.8 (0.85)1.8 – 3.0–––NYNR2.8 (0.75)1.5 (0.65)0.5 – 3.8–––Collection #4KSLA0.5 (0.50)1.5 (0.65)1.0 – 1.8–––Note: The experimental design was a randomized complete block with four replications. Means based on n=4.SE = Standard Error* Indicates a statistically significant difference was detected between KK179 and the conventional control (α=0.05) using ANOVA.– Indicates arthropod was not evaluated.1Reference range is the minimum and maximum mean valuesamong the conventional commercial reference varieties.2Parasitic wasps include braconids, ichneumons, and micro-parasitic Hymenoptera.Monsanto Company 12-AL-246U 375 of 407 

### Table G-14. Combined-Site Qualitative A ssessment: Abiotic St ressor Evaluations Using an Observational Severity Scale fo r KK179 and the Conventional Control in the Second Year (2011)

Note: The experimental design was a randomized complete block with four replications. Observations were made during each crop growth cycle, prior to harvest. Data were not subjected to statistical analysis. No differences were observed between KK179 and the conventional control during any observation for damage caused by any of the assessed abiotic stressors.

1 Includes wet soil and excess moisture. Abiotic Stressor Number of observations

across all sites in 2011 Number of observations with no

differences between KK179 and

the conventional control across

all sites in 2011

Total 129 129

Drought 22 22

Frost damage 3 3

Hail damage 8 8

Heat 19 19

Heaving 1 1

Nutrient deficiency 10 10

Soil compaction 6 6

Wet soil 1 22 22

Wind damage 30 30

Winter injury kill 8 8Monsanto Company 12-AL-246U 376 of 407 

### Table G-15. Combined-Site Qualitative Assessment: Disease Damage Evaluations Using an Observational Severity Scale fo r KK179 and the Conventional Control in the Second Year (2011)

Note: The experimental design was a randomized complete block with four replications. Observations were made during each crop growth cycle, prior to harves t. Data were not subjected to statistical analysis. No differences were observed between KK179 and the conventional control during any observation for damage caused by any of the assessed disease stressors.

1 Includes summer black stem.

2 Includes common leaf spot.

3 Includes Phytophthora. Disease Stressor Number of observations

across all sites in 2011 Number of observations with

no differences between KK179

and the conventional control

across all sites in 2011

Total 129 129

Anthracnose 7 7

Bacterial wilt 10 10

Black stem 1 12 12

Crown Rot 3 3

Downy mildew 28 28

Fusarium wilt 4 4

Leaf spots 2 36 36

Root rot 3 6 6

Sclerotinia crown and stem rot 4 4

Verticillium wilt 19 19Monsanto Company 12-AL-246U 377 of 407 

### Table G-16. Combined-Site Qualitative Assessment: Insect Stressor Evaluations Using an Observational Severity Scale fo r KK179 and the Conventional Control in the Second Year (2011)

Note: The experimental design was a randomized complete block with four replications. Observations were made during each crop growth cycle, prior to harves t. Data were not subjected to statistical analysis. No differences were observed between KK179 and the conventional control during any observation for damage caused by any of the assessed insect stressors.

1 Includes pea aphid. Insect Stressor Number of observations

across all sites in 2011 Number of observations with

no differences between

KK179 and the conventional

control across all sites in

2011

Total 129 129

Alfalfa caterpillar 5 5

Alfalfa leafminer 5 5

Alfalfa weevil 10 10

Aphid 1 38 38

Armyworm 3 3

Bean leaf beetle 1 1

Blister beetle 4 4

Cutworm 2 2

Grasshopper 16 16

Green cloverworm 1 1

Japanese beetle 1 1

Lygus bug 4 4

Meadow spittlebug 5 5

Plant bug 4 4

Potato leafhopper 11 11

Southern corn rootworm beetle 1 1

Spider mite 1 1

Thrips 17 17Monsanto Company 12-AL-246U 378 of 407 

### Table G-17. Individual-Site Analysis: Quantitative Assessment of Potato Leafhopper and Alfalfa Weevil Damage to KK179 Compared to the Conventional Control in the Second Year (2011)

Damage (0-10 rating scale)

Alfalfa weevil ( Hypera postica ) Potato leafhopper ( Empoasca fabae)

Site KK179 Mean (SE) Control Mean (SE) KK179 Mean (SE) Control Mean (SE)

Observation #1

ILCL 0.2 (0.08) 0.1 (0.06) 0.3 (0.10) 0.1 (0.13)

KSLA * 3.4 (0.09) 2.7 (0.17) * 2.2 (0.25) 1.7 (0.10)

NYNR † 0.0 (0.00) 0.0 (0.00) † 0.0 (0.00) 0.0 (0.00)

Observation #2

ILCL 0.6 (0.10) 0.7 (0.10) 0.4 (0.07) 0.3 (0.06)

KSLA † 0.0 (0.00) 0.0 (0.00) † 0.0 (0.00) 0.0 (0.00)

NYNR † 0.0 (0.00) 0.0 (0.00) † 0.0 (0.00) 0.0 (0.00)

Observation #3

ILCL 0.6 (0.10) 0.5 (0.18) 0.4 (0.03) 0.3 (0.12)

