Monsanto Company 12-AL-246U 1 of 407 

Petition for the Determination of Nonregulated 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., 1M. L. Breeze, M.S., 1M. S. Giovannini, M.S., 1M. D. Hall, Ph.D., 

1

T. H. Klusmeyer, Ph.D., 1S. Paul, M.S., 1K. A. Robinson, Ph.D., 1R. W. Schneider, B.S.,

1

Y. Wang, Ph.D. 

Contributors and/or Principal Investigators:

1

L. D. Barberis, B.S., 1E. L. Clawson, Ph.D., 1S. R. Eskelsen, Ph.D., 

1

S. N. Fitzpatrick, M.S., 1R. L. Girault, M.S., 1J. R. Groat, M.S., 1E. K. Hill, B.S., 

1

S. W. Hoi, M.S., 1K. M. Huizinga, Ph.D., 1K. R. Lawry, M.S., 1T. P. Perez, M.S., 

1

S. L. Phillips, M.S., 2M. S. S. Reddy, Ph.D., 1S. Shah, M.S., 1A. Skipwith, M.S., 

1

Z. Song, M.S., 2S. J. Temple, Ph.D., 1Q. Tian, M.D., Ph.D., 1J. M. Ward, Ph.D., 

2

D. J. Whalen, B.S., 1J. E. Whitsel, M.S., 1T. 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 understand 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 Company and Forage Genetics International 

expect that, in advance of the release of the document(s), USDA will provide Monsanto 

Company and Forage Genetics International with a copy of the 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 public 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 International do not authorize the release, 

publication or other distribution of this information without Monsanto Company's and 

Forage Genetics International’s prior notice and consent. 

© 2013 Monsanto Company and Forage Genetics International. 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 support of actions requested by Monsanto Company and Forage 

Genetics International. Any other use, copying, or transmission, including internet 

posting, of this document and the materials described in or accompanying this document, 

without prior consent of Monsanto Company and Forage Genetics International, is 

strictly prohibited; except that Monsanto Company and Forage Genetics International 

hereby grant such consent to the regulatory authority where required under applicable law 

or regulation. The intellectual property, information and materials described in or 

accompanying this document are owned by Monsanto Company and Forage Genetics 

International, which have filed for or been granted patents on those materials. By 

submitting this document and any accompanying materials, Monsanto and Forage 

Genetics International do not grant any party or entity any right or license to the 

information, material or intellectual property described or contained 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 30f407 
Monsanto Company 12-AL-246U 4 of 407 

EXECUTIVE SUMMARY 

The Animal and Plant Health Inspection Service (APHIS) of the United States (U.S.) 

Department of Agriculture (USDA) has responsibility 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 pests 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 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 FGI) are submitting this request to USDA-APHIS 

for a determination of nonregulated status for the new biotechnology-derived alfalfa 

product, KK179, any progeny derived from crosses between KK179 and conventional 

alfalfa, and any progeny derived from crosses of KK179 with biotechnology-derived 

alfalfa that have previously been granted nonregulated status 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 alfalfa. 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 growers 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 inverted repeat sequence 

produces double-stranded RNA (dsRNA) which suppresses endogenous CCOMT gene 

expression via the RNA interference (RNAi) pathway. Suppression of the CCOMT gene 

expression leads to lower CCOMT protein expression resulting in reduced synthesis of G 

lignin subunit compared to conventional alfalfa 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). 

Forage of KK179 and the conventional control were analyzed for monomeric lignin 

subunits, the building blocks for lignin molecules. 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 substantially affecting the levels of the other 

major lignin subunit, syringyl lignin (S lignin), or minor lignin subunits, as predicted by 

the mode-of-action. The result is a lower proportion of G lignin and a greater proportion 
Monsanto 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 approximately 22% and verifies that the reduction 

in the G lignin leads to a concurrent reduction in total lignin in KK179 forage compared 

to the conventional control harvested 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 and 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. Growers, therefore, must decide whether to 

harvest forage to obtain higher quality forage or higher yield (tonnage). 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 significant lignin accumulation 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 flexibility 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 meet or exceed the intended quality standard 

targeted by the grower; or 2) delay a harvest several days and obtain higher tonnage 

without sacrificing forage quality. At the same time, independently of the production 

decision on when to harvest, a grower will benefit from flexibility to accommodate 

unexpected delays in harvesting forage caused by adverse weather conditions, equipment 

failure, or competing farming activities. 

Data and Information Presented Confirm the Lack of Plant Pest Potential and the 

Feed and Food Safety of KK179 Compared to Conventional Alfalfa

The data and information presented in this petition demonstrate that KK179 is 

agronomically, phenotypically, and compositionally comparable to conventional alfalfa, 

with the exception of the introduced trait for reduced G lignin and total lignin. Moreover, 

the data and information presented demonstrate that KK179 is not expected to pose an 

increased plant pest risk, including 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 basis 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 by 
Monsanto Company 12-AL-246U 6 of 407 

eukaryotes, including plants, for endogenous gene suppression and pose no novel 

risks from a feed/food and environment perspective. 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 assessment supports the conclusion that KK179 

forage is compositionally equivalent, with 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 phenotypic 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 agriculture indicates that KK179 is not expected to have an 

effect on other organisms compared to conventional alfalfa under normal 

agricultural practices. 

 Evaluation of KK179 using current cultivation and management 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 agricultural 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 behaves as a weed, other than as a 

volunteer in agricultural settings. 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 species 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 LLC 
Monsanto Company 12-AL-246U 7 of 407 

Conventional Alfalfa is an Appropriate Comparator to KK179

A near-isogenic conventional alfalfa comparator was developed using an identical 

breeding path used for the development of KK179. The same breeding process used to 

develop subsequent generations of KK179 was then followed to develop subsequent 

generations of near-isogenic conventional alfalfa comparators for each generation of 

KK179. All generations of KK179 and corresponding generations of conventional alfalfa 

comparators used in safety assessments have closely related genetic backgrounds with the 

exception of the introduced trait for reduced G lignin and total lignin absent in the 

conventional comparator. Conventional commercial alfalfa varieties (referred to as 

conventional commercial reference varieties), were used to establish ranges of natural 

variability or responses representative 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 plasmid vector PV-MSPQ12633. The transformed 

plant was crossed with Ms208, an elite, male sterile, conventional alfalfa plant, to 

produce KK179. The PV-MSPQ12633 plasmid contains two separate T-DNAs, each 

delineated by Left and Right Border sequences to facilitate transformation. 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 isolate 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 characterization of KK179 by Southern blot analyses confirmed that one copy 

of the CCOMT suppression cassette (T-DNA I) was integrated into the alfalfa genome at 

a single locus. No T-DNA II or backbone DNA sequences from plasmid vector 

PV-MSPQ12633 were detected in KK179. The complete DNA sequence of the insert 

and adjacent genomic DNA sequence in KK179 confirmed the integrity of the inserted 

CCOMT suppression cassette within the inserted 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 was 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 decrease 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 function 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. Several 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, 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 translation initiation and protein 

synthesis, thus synthesis of the CCOMT protein or a putative polypeptide is highly 

unlikely. Based on this information, it is concluded 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-nutrients and secondary metabolites in KK179 

forage to levels in the conventional alfalfa control. Nutrients 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 (total bayogenin, total hederagenin, total 

medicagenic acid, total soyasapogenol B, total soyasapogenol E, total zanhic acid and 

total saponins). Secondary metabolites analyzed related to lignin biosynthesis and 

phenylpropanoid metabolism in alfalfa included p-coumaric acid, ferulic acid, sinapic 

acid, total polyphenols, free phenylalanine, and canavanine. 

Compositional analyses on forage samples were conducted to determine statistically 

significant differences (α = 0.05) between KK179 and the conventional control. 

Statistical results from combined-site data were evaluated using considerations relevant 

to the safety and nutritional quality of KK179 when compared to the conventional 

control. Considerations to assess the relevance of each statistically significant difference 

included: 1) the relative magnitude of the differences in the mean values of nutrient, antinutrient, and secondary metabolite components between KK179 and the conventional 

control; 2) whether the KK179 component mean values were within the range of 

variability of the components as represented by the 99% tolerance interval of the 
Monsanto Company 12-AL-246U 9 of 407 

conventional commercial reference varieties grown concurrently in the same field trial; 

and 3) an assessment of the differences within the context of natural variability of 

available commercial alfalfa composition published in the scientific literature. 

Analysis of the observed significant differences in nutrient, anti-nutrient, and secondary 

metabolite components with respect to magnitude 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 food safety and nutritional quality of this product 

are comparable to those of conventional alfalfa, which has a history of safe use and 

consumption. When KK179 is grown on a commercial scale and used as a source of 

feed, alfalfa products derived from KK179 are not expected to be compositionally 

different from the equivalent feeds originating from conventional alfalfa. 

KK179 Does Not Change Alfalfa Plant Pest Potential or Environmental Interactions

Plant pest potential of a biotechnology-derived 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 conventional plant whose biological properties and 

plant pest potential are well known. Familiarity considers the biology of the plant, the 

introduced trait, the receiving environment, and the interactions among these factors. 

This provides a basis for comparative risk assessment between a biotechnology-derived 

plant and the conventional control. Thus, the phenotypic, agronomic, and environmental 

interaction assessment of KK179 included the near-isogenic conventional control as a 

comparator. This evaluation used a weight of evidence approach and considered 

statistical differences between KK179 and the conventional control with respect to 

reproducibility, magnitude, and directionality. Characteristics assessed 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 field. 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 characterization indicated that KK179 seed had no 

changes relative to the conventional control in dormancy or germination characteristics 

that could be indicative of increased plant weediness or pest potential of KK179 

compared to the conventional control. Some statistically significant differences were 

detected in which KK179 had higher percent germinated seed and lower percent hard 

seed. These differences, however, were not considered 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 pollen or pollen grain diameter. Furthermore, no visual 
Monsanto Company 12-AL-246U 10 of 407 

differences in general pollen morphology were observed between KK179 and the 

conventional control, demonstrating that the introduced trait for reduced G lignin and 

total lignin did not alter the overall morphology or viability of pollen of KK179 

compared to that of the conventional control. Similarly, no statistically significant 

differences were detected between KK179 and the conventional control for morphology 

of flowers. 

The field evaluation of phenotypic, agronomic, and environmental characteristics of 

KK179 also supports the conclusion that KK179 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 conditions representative of commercial alfalfa 

production areas in North America. Trials were managed according to standard, local 

agronomic practices for forage production in order 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. In addition to phenotypic and agronomic 

characteristics, observations were also made for plant responses to abiotic stressors, 

diseases, and arthropod interactions. These studies were conducted over two complete 

growing seasons from 2010 to 2012. 

The phenotypic characteristics observed during forage production from 2010 to 2012 

across 10 field sites were comparable between KK179 and the conventional control. In a 

combined-site analysis of the phenotypic characteristics in the first growing season 

(2010-2011), data showed no statistically significant differences between KK179 and the 

conventional control for seedling emergence and early season vigor during stand 

establishment; no statistically 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 analysis of the phenotypic characteristics in 

the second growing season (2011-2012), data showed no statistically significant 

differences between KK179 and the conventional 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 combined-site and combined-year analysis, there 

were also no statistically significant differences for fall plant height, total forage yield, 

spring vigor, spring stand recovery, and spring stand count. 

The phenotypic characteristics observed during seed production at one field site in 2010 

were also comparable between KK179 and the conventional control. Data showed no 

statistically significant differences between 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 abiotic stressors, disease damage and arthropod 

damage. In the first year, no differences were observed between KK179 and the 
Monsanto Company 12-AL-246U 11 of 407 

conventional control for any of the 93 comparisons 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 quantitatively within each growing season over 

two years and included assessments of alfalfa weevil damage and potato leafhopper 

damage and pest- and beneficial-arthropod abundance. For alfalfa weevil or potato 

leafhopper damage, no statistically significant differences were detected in combined site 

analyses for either insect in 2010 and in 2011. For arthropod abundance, four differences 

out of 69 comparisons were detected at individual sites in the first year and one out of 89 

comparisons in the second year. At the sites where statistical differences were observed, 

the mean abundance values for pests and arthropods from KK179 were within the range 

of the conventional commercial reference varieties and/or the differences were not 

consistently detected across collection times or sites. Taken together, these data support 

the conclusion that compared to conventional alfalfa KK179 is no more susceptible to 

damage by alfalfa weevil or potato leafhopper and no more likely to promote increased 

abundance of these species. 

In summary, phenotypic, agronomic, and environmental interaction data were evaluated 

to characterize KK179 and to assess whether the introduced trait for reduced G lignin and 

total lignin in KK179 alters the plant pest potential compared to conventional alfalfa. 

The evaluation, using a weight of evidence approach, considered the reproducibility, 

magnitude, and direction of detected differences between KK179 and the conventional 

control, and comparison to the range of the conventional commercial reference varieties. 

Results from the phenotypic, agronomic, and environmental interactions assessment 

indicated that KK179 does not possess more or enhanced weediness characteristics, 

increased susceptibility or tolerance to specific abiotic stress, diseases, or arthropods, or 

characteristics that would confer a plant pest risk compared to conventional alfalfa. 

KK179 Will Not Have Effects On NTOs Including Those Beneficial To Agriculture

Evaluation of the impacts of KK179 on non-target organisms (NTOs) is a component of 

the plant pest risk assessment. Since KK179 does not possess pesticidal activity, all 

organisms that interact with KK179 are considered to be NTOs. The environmental 

interactions assessment conducted over two growing seasons under field conditions 

demonstrated that the introduction of the CCOMT suppression cassette for reduced G 

lignin and total lignin in KK179 does not unexpectedly alter plant-arthropod interactions, 

including beneficial arthropods, soil symbiont interactions, or alter disease susceptibility 

compared to the conventional 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 evaluation also considered product characterization 

information including molecular characterization and composition, and the history of 

environmental exposure to lignin. Taken together, 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 reproductive characteristics indicated that KK179 is not expected 

to outcross with sexually compatible species compared with conventional alfalfa. 

Additionally, under forage production conditions, 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 shown the introduced trait for reduced G lignin 

and total lignin in KK179 does not enhance weediness or plant-pest potential relative to 

conventional alfalfa. Therefore, the environmental consequence of pollen transfer 

between KK179 and feral alfalfa or a related species is considered negligible. 

Outcrossing to feral alfalfa populations and other species has been comprehensively 

reviewed by USDA-APHIS for Roundup Ready Alfalfa in its December 2010 Plant Pest 

Risk Assessment and Determination and associated Environmental Impact Statement. 

USDA-APHIS has concluded that outcrossing to feral 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 agronomic practices was conducted to determine 

whether the cultivation of KK179 has the potential to impact current alfalfa management 

practices. Alfalfa fields are typically highly managed agricultural 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 forage 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 introduction of KK179 and germplasm developers 

and seed producers will continue to use well-established industry practices 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 alfalfa or deregulated 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 Determination of Nonregulated Status under 

7 CFR § 340.6 ...............................................................................................................28

I.B. Rationale for the Development 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 Foreign 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 THE 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 .................................................................................64
Monsanto Company 12-AL-246U 14 of 407 

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

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

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

IV.C. Southern Blot Analysis to Determine 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 the 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 SAFETY 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 KK179 Expressed Products Summary and 

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

VI. COMPOSITIONAL ASSESSMENT OF KK179 ..................................................91

VI.A. Compositional Equivalence of KK179 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, AGRONOMIC, AND ENVIRONMENTAL 

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

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

VII.B. Interpretation of Phenotypic and Environmental Interactions Data ................114

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

VII.B.2. Interpretation of Environmental 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, Agronomic, and Environmental Interaction 

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

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

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

VII.D. Conclusions for Phenotypic, 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 Production 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 Characteristics 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 ........206
Monsanto Company 12-AL-246U 16 of 407 

IX.D.4. Transfer of Genetic Information 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 CONSEQUENCES 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 and Methods Used for Northern Blot Analyses of 

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

Appendix D: Materials, Methods, and Results for Intended Changes to 

Lignin Levels in KK179 Forage ....................................................................................247

Appendix E: Materials, Methods, and Results for Compositional Analysis of 

KK179 Forage ................................................................................................................263

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 Conditions 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 Methods for Symbiont Assessment..............................403
Monsanto 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 Genetic Elements in PV-MSPQ12633 ................................54

Table IV-1. Summary Chart of the Expected DNA Segments Based on 

Hybridizing Probes and Restriction Enzymes Used in KK179 Analysis ....................61

Table IV-2. Summary of Genetic 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 Alfalfa Forage Nutrients for KK179 vs. 

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

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

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

Table VI-4. Statistical Summary of Alfalfa 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 American Field Trials and Laboratory or 

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

Table VII-2. Comparison of Germination Characteristics of Non-scarified 

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

Table VII-3. Comparison of Germination Characteristics of Scarified Seed 

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

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

Table VII-5. Combined-Site Comparison 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 Comparison of KK179 to the Conventional 

Control for Phenotypic and Agronomic Characteristics in the Second Year 

(2011-2012) of Forage Production ................................................................................128
Monsanto Company 12-AL-246U 18 of 407 

Table VII-7. Combined-Site and Combined-Year Comparison of KK179 to 

the Conventional Control for Phenotypic and Agronomic Characteristics 

during Forage Production .............................................................................................130

Table VII-8. Summary of Qualitative Environmental Interactions 

Assessments in the First Year (2010) ...........................................................................133

Table VII-9. Summary of Qualitative 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) ............................................................................................................137

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 Characteristics during Seed Production ......................141

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

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

Table VII-15. Flower Characteristics 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 Production in the U.S. from 2000 to 20101 .....................152

Table VIII-2. Alfalfa and Alfalfa Mixtures Forage (Hay and Haylage) 

Production by State in 20101

. ........................................................................................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.S1. ..................................181

Table VIII-7. Alfalfa Tolerance and Herbicide Effectiveness Ratings on 

Direct Seedlings of Alfalfa1 ...........................................................................................184

Table VIII-8. Alfalfa Tolerance and Postemergence Herbicide Effectiveness 

Ratings on Common Weeds in Established Alfalfa Stands1. .....................................185

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

Volunteer Alfalfa1

. .........................................................................................................188

Table IX-1. Potential Scenarios for Pollen-mediated Gene Flow in Alfalfa 

(adapted from Van Deynze et al., 2008) .......................................................................202
Monsanto 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 Alfalfa Forage Lignin Subunits for KK179 

vs. Conventional Control (Conducted at Samuel Roberts Noble Foundation) ........251

Table D-4. Statistical Summary of Alfalfa Forage Total Lignin (ADL) Levels 

for KK179 vs. Conventional Control (Conducted 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 Site 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. Conventional 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 Site IARL Alfalfa Forage Secondary 

Metabolites for KK179 vs. Conventional 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. Conventional 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 Site KSLA Alfalfa Forage Secondary 

Metabolites for KK179 vs. Conventional 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 Site TXCL Alfalfa Forage Secondary 

Metabolites for KK179 vs. Conventional Control (Conducted at Covance 

Laboratories) ..................................................................................................................308
Monsanto 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 Site WIDL Alfalfa Forage Secondary 

Metabolites for KK179 vs. Conventional Control (Conducted at Covance 

Laboratories) ..................................................................................................................315

Table E -15. Statistical Summary of Alfalfa Forage Anti-Nutrients for 

KK179 vs. Conventional Control (Conducted 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 Phenotypic, Agronomic, and Environmental 

Interaction Assessment during Forage Production ....................................................334

Table G-2. Field and Planting Information ................................................................336

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

Table G-4. Definition of Morphological 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 Phenotypic Comparison of KK179 to the 

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

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

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

Table G-8. Combined-Site Qualitative Assessment: Abiotic Stressor 

Evaluations Using an Observational Severity Scale for KK179 and the 

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

Table G-9. Combined-Site Qualitative Assessment: Disease Damage 

Evaluations Using an Observational Severity Scale for KK179 and the 

Conventional Control in the First Year (2010) ...........................................................367

Table G-10. Combined-Site Qualitative Assessment: Insect Stressor 

Evaluations Using an Observational 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 Arthropods in Samples Collected from 

KK179, the Conventional Control, and the Conventional Commercial 

Reference Varieties in the First Year (2010) ...............................................................373
Monsanto Company 12-AL-246U 21 of 407 

Table G-14. Combined-Site Qualitative Assessment: Abiotic Stressor 

Evaluations Using an Observational Severity Scale for KK179 and the 

Conventional Control in the Second Year (2011) .......................................................375

Table G-15. Combined-Site Qualitative Assessment: Disease Damage 

Evaluations Using an Observational Severity Scale for KK179 and the 

Conventional Control in the Second Year (2011) .......................................................376

Table G-16. Combined-Site Qualitative Assessment: Insect Stressor 

Evaluations Using an Observational 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 Arthropods 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 Phenotypic and Agronomic Assessment during 

Seed Production .............................................................................................................388

Table H-2. Field and Planting 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 KK179, Conventional Control, and 

Conventional Commercial Reference Varieties Used in the Symbiont 

Assessment ......................................................................................................................406
Monsanto 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 Analysis 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 Generating 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 alfalfa variety breeding schematic ...............164
Monsanto 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 Untripped (Unopened) Alfalfa Flower ............................398

Figure J-2. General Morphology of Whole Racemes from KK179, the 

Conventional Control, and the Conventional Commercial Reference 

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

Figure J-3. General Morphology of Tripped Flowers from KK179, the 

Conventional Control, and the Conventional Commercial Reference 

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

ABBREVIATIONS AND DEFINITIONS1 

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 Tn7 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 Official 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 

C0 The single progeny plant selected from the cross of alfalfa 

plants R2336 and Ms208 to develop the near-isogenic 

conventional control 

C0-Syn1 The near-isogenic conventional control for KK179 

generation Syn1 

C0-Syn1 Adv The near-isogenic conventional 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 Tn5 of 

E. coli encoding neomycin phosphotransferase 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:shikimate: hydroxycinnamoyltransferase 

HGS Combined H lignin, G lignin, and S lignin 

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

Kb Kilobase 

kg Kilogram 

LOQ Limit of quantitation 

m Meter 

MBC Modified backcross 

Ms208 FGI proprietary conventional 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

P0 The single progeny plant selected from the cross of T0 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 database, 187.0 (Released January 30, 

2012) 

PV-MSPQ12633 Plasmid used to transform the alfalfa genome to produce 

KK179 

R2336 FGI proprietary conventional plant selected for ease of 
Monsanto 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 subunit divided by guaiacyl lignin subunit 

T-DNA Transfer DNA 

Syn1 First generation KK179 synthetic population 

Syn1 Adv Second generation KK179 synthetic population 

Tm Melting temperature 

T0 The transformed R2336 plant 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 stated 
Monsanto Company 12-AL-246U 28 of 407 

I. RATIONALE FOR THE DEVELOPMENT OF KK179 

I.A. Basis for the Request for a Determination of Nonregulated Status under 

7 CFR § 340.6 

The Animal and Plant Health Inspection Service (APHIS) of the United States (U.S.) 

Department of Agriculture (USDA) has responsibility, 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 pests 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 FGI) are submitting this request to USDA-APHIS 

for a determination of nonregulated status for the new biotechnology-derived alfalfa 

product, KK179, any progeny derived from crosses between KK179 and conventional 

alfalfa, and any progeny derived from crosses of KK179 with biotechnology-derived 

alfalfa that have previously been granted nonregulated status under 7 CFR Part 340. 

I.B. Rationale for the Development 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 between approximately $6.7 and $10.7 billion USD 

(USDA-NASS, 2011b) Alfalfa is the fourth largest 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 approximately 25 percent with grasses or another 

companion crop (USDA-APHIS, 2010). 

Alfalfa forage products are valued for their high protein content and highly digestible 

fiber for ruminants and horses (USDA-APHIS, 2010). The principal commercial product 

is hay, which is forage that has been dried and baled, or cubed 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 (Putnam et al., 2008b; USDA-APHIS, 2010). Other 

quality parameters evaluated include observations for the presence of weeds or molds, or 

anti-palatability factors such as poor texture, 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 regularly report average prices for alfalfa hay 

in major producing areas (USDA-AMS, 2010). Supreme 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 maturity, with heavy damage, and with minimal 
Monsanto 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, regrowth of stems and leaves initiates within a 

short time and the process of lignin accumulation 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 livestock. Therefore, forage producers and 

commodity purchasers desire alfalfa with lower lignin levels but without loss of 

nutritional components, including protein and fiber. 

Alfalfa is grown as a perennial forage crop that is repeatedly harvested throughout the 

growing season. After each harvest, alfalfa plants go through a period of recovery 

followed by regrowth. Harvesting of alfalfa forage from an established stand can take 

place from two to eleven times in a growing season depending on the climate and region. 

Deciding when to harvest forage is a critical decision made by the grower that determines 

both forage yield and quality, and the ultimate profitability and utility 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, alfalfa harvested at an 

immature or early maturity growth stage produces the highest level of quality in forage 

due to lower lignin levels but at a lower yield (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 forage, therefore, is a difficult management 

decision involving market considerations, agronomic factors (Orloff and Putnam, 2008), 

and weather. 

Monsanto and FGI have developed biotechnology-derived alfalfa, KK179, which has 

reduced levels of guaiacyl (G) lignin subunits (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 several days earlier under similar production 

conditions. As a result, growers will have an expanded harvest interval and experience 

the benefit of greater flexibility to dynamically manage harvest 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 remain the same as with conventional 

varieties. KK179 harvested at a typical crop cutting stage will produce alfalfa forage 
Monsanto Company 12-AL-246U 30 of 407 

with lower levels of lignin compared to conventional alfalfa harvested 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. The forage yield will be maintained at the 

same levels as with conventional 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 conventional alfalfa harvested at the same 

stage. 

 Maximizing forage yield: When aiming to maximize dry matter yield of KK179, a 

grower can delay harvest for several days to accumulate more forage biomass 

without significantly forfeiting quality. During the reproductive growth stage, 

alfalfa dry matter can increase at the 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 yield. 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 would provide a comparable yield, but the 

forage would have higher lignin content and, thus, lower quality. From a forageproduction perspective, the maximum potential yield of alfalfa attainable is not 

raised; rather, growers can more readily reach the higher end of the potential yield 

range while maintaining a targeted quality standard. 

 Tolerating unexpected harvest 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 delay period, forage quality often declines 

rapidly, leading to potential financial loss. A grower has more flexibility to 

withstand short delays in forage harvest 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 biosynthesis in alfalfa has provided the means to reduce 

lignin levels and slow the accumulation of lignin during the alfalfa growth cycle. Lignin 

is a high molecular weight, polymeric molecule composed principally 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 comprise up to 95% of the lignin subunits. In the lignin 

biosynthetic pathway, the formation of the G and 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 phenylpropanoids, 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-hydroxyconiferyl aldehyde to produce sinapyl aldehyde 
Monsanto 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: caffeoylCOMT: 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 lignin subunits to form complex lignin molecules 

(Boerjan et al., 2003). This specific reduction in G lignin 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 synthesis 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 vascularisation and displays a pattern of 

expression that corresponds with sites of lignin deposition in maturing plants (Guo et al., 

2001; Leyva et al., 1992). Thus, KK179 transgene expression correlates with tissues 

where higher lignin deposition is observed. The assembled CCOMT gene segments 

produce a transcript with an inverted repeat sequence to form double-stranded RNA 

(dsRNA), which works via the RNA interference 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 sequence 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 alternative 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 relative to all subunits. The 

decrease in G lignin also results in an increase 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 increase in the S:G lignin ratio, which is characteristic of 

CCOMT suppression (Chen 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 biosynthetic 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 alfalfa. 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 hybridized 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 probe 

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 Appendix 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 samples of forage tissue from the conventional 

control produced a strong hybridization 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 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 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 hybridization signal at ~1.5 kb, as expected for the actin

transcript. The hybridization signals from the forage tissue of the conventional control 

and KK179 of each replicate have similar intensities, indicating that the RNA loading, 

RNA quality, and hybridization within each replicate of the conventional control and 

KK179 were similar. When hybridized with the actin probe template, in addition to the 

expected ~0.5 kb band, a very faint ~1.0 kb band was detected (Figure 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 (Qiagen, 2008) that has 

partially reannealed in various confirmations (Kasuga et al., 2001). Since the actin probe 

template loaded in this lane served as a positive hybridization control 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 analysis. Therefore, the difference in the 

CCOMT hybridization signals between the conventional 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 samples of root tissue from the conventional 

control produced a strong hybridization 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 was 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 the 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 band detected (Figure I-4, 

Panel B, Lane 11) when hybridized with the actin probe template was not considered to 

have an impact on the conclusions drawn from this analysis. Therefore, the difference in 

the CCOMT hybridization signal intensities between the conventional control and KK179 
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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 were observed (Figure I-4, Panel B, Lanes 1, 

3, 5, and 7). These faint bands likely resulted from the incomplete removal of the 

CCOMT probe on the stripped blot prior to probing with the actin probe. The expected 

~1.5 kb actin transcript is larger 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

*
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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 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. 

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

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

As described in Section I.B.2.1, KK179 reduces G lignin levels through the suppression 

of caffeoyl CoA 3-O-methyltransferase (CCOMT), a key enzyme in the lignin 

biosynthetic pathway. Suppression of CCOMT acts to decrease 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 change in lignin subunit proportions 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 alfalfa (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 subunit compositional analyses were conducted. 

Forage samples were collected from KK179, a conventional alfalfa control, and 

conventional commercial alfalfa varieties grown in the United States from the first 

cutting of a 2011 field production, described in Appendix D. The conventional control 

(C0-Syn1) used as a comparator was a near-isogenic conventional alfalfa population with 

a genetic background similar to that of KK179. Fourteen different conventional 

commercial alfalfa varieties were included across the field production to provide data on 

the natural variability of each compositional component analyzed. Field production of 

forage was conducted in typical alfalfa growing regions at six sites located in California 

(CAPR); Iowa (IARL); Illinois (ILCY); Kansas (KSLA); Texas (TXCL); and Wisconsin 

(WIDL). KK179, conventional control and conventional commercial varieties were 

planted in a randomized complete block design with four replicated plots per site and 

grown under normal agronomic field conditions for their respective geographic regions. 

At the plant growth stage between 1 and 10% bloom, which is a normal stage for 

harvesting forage, samples of the whole alfalfa plant, 2-3 inches above the soil surface, 

were harvested at each site from the plants 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, generated values expressed as μmol/g cell wall 

residue (CWR). Two lignin subunits, caffeyl lignin and 5-hydroxyguaiacyl lignin, had 

more than 50% of the observations below the assay limit of quantitation (LOQ) and, as a 

result, were excluded from the statistical analyses. The S:G lignin ratio was calculated 

from the values of the individual components expressed 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 total 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 Forage Lab), a facility certified for analytical 
Monsanto Company 12-AL-246U 39 of 407 

assessments of forage quality by the National Forage Testing Association (NFTA). Dairy 

One Forage Lab used a semi-automated ANKOM-based methodology, described in 

Appendix D, as adopted by most commercial 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 lignin 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% compared 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 control (Table I-2). These results support the 

conclusion that suppression of CCOMT in KK179 decreases the production 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 utilizes standards and methods of analysis 

representative of those commonly adopted by the forage industry to measure forage 

quality-related parameters, including total lignin (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 commercial value of the forage as feed is determined. The ANKOM 

method, which has been adopted by most commercial forage testing laboratories, 

measures acid detergent lignin based on procedures 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, followed 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 total 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 

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

 Difference (Test minus Control)

Analytical Component (Units)1

KK179 

Mean (S.E.)2

(Range)

Control3

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence 

Interval

Significance

(p-Value)

Lignin Subunits (μmol/g CWR)Guaiacyl (G) lignin 68.10 (9.48) 83.72 (9.40) -15.62 (6.12) -29.16, -2.07 0.027 

(21.17 - 134.96) (33.11 - 131.40) (-39.11 - 27.03) 

Syringyl (S) lignin 55.96 (8.83) 50.41 (8.78) 5.55 (5.11) -5.82, 16.92 0.302 

(9.82 - 87.67) (12.20 - 91.89) (-18.80 - 43.57) 

p-Hydroxyphenyl (H) lignin 5.05 (0.45) 3.88 (0.43) 1.17 (0.60) -0.16, 2.50 0.077 

(2.20 - 10.84) (0.58 - 5.49) (-1.76 - 7.24) 

Syringyl to Guaiacyl Subunit ComparisonS:G Ratio 0.80 (0.061) 0.58 (0.060) 0.22 (0.027) 0.16, 0.29 <0.001 

(0.43 - 1.16) (0.35 - 0.70) (-0.16 - 0.50) 

1 CWR = Cell Wall Residue; S:G Ratio = Syringyl lignin subunit divided by Guaiacyl lignin subunit 2Mean (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. Ne
Monsanto Company 12-AL-246U 

Table I-2. Alfalfa Forage Lignin Subunit Levels as Percent of Total HGS for KK179 vs. Conventional Con

 Difference (Test minus Control)

Analytical Component (Units)1

KK179 

Mean (S.E.)2

(Range)

Control3

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence 

Interval

Significance

(p-Value)

Lignin Subunits (% Total HGS)Guaiacyl (G) lignin 53.69 (1.87) 61.69 (1.87) -8.00 (0.71) -9.42, -6.58 <0.001 

(44.92 - 63.78) (56.88 - 70.56) (-14.63 - 4.22) 

Syringyl (S) lignin 42.09 (2.35) 35.24 (2.35) 6.85 (0.75) 5.34, 8.36 <0.001 

(26.98 - 52.01) (24.60 - 40.26) (-6.77 - 13.61) 

p-Hydroxyphenyl (H) lignin 4.22 (0.54) 3.07 (0.54) 1.15 (0.28) 0.53, 1.76 0.001 

(2.04 - 9.78) (0.34 - 5.18) (-0.85 - 4.60) 

1Total HGS is the sum of Hydroxyphenyl (H), Guaiacyl (G) and Syringyl (S) lignin subunits (μmol/g CWR); CWR = Cell 2Mean (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. Nzero.
Monsanto Company 12-AL-246U 

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

Difference (Test minus Control)

Analytical Component (Units)1

KK179 

Mean (S.E.)2

(Range)

Control3

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

Relative % 

Difference

Significance

(p-Value)

Acid Detergent Lignin5(% dw)

5.39 (0.64) 6.93 (0.64) -1.53 (0.42) -22.15 0.004 

(2.73 - 7.60) (2.23 - 10.10) (-4.33 - 1.30) 

1dw = dry weight 2Mean (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. Nzero.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 Regulation of Biotechnology, the responsibility 

for regulatory oversight of biotechnology-derived crops falls primarily on three U.S. 

agencies: the U.S. Food and Drug Administration (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 oversight and review of this product. As a 

practical matter, KK179 cannot be released and marketed until FDA has completed its 

review and assessment under its 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 varieties, including those developed through 

biotechnology (U.S. FDA, 1992). In compliance with this policy, Monsanto and FGI 

have initiated a consultation with the FDA 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 Foreign Government Agencies 

Consistent with their commitments to the Biotechnology Industry Organization’s 

Excellence Through Stewardship Program, Monsanto and FGI intend to obtain import 

approvals from all key alfalfa import markets with functioning regulatory systems prior 

to commercial planting of KK179. As appropriate, notifications will be made to 

countries that import significant quantities of alfalfa and alfalfa products and do not have 

formal regulatory review processes for biotechnology-derived crops. 
Monsanto Company 12-AL-246U 44 of 407 

II. THE BIOLOGY OF ALFALFA 

The Canadian Food Inspection Agency document 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 and biology of alfalfa 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-APHIS, 2010). Taxonomic information for 

alfalfa is available in the U.S. Department of Agriculture’s Natural Resources 

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

To support the evaluation of the plant pest potential of KK179 relative to conventional 

alfalfa, additional information regarding several aspects of alfalfa biology can be found 

elsewhere in this petition. This includes: agronomic 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, Transcaucasia, Turkmenistan, and Iran. With the 

spread of agriculture, alfalfa became endemic throughout the Mediterranean region, 

North Africa, the Middle East, most of Europe, 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 for forage (Michaud et al., 1988). Due to its 

importance as an animal feed, it has spread 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 species, 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 karyotype with the ability 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 produced by crossing of the above mentioned 

subspecies, including subsp. varia, subsp. hemicycla, and subsp. tunetana are also 
Monsanto 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 tolerance, 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 world. A key milestone in advancing alfalfa 

breeding and development was reached in the early 20th century with the development of 

a system for classifying alfalfa germplasm into several distinct fall dormancy groups and 

selection of more winterhardy types within each group (Melton et al., 1988). This system 

made it possible to separate fall dormancy from winter hardiness. In recent years, a 

better understanding of autotetraploid genetics and its effects on breeding and variety 

synthesis has improved genetic gains for forage 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) greater yield potential; 3) improved stand persistence, and 4) 

increased forage quality (Putnam et al., 2008a; Undersander et al., 2011). 

Cultivated alfalfa is widely adapted, allowing production across varying climatic regions 

and geographies under both irrigated and non-irrigated systems. It is typically planted to 

establish perennial stands that remain in the field from three to seven years, depending 

upon geography and agronomic practice. Forage is harvested from two to eleven times 

per season, depending on the region and the system 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 livestock. As a legume, it is also desired for 

rotational use to improve soil characteristics such as nitrogen content (Undersander et al., 

2011). 

Alfalfa is among the most important forage crops in the United States and ranks as the 

fourth most widely grown crop by acreage, 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 acreages harvested in California, Colorado, 

Idaho, Iowa, Michigan, Minnesota, Montana, Nebraska, North Dakota, 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 also 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 production, which relies on treated seed and 

agronomic practices that are incompatible with non-forage purposes. Monsanto and FGI 
Monsanto 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 purchasers. 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 (T0) was crossed with Ms208, a conventional 

male-sterile alfalfa plant (propogated vegetatively via stem cuttings) to produce F1

progeny plants. A single plant (P0) was selected from these progeny plants 

(Figure IV-11) and used for molecular characterization of the F1 generation, as described 

in Section IV. 

Due to inbreeding depression and self-incompatibility in an outcrossing species like 

alfalfa, it is not possible to breed pure isogenic 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 P0 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 breeding step, known as a modified backcross (MBC), 

resulted in the first KK179 population with related individuals (MBC1). Repeating this 

step by hand crossing the MBC1 generation with the same 10 elite alfalfa genotypes with 

the FD4 phenotype resulted in the MBC2 generation, again using FD4 plants as the 

female seed parents. Finally, a population of MBC2 generation 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 commercial variety development. Analyses of 

the expressed products, described in Section 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 crossing in the KK179 synthetic populations. All 

hand-crosses were conducted in a greenhouse. 

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

The identical breeding process described above was followed using the C0 plant in order 

to produce a C0-Syn1 generation, which is a conventional synthetic population, to serve 

as the conventional comparator for the Syn1 generation, respectively. A single, nontransformed R2336 plant was crossed with Ms208 to produce conventional F1 progeny 

plants, from which a conventional alfalfa comparator C0 was selected. The same 

breeding process used to develop subsequent generations of KK179 was then followed to 

develop subsequent generations of near-isogenic conventional alfalfa comparators for 

each generation of KK179 as shown in Figure IV-11. As a result, all generations of 
Monsanto Company 12-AL-246U 47 of 407 

KK179 and the conventional controls used in the safety assessment studies have closely 

related genetic backgrounds, with the exception of the intended trait for reduced G lignin 

and total lignin. The C0 and C0-Syn1 plants are both referred to as the conventional 

control. R2336 and Ms208 are referred to as conventional parental controls. 

In addition, conventional commercial alfalfa varieties (referred to as conventional 

commercial reference varieties), were used to establish ranges of natural variability 

representative of commercial alfalfa varieties. The conventional commercial reference 

varieties used at each location were chosen based on their availability and agronomic 

adaptation for the respective geographic region. 
Monsanto Company 12-AL-246U 48 of 407 

III. DESCRIPTION OF THE GENETIC MODIFICATION 

Molecular analyses are an integral part of the characterization of crop products with new 

traits introduced by methods of biotechnology. Vectors and methods are selected for 

transformation to achieve high probability of obtaining the trait of interest 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 trait is integrated into the plant genome and 

facilitates the molecular characterization of the product. Information provided here 

allows for the identification of the genetic material 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 plant found in Section IV. 