KSLA † 0.0 (0.00) 0.0 (0.00) † 0.0 (0.00) 0.0 (0.00)

NYNR † 0.0 (0.00) 0.0 (0.00) † 0.0 (0.00) 0.0 (0.00)

Observation #4

ILCL 0.1 (0.05) 0.1 (0.05) 0.4 (0.05) 0.3 (0.03)

KSLA † 0.0 (0.00) 0.0 (0.00) * 0.1 (0.13) 0.7 (0.36)

NYNR † 0.0 (0.00) 0.0 (0.00) 1.5 (0.65) 1.5 (0.65)

Observation #5

KSLA † 0.0 (0.00) 0.0 (0.00) † 0.0 (0.00) 0.0 (0.00)

Note: The experimental design was a randomized complete block with four replications. Means based on

n=4.

SE = Standard Error * Indicates a statistically significan t difference was detected between KK179 and the conventional control ( α =0.05) using ANOVA. † No statistical comparisons were made due to lack of variability in the data.Monsanto Company 12-AL-246U 379 of 407Table G-18. Abundance of Pest Arthropods in SamplesCollected from KK179, the Conventional Control, and theConventional Commercial Reference Varieties in the Second Year (2011)Pest ArthropodAphid (Aphididae)Alfalfa weevil (Hypera postica)Armyworm (Spodopteraspp.)SiteKK179Mean (SE)ControlMean (SE)ReferenceRange1KK179Mean (SE)ControlMean (SE)ReferenceRange1KK179Mean (SE)ControlMean (SE)ReferenceRange1Collection #1ILCL52.8 (2.14)53.8 (16.04)60.0 - 100.8*0.3 (0.25)7.0 (5.05)0.0 – 1.0–––KSLA81.5 (11.38)75.3 (10.95)64.3 – 97.025.0 (7.69)29.8 (5.12)22.0 – 43.3–––NYNR185.8 (19.26)181.5 (38.49)220.0 – 252.044.3 (10.26)48.5 (6.74)35.5 – 64.3–––Collection #2ILCL–––––––––KSLA173.0 (24.19)177.0 (25.26)158.3 – 281.3––––––NYNR52.5 (5.07)52.3 (7.09)41.0 – 58.0––––––Collection #3ILCL73.5 (14.39)54.3 (7.38)76.3 – 87.8––––––KSLA186.5 (25.31)189.3 (31.98)134.3 – 236.0––––––NYNR114.0 (17.97)96.3 (8.40)62.3 – 112.0––––––Collection #4ILCL141.5 (29.22)112.8 (18.14)99.3 – 134.3––––––KSLA167.8 (35.46)232.8 (24.54)189.3 – 276.5––––––NYNR3.3 (1.60)4.8 (1.11)4.8 – 6.8–––0.3 (0.25)0.0 (0.00)0.0 – 0.8Collection #5KSLA11.0 (2.35)8.3 (1.31)6.5 – 8.0–––5.3 (2.21)3.8 (1.55)3.5 – 6.5Monsanto Company 12-AL-246U 380 of 407Table G-18 (continued). Abundance of Pest Arthropods in Samples Collected from KK179, the Conventional Control, and theConventional Commercial Reference Varieties in the Second Year (2011)Pest ArthropodFalse chinch bug (Nysius raphanus)Blister beetle (Meloidae)Green cloverworm (Plathypena scabra)SiteKK179Mean(SE)ControlMean(SE)ReferenceRangeKK179Mean(SE)ControlMean(SE)ReferenceRangeKK179Mean(SE)ControlMean(SE)ReferenceRangeCollection #1ILCL–––––––––KSLA1.5 (0.50)2.3 (1.03)2.0 – 4.8––––––NYNR–––––––––Collection #2ILCL–––––––––KSLA21.3 (5.22)20.3 (2.69)19.0 – 29.50.3 (0.25)0.5 (0.50)0.0 – 1.0NYNR–––––––––Collection #3ILCL–––––––––KSLA18.5 (7.92)17.3 (2.29)7.0 – 20.0––––––NYNR––––––1.3 (0.63)1.8 (0.75)0.0 – 1.3Collection #4ILCL–––––––––KSLA3.0 (0.41)5.8 (2.10)6.0 – 9.0––––––NYNR–––––––––Collection #5KSLA4.0 (1.73)3.3 (1.11)4.0 – 5.8––––––Monsanto Company 12-AL-246U 381 of 407Table G-18 (continued). Abundance of Pest Arthropods in Samples Collected from KK179, the Conventional Control, and theConventional Commercial Reference Varieties in the Second Year (2011)Pest ArthropodLygus bug (Lygus hesperus)Meadow spittlebug (Philaenus spumarius)Potato leafhopper (Empoasca fabae)SiteKK179Mean (SE)ControlMean (SE)ReferenceRangeKK179Mean (SE)ControlMean (SE)ReferenceRangeKK179Mean (SE)ControlMean (SE)ReferenceRangeCollection #1ILCL3.0 (1.08)2.5 (1.50)1.0 – 6.3––––––KSLA–––––––––NYNR9.8 (0.85)9.0 (4.83)6.3 – 11.32.0 (1.00)0.8 (0.75)0.3 – 2.53.0 (1.08)5.0 (0.71)2.3 – 7.5Collection #2ILCL14.5 (1.32)16.8 (5.53)9.5 – 16.5–––74.5 (11.53)69.0 (3.19)48.3 – 95.8KSLA–––––––––NYNR8.3 (2.93)11.8 (1.75)8.3 – 14.0–––49.3 (14.87)65.0 (14.96)37.3 – 52.8Collection #3ILCL6.8 (0.85)12.0 (3.72)5.8 – 12.0–––109.8 (21.26)129.5 (32.07)85.3 – 100.3KSLA––––––3.8 (1.49)2.5 (0.65)0.8 – 2.3NYNR21.5 (5.20)19.8 (3.12)13.3 – 16.3–––215.5 (83.92)169.3 (31.20)116.8 – 137.0Collection #4ILCL––––––22.0 (6.65)14.3 (2.02)9.8 – 17.0KSLA–––––––––NYNR2.0 (1.68)1.8 (0.25)2.3 – 3.8–––6.5 (1.04)3.5 (0.87)1.0 – 4.0Collection #5KSLA–––––––––Monsanto Company 12-AL-246U 382 of 407Table G-18 (continued). Abundance of Pest Arthropods in Samples Collected from KK179, the Conventional Control, and theConventional Commercial Reference Varieties in the Second Year (2011)Pest ArthropodThrips (Thysanoptera)Soybean looper (Pseudoplusia includens)SiteKK179 Mean (SE)Control Mean (SE)Reference RangeKK179 Mean (SE)Control Mean (SE)Reference RangeCollection #1ILCL32.0 (1.08)33.0 (12.73)27.8 – 48.3–––KSLA25.3 (2.50)37.5 (9.95)37.8 – 46.8–––NYNR–––Collection #2ILCL181.5 (33.26)177.0 (10.23)87.5 – 166.5–––KSLA739.3 (174.43)976.3 (118.06)796.5 – 1190.5–––NYNR––––––Collection #3ILCL––––––KSLA138.5 (39.86)172.8 (43.35)132.3 – 213.8–––NYNR––––––Collection #4ILCL52.5 (4.56)49.3 (5.62)36.3 – 58.0–––KSLA76.3 (7.04)137.3 (18.20)115.3 – 160.32.5 (0.96)2.5 (1.04)2.3 – 3.8NYNR12.0 (2.38)10.3 (2.46)8.0 – 14.0–––Collection #5KSLA174.0 (25.44)208.0 (18.52)157.3 – 223.0–––Note: The experimental design was a randomized complete block with four replications. Means based on n=4.SE = Standard Error* Indicates a statistically significant difference was detected between KK179 and the conventional control (α=0.05) using ANOVA.– Indicates arthropod was not evaluated.1Reference range is the minimum and maximum mean valuesamong the conventional commercial reference varieties.Monsanto Company 12-AL-246U 383 of 407Table G-19. Abundance of Beneficial Arthropods in Samples Collected from KK179, the Conventional Control, and theConventional Commercial Reference Varieties in the Second Year (2011)Beneficial ArthropodSpiders (Araneae)Ladybird beetle (Coccinellidae)Lacewing (Chrysopidae)SiteKK179Mean (SE)ControlMean (SE)ReferenceRange1KK179Mean (SE)ControlMean (SE)ReferenceRange1KK179Mean (SE)ControlMean (SE)ReferenceRange1Collection #1ILCL0.0 (0.00)0.3 (0.25)0.0 – 0.51.5 (0.29)3.0 (1.73)1.8 – 2.50.3 (0.25)0.0 (0.00)0.0 – 1.5KSLA0.5 (0.29)0.3 (0.25)0.3 – 0.51.0 (0.71)1.0 (0.71)0.8 – 2.3–––NYNR–––––––––Collection #2ILCL–––––––––KSLA0.8 (0.25)1.0 (0.71)0.8 – 1.52.3 (1.31)1.5 (0.65)1.5 – 2.0–––NYNR0.0 (0.00)0.3 (0.25)0.0 – 0.3––––––Collection #3ILCL0.8 (0.25)1.8 (0.25)1.0 – 2.3–––0.3 (0.25)0.3 (0.25)0.3 – 0.5KSLA2.8 (0.75)4.5 (1.44)3.3 – 4.01.3 (0.48)0.8 (0.25)0.5 – 2.80.5 (0.29)0.5 (0.50)0.5 – 1.0NYNR1.3 (0.25)0.8 (0.25)1.0 – 1.87.5 (1.66)6.0 (1.08)3.3 – 5.51.0 (0.41)0.8 (0.25)0.5 – 2.0Collection #4ILCL4.0 (1.47)1.3 (0.95)1.3 – 2.80.3 (0.25)0.5 (0.29)0.0 – 1.3–––KSLA2.0 (0.58)4.3 (0.48)2.8 – 4.8–––0.5 (0.29)0.8 (0.25)0.0 – 1.0NYNR0.0 (0.00)0.3 (0.25)0.3 – 0.80.0 (0.00)0.0 (0.00)0.0 – 0.3–––Collection #5KSLA3.0 (0.71)4.0 (1.08)3.3 – 6.30.3 (0.25)0.3 (0.25)0.0 – 0.51.5 (0.29)0.8 (0.48)0.5 – 1.3Monsanto Company 12-AL-246U 384 of 407Table G-19 (continued). Abundance of Beneficial Arthropods in Samples Collected from KK179, the Conventional Control,and the Conventional Commercial ReferenceVarieties in the Second Year (2011)Beneficial ArthropodNabids (Nabidae)Chalcid wasps (Chalcidoidea)SiteKK179 Mean (SE)Control Mean (SE)Reference RangeKK179 Mean (SE)Control Mean (SE)Reference RangeCollection #1ILCL––––––KSLA––––––NYNR––––––Collection #2ILCL––––––KSLA1.0 (0.71)2.3 (0.48)0.8 – 1.8–––NYNR0.5 (0.29)0.5 (0.29)0.0 – 0.8–––Collection #3ILCL0.8 (0.48)0.3 (0.25)0.0 – 0.5–––KSLA1.8 (1.44)0.3 (0.25)0.0 – 0.5–––NYNR0.8 (0.48)1.3 (0.25)0.0 – 1.5–––Collection #4ILCL––––––KSLA0.0 (0.00)0.5 (0.29)0.0 – 0.8–––NYNR––––––Collection #5KSLA0.8 (0.48)0.8 (0.48)0.8 – 1.59.5 (2.99)4.5 (0.65)3.8 – 7.8Note: The experimental design was a randomized complete block with four replications. Means based on n=4.SE = Standard Error* Indicates a statistically significant difference was detected between KK179 and the conventional control (α=0.05) using ANOVA.– Indicates arthropod was not evaluated.1Reference range is the minimum and maximum mean valuesamong the conventional commercial reference varieties.Monsanto Company 12-AL-246U 385 of 407 