KK179 was developed through Agrobacterium tumefaciens-mediated transformation of 

conventional alfalfa R2336 leaf tissue utilizing plasmid 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, transfer DNA (T-DNA) refers to DNA that is 

transferred to the plant during transformation. The suppression cassette refers to the 

sequences and regulatory elements necessary 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 transformation 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 Left and Right Border regions to facilitate 

transformation. The first T-DNA, designated as T-DNA I, 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 (Section IV.B.) where T-DNA II, containing the 

nptII expression cassette, functioned as a marker gene for the selection of transformed 

plantlets. Subsequently, traditional breeding methods and 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 bacterial selectable marker gene (aadA), and a coding sequence for 

repressor of primer (ROP) protein for the 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-MSPQ12633 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 Hildebrandt, 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 alfalfa plant, selected for regenerability from 

an elite, high yielding, fall dormant alfalfa 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 calli developed with somatic embryos. The 

somatic embryos were placed in media conducive to shoot and root development. Rooted 

plants (hereafter called T0 plants) with normal phenotypic characteristics were selected 

and transferred to soil for growth and further assessment. 

The T0 plants were crossed with Ms208, a conventional male sterile plant selected from a 

population with a fall dormancy (FD4) phenotype, to produce F1 plants, in which the 

unlinked insertions of T-DNA I and T-DNA II were 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 (P0) was selected as the lead 

event based on superior phenotypic characteristics and its molecular profile. P0 is an 

individual F1 plant produced from crossing T0 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 process was the production of marker-free alfalfa 

KK179. 

 
Monsanto Company 12-AL-246U 50 of 407 

Figure III-1. Circular Map of PV-MSPQ12633 Showing Probe 1 through Probe 9 

A circular map of PV-MSPQ12633 used to develop KK179 is shown. PV-MSPQ12633 contains 

two T-DNAs, designated as T-DNA I and T-DNA II. Genetic 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 Southern analyses are shown on the interior of the 

map. The dashed arcs indicate that probes 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

CS-rop OR-ori-pUC

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

8 7

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 1.2 
Monsanto 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 T0 plants were crossed to male sterile clone Ms208

Screening of F1 plants for the presence of T-DNA I and the absence of 

T-DNA II
Monsanto 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 inverted 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 inverted repeat produces dsRNA that, via an RNA interference 

(RNAi) pathway (Siomi and Siomi, 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 Tn5 of E. coli encoding the NPT II protein (Beck 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 process. Plants that did not contain the nptII

expression cassette were isolated through traditional cross-pollinated breeding methods 

and segregation, along with a combination 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 results in a pattern of 

expression that closely mirrors deposition of lignin as the plant matures (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 polyadenylation 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) (Odell 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 III-1 and Table III-1) 

that were derived from Agrobacterium tumefaciens (Barker et al., 1983; Depicker et al., 

1982; Zambryski et al., 1982). The 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. Because PV-MSPQ12633 is a 2-T-DNA vector, it 

contains two Left Border regions and two Right Border regions, where one border region 

set flanks T-DNA I and the other border 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 exist 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 Tn7 that confers spectinomycin and streptomycin 

resistance (Fling et al., 1985) in E. coli and Agrobacterium during molecular cloning. 

Because these elements are outside the border 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 Genetic Elements in PV-MSPQ12633 

Genetic Element 

Location in 

Plasmid Function (Reference) 

T-DNA I 

B1-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 

P2-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 

T3-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 cloning

 
Monsanto 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)-Onucleotidyltransferase from the transposon Tn7 (Fling et 

al., 1985) that confers spectinomycin and streptomycin 

resistance

Intervening 

Sequence 4089-4618 Sequence used in DNA cloning

OR4-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

CS5-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 cloning

 
Monsanto 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 Tn5 of 

E. coli encoding neomycin phosphotransferase 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 containing 

the 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. 
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IV. CHARACTERIZATION OF THE GENETIC MODIFICATION 

This section contains a comprehensive molecular characterization of the genetic 

modification present in KK179. It provides information on the DNA insertion into the 

plant genome of KK179, and additional information relative to the arrangement and 

stability of the introduced genetic material. The information provided in this section 

addresses the relevant factors in Codex Plant 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 stably 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 vector backbone sequences are not detected 

in KK179. These conclusions were based on several lines of evidence: 1) Southern blot 

analyses assayed the entire alfalfa genome 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 single copy of T-DNA I was inserted at a 

single locus; 2) DNA sequence analyses to determine 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 demonstrated the lack of any rearrangements that 

occurred at the insertion site during transformation; 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 inheritance. Taken together, the characterization 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 determine the number of copies, to characterize the 

insertion site of T-DNA I, as well as to assess the presence or absence of T-DNA II and 

plasmid vector backbone sequences. The Southern blot strategy was designed to ensure 

that all potential inserted segments would be identified. The entire alfalfa genome was 

assayed with probes that spanned the complete 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 potential segments from the plasmid vector 

PV-MSPQ12633. The restriction enzyme sets were chosen such that each enzyme set 

cleaves once within the inserted T-DNA and at least once within 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 entire insert sequence and adjacent 5′ or 3′ flanking 

DNA sequence. Therefore, at least one segment containing a portion of the insert with 
Monsanto 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 another predictable size segment containing 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 genome 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 vector backbone sequences, duplicated samples 

that consisted of equal amounts 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 long 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 run lanes of the Southern blots. Southern blot results 

demonstrated that KK179 contains a single copy of T-DNA I at a single insertion site in 

the alfalfa genome, and no T-DNA II or backbone 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. Furthermore, 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 insertion site in conventional alfalfa. 

The stability of the T-DNA I present in KK179 across multiple generations was 

demonstrated by Southern blot fingerprint analysis. Genomic DNA from four 

generations (P0, MBC1, MBC2, and Syn1) of KK179 (Figure 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 restriction segments that encompass the entire T-DNA I insert 

(Figure IV-1). This fingerprint strategy consists of two border segments that assess not 

only the stability of T-DNA I, but also the stability of genomic DNA directly adjacent to 

T-DNA I. Generational stability analysis demonstrated 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 establishes 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 used 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 the insert, a table summarizing the expected 

DNA segments for Southern analyses is presented in Table IV-1. The genetic elements 

within the KK179 insert are summarized in Table IV-2. The results from the Southern 

blot analyses are presented in Figure IV-2 through Figure IV-8. PCR amplification of the 

KK179 insert and the insertion site in conventional control for DNA sequence analysis is 

shown in Figure IV-9 and Figure IV-10, respectively. The generations used in the 

generational stability analysis are depicted in the breeding history shown in Figure IV-11 

and the results from the generational stability analysis are presented in Figure IV-12. The 

breeding path for the segregation data is shown 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 

Figure IV-1. Schematic Representation of the Insert and Flanking DNA in KK179 DNA derived from T-DNA I of PV-MSPQ12633 integrated in KK179. Right-angled arrows indicate the eT-DNA I and the beginning of the flanking sequence. Identified on the map are genetic elements within 

restriction sites with positions relative to the size of the DNA sequence (flanks and insert). The relative size

T-DNA I probes and the expected sizes of restriction fragments are indicated. This schematic diagram is Locations of genetic elements and T-DNA I probes are approximate. Probes are describ

Swa I 1017

5' Flank 3' Flank 

Dra III 2165 Xba I 2986 Swa I 4345

1 4

Probe 1

Probe 2

Probe 3 Probe 4

B-Left Border Region r1

P-Pal2

CCOMT

T-nos

B-Right Border Region r1

CCOMT

Xba I and Swa I

~ 2.0 kb ~ 1.4 kb

Xmn I and Dra III

≥2.2 kb ~ 2.0 kb

Xmn I 4186

Xmn I 
Monsanto Company 12-AL-246U 

Table IV-1. Summary Chart of the Expected DNA Segments Based on Hybridizing Probes and RestrictKK179 Analysis 

Southern Blot Analysis T-DNA I T-DNA II 

Figure V-2 V-3 V-4 V-5 V-6 V

Probe Used

1 2, 4 3 3 5 6

Probing Target Digestion enzyme Expected Band Sizes on each Southern Blot

PV-MSPQ12633 

Xba I ~6.9 kb 

~3.7 kb 

~6.9 kb 

~3.7 kb ~~2 ~~2 ~6.9 kb 

~6.~3.

Eco RI

~~2 ~~2 ~2.2 kb ~2.2 kb ~~2 ~

Probe Templates1 N/A 

--3

~0.8 kb 

~0.7 kb --3 --3 --3

~1.~1.

KK179 

Xba I and 

Swa I ~2.0 kb

~2.0 kb ~1.4 kb 

~2.0 kb ~1.4 kb ~~2 ~1.4 kb N

Xmn I and 

Dra III

≥2.2 kb 

≥2.2 kb 

~2.0 kb ~~2 ~2.0 kb ~2.0 kb N

1 probe template spikes were used as positive hybridization controls in Southern blot analyses when multiple probes wer

simultaneously 

2 ‘~~’ indicates that this digest was not performed. 3 ‘--’ indicates that probe templates were not used. 4 Not Applicable. 
Monsanto Company 12-AL-246U 62 of 407 

Table IV-2. Summary of Genetic Elements in KK179 

Genetic Element Location in Sequence Function (Reference) 

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

insert 

B1-Left Border Regionr1 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 

P2-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 

T3-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 Regionr1 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 

r1Superscript in Left Border and Right Border regions 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. 
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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 P0 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 blots with probes that span 

the T-DNA I (Figure III-1). Each restriction digest is expected to produce a specific 

banding pattern on the Southern blots (Table IV-1). Any additional copies and/or 

integration sites would be detected as additional bands. 

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

once within the known 3′ flanking sequence 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 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 within the inserted DNA and 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 segments 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 Southern 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, C0, is derived from a cross of the untransformed R2336 with 

the elite conventional male sterile plant Ms208, resulting in a near isogenic line 

comparator to KK179. Alfalfa is an autotetraploid (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 patterns due to random segregation of the 

endogenous CCOMT gene. Therefore, for blots that were probed with CCOMTcontaining sequences (Probe 3), the conventional parental plants R2336 and Ms208 were 

also included as negative controls. Conventional 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 are shown in Figures IV-2 through 

IV-8. 

IV.A.1. Probe 1 

Conventional control genomic DNA digested 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 (Figure III-1) showed no detectable 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 (Figure IV-2, Lane 6 and Lane 7). The 

~3.7 kb band and ~6.9 kb band were both detected because the Left Border region 
Monsanto Company 12-AL-246U 64 of 407 

contained in Probe 1 sequence is present in both the ~3.7 kb and the ~6.9 kb Xba I 

segments from PV-MSPQ12633. Detection of the 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) produced 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 digested 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 Probe 2 and Probe 4 (Figure III-1) showed no 

detectable hybridization bands, as expected. 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 control genomic DNA digested with a combination 

of Xmn I and Dra III and spiked with probe templates 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 expected 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) produced 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 indicate that the sequence covered by Probe 2 and Probe 4 

resides as one copy at a single detectable 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 sequences 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 parental controls in addition to conventional 

control C0 when probed with Probe 3. A hybridization 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 parental 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 conventional parental controls. All observed bands in the 

conventional and conventional parental controls represent 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 (Figure 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 and ~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 Ms208, as expected, the hybridization bands 

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

The conventional control genomic 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 the positive control indicates 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 hybridization 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 of 
Monsanto 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 genomic DNA digested with Xmn I and Dra III (Figure IV-5, 

Lane 1 and Lane 8) and hybridized with Probe 3 (Figure III-1) displayed hybridization 

bands at ~6.9 kb, ~7.9 kb, ~11.0 kb, ~14.0 kb, and ~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 (Figure 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 cross between R2336 and Ms208, as expected, the hybridization 

bands detected in the conventional control are present 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 hybridized 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 the 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 indicates that the sequence covered 

by Probe 3 resides as one copy at a single detectable 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 32P-labeled probe that spanned a portion of the 

T-DNA I sequence (Figure IV-1, Probe 1). 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 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. 

1 4 5 6 7 8 9 11 2 3 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 Run
Monsanto 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 32P-labeled probes that spanned 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 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 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, 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

40 20 15 10 8.1

5.1

3.1

2.0

1.6

1.0

0.5

6.1

4.1

7.1

1 2 3 4 5 6 11 78 9 10

Long Run Short Run
Monsanto 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 32P-labeled probe that spanned 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. Lane 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 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

1 4 5 6 7 8 9 11 2 3 10

Long Run Short Run
Monsanto 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 32P-labeled probe that spanned 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. Lane 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 Extension 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

1 3 4 5 6 7 8 9 11 2 10

Long Run Short Run
Monsanto Company 12-AL-246U 71 of 407 

IV.B. Southern Blot Analysis to Determine the Presence or Absence of T-DNA II 

Sequences in KK179 

The presence or absence of T-DNA II sequences in the KK179 alfalfa genome was 

evaluated by digesting the P0 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 (Figure III-1, Probe 5). Since the 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 detection 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 detection of unique hybridization 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 Probe 5 (Figure III-1) showed no detectable 

hybridization bands, as expected. Conventional control genomic 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 indicates 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) produced the expected band at ~2.0 kb only 

visible in the longer exposure (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 (Figure 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 expected 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 detectable 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 Detect the Presence or Absence of T-DNA II 

Sequences in KK179: Probe 5 

The blot was hybridized with one 32P-labeled probe that spanned a portion of the 

T-DNA II sequence (Figure IV-1, Probe 5). 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 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

1 2 3 4 5 6 11 78 9 10

Long Run Short Run
Monsanto 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 backbone sequences in the alfalfa genome 

was evaluated by digesting the P0 generation of KK179 and the 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 detection of hybridization bands on the Southern 

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

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

Conventional control genomic DNA digested 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 Probe 6 and Probe 8 (Figure IV-1) showed no 

detectable hybridization bands, as expected. 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 Probe 9 (Figure III-1). 

Conventional control genomic DNA digested 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 Probe 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 digested 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 Probe 7 and Probe 9 (Figure III-1) showed no 

detectable hybridization bands, as expected. 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 digested 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 probes 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 32P-labeled probes that spanned 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 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 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/Hind 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 Run
Monsanto Company 12-AL-246U 76 of 407 

Figure IV-8. Southern Blot Analysis to Determine the Presence or Absence of 

PV-MSPQ12633 Backbone Sequences in KK179: Probe 7 and Probe 9 

The blot was hybridized with two 32P-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 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 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 7 and Probe 9 [~1.0 

genome equivalent]

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/Hind 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

1 3 4 5 6 7 8 9 11 2 10

Long Run Short Run
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IV.D. Organization and Sequence of the Insert and Adjacent Genomic DNA in 

KK179 

PCR and sequence analyses were performed 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 span 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 KK179 insert is 2582 bp long (Table IV-2) and 

is identical to the corresponding T-DNA I sequence of PV-MSPQ12633 as described in 

Table III-1. From the sequence analyses, 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 Analysis across the Insert in KK179 

PCR was performed on both parental control genomic DNA and KK179 genomic DNA 

using five pairs of primers to generate overlapping PCR 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 illustration of the insert in KK179 that appears at the bottom of the figure. 

This figure is a representative of the data generated in the study. Lane designations 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 the DNA, in kilobase pairs, obtained from the 1 kb 

DNA Extension Ladder (Invitrogen) on the ethidium bromide stained gel. 

1 4 5 15 18 2 3 14 1617 6 11 7 8 9 10 12 13 19 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′ Flank B-Left Border Region r1 P-Pal2 CCOMT T-nos B-Right Border Region r1 CCOMT

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

E
Monsanto Company 12-AL-246U 79 of 407 

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

PCR and sequence analyses were performed 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 performed with a forward primer 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 conventional 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. The alignment between the 3′ end of the KK179 insert and the

conventional parental control sequence showed that the conventional 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 the KK179 insert and is a 

A/G heterozygote. These two heterozygotes were most likely caused by single 

nucleotide polymorphisms segregating in the autotetraploid alfalfa population (Yang et 

al., 2009). The alignment analyses also indicated 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 break-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 

KK179. 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′ Flank B-Left Border Region r1 P-Pal2 CCOMT T-nos B-Right Border Region r1 CCOMT

Primer A Primer B 

Primer A Primer B 
Monsanto Company 12-AL-246U 81 of 407 

IV.F. Southern Blot Analysis to Examine Insert 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 generations 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 (Figure 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 the 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 C0 genomic DNA digested with Xmn I and Dra III (Figure IV-12, 

Lane 1) and hybridized with Probe 2 and Probe 4 (Figure III-1) showed no detectable 

hybridization bands, as expected. Conventional control genomic 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 (Figure IV-12, Lane 2), as expected. 

Conventional control genomic DNA digested 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 additional faint ~1.4 kb band in the probe templates 

control lane (Figure IV-12, Lane 3) was observed and is most likely single stranded DNA 

formed during purification (Qiagen, 2008) that has partially reannealed in various 

conformations (Kasuga et al., 2001). Since this ~1.4 kb band was detected 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 analysis on KK179. Detection of the spiked 

controls indicates that the probes hybridized to their target sequences. 

KK179 DNA extracted from four generations (P0, 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 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 genomic DNA flanking the 3′ end of the insert.  The 

presence of ~4.5 kb and ~2.0 kb bands in the P0, 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 P0 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 

T0: the original transformed R2336 plant; 

Ms208: conventional mal

P0: 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

breeding 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 KK179 

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 male sterile alfalfa plant; 

the single KK179 plant selected from the progeny of T0 × Ms208, 

10 elite alfalfa genotypes with fall dormancy rating 4 phenotype;

KK179 generation produced from crossing P0 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 each 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 P0, MBC1, 

generations were used for the insert stability of KK179 reported 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 Syn1 
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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 32P-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 (P0) (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, obtained from the 1 kb DNA Extension 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

1 4 56 78 2 3
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IV.G. Inheritance of the Genetic Insert in KK179 

During development of KK179, segregation data were generated to assess the heritability 

and stability of the T-DNA I present in KK179 using Chi square (χ2) analysis over several 

generations. The Chi square analysis is based on comparing the observed segregation 

ratio to the expected segregation ratio according to Mendelian principles. 

The KK179 breeding path for generating segregation data is described in Figure IV-13. 

The transformed T0 plant was cross-pollinated to an elite male sterile alfalfa plant, 

Ms208, to produce F1 seed. From the F1 segregating population, an individual plant 

(designated as P0) 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 P0 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 pollen 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 produce 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 determine the presence or absence of the KK179 

insert in the generations evaluated. The MBC2 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 (χ2) analysis was performed using the statistical program 

R Version 2.12.0 (2010-10-15) to compare the observed segregation ratios to the 

expected ratios according to Mendelian inheritance principles. The χ2 was calculated as: 

χ 2

 = ∑ [( | o – e | )2 / e] 

where o = observed frequency of the phenotype and e = expected frequency of the 

phenotype. The level of statistical significance was predetermined to be 5% (α = 0.05). 

The results of the χ2 analysis of the segregating progeny of KK179 are presented in Table 

IV-3. The χ2 values in the MBC2 and MBC3 populations indicated no statistically 

significant difference between the observed and expected 1:1 segregation ratio (KK179 

positive:KK179 negative) of the KK179 insert. Likewise, the χ

2 value in the Syn1 

population indicated no statistically significant difference between the observed and 

expected 3:1 segregation ratio (KK179 positive:KK179 negative) of the KK179 insert. 
Monsanto Company 12-AL-246U 85 of 407 

These results support the conclusion 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 are 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 

T0: the original transformed R2336 plant;

Ms208: conventional male sterile alfalfa 

F1: KK179 progeny produced from the cross of T

P0: 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 T0 and Ms208. 

the single KK179 plant selected from the progeny of T0 × Ms208; 

10 elite alfalfa genotypes with fall dormancy rating 4 phenotype;

KK179 generation produced from crossing P0 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 

Table IV-3. Segregation of the KK179 Insert During Mendelian Inheritance Testing  1:1 Segregation Generation Total Plants1 Observed # Positives Observed # Negatives Expected # Positives Expected # Negatives 

χ2 Probability 

MBC2 261 119 142 130.5 130.5 2.027 0.1545 MBC3 263 132 131 131.5 131.5 0.004 0.9508  3:1 Segregation Generation Total Plants1 Observed # Positives Observed # Negatives Expected # Positives Expected # Negatives 

χ 2 Probability 

Syn1 504 376 128 378 126 0.042 0.8370 1 Plants were tested for the presence of the KK179 insert by Endpoint TaqMan analysis. “Total plants” refers to the total number of pl

or absence of the insert could be determined using the assay. 
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IV.H. Characterization of the Genetic Modification Summary and Conclusion 

Molecular characterization of KK179 by Southern blot analyses confirmed that one copy 

of the CCOMT suppression cassette was integrated into the alfalfa genome at a single 

locus. No T-DNA II or backbone DNA sequence from plasmid vector PV-MSPQ12633 

was detected in KK179. 

PCR and DNA sequence analyses performed on KK179 and conventional parental 

control R2336 determined the following: the complete DNA sequence of the insert and 

adjacent DNA sequences in KK179; the organization 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 junctions. 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 analysis also identified a 

102 base pair deletion that occurred at the insertion site in KK179. 

Southern blot analysis of KK179 demonstrated that the inserted DNA has been 

maintained through multiple generations of breeding, thereby confirming the stability of 

the insert. Results from segregation analyses show heritability and stability of the insert 

occurred as expected across multiple generations, which corroborates the molecular insert 

stability analysis and establishes the genetic behavior of the T-DNA I inserted in KK179 

at a single chromosomal locus. 
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V. CHARACTERIZATION AND SAFETY ASSESSMENT OF KK179 

EXPRESSED PRODUCTS 

A multistep approach is used to assess the safety 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 protein. Information pertaining to the mode-ofaction for RNA-based suppression of the expression of an endogenous plant gene can be 

found in Secton I.A. Section V.A. describes the 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 intended reduction of G 

lignin and total lignin in KK179, is mediated by dsRNA molecules. These dsRNA 

molecules, which are produced from assembled 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). Nucleic acids have a long history of safe 

consumption and are considered GRAS (Generally 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). 

Several 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 dsRNA 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 consequence, 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 flanking 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 documented evidence for 

toxicity or allergenicity of dietary RNA, and the lack of evidence of any expressed 
Monsanto Company 12-AL-246U 90 of 407 

protein from the DNA inserted into KK179, 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 address the history of safe use for RNA-based 

gene suppression. In Section I.B.2.2., KK179 northern 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, demonstration of mode-of-action through 

CCOMT RNA suppression, the history of safe consumption of RNA, and the apparent 

lack of toxicity or allergenicity of dietary RNA, the RNA-based suppression technology 

used in KK179 poses no novel risks as a result of expressed products. 

 
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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 (66FR 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 commodities of the biotechnology-derived crop are 

compared to the appropriate conventional counterpart 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 biotechnology-derived crop varieties, nine countries, 

and eleven growing seasons, concluded that incorporation of biotechnology-derived 

agronomic traits has had little impact on natural 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 reflect the influence of environmental and 

genetic factors as well as extensive conventional breeding efforts to improve nutrition, 

agronomics, and yield (Harrigan et al., 2010). 

Compositional equivalence between biotechnology-derived and conventional crops 

supports an “equal or increased assurance of the safety of foods derived from genetically 

modified plants” (OECD, 2002). OECD consensus documents on compositional 

considerations for new crop varieties emphasize quantitative measurements of essential 

nutrients and known anti-nutrients. These quantitative measurements effectively discern 

any compositional changes that imply 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 compared to: 1) corresponding levels in a 

conventional comparator, a genetically similar conventional line, grown concurrently 

under similar field conditions; and 2) ranges generated from an evaluation of 

conventional commercial reference varieties grown concurrently, and from data 

published in the scientific literature. 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-documented variation in the concentrations of crop nutrients, antinutrients, and secondary metabolites. 

This section provides analyses of concentrations 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 included in the composition 

analyses to establish a range of natural variability for each component. The production of 

forage for compositional analyses used field designs (randomized complete block design 

with four blocks) and sensitive analytical methods that allow accurate assessments of 
Monsanto 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 KK179 Forage to Conventional Alfalfa 

Forage samples were collected from the first cutting of KK179, a conventional alfalfa 

control, and conventional commercial alfalfa varieties grown in a 2011 field production. 

The conventional control (C0-Syn1) used as a comparator was a near-isogenic 

conventional alfalfa population with a genetic background similar to that of KK179. 

Fourteen different conventional commercial alfalfa reference varieties were included 

across the field production sites 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 commercial varieties were planted in a 

randomized complete block design with four replicated plots per site and were grown 

under normal agronomic field conditions for their respective geographic regions. At the 

plant growth stage between 1 and 10% bloom, 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 the 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 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-essential). Anti-nutrient and secondary metabolites 

included daidzein, glycitein, genistein, coumesterol, formononetin, biochanin A, saponins 

(total bayogenin, total hederagenin, total medicagenic 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 suppression on the lignin biosynthetic 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 field production were measured by Dairy One 

Lab as described in Section I.B.3. Dairy One Forage Lab used a semi-automated 

ANKOM-based methodology, described in Appendix D, as adopted by most commercial 

forage testing laboratories. Covance Laboratories, Inc., which conducted the 

compositional analyses of key nutrients, anti-nutrients, and secondary metabolites 
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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 biochanin A) and one secondary metabolite 

(sinapic acid) had more than 50% of the observations below the assay limit of 

quantitation (LOQ) and, as a result, were excluded from statistical analyses. Therefore, 

47 components were statistically assessed using a mixed-model analysis of variance 

method. Values for all components were expressed 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. Compositional 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 biological relevance from a feed/food safety or 

nutritional perspective (EFSA, 2011). Considerations used to assess the relevance of 

each statistically significant combined-site difference included: 1) the relative magnitude 

of the difference in the mean values of nutrient, anti-nutrient, and secondary metabolite 

components of KK179 and the conventional control; 2) whether the KK179 component 

mean value is within the range of natural variability 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 assessment 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 metabolites 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 and comparisons of mean values to the 99 % 

tolerance interval and literature values, indicated that they were not biologically 

meaningful from a feed/food safety or nutritional perspective. Further assessment of 

statistically significant differences observed 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. 
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VI.B. Nutrients, Anti-Nutrients, and Secondary Metabolites in KK179 Forage 

The means and ranges for nutrients, anti-nutrients, and secondary metabolites from 

KK179 were consistent with values established from the conventional alfalfa control 

(Tables VI-2, VI-3, and VI-4). No significant 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 significant differences for ash, canavanine, 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 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 0.41% dw, which is a relative 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 difference in ash in KK179 forage compared to the 

conventional control is not considered biologically meaningful from a feed/food safety 

and nutritional perspective. 

The mean level of canavanine, an anti-nutrient, 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 16.94 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 varieties and within the range of values found in 

the published literature (Table VI-5). Lower levels of canavanine would not be adverse 

as it is considered an anti-nutrient in leguminous plants. Therefore, the difference in 

canavanine in KK179 forage compared to the conventional control is not considered 

biologically meaningful from a feed/food safety and nutritional perspective. 

The mean level of ferulic acid was significantly higher (p<0.05) in KK179 forage than 

the conventional control in the combined-site analysis (Table 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 the 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 meaningful from a feed/food 

safety and nutritional perspective. 

Although the mean level of total lignin (ADL) in KK179 was not significantly lower as 

reported in Section I.B.3., it was numerically lower in KK179 forage compared to the 

conventional control in the combined-site analysis (Table VI-2). The absolute difference 

in magnitude was 0.32% dw, which is a relative difference of -4.89%. The use of 

different methods, semi-automated ANKOM-based assay by Dairy One Forage Lab and 
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the manual assay by Covance Lab, likely contributed to the variability in total lignin 

(ADL) values determined by the two laboratories. Both methods did confirm a decrease 

in total lignin in KK179, with Dairy One Forage Lab reporting a significant decrease of 

22.15% and Covance a numerical decrease of 4.89%. 

Assessment of these compositional results supports the overall conclusion that, with the 

exception of the intended reduction 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 metabolites. 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 population of conventional commercial reference 

varieties and within the range of values found in the published literature (Table VI-5). 

Therefore, these differences are not considered biologically meaningful from a feed/food 

safety and nutritional perspective. 
Monsanto Company 12-AL-246U 

Table VI-1. Summary of Differences Observed in the Combined-Site Anal

ysis (p<0.05) of Alfalfa Fora

g

for KK179 vs. Conventional Control 

 Mean Difference 

(Test minus Control)

Analytical Component (Units)1

KK179 

Mean2

Control3

Mean

Mean Difference 

(% of Control)

Significance

(p-Value)

Test 

Range

Statistical Differences Observed in Combined-Site AnalysisForage Proximate (% dw)Ash 10.38 10.79 -3.77 0.034 8.43 - 13.2Forage MetaboliteCanavanine (ppm dw) 40.30 57.24 -29.60 0.013 11.42 - 87.8Ferulic Acid (ppm dw) 1596.41 1485.81 7.44 0.008 1389.38 - 1881dw = dry weight 2Mean = least-square mean 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 referenlimits set to zero.
Monsanto Company 12-AL-246U 

Table VI-2. Statistical Summary of Alfalfa Forage Nutrients for KK179 vs. Conventional Control 

 Difference (Test minus Control)

Analytical Component (Units)1

KK179 

Mean (S.E.)2

(Range)

Control3

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significa

(p-Valu

Proximate (% dw)Ash 10.38 (0.53) 10.79 (0.52) -0.41 (0.19) -0.78, -0.030 0.034 

(8.43 - 13.26) (8.79 - 12.95) (-1.80 - 1.06) 

Carbohydrates 66.55 (1.71) 65.97 (1.70) 0.58 (0.49) -0.55, 1.71 0.272 

(57.73 - 73.90) (59.94 - 72.91) (-3.45 - 8.36) 

Moisture (% fw) 78.26 (1.54) 78.15 (1.54) 0.11 (0.33) -0.64, 0.86 0.748 

(73.70 - 84.60) (70.50 - 83.70) (-2.70 - 3.60) 

Protein 20.83 (1.36) 21.02 (1.35) -0.19 (0.39) -0.98, 0.60 0.636 

(15.50 - 29.03) (15.98 - 27.30) (-6.67 - 3.19) 

Total Fat 2.28 (0.17) 2.28 (0.17) 0.0039 (0.16) -0.31, 0.32 0.980 

(0.84 - 3.98) (1.08 - 3.38) (-1.30 - 1.46) 

Fiber (% dw)Acid Detergent Fiber 27.03 (2.45) 27.02 (2.44) 0.015 (0.96) -2.12, 2.15 0.987 

(15.71 - 37.26) (10.96 - 36.11) (-10.08 - 9.57) 
Monsanto Company 12-AL-246U 

Table VI-2 (continued). Statistical Summary of Alfalfa Forage Nutrients for KK179 vs. Conventional Con

 Difference (Test minus Control)

Analytical Component (Units)1

KK179 

Mean (S.E.)2

(Range)

Control3

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significa

(p-Valu

Fiber (% dw)Acid Detergent Lignin 6.22 (0.60) 6.54 (0.59) -0.32 (0.27) -0.91, 0.28 0.265 

(2.72 - 10.31) (3.58 - 8.26) (-2.08 - 3.39) 

Neutral Detergent Fiber 33.95 (2.64) 34.46 (2.63) -0.52 (0.97) -2.67, 1.63 0.605 

(18.57 - 48.67) (18.94 - 43.32) (-9.74 - 11.84) 

Amino Acid (% dw)Alanine 1.11 (0.074) 1.13 (0.074) -0.017 (0.020) -0.062, 0.028 0.417 

(0.84 - 1.52) (0.87 - 1.39) (-0.19 - 0.13) 

Arginine 0.99 (0.065) 1.01 (0.065) -0.020 (0.020) -0.065, 0.024 0.326 

(0.73 - 1.35) (0.75 - 1.28) (-0.17 - 0.11) 

Aspartic acid 2.77 (0.28) 2.74 (0.28) 0.027 (0.071) -0.13, 0.19 0.711 

(1.97 - 4.65) (2.04 - 4.08) (-0.64 - 0.71) 

Cystine 0.21 (0.012) 0.21 (0.011) 0.00079 (0.0074) -0.016, 0.017 0.916 

(0.15 - 0.30) (0.15 - 0.29) (-0.041 - 0.062) 
Monsanto Company 12-AL-246U 

Table VI-2 (continued). Statistical Summary of Alfalfa Forage Nutrients for KK179 vs. Conventional Con

 Difference (Test minus Control)

Analytical Component (Units)1

KK179 

Mean (S.E.)2

(Range)

Control3

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significa

(p-Valu

Amino Acid (% dw)Glutamic acid 1.85 (0.12) 1.91 (0.12) -0.053 (0.035) -0.13, 0.024 0.156 

(1.40 - 2.55) (1.39 - 2.36) (-0.31 - 0.22) 

Glycine 0.95 (0.055) 0.97 (0.055) -0.018 (0.013) -0.047, 0.011 0.190 

(0.75 - 1.21) (0.73 - 1.14) (-0.10 - 0.085) 

Histidine 0.43 (0.020) 0.44 (0.020) -0.0064 (0.0058) -0.018, 0.0053 0.276 

(0.35 - 0.55) (0.36 - 0.51) (-0.053 - 0.059) 

Isoleucine 0.86 (0.053) 0.88 (0.053) -0.016 (0.014) -0.048, 0.016 0.284 

(0.67 - 1.15) (0.66 - 1.07) (-0.12 - 0.12) 

Leucine 1.43 (0.089) 1.47 (0.089) -0.033 (0.023) -0.086, 0.019 0.187 

(1.09 - 1.90) (1.09 - 1.78) (-0.22 - 0.16) 

Lysine 1.14 (0.067) 1.17 (0.067) -0.024 (0.017) -0.058, 0.0094 0.153 

(0.93 - 1.55) (0.92 - 1.44) (-0.15 - 0.12) 
Monsanto Company 12-AL-246U 

Table VI 2 (continued). Statistical Summary of Alfalfa Forage Nutrients for KK179 vs. Conventional Cont

 Difference (Test minus Control)

Analytical Component (Units)1

KK179 

Mean (S.E.)2

(Range)

Control3

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significa

(p-Valu

Amino Acid (% dw)Methionine 0.25 (0.024) 0.24 (0.024) 0.0083 (0.012) -0.017, 0.033 0.508 

(0.15 - 0.39) (0.15 - 0.37) (-0.12 - 0.14) 

Phenylalanine 0.98 (0.061) 1.00 (0.061) -0.025 (0.016) -0.060, 0.0097 0.138 

(0.75 - 1.27) (0.74 - 1.21) (-0.15 - 0.083) 

Proline 0.89 (0.054) 0.92 (0.053) -0.028 (0.021) -0.075, 0.018 0.199 

(0.71 - 1.18) (0.71 - 1.21) (-0.28 - 0.11) 

Serine 0.87 (0.044) 0.88 (0.044) -0.0061 (0.017) -0.045, 0.033 0.733 

(0.68 - 1.16) (0.68 - 1.05) (-0.16 - 0.13) 

Threonine 0.86 (0.050) 0.88 (0.050) -0.018 (0.016) -0.053, 0.018 0.288 

(0.66 - 1.12) (0.67 - 1.05) (-0.13 - 0.10) 

Tryptophan 0.37 (0.020) 0.37 (0.020) 0.0036 (0.0082) -0.013, 0.020 0.663 

(0.30 - 0.48) (0.27 - 0.45) (-0.056 - 0.065) 
Monsanto Company 12-AL-246U 

Table VI-2 (continued). Statistical Summary of Alfalfa Forage Nutrients for KK179 vs. Conventional Con

 Difference (Test minus Control)

Analytical Component (Units)1

KK179 

Mean (S.E.)2

(Range)

Control3

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significa

(p-Valu

Amino Acid (% dw)Tyrosine 0.71 (0.042) 0.71 (0.042) 0.0012 (0.015) -0.033, 0.035 0.939 

(0.55 - 0.94) (0.53 - 0.89) (-0.098 - 0.10) 

Valine 1.05 (0.061) 1.07 (0.061) -0.017 (0.015) -0.048, 0.014 0.280 

(0.79 - 1.38) (0.81 - 1.32) (-0.16 - 0.13) 

MineralCalcium (% dw) 1.68 (0.16) 1.72 (0.16) -0.037 (0.037) -0.12, 0.045 0.336 

(1.12 - 2.62) (1.09 - 2.53) (-0.41 - 0.28) 

Copper (mg/kg dw) 8.86 (0.85) 8.34 (0.85) 0.52 (0.34) -0.16, 1.20 0.131 

(5.14 - 13.16) (5.18 - 11.93) (-1.62 - 4.04) 

Iron (mg/kg dw) 272.00 (31.45) 315.74 (30.93) -43.74 (24.69) -98.55, 11.07 0.106 

(123.38 - 473.91) (163.92 - 547.83) (-279.88 - 115.21) 

Magnesium (% dw) 0.22 (0.023) 0.23 (0.023) -0.0082 (0.0050) -0.018, 0.0019 0.108 

(0.12 - 0.31) (0.15 - 0.32) (-0.048 - 0.037) 
Monsanto Company 12-AL-246U 

Table VI-2 (continued). Statistical Summary of Alfalfa Forage Nutrients for KK179 vs. Conventional Con

 Difference (Test minus Control)

Analytical Component (Units)1

KK179 

Mean (S.E.)2

(Range)

Control3

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significa

(p-Valu

MineralManganese (mg/kg dw) 52.56 (6.30) 52.45 (6.27) 0.11 (2.65) -5.86, 6.09 0.966 

(30.52 - 106.47) (30.92 - 77.32) (-15.31 - 37.75) 

Phosphorus (% dw) 0.29 (0.019) 0.28 (0.019) 0.0037 (0.0057) -0.0079, 0.015 0.523 

(0.22 - 0.40) (0.20 - 0.38) (-0.040 - 0.071) 

Potassium (% dw) 2.35 (0.052) 2.41 (0.051) -0.055 (0.051) -0.17, 0.059 0.307 

(2.16 - 2.65) (2.18 - 2.71) (-0.45 - 0.21) 

Sodium (% dw) 0.089 (0.024) 0.077 (0.024) 0.013 (0.0076) -0.0026, 0.028 0.102 

(0.020 - 0.22) (0.018 - 0.15) (-0.056 - 0.083) 

Zinc (mg/kg dw) 27.83 (2.11) 26.81 (2.09) 1.02 (1.42) -2.15, 4.19 0.489 

(18.40 - 39.22) (17.38 - 40.42) (-5.64 - 11.08) 

1dw = dry weight; fw = fresh weight 2Mean (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 referenlimits set to zero.
Monsanto Company 12-AL-246U 

Table VI-3. Statistical Summary of Alfalfa Forage Secondary Metabolites for KK179 vs. Conventional Co

 Difference (Test minus Control)

Analytical Component (Units)1

KK179 

Mean (S.E.)2

(Range)

Control3

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significa

(p-Valu

MetaboliteCanavanine (ppm dw) 40.30 (13.53) 57.24 (13.51) -16.94 (5.69) -29.62, -4.27 0.013 

(11.42 - 87.83) (12.69 - 134.50) (-79.53 - 5.32) 

Ferulic Acid (ppm dw) 1596.41 (59.57) 1485.81 (58.83) 110.59 (40.34) 29.36, 191.83 0.008 

(1389.38 - 1884.17) (1103.96 - 2007.38) (-301.26 - 503.18) 

Free Phenylalanine (ppm dw) 266.99 (28.84) 283.70 (28.69) -16.71 (12.62) -42.11, 8.69 0.192 

(111.86 - 409.20) (154.07 - 457.63) (-125.58 - 75.77) 

Total Polyphenols (mg/g dw) 8.19 (0.34) 7.99 (0.34) 0.20 (0.23) -0.30, 0.71 0.390 

(6.35 - 10.19) (6.57 - 10.21) (-2.13 - 1.72) 

p-Coumaric Acid (ppm dw) 639.50 (37.62) 623.54 (37.34) 15.97 (19.93) -28.25, 60.18 0.441 

(458.33 - 870.13) (442.08 - 819.59) (-112.64 - 226.29) 

1dw = dry weight; fw = fresh weight 2Mean (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 referenlimits set to zero.
Monsanto Company 12-AL-246U 

Table VI-4. Statistical Summary of Alfalfa Forage Anti-Nutrients for KK179 vs. Conventional Control 

 Difference (Test minus Control)

Analytical Component (Units)1

KK179 

Mean (S.E.)2

(Range)

Control3

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significa

(p-Valu

Saponins (response units/μg)Total Bayogenin 5.10 (0.76) 5.67 (0.76) -0.57 (0.47) -1.53, 0.38 0.23

(2.54 - 13.97) (2.20 - 11.28) (-2.85 - 5.81) 

Total Hederagenin 2.94 (0.35) 3.47 (0.35) -0.53 (0.32) -1.24, 0.19 0.13

(1.70 - 5.80) (1.58 - 6.85) (-3.51 - 1.21) 

Total Medicagenic Acid 21.88 (2.44) 23.39 (2.44) -1.51 (2.51) -6.57, 3.55 0.55

(9.09 - 45.08) (9.43 - 51.04) (-22.95 - 15.92) 

Total Soyasapogenol B 22.17 (3.02) 24.53 (3.02) -2.36 (1.44) -5.56, 0.84 0.13

(9.68 - 40.48) (7.05 - 41.93) (-12.47 - 8.72) 

Total Soyasapogenol E 2.77 (0.54) 3.08 (0.54) -0.31 (0.26) -0.84, 0.22 0.24

(1.20 - 5.02) (0.84 - 8.89) (-4.99 - 1.87) 
Monsanto Company 12-AL-246U 

Table VI-4 (continued). Statistical Summary of Alfalfa Forage Anti-Nutrients for KK179 vs. Conventional C

 Difference (Test minus Control)

Analytical Component (Units)1

KK179 

Mean (S.E.)2

(Range)

Control3

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Signi

(p-V

Saponins (response units/μg)Total Zanhic Acid 4.59 (0.58) 5.16 (0.58) -0.57 (0.45) -1.48, 0.33 0.210 

(2.25 - 12.08) (2.62 - 8.69) (-3.97 - 3.69) 

Total Saponins 59.30 (4.94) 65.58 (4.94) -6.28 (4.35) -15.05, 2.49 0.156 

(36.00 - 122.44) (29.20 - 96.50) (-32.96 - 25.94) 

1dw = dry weight; fw = fresh weight 2Mean (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 limits set to zero.
Monsanto Company 12-AL-246U 106 of 407 

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

Forage 

Components1

 Literature Range2 OECD Range3

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 - 36.0
Monsanto Company 12-AL-246U 107 of 407 

Table VI-5 (continued). Literature and OECD Ranges for Components in Alfalfa 

Forage 

Components1 Literature Range2

 OECD Range3

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 available
Monsanto Company 12-AL-246U 108 of 407 

VI.C. Compositional Assessment of KK179 Conclusion 

Analyses of nutrient, anti-nutrient, and secondary 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-derived 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 the U.S. that are representative of normal 

agricultural regions for alfalfa production. The compositional analysis, based on OECD 

consensus document for alfalfa, also included measurement of nutrients, anti-nutrients 

and secondary metabolites in conventional commercial reference varieties to provide data 

on the natural variability of each compositional component analyzed. 