### References for Appendix G

Drees, B.M. and M.E. Rice. 1985. The vertical beat sheet: A new device for sampling soybeans insects. Journal of Economic Entomology 78:1507-1510.

Kalu, B.A. and G.W. Fick. 1981. Quantifying morphological development of alfalfa for studies of herbage quality. Crop Science 21: 267-271.

Tesar, M.B. and V.L. Marble. 1988. Alfalfa es tablishment. Pages 303-332 in Alfalfa and Alfalfa Improvement. American Society of Agronomy, Inc., Crop Science Society of America, Inc., Soil Science Society of America, Inc., Madison, Wisconsin

SAS. 2008. SAS/STAT software version 9.2. SAS Institute, Inc., Cary, North Carolina.Monsanto Company 12-AL-246U 386 of 407 

### Appendix H: Materials and Method s from Phenotypic and Agronomic

Assessment of KK179 under Field Condi tions during Seed Production

H.1. Materials

Agronomic and phenotypic char acteristics were assessed for KK179, the conventional control ( C 0 -Syn1) 

### , and seven conventional commercial reference varieties grown under similar agronomic conditions of seed production (Table H-1).

H.2. Characterization of the Materials

The presence or absence of KK179 in th e starting seed of KK179, the conventional control, and three of the conventional co mmercial reference varieties, which were produced in the same production with KK179 a nd the conventional control, was verified by event-specific PCR analyses.

H.3. Field Sites and Plot Design

A seed production trial was established in 2010 at a single locat ion in Canyon County, ID. Seed of KK179, the conventional contro l, seven conventional commercial reference varieties, and one othe r material was planted in a randomi zed complete block design with six replications. Each plot consisted of two 20 foot-long rows spaced 22 inches apart.

H.4. Planting and Field Operations

Planting information, soil description, and croppi ng history of the study area are listed in Table H-2. Prior to planting, th e Principal Investigator at th e site prepared a proper seed bed according to local agronomic practices, including tillage, fertilization and pH adjustment, and pest management. After pl anting, all plots were thinned to a uniform density of 200 plants per plot. During the gr owing season, the field site was scouted for agronomic conditions and pest populations, including pest arthropods, diseases, and weeds. Fertilizer, irrigation, agricultural ch emicals, and other management treatments were applied as necessary. All maintenance operations were performed uniformly across the entire plot area.

H.5. Study Management

The field trial was uniformly managed under the local agronomic practices for seed production. Plots were not harves ted for forage. Bees were introduced into the field in two separate releases in July. Irrigation was restricted following bee release. All plots were swathed September 13, 2010, and seed was harvested on September 17, 2010.

H.6. Phenotypic Observations

KK179 was compared to the conventional cont rol for seedling esta blishment, seedling vigor, lodging, seed weight, seeds per pod, split pods, and seed yield. A description of each phenotypic characteristic assess ed is listed in Table VII-1.Monsanto Company 12-AL-246U 387 of 407 

### H.7. Data Assessment

Experienced scientists familiar with the expe rimental design and evaluation criteria were involved in all components of data collection, summariza tion, and analysis. Study personnel assessed that measurements were ta ken properly, data were consistent with expectations based on experi ence with the crop, and the experiment was carefully monitored. Prior to analysis, the overall dataset was evaluated for evidence of biologically-relevant changes and for possible evidence of an unexpected plant response. Any unexpected observations or issues dur ing the study that would impact the study objectives were noted. Data were then subj ected to statistical analysis as indicated

below.