Compositional analyses based on the OECD consensus document confirmed that, other 

than the intended reduction in G lignin and total 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 conventional alfalfa control. Of the 47 components 

statistically assessed, only three (ash, canavanine, 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 ferulic acid) are less than 10% in relative 

magnitude. The mean values for all three components with statistically significant 

differences between KK179 and the conventional 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 published in the scientific literature. 

These analyses provide a comprehensive comparative assessment of the levels of key 

nutrients, anti-nutrients, and secondary metabolites in forage of KK179 compared to the 

conventional control. These results support the overall conclusion that, with the 

exception of the intended reduction in G lignin and total lignin, forage of KK179 is 

compositionally equivalent to that of conventional alfalfa; therefore, the feed/food safety 

and nutritional quality of this product is comparable to conventional alfalfa. 

The processing of KK179 forage into animal feed 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 demonstrated 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 originating from conventional alfalfa. 
Monsanto Company 12-AL-246U 109 of 407 

VII. PHENOTYPIC, AGRONOMIC, AND ENVIRONMENTAL 

INTERACTIONS ASSESSMENT 

This section provides a comparative assessment of the phenotypic, agronomic, and 

environmental interaction characteristics 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 the trait for reduced G lignin 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 interaction characteristics of KK179 were 

evaluated in a comparative manner to assess plant pest potential. These assessments 

included evaluation of seed germination characteristics, plant growth and development 

characteristics, observations of plant responses to abiotic stress, plant-disease and plantarthropod interactions, pollen characteristics, flower characteristics, and plant-symbiont 

interactions. Results from these assessments demonstrate that KK179 does not possess 

1) increased weediness characteristics; 2) increased 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. Characteristics Measured for Assessment 

In the phenotypic, agronomic, and environmental interactions assessment 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, including differences from the unmodified 

recipient organism that would “substantiate that the regulated article is unlikely to pose a 

greater plant pest risk than the unmodified organism from which it was derived.” As part 

of the characterization of KK179, data were collected 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) vegetative growth; 3) winter survival; 4) 

reproductive development, including pollen, flower, and seed characteristics; 5) lodging 

and seed retention on the plant; 6) plant response to abiotic stress and interactions with 

diseases and arthropods; and 7) plant-symbiont interactions. 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, C0-Syn1, which is a near-isogenic population produced via an 
Monsanto Company 12-AL-246U 110 of 407 

identical breeding path as KK179, with the exception 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 Appendices F-K and Tables F-1, G-1, H-1, I-1, J-1, and K-1) 

were included to provide a range of comparative values for each characteristic that 

represent the variability in existing conventional commercial alfalfa varieties. Data 

collected for the various characteristics from the conventional commercial reference 

varieties provide context for interpreting experimental results. 
Monsanto Company 12-AL-246U 111 of 407 

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

Characteristics Evaluated in North American Field Trials and Laboratory or 

Greenhouse Studies 

Data 

Category 

Characteristics 

measured 

(associated section 

where discussed) 

Evaluation timing1

(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 test2

)

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 test2

) 

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 plot1

 (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 soil surface to the 

uppermost node on the main stem of 

five representative plants 
Monsanto Company 12-AL-246U 112 of 407 

Table VII-1 (continued). Phenotypic, Agronomic, 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 

plot 
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Table VII-1 (continued). Phenotypic, Agronomic, 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 biotechnology-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-derived plant is developed from a conventional 

crop whose biological properties and plant pest potential are well-known. Familiarity 

considers the biology of the crop, the introduced trait, the receiving environment, and the 

interaction of these factors, and provides a basis for comparative environmental risk 

assessment between a biotechnology-derived plant and its conventional counterpart. 

Expert knowledge and experience with conventionally bred alfalfa were the basis for 

selecting appropriate endpoints and estimating the range of responses that would be 

considered typical for alfalfa. As such, KK179 was compared to the conventional 

control, C0-Syn1, in the assessment of phenotypic, agronomic, and environmental 

interaction characteristics. An overview of the characteristics assessed is presented in 

Table VII-1. A subset of the data relating 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 compared 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 evaluated for possible evidence of biologicallyrelevant changes and unexpected plant responses. No unexpected observations or issues 

were identified. Based on all of the data collected, 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 characterization data between a biotechnology-derived crop and the 

conventional control are interpreted in the context of contributions to increased plant 

pest/weed potential as assessed by USDA-APHIS. Under the framework of familiarity, 

characteristics for which no differences are detected support a conclusion of no increased 

plant pest/weed potential of the biotechnology-derived crop compared to the conventional 

crop. Characteristics for which differences are detected are considered in a step-wise 

method (Figure VII-1) or in a similar fashion. All detected differences for a 

characteristic are considered in the context 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: 

 
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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 answer is “yes” or “uncertain,” the subsequent 

step is considered.

Steps 1 and 2 - Evaluate Detected Statistically Significant Differences 

Data on each measured characteristic are statistically 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 multiple environments are pooled in the combinedsite analysis are considered not biologically meaningful in terms of plant pest/weed 

potential and, therefore, are not further considered in subsequent steps. 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 detected 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 1
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characteristic is assessed relative 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 characteristic for a biotechnology-derived crop is outside the 

variation of the conventional commercial reference varieties, the mean value of the 

biotechnology-derived crop is assessed relative to known values common for the crop, 

e.g., published values. 

Step 5 - Plant Pest Potential 

If the mean value of the characteristic for a biotechnology-derived crop is outside the 

range of values common for the crop, the detected 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 detected in other measured characteristics, and 

potential for and effects of trait introgression into any populations growing outside of 

cultivated environments or into a sexually-compatible species. 

VII.B.2. Interpretation of Environmental Interactions Data 

Plant responses to abiotic stress, disease damage, and arthropod damage were 

qualitatively assessed using a continuous scale of increasing damage severity to 

determine a range of responses observed across four replications. The biotechnologyderived crop and the conventional control were considered not different in plant response 

if the range of damage symptoms observed for the biotechnology-derived crop 

overlapped with the range of symptoms observed for the conventional control at a site. 

Any observed differences between the biotechnology-derived crop and 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 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 (alfalfa weevil and potato leafhopper) was further 

assessed quantitatively, and data were analyzed within individual sites and pooled across 

sites in a combined-site analysis. Statistically significant differences detected between 

the biotechnology-derived crop and the conventional 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 and sites. Statistically significant differences 

between the biotechnology-derived crop and the 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 other observation times and sites. 
Monsanto Company 12-AL-246U 117 of 407 

Differences that were not consistently detected 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 Phenotypic, Agronomic, and Environmental 

Interaction Characteristics of KK179 

This section provides the results of comparative assessments conducted in replicated 

laboratory, greenhouse, and field studies to provide a detailed phenotypic, agronomic, 

and environmental interactions description of KK179. The KK179 characteristics 

evaluated in these assessments included: seed germination and dormancy characteristics 

(Section VII.C.1.); plant phenotypic and environmental interaction observations under 

field conditions of plants managed for forage production (Section VII.C.2.1. and Section 

VII.C.2.2); plant phenotypic observations under field conditions of plants managed for 

seed production (Section VII.C.2.3.); reproductive characteristics (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 weediness 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, and several dormancy mechanisms can occur in 

seeds (Anderson, 1996; Copeland and McDonald, 2001). Information on germination 

and dormancy characteristics is therefore useful when assessing a plant for increased 

weediness potential. To assess germination characteristics, standardized germination 

assays are routinely used. The Association of Official Seed Analysts (AOSA), an 

internationally recognized seed testing organization, recommends a temperature of 20 °C 

as optimal for testing the germination and dormancy characteristics 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 impermeable to water (Bass et al., 1988; Rolston, 

1978). The production environment has a great impact 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 seed and poor germination, i.e., below 70%, 

is unlikely to be consistently observed in the field (AOSCA, 2009). 

A comparative assessment of seed germination and dormancy characteristics was 

conducted on KK179 and the conventional control. Four conventional commercial 

reference varieties were also included to provide a range of germination and dormancy 
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characteristic values representative of commercial alfalfa varieties. Descriptions of the 

evaluated germination and dormancy characteristics 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 evaluation are presented in Appendix F. 

All starting seed were produced in the artificial 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-scarified and scarified seed were evaluated. 

Non-scarified seed were used to assess the germination and dormancy characteristics of 

KK179, without regard to a specific mechanism of dormancy. Scarified seed were used 

to assess whether any other seed dormancy effects, besides a hard seed coat, were 

introduced with the trait for reduced G lignin and total lignin. In addition to the AOSArecommended temperature of 20 °C, non-scarified 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 percent abnormal germinated, dead, and viable firm-swollen 

seed (Table VII-2). KK179 had a higher percent normal germinated seed than the 

conventional control (86.0% vs. 72.5%), and the mean for KK179 for percent normal 

germinated seed was slightly above the range 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 (See Figure VII-1, Step 4, answer “no”). KK179 also had a 

lower percent viable hard seed than the control (13.0% vs. 25.3%) at 20 °C, although the 

mean for percent viable hard seed was within the reference range (12.5% – 24.4%). 

Therefore, the difference in percent viable hard seed at 20 °C is not expected to be 

biologically meaningful in terms of plant pest/weed potential (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 and viable firm-swollen seed. At 10 °C, KK179 had higher 

percent germinated seed than the conventional control (86.5% vs. 74.1%) and 

concurrently lower percent viable hard seed than the conventional 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 commercial reference varieties (75.8% – 88.8% 

and 11.2% – 23.0%, respectively). At 30 °C, KK179 had higher percent germinated seed 

than the conventional control (87.3% vs. 74.9%) and concurrently lower percent viable 

hard seed than the conventional 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%, respectively). Therefore, the differences in percent 

germinated and viable hard seed at 10 °C and 30 °C are not expected to be biologically 

meaningful in terms of plant pest/weed potential (See Figure VII-1, Step 3, answer “no”). 
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Increased germination and reduced seed dormancy, regardless of the specific mechanism 

of dormancy, i.e., hard seed coat or other mechanism, would likely shorten the 

persistence of seed in the soil. Long-term persistence of seed in the soil is a characteristic 

common to some weed species, and for some perennial weeds, has been associated with a 

high percentage of dormant seed (Conn and Werdin-Pfisterer, 2010). Short-term 

persistence, on the other hand, has been associated with a high initial rate of germination 

(Hesse et al., 2007). Increased germination and reduced dormancy would reduce longterm persistence and thereby reduce the potential for weediness. Moreover, increased 

germination and reduced seed dormancy 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 germination (Bass et al., 1988; Klos 

and Brummer, 2000). Therefore, the differences 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 significantly higher percent normal germinated seed than the 

conventional control (93.5% vs. 89.8%) and this value was slightly lower than the 

reference range (93.8% – 98.0%). However, the mean for KK179 for percent 

germination of scarified seed is consistent with published germination rates of scarified 

alfalfa seed (93 – 98%) (Hall et al., 1993). Therefore, the difference in percent 

germinated seed at 20 °C is not expected to be biologically meaningful 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 dormancy effects introduced with the trait. KK179 and the 

conventional control did not exhibit consistent differences in any of the assessed 

characteristics across different temperature regimes following scarification. Therefore, it 

is not expected that additional dormancy effects were introduced with the trait for 

reduced G lignin and total lignin. 

While the exact nature of seed coat impermeability is not known for alfalfa, lignin is 

known to be present in the seed coat (Rolston, 1978); and if altered, could be 

hypothesized to alter the germination and dormancy characteristics of alfalfa seed. For 

example, a study with another 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 the 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 characteristics 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 increased plant pest/weed potential compared to 

conventional alfalfa. 
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Table VII-2. Comparison of Germination Characteristics of Non-scarified Seed of 

KK179 and the Conventional Control 

Temperature Germination Mean %1 (SE)

Regime Category KK179 Control Reference Range2

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., scarified 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. 

 
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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 Range2

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 control, or a conventional commercial reference variety) 

and treatment (i.e., scarified 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.
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VII.C.2. Field Phenotypic, Agronomic, and Environmental Interaction 

Characteristics 

Phenotypic, agronomic, and environmental interactions of plants managed for forage and 

seed production were evaluated under field conditions of forage and seed production as 

part of the plant characterization assessment of KK179. These data were developed to 

provide USDA-APHIS with a detailed description 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 seek deregulation, a petitioner must submit “a 

detailed description of the phenotype of the regulated article.” This information is being 

provided to assess whether there are phenotypic differences between KK179 and the 

conventional control that may impact its plant pest/weed potential. Specific 

characteristics that are related to weediness, e.g., lodging, and split pods, were used to 

assess whether there is a potential increase in weediness of KK179 compared to 

conventional alfalfa. Environmental interactions including plant response to abiotic 

stress, disease damage, specific arthropod-related 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 and seed production demonstrated that the 

introduction of the trait for reduced G lignin and total lignin did not meaningfully alter 

the plant pest/weed potential of KK179 compared to conventional alfalfa. Furthermore, 

the lack of meaningful differences in environmental interactions 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 Agronomic Characteristics of KK179 Managed for 

Forage Production 

A field study was conducted, beginning in 2010 and continuing through 2012, to evaluate 

phenotypic and agronomic characteristics of KK179 compared to the conventional 

control when managed under the agronomic practices 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 characteristics of KK179 compared to the conventional 

control. 

Phenotypic and agronomic data were 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 replications. 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 control, and five conventional 
Monsanto Company 12-AL-246U 123 of 407 

commercial reference varieties were evaluated. A total of twelve unique conventional 

commercial reference varieties were evaluated among the 10 sites (Table G-1). 

The trials at each site were managed according 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 season at a site depends on numerous factors 

including agro-ecological environment, 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 planting in 2010 through the winter survival 

assessment in the spring of 2011. The second year 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 within each crop growth cycle, i.e., the period of forage 

growth between cuttings, and at each cutting. 

Descriptions of the evaluated phenotypic characteristics and the timing 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 below. The results of the agronomic and 

phenotypic assessment demonstrate that there 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 pest/weed potential of KK179 compared to 

conventional alfalfa. 
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Table VII-4. Field Phenotypic Evaluation Sites for KK179 during 2010-2012 

Site Code County, State/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. 

 
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In the combined-site analysis of the phenotypic characteristics assessed in the first year 

(2010-2011), no statistically significant differences (5% level of significance) were 

detected between KK179 and the conventional control for any of the assessed 

characteristics, including seedling emergence, early season vigor; lodging, crop growth 

stage, or regrowth after cutting for cutting #1; lodging, crop growth stage, forage yield or 

regrowth after cutting for cutting #2; lodging, crop growth stage, or forage yield for 

cutting #3; fall plant height, total forage yield; spring vigor, 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 combined-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 phenotypic characteristics assessed in the second 

year (2011-2012), no statistically significant differences were detected between KK179 

and the conventional control for any of the assessed characteristics, 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 recovery, 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 significant differences 

were detected between KK179 and the conventional control for any of the assessed 

characteristics in the combined-year analysis (Table VII-7). 

Lodging was a characteristic used to specifically assess the potential weediness of 

KK179. No differences were observed between KK179 and the conventional control for 

lodging within any crop growth cycle assessed over the two years. 

Based on the assessed phenotypic and agronomic characteristics within each individual 

year and across years, the results demonstrate that there were no unexpected changes in 

phenotype indicative of increased plant pest/weed potential of KK179 compared to the 

conventional control (See Figure VII-1, Step 2, answer “no”). 

 
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Table VII-5. Combined-Site Comparison of KK179 to the Conventional Control 

for Phenotypic and Agronomic Characteristics in the First Year (2010-2011) of 

Forage Production 

KK179 Control Reference Range1

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 4.4
Monsanto Company 12-AL-246U 127 of 407 

Table VII-5. Combined-Site Comparison of KK179 to the Conventional Control 

for Phenotypic and Agronomic Characteristics in the First Year (2010-2011) of 

Forage Production (continued) 

KK179 Control Reference Range1

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 early 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 detected between KK179 and the conventional control (α=0.05) 

using ANOVA.

† No statistical comparisons were made because 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 Characteristics in the Second Year (2011-2012) of 

Forage Production 

KK179 Control Reference Range1

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 4.9

 
Monsanto Company 12-AL-246U 129 of 407 

Table VII-6 (continued). Combined-Site Comparison of KK179 to the Conventional 

Control for Phenotypic and Agronomic Characteristics in the Second Year (2011-

2012) of Forage Production 

KK179 Control Reference Range1

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 detected between KK179 and the conventional control (α=0.05) 

using ANOVA.

1

Reference range is the minimum and maximum mean values among the conventional commercial 

reference varieties. 
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Table VII-7. Combined-Site and Combined-Year Comparison of KK179 to the 

Conventional Control for Phenotypic and Agronomic Characteristics during Forage 

Production 

KK179 Control Reference Range1

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 detected between KK179 and the 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 Characteristics 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 environmental interactions were conducted as part of 

the plant characterization of KK179 managed under forage production conditions. In the 

2010-2012 field trials conducted for evaluation of phenotypic and agronomic 

characteristics of KK179, data were also collected 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 environmental 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 conventional control. In addition, multiple 

conventional commercial reference 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-related damage, alfalfa weevil damage, potato 

leafhopper damage, and pest- and beneficial-arthropod abundance support 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 or to have an effect on the environment for KK179 

compared to conventional alfalfa. 

VII.C.2.2.1. Qualitative Environmental Interactions Assessment 

KK179 was compared to the conventional control for qualitative environmental 

interactions in 2010 and 2011. Qualitative assessments were conducted at 10 sites and 

included plant responses to abiotic stressors, disease damage, and arthropod damage. 

The number of observation times at a site depended on the number of cuttings at that site. 

Assessments were conducted three to four times during the 2010 growing season and four 

to five times during the 2011 growing season. 

Plant responses to abiotic stressors, disease damage, and 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 collected from each plot 

using a 0 – 9 scale of increasing severity of observed damage for each stressor. This 

scale was utilized to allow for the evaluation 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-specific 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 replications at a site (e.g, none to slight). For 

a particular stressor, all comparisons of the range of responses for KK179 to the range of 

responses for the conventional control across all observation times and sites are reported. 
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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 interactions assessments, and detailed results of 

the qualitative data comparisons are presented 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 responses were observed between KK179 and the 

conventional control for any of the 93 comparisons of plant response to abiotic stressors, 

including drought, flood, frost, hail, heat, nutrient 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 the conventional control for any of the 93 comparisons for 

plant damage caused by diseases, including Anthracnose, bacterial wilt, black stem, 

damping-off, downy mildew, Fusarium wilt, leaf spots, root rot, Sclerotinia crown and 

stem rot, stem nematode, and Verticillium wilt (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 comparisons for plant damage caused by 

arthropods, including alfalfa caterpillar, alfalfa weevil, aphid, armyworm, blister beetle, 

cutworm, grasshopper, meadow spittlebug, plant bug, potato leafhopper, spider mite, and 

thrips (Table VII-8 and Table G-10). 

In 2011, no differences in the range of responses were observed between KK179 and the 

conventional control for any of the 129 comparisons of plant response to abiotic stressors, 

including drought, frost, hail, heat, heaving, 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 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 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 thrips (Table VII-9 and 

Table G-16). 

The lack of differences observed between KK179 and the conventional control for plant 

responses to abiotic stressors, disease damage, and arthropod-related damage in multiple 

environments across the U.S. and Canada supports the conclusion that the introduction of 

the trait for reduced G lignin and total lignin 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 conventional control (See Section VII.B.2.) 

. 
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Table VII-8. Summary of Qualitative Environmental 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 

20101

Abiotic stressors 93 93 

Disease damage 93 93 

Arthropod-related damage 96 96 

Total 282 282 
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Table VII-9. Summary of Qualitative Environmental 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 

20111

Abiotic stressors 129 129 

Disease damage 129 129 

Arthropod-related damage 129 129 

Total 387 387 
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VII.C.2.2.2. Quantitative Environmental Interactions Assessment 

Quantitative arthropod assessments were conducted at three sites (ILCL, KSLA, NYNR) 

and included alfalfa weevil damage, potato leafhopper damage, and pest- and beneficialarthropod abundance. Each quantitative assessment occurred three to four times in 2010 

and four to five times in 2011 based on the number of cuttings at each site. Alfalfa 

weevil and potato leafhopper damage were assessed in each plot from 10 nonsystematically selected 6 × 6 inch areas using an arthropod-specific 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 analysis and a combined-site analysis were 

conducted, in which the data were pooled among the three sites for each single year 

separately. 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 specific arthropod damage assessments, and detailed results of the 

individual-site data comparisons are presented and discussed in Appendix G (Tables 

G-11 and G-17). The results of the combined-site analysis are summarized below. 

In the combined-site analyses in both 2010 and in 2011, no statistically significant 

differences were detected between KK179 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 difference 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 were also collected at the three sites in 

2010 and in 2011. Variations in temporal activity and geographical distribution of 

arthropod taxa occur between sites, therefore, only individual-site analyses were 

conducted for arthropod abundance data in each year. Additional details of the arthropod 

abundance assessments and detailed results of the individual-site data comparisons are 

provided in Appendix G (Tables G-12, G-13, G-18, and G-19). The results of these 

analyses are summarized below 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, alfalfa looper, false chinch bug, green cloverworm, garden 

webworm, thrips, damsel bug, ladybird beetle, parasitic wasps, and lacewing. No 

statistically significant differences were detected between KK179 and the conventional 

control for 65 out of 69 comparisons, including 39 pest-arthropod comparisons and 30 

beneficial-arthropod comparisons (Tables G-12 and G-13). Of the four differences 

detected, there were two statistically significant differences (α=0.05) in pest-arthropod 

abundance and two statistically significant differences in beneficial-arthropod abundance. 
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In the assessment of pest arthropod-abundance, 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 Lygus 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 conventional commercial reference 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 arthropod abundance were not considered 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 control. 

In the assessment of beneficial-arthropod abundance, KK179 had significantly lower 

abundance of spiders than the conventional control 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 reference 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 biologically meaningful in terms of plant pest/weed potential 

of KK179 compared to the conventional 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, blister beetle, green cloverworm, Lygus bug, 

meadow spittlebug, potato leafhopper, thrips, soybean looper, spiders, ladybird beetle, 

lacewing, nabids, and chalcid wasps. No statistically significant differences were 

detected between KK179 and the conventional 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 the 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 considered 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 control (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 Conventional Control in the First Year (2010) 

Damage (0-10 rating scale)

KK179 Control Reference Range1

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 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. 

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 Range1

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 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. 

1

Reference range is the minimum and maximum mean values observed among the four conventional 

commercial reference varieties.
Monsanto Company 12-AL-246U 

Table VII-12. Summary of Arthropod Abundance Assessments and Detected Differences in 2010 and 201Summary of Statistical Comparisons1 Summary of Detected Differences2Arthropod Abundance Assessment 

Number of sites 

Number of comparisons across sites 

Number of comparisons where no differences were detected Arthropod Site 

Collection 

Number 

Withirefererange

2010 2010Pest 3 39 37 Potato leafhopper ILCL 1 No 

Lygus bug KSLA 2 No 

Beneficial 3 30 28 Spiders ILCL 2 Yes 

 KSLA 2 Yes 

2011 2011

Pest 3 50 49 Alfalfa weevil ILCL 1 Yes 

Beneficial 3 33 33 – – – – 

1Quantitative arthropod abundance assessments were statistically analyzed at 

α=0.05 using ANOVA. 2Five statistically significant differences were detected. These differences are further 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 Agronomic Characteristics of KK179 Managed for 

Seed Production 

Alfalfa seed production requires different environmental conditions and agronomic 

practices than alfalfa grown for forage. Alfalfa seed production is limited globally to 

more arid regions, and more than 85% of all 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 are typically planted in rows and are seeded 

at a much lower density than forage production 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 characteristics 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 established in 2010 in Canyon County, Idaho, which 

represents an environment that is appropriate 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 control, and seven conventional 

commercial reference varieties were uniformly managed under the agronomic practices 

for seed production, in order to assess whether the introduction of the trait for reduced G 

lignin and total lignin altered the phenotypic and agronomic characteristics of KK179 

compared to the conventional control. 

Descriptions of the evaluated phenotypic characteristics and the timing of the evaluations 

during seed production are listed in Table VII-13. The details of the materials and 

experimental methods used in this evaluation are presented in Appendix H. 

No statistically significant differences (α=0.05) were detected between KK179 and the 

conventional control for seedling establishment, seedling vigor, seed weight, seed per 

pod, or seed yield (Table VII-13). Additionally, no statistically significant differences 

were detected between KK179 and the conventional control for lodging or split pods, 

both characteristics related to weediness. Thus, the results demonstrate that there were 

no unexpected changes in phenotype indicative of increased plant pest/weed potential or 

potential to have an effect on the environment for KK179 compared to the conventional 

control (See Figure VII-1, Step 1, answer “no”). 

The results of the agronomic and phenotypic assessment 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 result in increased plant 

pest/weed potential of KK179 compared to conventional alfalfa. 
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Table VII-13. Comparison of KK179 to the Conventional Control for Phenotypic 

and Agronomic Characteristics during Seed Production 

Characteristic (units)

Mean (SE) Reference Range1

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 the receiving species. 

Information on reproductive characteristics including pollen morphology and viability 

and flower morphology are pertinent to this assessment, and therefore were assessed for 

KK179. In addition, characterizations of pollen and flowers produced by KK179 and the 

conventional control are relevant to the plant pest 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 collected from KK179 compared to that of the 

conventional control was also assessed. KK179, the conventional control, and four 

conventional commercial reference varieties were grown under similar agronomic 

conditions in a greenhouse. The trial was arranged in a randomized complete block 

design with four replications, with 10 plants of each material per replication. 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 and morphology characteristics and the timing of 

the evaluations are listed in Table VII-1. The details of the materials and experimental 

methods used in this evaluation are presented in Appendix I. 

No statistically significant 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 general pollen morphology were observed between 

KK179 and the conventional control (Figure I-1). 

The pollen 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 neither pollen viability nor morphology of KK179 were 

altered compared to conventional alfalfa. 
Monsanto Company 12-AL-246U 143 of 407 

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

Control 

KK179 Control Reference Range1 

Pollen Characteristic Mean (SE) Mean (SE) Minimum Maximum 

Viability2 (%) 89.2 (2.72) 88.4 (1.06) 86.0 89.9 

Diameter3 (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 assess the morphology of flowers collected from 

KK179 compared to those of the conventional control. Flowers were collected from 

KK179, the conventional control, and four conventional commercial reference varieties 

grown under similar agronomic conditions in a Wisconsin field trial. The field trial was 

arranged in a randomized complete block design with four replications. Flowers were 

collected from the field once all plots had reached approximately 10% bloom. 

Descriptions of the evaluated phenotypic characteristics and the timing of the evaluations 

are listed in Table VII-1. The details of the materials and experimental methods used in 

this evaluation are presented in Appendix J. 

No statistically significant 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, keel petal length, calyx tube diameter, sexual 

column length, and wing petal length (Table VII-15). Furthermore, no visual differences 

in flower color class, gross raceme morphology, or gross flower morphology were 

observed between KK179 and the conventional 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 morphology 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 Range2

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 class3 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, standard 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 differences 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 statistically analyzed is the minimum and maximum mean 

values from among the conventional commercial reference varieties. The reference range for those 

characteristics that were not statistically analyzed consists of the minimum and maximum observed ratings 

from among the conventional commercial reference varieties. 

3

Flower color classification (USDA-ARS, 1972). 
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VII.C.4. Symbiont Interactions 

As part of the plant pest risk assessment, USDA-APHIS considers the impact of the 

biotechnology-derived crop on plant pest potential and the environment as well as on 

agricultural or cultivation practices compared to its conventional counterpart. Potential 

changes in the symbiotic relationship between 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 requirement (Lanyon and Griffith, 

1988). Thus, the purpose of this evaluation was to assess whether the introduction of the 

trait for reduced G lignin and total lignin altered the symbiotic interaction of KK179 with 

Sinorhizobium meliloti compared to that of the conventional control. 

KK179, the conventional control, and six conventional commercial reference varieties 

were grown under similar agronomic conditions in a greenhouse. Descriptions of the 

evaluated phenotypic characteristics are listed in Table VII-16. The details of the 

materials and experimental methods used are presented in Appendix K. 

No statistically significant differences (α=0.05) were detected between KK179 and the 

conventional control for each measured parameter, including dry weight of nodules, root 

material, shoot material, shoot percent total nitrogen, and shoot total nitrogen (Table 

VII-16). 

Based on the assessed characteristics, the lack of statistical differences between KK179 

and the conventional control indicate that introduction of the trait for reduced G lignin 

and total lignin did not alter the symbiotic relationship between S. meliloti and KK179 

compared to that of conventional alfalfa. Thus, the results demonstrate that there were no 

unexpected changes indicative of increased plant pest/weed potential or potential to have 

an effect on the environment for KK179 compared to the conventional control. 
Monsanto Company 12-AL-246U 147 of 407 

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

Control 

Mean (SE) Reference Range1

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 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 six conventional 

commercial reference varieties.
Monsanto Company 12-AL-246U 148 of 407 

VII.D. Conclusions for Phenotypic, Agronomic, and Environmental Interactions 

Evaluations 

Comparative plant characterization data between a biotechnology-derived crop and the 

conventional control are interpreted in the context of contributions to increased plant 

pest/weed potential as assessed by USDA-APHIS. Under the framework of familiarity, 

characteristics for which no differences are detected support a conclusion 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 their significance. 

An extensive and robust set of agronomic, phenotypic, and environmental interactions 

data, including specific weedy characteristics, 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, considered within the context 

of the variation among the conventional commercial reference varieties. These 

assessments included seven general data categories: 1) seed germination, dormancy, and 

emergence; 2) vegetative growth; 3) winter survival; 4) reproductive development 

including pollen, flower, and seed characteristics; 5) lodging and seed retention on the 

plant; 6) plant response to abiotic stress and interactions with diseases 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 increase in weediness of KK179 compared to 

conventional alfalfa. 

Results from the phenotypic, agronomic, and environmental interactions assessments 

comparing KK179 and the conventional control demonstrate that KK179 does not 

possess: 1) increased weediness characteristics; 2) increased susceptibility or tolerance to 

specific abiotic stress, diseases, or arthropods; or 3) characteristics that would confer a 

plant pest risk compared to conventional alfalfa. Therefore, based on the results of 

multiple assessments discussed above and presented in the appendices, the weight of 

evidence indicates that KK179 is not meaningfully 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 compared to conventional alfalfa. 
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VIII. U.S. AGRONOMIC PRACTICES 

VIII.A. Introduction 

As part of the plant pest assessment required by 7 CFR § 340.6(c)(4), impacts of 

deregulation on agricultural and cultivation practices 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 include the importance of alfalfa as a crop; 

alfalfa production; seed production; plant growth and development; practices for the 

establishment, management, and termination of alfalfa stands; pest management 

practices; alfalfa rotational crops; and volunteer and feral alfalfa management. 

Information on U.S. alfalfa agronomic practices can be found in the published literature 

and in USDA-APHIS Environmental Impact Statement for Roundup Ready alfalfa 

(Section III and Appendices G, H, and J) (USDA-APHIS, 2010). The overview 

presented here in Section VIII collectively discusses the agricultural practices from those 

sources. 

Information presented in Section VII demonstrated 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 magnitude 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 the 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 soils 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 production has two distinct growing phases: the 

establishment phase and the production phase associated with established stands (USDAAPHIS, 2010). Proper seedbed preparation, correct variety selection, optimum planting 

dates, optimum plant population, and good integrated pest management practices are 

important for optimizing the yield potential 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 established stand or seed that have germinated 

and emerged unintentionally in a subsequent rotational crop. Mechanical and chemical 

methods are also effective in removing or taking out old, thinning stands when rotating to 

other crops. Due to the lack of differences between KK179 and conventional alfalfa in 

phenotypic, agronomic, and environmental interaction 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 plants in rotational crops such as corn, soybean, sorghum, 

and wheat. The numerous control measures that are effective on conventional 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 for reduced G lignin and 

total lignin, no biologically meaningful phenotypic, compositional, or environmental 

interaction differences between KK179 and conventional alfalfa have been observed. 

Therefore, it is not anticipated that commercialization of KK179 in the U.S. would have a 

notable impact on current alfalfa 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 missionaries from Mexico into Texas and the 

Southwest region, and later into the British colonies 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 seed production. Alfalfa is generally regarded 

as the “queen of forages” because of its high 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 (USDA-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-lactating dairy cows. Alfalfa forage is stored as hay (bales 

at 18 to 20% moisture), haylage (round bale silage, baled at 50 to 60% moisture and 

wrapped in plastic), and silage (chopped and stored in silos) (USDA-APHIS, 2010). 

Grazing alfalfa in the vegetative state is practiced sometimes for dormant-season alfalfa 

stubble, a substitute for early or late season cutting, and rotational grazing during the 

season. However, grazing can cause gastrointestinal bloating in animals and result in 

stand maintenance problems with over-grazing. 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 consumption 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 variety of non-agricultural purposes, including 

rehabilitation of over-grazed rangelands, erosion-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. exports of alfalfa hay were valued (average 
Monsanto Company 12-AL-246U 151 of 407 

prices × volumes) at approximately $354 million 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 major importers of alfalfa hay and cubes 

(compressed bales of hay) from the western U.S. (Klonsky et al., 2008). 

Alfalfa is a deep-rooted and short-lived perennial plant with a long growing season. 

Alfalfa grows from early spring until late fall or early winter. Growth begins when the 

average temperature reaches 50°F and continues 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 ideal (Kansas State University, 1998). Finetextured soils are usually difficult to manage. In deep, well-aerated soil, roots may 

extend 8 to 12 feet deep (Kansas State University, 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 considered naturally drought-tolerant. It has a high 

water requirement under normal conditions, using in excess of 40 inches of water during 

the season (Kansas State University, 1998). During the summer months, the water use is 

6 to 7 acre-inches per ton of forage. The water 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 development of root and crown diseases, and shallow 

water tables limit root growth (Kansas State University, 1998). 

Alfalfa is grown for forage in all continental 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). Currently, 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 alfalfa hay have been harvested annually over 

the past 10 years (Table VIII-1). Approximately 2.5 to 3.3 million acres (12-13% of the 

harvested acres) are seeded annually for new 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 estimate based on multiplying average prices with 

production volumes and does not correspond to actual 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 alfalfa hay harvested acres in the U.S. 

Source: (USDA-NASS, 2010). 

Table VIII-1. Alfalfa Hay Production in the U.S. from 2000 to 20101

Year 

Seeded Alfalfa 

Acres (000) 

Harvested 

Acres (000) 

Production 

(000 tons) 

Yield 

(tons/acre) 

Value of 

Production2

($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 discussion, alfalfa production (alfalfa and alfalfa 

mixtures) is divided into six major alfalfa growing regions: North 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), Pacific 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 haylage were harvested in 2010. The largest 

acreage of alfalfa (hay and haylage) is grown 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 reported in the Pacific Northwest 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 Central 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 value 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 20101. 

Region/State 

Harvest Acres 

(000) 

Average 

Yield 

(tons/acre) 

Production 

(000 tons) 

Value of Hay 

Production2

($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 3.24 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 

States3

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 3.34 13,982 1,350,162 
Monsanto Company 12-AL-246U 155 of 407 

Table VIII-2 (continued). Alfalfa and Alfalfa Mixtures Forage (Hay and 

Haylage) Production by State in 20101. 

Region/State 

Harvest Acres 

(000) 

Average 

Yield 

(tons/acre) 

Production 

(000 tons) 

Value of 

Production2

($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 3.94 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 2.84 11,127 1,100,551 

Pacific Northwest Region 

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 4.64 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 6.84 10,027 1,321,460 

 

U.S. Totals 22,070 3.64 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 regions as a low-value rotation or pasture crop 

and used primarily as a supplement to other 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, especially in the high 

production areas of California. Dairy producers 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 categories 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 mixtures. 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-quality hay are frequently acceptable for nonlactating dairy cows and meat-producing livestock. 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). Whereas in the western regions of the U.S., 

over 95 percent of the alfalfa 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 including acid detergent 

fiber (i.e., cellulose and lignin content), neutral detergent fiber (i.e., lignin, cellulose and 

hemi-cellulose content), crude protein, total digestible nutrients, and relative feed value 

(Putnam et al., 2008b; USDA-APHIS, 2010). Certain 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 heating, or unpleasant odor. These 

factors can significantly affect nutritional value and overall forage quality. The USDA’s 

Agricultural Marketing Service (USDA-AMS) uses the grades of supreme, premium, 

good, fair, and utility to regularly report average prices for alfalfa in various states 

(USDA-APHIS, 2010). Prices vary significantly by quality grade and region. Prices paid 

for supreme grade alfalfa hay averaged $160.63/ton, while fair grade 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-marketing strategy targets certain markets such as 

the dairy industry to get top dollar for a consistently high-quality alfalfa product. 