H.8. Statistical Analysis

An analysis of variance was conducted accord ing to a randomized complete block design using SAS  

### (SAS, 2008) to compare KK179 and the conventional control for seedling establishment, seedling vigor, lodging, seed weight, seed per pod, split pods, and seed yield. The level of statistical signif icance was predetermined to be 5% ( α =0.05). KK179

and the conventional control were not sta tistically compared to the conventional

commercial reference varieties. Minimum a nd maximum mean values were calculated

for each characteristic from the seven conven tional commercial reference varieties.Monsanto Company 12-AL-246U 388 of 407 

### Table H-1. Starting Seed for Phenotypic and Agronomic Assessment during Seed

Production

Material Name Genotype / Phenotype Monsanto Lot Number

KK179 Reduced Lignin; Syn1 population 11265471

C 0 -Syn1 Conventional; Syn1 population 11265474

WL 319HQ Conventional 11265484

DKA50-18 Conventional 11265481

Vernal Conventional 11265487

Dow/Dairyland Hybriforce 400 Conventional 11265485

Ranger Conventional 11265488

Pioneer 54V09 Conventional 11265480

Pioneer 54H11 Conventional 11265478

Note: KK179 was grown under USDA-APHIS Permit 09-308-108rm. The field trial included one additional material that is outside of the scope of objectives of this evaluation of KK179.Monsanto Company 12-AL-246U 389 of 407Table H-2. Field andPlanting InformationPlot area (ft2)Rows (#/plot)Planting datePlanting density1(lb/A)Soil SeriesOM2(%)2009 Crop73.324/20/20102.6Sandy Loam1.5Corn1All plots were thinned to a uniform density of 200 plants per plot after seedlingestablishment and seedling vigor data were collected.2OM =Organic MatterMonsanto Company 12-AL-246U 390 of 407 

### References for Appendix H

SAS. 2008. SAS/STAT software version 9.2. SAS Institute, Inc., Cary, North Carolina.Monsanto Company 12-AL-246U 391 of 407 

### Appendix I: Materials and Methods for Pollen Morphology and Viability

Assessment

I.1. Plant Production

KK179, the conventional control ( C 0 -Syn1) 

### , and four conventional commercial reference varieties were grown under similar agronomic conditions in a gree nhouse (Table I-1). Approximately 1-2 weeks after planting, seed lings of KK179 were sampled and tested using event-specific PCR analyses. Plants not containing KK179 were removed from the trial. The greenhouse trial was arranged in a randomized complete block design with four replications, with 10 plants of each material per replication.

I.2. Flower Collection

A minimum of eight racemes were collected from each entry in each replication. Each set of racemes was placed in a labeled cont ainer and stored on wet ice for transport and storage in the microscope laboratory. A mi nimum of 15 mature fl owers were removed

from the collected racemes of each entry within a replication.

I.3. Pollen Sample Preparation

From the collected flowers, five individual s ub-samples were prepared. Each sub-sample consisted of the pollen from three to five flowers. For each sub-sample, the pollen was

then stained with Alexander’s st ain diluted 1:5 (Alexander, 1980).

I.4. Data Collection

Pollen characteristics were assessed by vi ewing samples under an Olympus BX51 light microscope equipped with an Olympus DP70 di gital color camera. The microscope and camera were connected to a computer runni ng Microsoft Windows 2000 Professional (© 1981-1999, Microsoft Corp.) and installed with associated DP Controller v1.2.1.108 and DP Manager v1.2.1.107 camera software (© 2001-2003, Olympus Optical Co., Ltd.) and Image-Pro Plus v6.2.1.491 imaging software (© 1993-2007, Media Cybernetics, Inc.).

I.4.1. Pollen Viability

When exposed to the staining solution, viable pollen grains stained red to purple due to the presence of living cytoplasmic content. N on-viable pollen grains stained light blue to green or colorless and may have appeared round to collapsed in shape, depending on the degree of hydration. For each pollen sample, the number of viable and non-viable pollen grains was counted from a ra ndom field of view under the microscope. A minimum of 75 pollen grains were counted for each sample. Dense clusters of pollen or pollen grains adhering to flower parts were not counted because they may not have absorbed the staining solution uniformly.Monsanto Company 12-AL-246U 392 of 407 

### I.4.2. Pollen Diameter

Pollen grain diameter was measured along tw o perpendicular axes for 10 representative pollen grains per replication. Mean pollen di ameter for each replicate was calculated

from the 20 total measurements as shown in Table VII-14.

I.4.3. General Pollen Morphology

General pollen morphology of KK179, the conve ntional control, and the conventional

commercial reference varieties was ob served as shown in Figure I-1.

I.5. Statistical Analysis

An analysis of variance was conducted accord ing to a randomized complete block design using SAS  

### (SAS, 2008). The level of statistical significance was predetermined to be 5% ( α =0.05). KK179 was compared to the conven tional control for percent viable pollen and pollen grain diameter. KK179 and the co nventional control were not statistically compared to the conventional commercial re ference varieties. Minimum and maximum

mean values were calculated for each ch aracteristic from the four conventional commercial reference varieties. General po llen morphology was qualitative; therefore, no statistical analysis was c onducted on these observations.Monsanto Company 12-AL-246U 393 of 407 

### Table I-1. Starting Seed for Pollen Morphology and Viability Assessment

Material Genotype / Phenotype Monsanto Lot Number

KK179 Reduced Lignin; Syn1 population 11265471

C 0 -Syn1 Conventional; Syn1 population 11265474

Pioneer 54H11 Conventional 11265478

WL 319HQ Conventional 11265484

DKA50-18 Conventional 11265481

Dow/Dairyland HybriForce 400 Conventional 11265485Monsanto Company 12-AL-246U 394 of 407 

### Figure I-1. General Morphology of Po llen from KK179, the Control, and

Conventional Commerical Reference Varieties under 200X Magnification

The alfalfa pollen samples were stained with Al exander’s stain diluted 1:5. Viable pollen grains stained red to purple, while non-viable polle n grains stained blue to green and may appear round to collapsed depending on the de gree of hydration. No visual differences

were observed between KK179 and the conventi onal control or conventional commercial

reference alfalfa varieties.Monsanto Company 12-AL-246U 395 of 407 

### References for Appendix I

Alexander, M.P. 1980. A versat ile stain for pollen fungi, yeast and bacteria. Stain Technology 55: 13-18.

SAS. 2008. SAS/STAT software version 9.2. SAS Institute, Inc., Cary, North Carolina.Monsanto Company 12-AL-246U 396 of 407 

### Appendix J: Materials and Methods for Flower Morphology Assessment

J.1. Plant Production

Transplants of KK179, the conventional control ( C 0 -Syn1) 

### , and four conventional commercial reference varieties were grown unde r similar agronomic conditions in a field

trial in 2010 in Walworth C ounty, WI (WIDL). Prior to transplantation, seedlings of KK179 were sampled and tested using even t-specific PCR analyses. Plants not containing KK179 were not used in the fiel d trial. The trial was arranged in a randomized complete block design with four re plications. Each plot consisted of seven 3.5-foot rows, with seven plants per row, for a total of 49 plants per plot.

J.2. Flower Collection

Racemes ( i.e. , a type of inflorescence) were collected from a single plot at a time, in plot sequence, at approximately the 10% bloom stage. From each plot, a minimum of 10 racemes were collected from among the i nner 25 plants and were non-systematically selected from the top, middle, or bottom of a minimum of 10 separate plants. Racemes were placed in a single clean container th at was pre-labeled with entry number, entry name, and plot number from which the sa mple originated. Each raceme had approximately 5-40 flowers, with not less th an half of the individual flowers fully opened. The containers were kept on wet i ce from collection until the photographs and

measurements were taken.

J.3. Flower Sample Preparation

Ten racemes were taken out from the labeled plastic container from each plot using a forceps and placed on a white paper for detail ed evaluation. These 10 racemes of each plot were arranged in a group (2 rows × 5 plants) and were photographed, with the plot number, location, date, and a scale bar identified in each photograph.

From each raceme, one individual non-senesced flower was detached from the rachis ( i.e. , main stem of the raceme) with a forceps, for a total of 10 flowers per plot. Each flower was tripped using a forceps by pushing the fused keel petals and then evaluated. A single flower from each plot was then non-systematically selected and photographed.

J.4. Data Collection

KK179 was compared to the conventional contro l for flowers per raceme, standard petal length, keel petal length, calyx tube diamet er, sexual column length, wing petal length, flower color class (USDA-ARS, 1972), gross raceme morphology, and gross flower morphology (Table VII-15). Flower characte ristics were assessed by viewing samples with an Olympus  

### SZ60 stereo-zoom microsc ope equipped with a Nikon  

### D5000 digital

 Olympus is a registered trademark of Olympus Corporation, Olympus America Inc.

 2011 Nikon Corporation.Monsanto Company 12-AL-246U 397 of 407 

### color camera. Descriptions of each characteri stic evaluated are listed in Table VII-1 and are shown in Figure J-1.

J.5. General Flower Morphology

Gross raceme morphology and gross flower morphology were observed from digital images of whole racemes and individual flow ers of KK179, the conventional control, and

the conventional commercial referen ce varieties (Figures J-2 and J-3).

J.6. Statistical Analysis

An analysis of variance was conducted accord ing to a randomized complete block design using SAS (SAS, 2008) to compare KK179 and the conventional control for the characteristics: flowers per raceme, standard petal length, keel peta l length, calyx tube diameter, sexual column length, and wing pe tal length. The level of statistical significance was predetermined to be 5% ( α =0.05). KK179 and the conventional control were not statistically compar ed to the conventional commer cial reference varieties.

Minimum and maximum mean values were ca lculated for each characteristic from the

four conventional commercial reference vari eties. Analysis of variance and the calculation for quantitative descriptive statis tics was not appropria te for flower color class, gross raceme morphology, and gross flow er morphology due to the nominal nature of the responses. For these characteristics, the rating observed most often is reported for

KK179 and the conventional cont rol, and the reference range consists of the minimum and maximum observed ratings from among the conventional commercial reference

varieties.Monsanto Company 12-AL-246U 398 of 407 

### Figure J-1 Diagram of an Untri pped (Unopened) Alfalfa Flower

a -sepals, b -standard petal, c -wing petals, and d -fused keel petals, which conceal the

sexual column.