Differentiation can include designations such as certified weed-free hay, certified organic 

hay, special cubed alfalfa containing grasses for horses, and low-potassium hay. The 

additional service strategy involves providing special service for the customer such as 

making hay available to the customer on demand, meeting specific baling requirements, 

developing favorable payment or delivery contracts, 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 perennial crop and harvested over several years, the analysis of 

production costs for alfalfa are generally presented in two parts: 1) costs associated with 

stand establishment, and 2) the annual costs during the production years. Table VIII-3 

provides a summary of the variable and fixed costs for producing non-irrigated, 

conventional alfalfa in the North Central region based on the Ag Decision Maker 

spreadsheet by Iowa State University (Johanns, 2012). Assuming two cuttings and two 

tons yield per acre from a spring seeding, variable and fixed costs were $268.70 and 

$199.00 per acre, respectively, during the establishment year, with a total cost of $467.70 

per acre. There was a net return of $31.30 per acre over variable costs, but a negative net 

return of $167.70 over total costs. Assuming three cuttings, four tons yield per acre per 

year, and three years of production after establishment year, variable and fixed costs were 

$225.30 and $189.50 per acre, respectively, during 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 costs and gross returns for both establishment 

and production (four years) net return per year 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 Valley in 2008 (Mueller et al., 2008a). These 

data are presented in Table VIII-4. In these studies, the fall-seeded alfalfa was assumed 

to produce a total of nine cuttings yielding eight tons of hay and three green tons (one-ton 

hay equivalent) of haylage per acre. Operating costs and total overhead costs were $385 

and $375 per acre ($760 total), respectively, for establishing alfalfa. Annual operating 

costs and total overhead costs were $708 and $654 per acre ($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 production 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 equivalent) 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 years hay production after establishment year; two 

cuttings and 2 tons/acre in establishment year; three cuttings and 4 tons/acre 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). Supporting information: 1200 acre farm with 300 acres of alfalfa; fall 

seeding; 3-year stand life for alfalfa; irrigation - 8 acre-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. agriculture. 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 varieties that were distributed to 

growers for use. Seed was saved by growers and later sold to neighbors; however, the 

desirable traits of the varieties often were lost through random genetic changes and 

contamination with other crop and weed seed (Sundstrom et al., 2002). The value of seed 

quality (including genetic purity, vigor, and presence 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 certification agencies to ensure that purchasers 

who received certified seed could be assured that the seed met established seed quality 

standards (Bradford, 2006). The federal government passed the U.S. Federal Seed Act of 

1939 to recognize seed certification and the establishment of official certifying agencies. 

Regulations first adopted in 1969 under the Federal Seed Act recognize land history, field 

isolation, and varietal purity standards for foundation, registered, and certified seed. 

Under international agreements such as the Organisation for Economic Co-operation and 

Development (OECD), the U.S. and other countries mutually recognize minimum seed 

quality standards (Bradford, 2006). The Association of Official Seed Certifying 

Agencies (AOSCA) represents state and private 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 classes: 1) breeder, 2) foundation, 3) registered, 

and 4) certified (AOSCA, 2011). Breeder seed is seed that is directly controlled by the 

originating or sponsoring plant 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 seed 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 seed. While not all alfalfa seed sold to 

growers is officially certified, commercial alfalfa 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 overview 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 alfalfa varietal seed for commercial planting. The need for 

such systems arose from the recognition that the quality of improved varieties quickly 

deteriorated in the absence of monitoring for quality and genetic purity (CAST, 2007). 

Seed certification programs were initiated in the 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 inspections, and seed labeling standards for seed 

certification. Seed certification services are available through various state agencies 

affiliated with AOSCA. Large seed producers implement their own seed quality 

assurance programs that go beyond mandated quality standards. However, large seed 
Monsanto Company 12-AL-246U 161 of 407

producers often will utilize the services of state certifying agencies as a third party source 

to perform certain field inspections and audits. 

In contrast to the broad geographic distribution 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 approximately 62 million pounds of seed with an 

average yield of approximately 510 pounds per acre. Historically, California has been 

and remains the largest producer of alfalfa seed, but acreage has declined in recent years 

due to economics, environmental constraints, and regulatory issues (USDA-APHIS, 

2010). Ninety-five percent of the seed produced in California is of non-dormant varieties 

(FD 7-10) while the Pacific Northwest produces 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-certified alfalfa seed, respectively, were exported from the 

U.S. in 2011 (WASGA, 2011 ). The largest quantities 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 production 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 production 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 consumption as alfalfa sprouts (USDA-APHIS, 

2010). Alfalfa seed is not consumed as a grain 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-pollinate (Viands et al., 1988). Alfalfa is adversely affected 

by inbreeding, i.e., self-fertilized plants commonly demonstrate 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 deleterious recessive alleles 

that occur as a result of self-pollination and/or pollination among close relatives. 

Therefore, commercial alfalfa breeding programs are structured to avoid significant 

inbreeding and the resulting negative effects of inbreeding depression (Rumbaugh et al., 

1988). 

Alfalfa varieties are primarily bred for forage 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 isolation to form the 

breeder generation seed (Figure VIII-2). The breeder seed of commercial alfalfa varieties 

is produced by the random intercrossing (open pollination) of all parent plants. An 

alfalfa variety is maintained through multiple seed generations beyond breeder seed via 

the open pollination of their progeny in isolation from other alfalfa varieties or pollen 

sources. Plant varieties bred in this way are called synthetic varieties (Rumbaugh et al., 

1988). 

Individual plants within a synthetic variety are genotypically and phenotypically 

heterogeneous, i.e., no two individuals within the variety are exactly alike. Synthetic 

alfalfa varieties are closed populations that segregate, within a defined range, for most 

morphological traits and naturally occurring genetic markers. Because alfalfa varieties 

are segregating heterogeneous populations, alfalfa varieties are routinely described in 

terms appropriate to populations (mean or % trait expression). For example, alfalfa 

variety registration agencies require that the pest resistance of a variety be described as 

the mean percent of plants that express the segregating trait when the population is tested 

under standardized conditions. 
Monsanto Company 12-AL-246U 164 of 407

Figure VIII-2. Commercial synthetic alfalfa 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 alfalfa 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. The 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 intercross of Syn1 plants; and -3) Syn3 seed is 

produced from a random, isolated intercross of Syn2 plants. Breeder, foundation, 

registered, and certified seed classes are defined at the discretion of the plant breeder 

during the variety registration process. Breeder and foundation seed are typically Syn1 

and Syn2 generations, respectively. Commercial 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 

Population 1 Population 2 

“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 process 
Monsanto Company 12-AL-246U 165 of 407

Commercial variety testing and registration by alfalfa breeders typically use Syn1 and 

Syn2 generation seed to establish variety testing 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 generation test data are also 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 development, enhancement, and the use of standard 

breeding techniques typical for cross-pollinated crops. 

Alfalfa seed is produced by a number of companies that produce and sell seed, such as 

Forage Genetics International, Pioneer Hi-Bred, Dairyland Seed, and Cal/West Seeds. 

Seed companies in turn contract 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. Seed companies have 

processing facilities to clean, condition, and bag the harvested 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) certification standards, and therefore 

include internal and external audits (ISO, 2012). ISO standards ensure desirable 

characteristics of seeds and services, such as quality, safety, reliability, and efficiency. 

The ISO standards represent an international consensus on good management practices 

with the aim of ensuring that the organization can consistently deliver excellent products 

or services. The standards not only must meet customers’ and applicable seed regulatory 

requirements, but must aim to enhance customer satisfaction and achieve continual 

improvement of its performance in pursuit of these objectives (ISO, 2012). 

While many of the field operations and management practices for producing alfalfa seed 

are similar to normal alfalfa forage production, there are significant differences. Special 

attention is needed in certain areas to optimize yields and to produce seed with high 

quality, high germination rates, and high genetic purity. Fields selected must not have 

grown alfalfa on the land for a four-, three-, 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 region. This restriction 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 alfalfa plants the year prior to seed production 

(AOSCA, 2011). 

Successful seed production begins with proper stand 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 variety of the seed is documented (USDA-APHIS, 2010). 

Depending upon the relative market prices for forage versus common or variety not 

stated (VNS) alfalfa seed, a minor proportion of the U.S. alfalfa seed crop is produced 
Monsanto 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. This practice occurs sporadically in the Plains, 

Pacific Northwest, and Desert Southwest. “Catch-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 crop (Hower et al., 1999). Recommended 

planting dates are similar to forage production. Fall establishment predominates (58%) 

because first year seed yields in fall-established stands are greater than for spring 

seedings (Hower et al., 1999; Mueller, 2008). 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 irrigation and early cultivation for weed control (Mueller, 2008). The 

plant density required to optimize seed yields depends on row spacing and soil type, 

which influence growth and final size of the alfalfa plant. Successful stands have been 

established with seeding 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 yields with thinner stands are attributed to 

improved water use efficiency, pest control, and pollination. 

After stand establishment, fields are clipped in the spring. The clipback 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 controlling the alfalfa seed chalcid (Bruchophagus 

roddi) insect pest (Mueller, 2008). 

Field inspections are vital to ensure the alfalfa seed meets seed certification requirements, 

ISO certification standards, regulatory standards, and licensing agreement standards. 

Field inspections are conducted on seed production fields throughout the growing season 

to visually evaluate variety purity, ensure alfalfa plants are developing properly, and 

fields are maintained free of weeds, insects, and diseases. The fields are also mapped to 

ensure the seed field has the minimum isolation requirement as a physical barrier. 

Foundation seed fields must be isolated from alfalfa of different 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 certified seed. Since alfalfa for seed is often grown in wide 

rows, there is greater potential for weed invasion. All available weed control methods, 
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including herbicides, mechanical cultivation, and hand weeding are used to eliminate 

weeds including volunteer alfalfa during the growing season (Mueller, 2008). Herbicide 

use on alfalfa seed acres averaged 78% from 1988 to 1992 (Hower et al., 1999) and, 

undoubtedly, has increased in recent 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 control than post-harvest screening 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 herbicides 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 seed 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). Lygus 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 insect pest to manage in alfalfa seed fields. 

They feed on the reproductive parts of alfalfa plants causing premature drop of buds and 

flowers, seed deformation, and reduced seed viability. Seed producers monitor insect 

pest and beneficial insect populations throughout the growing season and implement 

appropriate insect management strategies. Multiple pesticide applications are usually 

required during the bloom and pollination period. Applicators must exercise special 

precautions and follow special label instructions to protect bee colonies that are present to 

facilitate the pollination necessary for seed production. 

Alfalfa flowers require tripping and cross-pollination for maximum 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). Alfalfa 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. Bee colonies are usually placed 

around seed fields when the alfalfa 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 plant material. This is accomplished by cutting 

and windrow curing or chemically desiccating 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 conditions. Seed is typically harvested once per 

year. 
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Harvested seed is conditioned to remove soil, weed seeds, and other debris. Separating 

machines utilizing the differences in physical characteristics of alfalfa seeds and the nonseed fractions complete the cleaning process. Seed scarification (mechanical or 

chemical) is performed on seed lots that contain high percentages of hard seed to improve 

germination (Mueller, 2008). All seed lots are tested for purity, germination, and noxious 

weed seed content before bagging and sale. 

Commercial certified alfalfa seed must meet state and federal seed standards and seed 

bag tag labeling requirements. AOSCA standards for certified alfalfa 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% other varieties (maximum) and 0.05% other 

kinds (maximum), and 80% germination (minimum) (AOSCA, 2011). The alfalfa 

industry historically sets a higher minimum germination rate for labeling purposes. State 

seed certification standards vary slightly from state to state and can be more restrictive 

than the seed standards of AOSCA. 

The phenotypic, agronomic, and environmental interaction assessments presented in 

Section VII have demonstrated that KK179 is not meaningfully different from 

conventional alfalfa. Therefore, if deregulated, KK179 seed would be produced in the 

same manner as certified alfalfa seed, such that it will meet all state and federal seed 

standards and labeling requirements. 

VIII.C. Alfalfa Forage Production 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 advance of planting the alfalfa crop. Many of the 

decisions in this area are made prior to or immediately after harvest of the previous crop. 

Rotations with other crops should be an integral part of a farm management program. 

Alfalfa provides soil improvement and other benefits to subsequent crops (Mueller and 

Teuber, 2008). Alfalfa is commonly rotated with wheat, corn, cotton, sugar beets, and 

vegetables. Alfalfa assimilates nitrogen through nitrogen fixation and can provide levels 

of nitrogen ranging from 40 to 60 pounds per acre to the crop that follows alfalfa in the 

rotation (Orloff, 2008). Vegetable growers realize the added value from alfalfa in the 

rotation by improving water infiltration, 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 alfalfa seedlings. Production costs, relative rate 

of return, and current market conditions will dictate which crops to rotate with alfalfa. 

Profitable alfalfa production begins with the selection of the proper sites or fields to plant 

alfalfa. Sites that provide adequate rooting depth, nutrition, aeration, and water, and have 

no salinity or alkalinity problems, can produce good forage yields with good 

management. Alfalfa is adaptable to a wide range of soil types, but requires a welldrained soil for optimum production (Orloff, 2008). Therefore, fields should be selected 
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that have good surface drainage and have soils with good internal drainage and lack 

subsoil impediments (Orloff, 2008; Undersander et al., 2011). Wet soils create 

conditions suitable for diseases that may kill seedlings, reduce forage yield, and kill 

established plants. Of major importance in the western alfalfa growing regions, is 

selecting sites that have an adequate supply of quality water available for season-long 

irrigation. In the arid Southwest region, irrigation supplies between 70 and 100% of the 

total crop water requirement (Hanson et al., 2008). Site selection should also take into 

account the previous crop and any soil residual herbicides that 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 traffic compaction from the previous season, 

improve water filtration and soil aeration, control weeds, loosen the soil for root 

penetration, and provide a suitable environment for the planting and germination of the 

seed (Hake et al., 1996; Mueller et al., 2008b). Proper field preparation before planting is 

critical because the alfalfa stand will be intensively managed and harvested for three to 

five years, or longer in some areas. Deep tillage 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 potential benefit to determine whether it is 

economically feasible. Land leveling 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 erosion on slopes using conventional and deep tillage systems, 

there is a great deal of interest in reduced-tillage alfalfa establishment in the North and 

East Central regions (Undersander 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 in practicing reduced-tillage 

seeding. No-till seeders are recommended in reduced tillage and no-tillage systems with 

residue levels above 35% (Undersander et al., 2011). Cultipacker seeders, which 

broadcast the seed on the soil surface and then press the seed into the soil with rollers, 

have been used extensively in conventional tillage systems because they provide 

consistently even seed depth placement and good seed-soil contact. However, 

cultipacker seeders do not perform well in residue levels above 35% (Undersander et al., 

2011). Weed control is more difficult in reduced-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, there is some interest in alfalfa establishment 

with no tillage because of the high potential for soil erosion on slopes with conventional 

tillage. Special attention must be given to soil fertility and pH in no-till alfalfa 

establishment. Fertilizer and lime requirements should be incorporated into the soil well 

in advance of the alfalfa establishment year in the cropping system. 

Maintaining optimum crop nutrition and proper lime 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 alfalfa to compete with weeds, and strengthens 

disease and insect resistance (Undersander et al., 2011). Seventeen elements are essential 
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in varying amounts for plant growth (Meyer 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. Cobalt is essential 

to legumes for nitrogen fixation. Soil testing 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 testing 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 potassium, sulfur, molybdenum, and boron. 

Adequate levels of nitrogen are almost always provided by symbiotic nitrogen-fixing 

bacteria (e.g., Rhizobium meliloti). The most common causes of nitrogen deficiency are 

poor inoculation and nodule formation. Alfalfa seed should be inoculated when planting 

into fields without a history of alfalfa production. Small amounts of nitrogen fertilizer 

(25 to 30 pounds per acre) have been shown to enhance establishment and first year 

yields when direct-seeding on low organic matter, coarse-textured soils (Undersander et 

al., 2011). 

Maximum nutrient availability for most crops 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 are not recommended for 

alfalfa (Orloff, 2008). Lime should be applied during the production years of rotational 

crops following alfalfa in the rotation sequence to allow time for the lime to react with 

the soil and be mixed into the soil with tillage prior to replanting the next alfalfa crop. 

Variety selection is an important preseason decision 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 varieties 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 range 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 important economic factor with regard to variety 

selection. Growers are advised 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 should 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 northern locations depends primarily on the winter-hardiness of 

the variety, while persistence in southern locations depends more on pest resistance. 

Winter-hardiness is a measure (1 to 6 scale, 1 being the most hardy) of the alfalfa plant’s 
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ability to survive the winter without injury. Fall dormancy measures the degree of 

growth (plant height) of alfalfa in the fall. Varieties with fall dormancy (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 varieties that meet their winter survival 

requirement. These varieties will green up earlier in the spring and recover more quickly 

between cuttings to give higher total yields for the season. Plant breeders have 

successfully incorporated resistance to certain diseases, nematodes, and insects into 

alfalfa varieties over the past 40 years (Putnam et al., 2008a). Growers need to determine 

the most significant diseases, nematodes, and insect pests in their region to decide which 

variety has the best pest-resistance package. Although agronomic practices such as 

cutting schedule and weed control influence forage quality to a greater degree than 

variety selection, varieties should also be assessed on forage quality by evaluating ratings 

on digestibility, intake, and relative feed value (Undersander et al., 2011). Since the 

introduction of alfalfa varieties with the biotechnology-enhanced glyphosate-tolerant trait 

(Roundup Ready), growers also need to weigh the weed management benefits and costs 

this trait will provide to their alfalfa production program. 

VIII.C.2. Planting and Stand Establishment 

Stand establishment is a critical step in alfalfa production that can impact 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 portion 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 indicates 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 spring-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 Central 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 germination and stand establishment and ultimately 

determine which planting time is most successful and provide the highest alfalfa yields 

for a given area. Spring seeding begins as soon as the potential for damage from spring 

frosts is over which is normally April and May in the northern areas of the North and 

East Central regions (Undersander et al., 2011). Although alfalfa is very tolerant of cold 

temperatures at emergence, seedlings at the second trifoliate 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). Fall seeding of alfalfa requires at least six weeks of growth 
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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 September to early October (Mueller et al., 2008b). 

Pre-irrigation or overhead irrigation after seeding 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 companion crop (or nurse crop) such as annual ryegrass, oats, 

spring barley, and rye is often practiced with spring seeding to help control erosion on 

steep slopes, reduce seedling damage from wind erosion on sandy soils, and reduce weed 

competition during alfalfa establishment (Undersander et al., 2011; USDA-APHIS, 

2010). Companion crops also provide additional forage or grain, and the straw can be 

harvested for livestock bedding. Fall seeding of alfalfa with a companion 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 alfalfa (seeding without a 

companion crop) can produce up to two extra cuttings of alfalfa and produce higher 

quality forage in the seeding year (Undersander et al., 2011). However, total forage 

production may be less than that of companion-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 herbicide 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 without 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 southern 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 nutrients, 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 fields are direct (solo) seeded to exploit the 

benefits of pure-stand alfalfa. 

Alfalfa is a small-seeded crop (220,000 seeds per pound) and is recommended to be 

seeded into a fine, firm seedbed that provides good seed-to-soil contact (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 (Mueller et al., 2008b). Poor seedbed 

conditions, poor seeding depth control, insufficient or excessive moisture, poor seed 

germination, seedling diseases, and inclement 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 of 
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alfalfa Rhizobium bacteria when seeding into soils without a recent history (within the 

past 10 years) of alfalfa (Mueller et al., 2008b). Within four weeks after germination, 

Rhizobium bacteria create nodules on alfalfa roots, allowing the bacteria to fix adequate 

nitrogen for the life of the alfalfa stand. Seed treatment with a fungicide (metalaxyl) is 

also recommended to protect the seed from seedling diseases (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 mutifoliolate leaves as well as secondary stems until it develops into a 

mature plant (Mueller and Teuber, 2008). Unique to early alfalfa development is 

contractile growth or the formation of the crown during stand establishment. 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 categories and ten growth stages – vegetative 

stages, bud stages, flowering stages, and seed development stages (Mueller and Teuber, 

2008). There are three vegetative stages (Stage 0, 1, and 2), distinguished by stem length. 

Flowering stages are early flower (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 development of the alfalfa plant, the proportion 

of leaves and stems changes. Stems lengthen and become more fibrous, increasing their 

total proportion in the forage. Overall forage quality declines because there is no 

concomitant increase in leaf percentage, and the forage quality of leaves is higher than 

stems. The stems of alfalfa plants develop 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 Development” method is utilized to more accurately 

relate relative maturity to forage quality in a field. The Mean Stage method involves 

examining individual stems and classifying them according to the staging system outlined 

in a detailed protocol defined by Kalu and Fick (1981). 

Alfalfa fields should be monitored routinely 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 implementing 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 control methods, insect pests can 

reach economic threshold levels requiring the use of insecticides. Unlike many field 
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crops, economic threshold values with alfalfa forage production must take into 

consideration both yield and quality losses. 

Management practices need to maximize forage yield while achieving a level of quality 

that meets the nutritional requirement for the intended use or market. The maturity of 

alfalfa plants at harvest has the greatest impact on forage quality. Prior to flowering, 

forage yield generally increases faster than quality declines. However, during the 

flowering period, reduction in quality 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 alfalfa crop. Identifying the best harvesting or cutting schedule is one 

of the most difficult management decisions because it involves market considerations in 

addition to several agronomic factors (Orloff and Putnam, 2008) Several cutting 

strategies can be considered or implemented including harvesting on a calendar basis, 

certain growth stage, growing-degree day models, numerical staging, and Predictive 

Equations for Alfalfa Quality (PEAQ) (Orloff 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 most 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 challenging 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 related 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 (short interval between cuttings) results in 

relatively low yield but high forage quality (Orloff and Putnam, 2008). Conversely, the 

cutting of alfalfa at a mature growth stage (long interval between cuttings) results in high 

yield but low quality forage. The growth stage 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 appearance of alfalfa hay, alfalfa hay 

quality is defined by a number of nutritional traits including acid detergent fiber (i.e., 

cellulose and lignin content), neutral detergent fiber (i.e., lignin, cellulose and hemicellulose content), crude protein, total digestible 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 horses 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 immature growth stages (pre-bud to bud) shortens 

stand life because it does not allow sufficient time for the alfalfa plants to replenish root 

reserves (Orloff and Putnam, 2008). Stand loss can lead to the invasion of weeds that 

compete for available resources (e.g., water, nutrients, light, and space) and negatively 
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impact forage quality. Additionally, cutting schedules influence 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 studies have shown the opposite to be true (Orloff and Putnam, 

2008). nine to ten times at pre-bud (harvest interval of 21 days) resulted in a 3-year 

average yield of 7.5 tons per acre in the Central Valley. In comparison, harvesting at full 

bloom (harvest interval of 37 days) with a total of five to six cuttings per year yielded an 

average of 11.6 tons per acre. Most California growers harvest during early to late-bud 

stage to obtain forage with lower lignin content, because most of their alfalfa production 

is targeted for the dairy industry (Orloff and Putnam, 2008). The most common cutting 

schedule in the Low Desert and Mediterranean regions of California 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 and a 30- to 35-day harvest interval is 

recommended for the remaining cuttings (Undersander et al., 2011). 

Pest management decisions may impact cutting schedules. Harvesting early can provide 

effective management of alfalfa weevil in the spring and worm management in the 

summer (Summers et al., 2008). Also, longer harvest intervals can enable the alfalfa crop 

to compete more vigorously with weeds. 

When cutting alfalfa, university alfalfa specialists recommend leaving no more than 2-4 

inches of stubble height (Orloff and Putnam, 2008). Studies have shown that cutting 

above two inches results in a yield reduction of 0.5 ton per acre per year per inch of 

additional cutting height. However, raising the cutting height does increase forage 

quality. 

High alfalfa forage yields and stand life depend 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 several uncontrollable environmental factors that 

impact the risk of winter injury (Undersander 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 injured 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 weather (40°F or higher) and midwinter thaws 

cause alfalfa to break dormancy and have less resistance to freezing temperatures. 

However, growers are able to utilize several 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. 
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Harvesting hay is a four step process: 1) cutting the forage, 2) raking the partially cured 

hay into windrows, 3) baling the dry hay, and 4) storing the hay (Orloff and Mueller, 

2008). The moisture content of alfalfa growing in the field is generally about 80% 

(Undersander et al., 2011). Soluble sugars and proteins are dissolved in the forage liquid. 

When forage is dried to hay before being baled, water in the 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 the quality of the hay. 

Rapid drying is important to minimize quality losses caused by bleaching, respiration, 

leaf loss, and rain damage (Orloff and Mueller, 2008). Weather conditions can make 

harvesting hay a real challenge. Rainy weather causes delays in harvest which increases 

the neutral detergent fiber (NDF) and acid detergent fiber (ADF) and decreases 

digestibility and crude protein of the hay (Undersander et al., 2011). In addition, rain on 

hay before baling leaches soluble nutrients (protein and carbohydrates). The following 

management practices are recommended for harvesting hay to accelerate drying and 

minimize losses: 1) cut the forage early in the 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 alfalfa during the cutting operation is a widely 

accepted practice to accelerate curing (Orloff and Mueller, 2008). The purpose of 

crimping is to facilitate water loss from the stems, making the drying rate of stems more 

similar with that of the leaves. This can hasten the drying process by as much as 30% 

(Orloff and Mueller, 2008). Chemical conditioning or use of drying agents can also be 

used to speed the drying process. Drying agents 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 specialists do not believe drying agents are cost 

effective. 

Preservatives are used to a limited degree to allow storage of alfalfa hay baled at moisture 

contents higher than would ordinarily be considered safe. Hay baled and stored at too 

high a moisture content without a preservative is subject to problems with mold, 

discoloration, and even spontaneous combustion (Orloff and Mueller, 2008). Hay 

preservatives are organic acids, primarily propionic 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 insect 
Monsanto 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 sporadic and less frequent with other insect 

pests. 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 implementing control strategies that minimize effects to natural enemies and 

other nontarget species (Summers 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 minimum of 50% of the plant population are resistant (Summers 

et al., 2008). Accelerating or delaying cutting schedules is also an important cultural 

method of managing some insect pests. In irrigated alfalfa areas, border-strips are 

utilized to serve as refuge for natural enemies 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 production must take into consideration 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 under severe infestations can 

completely defoliate the plant. Additionally, the adult weevils feed on alfalfa stems. 

Damage is usually greatest on the first cutting, 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 effective, although several parasitic wasps are 

effective in some growing regions to suppress weevil population buildup (Kansas State 

University, 1998). However, an alfalfa weevil-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 environmental interaction assessments presented in 

Section VII have demonstrated that KK179 is not meaningfully different from 

conventional alfalfa. Therefore, if deregulated, 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; Undersander 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 nematodes. Selecting diseaseresistant varieties is one of the most effective methods to managing many of the alfalfa 

diseases. Other integrated strategies and techniques utilized to manage alfalfa diseases 

include irrigation management, planting methods, 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 adverse temperatures for seedling growth. Seedling diseases 

can be devastating to the long-term productivity and life of a new alfalfa seeding. 

Cultural and chemical control measures are generally effective in managing seedling 

diseases. 

Foliar diseases reduce photosynthesis and defoliate alfalfa plants subsequently decreasing 

yield and forage quality (Frate and Davis, 2008). Some important 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 techniques are usually utilized to manage the 

foliar diseases. 

Root, stem, and crown diseases are less obvious 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 alfalfa (Frate and Davis, 2008; Undersander et 

al., 2011). the exception of Rhizoctinia 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 important wilt diseases 
Monsanto Company 12-AL-246U 179 of 407

(Frate and Davis, 2008). The pathogens for wilt diseases invade the vascular system of 

plants which leads to the plant wilting and usually dying. Resistant varieties are the first 

line of defense for the diseases in this group. 

Parasitic nematodes are microscopic, unsegmented roundworms that reduce yields and 

cause considerable economic losses in alfalfa (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. Nematodes 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 stress the plants and act in conjunction with 

other stress factors in the field to reduce growth and yield. The key practices to manage 

nematodes include site selection, crop rotation, variety selection, certified seed, fallow, 

chemical nematicides, and other management techniques that prevent nematodes from 

spreading, such as using clean equipment and irrigation water (Westerdahl and Frate, 

2008). 

The phenotypic, agronomic, and environmental interaction assessments presented in 

Section VII have demonstrated that KK179 is not meaningfully different from 

conventional alfalfa. Therefore, if deregulated, KK179 would require the same 

management practices for diseases and other pests as for conventional alfalfa. 

VIII.F. Weed Management 

Annual and perennial weeds reduce alfalfa yield and quality and cause serious economic 

losses in alfalfa because they compete for the 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 grow slowly and compete poorly with weeds. 

Forage yield losses due to the presence of weeds 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 when weeds are left uncontrolled (Canevari et 

al., 2008). However, the feeding value or nutritional 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 low as 9% in hay that contained 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 weed species present. Annual 

grasses have a significant impact on quality because they have high fiber content and 

decrease livestock intake (Undersander et al., 2011). Annual broadleaf weeds, such as 

curly dock, hoary alyssum, and yellow rocket are unpalatable and decrease 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 Canada 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. are presented in Table 

VIII-6. Tillage is an important part of a weed management program when establishing 

alfalfa. Tillage controls emerged annual weeds 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 established (Undersander et al., 2011). Practices that encourage 

alfalfa germination and vigorous seedling growth are effective for weed management. A 

dense, vigorous alfalfa stand is an effective 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 control throughout this section are focused on 

conventional weed control programs. 

 

® Roundup is a registered trademark of Monsanto Technology LLC. 
Monsanto Company 12-AL-246U 181 of 407

Table VIII-6. Common Weeds Present in Alfalfa in the U.S1

. 

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 conventional alfalfa. According to 

an extensive survey conducted by the USDA from 1988 to 1992, herbicides are used on 

approximately 17% of all alfalfa acreage (seedling establishment and established stands) 

grown for forage (USDA-APHIS, 2010). Herbicides 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 the 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 (Hower et al., 1999). However, since more 

than one herbicide and more than one application 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 (USDA-APHIS, 2010). Companion crops were utilized on 49% 

and 3% of the spring- and fall -seeded alfalfa, respectively (Hower et al., 1999). 

There are several herbicides available for effective weed control during seedling 

establishment, including 2,4-DB, benefin, bromoxinil, clethodim, hexazinone, imazamox, 

imazethapyr, sethoxydim, paraquat, and pronamide (USDA-APHIS, 2010). Table VIII-7 

provides a summary of the crop tolerance and 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 addition, the restrictions 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 production 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 fertility, disease, insect problems, and winter injury 

(Undersander et al., 2011). The decision to apply an herbicide to remove undesirable 

weeds in established alfalfa will depend on the weed density, weed species, stand density, 

and intended use of the hay (Undersander et al., 2011). It is important to note that alfalfa 

does not spread into the open areas previously 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 existence of alfalfa-grass mixtures, the low economic return 

from current herbicides, the ineffectiveness 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 established stands of alfalfa including: 2,4-DB, 

clethodim, diuron, EPTC, hexazinone, metribuzin, norfluzaon, paraquat, pendimethalin, 

sethoxydim, terbacil, and trifluralin (USDA-APHIS, 2010). Table VIII-8 provides a 

summary of the crop tolerance and effectiveness ratings of herbicides on common weeds 

in established stands of alfalfa. The spectrum of weed species controlled varies 

significantly among these herbicides. In addition, some of the herbicides are applied for 

residual control, some control emerged weeds, and some have residual and 
Monsanto 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 environmental interaction assessments presented in 

Section VII have demonstrated that KK179 is not meaningfully different from 

conventional alfalfa. Therefore, if deregulated, KK179 would require the same weed 

management practices as conventional alfalfa. 
Monsanto Company 12-AL-246U 

Table VIII-7. Alfalfa Tolerance and Herbicide Effectiveness Ratings on Direct Seedlings of Alfalfa1

Preplant Incorporated Herbicides 

Postemergence Herbicides 

EPTC Trifluralin Bromoxynil 

2,4-

DB Glyphosate Sethoxydim Pendimethalin Imazethapyr 

Alfalfa Tolerance 

F/G G F/G G E2 E E G 

Grasses

Barnyardgrass 

G/E G/E P N E G/E G/E G 

Foxtails G/E G/E P N E E G/E G Quackgrass P/F P N N G/E F/G P P 

Broadleaves

C. chickweed F P P P E N F/G G 

C. lambsquarters 

F F/G G/E G/E G/E

N G/E F/G

C. ragweed F P G/E G/E E N P F Eastern Black nightshade F P G/E F E N P E Pennycress P P G/E F E N P G/E Pigweed spp. F/G G/E F G/E E N G/E E Shepherd’s 

purse P P E F/G E N G G/E 

Smartweed spp. P P G P G/E N P G Velvetleaf F/G N G G/E G/E N N G/E Wild mustard P/F P G F/G E N N G/E Wild radish P/F N G F E N N G 

Herbicide 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 in Roundup Ready alfalfa varieties. 
Monsanto Company 12-AL-246U 

Table VIII-8. Alfalfa Tolerance and Postemergence Herbicide Effectiveness Ratings on Common Weeds iStands1. 

2,4-DB Flumioxazin Glyphosate2 Metribuzin Sethox- ydim Pendi- methalin Imazeth- apyr Imazamox C

Alfalfa Tolerance G G/E E2 F/G E E G G Annuals 

Common chickweed P E E E N P G G 

Field pennycress F/G E E G/E N P E E Foxtails spp. N G/E E F E G/E F/G G/E Shepherd’s purse F/G E E E N F/G G/E G/E 

Perennials 

Broadleaf plantain F/G - G/E P N P F F 

Canada thistle N P G P N P P P/F C. dandelion P P G G/E N P P P/F Curly dock P/F P - F N P P/F P/F Hemp dogbane N - E P N P P - Hoary alyssum F P3 - F/G N P F F Perennial sowthistle N - E P N - G G Quackgrass N P G/E F/G F/G P P P/F White cockle P P3 F/G G N P P P Wirestem muhly N - E P F/G - P P Yellow nutsedge N P F F N P P/F F Yellow rocket P P3 G/E G N P F/G F/G 

Herbicide 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 applications of 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 postemergence 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 provide a summary of the crop tolerance and 

effectiveness ratings of several herbicides, including glyphosate, on common weeds in 

direct-seeded and established alfalfa stands. Glyphosate is rated excellent for Roundup 

Ready alfalfa tolerance, while several other herbicides are only rated good or fair to good 

for alfalfa tolerance. Glyphosate is rated good to excellent on almost all the common 

weeds listed in direct seedings and established stands of alfalfa. Postemergence 

application of glyphosate provides 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 products 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 established stands must not exceed 4.64 lbs a.e. per 

acre. The combined total per year for all applications, including preplant during the year 

of establishment, must not exceed 6.0 lbs a.e. of glyphosate (Monsanto Company, 2012). 

Since glyphosate controls only emerged weeds with no residual control, it is important to 

apply Roundup agricultural herbicide 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). Earlier 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 mixes of glyphosate with 

other mode-of-action herbicides can minimize the risk of weed species shifts or weeds 

developing resistance to glyphosate (Canevari et al., 2008). 

VIII.G. Alfalfa Stand Termination 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 Putnam, 2008). Eventually, the forage yield 

and quality decline to the point that the stand must be removed and the field rotated to 

another crop. In some cases, it may be economically feasible to extend the life of the 
Monsanto Company 12-AL-246U 187 of 407

stand by overseeding with grasses and other legumes. However, there must be a potential 

market for the alfalfa-grass mixture. The plant 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 (Canevari and Putnam, 2008). 

Alfalfa stand removal or termination is achieved 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, termination 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 killed 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 production fields, volunteers may be either from 

surviving or newly germinated plants. Growers 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 glufosinate. Glphosate only provides partial 

control of conventional alfalfa and is not recommended for volunteer management. 

Research has demonstrated that 2,4-D, chlorpyralid, 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 stand 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 for stand termination or volunteer control 

preplant to the crop, but no herbicides are labeled 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 environmental interaction assessments presented in 

Section VII have demonstrated that KK179 is not meaningfully different from 

conventional alfalfa. Therefore, if deregulated, stand termination and volunteer control 

of KK179 would use the same management practices as utilized for conventional alfalfa. 

 
Monsanto Company 12-AL-246U 188 of 407

Table VIII-9. Herbicides for Alfalfa Stand Termination and Control of Volunteer 

Alfalfa1. 

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 √ √

Chlorpyralid 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 and 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 locations (USDA-APHIS, 

2010). The Medicago sativa subspecies sativa (purple-flowered alfalfa used in 

cultivation) has naturalized populations in all 50 states (USDA-APHIS, 2010). M. sativa

subspecies falcata (yellow-flowered or Siberian alfalfa) is naturalized in the northern and 

western states and is being promoted as a rangeland enhancer for grazing. Like alfalfa 

under cultivation, feral alfalfa originated from introduced varieties. Feral alfalfa can be 

found in air fields, canals, cemeteries, ditch banks, fence rows, highways, irrigation 

ditches, pipelines, railroads, rangeland, rights-of-way, roadsides, wasteland, preserves, 

parks, and recovery areas. Feral populations exist near locations used for alfalfa seed and 

forage production (USDA-APHIS, 2010). 

In the event of the trait for reduced G lignin and total lignin being transferred from 

KK179 to feral populations by cross-pollination, no environmental consequences are 

expected to occur. Furthermore, evaluations have shown that the introduced trait for 

reduced G lignin and total lignin in KK179 does 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 could confer a selective advantage or to be 

selected for in an unmanaged setting. 

 In general, alfalfa is not a species specifically targeted for weed control in unmanaged 

areas or in roadsides. In fact, alfalfa may 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 controlled or discouraged using physical or 

chemical methods. Herbicides, such as 2,4-D, dicamba, or a combination of these 

herbicides, effectively control 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 effective on conventional alfalfa. 

The phenotypic, agronomic, and environmental interaction assessments presented in 

Section VII have demonstrated that KK179 is not meaningfully different from 

conventional alfalfa. Therefore, if deregulated, control of KK179 would use the same 

physical and herbicide management practices as utilized for conventional alfalfa. 
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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 erosion, avoid pathogen and pest buildup, and 

adjust to market conditions (USDA-APHIS, 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 channels 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 which greatly benefits the growth and yield of 

subsequent crops, such as corn, tomato, wheat, or specialty crops. Alfalfa fixes 

atmospheric nitrogen through the symbiotic relationship with Rhizobium bacteria which 

can provide from 40 to 60 pounds of nitrogen per acre to crops that follow alfalfa (Orloff, 

2008). In turn, rotations with other crops benefit alfalfa by breaking 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 inefficient use of residual soil 

nitrogen credits. Approximately 18% of existing alfalfa 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 the plant stem without leakage. As a 

component of plant cell walls, lignin molecules bind with hemicellulose and fill the 

spaces between cellulose, hemicellulose, and pectins. 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 desire alfalfa forage with lower lignin values 

as well as high protein content. 

KK179 alfalfa is a biotechnology-derived alfalfa with an intended reduction in G lignin 

and total lignin compared to conventional alfalfa at the same stage of growth. Because of 

its lower lignin content, KK179 will provide growers with production benefits, including: 

1) greater flexibility in harvesting schedules and 2) potentially lower production costs. 