Intact flowers were tripped to reveal the se xual column and measurements were recorded

for: A - diameter of calyx, B - length of keel petals, C -length of sexual column after release from keel petals (sexual colu mn is not shown in the diagram) and, D - length of standard petal.

The figure was produced by Dr. Larry Teuber of the University of California-Davis.Monsanto Company 12-AL-246U 399 of 407Table J-1. Starting Plants for Flower Morphology AssessmentMaterial NameGenotype / PhenotypeMonsanto Seed Lot NumberMonsanto Transplant Lot Number1KK179Reduced Lignin; Syn1 population1126547111266289C0-Syn1Conventional; Syn1 population1126547411266292Pioneer 54V54 Conventional11265475 11266293Croplan LegenDairy 5.0 Conventional11265476 11266294Dow/Dairyland HybriForce 400 Conventional11265485 11266303Vernal Conventional11265487 11266305Note: The field production source of flowers evaluated also included two additional materials that are outside of the scope ofthe objectives of this evaluation ofKK179.1Transplants were produced in a greenhouse and were analyzed by PCR to verify identity.Monsanto Company 12-AL-246U 400 of 407Figure J-2. General Morphology of Whole Racemes from KK179, the Conventional Control, and the ConventionalCommercial Reference VarietiesNo visual differences were observed betweenKK179 and the conventional control or theconventional commercial reference varieties.KK179 ControlReferenceReference ReferenceReferenceMonsanto Company 12-AL-246U 401 of 407 

### Figure J-3. General Morphology of Tripped Flowers from KK179, the Conventional Control, and the Conven tional Commercial Reference Varieties

No visual differences were observed between KK179 and the conventio nal control or the

conventional commercial reference varieties. 

### KK179 Control

Reference Reference

Reference ReferenceMonsanto Company 12-AL-246U 402 of 407 

### References for Appendix J

SAS. 2008. SAS/STAT software version 9.2. SAS Institute, Inc., Cary, North Carolina.

USDA-ARS. 1972. A system for visually classi fying alfalfa flower color. Agriculture Handbook No. 424. U.S. Department of Agricultu re, Agricultural Research Service, Washington, D.C. http://www.naaic.org/ Resources/colorguide/flowercolor.html [Accessed April 4, 2012].Monsanto Company 12-AL-246U 403 of 407 

### Appendix K: Materials and Methods for Symbiont Assessment

K.1. Background

Members of the bacterial families Rhizobiaceae and Bradyrhizobiaceae form a highly complex and specific symbiotic relationship with leguminous pl ants and fix a substantial portion of the world’s supply of organic n itrogen (Gage, 2004). The nitrogen-fixing plant-microbe symbiosis results in the fo rmation of root nodules, which provide an

environment in which differentiated bacteria called bacteroids are capable of reducing or “fixing” atmospheric nitrogen. The product of nitrogen fixation, ammonia, can then be utilized by the plant. In alfalfa, atmosphe ric nitrogen is fixed into organic nitrogen through a symbiotic association with the bacterium Sinorhizobium meliloti which can contribute up to 64% of alfalfa’s nitr ogen requirement (Lanyon and Griffith, 1988).

The relative effectiveness of the symbiotic relationship between a leguminous plant and its rhizobial symbiont can be assessed by various methods. Measurement of nodule dry weight along with plant growth and nitr ogen status are commonl y used to assess differences in the symbiotic relationship between a legume and its associated rhizobia (Israel et al., 1986).

K.2. Materials

KK179, the conventional control ( C 0 -Syn1) 

### , and six conventional commercial reference varieties were used in a sy mbiont interaction assessment (Table K-1). Nodules, root tissue, and shoot tissue collected from KK179, the conventional control, and the

conventional commercial referen ce varieties were evaluated.

K.3. Characterization of the Materials

The presence or absence of KK179 in the starting seed of KK179 and the conventional control was verified by PCR analyses. Appr oximately 1-2 weeks after planting, seedlings of KK179 were sampled and tested using ev ent-specific PCR analyses. Plants not containing KK179 were removed from the trial.

K.4. Greenhouse Phase and Experimental Design

Starting seed of KK179, the conventional control, and six conventional commercial reference varieties were planted in 6-inch pots containing a custom blend of nitrogen deficient, peat-based potting medium (1⁄2 ba g each of Premier Horticulture Promix PGX and Promix BX, 1⁄4 cup lime and 2 cups sand) . Plants were grow n in a greenhouse where temperatures ranged from approximately 20 °C to 35 °C. Prior to planting, pots were

saturated with water and allowed to drain fo r a minimum of four hours. Following this, approximately 500 ml of nitrogen-free nutri ent solution was added to each pot. Ten replicate pots were planted with 10 seeds per pot for each of KK179, the conventional control, and the six conventional commercial re ference varieties. At planting, each seed was inoculated with a dry, clay-based inoculant containing a minimum of 7 × 10 7 

### cells of

S. meliloti (Dormal, Becker Underwood, Ames, IA). Pots were arranged in ten replicatedMonsanto Company 12-AL-246U 404 of 407 

### blocks containing one pot each of KK179, the conventional control, and the six

conventional commercial reference varieties using a randomized complete block design.