Growers will have the flexibility at each forage 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 growth stage at harvest. When implementing the first 

production strategy (maximizing forage quality while maintaining yield), the cutting 
Monsanto 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 was producing high quality alfalfa hay for dairy 

cows, the grower would typically harvest alfalfa at the early 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 without forfeiting forage quality compared 

to conventional alfalfa. KK179 can be harvested 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 this plant growth stage can increase 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. Additionally, when rain or adverse weather 

conditions coincide with a planned harvest interval, 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 maintaining 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 stand. 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 cutting 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 longer harvest intervals and resulting higher 

forage yields with each cutting. Fewer harvests also means fewer trips across the field, 

resulting in less labor, fuel consumption and soil compaction, plus potentially less crown 

damage to alfalfa plants in established stands. In addition, 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 environmental interaction assessments presented in 

Section VII have demonstrated that KK179 is not meaningfully different from 

conventional alfalfa. Therefore, KK179 will utilize the same agronomic practices as 

conventional alfalfa production, including: tillage operations, seedbed preparation, pest 

management, and harvesting procedures. In addition, KK179 alfalfa growers will have 

increased flexibility in their forage harvest schedules, but the resulting hay quality will 

continue to meet the established USDA Agricultural Marketing Services’ quality 

classifications of supreme, premium, good, fair, and utility. These classifications are 

based on several factors, including: the presence of weeds, fiber content, protein content, 

general color, and presence of mold (USDA-APHIS, 2010). 

KK179 will be combined with Roundup Ready alfalfa utilizing traditional breeding 

techniques. The combined traits will allow growers planting Roundup Ready × KK179 

alfalfa to take advantage of the weed management benefits of the Roundup Ready weed 

control system: broad spectrum weed control and excellent crop safety with greater 
Monsanto Company 12-AL-246U 192 of 407

application flexibility and simplicity. These growers will also have the flexibility to 

choose the production strategy that improves forage quality or yield and maximizes the 

profitability of alfalfa production for their farming operation. Increased flexibility will 

allow growers to better manage the yield-quality relationship and harvesting schedules to 

meet market needs and intended on-farm uses for their alfalfa forage production. 

VIII.K. Stewardship of KK179 

Monsanto and FGI develop effective alfalfa products and technologies and are committed 

to assuring that their products and technologies are safe and environmentally responsible. 

Monsanto and FGI demonstrate this commitment by implementing product stewardship 

processes throughout the lifecycle of a product and by participation in the Excellence 

Through Stewardship® (ETS) Program1

 and the USDA-APHIS Biotechnology Quality 

Management System (BQMS)2

. These policies and practices include rigorous field 

compliance and quality management systems and verification through auditing. 

Stewardship Principles are also articulated in Technology Use Guides (TUG) distributed 

annually to growers who utilize 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 Stewardship3

. Monsanto and FGI will 

continue to monitor other countries 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 alfalfa producers, processors, and buyers. 

 

® Excellence Through Stewardship is a registered servicemark of the Excellence Through Stewardship 

Organisation, Washington, DC. 

1 http://www.excellencethroughstewardship.org/. 2 http://www.aphis.usda.gov/biotechnology/bqms_main.shtml

3

http://www.excellencethroughstewardship.org/facts/documents/Guide%20for%20Product%20Launch%20

Stewardship.pdf. 
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IX. ENVIRONMENTAL ANALYSIS 

IX.A. Introduction 

This section provides a brief review and assessment 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 Act (PPA) (7 U.S.C. § 7701-7772), to prevent 

the introduction and dissemination of plant pests into the U.S. 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 should 

no longer 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. According to the PPA, the definition of “plant pest” includes the living 

stage of any of the following, or a similar article that can directly or indirectly 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 viroid; or (G) 

an infectious agent or other pathogens. (7 U.S.C. § 7702[14]). 

The regulatory endpoint under the PPA for biotechnology-derived crop products does not 

involve zero risk, but rather a determination that the regulated article is not expected to 

pose a plant pest risk. Information in this petition related to plant pest risk characteristics 

of KK179 include: 1) mode-of-action and changes to plant metabolism; 2) composition; 

3) characteristics of the expressed product; 4) potential 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) impacts 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 exception 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 beneficial 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 analysis 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, 
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environmental interaction characteristics 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 conventional alfalfa R2336 leaf tissue using the 

plasmid vector PV-MSPQ12633. PV-MSPQ12633 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 isolate 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 into the alfalfa genome at a single locus. No T-DNA II or 

backbone DNA sequences from PV-MSPQ12633 were detected in KK179. Recorded 

data confirmed the organization and sequence of the insert, 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 inverted repeat sequence 

produces double-stranded RNA (dsRNA) which suppresses endogenous CCOMT gene 

expression via the RNA interference (RNAi) pathway. Suppression of the CCOMT gene 

expression leads to lower CCOMT protein expression resulting in reduced production of 

G lignin subunit compared to conventional alfalfa 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 blocks of lignin molecules, confirms that the suppression of 

CCOMT acts to specifically reduce the level of one major lignin subunit, G lignin, while 

not substantially affecting the levels of the other major lignin subunit, syringyl lignin (S 

lignin), or a minor lignin subunit, p-hydroxyphenyl lignin (H lignin), as predicted by the 
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mode-of-action. The result is a lower proportion of G lignin and a greater proportion of S 

lignin, shown by an increase in the S to G lignin ratio. Analysis of total lignin levels by a 

commercial forage testing lab, measured as acid detergent lignin (ADL), verifies that the 

reduction in the G lignin leads to a concurrent reduction in total lignin 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 eukaryotes, including plants, for endogenous 

gene suppression. Nucleic acids have a long history of safe consumption and are 

considered GRAS by the U.S. FDA as there is no evidence to suggest dietary 

consumption of RNA is associated with mammalian toxicity or allergenicity. 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 lack of toxicity or allergenicity of dietary 

RNA, the RNA-based suppression of endogenous CCOMT gene expression in KK179 

poses no novel risks as a result of exposure to expressed products of the DNA insert. 

IX.B.2. Compositional Characteristics of KK179 

Compositional comparisons based on OECD guidance were presented in Section VI.B. to 

assess whether levels of nutrients, anti-nutrients, and secondary metabolites in forage 

derived from KK179 are comparable to levels in the conventional control and several 

conventional commercial reference varieties for which there is an established history of 

safe consumption. Nutrients assessed in this analysis included 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 non-essential). Anti-nutrients assessed 

included daidzein, glycitein, genistein, coumesterol, formononetin, biochanin A, and 

saponins (total bayogenin, total hederagenin, total medicagenic acid, total soyasapogenol 

B, total soyasapogenol E, total zanhic acid, and total saponins). Secondary metabolites 

assessed included p-coumaric acid, ferulic acid, sinapic acid, total polyphenols, free 

phenylalanine, and canavanine. 

Compositional and nutritional comparisons were conducted to determine statistically 

significant differences at the 5% level of significance between KK179 and the 

conventional control. The relevance of each statistically significant difference observed 

was assessed through evaluation of: 1) the magnitude 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 assessment of the differences within the context 

of natural variability of available commercial alfalfa composition published in the 

scientific literature. 
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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 exception 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 varieties grown concurrently. While compositional 

analysis confirmed that the intended reduction in G lignin and total lignin, this 

compositional change does not pose meaningful or novel feed/food safety 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 total lignin, these results support the overall 

conclusion that KK179 forage is compositionally equivalent to conventional alfalfa in 

accordance with OECD guidelines. 

IX.B.3. Phenotypic and Agronomic and Environmental 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, and environmental interaction 

characteristics of KK179 were evaluated and compared to those of the conventional 

control and considered within the variation among conventional commercial reference 

varieties. These assessments included seven general data categories: 1) seed 

germination, dormancy, and emergence; 2) vegetative growth; 3) winter survival; 4) 

reproductive growth including pollen, flower, and seed characteristics; 5) lodging and 

seed retention on the plant; 6) plant response to abiotic stress and interactions with 

diseases and arthropods; and 7) plant-symbiont interactions. Results from the 

phenotypic, agronomic, and environmental interaction assessment demonstrate that 

KK179 does not possess: 1) increased weedy characteristics; 2) increased susceptibility 

or tolerance to specific abiotic stresses, diseases 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 characteristic that is often associated with plants 

considered weeds (Anderson, 1996; Lingenfelter 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 conventional 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 germination characteristics were 

observed in either scarified or non-scarified seed that would be indicative of increased 

plant weediness or plant pest potential of KK179 compared to the conventional control as 

described in Section VII.C.1. 
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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 characteristics including emergence, early 

season vigor, lodging, crop growth stage, regrowth after forage harvest, fall plant height, 

total forage yield, spring vigor, spring stand recovery, and spring stand count. Also 

assessed were phenotypic characteristics during seed production 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 control, no statistically 

significant differences were detected at a 5% level of significance (α = 0.05). The data 

support the conclusion that there is no biologically meaningful change in terms of 

weediness or plant pest potential 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 conventional 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 compared to conventional 

alfalfa. 

IX.B.3.4. Reproductive Characteristics 

Evaluations of pollen morphology and viability and flower morphology in KK179 and 

the conventional control as described in Section VII.C.3 provide information useful in a 

plant pest risk assessment as it relates to the potential for gene flow and introgression of 

the biotechnology-derived trait into other alfalfa varieties and sexually compatible related 

species. Pollen morphology and viability 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 weediness or plant pest potential compared to 

conventional alfalfa. 

IX.B.3.5. Interactions with Non-target Organisms Including Those Beneficial to 

Agriculture 

Evaluation of KK179 for potential effects on NTOs is a component of the plant pest risk 

assessment and was evaluated using a combination of biochemical information and 

experimental data. The information and data included molecular characterization, safety 

of expressed products, the history of environmental exposure to reduced lignin, results 

from the environmental interactions assessment described earlier, and the demonstration 

of compositional, agronomic, and phenotypic equivalence to conventional alfalfa. The 

nature of KK179 as a product with no pesticidal activity means all exposed organisms 

can be considered NTOs. Observational data on environmental interactions for KK179 
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and a conventional alfalfa control were collected 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 natural variability for each characteristic. The 

environmental interactions evaluation, described in Section VII.C., included qualitative 

assessment of plant response to insects and disease and quantitative assessment of 

arthropod pests and abundance. The results of these assessments indicated that the 

presence of the trait for reduced G lignin and total lignin does not alter plant-insect 

interactions, including beneficial arthropods and pests, nor does it alter disease 

susceptibility of KK179 compared to conventional alfalfa. The results also indicated that 

reduction of lignin to levels already present in the environment does not alter plant-insect 

interactions or disease 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 detected between KK179 and the control for, 

indicating no impact on either the symbiotic relationship or the symbiotic nitrogen-fixing 

bacteria. This assessment demonstrated that the symbiosis between nitrogen fixing 

bacteria and alfalfa was not altered as a result of the introduction of the trait for reduced 

G lignin and total lignin (Section VII). 

Taken together, these data combined with 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 fertile, 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 states where alfalfa is cultivated extensively, 

including Arizona, California, Idaho, Oregon, 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 lists available in the USDA 

PLANTS Database (USDA-NRCS, 2012), Medicago sativa is found in only one weed 

identification guide by the Southern Weed Science Society (SWSS). The author of the 

SWSS entry for alfalfa has clarified that alfalfa 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 outside of agricultural 

settings. These plants originated from introduced varieties 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-agricultural purposes including: rehabilitation 

of overgrazed rangelands to improve wildlife habitat and for livestock; erosion-control 

projects in forest interiors; improvement of compacted soils; use in seed mixes for 

USDA’s Conservation Reserve Program (CRP); revegetation of areas damaged by 
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wildfire; and erosion reduction in mined soils (USDA-APHIS, 2010). These uses have in 

some cases led to establishment of feral alfalfa populations. Surveys have confirmed that 

minor feral populations exist in six major alfalfa-producing states, in areas where alfalfa 

seed or forage is produced (Rogan and Fitzpatrick, 2004). The falcata subspecies of 

alfalfa (yellow-flowered) is naturalized on rangelands in the northern and western States, 

where it has been promoted as a rangeland 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 Sections VIII.G and VIII.H. 

Feral alfalfa can be found in various soils and environments in coexistence with many 

other plant species. As expected, feral alfalfa populations are not managed and lack 

regular external inputs like irrigation, herbicides, insecticides, and fertilizers. Thus, they 

are exposed to numerous environmental factors 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 pests are largely absent, feral alfalfa is more 

negatively impacted by endemic pests than alfalfa under cultivation (Bagavathiannan et 

al., 2011b). As a result, the competitiveness 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 often associated with plants that are weeds 

(Anderson, 1996). Dormancy mechanisms, including hard seed, vary with species and 

are usually based on complex processes. For most crops, the number of hard seed is 

negligible or nonexistent. However, when alfalfa seed is produced, a portion of the seed 

is "hard," that is, the seeds do not absorb water after a prescribed period of time because 

of an impermeable seed coat (AOSA, 2007b). The percentage of hard seed in alfalfa 

varies widely, and depends largely on environmental conditions during and after seed 

maturation, harvesting and seed conditioning methods and on genetic factors (Bass et al., 

1988). Seed aging, weathering or mechanical scarification make the seed coat permeable 

to water and allows rapid germination under favorable conditions. Apart from an 

impervious seed coat, alfalfa has no physiological 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 soil 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 rotational crop. However, problems controlling 

volunteer alfalfa are not common (Van Deynze et al., 2008). Volunteers, including ones 

with herbicide-tolerant traits, can be managed with pre-plant or selective post-emergent 

herbicide applications or by mechanical means as described in Section VIII.G. 

In comparative studies described in Section VII.C. between KK179 and the conventional 

control, phenotypic, agronomic, and environmental interaction data were evaluated for 

changes that would impact the plant pest potential, in particular, weediness potential. 

Results of these evaluations show that there are no biologically significant differences 
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between KK179 and the conventional control for 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 interactions 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 conclusion that KK179 has no 

increased weediness potential compared to alfalfa and is not expected to become a weed. 
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IX.D. Potential for Gene Flow and Introgression 

Gene flow refers to all mechanisms that may result in the movement of genes between 

populations. Pollen-mediated gene flow, also 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 process whereby one or more genes successfully incorporate into 

the genome of a recipient plant. Gene introgression must be considered in the context of 

the transgenes inserted into the biotechnology-derived plant and the likelihood that the 

presence of the transgenes and their subsequent transfer to recipient plants and plant 

populations will result in increased plant pest potential. 

The baseline potential for gene flow and introgression in conventional alfalfa in 

agricultural and feral settings has been comprehensively reviewed by USDA-APHIS 

(USDA-APHIS, 2010) and is relevant to the environmental analysis of KK179. The lack 

of differences between the KK179 and conventional control with respect to plant pest 

potential, described in Section VII.C., includes 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-pollination by insects; therefore, pollen-mediated gene flow 

between different alfalfa populations is possible. Many factors influence the probability 

of successful gene flow between alfalfa populations, including: timing and degree of 

flowering; relative abundance of pollen sources; the presence and activity of pollinators; 

proximity of alfalfa populations; physical barriers between populations; the relative scale 

of the alfalfa populations; probability of seed maturation and germination; 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 alfalfa industry with respect to potential 

impacts on commercial alfalfa cultivation and 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; Teuber 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). 
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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 provided 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 previous studies and reviews have provided a 

comprehensive assessment of gene flow as a commercial and environmental risk factor 

(USDA-APHIS, 2010; Van Deynze et al., 2008). More recent studies have confirmed 

previous findings (Hagler et al., 2011; Teuber et al., 2011). 

For purposes of analyzing gene flow, alfalfa populations can be categorized into three 

major types as sources and recipients of pollen: 1) alfalfa fields intended for hay 

production; 2) alfalfa fields intended for seed production; and 3) naturalized or feral 

alfalfa populations existing outside of managed 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 production typically requires the intentional introduction of 

large numbers of bee colonies in or near fields 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 any stage. The primary pollinators used are 

leafcutter bees, honey bees, and to a lesser extent alkali 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 and Cane, 2011). Leafcutter bees have a 

foraging range under 1 mile while honey bees range up to 3 miles and alkali bees even 

further (Beekman and Ratnieks, 2000; Hagler et al., 2011; Pitts-Singer and Cane, 2011). 

The studies show that cross-pollination rates decrease with increasing distance from the 

source of pollen and vary with the predominant 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, however, 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 vegetative and early flower stages when little 
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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 competition and natural autotoxicity of the alfalfa 

prevents new seedlings resulting from rare outcrossing 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 seed-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 varieties for sowing alfalfa stands. High quality 

seed decreases the risk of seed-mediated gene flow during stand establishment and the 

introduction of varietal mixtures 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 practices, which include multiple harvests per 

year of the hay field, coupled with physical isolation distance requirements of certified 

alfalfa seed production fields keep the potential for gene flow from seed production fields 

to hay production fields very low (USDA-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 natural autotoxicity would reduce the probability 

of successful germination and survival 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 common, managable, and measurable occurrence 

for conventional cultivars of most outcrossing crops including alfalfa. This scenario 

applies to a much smaller area 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 production 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 limit 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 many decades on physical isolation to minimize 

and mitigate pollen-mediated gene flow in order to manage genetic purity of commercial 

varieties (Brown et al., 1986; Dunkle, 2011; Kalaitzandonakes, 2011; Van Deynze, 

2011). Gene flow studies of alfalfa seed production fields have confirmed that current 

AOSCA and OECD seed isolation and production standards can be used to meet Federal 

Seed Act standards (7 CFR § 201.76) (AOSCA, 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 greater purity is desired, seed producers use 

additional isolation or sanitation to ensure high seed purity. 
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Current AOSCA isolation requirements for certified alfalfa seed production have also 

been shown to effectively mitigate gene flow from hay production fields into seed fields 

(Teuber et al., 2007). The normal forage production practice of harvesting a hay field at 

or before 10 percent bloom during the seed production pollination period is sufficient to 

ensure that pollen-mediated gene flow from hay production fields into seed production 

fields is low. Furthermore, this practice is a stewardship requirement for Roundup Ready 

alfalfa hay growers, under terms of the technology use agreement (Monsanto Company, 

2012). Research has shown that harvesting hay at stages of 20 to 50% bloom does not 

significantly raise the potential 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 commercial alfalfa seed grower conditions have shown that 

the use of minimum isolation distances specific to pollinator species and identitypreserved production protocols successfully mitigate gene flow to levels observed for 

other biotechnology-derived crop species to produce seeds of high genetic purity 

(>99 %). Adventitious presence levels can be managed to less 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; Fitzpatrick et al., 2007). 

The information from these studies has allowed 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-mediated gene flow to their 

commercially-sensitive seed production crops from hay production fields. The strategies 

include FGI Best Practices for Stewardship in Roundup Ready Seed Production, NAFA 

Best Management Practices for Roundup Ready 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 Seed Stewardship Program (ASSP) (AOSCA, 

2012). The protocols in these strategies require 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 ASSP was launched in 2010 by participating state 

seed certification agencies to offer a voluntary 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 production areas since 2011 has facilitated local 

coordination of coexistence efforts of alfalfa seed growers for both conventional and 

biotechnology-derived alfalfa (NAFA, 2011a). 

Gene Flow via Feral Alfalfa 

As described in Section IX.C, populations of feral alfalfa have existed in the U.S. since 

its introduction due to natural dispersal from cultivated alfalfa and from intentional 

introductions for rangeland development and other purposes (Kendrick et al., 2005; 

USDA-APHIS, 2010). Gene flow to feral alfalfa plants from large-scale seed or hay 
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production fields of conventional alfalfa and biotechnology-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 production 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 production requires minimum isolation from all sources of alfalfa 

including feral populations (AOSCA, 2011). Removing or mowing feral alfalfa plants 

near cultivated fields prevents synchronous bloom and reduces the risk of gene flow to 

near zero (USDA-APHIS, 2010). Seed producers are known to control these populations 

as there are fewer feral populations in areas of intensive alfalfa seed production compared 

to hay production areas (Kendrick et al., 2005). This practice also lowers the potential 

for gene flow into as well as from feral populations (Van Deynze et al., 2008). As 

explained previously, normal forage production practices also restrict gene flow from 

feral populations to hay production fields 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). First, 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, sinks or bridges for gene flow. Third, feral populations, as 

described in Section IX.C., are not managed, and therefore are more susceptible to 

environmental stresses. Under these conditions, pollen and seed production on feral 

plants are expected to be considerably less prolific 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 introgressed into a feral population, 

including a biotechnology-derived gene, would be present at a limited frequency unless 

there were pressure from a selection mechanism 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 KK179 would not confer a selective advantage to 

a feral alfalfa plant. These factors taken together lead to the conclusion that gene flow 

from KK179 under scenarios involving feral populations 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., comparative studies between KK179 and the 

conventional control to evaluate 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 conclusion that 

KK179 has no increased potential compared to conventional alfalfa to outcross or 

hybridize with cultivated alfalfa under hay production or seed production conditions. 
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IX.D.2. Hybridization with Annual Species of Subgenus Medicago

Medicago sativa is very distantly related to the annual members of Medicago (Lesins and 

Lesins, 1979). No annual species 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 legume seed crops. Crosses between the annual 

and perennial species do not occur naturally, and even artificial cross-fertilization is 

unsuccesful (Fridriksson and Bolton, 1963; Sangduen et al., 1983). Significant biological 

barriers exist between annual and perennial species, which prevent successful unassisted 

hybridization. Annual species are self-pollinating, while perennial species outcross and 

require bees to facilitate pollination. Ploidy and karyotype differences between alfalfa 

and the annual Medicago species also prevent successful hybridization. Additional 

reproductive barriers include both pre- and post-fertilization abnormalities, such as 

abnormal pollen tube growth (Sangduen et al., 1983) and post-fertilization 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 two species to hybridize is disputed (USDAAPHIS, 2010). Expert opinion concludes that no annual species is known to naturally 

hybridize with M. sativa (McCoy and Bingham, 1988; Quiros and Bauchan, 1988). 

USDA-APHIS has also concluded, due to lack of confirmatory evidence, hybridization 

between M. lupulina and M. sativa is very unlikely to occur (USDA-APHIS, 2010). 

Recent research further supports that M. lupulina and M. sativa hybridization is unlikely 

(Chandra et al., 2011; Steele 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 hybrid 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 South Africa (Quiros and Bauchan, 1988; USDAAPHIS, 2010). therefore, no risk of interspecific 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 Information to Species with which Alfalfa Cannot 

Interbreed (Horizontal Gene Flow) 

Monsanto and FGI are not aware of any reports confirming the transfer of genetic 

material from alfalfa to other sexually incompatible plant species. The probability for 

horizontal gene flow to occur is judged to be exceedingly small. The ecological risk 

associated with gene flow and introgression from KK179 derives from the presence of 
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the CCOMT gene segments. The consequences of the introgression of the CCOMT gene 

segments from KK179 into a sexually compatible species are neglible since, as data 

presented in this petition confirm, 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 alfalfa cultivation practices and management 

as described in Section VIII. Typically, alfalfa 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-established 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 will utilize the same agronomic practices as 

conventional alfalfa production, including tillage operations, seedbed preparation, pest 

management, and harvesting procedures. Therefore, no impacts on current cultivation 

and management practices for alfalfa are expected following the introduction of KK179. 

KK179 will allow growers the flexibility to alter 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 value of alfalfa production on their farm. Either they can 1) 

maximize forage quality while maintaining yield or 2) maximize yield while maintaining 

forage quality, depending on plant growth stage at harvest. When implementing the first 

production strategy, the cutting schedules and the timing of harvest remains the same as 

those used with conventional alfalfa. KK179 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 more likely to meet or exceed the intended quality standard 

targeted by the grower. The second production strategy involves delaying harvest to 

maximize yield without forfeiting forage quality compared to conventional alfalfa. 

KK179 can be harvested later and still produce high quality forage that is comparable to 

earlier harvest timings with conventional alfalfa. This strategy is expected to result in 

increased forage tonnage or yield from the harvest. 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., three 
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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 similar or even higher because of the longer cutting intervals and 

resulting higher forage yields with each cutting. 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 Protection Act, are the living stage of any of the 

following, or a similar article, that can directly or indirectly 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 viroid; 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 reduced G lignin and total 

lignin in forage compared to conventional alfalfa cut at the same stage of growth, is not 

significantly different from conventional alfalfa, in terms of plant pest potential. 

Monsanto and FGI are not aware of any study results or observations 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 compared to a conventional control; 4) 

phenotypic and agronomic characteristics demonstrating no increased plant pest 

potential; 5) negligible risk to NTOs including organisms beneficial to agriculture; 6) 

familiarity with alfalfa as a cultivated crop and 7) no greater likelihood to impact 

agronomic practices, including land use, cultivation 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 conventional alfalfa and previously deregulated Roundup Ready 

alfalfa products, KK179 is not expected to be a plant pest. Results also support a 

conclusion of no increased weediness potential of KK179 compared to conventional 

alfalfa. Therefore, Monsanto and FGI request a determination from USDA-APHIS that 

KK179 and any progeny derived from crosses between KK179 and other commercial 

alfalfa be granted non-regulated status under 7 CFR § 340. 
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X. ADVERSE CONSEQUENCES OF INTRODUCTION 

Monsanto and FGI are not aware of any study results or observations associated with 

KK179 indicating that there would be unexpected environmental consequences from the 

introduction of KK179. KK179 contains an DNA insert with gene segments from the 

endogenous CCOMT enzyme gene under the control 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 sequence that forms double 

stranded RNA (dsRNA), which specifically results in the degradation of CCOMT

transcripts via an RNA interference 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 phenotypic difference between KK179 and conventional alfalfa 

is reduction in G lignin and total lignin. 

The data and information presented in this 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 characterization evaluation included molecular 

analyses, which confirmed the insertion of a single functional copy of the CCOMT 

suppression cassette at a single locus within the alfalfa genome. Additionally, there is a 

history of safe use of RNA as an expressed product and no evidence of protein 

production from the CCOMT suppression cassette. 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 evaluations of KK179, including an assessment of 

seed germination and dormancy characteristics, plant growth and development 

characteristics, reproductive characteristics, 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 including 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 anticipated conditions of use. 

The introduction of KK179 will not result in changes to cultivation practices or the 

management of weeds, diseases and insects in alfalfa production systems. Growers 

familiar with cultivation of conventional or Roundup Ready alfalfa will continue to 

employ the same crop rotational practices, weed control practices and/or volunteer 

control measures currently in place for conventional or Roundup Ready alfalfa products. 
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APPENDICES 
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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, some final reports, mainly from the 2010-12 

seasons, are still in preparation. A list of trials conducted under USDA permits and the 

status of the final reports for these trials are provided in Table A-1. 
Monsanto Company 12-AL-246U 231 of 407

Table A-1. USDA Notifications and Permits 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 USDA
Monsanto 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 Progress
Monsanto 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 Progress
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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

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

B.1. Materials 

The genomic DNA used in molecular analyses was isolated from leaf tissues of the P0

generation of KK179, the conventional control C0, and conventional parental controls 

R2336 and Ms208. For generational stability analysis, genomic DNA was extracted from 

leaf tissue of the P0, MBC1, MBC2, and Syn1 generations of KK179. PV-MSPQ12633 

(Figure III-1) was used as a positive hybridization control in Southern blot analyses. 

Probe templates generated from PV-MSPQ12633 were used as additional positive 

hybridization controls. Additional reference standards, the 1 kb DNA Extension Ladder, 

and λ DNA/Hind III Fragments from Invitrogen (Carlsbad, CA) were used for size 

estimations on Southern blots 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 Southern Blot and PCR Analyses 

KK179, conventional control C0, and conventional parental controls R2336 and Ms208 

genomic DNA samples were isolated from alfalfa leaf tissue. Prior to extraction, leaf 

tissue was processed to a fine powder by mortar and pestle 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 μg 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 centrifuged at ~10,300 × g for 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 was 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 μl of TE buffer (10 mM Tris HCl, 1 mM 

EDTA, pH 8.0). All extracted DNA was stored in a 4°C refrigerator 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 QubitTM Fluorometer (Invitrogen, Carlsbad, CA) according to manufacturer’s 

instruction. 

B.5. Restriction Enzyme Digestion of Genomic DNA 

Approximately ten micrograms (μg) of genomic DNA extracted from the test and control 

substances were digested with the appropriate 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 (NEB) 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 hours at room temperature. For the purpose of 

running positive hybridization 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 run/short run format. The long run allows 

for greater resolution of large molecular weight 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 stability analysis, individual digests of ~10 μg 

of genomic DNA extracted from leaf tissue across multiple generations of KK179 were 

loaded on the agarose gel in a single run format. 

B.7. DNA Probe Preparation for Southern Blot Analyses 

Probe templates were prepared by PCR amplification using PV-MSPQ12633 as the 

template. The PCR products were separated on an agarose gel by electrophoresis and 

purified from the gel using QIAquick Gel Extraction Kit (Qiagen, Valencia, CA) 

according to the manufacturer’s instruction. The probe templates were designed based on 

the nucleotide composition (% GC) of the sequence, 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

[α32P]-deoxycytidine triphosphate (dCTP) (6000 Ci/mmol) or [α32P]-deoxyadenosine 

triphosphate (dATP) (6000 Ci/mmol) using the random priming 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 control 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 equivalent of the PV-MSPQ12633 DNA previously 

digested with Xba I or Eco RI (NEB) was added to digested conventional control 

genomic DNA to serve as a positive hybridization control. In Southern blots hybridized 

with multiple probes, ~1.0 genome equivalent of the PV-MSPQ12633 DNA previously 

digested with Xba I or Eco RI (NEB), as well as ~0.1 and ~1.0 genome equivalent of the 

appropriate probe templates were added to digested conventional control genomic DNA 

to serve as positive hybridization controls. The DNA was then separated by agarose gel 

electrophoresis and transferred onto a nylon membrane. Southern blots were hybridized 

and washed at 55°C or 60°C, depending 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 conjunction 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 Product A, Product B, Product C, Product D, and 

Product E were generated to span the insert and adjacent 5′ and 3′ flanking DNA 

sequences in KK179 (Figure V-9). These products 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 MgCl2, 0.2 μM of each primer, 0.08 mM of each dNTP, and 

0.02 units/μl of Phusion Hot Start II High Fidelity 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 trademark ofTakara Bio Inc., Shiga, Japan. 

Probe Probe Type Labeling 

Method 

Probe labeled 

with dNTP 

(

32P) 

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 60 
Monsanto Company 12-AL-246U 240 of 407

The amplification of Product B 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 30°seconds, 

and 63°C for one minute; one cycle at 63°C for five minutes. 

The amplification of Product C was performed 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 performed 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 were 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. This consensus sequence was aligned to the 

PV-MSPQ12633 sequence to determine the integrity 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 conventional alfalfa, PCR and sequence analyses 

were performed on genomic DNA from both KK179 and the conventional parental 

control alfalfa R2336 (Figure V-10). The primers used in this 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 contains a final concentration of 1.5 mM MgCl2,

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 (Finnzymes, 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 the PCR products were the expected size prior 
Monsanto Company 12-AL-246U 241 of 407

to sequencing. Only the verified PCR product from the conventional parental control 

R2336 was purified with the QIAquick PCR Purification 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 performed 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. This 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 specific sequences among DNA fragments separated 

by gel electrophoresis. Journal of Molecular Biology 98:503-517. 
Monsanto Company 12-AL-246U 243 of 407

Appendix C: Materials and Methods Used 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 generation of KK179 and the conventional control C0. 

Probe templates were used as positive hybridization controls in northern blot analyses. 

Prior to probe generation, DNA fragments 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 sequence of the plasmid DNA was confirmed by 

sequencing and comparison to the expected sequences 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 template was generated by PCR from conventional 

alfalfa genomic DNA using a forward primer and a reverse primer specific to the partialcoding region of the alfalfa actin gene. RiboRulerTM RNA ladders (high range) from 

Fermentas Co (Glen Burnie, MD) were used for size estimations on northern blots and 

formaldehyde/agarose gels. The unique identities 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 for 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 and control substances was confirmed by eventspecific PCR; the raw data were archived with the study. The study director reviewed the 

chain of custody (COC) documentation, which confirmed the identity 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 identities of the probes. The raw data are archived at Monsanto. 

Extracted RNA and RNA markers were stored 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 interpretable signals on northern blots. 

 

® pCR is a registered trademark 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 samples 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 nitrogen. 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 vortexer 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. Approximately 6 ml of chloroform was added to the 

samples, mixed by vortexing for one to two minutes, and incubated at room temperature 

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. 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 centrifuged at ~12,000 × g for five minutes at 4°C for 

additional purification of RNA. The upper aqueous phase was transferred 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 temperature 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 was precipitated with approximately 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 were then centrifuged at ~12,000 × g for 15-20 

minutes at 4°C. The RNA pellets were washed 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 extracted RNA was stored in a -80°C freezer. 

C.4. Quantification of RNA 

Total RNA was quantified using a Beckman Coulter DU-650 Spectrophotometer 

(Beckman Coulter, Inc. Atlanta, GA) according to manufacturer’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 control total RNA samples was extracted 

using the Poly(A) Purist MAG Kit (Ambion Co., Austin, TX) according to the 

manufacturer's instruction with minor modifications. Briefly, ~420 μg of total RNA was 

placed in a 2 ml microcentrifuge tube and Rnase-free water was added to obtain a final 

RNA concentration of ~600 μg/ml. An equal 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 mixture was incubated in a 65°C water bath for 

five minutes and then incubated 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 the side of the tube. The supernatant was 
Monsanto 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 two washes with an equal volume (to the 

starting RNA solution) of Wash Solution 2, discarding 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 beads. 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 1/10th 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. The pellets were briefly air dried, and then 

resuspended in an appropriate volume of prewarmed 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 conventional 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 onto nylon membranes and probing with radiolabeled DNA. 

CCOMT hybridization signals were stripped from the blots and the stripped blots were 

hybridized to a 32P-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 × 106

 cpm/ml of the 

hybridization solution. Northern blots were hybridized 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 Kodak, Rochester, NY) in conjunction with at 

least one Kodak Biomax MS-intensifying screen in a -80°C freezer. 

C.8. Positive Hybridization Controls and Probe Template 

DNA probe templates were run on the formaldehyde/agarose gels to serve as positive 

controls for hybridization. On northern blots 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 probe to an RNA ladder. The following table 

lists the DNA probes, the positive hybridization controls, and 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 55oC 
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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 11266292). The conventional control has 

background genetics similar to that of KK179. The conventional commercial reference 

varieties were 14 conventional alfalfa 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 further confirm the identities of KK179, the 

conventional C0-Syn1, and conventional commercial reference varieties, event-specific 

polymerase chain reaction (PCR) analyses were conducted on the 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 from each of four replicated plots from six 

field sites [Tulare County, California (CAPR); Jefferson County, Iowa (IARL); Clinton 

County, Illinois (ILCY); Pawnee County, Kansas (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 was verified using PCR. The field plots were planted in a 

randomized complete block design. All samples at the field sites were grown under 

normal agronomic field conditions for their respective geographic regions. Between 1 
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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 the 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 temperature to Samuel Roberts Noble Foundation 

(Ardmore, Oklahoma) for analysis of lignin 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 lignin), caffeoyl lignin, 

guaiacyl lignin (G lignin), 5-hydroxyguaiacyl lignin, and syringyl lignin (S lignin) 

subunits by the Samuel Roberts Noble Foundation to demonstrate that suppression of the 

caffeoyl coenzyme A 3-O-methyltransferase (CCOMT) gene by dsRNA produced by 

KK179 resulted in the intended reduction of the G lignin subunit, and thereby total lignin. 

Forage samples were also analyzed by Dairy 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 Thioacidolysis (H, G and S Lignin) (Conducted at 

Samuel Roberts Noble Foundation) 

The lyophilized, ground samples were extracted 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 manifold freeze-drier. About 18 mg of dried 

samples were weighed into 12 ml glass tubes; 3 ml of thioacidolysis reaction mixture 

were added, and the reaction was conducted 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 gas 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 comparable to the conventional crucible method 

(Weston et al., 2006). Briefly, samples were boiled in acid detergent fiber (ADF) 

solution, rinsed with water and acetone, and hydrolyzed in sulfuric acid. Following acid 

hydrolysis, samples were rinsed in water and soaked in acetone before being dried to a 

constant weight. The dried samples are weighed and then ashed in individual crucibles at 
Monsanto Company 12-AL-246U 249 of 407

525oC for 3 hours. After ashing, samples are cooled in a desiccator 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 Monsanto Company (St. Louis, Missouri) for 

review. Data were then transferred 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 greater than the assay limit of 

quantitation (LOQ). Components with more than 50% of observations below the assay 

LOQ were excluded from summaries and analysis. The following two components with 

more than 50% of the observations below the assay LOQ were excluded: caffeoyl lignin 

and 5-hydroxyguaiacyl lignin. 

The data were assessed for potential outliers using a studentized PRESS residuals 

calculation. A PRESS residual is the difference 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 tend 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 studentized PRESS residual range are considered 

for exclusion as outliers from the final analyses. In lignin units outliers as a proportion of 

HGS lignin were indentified in three conventional 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 were statistically analyzed using a mixed 

model analysis. The six replicated field sites were analyzed as a combined data set. 

These combined-site analyses used model (1). 

(1) Yijk = U + Ti + Lj + B(L)jk + LTij + eijk, 

where Yijk = unique individual observation, U = overall mean, Ti = substance effect, Lj = 

random location effect, B(L)jk = random block within location effect, LTij = random 

location by substance interaction effect, and eijk = residual error. 

A supplementary individual site analysis used model (2). 

(2) Yij = U + Ti + Bj + eij, 

where Yij = unique individual observation, U = overall mean, Ti = substance effect, Bj = 

random block effect, and eij = 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 specified proportion, p, of an entire 

sampled population for the parameter measured. The calculated tolerance intervals are 

expected to contain, with 95% confidence, 99% of the quantities expressed in the 

population of conventional alfalfa. Each tolerance 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. 