Replicates 1, 2, 3, and 4 were planted on September 6, 2011; replicates 5, 6, and 7 were planted on September 7, 2011; and replicates 8, 9 and 10 were planted September 8, 2011. A nitrogen-free nutrient solution (targe t of 250 ml) was added weekly after plant emergence. In addition, the plant developmental growth stage was evaluated and documented once per week.

Plants from all replicates were thinned to three seedlings per pot on September 27, 2011. Replicates 1, 2, 3, and 4 plants were harv ested on October 25, 2011; replicates 5, 6, and 7 were harvested on October 26, 2011; and replicates 8, 9, and 10 were harvested on October 27, 2011. Ten pots containing three plants each were harvested for each of KK179, the conventional control, and the six conventional co mmercial reference varieties with the following exceptions : four pots had an extra plan t emerge after thinning, which was not noticed until harvest, and one pot contai ned plants that were dead at harvest time. These pots were excluded from the statistical analysis.

K.5. Plant Harvesting/Data Collection

Six weeks after emergence, plants were exci sed at the surface of the potting medium and shoot and root plus nodule mate rial were removed from the pot s. The shoot material was cut into smaller pieces and placed in labeled bags. The plant roots with nodules were separated from the potting medium by washi ng with water. Excess moisture was removed using absorbent paper towels, and the roots plus no dules were placed in labeled bags. The same day that plants were harv ested, nodules were removed by hand from the roots of each plant and weighed to determin e the fresh weight (fwt) of the nodules.

Nodules were not enumerated as the alfalfa nodules were bran ched and clustered, making it impossible to record an accurate count. This was due to the fact that alfalfa nodules are indeterminate and continually form new nodule tissue (Gage, 2004). The remaining root and shoot fresh weight was recorded for each pl ant. Nodules as well as root and shoot material were placed in a drying oven on the sa me day as collected. The plant material was dried for at least 72 hours at approximately 65 °C to determine dry weight (dwt).

After drying, the shoot tissue was ground with a Harbil 5G high-speed paint shaker and stored at -20 °C prior to shipping to Agvise Laboratories for total nitrogen analysis. Shoot percent total nitrogen was determined by combustion using a nitrogen analyzer according to a study-specific work procedure. Shoot total nitrogen (g) was calculated using the shoot dwt and shoot percent of total nitrogen valu es. Any remaining seeds and plants were devitalized pe r USDA-APHIS requirements.Monsanto Company 12-AL-246U 405 of 407 

### K.6. Statistical Analysis

The data analyzed consisted of five measurem ents taken at the six- week sampling period: nodule dwt (g), shoot dwt (g), root dwt (g), shoot percent total nitrogen, and shoot total nitrogen (g) (Tab le VII-16).

An analysis of variance was conducted usi ng a randomized complete block design with ten replications for each of KK179, the conventional contro l, and the six conventional

commercial reference varieties. Data were analyzed using SAS (SAS, 2008) with the level of statistical significan ce predetermined to be 5% ( α =0.05). The means of KK179 and the conventional control were compared to each other. Minimum and maximum values (reference range) were determined fo r the six conventional commercial reference

varieties. No statistical comparisons were made between KK179 and the conventional

commercial reference varieties.Monsanto Company 12-AL-246U 406 of 407 

### Table K-1. Starting Seed of KK179, Co nventional Control, and Conventional Commercial Reference Varieties Used in the Symbiont Assessment

Starting Seed Name Genotype / Phenotype Monsanto Lot Number

KK179 Reduced Lignin; Syn1 population 11265471

C 0 -Syn1 Conventional; Syn1 population 11265474

DKA50-18 Conventional 11265481

FG78T803 Conventional 11293630

FG98M817 Conventional 11293629

Ranger Conventional 11265488

Vernal Conventional 11265487

WL 319HQ Conventional 11265484Monsanto Company 12-AL-246U 407 of 407 

### References for Appendix K

Gage, D.J. 2004. Infection and invasion of roots by symbiotic, nitrogen-fixing rhizobia during nodulation of temperate legumes. Microbiology and Molecular Biology Reviews

68: 280-300.

Israel, D.W., J.N. Mathis, W.M. Barbour and G.H. Elkan. 1986. Symbiotic effectiveness

and host-strain interactions of Rhizobium fredii USDA 191 on different soybean cultivars. Applied and Environmental Microbiology 51: 898-903.

Lanyon, L.E. and W.K. Griffith. 1988. Nutriti on and fertilizer use. Pages 333-372 in Alfalfa and Alfalfa Improvement. A.A. Hanso n, D.K. Barnes, and R.R. Hill, Jr. (eds.). American Society of Agronomy, Inc., Crop Sc ience Society of America, Inc., Soil Science Society of America, Inc., Madison, Wisconsin.

SAS. 2008. SAS/STAT software version 9.2. SAS Institute, Inc., Cary, North Carolina.