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 Statistical Analysis of Composition Data 

Component From (X) To Formula1

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 

Table D-3. Statistical Summary of Alfalfa Forage Lignin Subunits for KK179 vs. Conventional Control (C

Roberts Noble Foundation) 

 Difference (Test minus Control)

Analytical Component (Units)1

Test2 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence 

Interval

Signific

(p-Val

Site CAPR

Lignin Subunits (μmol/g CWR)Guaiacyl lignin unit 72.86 (11.69) 92.84 (8.27) -19.99 (14.32) -53.85, 13.88 0.20

(70.00 - 75.71) (66.63 - 113.14) (-35.42 - -10.46) 

Hydroxyphenyl lignin unit 5.41 (0.62) 4.40 (0.42) 1.01 (0.66) -0.78, 2.79 0.19

(5.17 - 6.05) (2.78 - 5.26) (0.27 - 1.39) 

Syringyl lignin unit 73.27 (10.15) 62.98 (6.72) 10.29 (10.99) -19.01, 39.59 0.39

(63.21 - 87.67) (46.77 - 76.24) (6.87 - 15.10) 
Monsanto Company 12-AL-246U 

Table D-3 (continued). Statistical Summary of Alfalfa Forage Lignin Subunits for KK179 vs. Conventionaat Samuel Roberts Noble Foundation) 

 Difference (Test minus Control)

Analytical Component (Units)1

Test2 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence 

Interval

Signific

(p-Val

Site CAPR

Lignin Subunits (% Total HGS)Guaiacyl lignin unit 47.59 (1.64) 57.82 (1.16) -10.23 (2.01) -14.97, -5.48 0.00

(44.92 - 50.27) (56.88 - 58.92) (-14.01 - -6.61) 

Hydroxyphenyl lignin unit 3.70 (0.35) 2.75 (0.22) 0.96 (0.40) -0.038, 1.95 0.05

(3.07 - 4.34) (2.39 - 3.29) (0.47 - 1.05) 

Syringyl lignin unit 48.70 (1.83) 39.43 (1.30) 9.27 (2.24) 3.96, 14.57 0.00

(45.39 - 52.01) (38.48 - 40.26) (5.56 - 13.54) 

RatioS:G Ratio 1.03 (0.065) 0.68 (0.046) 0.35 (0.080) 0.16, 0.54 0.00

(0.90 - 1.16) (0.65 - 0.70) (0.20 - 0.50) 

Site IARL

Lignin Subunits (μmol/g CWR)Guaiacyl lignin unit 37.57 (6.10) 37.48 (6.10) 0.088 (8.63) -19.44, 19.61 0.99

(21.17 - 65.54) (33.11 - 43.31) (-13.83 - 27.03) 
Monsanto Company 12-AL-246U 

Table D-3 (continued). Statistical Summary of Alfalfa Forage Lignin Subunits for KK179 vs. Conventionaat Samuel Roberts Noble Foundation) 

 Difference (Test minus Control)

Analytical Component (Units)1

Test2 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence 

Interval

Signific

(p-Val

Site IARL

Lignin Subunits (μmol/g CWR)Hydroxyphenyl lignin unit 4.88 (1.08) 2.69 (1.08) 2.19 (1.52) -1.25, 5.64 0.18

(2.20 - 10.14) (2.40 - 3.06) (-0.20 - 7.24) 

Syringyl lignin unit 17.46 (2.63) 13.74 (2.63) 3.72 (3.72) -4.69, 12.13 0.34

(9.82 - 27.96) (12.20 - 15.36) (-2.38 - 13.42) 

Lignin Subunits (% Total HGS)Guaiacyl lignin unit 62.80 (0.81) 69.50 (0.81) -6.70 (1.06) -9.30, -4.11 <0.00

(60.80 - 63.78) (68.47 - 70.56) (-9.36 - -5.09) 

Hydroxyphenyl lignin unit 7.55 (0.45) 4.99 (0.45) 2.56 (0.61) 1.07, 4.05 0.00

(6.39 - 9.78) (4.84 - 5.18) (1.43 - 4.60) 

Syringyl lignin unit 29.65 (0.90) 25.51 (0.90) 4.14 (1.03) 1.63, 6.65 0.00

(26.98 - 31.80) (24.60 - 26.57) (0.99 - 6.92) 
Monsanto Company 12-AL-246U 

Table D-3 (continued). Statistical Summary of Alfalfa Forage Lignin Subunits for KK179 vs. Conventionaat Samuel Roberts Noble Foundation) 

 Difference (Test minus Control)

Analytical Component (Units)1

Test2 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence 

Interval

Signific

(p-Val

Site IARL

RatioS:G Ratio 0.47 (0.019) 0.37 (0.019) 0.11 (0.022) 0.051, 0.16 0.00

(0.43 - 0.52) (0.35 - 0.39) (0.049 - 0.17) 

Site ILCY

Lignin Subunits (μmol/g CWR)Guaiacyl lignin unit 87.60 (12.13) 104.06 (12.13) -16.46 (13.04) -48.36, 15.44 0.25

(52.96 - 134.96) (77.34 - 131.40) (-29.52 - 3.56) 

Hydroxyphenyl lignin unit 6.19 (1.03) 4.14 (1.03) 2.05 (1.38) -1.34, 5.44 0.18

(3.16 - 10.84) (2.92 - 5.49) (0.24 - 5.35) 

Syringyl lignin unit 67.00 (7.82) 63.39 (7.82) 3.61 (9.39) -19.37, 26.59 0.71

(49.44 - 85.83) (47.39 - 91.89) (-18.80 - 27.05) 

Lignin Subunits (% Total HGS)Guaiacyl lignin unit 53.58 (1.89) 60.96 (1.89) -7.37 (2.68) -13.43, -1.31 0.02

(49.12 - 61.66) (57.44 - 63.59) (-13.10 - 4.22) 
Monsanto Company 12-AL-246U 

Table D-3 (continued). Statistical Summary of Alfalfa Forage Lignin Subunits for KK179 vs. Conventionaat Samuel Roberts Noble Foundation) 

 Difference (Test minus Control)

Analytical Component (Units)1

Test2 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence 

Interval

Signific

(p-Val

Site ILCY

Lignin Subunits (% Total HGS)Hydroxyphenyl lignin unit 3.69 (0.30) 2.40 (0.30) 1.29 (0.43) 0.32, 2.25 0.01

(2.99 - 4.95) (2.29 - 2.54) (0.58 - 2.55) 

Syringyl lignin unit 42.73 (2.13) 36.64 (2.13) 6.09 (3.02) -0.74, 12.91 0.07

(33.39 - 47.77) (34.02 - 40.17) (-6.77 - 12.52) 

RatioS:G Ratio 0.81 (0.063) 0.60 (0.063) 0.21 (0.089) 0.0083, 0.41 0.04

(0.54 - 0.97) (0.54 - 0.70) (-0.16 - 0.41) 

Site KSLA

Lignin Subunits (μmol/g CWR)Guaiacyl lignin unit 62.88 (3.72) 82.54 (3.72) -19.66 (5.25) -31.54, -7.77 0.00

(59.88 - 66.12) (65.03 - 91.43) (-31.46 - 1.09) 

Hydroxyphenyl lignin unit 4.38 (0.28) 4.38 (0.28) 0.0050 (0.39) -0.88, 0.89 0.99

(4.14 - 4.60) (3.79 - 5.02) (-0.70 - 0.35) 
Monsanto Company 12-AL-246U 

Table D-3 (continued). Statistical Summary of Alfalfa Forage Lignin Subunits for KK179 vs. Conventiona(Conducted at Samuel Roberts Noble Foundation) 

 Difference (Test minus Control)

Analytical Component (Units)1

Test2 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence 

Interval

Signific

(p-Val

Site KSLA

Lignin Subunits (μmol/g CWR)Syringyl lignin unit 51.49 (3.43) 52.06 (3.43) -0.58 (4.85) -11.54, 10.39 0.90

(44.58 - 54.81) (40.48 - 62.77) (-18.19 - 14.33) 

Lignin Subunits (% Total HGS)Guaiacyl lignin unit 53.00 (0.72) 59.46 (0.72) -6.46 (1.02) -8.77, -4.15 <0.00

(52.15 - 54.91) (57.56 - 61.36) (-9.21 - -2.65) 

Hydroxyphenyl lignin unit 3.70 (0.25) 3.19 (0.25) 0.52 (0.36) -0.30, 1.33 0.18

(3.49 - 4.22) (2.82 - 3.76) (-0.19 - 1.33) 

Syringyl lignin unit 43.30 (0.82) 37.35 (0.82) 5.94 (1.16) 3.33, 8.56 <0.00

(40.88 - 44.36) (35.81 - 39.56) (1.32 - 8.54) 

RatioS:G Ratio 0.82 (0.024) 0.63 (0.024) 0.19 (0.034) 0.11, 0.27 <0.00

(0.74 - 0.85) (0.58 - 0.69) (0.057 - 0.27) 
Monsanto Company 12-AL-246U 

Table D-3 (continued). Statistical Summary of Intended Changes in Forage Lignin Subunits for KK179 vsControl (Conducted at Samuel Roberts Noble Foundation) 

 Difference (Test minus Control)

Analytical Component (Units)1

Test2 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence 

Interval

Signific

(p-Val

Site TXCL

Lignin Subunits (μmol/g CWR)Guaiacyl lignin unit 64.68 (9.26) 91.11 (9.26) -26.44 (12.16) -56.19, 3.32 0.07

(54.72 - 74.33) (71.43 - 105.30) (-39.11 - -9.45) 

Hydroxyphenyl lignin unit 4.18 (0.91) 4.55 (0.91) -0.37 (1.28) -3.27, 2.53 0.77

(2.74 - 5.41) (4.24 - 4.94) (-1.76 - 1.17) 

Syringyl lignin unit 59.13 (9.52) 55.21 (9.52) 3.92 (12.79) -27.37, 35.22 0.76

(45.79 - 70.15) (45.73 - 63.25) (-17.46 - 15.72) 

Lignin Subunits (% Total HGS)Guaiacyl lignin unit 50.67 (1.07) 60.26 (1.07) -9.59 (1.51) -13.01, -6.16 <0.00

(49.59 - 52.63) (58.06 - 64.22) (-14.63 - -5.43) 

Hydroxyphenyl lignin unit 3.28 (0.53) 3.08 (0.53) 0.20 (0.75) -1.49, 1.88 0.79

(2.04 - 4.13) (2.59 - 4.05) (-0.85 - 1.02) 
Monsanto Company 12-AL-246U 

Table D-3 (continued). Statistical Summary of Intended Changes in Forage Lignin Subunits for KK179 vsControl (Conducted at Samuel Roberts Noble Foundation) 

 Difference (Test minus Control)

Analytical Component (Units)1

Test2 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence 

Interval

Signific

(p-Val

Site TXCL

Lignin Subunits (% Total HGS)Syringyl lignin unit 46.05 (0.99) 36.66 (0.99) 9.39 (1.38) 6.00, 12.78 <0.00

(44.04 - 47.67) (33.20 - 39.14) (4.90 - 13.61) 

RatioS:G Ratio 0.91 (0.031) 0.61 (0.031) 0.30 (0.043) 0.20, 0.40 <0.00

(0.84 - 0.95) (0.52 - 0.67) (0.16 - 0.43) 

Site WIDL

Lignin Subunits (μmol/g CWR)Guaiacyl lignin unit 84.34 (5.42) 94.31 (5.42) -9.97 (7.66) -27.30, 7.36 0.22

(77.36 - 88.36) (69.76 - 111.31) (-24.67 - 18.60) 

Hydroxyphenyl lignin unit 5.24 (0.69) 3.10 (0.69) 2.15 (0.78) 0.23, 4.07 0.03

(4.34 - 6.22) (0.58 - 5.48) (-0.38 - 3.76) 

Syringyl lignin unit 69.11 (4.76) 55.09 (4.76) 14.02 (6.73) -1.20, 29.25 0.06

(63.76 - 78.51) (34.94 - 64.85) (-0.86 - 43.57) 
Monsanto Company 12-AL-246U 

Table D-3 (continued). Statistical Summary of Intended Changes in Forage Lignin Subunits for KK179 vsControl (Conducted at Samuel Roberts Noble Foundation) 

 Difference (Test minus Control)

Analytical Component (Units)1

Test2 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence 

Interval

Signific

(p-Val

Site WIDL

Lignin Subunits (% Total HGS)Guaiacyl lignin unit 53.18 (1.25) 62.12 (1.25) -8.94 (1.76) -12.92, -4.95 <0.00

(51.32 - 54.77) (57.24 - 65.32) (-14.01 - -2.47) 

Hydroxyphenyl lignin unit 3.33 (0.42) 2.04 (0.42) 1.29 (0.49) 0.088, 2.49 0.03

(2.67 - 4.22) (0.34 - 3.33) (0.0067 - 2.33) 

Syringyl lignin unit 43.49 (1.03) 35.84 (1.03) 7.65 (1.46) 4.35, 10.94 <0.00

(41.89 - 45.60) (32.72 - 39.43) (2.46 - 12.88) 

RatioS:G Ratio 0.82 (0.032) 0.58 (0.032) 0.24 (0.045) 0.14, 0.34 <0.00

(0.76 - 0.89) (0.50 - 0.69) (0.076 - 0.39) 

1Total HGS is the sum of Hydroxyphenyl, Guaiacyl and Syringyl lignin subunits (μmol/g CWR); S:G Ratio = Syringyl lignGuaiacyl lignin unit. CWR = Cell Wall Residue 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 referenlimits set to zero.
Monsanto Company 12-AL-246U 

Table D-4. Statistical Summary of Alfalfa Forage Total Lignin (ADL) Levels for KK179 vs. Conventional at Dairy One Forage Lab) 

 Difference (Test minus Control)

Analytical Component (Units)1

Test2 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence 

Interval

Signific

(p-Val

Site CAPR

Acid Detergent Lignin (% dw) 5.79 (0.44) 8.38 (0.44) -2.59 (0.54) -3.91, -1.27 0.00

(4.93 - 6.57) (7.30 - 10.10) (-3.53 - -1.23) 

Site IARL

Acid Detergent Lignin (% dw) 2.79 (0.46) 3.70 (0.46) -0.91 (0.65) -2.37, 0.55 0.19

(2.73 - 2.87) (2.23 - 5.20) (-2.47 - 0.63) 

Site ILCY

Acid Detergent Lignin (% dw) 6.87 (0.60) 6.87 (0.60) 0 (0.84) -1.91, 1.91 1.00

(6.43 - 7.60) (5.67 - 8.63) (-2.20 - 1.30) 

Site KSLA

Acid Detergent Lignin (% dw) 5.35 (0.49) 8.35 (0.49) -3.00 (0.70) -4.58, -1.42 0.00

(3.47 - 6.53) (7.60 - 9.77) (-4.33 - -1.07) 
Monsanto Company 12-AL-246U 

Table D-4 (continued). Statistical Summary of Alfalfa Forage Total Lignin (ADL) Levels for KK179 vs. Co(Conducted at Dairy One Forage Lab) 

 Difference (Test minus Control)

Analytical Component (Units)1

Test2 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence 

Interval

Signific

(p-Val

Site TXCL

Acid Detergent Lignin (% dw) 4.65 (0.26) 7.32 (0.26) -2.67 (0.37) -3.58, -1.76 <0.00

(4.07 - 5.03) (6.40 - 8.10) (-3.17 - -1.40) 

Site WIDL

Acid Detergent Lignin (% dw) 6.92 (0.29) 6.96 (0.29) -0.042 (0.37) -0.94, 0.86 0.91

(6.70 - 7.07) (5.97 - 7.87) (-1.17 - 0.87) 

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 referenlimits 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. Rolando. 1995. New Insights into the Molecular 

Architecture of Hardwood Lignins by Chemical Degradative Methods. Research on 

Chemical Intermediates 21: 397-412. 

Weston, T.R., V. Nayigihugu and B.W. Hess. 2006. Comparison of techniques for 

quantitative analysis of acid detergent lignin 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 11266289) and the conventional control C0-Syn1 

(Seed Lot Number 11266292) was evaluated. The conventional control C0-Syn1has 

background genetics similar to that of KK179. The conventional commercial reference 

varieties were 14 conventional alfalfa 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 further confirm the identities of KK179, the 

conventional control C0-Syn1, and conventional commercial reference varieties, eventspecific polymerase chain reaction (PCR) analyses were conducted on the forage from 

each site to confirm the presence 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 from each of four replicated plots from six 

field sites [Tulare County, California (CAPR); Jefferson County, Iowa (IARL); Clinton 

County, Illinois (ILCY); Pawnee County, Kansas (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 was verified using PCR. The field plots were planted in a 

randomized complete block design. All samples at the field sites were grown under 

normal agronomic field conditions for their respective geographic regions. Between 1 
Monsanto 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 individual plot. A subsample (about 680g) of 

plants was randomly collected from all harvested plants in each plot. 

Forage samples were shipped frozen from the 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 the Samuel Roberts Noble Foundation were 

conducted to establish the compositional equivalence of KK179 compared to 

conventional alfalfa using the principles and analytes outlined in the OECD consensus 

document for alflafa composition (OECD, 2005). Forage samples analyzed by Covance 

Laboratories Inc. included nutrients, proximates (ash, fat, moisture, and protein), 

carbohydrates by calculation, acid detergent fiber (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-nutrients assessed by Covance included daidzein, 

glycitein, genistein, coumesterol, formononetin, 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 saponins, total bayogenin, 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 the process of removal of proteins, 

carbohydrates, and ash. Fats and pigments were removed with an acetone 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 (including asparagine) 

 Total cystine (including cysteine) 

 Total glutamic acid (including glutamine) 

 Total glycine 
Monsanto 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 and cysteine are converted to S-2-

carboxyethylthiocysteine by the addition of dithiodipropionic acid. Tryptophan was 

hydrolyzed from proteins by heating at approximately 110oC 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; Schuster, 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 Monohydrochloride, 100%, Lot Number 1361811 

 Sigma-Aldrich, L-Aspartic Acid, 100.6%, Lot Number BCBB9274 

 Sigma-Aldrich, L-Cystine, 99.9%, Lot Number 1418036 

 Sigma-Aldrich, L-Glutamic Acid, 100.2%, 1423805 

 Sigma-Aldrich, Glycine, 100%, Lot Number 1119375 

 Sigma-Aldrich, L-Histidine Monohydrochloride Monohydrate, 99.9%, Lot Number 

BCBB1348 

 Sigma-Aldrich, L-Isoleucine, 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-Phenylalanine, 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-Threonine, 100%, Lot Number 1402329 

 Sigma-Aldrich, L-Tryptophan, 99.8%, BCBB1284 
Monsanto 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 furnace at 550°C and ignited. The nonvolatile 

matter remaining was quantitated gravimetrically 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 quantitated using isocratic reverse phase highperformance liquid chromatography (HPLC) with UV detection (Chen et al., 2010; 

Saloniemi et al., 1995). The limit 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 and purified with ultrafiltration. Analysis 

was performed with an automated amino acid 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 (Conducted at Covance Laboratories Inc.) 

The samples were extracted with 0.02N HCL and purified with ultrafiltration. Analysis 

was performed with an automated amino acid analyzer (Pettersson and Kiessling, 1984; 

Saloniemi et al., 1995). The limit 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 evaporated, 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 cystine 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 Spectrometry (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-ashed with nitric acid, treated with 

hydrochloric acid, taken to dryness, 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, measured on the inductively coupled plasma 

spectrometer, with the emission of the standard solutions (AOAC, 2011c; 2011h). 
Monsanto Company 12-AL-246U 268 of 407

Reference Standards: 

Inorganic Ventures Reference Standards 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 0.400 
Monsanto 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 brought to volume, diluted, and centrifuged. An 

aliquot of the supernatant was placed onto a C18 solid-phase extraction column. 

Unwanted components of the matrix were rinsed off with 20% methanol and then the 

isoflavones were eluted with 80% methanol. 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 conventional crucible method that is a manual 

method (Goering and Van Soest, 1970). The 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 quantitation was calculated and reported on 

fresh weight basis. The limit 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 percent 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 removal of protein, 

carbohydrate, and ash. Fats and pigments were removed with an acetone 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 limit of quantitation was 0.100%.

E.4.15. p-Coumaric Acid, Ferulic Acid, Sinapic 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 ultraviolet 
Monsanto 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-Hydroxycinnamic acid (Sinapic Acid), 99%, Lot 

Number 040M1943 

 ACROS Organics, 4-Hydroxy-3-methoxycinnamic 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 sulfuric acid containing a catalyst mixture. The acid digest was 

made alkaline. The ammonia was distilled and then titrated with a previously 

standardized acid. The percent nitrogen was calculated and 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 extracted with 80% methanol solution that 

contained the Internal Standard (Huhman et al., 2005; Huhman and Sumner, 2002). 

Samples were analyzed with a Waters Acquity Ultra Performance Liquid 

Chromotography (UPLC) system (Waters Corporation, Milford, Massachusetts) fitted 

with a Quadrupole Time of Flight (QTOF) 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, Milford, Massachusetts). The mobile phase 

consisted of eluent A (0.1% [v/v] CH3COOH/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 48 
Monsanto 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-GlcUA- 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 Lab, Gal-GlcUA- Soyasaponin B, 10.5 ng/inj (13.1 pmol), 

 Mark A. Berhow USDA Lab, 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 produce a color that can be measured 

spectrophotometrically at 760 nm. Results are 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 Monsanto Company (St. Louis, Missouri) for 

review. Data were then transferred 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 greater than the assay limit of 

quantitation (LOQ). Components with more than 50% of observations below the assay 
Monsanto Company 12-AL-246U 272 of 407

LOQ were excluded from summaries and analysis. The following seven components 

with more than 50% of the observations below the assay LOQ were excluded: biochanin 

A, coumesterol, daidzein, formononetin, genistein, glycitein, and sinapic acid. 

If less than 50% of the observations for a component were below the LOQ, individual 

analyses that were below the LOQ were assigned a value equal to one-half the LOQ. In 

this study 56 values for sodium were assigned 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 outliers using a studentized PRESS residuals 

calculation. A PRESS residual is the difference 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 tend 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 studentized 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 were statistically analyzed using a mixed 

model analysis. The six replicated field sites were analyzed as a combined data set. 

These combined-site analyses used model (1). 

(1) Yijk = U + Ti + Lj + B(L)jk + LTij + eijk, 

where Yijk = unique individual observation, U = overall mean, Ti = substance effect, Lj = 

random location effect, B(L)jk = random block within location effect, LTij = random 

location by substance interaction effect, and eijk = residual error. 

A supplementary individual site analysis used model (2). 

(2) Yij = U + Ti + Bj + eij, 

where Yij = unique individual observation, U = overall mean, Ti = substance effect, Bj = 

random block effect, and eij = 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 specified proportion, p, of an entire 

sampled population for the parameter measured. The calculated tolerance intervals are 

expected to contain, with 95% confidence, 99% of the quantities expressed in the 

population of conventional alfalfa. Each tolerance 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 Statistical Analysis of Composition Data 

Component From (X)1 To Formula2

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/(104

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 103(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 

Table E-3. Statistical Summary of Site CAPR Alfalfa Forage Nutrients for KK179 vs. Conventional Co

Covance Laboratories) 

 Difference (Test minus Control)

Analytical Component (Units)1

KK179 

Mean (S.E.) 2

(Range)

Control3

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence 

Interval

Signific

(p-Val

Proximate (% dw)Ash 8.82 (0.54) 9.85 (0.36) -1.04 (0.58) -2.59, 0.52 0.142 

(8.73 - 9.18) (8.79 - 10.59) (-1.80 - -0.32) 

Carbohydrates 69.61 (0.98) 66.41 (0.69) 3.20 (1.20) 0.37, 6.03 0.031 

(68.88 - 70.34) (64.59 - 68.12) (1.55 - 5.75) 

Moisture (% fw) 78.40 (0.92) 79.90 (0.65) -1.50 (1.13) -4.17, 1.17 0.225 

(76.40 - 80.40) (79.10 - 81.40) (-2.70 - 0.60) 

Protein 19.69 (0.55) 21.86 (0.46) -2.17 (0.46) -3.42, -0.92 0.008 

(19.41 - 20.41) (20.72 - 22.47) (-2.75 - -1.67) 

Total Fat 1.54 (0.40) 1.92 (0.28) -0.38 (0.49) -1.54, 0.78 0.463 

(1.47 - 1.62) (1.31 - 2.67) (-1.19 - 0.31) 

Fiber (% dw)Acid Detergent Fiber 29.98 (2.64) 27.75 (1.58) 2.23 (2.95) -5.69, 10.15 0.489 

(27.91 - 32.12) (24.31 - 30.69) (-2.78 - 7.81) 
Monsanto Company 12-AL-246U 

Table E-3 (continued). Statistical Summary of Site CAPR Alfalfa Forage Nutrients for KK179 vs. Conven(Conducted at Covance Laboratories) 

 Difference (Test minus Control)

Analytical Component (Units)1

KK179 

Mean (S.E.) 2

(Range)

Control3

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence 

Interval

Signific

(p-Val

Fiber (% dw)Acid Detergent Lignin 6.50 (0.43) 6.99 (0.31) -0.50 (0.53) -1.75, 0.76 0.380 

(5.92 - 7.07) (6.31 - 7.54) (-1.63 - 0.012) 

Neutral Detergent Fiber 36.38 (1.98) 36.43 (1.65) -0.043 (1.63) -4.55, 4.46 0.980 

(37.14 - 38.14) (30.91 - 40.74) (-3.60 - 1.72) 

Amino Acid (% dw)Alanine 1.06 (0.022) 1.17 (0.015) -0.11 (0.026) -0.17, -0.047 0.004 

(1.06 - 1.07) (1.12 - 1.21) (-0.095 - -0.063) 

Arginine 0.98 (0.027) 1.10 (0.017) -0.12 (0.031) -0.20, -0.047 0.008 

(0.95 - 0.99) (1.09 - 1.11) (-0.16 - -0.094) 

Aspartic acid 2.69 (0.13) 3.06 (0.091) -0.36 (0.16) -0.74, 0.010 0.055 

(2.44 - 2.94) (2.95 - 3.14) (-0.64 - -0.012) 

Cystine 0.22 (0.0097) 0.23 (0.0069) -0.010 (0.012) -0.039, 0.018 0.411 

(0.20 - 0.23) (0.21 - 0.24) (-0.023 - -0.014) 
Monsanto Company 12-AL-246U 

Table E-3 (continued). Statistical Summary of Site CAPR Alfalfa Forage Nutrients for KK179 vs. Conven(Conducted at Covance Laboratories) 

 Difference (Test minus Control)

Analytical Component (Units)1

KK179 

Mean (S.E.) 2

(Range)

Control3

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence 

Interval

Signific

(p-Val

Amino Acid (% dw)Glutamic acid 1.82 (0.044) 2.05 (0.031) -0.23 (0.054) -0.36, -0.10 0.003 

(1.77 - 1.88) (2.01 - 2.11) (-0.31 - -0.14) 

Glycine 0.94 (0.020) 1.01 (0.015) -0.076 (0.019) -0.13, -0.025 0.014 

(0.90 - 0.94) (0.99 - 1.05) (-0.10 - -0.046) 

Histidine 0.43 (0.013) 0.46 (0.0096) -0.026 (0.012) -0.059, 0.0074 0.097 

(0.42 - 0.43) (0.43 - 0.48) (-0.038 - -0.0067) 

Isoleucine 0.86 (0.025) 0.94 (0.017) -0.080 (0.027) -0.15, -0.0084 0.035 

(0.83 - 0.87) (0.91 - 0.98) (-0.097 - -0.039) 

Leucine 1.42 (0.043) 1.56 (0.027) -0.15 (0.048) -0.27, -0.022 0.029 

(1.38 - 1.43) (1.51 - 1.63) (-0.16 - -0.076) 

Lysine 1.13 (0.054) 1.24 (0.034) -0.11 (0.059) -0.27, 0.049 0.129 

(1.10 - 1.12) (1.15 - 1.34) (-0.14 - -0.046) 
Monsanto Company 12-AL-246U 

Table E-3 (continued). Statistical Summary of Site CAPR Alfalfa Forage Nutrients for KK179 vs. Conven(Conducted at Covance Laboratories)

 Difference (Test minus Control)

Analytical Component (Units)1

KK179 

Mean (S.E.) 2

(Range)

Control3

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence 

Interval

Signific

(p-Val

Amino Acid (% dw)Methionine 0.21 (0.018) 0.24 (0.011) -0.034 (0.020) -0.086, 0.018 0.159 

(0.20 - 0.21) (0.21 - 0.27) (-0.019 - -0.0094) 

Phenylalanine 0.96 (0.028) 1.06 (0.020) -0.10 (0.035) -0.18, -0.018 0.023 

(0.95 - 0.97) (1.01 - 1.11) (-0.11 - -0.045) 

Proline 0.85 (0.034) 0.95 (0.021) -0.10 (0.037) -0.20, -0.0018 0.047 

(0.83 - 0.84) (0.91 - 1.01) (-0.13 - -0.064) 

Serine 0.84 (0.022) 0.92 (0.015) -0.087 (0.027) -0.15, -0.024 0.014 

(0.80 - 0.88) (0.90 - 0.96) (-0.16 - -0.023) 

Threonine 0.84 (0.024) 0.94 (0.014) -0.10 (0.026) -0.17, -0.029 0.016 

(0.82 - 0.85) (0.91 - 0.97) (-0.12 - -0.064) 

Tryptophan 0.34 (0.028) 0.38 (0.018) -0.033 (0.032) -0.11, 0.047 0.337 

(0.34 - 0.35) (0.35 - 0.39) (-0.056 - -0.00099) 
Monsanto Company 12-AL-246U 

Table E-3 (continued). Statistical Summary of Site CAPR Alfalfa Forage Nutrients for KK179 vs. Conven(Conducted at Covance Laboratories)

 Difference (Test minus Control)

Analytical Component (Units)1

KK179 

Mean (S.E.) 2

(Range)

Control3

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence 

Interval

Signific

(p-Val

Amino Acid (% dw)Tyrosine 0.70 (0.017) 0.78 (0.012) -0.081 (0.021) -0.13, -0.033 0.005 

(0.69 - 0.70) (0.76 - 0.80) (-0.098 - -0.053) 

Valine 1.03 (0.027) 1.13 (0.019) -0.098 (0.033) -0.18, -0.019 0.021 

(1.00 - 1.06) (1.09 - 1.18) (-0.12 - -0.028) 

MineralCalcium (% dw) 1.42 (0.092) 1.58 (0.065) -0.16 (0.11) -0.43, 0.11 0.196 

(1.29 - 1.55) (1.42 - 1.72) (-0.36 - 0.13) 

Copper (mg/kg dw) 9.98 (1.09) 10.79 (0.77) -0.81 (1.33) -3.96, 2.34 0.561 

(9.19 - 10.77) (10.10 - 11.93) (-1.62 - 0.67) 

Iron (mg/kg dw) 256.68 (78.54) 348.49 (51.22) -91.81 (85.46) -320.55, 136.92 0.337 

(236.86 - 316.33) (174.40 - 516.75) (-279.88 - 5.44) 

Magnesium (% dw) 0.14 (0.0085) 0.15 (0.0060) -0.016 (0.0089) -0.040, 0.0070 0.129 

(0.12 - 0.15) (0.15 - 0.16) (-0.028 - -0.00009) 
Monsanto Company 12-AL-246U 

Table E-3 (continued). Statistical Summary of Site CAPR Alfalfa Forage Nutrients for KK179 vs. Conven(Conducted at Covance Laboratories)

 Difference (Test minus Control)

Analytical Component (Units)1

KK179 

Mean (S.E.) 2

(Range)

Control3

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence 

Interval

Signific

(p-Val

MineralManganese (mg/kg dw) 35.15 (4.47) 40.72 (3.17) -5.57 (4.58) -18.07, 6.94 0.288 

(36.36 - 37.65) (30.92 - 48.33) (-11.97 - -5.81) 

Phosphorus (% dw) 0.26 (0.010) 0.30 (0.0091) -0.038 (0.0063) -0.055, -0.021 0.003 

(0.27 - 0.28) (0.29 - 0.31) (-0.040 - -0.033) 

Potassium (% dw) 2.19 (0.078) 2.36 (0.055) -0.17 (0.096) -0.40, 0.054 0.114 

(2.18 - 2.20) (2.31 - 2.48) (-0.13 - -0.11) 

Sodium (% dw) 0.16 (0.013) 0.14 (0.0093) 0.025 (0.013) -0.0086, 0.059 0.108 

(0.15 - 0.16) (0.11 - 0.15) (0.0075 - 0.039) 

Zinc (mg/kg dw) 27.03 (1.05) 29.30 (0.94) -2.28 (0.71) -4.23, -0.32 0.031 

(25.38 - 26.99) (27.27 - 30.81) (-3.26 - -1.89) 

1dw = dry weight; fw = fresh 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 referen Negative limits set to zero.
Monsanto Company 12-AL-246U 

Table E-4. Statistical Summary of Site CAPR Alfalfa Forage Secondary Metabolites for KK179 vs. C(Conducted at Covance Laboratories) 

 Difference (Test minus Control)

Analytical Component (Units)1

KK179 

Mean (S.E.) 2

(Range)

Control3

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence 

Interval

Signific

(p-Val

MetaboliteCanavanine (ppm dw) 44.03 (9.98) 69.09 (7.06) -25.05 (12.22) -53.96, 3.85 0.079 

(32.97 - 55.10) (58.60 - 79.23) (-26.84 - -23.61) 

Ferulic Acid (ppm dw) 1509.08 (114.39) 1350.71 (80.89) 158.37 (140.10) -172.92, 489.65 0.295 

(1411.02 - 1607.14) (1103.96 - 1596.77) (80.87 - 503.18) 

Free Phenylalanine (ppm dw) 165.37 (43.57) 204.65 (30.81) -39.28 (53.36) -165.45, 86.89 0.485 

(111.86 - 218.88) (154.07 - 278.22) (-59.34 - -42.20) 

Total Polyphenols (mg/g dw) 8.51 (0.39) 7.45 (0.25) 1.06 (0.46) -0.070, 2.18 0.061 

(8.31 - 8.72) (7.20 - 7.66) (0.65 - 1.40) 

p-Coumaric Acid (ppm dw) 567.23 (57.51) 514.33 (40.66) 52.91 (70.43) -113.64, 219.45 0.477 

(466.10 - 668.37) (442.08 - 645.16) (-31.51 - 226.29) 

1dw = dry weight; fw = fresh 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 referen Negative limits set to zero.
Monsanto Company 12-AL-246U 

Table E-5. Statistical Summary of Site IARL Alfalfa Forage Nutrients for KK179 vs. Conventional CoCovance Laboratories) 

 Difference (Test minus Control)

Analytical Component (Units)1

KK179 

Mean (S.E.) 2

(Range)

Control3

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence 

Interval

Signific

(p-Val

Proximate (% dw)Ash 10.05 (0.36) 10.63 (0.36) -0.58 (0.51) -1.74, 0.57 0.281 

(9.88 - 10.39) (10.29 - 11.39) (-1.52 - 0.095) 

Carbohydrates 59.32 (0.78) 61.15 (0.78) -1.83 (0.89) -4.01, 0.34 0.084 

(57.73 - 62.20) (59.94 - 62.42) (-3.45 - 1.12) 

Moisture (% fw) 84.13 (0.24) 83.38 (0.24) 0.75 (0.34) -0.025, 1.53 0.056 

(83.60 - 84.60) (83.00 - 83.70) (0.20 - 1.60) 

Protein 27.34 (0.72) 25.71 (0.72) 1.64 (0.59) 0.19, 3.08 0.032 

(24.51 - 29.03) (24.73 - 27.30) (-0.58 - 3.19) 

Total Fat 3.25 (0.30) 2.59 (0.30) 0.66 (0.28) -0.011, 1.34 0.052 

(2.58 - 3.98) (1.72 - 3.38) (-0.21 - 1.07) 

Fiber (% dw)Acid Detergent Fiber 16.65 (1.42) 15.76 (1.42) 0.89 (1.58) -2.96, 4.75 0.591 

(15.71 - 17.85) (10.96 - 19.18) (-3.20 - 6.12) 
Monsanto Company 12-AL-246U 

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

 Difference (Test minus Control)

Analytical Component (Units)1

KK179 

Mean (S.E.) 2

(Range)

Control3

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence 

Interval

Signific

(p-Val

Fiber (% dw)Acid Detergent Lignin 3.22 (0.17) 3.85 (0.17) -0.62 (0.24) -1.21, -0.041 0.039 

(2.72 - 3.69) (3.58 - 4.23) (-1.51 - -0.16) 

Neutral Detergent Fiber 22.65 (2.23) 22.89 (2.23) -0.24 (3.16) -7.38, 6.90 0.941 

(18.57 - 30.78) (18.94 - 26.83) (-8.11 - 11.84) 

Amino Acid (% dw)Alanine 1.40 (0.034) 1.36 (0.034) 0.038 (0.032) -0.041, 0.12 0.281 

(1.31 - 1.52) (1.30 - 1.39) (-0.042 - 0.13) 

Arginine 1.28 (0.029) 1.23 (0.029) 0.052 (0.022) -0.0012, 0.10 0.053 

(1.23 - 1.35) (1.19 - 1.28) (0.0092 - 0.11) 

Aspartic acid 4.05 (0.17) 3.85 (0.17) 0.20 (0.16) -0.19, 0.59 0.253 

(3.74 - 4.65) (3.47 - 4.08) (-0.29 - 0.57) 

Cystine 0.26 (0.013) 0.26 (0.013) 0.0044 (0.010) -0.021, 0.029 0.685 

(0.23 - 0.30) (0.24 - 0.29) (-0.018 - 0.019) 
Monsanto Company 12-AL-246U 

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

 Difference (Test minus Control)

Analytical Component (Units)1

KK179 

Mean (S.E.) 2

(Range)

Control3

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence 

Interval

Signific

(p-Val

Amino Acid (% dw)Glutamic acid 2.39 (0.045) 2.31 (0.045) 0.079 (0.038) -0.013, 0.17 0.080 

(2.24 - 2.55) (2.27 - 2.36) (-0.026 - 0.22) 

Glycine 1.15 (0.020) 1.12 (0.020) 0.026 (0.020) -0.022, 0.075 0.230 

(1.10 - 1.21) (1.10 - 1.14) (-0.022 - 0.085) 

Histidine 0.52 (0.0099) 0.50 (0.0099) 0.015 (0.012) -0.015, 0.044 0.270 

(0.49 - 0.55) (0.49 - 0.51) (-0.012 - 0.059) 

Isoleucine 1.08 (0.021) 1.05 (0.021) 0.032 (0.028) -0.037, 0.10 0.293 

(1.02 - 1.15) (1.03 - 1.07) (-0.048 - 0.12) 

Leucine 1.80 (0.031) 1.74 (0.031) 0.061 (0.030) -0.012, 0.13 0.085 

(1.73 - 1.90) (1.71 - 1.78) (-0.011 - 0.16) 

Lysine 1.44 (0.034) 1.40 (0.034) 0.038 (0.021) -0.014, 0.091 0.124 

(1.37 - 1.55) (1.34 - 1.44) (-0.0052 - 0.12) 
Monsanto Company 12-AL-246U 

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

 Difference (Test minus Control)

Analytical Component (Units)1

KK179 

Mean (S.E.) 2

(Range)

Control3

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence 

Interval

Signific

(p-Val

Amino Acid (% dw)Methionine 0.37 (0.015) 0.34 (0.015) 0.031 (0.017) -0.010, 0.072 0.117 

(0.34 - 0.39) (0.30 - 0.37) (0.0086 - 0.062) 

Phenylalanine 1.22 (0.020) 1.18 (0.020) 0.034 (0.019) -0.013, 0.080 0.127 

(1.16 - 1.27) (1.15 - 1.21) (-0.027 - 0.083) 

Proline 1.09 (0.030) 1.07 (0.030) 0.020 (0.022) -0.033, 0.072 0.401 

(1.02 - 1.18) (1.05 - 1.11) (-0.024 - 0.10) 

Serine 1.09 (0.023) 1.01 (0.023) 0.076 (0.018) 0.030, 0.12 0.006 

(1.03 - 1.16) (0.95 - 1.05) (0.019 - 0.13) 

Threonine 1.07 (0.017) 1.02 (0.017) 0.048 (0.016) 0.0075, 0.088 0.027 

(1.02 - 1.12) (0.99 - 1.05) (0.0064 - 0.10) 

Tryptophan 0.45 (0.012) 0.43 (0.012) 0.015 (0.016) -0.025, 0.055 0.399 

(0.41 - 0.48) (0.40 - 0.45) (-0.027 - 0.038) 
Monsanto Company 12-AL-246U 

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

 Difference (Test minus Control)

Analytical Component (Units)1

KK179 

Mean (S.E.) 2

(Range)

Control3

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence 

Interval

Signific

(p-Val

Amino Acid (% dw)Tyrosine 0.90 (0.023) 0.86 (0.023) 0.040 (0.028) -0.028, 0.11 0.200 

(0.86 - 0.94) (0.84 - 0.89) (-0.0025 - 0.10) 

Valine 1.31 (0.024) 1.27 (0.024) 0.040 (0.033) -0.041, 0.12 0.274 

(1.24 - 1.38) (1.24 - 1.32) (-0.073 - 0.13) 

MineralCalcium (% dw) 1.67 (0.041) 1.76 (0.041) -0.093 (0.058) -0.22, 0.038 0.142 

(1.63 - 1.71) (1.69 - 1.86) (-0.20 - -0.0096) 

Copper (mg/kg dw) 9.38 (0.86) 8.57 (0.86) 0.80 (0.98) -1.60, 3.21 0.444 

(8.54 - 11.23) (8.20 - 8.83) (0.073 - 2.40) 

Iron (mg/kg dw) 169.80 (36.93) 310.65 (36.93) -140.85 (52.22) -258.99, -22.71 0.024 

(123.38 - 207.36) (202.40 - 386.50) (-263.13 - 1.26) 

Magnesium (% dw) 0.30 (0.0095) 0.31 (0.0095) -0.011 (0.014) -0.041, 0.020 0.452 

(0.29 - 0.31) (0.30 - 0.32) (-0.023 - -0.0023) 
Monsanto Company 12-AL-246U 

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

 Difference (Test minus Control)

Analytical Component (Units)1

KK179 

Mean (S.E.) 2

(Range)

Control3

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence 

Interval

Signific

(p-Val

MineralManganese (mg/kg dw) 31.84 (2.81) 39.98 (2.81) -8.14 (3.97) -17.12, 0.84 0.070 

(30.52 - 34.57) (34.37 - 42.39) (-11.42 - 0.20) 

Phosphorus (% dw) 0.37 (0.018) 0.35 (0.018) 0.016 (0.025) -0.040, 0.072 0.535 

(0.33 - 0.40) (0.33 - 0.38) (-0.035 - 0.071) 

Potassium (% dw) 2.45 (0.11) 2.53 (0.11) -0.077 (0.15) -0.42, 0.27 0.624 

(2.26 - 2.62) (2.41 - 2.71) (-0.45 - 0.18) 

Sodium (% dw) 0.050 (0.010) 0.041 (0.010) 0.0087 (0.014) -0.023, 0.041 0.557 

(0.030 - 0.069) (0.029 - 0.075) (-0.044 - 0.039) 

Zinc (mg/kg dw) 36.08 (1.99) 37.26 (1.99) -1.18 (2.32) -6.86, 4.51 0.630 

(33.90 - 39.22) (34.12 - 40.42) (-5.64 - 2.31) 

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 referen Negative limits set to zero.
Monsanto Company 12-AL-246U 

Table E-6. Statistical Summary of Site IARL Alfalfa Forage Secondary Metabolites for KK179 vs. C(Conducted at Covance Laboratories) 

 Difference (Test minus Control)

Analytical Component (Units)1

KK179 

Mean (S.E.) 2

(Range)

Control3

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence 

Interval

Signific

(p-Val

MetaboliteCanavanine (ppm dw) 15.76 (0.24) 15.04 (0.24) 0.72 (0.33) -0.035, 1.48 0.059 

(15.24 - 16.23) (14.71 - 15.34) (0.19 - 1.53) 

Ferulic Acid (ppm dw) 1591.83 (78.25) 1390.17 (78.25) 201.66 (94.83) -30.37, 433.70 0.077 

(1402.60 - 1779.22) (1181.82 - 1580.84) (22.23 - 361.57) 

Free Phenylalanine (ppm dw) 378.02 (21.12) 386.96 (21.12) -8.94 (26.59) -74.01, 56.13 0.748 

(345.73 - 409.20) (370.30 - 422.94) (-29.72 - 38.90) 

Total Polyphenols (mg/g dw) 9.68 (0.22) 8.86 (0.22) 0.82 (0.31) 0.11, 1.52 0.027 

(8.96 - 10.19) (8.47 - 9.22) (0.048 - 1.72) 

p-Coumaric Acid (ppm dw) 748.40 (40.83) 732.14 (40.83) 16.26 (57.74) -114.36, 146.89 0.784 

(625.77 - 870.13) (660.61 - 784.43) (-79.99 - 128.95) 

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 referenNegative limits set to zero.
Monsanto Company 12-AL-246U 

Table E-7. Statistical Summary of Site ILCY Alfalfa Forage Nutrients for KK179 vs. Conventional Co

Covance Laboratories) 

 Difference (Test minus Control)

Analytical Component (Units)1

KK179 

Mean (S.E.) 2

(Range)

Control3

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence 

Interval

Signific

(p-Val

Proximate (% dw)Ash 9.85 (0.31) 10.33 (0.31) -0.48 (0.28) -1.17, 0.21 0.139 

(8.81 - 10.83) (9.80 - 11.22) (-1.35 - -0.025) 

Carbohydrates 69.37 (1.34) 67.53 (1.34) 1.84 (1.89) -2.44, 6.13 0.355 

(66.84 - 71.14) (61.73 - 71.28) (-2.74 - 8.36) 

Moisture (% fw) 79.73 (0.43) 80.45 (0.43) -0.73 (0.49) -1.93, 0.48 0.192 

(78.10 - 81.30) (80.30 - 80.60) (-2.20 - 0.70) 

Protein 18.87 (1.15) 20.06 (1.15) -1.18 (1.62) -4.86, 2.49 0.485 

(17.41 - 21.12) (16.56 - 25.00) (-6.67 - 2.15) 

Total Fat 1.99 (0.36) 2.22 (0.36) -0.23 (0.50) -1.45, 0.99 0.662 

(0.84 - 2.62) (1.31 - 3.23) (-1.30 - 0.93) 

Fiber (% dw)Acid Detergent Fiber 27.25 (1.40) 32.57 (1.40) -5.32 (1.84) -9.82, -0.82 0.027 

(20.53 - 29.59) (30.62 - 33.67) (-10.08 - -2.75) 
Monsanto Company 12-AL-246U 

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

 Difference (Test minus Control)

Analytical Component (Units)1

KK179 

Mean (S.E.) 2

(Range)

Control3

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence 

Interval

Signific

(p-Val

Fiber (% dw)Acid Detergent Lignin 5.88 (0.31) 6.81 (0.31) -0.94 (0.41) -1.94, 0.069 0.062 

(5.51 - 6.32) (5.89 - 7.50) (-1.99 - -0.36) 

Neutral Detergent Fiber 34.67 (1.47) 39.92 (1.47) -5.25 (1.62) -9.21, -1.29 0.017 

(30.00 - 38.58) (38.26 - 43.32) (-9.74 - 0.21) 

Amino Acid (% dw)Alanine 0.99 (0.027) 1.01 (0.027) -0.020 (0.038) -0.11, 0.066 0.610 

(0.91 - 1.06) (0.94 - 1.07) (-0.096 - 0.074) 

Arginine 0.89 (0.024) 0.90 (0.024) -0.0090 (0.033) -0.089, 0.071 0.792 

(0.83 - 0.94) (0.87 - 0.96) (-0.043 - 0.039) 

Aspartic acid 2.68 (0.11) 2.50 (0.11) 0.18 (0.15) -0.16, 0.52 0.258 

(2.36 - 3.13) (2.42 - 2.61) (-0.25 - 0.71) 

Cystine 0.20 (0.0081) 0.20 (0.0081) 0.0071 (0.011) -0.019, 0.033 0.550 

(0.19 - 0.23) (0.18 - 0.21) (-0.021 - 0.018) 
Monsanto Company 12-AL-246U 

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

 Difference (Test minus Control)

Analytical Component (Units)1

KK179 

Mean (S.E.) 2

(Range)

Control3

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence 

Interval

Signific

(p-Val

Amino Acid (% dw)Glutamic acid 1.66 (0.046) 1.70 (0.046) -0.043 (0.065) -0.19, 0.10 0.524 

(1.59 - 1.78) (1.59 - 1.80) (-0.16 - 0.14) 

Glycine 0.84 (0.022) 0.88 (0.022) -0.033 (0.032) -0.11, 0.038 0.320 

(0.80 - 0.90) (0.82 - 0.93) (-0.096 - 0.053) 

Histidine 0.41 (0.0095) 0.40 (0.0095) 0.0035 (0.013) -0.027, 0.034 0.800 

(0.38 - 0.43) (0.38 - 0.43) (-0.012 - 0.032) 

Isoleucine 0.76 (0.023) 0.80 (0.023) -0.038 (0.032) -0.11, 0.034 0.260 

(0.73 - 0.82) (0.74 - 0.85) (-0.094 - 0.050) 

Leucine 1.27 (0.037) 1.33 (0.037) -0.061 (0.052) -0.18, 0.057 0.270 

(1.19 - 1.36) (1.23 - 1.41) (-0.15 - 0.075) 

Lysine 1.04 (0.028) 1.05 (0.028) -0.0074 (0.040) -0.097, 0.082 0.856 

(0.99 - 1.11) (0.97 - 1.14) (-0.11 - 0.11) 
Monsanto Company 12-AL-246U 

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

 Difference (Test minus Control)

Analytical Component (Units)1

KK179 

Mean (S.E.) 2

(Range)

Control3

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence 

Interval

Signific

(p-Val

Amino Acid (% dw)Methionine 0.26 (0.023) 0.23 (0.023) 0.032 (0.032) -0.041, 0.10 0.350 

(0.17 - 0.32) (0.22 - 0.24) (-0.053 - 0.10) 

Phenylalanine 0.86 (0.023) 0.91 (0.023) -0.050 (0.032) -0.12, 0.022 0.153 

(0.82 - 0.90) (0.84 - 0.96) (-0.10 - 0.022) 

Proline 0.84 (0.025) 0.81 (0.025) 0.031 (0.035) -0.047, 0.11 0.390 

(0.81 - 0.87) (0.75 - 0.88) (-0.064 - 0.11) 

Serine 0.83 (0.023) 0.83 (0.023) 0.0042 (0.032) -0.068, 0.077 0.898 

(0.77 - 0.90) (0.79 - 0.87) (-0.055 - 0.099) 

Threonine 0.79 (0.022) 0.80 (0.022) -0.013 (0.032) -0.085, 0.059 0.695 

(0.74 - 0.84) (0.76 - 0.85) (-0.058 - 0.061) 

Tryptophan 0.35 (0.010) 0.35 (0.010) 0.0032 (0.015) -0.030, 0.036 0.831 

(0.32 - 0.38) (0.32 - 0.37) (-0.047 - 0.035) 
Monsanto Company 12-AL-246U 

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

 Difference (Test minus Control)

Analytical Component (Units)1

KK179 

Mean (S.E.) 2

(Range)

Control3

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence 

Interval

Signific

(p-Val

Amino Acid (% dw)Tyrosine 0.64 (0.015) 0.65 (0.015) -0.0087 (0.022) -0.058, 0.040 0.698 

(0.60 - 0.68) (0.63 - 0.67) (-0.024 - 0.025) 

Valine 0.95 (0.023) 0.99 (0.023) -0.040 (0.033) -0.11, 0.035 0.263 

(0.89 - 0.99) (0.91 - 1.04) (-0.090 - -0.00058) 

MineralCalcium (% dw) 1.45 (0.085) 1.46 (0.085) -0.013 (0.12) -0.30, 0.27 0.912 

(1.27 - 1.62) (1.25 - 1.66) (-0.12 - 0.062) 

Copper (mg/kg dw) 7.04 (0.57) 6.05 (0.57) 0.99 (0.77) -0.88, 2.86 0.243 

(5.21 - 8.50) (5.18 - 7.32) (0.028 - 1.51) 

Iron (mg/kg dw) 212.81 (25.68) 212.29 (25.68) 0.52 (35.24) -85.70, 86.74 0.988 

(173.97 - 230.88) (163.92 - 313.78) (-82.89 - 52.13) 

Magnesium (% dw) 0.17 (0.010) 0.18 (0.010) -0.0085 (0.012) -0.038, 0.021 0.514 

(0.13 - 0.20) (0.17 - 0.19) (-0.040 - 0.019) 
Monsanto Company 12-AL-246U 

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

 Difference (Test minus Control)

Analytical Component (Units)1

KK179 

Mean (S.E.) 2

(Range)

Control3

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence 

Interval

Signific

(p-Val

MineralManganese (mg/kg dw) 77.86 (7.33) 69.83 (7.33) 8.02 (10.37) -15.89, 31.94 0.461 

(59.82 - 106.47) (58.16 - 77.32) (-15.31 - 37.75) 

Phosphorus (% dw) 0.29 (0.0074) 0.30 (0.0074) -0.0078 (0.010) -0.033, 0.018 0.480 

(0.28 - 0.31) (0.29 - 0.31) (-0.032 - 0.0061) 

Potassium (% dw) 2.37 (0.055) 2.51 (0.055) -0.14 (0.077) -0.32, 0.031 0.096 

(2.25 - 2.58) (2.44 - 2.60) (-0.35 - 0.082) 

Sodium (% dw) 0.090 (0.013) 0.091 (0.013) -0.0014 (0.019) -0.043, 0.041 0.942 

(0.065 - 0.14) (0.074 - 0.13) (-0.056 - 0.068) 

Zinc (mg/kg dw) 25.77 (1.88) 20.32 (1.88) 5.45 (2.58) -0.87, 11.77 0.079 

(18.40 - 32.73) (17.38 - 22.50) (-1.34 - 11.08) 

1dw = dry weight; fw = fresh 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 referen Negative limits set to zero.
Monsanto Company 12-AL-246U 

Table E-8. Statistical Summary of Site ILCY Alfalfa Forage Secondary Metabolites for KK179 vs. C(Conducted at Covance Laboratories) 

 Difference (Test minus Control)

Analytical Component (Units)1

KK179 

Mean (S.E.) 2

(Range)

Control3

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence 

Interval

Signific

(p-Val

MetaboliteCanavanine (ppm dw) 12.37 (0.26) 12.79 (0.26) -0.42 (0.30) -1.15, 0.31 0.210 

(11.42 - 13.37) (12.69 - 12.89) (-1.27 - 0.48) 

Ferulic Acid (ppm dw) 1617.62 (75.96) 1605.38 (75.96) 12.24 (107.43) -230.77, 255.26 0.911 

(1508.02 - 1840.80) (1515.31 - 1809.28) (-301.26 - 292.08) 

Free Phenylalanine (ppm dw) 301.46 (23.08) 297.57 (23.08) 3.89 (32.03) -74.49, 82.27 0.907 

(232.62 - 362.69) (245.36 - 317.44) (-41.81 - 45.25) 

Total Polyphenols (mg/g dw) 8.43 (0.36) 8.74 (0.36) -0.31 (0.51) -1.46, 0.84 0.555 

(7.86 - 9.00) (8.11 - 10.21) (-2.13 - 0.87) 

p-Coumaric Acid (ppm dw) 696.28 (24.51) 715.16 (24.51) -18.88 (31.57) -96.12, 58.37 0.571 

(661.76 - 736.32) (649.75 - 819.59) (-108.36 - 44.01) 

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 referenNegative limits set to zero.
Monsanto Company 12-AL-246U 

Table E-9. Statistical Summary of Site KSLA Alfalfa Forage Nutrients for KK179 vs. Conventional Co

Covance Laboratories) 

 Difference (Test minus Control)

Analytical Component (Units)1

KK179 

Mean (S.E.) 2

(Range)

Control3

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence 

Interval

Signific

(p-Val

Proximate (% dw)Ash 11.97 (0.36) 12.43 (0.36) -0.46 (0.50) -1.69, 0.77 0.398 

(11.27 - 13.26) (12.11 - 12.88) (-1.04 - 0.83) 

Carbohydrates 64.61 (0.56) 64.29 (0.56) 0.32 (0.80) -1.49, 2.12 0.698 

(63.04 - 65.40) (62.84 - 65.77) (-2.72 - 2.22) 

Moisture (% fw) 76.90 (0.28) 77.53 (0.28) -0.63 (0.37) -1.53, 0.28 0.141 

(76.30 - 77.20) (77.00 - 78.20) (-1.10 - 0.10) 

Protein 21.08 (0.56) 20.94 (0.56) 0.14 (0.79) -1.64, 1.92 0.860 

(20.57 - 21.40) (19.91 - 22.16) (-1.59 - 1.31) 

Total Fat 2.35 (0.32) 2.31 (0.32) 0.043 (0.42) -0.98, 1.07 0.921 

(1.90 - 2.77) (1.08 - 2.92) (-0.71 - 1.46) 

Fiber (% dw)Acid Detergent Fiber 27.84 (1.43) 28.97 (1.43) -1.14 (1.14) -3.92, 1.65 0.356 

(25.19 - 30.13) (23.70 - 31.42) (-4.13 - 1.49) 
Monsanto Company 12-AL-246U 

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

 Difference (Test minus Control)

Analytical Component (Units)1

KK179 

Mean (S.E.) 2

(Range)

Control3

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence 

Interval

Signific

(p-Val

Fiber (% dw)Acid Detergent Lignin 6.43 (0.35) 7.44 (0.35) -1.02 (0.49) -2.13, 0.097 0.068 

(5.47 - 7.27) (6.65 - 8.17) (-2.08 - 0.61) 

Neutral Detergent Fiber 34.37 (0.83) 36.31 (0.83) -1.94 (1.17) -4.59, 0.71 0.132 

(31.79 - 37.37) (35.86 - 37.09) (-4.22 - 1.08) 

Amino Acid (% dw)Alanine 1.11 (0.032) 1.09 (0.032) 0.021 (0.045) -0.082, 0.12 0.654 

(1.06 - 1.17) (1.03 - 1.14) (-0.083 - 0.070) 

Arginine 0.98 (0.040) 0.99 (0.040) -0.0086 (0.057) -0.14, 0.12 0.883 

(0.96 - 1.00) (0.90 - 1.06) (-0.10 - 0.079) 

Aspartic acid 2.72 (0.081) 2.61 (0.081) 0.10 (0.11) -0.16, 0.37 0.369 

(2.55 - 3.02) (2.41 - 2.72) (-0.14 - 0.31) 

Cystine 0.21 (0.013) 0.21 (0.013) 0.00044 (0.018) -0.040, 0.041 0.981 

(0.18 - 0.25) (0.19 - 0.23) (-0.041 - 0.062) 
Monsanto Company 12-AL-246U 

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

 Difference (Test minus Control)

Analytical Component (Units)1

KK179 

Mean (S.E.) 2

(Range)

Control3

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence 

Interval

Signific

(p-Val

Amino Acid (% dw)Glutamic acid 1.89 (0.051) 1.87 (0.051) 0.020 (0.073) -0.14, 0.18 0.789 

(1.83 - 1.97) (1.75 - 1.95) (-0.12 - 0.13) 

Glycine 0.98 (0.025) 0.98 (0.025) 0.0034 (0.035) -0.076, 0.083 0.924 

(0.97 - 1.02) (0.93 - 1.04) (-0.071 - 0.048) 

Histidine 0.43 (0.012) 0.43 (0.012) -0.0062 (0.017) -0.046, 0.033 0.730 

(0.41 - 0.45) (0.41 - 0.45) (-0.022 - 0.0075) 

Isoleucine 0.87 (0.023) 0.86 (0.023) 0.0069 (0.033) -0.068, 0.081 0.839 

(0.83 - 0.91) (0.82 - 0.90) (-0.040 - 0.056) 

Leucine 1.45 (0.041) 1.45 (0.041) 0.0010 (0.057) -0.13, 0.13 0.986 

(1.42 - 1.53) (1.36 - 1.53) (-0.10 - 0.085) 

Lysine 1.12 (0.034) 1.15 (0.034) -0.031 (0.048) -0.14, 0.077 0.536 

(1.09 - 1.16) (1.10 - 1.19) (-0.062 - -0.0039) 
Monsanto Company 12-AL-246U 

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

 Difference (Test minus Control)

Analytical Component (Units)1

KK179 

Mean (S.E.) 2

(Range)

Control3

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence 

Interval

Signific

(p-Val

Amino Acid (% dw)Methionine 0.22 (0.020) 0.23 (0.020) -0.0029 (0.028) -0.066, 0.061 0.918 

(0.19 - 0.25) (0.18 - 0.30) (-0.089 - 0.064) 

Phenylalanine 1.00 (0.029) 0.99 (0.029) 0.0049 (0.041) -0.088, 0.097 0.907 

(0.97 - 1.04) (0.93 - 1.05) (-0.063 - 0.057) 

Proline 0.88 (0.024) 0.88 (0.024) -0.0029 (0.034) -0.081, 0.075 0.933 

(0.84 - 0.92) (0.85 - 0.91) (-0.075 - 0.038) 

Serine 0.86 (0.021) 0.86 (0.021) 0.0036 (0.030) -0.064, 0.071 0.906 

(0.84 - 0.90) (0.82 - 0.89) (-0.030 - 0.034) 

Threonine 0.87 (0.024) 0.87 (0.024) 0.0042 (0.034) -0.074, 0.082 0.905 

(0.86 - 0.90) (0.81 - 0.91) (-0.049 - 0.052) 

Tryptophan 0.38 (0.017) 0.38 (0.017) 0.00096 (0.024) -0.054, 0.056 0.969 

(0.34 - 0.42) (0.36 - 0.39) (-0.050 - 0.065) 
Monsanto Company 12-AL-246U 

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

 Difference (Test minus Control)

Analytical Component (Units)1

KK179 

Mean (S.E.) 2

(Range)

Control3

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence 

Interval

Signific

(p-Val

Amino Acid (% dw)Tyrosine 0.71 (0.034) 0.70 (0.034) 0.0084 (0.048) -0.10, 0.12 0.864 

(0.68 - 0.73) (0.63 - 0.77) (-0.053 - 0.060) 

Valine 1.05 (0.027) 1.04 (0.027) 0.0063 (0.039) -0.081, 0.094 0.874 

(1.01 - 1.09) (0.98 - 1.09) (-0.051 - 0.070) 

MineralCalcium (% dw) 2.04 (0.048) 1.95 (0.048) 0.093 (0.037) 0.0021, 0.18 0.046 

(1.98 - 2.09) (1.85 - 2.10) (-0.017 - 0.15) 

Copper (mg/kg dw) 9.13 (0.52) 9.18 (0.52) -0.053 (0.74) -1.72, 1.61 0.943 

(8.31 - 9.96) (8.60 - 9.59) (-0.98 - 1.35) 

Iron (mg/kg dw) 362.07 (58.09) 451.45 (58.09) -89.38 (82.16) -275.23, 96.47 0.304 

(269.62 - 473.91) (375.69 - 547.83) (-278.21 - 67.61) 

Magnesium (% dw) 0.26 (0.0067) 0.26 (0.0067) -0.0077 (0.0078) -0.027, 0.011 0.359 

(0.24 - 0.27) (0.25 - 0.27) (-0.0094 - -0.0063) 
Monsanto Company 12-AL-246U 

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

 Difference (Test minus Control)

Analytical Component (Units)1

KK179 

Mean (S.E.) 2

(Range)

Control3

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence 

Interval

Signific

(p-Val

MineralManganese (mg/kg dw) 50.02 (1.51) 49.82 (1.51) 0.20 (1.50) -3.48, 3.88 0.899 

(47.68 - 53.48) (46.79 - 53.15) (-4.49 - 3.28) 

Phosphorus (% dw) 0.25 (0.0060) 0.25 (0.0060) 0.0036 (0.0083) -0.017, 0.024 0.682 

(0.24 - 0.26) (0.23 - 0.26) (-0.0061 - 0.018) 

Potassium (% dw) 2.21 (0.041) 2.23 (0.041) -0.019 (0.054) -0.15, 0.11 0.732 

(2.16 - 2.28) (2.18 - 2.29) (-0.12 - 0.071) 

Sodium (% dw) 0.17 (0.019) 0.14 (0.019) 0.035 (0.026) -0.028, 0.099 0.223 

(0.13 - 0.22) (0.12 - 0.15) (0.0023 - 0.083) 

Zinc (mg/kg dw) 24.75 (0.88) 22.94 (0.88) 1.81 (1.18) -1.08, 4.71 0.176 

(23.28 - 27.57) (21.91 - 24.13) (-0.85 - 3.92) 

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 referen Negative limits set to zero.
Monsanto Company 12-AL-246U 

Table E-10. Statistical Summary of Site KSLA Alfalfa Forage Secondary Metabolites for KK179 vs. C(Conducted at Covance Laboratories) 

 Difference (Test minus Control)

Analytical Component (Units)1

KK179 

Mean (S.E.) 2

(Range)

Control3

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence 

Interval

Signific

(p-Val

MetaboliteCanavanine (ppm dw) 80.13 (7.01) 111.94 (7.01) -31.81 (7.01) -48.97, -14.65 0.003 

(64.19 - 87.83) (87.61 - 134.50) (-53.36 - -23.42) 

Ferulic Acid (ppm dw) 1584.79 (68.95) 1438.26 (68.95) 146.54 (97.51) -74.04, 367.11 0.167 

(1392.41 - 1729.26) (1334.86 - 1582.61) (-190.20 - 394.40) 

Free Phenylalanine (ppm dw) 225.68 (24.61) 252.06 (24.61) -26.39 (34.02) -109.64, 56.86 0.467 

(172.15 - 253.48) (174.67 - 327.98) (-101.34 - 75.77) 

Total Polyphenols (mg/g dw) 7.25 (0.28) 7.27 (0.28) -0.020 (0.40) -0.93, 0.88 0.960 

(6.62 - 8.34) (6.93 - 7.55) (-0.93 - 1.41) 

p-Coumaric Acid (ppm dw) 630.59 (26.98) 595.81 (26.98) 34.78 (38.15) -51.53, 121.09 0.385 

(544.30 - 716.16) (545.87 - 626.13) (-81.78 - 170.29) 

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 referenNegative limits set to zero.
Monsanto Company 12-AL-246U 

Table E-11. Statistical Summary of Site TXCL Alfalfa Forage Nutrients for KK179 vs. Conventional CoCovance Laboratories) 

 Difference (Test minus Control)

Analytical Component (Units)1

KK179 

Mean (S.E.) 2

(Range)

Control3

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence 

Interval

Signific

(p-Val

Proximate (% dw)Ash 12.12 (0.30) 12.27 (0.30) -0.15 (0.42) -1.10, 0.79 0.725 

(11.33 - 12.93) (11.53 - 12.95) (-1.32 - 1.06) 

Carbohydrates 65.05 (0.68) 64.63 (0.68) 0.42 (0.90) -1.79, 2.62 0.659 

(62.89 - 66.54) (63.47 - 65.69) (-2.80 - 3.07) 

Moisture (% fw) 74.18 (0.50) 72.60 (0.50) 1.58 (0.71) -0.021, 3.17 0.052 

(73.70 - 74.50) (70.50 - 74.40) (0.10 - 3.60) 

Protein 20.42 (0.46) 20.58 (0.46) -0.17 (0.65) -1.63, 1.30 0.801 

(18.86 - 21.17) (19.56 - 21.90) (-1.55 - 1.61) 

Total Fat 2.43 (0.29) 2.54 (0.29) -0.11 (0.29) -0.82, 0.60 0.713 

(1.45 - 2.99) (1.63 - 3.15) (-0.54 - 0.49) 

Fiber (% dw)Acid Detergent Fiber 26.32 (1.02) 26.16 (1.02) 0.16 (1.32) -3.06, 3.38 0.907 

(24.36 - 29.88) (24.75 - 27.66) (-2.63 - 3.36) 
Monsanto Company 12-AL-246U 

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

 Difference (Test minus Control)

Analytical Component (Units)1

KK179 

Mean (S.E.) 2

(Range)

Control3

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence 

Interval

Signific

(p-Val

Fiber (% dw)Acid Detergent Lignin 7.43 (0.66) 6.64 (0.66) 0.80 (0.66) -0.82, 2.41 0.273 

(5.86 - 10.31) (5.88 - 7.28) (-0.11 - 3.39) 

Neutral Detergent Fiber 32.47 (1.21) 31.92 (1.21) 0.55 (0.97) -1.83, 2.93 0.591 

(28.34 - 36.00) (30.64 - 33.71) (-2.30 - 2.82) 

Amino Acid (% dw)Alanine 1.22 (0.049) 1.22 (0.049) -0.0012 (0.070) -0.16, 0.16 0.986 

(1.19 - 1.28) (1.09 - 1.39) (-0.19 - 0.13) 

Arginine 1.02 (0.036) 1.02 (0.036) 0.0031 (0.051) -0.11, 0.12 0.952 

(0.97 - 1.05) (0.95 - 1.08) (-0.031 - 0.063) 

Aspartic acid 2.11 (0.060) 2.27 (0.060) -0.16 (0.084) -0.35, 0.031 0.090 

(1.97 - 2.23) (2.04 - 2.42) (-0.30 - 0.19) 

Cystine 0.21 (0.0065) 0.20 (0.0065) 0.0073 (0.0088) -0.014, 0.029 0.437 

(0.19 - 0.22) (0.18 - 0.22) (-0.0093 - 0.024) 
Monsanto Company 12-AL-246U 

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

 Difference (Test minus Control)

Analytical Component (Units)1

KK179 

Mean (S.E.) 2

(Range)

Control3

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence 

Interval

Signific

(p-Val

Amino Acid (% dw)Glutamic acid 1.86 (0.065) 1.96 (0.065) -0.10 (0.092) -0.31, 0.11 0.302 

(1.80 - 1.92) (1.83 - 2.06) (-0.17 - 0.011) 

Glycine 1.03 (0.026) 1.05 (0.026) -0.021 (0.037) -0.10, 0.062 0.583 

(1.00 - 1.05) (0.98 - 1.10) (-0.077 - 0.042) 

Histidine 0.44 (0.011) 0.45 (0.011) -0.012 (0.014) -0.047, 0.023 0.421 

(0.43 - 0.44) (0.43 - 0.48) (-0.053 - 0.0095) 

Isoleucine 0.90 (0.028) 0.91 (0.028) -0.0062 (0.039) -0.095, 0.082 0.877 

(0.87 - 0.95) (0.84 - 0.97) (-0.070 - 0.051) 

Leucine 1.51 (0.046) 1.53 (0.046) -0.023 (0.065) -0.17, 0.12 0.734 

(1.47 - 1.57) (1.42 - 1.62) (-0.11 - 0.057) 

Lysine 1.19 (0.035) 1.20 (0.035) -0.0061 (0.049) -0.12, 0.11 0.904 

(1.16 - 1.24) (1.12 - 1.30) (-0.14 - 0.11) 
Monsanto Company 12-AL-246U 

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

 Difference (Test minus Control)

Analytical Component (Units)1

KK179 

Mean (S.E.) 2

(Range)

Control3

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence 

Interval

Signific

(p-Val

Amino Acid (% dw)Methionine 0.24 (0.034) 0.21 (0.034) 0.025 (0.049) -0.085, 0.13 0.623 

(0.19 - 0.29) (0.15 - 0.31) (-0.12 - 0.14) 

Phenylalanine 1.04 (0.029) 1.06 (0.029) -0.019 (0.041) -0.11, 0.075 0.663 

(1.01 - 1.07) (0.99 - 1.12) (-0.057 - 0.034) 

Proline 0.92 (0.039) 1.03 (0.039) -0.11 (0.055) -0.23, 0.015 0.078 

(0.88 - 0.94) (0.93 - 1.21) (-0.28 - -0.0051) 

Serine 0.85 (0.031) 0.89 (0.031) -0.037 (0.044) -0.14, 0.063 0.425 

(0.80 - 0.89) (0.83 - 0.93) (-0.10 - 0.031) 

Threonine 0.90 (0.031) 0.93 (0.031) -0.031 (0.044) -0.13, 0.068 0.495 

(0.87 - 0.93) (0.86 - 0.99) (-0.055 - 0.041) 

Tryptophan 0.40 (0.016) 0.40 (0.016) 0.0054 (0.023) -0.047, 0.058 0.819 

(0.38 - 0.43) (0.36 - 0.45) (-0.026 - 0.035) 
Monsanto Company 12-AL-246U 

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

 Difference (Test minus Control)

Analytical Component (Units)1

KK179 

Mean (S.E.) 2

(Range)

Control3

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence 

Interval

Signific

(p-Val

Amino Acid (% dw)Tyrosine 0.75 (0.025) 0.71 (0.025) 0.040 (0.035) -0.040, 0.12 0.282 

(0.71 - 0.78) (0.66 - 0.78) (-0.017 - 0.066) 

Valine 1.08 (0.033) 1.09 (0.033) -0.0079 (0.047) -0.11, 0.099 0.870 

(1.04 - 1.13) (1.01 - 1.14) (-0.065 - 0.044) 

MineralCalcium (% dw) 2.34 (0.091) 2.35 (0.091) -0.012 (0.13) -0.30, 0.28 0.927 

(2.12 - 2.62) (2.05 - 2.53) (-0.41 - 0.28) 

Copper (mg/kg dw) 11.09 (0.76) 9.67 (0.76) 1.42 (1.01) -1.32, 4.16 0.228 

(8.52 - 13.16) (8.75 - 11.64) (-0.67 - 4.04) 

Iron (mg/kg dw) 338.53 (28.04) 308.71 (28.04) 29.82 (35.02) -55.86, 115.50 0.427 

(221.18 - 417.97) (270.51 - 356.46) (-60.07 - 115.21) 

Magnesium (% dw) 0.24 (0.015) 0.25 (0.015) -0.013 (0.018) -0.056, 0.030 0.486 

(0.21 - 0.28) (0.23 - 0.29) (-0.048 - 0.037) 
Monsanto Company 12-AL-246U 

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

 Difference (Test minus Control)

Analytical Component (Units)1

KK179 

Mean (S.E.) 2

(Range)

Control3

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence 

Interval

Signific

(p-Val

MineralManganese (mg/kg dw) 61.36 (2.91) 59.33 (2.91) 2.02 (4.05) -7.89, 11.94 0.635 

(49.41 - 71.48) (57.63 - 61.25) (-9.18 - 11.63) 

Phosphorus (% dw) 0.23 (0.0056) 0.21 (0.0056) 0.014 (0.0079) -0.0040, 0.032 0.113 

(0.22 - 0.24) (0.20 - 0.23) (-0.00067 - 0.030) 

Potassium (% dw) 2.45 (0.067) 2.47 (0.067) -0.017 (0.079) -0.21, 0.18 0.837 

(2.29 - 2.65) (2.37 - 2.54) (-0.085 - 0.10) 

Sodium (% dw) 0.052 (0.010) 0.033 (0.010) 0.019 (0.014) -0.013, 0.051 0.217 

(0.040 - 0.069) (0.018 - 0.058) (-0.013 - 0.049) 

Zinc (mg/kg dw) 23.52 (3.82) 22.92 (3.82) 0.59 (5.40) -11.62, 12.80 0.915 

(19.89 - 27.30) (20.58 - 25.09) (-5.21 - 6.72) 

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 referen Negative limits set to zero.
Monsanto Company 12-AL-246U 

Table E-12. Statistical Summary of Site TXCL Alfalfa Forage Secondary Metabolites for KK179 vs. C(Conducted at Covance Laboratories) 

 Difference (Test minus Control)

Analytical Component (Units)1

KK179 

Mean (S.E.) 2

(Range)

Control3

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence 

Interval

Signific

(p-Val

MetaboliteCanavanine (ppm dw) 44.54 (7.37) 84.96 (7.37) -40.42 (10.42) -63.99, -16.85 0.003 

(29.96 - 53.52) (70.11 - 109.49) (-79.53 - -22.46) 

Ferulic Acid (ppm dw) 1831.12 (68.79) 1763.27 (68.79) 67.86 (76.96) -120.45, 256.17 0.411 

(1737.25 - 1884.17) (1594.89 - 2007.38) (-148.06 - 248.86) 

Free Phenylalanine (ppm dw) 290.05 (26.29) 346.46 (26.29) -56.41 (20.60) -106.82, -6.01 0.033 

(244.11 - 332.05) (298.89 - 457.63) (-125.58 - -6.99) 

Total Polyphenols (mg/g dw) 8.61 (0.36) 8.22 (0.36) 0.39 (0.46) -0.74, 1.52 0.429 

(7.99 - 9.35) (7.41 - 9.12) (-1.13 - 1.65) 

p-Coumaric Acid (ppm dw) 676.49 (34.94) 700.57 (34.94) -24.08 (43.71) -131.03, 82.87 0.601 

(600.00 - 726.56) (644.53 - 797.05) (-112.64 - 40.43) 

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 referenNegative limits set to zero.
Monsanto Company 12-AL-246U 

Table E-13. Statistical Summary of Site WIDL Alfalfa Forage Nutrients for KK179 vs. Conventional CoCovance Laboratories) 

 Difference (Test minus Control)

Analytical Component (Units)1

KK179 

Mean (S.E.) 2

(Range)

Control3

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence 

Interval

Signific

(p-Val

Proximate (% dw)Ash 9.05 (0.27) 9.21 (0.27) -0.16 (0.38) -1.03, 0.71 0.685 

(8.43 - 10.13) (8.99 - 9.63) (-0.79 - 1.01) 

Carbohydrates 72.45 (0.51) 71.82 (0.51) 0.63 (0.73) -1.01, 2.27 0.408 

(70.80 - 73.90) (70.54 - 72.91) (-2.11 - 2.08) 

Moisture (% fw) 75.83 (0.42) 75.08 (0.42) 0.75 (0.56) -0.62, 2.12 0.230 

(74.80 - 77.40) (74.20 - 75.90) (-0.20 - 2.50) 

Protein 16.75 (0.41) 16.97 (0.41) -0.23 (0.58) -1.53, 1.08 0.705 

(15.50 - 17.65) (15.98 - 18.18) (-1.51 - 1.68) 

Total Fat 1.86 (0.18) 2.07 (0.18) -0.22 (0.26) -0.80, 0.36 0.421 

(1.60 - 2.14) (2.01 - 2.17) (-0.47 - 0.13) 

Fiber (% dw)Acid Detergent Fiber 34.76 (1.41) 30.88 (1.41) 3.88 (1.99) -0.63, 8.39 0.083 

(31.41 - 37.26) (27.69 - 36.11) (-4.71 - 9.57) 
Monsanto Company 12-AL-246U 

Table E-13 (continued). Statistical Summary of Site WIDL Alfalfa Forage Nutrients for KK179 vs. Conven(Conducted at Covance Laboratories) 

 Difference (Test minus Control)

Analytical Component (Units)1

KK179 

Mean (S.E.) 2

(Range)

Control3

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence 

Interval

Signific

(p-Val

Fiber (% dw)Acid Detergent Lignin 7.88 (0.39) 7.51 (0.39) 0.37 (0.53) -0.93, 1.66 0.516 

(6.83 - 8.58) (6.15 - 8.26) (-0.77 - 2.24) 

Neutral Detergent Fiber 42.99 (1.53) 39.31 (1.53) 3.68 (1.61) -0.26, 7.63 0.062 

(37.47 - 48.67) (36.67 - 40.64) (-1.88 - 8.04) 

Amino Acid (% dw)Alanine 0.87 (0.024) 0.93 (0.024) -0.055 (0.033) -0.13, 0.021 0.133 

(0.84 - 0.90) (0.87 - 1.03) (-0.19 - 0.014) 

Arginine 0.76 (0.020) 0.82 (0.020) -0.060 (0.029) -0.12, 0.0046 0.065 

(0.73 - 0.78) (0.75 - 0.90) (-0.17 - 0.014) 

Aspartic acid 2.25 (0.070) 2.15 (0.070) 0.10 (0.099) -0.13, 0.32 0.341 

(2.12 - 2.51) (2.09 - 2.23) (-0.035 - 0.28) 

Cystine 0.17 (0.0084) 0.18 (0.0084) -0.0082 (0.010) -0.034, 0.017 0.461 

(0.15 - 0.19) (0.15 - 0.20) (-0.030 - 0.017) 
Monsanto Company 12-AL-246U 

Table E-13 (continued). Statistical Summary of Site WIDL Alfalfa Forage Nutrients for KK179 vs. Conven(Conducted at Covance Laboratories) 

 Difference (Test minus Control)

Analytical Component (Units)1

KK179 

Mean (S.E.) 2

(Range)

Control3

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence 

Interval

Signific

(p-Val

Amino Acid (% dw)Glutamic acid 1.47 (0.040) 1.54 (0.040) -0.075 (0.057) -0.20, 0.054 0.222 

(1.40 - 1.52) (1.39 - 1.70) (-0.30 - 0.065) 

Glycine 0.77 (0.016) 0.78 (0.016) -0.015 (0.023) -0.067, 0.038 0.545 

(0.75 - 0.79) (0.73 - 0.85) (-0.099 - 0.022) 

Histidine 0.36 (0.0056) 0.38 (0.0056) -0.013 (0.0068) -0.029, 0.0040 0.113 

(0.35 - 0.38) (0.36 - 0.40) (-0.033 - 0.00032) 

Isoleucine 0.70 (0.018) 0.72 (0.018) -0.018 (0.026) -0.078, 0.041 0.497 

(0.67 - 0.73) (0.66 - 0.78) (-0.12 - 0.024) 

Leucine 1.14 (0.031) 1.19 (0.031) -0.051 (0.043) -0.15, 0.047 0.272 

(1.09 - 1.18) (1.09 - 1.31) (-0.22 - 0.023) 

Lysine 0.93 (0.021) 0.98 (0.021) -0.053 (0.027) -0.12, 0.014 0.100 

(0.93 - 0.93) (0.92 - 1.08) (-0.15 - 0.0086) 
Monsanto Company 12-AL-246U 

Table E-13 (continued). Statistical Summary of Site WIDL Alfalfa Forage Nutrients for KK179 vs. Conven(Conducted at Covance Laboratories) 

 Difference (Test minus Control)

Analytical Component (Units)1

KK179 

Mean (S.E.) 2

(Range)

Control3

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence 

Interval

Signific

(p-Val

Amino Acid (% dw)Methionine 0.17 (0.017) 0.18 (0.017) -0.015 (0.021) -0.066, 0.036 0.506 

(0.15 - 0.21) (0.15 - 0.22) (-0.062 - 0.011) 

Phenylalanine 0.77 (0.020) 0.81 (0.020) -0.038 (0.029) -0.10, 0.027 0.223 

(0.75 - 0.80) (0.74 - 0.89) (-0.15 - 0.018) 

Proline 0.73 (0.015) 0.75 (0.015) -0.022 (0.021) -0.069, 0.024 0.308 

(0.71 - 0.76) (0.71 - 0.80) (-0.092 - 0.051) 

Serine 0.73 (0.022) 0.75 (0.022) -0.012 (0.032) -0.083, 0.060 0.721 

(0.68 - 0.80) (0.68 - 0.81) (-0.13 - 0.055) 

Threonine 0.69 (0.017) 0.72 (0.017) -0.029 (0.024) -0.083, 0.026 0.266 

(0.66 - 0.73) (0.67 - 0.79) (-0.13 - 0.019) 

Tryptophan 0.31 (0.0090) 0.29 (0.0090) 0.015 (0.013) -0.014, 0.044 0.264 

(0.30 - 0.33) (0.27 - 0.31) (-0.011 - 0.062) 
Monsanto Company 12-AL-246U 

Table E-13 (continued). Statistical Summary of Site WIDL Alfalfa Forage Nutrients for KK179 vs. Conven(Conducted at Covance Laboratories) 

 Difference (Test minus Control)

Analytical Component (Units)1

KK179 

Mean (S.E.) 2

(Range)

Control3

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence 

Interval

Signific

(p-Val

Amino Acid (% dw)Tyrosine 0.57 (0.013) 0.58 (0.013) -0.013 (0.019) -0.056, 0.030 0.504 

(0.55 - 0.58) (0.53 - 0.63) (-0.053 - 0.024) 

Valine 0.85 (0.026) 0.87 (0.026) -0.023 (0.036) -0.11, 0.059 0.543 

(0.79 - 0.90) (0.81 - 0.96) (-0.16 - 0.046) 

MineralCalcium (% dw) 1.14 (0.031) 1.22 (0.031) -0.082 (0.044) -0.18, 0.018 0.096 

(1.12 - 1.15) (1.09 - 1.31) (-0.17 - 0.035) 

Copper (mg/kg dw) 5.78 (0.18) 5.74 (0.18) 0.047 (0.25) -0.52, 0.61 0.854 

(5.14 - 6.33) (5.42 - 5.93) (-0.65 - 0.91) 

Iron (mg/kg dw) 258.80 (29.54) 262.86 (29.54) -4.05 (37.93) -96.85, 88.74 0.918 

(200.00 - 357.52) (210.08 - 366.93) (-46.89 - 41.91) 

Magnesium (% dw) 0.23 (0.0061) 0.22 (0.0061) 0.0043 (0.0086) -0.015, 0.024 0.627 

(0.22 - 0.25) (0.21 - 0.24) (-0.025 - 0.022) 
Monsanto Company 12-AL-246U 

Table E-13 (continued). Statistical Summary of Site WIDL Alfalfa Forage Nutrients for KK179 vs. Conven(Conducted at Covance Laboratories) 

 Difference (Test minus Control)

Analytical Component (Units)1

KK179 

Mean (S.E.) 2

(Range)

Control3

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence 

Interval

Signific

(p-Val

MineralManganese (mg/kg dw) 56.24 (3.32) 55.01 (3.32) 1.24 (4.65) -10.13, 12.60 0.799 

(49.40 - 65.93) (44.94 - 62.15) (-8.50 - 5.20) 

Phosphorus (% dw) 0.30 (0.0075) 0.29 (0.0075) 0.011 (0.0098) -0.013, 0.035 0.287 

(0.28 - 0.32) (0.27 - 0.31) (-0.025 - 0.035) 

Potassium (% dw) 2.39 (0.063) 2.34 (0.063) 0.055 (0.060) -0.092, 0.20 0.393 

(2.30 - 2.44) (2.23 - 2.56) (-0.13 - 0.21) 

Sodium (% dw) 0.021 (0.012) 0.020 (0.012) 0.00065 (0.017) -0.040, 0.042 0.970 

(0.020 - 0.022) (0.019 - 0.021) (-0.00016 - 0.0022) 

Zinc (mg/kg dw) 29.54 (0.95) 28.13 (0.95) 1.42 (1.34) -1.61, 4.44 0.316 

(28.89 - 30.27) (25.82 - 32.16) (-2.53 - 4.45) 

1dw = dry weight; fw = fresh 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 referen Negative limits set to zero.
Monsanto Company 12-AL-246U 

Table E-14. Statistical Summary of Site WIDL Alfalfa Forage Secondary Metabolites for KK179 vs. C(Conducted at Covance Laboratories) 

 Difference (Test minus Control)

Analytical Component (Units)1

KK179 

Mean (S.E.) 2

(Range)

Control3

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence 

Interval

Signific

(p-Val

MetaboliteCanavanine (ppm dw) 43.03 (3.39) 49.63 (3.39) -6.60 (4.79) -17.44, 4.24 0.201 

(38.76 - 46.25) (39.92 - 59.92) (-21.16 - 5.32) 

Ferulic Acid (ppm dw) 1443.73 (38.85) 1367.10 (38.85) 76.63 (54.94) -47.65, 200.92 0.196 

(1389.38 - 1479.17) (1314.74 - 1403.10) (33.41 - 117.65) 

Free Phenylalanine (ppm dw) 224.65 (15.68) 214.48 (15.68) 10.17 (20.04) -38.87, 59.21 0.629 

(190.36 - 267.26) (155.04 - 246.15) (-55.79 - 58.45) 

Total Polyphenols (mg/g dw) 6.90 (0.43) 7.38 (0.43) -0.48 (0.60) -1.84, 0.88 0.447 

(6.35 - 7.35) (6.57 - 8.22) (-1.87 - 0.77) 

p-Coumaric Acid (ppm dw) 526.67 (25.69) 483.23 (25.69) 43.44 (35.63) -43.74, 130.63 0.268 

(458.33 - 566.37) (469.64 - 503.88) (-22.99 - 88.28) 

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 referenNegative limits set to zero.
Monsanto Company 12-AL-246U 

Table E -15. Statistical Summary of Alfalfa Forage Anti-Nutrients for KK179 vs. Conventional Control (CRoberts Noble Foundation) 

 Difference (Test minus Control)

Analytical Component (Units)1

KK179 

Mean (S.E.) 2

(Range)

Control3

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence 

Interval

Signific

(p-Val

Saponins (response units/μg)

Site CAPR

Total Bayogenin 8.30 (2.52) 4.98 (1.66) 3.33 (2.85) -3.84, 10.49 0.29

(3.00 - 13.97) (3.58 - 8.16) (-1.44 - 5.81) 

Total Hederagenin 4.16 (0.64) 3.25 (0.53) 0.91 (0.54) -0.55, 2.37 0.16

(3.01 - 5.80) (2.14 - 4.59) (-0.11 - 1.21) 

Total Medicagenic Acid 38.13 (8.20) 26.51 (5.15) 11.63 (9.45) -12.14, 35.39 0.26

(31.41 - 45.08) (9.43 - 40.45) (4.64 - 11.57) 
Monsanto Company 12-AL-246U 

Table E-15 (continued). Statistical Summary of Alfalfa Forage Anti-Nutrients for KK179 vs. C0-Syn1 (ConRoberts Noble Foundation) 

 Difference (Test minus Control)

Analytical Component (Units)1

KK179 

Mean (S.E.) 2

(Range)

Control3

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence 

Interval

Signific

(p-Val

Saponins (response units/μg)

Site CAPR

Total Soyasapogenol B 26.47 (5.31) 24.88 (3.97) 1.59 (5.27) -12.28, 15.46 0.77

(15.68 - 40.48) (17.41 - 31.77) (-10.25 - 8.72) 

Total Soyasapogenol E 3.42 (0.77) 2.74 (0.49) 0.68 (0.90) -1.56, 2.91 0.48

(1.84 - 5.02) (2.12 - 3.16) (-1.30 - 1.87) 

Total Zanhic Acid 7.14 (2.29) 5.03 (1.52) 2.11 (2.56) -4.36, 8.57 0.44

(2.58 - 12.08) (3.66 - 8.39) (-1.45 - 3.69) 

Total Saponins 87.15 (16.16) 67.38 (11.60) 19.76 (16.73) -23.88, 63.40 0.29

(57.51 - 122.44) (38.88 - 96.50) (-2.99 - 25.94) 

Site IARL

Total Bayogenin 3.55 (0.41) 2.80 (0.57) 0.74 (0.70) -0.92, 2.41 0.32

(3.06 - 4.44) (2.20 - 3.40) (-0.034 - 1.11) 
Monsanto Company 12-AL-246U 

Table E-15 (continued). Statistical Summary of Alfalfa Forage Anti-Nutrients for KK179 vs. C0-Syn1 (ConRoberts Noble Foundation) 

 Difference (Test minus Control)

Analytical Component (Units)1

KK179 

Mean (S.E.) 2

(Range)

Control3

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence 

Interval

Signific

(p-Val

Saponins (response units/μg)

Site IARL

Total Hederagenin 2.37 (0.25) 1.91 (0.35) 0.47 (0.43) -0.54, 1.48 0.30

(1.83 - 3.09) (1.58 - 2.23) (-0.40 - 0.73) 

Total Medicagenic Acid 18.75 (1.98) 20.09 (2.98) -1.33 (3.28) -9.78, 7.12 0.70

(15.66 - 21.15) (14.90 - 24.08) (-8.41 - 2.34) 

Total Soyasapogenol B 12.72 (1.53) 9.61 (2.16) 3.11 (2.65) -3.16, 9.38 0.27

(9.68 - 18.28) (7.05 - 12.16) (-0.61 - 4.31) 

Total Soyasapogenol E 1.40 (0.12) 1.10 (0.17) 0.30 (0.21) -0.19, 0.79 0.18

(1.20 - 1.73) (0.84 - 1.35) (-0.15 - 0.38) 

Total Zanhic Acid 2.80 (0.41) 3.15 (0.59) -0.35 (0.72) -2.05, 1.35 0.64

(2.25 - 4.21) (2.62 - 3.67) (-1.29 - -0.27) 
Monsanto Company 12-AL-246U 

Table E-15 (continued). Statistical Summary of Alfalfa Forage Anti-Nutrients for KK179 vs. C0-Syn1 (ConRoberts Noble Foundation) 

 Difference (Test minus Control)

Analytical Component (Units)1

KK179 

Mean (S.E.) 2

(Range)

Control3

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence 

Interval

Signific

(p-Val

Saponins (response units/μg)

Site IARL

Total Saponins 41.59 (4.25) 38.05 (6.01) 3.54 (7.36) -13.86, 20.93 0.64

(36.00 - 52.91) (29.20 - 46.90) (-10.90 - 8.59) 

Site ILCY

Total Bayogenin 3.79 (0.55) 5.23 (0.55) -1.45 (0.59) -2.89, -0.0043 0.04

(2.81 - 4.52) (2.87 - 6.77) (-2.25 - -0.061) 

Total Hederagenin 2.59 (0.40) 3.10 (0.40) -0.50 (0.48) -1.68, 0.67 0.33

(1.89 - 3.66) (2.37 - 4.68) (-1.82 - 0.83) 

Total Medicagenic Acid 27.83 (4.68) 30.73 (4.68) -2.90 (5.40) -16.12, 10.31 0.61

(19.21 - 36.75) (19.96 - 51.04) (-14.28 - 9.18) 

Total Soyasapogenol B 16.69 (2.17) 21.58 (2.17) -4.89 (2.34) -10.61, 0.83 0.08

(14.30 - 22.25) (13.56 - 26.38) (-11.46 - 0.75) 
Monsanto Company 12-AL-246U 

Table E-15 (continued). Statistical Summary of Alfalfa Forage Anti-Nutrients for KK179 vs. C0-Syn1 (ConRoberts Noble Foundation) 

 Difference (Test minus Control)

Analytical Component (Units)1

KK179 

Mean (S.E.) 2

(Range)

Control3

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence 

Interval

Signific

(p-Val

Saponins (response units/μg)

Site ILCY

Total Soyasapogenol E 1.94 (0.26) 2.58 (0.26) -0.64 (0.23) -1.21, -0.073 0.03

(1.47 - 2.72) (1.82 - 3.18) (-1.12 - -0.28) 

Total Zanhic Acid 3.49 (0.42) 4.35 (0.42) -0.86 (0.52) -2.14, 0.42 0.15

(2.80 - 3.88) (2.67 - 5.75) (-1.87 - 0.13) 

Total Saponins 56.32 (7.09) 67.57 (7.09) -11.25 (7.52) -29.65, 7.16 0.18

(42.54 - 68.38) (43.25 - 94.51) (-30.05 - 1.39) 

Site KSLA

Total Bayogenin 4.83 (0.49) 6.30 (0.49) -1.48 (0.59) -2.91, -0.039 0.04

(3.83 - 6.60) (5.45 - 6.71) (-2.39 - -0.12) 

Total Hederagenin 2.44 (0.53) 4.04 (0.53) -1.59 (0.68) -3.26, 0.073 0.05

(2.09 - 3.13) (2.97 - 4.62) (-2.10 - -0.88) 
Monsanto Company 12-AL-246U 

Table E-15 (continued). Statistical Summary of Alfalfa Forage Anti-Nutrients for KK179 vs. C0-Syn1 (ConRoberts Noble Foundation) 

 Difference (Test minus Control)

Analytical Component (Units)1

KK179 

Mean (S.E.) 2

(Range)

Control3

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence 

Interval

Signific

(p-Val

Saponins (response units/μg)

Site KSLA

Total Medicagenic Acid 12.60 (3.67) 18.16 (3.67) -5.57 (5.15) -18.16, 7.03 0.32

(9.73 - 19.65) (15.48 - 20.96) (-8.70 - -1.31) 

Total Soyasapogenol B 24.34 (2.47) 30.71 (2.47) -6.38 (2.62) -12.79, 0.037 0.05

(18.83 - 28.51) (23.91 - 34.73) (-10.72 - -0.70) 

Total Soyasapogenol E 4.05 (0.30) 4.01 (0.30) 0.045 (0.35) -0.82, 0.91 0.90

(3.30 - 4.90) (3.43 - 4.31) (-1.01 - 0.70) 

Total Zanhic Acid 3.66 (0.42) 5.49 (0.42) -1.83 (0.41) -2.83, -0.82 0.00

(2.60 - 4.67) (4.88 - 6.55) (-2.62 - -0.21) 

Total Saponins 51.92 (7.16) 68.72 (7.16) -16.79 (8.43) -37.41, 3.83 0.09

(40.39 - 67.46) (62.00 - 76.11) (-22.46 - -8.65) 
Monsanto Company 12-AL-246U 

Table E-15 (continued). Statistical Summary of Alfalfa Forage Anti-Nutrients for KK179 vs. C0-Syn1 (ConRoberts Noble Foundation) 

 Difference (Test minus Control)

Analytical Component (Units)1

KK179 

Mean (S.E.) 2

(Range)

Control3

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence 

Interval

Signific

(p-Val

Saponins (response units/μg)

Site TXCL

Total Bayogenin 7.45 (0.76) 9.09 (0.76) -1.64 (1.08) -4.07, 0.79 0.16

(5.90 - 8.43) (7.46 - 11.28) (-2.85 - 0.18) 

Total Hederagenin 3.66 (0.64) 4.71 (0.64) -1.05 (0.90) -3.10, 0.99 0.27

(2.51 - 4.92) (3.28 - 6.85) (-3.51 - 0.55) 

Total Medicagenic Acid 17.77 (3.55) 17.59 (3.55) 0.19 (5.02) -11.17, 11.54 0.97

(9.09 - 35.00) (14.06 - 20.31) (-7.33 - 15.92) 

Total Soyasapogenol B 27.51 (3.31) 31.83 (3.31) -4.32 (3.87) -13.80, 5.15 0.30

(21.98 - 31.25) (25.02 - 41.93) (-12.47 - 3.21) 

Total Soyasapogenol E 3.80 (1.10) 5.00 (1.10) -1.20 (1.21) -4.16, 1.75 0.35

(2.42 - 4.49) (3.29 - 8.89) (-4.99 - 1.20) 
Monsanto Company 12-AL-246U 

Table E-15 (continued). Statistical Summary of Alfalfa Forage Anti-Nutrients for KK179 vs. C0-Syn1 (ConRoberts Noble Foundation) 

 Difference (Test minus Control)

Analytical Component (Units)1

KK179 

Mean (S.E.) 2

(Range)

Control3

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence 

Interval

Signific

(p-Val

Saponins (response units/μg)

Site TXCL

Total Zanhic Acid 6.73 (0.92) 6.77 (0.92) -0.037 (1.18) -2.93, 2.86 0.97

(3.08 - 9.33) (6.16 - 7.89) (-3.39 - 2.76) 

Total Saponins 66.92 (8.31) 74.99 (8.31) -8.07 (11.75) -34.65, 18.51 0.50

(44.97 - 91.65) (59.72 - 95.91) (-31.55 - 19.44) 

Site WIDL

Total Bayogenin 4.16 (0.56) 5.09 (0.56) -0.93 (0.79) -2.73, 0.86 0.27

(2.54 - 5.52) (3.70 - 6.54) (-2.10 - -0.035) 

Total Hederagenin 2.79 (0.40) 3.42 (0.40) -0.63 (0.56) -1.90, 0.65 0.29

(1.70 - 3.67) (1.99 - 4.22) (-1.42 - 0.29) 

Total Medicagenic Acid 23.23 (5.18) 26.30 (5.18) -3.07 (7.32) -19.63, 13.49 0.68

(14.06 - 34.94) (15.02 - 42.64) (-22.95 - 12.43) 
Monsanto Company 12-AL-246U 

Table E-15 (continued). Statistical Summary of Alfalfa Forage Anti-Nutrients for KK179 vs. C0-Syn1 (ConRoberts Noble Foundation) 

 Difference (Test minus Control)

Analytical Component (Units)1

KK179 

Mean (S.E.) 2

(Range)

Control3

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence 

Interval

Signific

(p-Val

Saponins (response units/μg)

Site WIDL

Total Soyasapogenol B 26.38 (2.10) 26.31 (2.10) 0.073 (2.97) -6.65, 6.80 0.98

(21.42 - 29.70) (19.10 - 31.46) (-1.95 - 2.31) 

Total Soyasapogenol E 2.40 (0.23) 2.80 (0.23) -0.39 (0.31) -1.15, 0.36 0.24

(2.08 - 3.10) (2.00 - 3.45) (-0.73 - 0.081) 

Total Zanhic Acid 4.96 (0.81) 5.90 (0.81) -0.93 (1.14) -3.52, 1.65 0.43

(2.79 - 6.54) (3.77 - 8.69) (-3.97 - 1.86) 

Total Saponins 63.92 (7.99) 69.81 (7.99) -5.89 (11.29) -31.43, 19.66 0.61

(44.58 - 81.55) (45.57 - 91.36) (-32.96 - 10.64) 

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 r Negative limits set to zero.
Monsanto Company 12-AL-246U 325 of 407

References for Appendix E 

AACC. 1998. Approved methods of the American Association of Cereal Chemists. 

American Association of Cereal Chemists, Inc., St. Paul, Minnesota. 

AOAC. 2010a. AOAC official method 922.06: Fat in flour. Association of Analytical 

Communities International, Gaithersburg, Maryland. 

AOAC. 2010b. AOAC official 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: Nitrogen (total) in fertilizers. Association 

of Analytical Communities International, Gaithersburg, Maryland. 

AOAC. 2011a. AOAC official method 982.30: Protein efficiency ratio. Association of 

Analytical Communities International, Gaithersburg, Maryland. 

AOAC. 2011b. AOAC official method 926.08: Loss on drying (moisture) in cheese. 

Association of Analytical Communities International, Gaithersburg, Maryland. 

AOAC. 2011c. AOAC official method 985.01: Metals and other elements in plants and 

pet foods. Association of Analytical Communities International, Gaithersburg, Maryland. 

AOAC. 2011d. AOAC official method 925.09: Solids (total) and loss on drying 

(moisture) in flour. Association of Analytical 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 official 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 zinc 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 after 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). Agriculture Handbook No. 379. U.S. Department of 

Agriculture, Agricultural Research 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. Sumner. 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. Van Soest. 1993. A comparison of methods for 

determining ADF using the Filter Bag Technique versus conventional filtration. Journal 

of Animal Science 72 114. 

Komarek, A.R., J.B. Robertson and P.J. Van 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 America, Inc., Soil Society of America, Inc., 

Madison, Wisconsin. 

OECD. 2005. Consensus document on compositional considerations for new varieties of 

alfalfa and other temperate forage legumes: Key feed nutrients, anti-nutrients and 

secondary plant metabolites. Organisation for Economic Co-operation and Development, 

Paris, France. 

Pettersson, H. and K.H. Kiessling. 1984. Liquid 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 acids in biological pharmaceutical plant and 

food samples by automated precolumn derivatization and high-performance liquid 

chromatography. Journal of Chromatography 431: 271-284. 
Monsanto Company 12-AL-246U 327 of 407

Saloniemi, H., K. Wahala, P. Nykanenkurki, K. Kallela and I. Saastamoinen. 1995. 

Phytoestrogen Content and Estrogenic Effect of Legume Fodder. Proceedings of the 

Society for Experimental 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, Agricultural 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 characteristics were assessed on seed from KK179, the 

conventional control, and four conventional commercial reference varieties produced in a 

greenhouse at a single location in West Salem, WI in 2010. Both non-scarified and 

scarified seed were assessed. Uniform, mechanical 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 the starting seed from KK179, the conventional 

control (C0-Syn1), and the conventional commercial reference varieties was verified by 

event-specific polymerase chain reaction (PCR) analyses. 

F.3. Germination Testing Facility and Experimental Methods 

Seed germination and dormancy evaluations were conducted at BioDiagnostics, Inc. in 

River Falls, WI. The principal investigator was qualified to conduct seed germination 

and dormancy testing consistent with the standards established by the Association of 

Official Seed Analysts (AOSA), a seed trade association (AOSA, 2007, 2010a; b). 

Seed of KK179, the conventional control, and four conventional commercial reference 

varieties was tested under three different temperature regimes. Three germination 

chambers were maintained under dark conditions with one of the following temperature 

regimes: constant temperature of approximately 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. Additional 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 germination towels, placed into a single, labeled 

bucket, for a total of four buckets per temperature 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 reference variety) and treatment (i.e., scarified or nonscarified). 
Monsanto Company 12-AL-246U 329 of 407

A description of each germination characteristic evaluated and the timing of evaluations 

are presented in Table VII-1. Seed placed in the AOSA-recommended temperature 

regime (i.e., 20 °C) was evaluated according to AOSA standards as normal germinated, 

abnormal germinated, hard, dead, or firm-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 regimes of 10 °C and 30 °C were evaluated as 

germinated, hard, dead, or firm-swollen. Because temperature extremes could affect the 

development of seedlings, AOSA standards were 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 classified as germinated. 

The calculation of percent seed 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 temperature 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 number of dead seed 

counted on both collection dates to determine the total percent dead seed. Total counts 

for percent viable hard and viable firm-swollen 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 established in a two-way factorial design with 

four replications. The two factors 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 characteristics analyzed included percent of 

germinated seed, percent of viable 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 the AOSA temperature regime. The level of 

statistical significance was predetermined to be 5% (α=0.05). KK179 was not 

statistically compared to the conventional commercial reference 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, Control and Conventional Commercial Alfalfa 

Reference Varieties Used in Dormancy Assessment 

Material Name Genotype / Phenotype Treatment1

Monsanto Lot 

Number

KK179 Reduced Lignin; Syn1 

population Non-scarified 11287366

C0-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

C0-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 imbibition of water. Scarification was performed using a 

Forsberg manual seed scarifier. Each 7 g seed lot 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. Contribution 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 Analysts, 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 Individual Site Results from Phenotypic, 

Agronomic, and Environmental Interaction Assessment of KK179 under Field 

Conditions during Forage Production 

G.1. Materials 

Agronomic, phenotypic, and environmental interaction characteristics were assessed for 

KK179, the conventional control, and 12 conventional commercial reference 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 the starting seed of KK179, the conventional 

control (C0-Syn1), and three of the conventional commercial 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 provided 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 the alfalfa characteristics. 

At the nine U.S. sites, seed of KK179, the conventional control, four conventional 

commercial reference varieties, and one other material were planted in a randomized 

complete block design with four replications. The planted plot dimensions varied 

between sites, due to variability in available 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 were 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 six 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 consisted of 10 rows. At ILCL, rows were 

spaced six inches apart, with an additional six 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, and 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 environmental interactions data collection. 
Monsanto Company 12-AL-246U 333 of 407

At the Canadian site, CANA, seed of KK179, the conventional control, and five 

conventional commercial reference varieties were planted in a randomized complete 

block design with four replications. Each plot at CANA consisted 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 different 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 alfalfa variety. Alleys between 

replicates were planted with approximately 3-8 feet of a conventional commercial alfalfa 

variety. The purpose of the planted border and alleys was to create a continuous alfalfa 

stand across the entire trial area to ensure collection of more robust arthropod abundance 

data within each plot. 

G.4. Planting and Field Operations 

Planting information, soil description, and cropping history of the trial area are listed in 

Table G-2. Prior to planting, the Principal Investigator at each site prepared a proper 

seedbed according to local agronomic practices, 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 harvest forage at 1-10% bloom throughout both 

the 2010 and 2011 growing seasons. The number of cuttings per site 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 C0-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 11265477

 
Monsanto Company 12-AL-246U 335 of 407

Table G-1. Starting Seed for Phenotypic, 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 

Table G-2. Field and Planting Information 1A broad range of seeding rates is acceptable to produce adequate stands of alfalfa. Stands naturally self-thin over time, primarily due t

seedlings, and seedling survival further decreases with increasing seeding rate (Tesar and Marble, 1988). Plants can also compensate fo

increasing the number of stems per plant. Stand and yield are ultimately influenced by the number of stems, not the number of plants, p

2OM = organic matter 3CANA harvested a smaller area in 2010 and cutting #1 of 2011 due to potential impacts to outer rows from heavy rain immediately aft

was 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.

Site1 Harvested plot area (ft2

) Rows (#/plot) Planting rate (lb/A)1 Soil series OM2 (%) 

CADV 35 6 18.0 Clay loam 1.8 CANA3 54.9 / 76.9 7 9.6 Brant silt loam 1.3 

IABN 40 5 18.0 Nicollet loam 4.0 IDNP 32.5 5 15.0 Power silt loam 1.0 ILCL 66 10 19.2 Cisne silt loam 2.7 KSLA 99 10 16.4 Loam silt loam 2.6 NYNR4 50.4 / 78.4 10 13.8 Elnora sand 2.5 PACO 40 5 20.1 Hagerstown silt loam 1.2 WATC 40 5 14.6 Touchet silt loam 1.3 WIWS 40 5 21.4 Toddville silt loam 3.7 
Monsanto Company 12-AL-246U 

Table G-3. Study Site Management NA 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.

Dates1 by Site

Activity CADV CANA IABN IDNP ILCL KSLA NYNR PACO First YearPlanting 3/29/10 6/2/10 4/20/10 4/26/10 4/19/10 4/21/10 4/23/10 4/12/10 Cutting #1 6/8/10 7/19/10 6/30/10 7/7/10 6/25/10 6/21/10 7/1/10 6/18/10 Cutting #2 7/16/10 8/10/10 7/29/10 8/13/10 8/6/10 7/20/10 8/3/10 7/28/10 Cutting #3 9/3/10 9/23/10 9/8/10 10/1/10 9/20/10 8/19/10 9/22/10 8/31/10 Cutting #4 11/6/10 NA NA NA NA 9/20/10 NA NA 

Second Year 

Cutting #1 4/22/11 6/10/11 6/6/11 6/6/11 5/17/11 5/23/11 6/3/11 5/24/11 Cutting #2 5/27/11 7/8/11 7/7/11 7/12/11 6/20/11 6/22/11 7/6/11 6/24/11 Cutting #3 7/5/11 7/28/11 8/9/11 8/16/11 7/21-22/112 7/22/11 8/18/11 7/27/11 Cutting #4 8/3/11 8/25/11 9/16/11 9/21/11 9/1/11 8/22/11 10/14/11 9/1/11 Cutting #5 9/9/11 9/29/11 NA NA NA 9/21/11 NA 10/5/11 
Monsanto Company 12-AL-246U 338 of 407

G.6. Phenotypic Observations 

The descriptions of the phenotypic characteristics assessed are listed in Table VII-1. 

After planting, KK179 was compared to the conventional 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 stand recovery, and spring stand count. 

Phenotypic characteristics were assessed over each complete year. The first year was 

considered to be from planting in 2010 through the winter survival assessment in the 

spring of 2011. The second year 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 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 Morphological Stages of Development for Individual 

Alfalfa Stems Used to Calculate 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 collected at each site during each crop growth cycle, prior to harvest. 

Approximately 35-45 stems from each plot were non-systematically selected and ranked by growth stage. 

The MSC was calculated for each plot using 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 crop 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, specific 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 evaluated at all sites for plant response to 

abiotic stressors, disease damage, and arthropod damage. Three abiotic stressors, three 

diseases, and three arthropod pests were evaluated 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 stressors, 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 type of abiotic stressors, diseases, and 

arthropod pests assessed vary between observation times at a site, between sites, and 

between years. 

Abiotic stressors, disease damage, and arthropod 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 scale 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 replications 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 injury symptoms across 

all four replications overlapped between KK179 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 Damage 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 second 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. The 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 feeding damage between the veins 

4 51-90% foliage with skeletonized appearance 

5 >90 % foliage with skeletonized appearance 

For potato leafhopper damage, 10 non-systematically 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 puckering (stunting of plant is evident) 

5 >90% foliage with yellowing and puckering (severe stunting of plant) 

Pest- and beneficial-arthropods were collected at ILCL, KSLA, and NYNR zero to 10 

days prior to each cutting, which occurred three to four times during the first growing 

season (2010) and four to five times during the second growing season (2011). 

Arthropods were collected using a vertical beat sheet sampling method (Drees and Rice, 

1985). The beat sheet was approximately a 36 × 36 inch sheet constructed 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 from the same row, approximately two feet 

from the first sub-sample. Two sub-samples were collected from each rows #8 and 9 in 
Monsanto 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 maximum of five pest- and five beneficialarthropods were enumerated. For each individual collection (e.g., collection #1 at 

KSLA), four non-systematically selected samples were examined to determine presence 

and relative abundance of up to five pest- and beneficial-arthropods to be enumerated for 

that particular collection and site. Thus, the suite of pest- and beneficial-arthropods 

assessed often varied between collections from a site and between sites due to differences 

in temporal activity and geographical distribution of arthropod taxa. 

G.10. Data Assessment 

Experienced scientists familiar with the experimental design and evaluation criteria were 

involved in all components of data collection, summarization, and analysis. Study 

personnel assessed that measurements were taken properly, data were consistent with 

expectations based on experience 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 Evaluation Criteria for Qualitative Data 

The qualitative environmental interactions data, including plant responses to abiotic 

stressors, disease damage, and arthropod damage were categorical and, therefore, were 

not subjected to statistical analysis. KK179 and the conventional control were considered 

different in plant responses if the range of injury symptoms across all four replications 

did not overlap between KK179 and the conventional control. Any observed differences 

between KK179 and the conventional control were assessed for biological significance in 

the context of the range of the conventional commercial reference varieties at a 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 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 according to a randomized complete block design 

using SAS® to compare KK179 and the conventional control for the phenotypic 

characteristics listed in Table VII-1. For each year, comparisons of KK179 and the 

conventional control were conducted within sites (individual-site analysis) and in a 

combined-site analysis in which the data were pooled among the sites. KK179 was also 

compared to the conventional control for a subset of phenotypic characteristics in a 

combined-year analysis in which data were pooled across years. The level of statistical 
Monsanto Company 12-AL-246U 343 of 407

significance was predetermined to be 5% (α=0.05). KK179 and the conventional control 

were not statistically compared to the conventional commercial reference materials. 

Minimum and maximum mean values were determined for each characteristic from the 

12 unique conventional commercial reference varieties that were included at all sites. 

G.12.2 Statistical Analysis for Specific Arthropod Damage 

An analysis of variance was conducted according to a randomized complete block design 

using SAS to compare KK179 and the conventional 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 three sites, a combined-site analysis in which 

the data were pooled among the sites was also conducted for each year. The level of 

statistical significance was predetermined to be 5% (α=0.05). KK179 and the 

conventional control were not statistically compared to the conventional commercial 

reference varieties. Minimum and maximum mean values were determined for each 

characteristic from the nine unique conventional commercial reference varieties that were 

included at the three sites. 

G.12.3 Statistical Analysis for Arthropod Abundance 

An analysis of variance was conducted according 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 geographical distribution of arthropod taxa occur between sites. 

Therefore, comparisons of KK179 and the conventional control were conducted only 

within sites (individual-site analysis) for both years. The level of statistical significance 

was predetermined to be 5% (α=0.05). KK179 and the conventional control were not 

statistically compared to the conventional commercial reference materials. Minimum and 

maximum mean values were determined for arthropod abundance from the conventional 

commercial reference varieties at each site and collection time. 

Data excluded from analysis for 2010 through 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 

Table G-5. Data Missing or Excluded from Analysis Site Plots Entry type Data Reason First Year All All All Forage yield #1 No yield data were collected from 

site due to variability in establishm

CADV, IABN, IDNP, WATC, WIWS, CADV 

All All Forage quality sample weights 

Not all sites weighed the quality sto harvest; therefore, this weight wplot weight of forage yield per cut

CANA, IABN, IDNP, ILCL, NYNR, PACO, WATC 

All All Regrowth after cutting #3 Rating was not required following 

season. 

CADV, IDNP, WATC All All Fall plant height-Yr 1: 1st rating 

Fall plant height data were collectsites, as is regionally appropriate. started going dormant at the first r

CADV All All Percent stand Data collected by PI were not requCADV All All Regrowth after cutting #1 and #4 

Regrowth after cutting #1 was not appropriate time and regrowth afterequired following the last harvest 
Monsanto Company 12-AL-246U 

Table G-5 (continued). Data Missing or Excluded from Analysis Site Plots Entry type Data Reason CADV 107 Control Forage yield #4 The forage weight for plot 107 wa

malfunction. 

CANA All All Spring stand count- Yr 2 Plants per foot were counted inste

requested. 

IABN All All Abiotic Stressor Observation #1, rating for stressor #3 Plots were evaluated for winter in

yet to be exposed to winter. 

IABN All All Abiotic Stressor, Disease Damage, and Arthropod Damage Observation #3 Ratings were not made at the appr

specified in the protocol. 

KSLA 305 Reference Arthropod collection #1 Collection jar broke and the sampNYNR All All Spring stand rating and spring vigor rating- Yr 2 Data 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 sitePACO All All Disease Damage Observation #1 PI did not complete evaluation or 

instructions. 

WATC All All 

2nd Seedling emergence rating; Early 

season vigor; Mean stage by count #1; Lodging #1 

Seedling emergence was collected rating was unnecessary. Timing oEarly season vigor and Mean stagfollow the protocol and Lodging #collected. 

WATC 101, 102, 103 References, additional material All Frost event in April 2010 killed m

plots. 
Monsanto Company 12-AL-246U 

Table G-5 (continued). Data Missing or Excluded from Analysis Site Plots Entry type Data Reason WIWS All All Lodging #2 

Heavy rains immediately precedinforage in all plots to be weighed dtherefore not an accurate assessme

All All All All mean stage by count evaluations Any plots at any site for which few

evaluated per plot. 

Second Year CADV, CANA, KSLA, PACO All All Regrowth after cutting #5 Rating was not required following 

season. 

IABN, IDNP, ILCL, NYNR, WIWS 

All All Regrowth after cutting #4 Rating was not required following 

season. 

CADV All All Forage yield #21

Plot weight data could not be recothe data collector is no longer empthus unavailable to provide clarifi

CADV 201 Control Forage yield #1 and #31 There was gopher damage to a sig

plants in the plot. 

CADV 207 Reference Forage yield #51 Plot weight was not collected due 

harvesting equipment operation. 

CADV All All Forage yield #6 Plot weight data were collected thCADV 

301, 302, 304, 305, 305, 306 

All Arthropod Damage Observation #4 Entries on paper were not readabl

CADV All All Abiotic Stressor, Disease Damage, and Arthropod Damage Observation #2 PI did not complete data evaluatio

instructions. 
Monsanto Company 12-AL-246U 

Table G-5 (continued). Data Missing or Excluded from Analysis Site Plots Entry type Data Reason CADV 301 Test Spring vigor, Spring stand recovery, and Spring stand count There was significant gopher damCANA All All Fall plant height Yr 2 Data were not collected at the appCANA 101 Test Arthropod Damage Observation #5, stressor 1 and 2 Raw data were not available. IDNP 307 Reference Mean stage by count #4 Raw data were not available. KSLA All All Mean stage by count #5 The data were lost due to an elect

malfunction. 

KSLA 403, 407 Control, reference Fall plant height-Yr 2, plant number 9 An extra plant was measured. 

KSLA 203, 304, 405 References, additional material Arthropod collection #3 Collection samples were of poor q

enumerated. 

NYNR All All Mean stage by count # 1-4 PI did not take representative stem NYNR All All Fall plant height-Yr 2 Data were not collected at the appPACO All All Lodging #1 Data were not collected at the appPACO All All Mean stage by count # 1 and 2 Data were not collected at this sitePACO 303 Test Forage yield #31 There was groundhog damage to a 

plants in the plot. 

PACO All All Fall plant height- Yr 2, plant numbers 6-10 The protocol only required five pl
Monsanto Company 12-AL-246U 

Table G-5 (continued). Data Missing or Excluded from Analysis Site Plots Entry type Data Reason PACO All All Abiotic Stressor, Disease Damage, and Arthropod Damage Observation #1 PI did not complete data evaluatio

instructions. 

WATC All All Mean stage by count #2 Insect (Lygus) stripping of buds in

growth stage evaluation. 

WATC 402 Reference Forage yield #31 Gopher mound caused excessive s

affecting plot weight. 

All All All All mean stage by count evaluations Any plots at any site for which few

evaluated 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 w

sample weight was also excluded. 
Monsanto Company 12-AL-246U 349 of 407

G.13. Individual Field Site Plant Growth and Development Results and Discussion 

G.13.1. Individual Field Site Plant Growth 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 conventional control (Table G-6). No statistical 

comparisons could be made in some instances due to lack of variability in the data. 

At the beginning of the 2010 season, one statistically significant difference was detected 

between KK179 and the conventional control for seedling emergence. Seedling 

emergence was lower for KK179 than the conventional control at ILCL (20.0 vs. 25.5 

plants/ft). 

At cutting #1, three statistically significant differences were detected between KK179 and 

the conventional control. KK179 had a lower 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 differences were detected between KK179 and 

the conventional control. KK179 had a lower lodging rating than the conventional 

control at IABN (7.8 vs. 8.5 rating). 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 differences 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 statistically significant difference was 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 statistically 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 significantly 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 conducted in spring 2011, one statistically significant 

difference was detected between KK179 and the conventional control. KK179 had lower 

spring vigor than the conventional control at WATC (8.5 vs. 10.0 rating). 

None of the statistical differences detected in the individual-site analysis were detected in 

the combined-site analysis, suggesting these differences were not indicative of a 

consistent plant response associated with the trait and were not considered biologically 
Monsanto Company 12-AL-246U 350 of 407

meaningful in terms of plant pest/weed potential of KK179 compared to the conventional 

control (Figure VII-1, Step 2, answer “no”). 

G.13.2. Individual Field Site Plant Growth 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 conventional control (Table G-7). No statistical 

comparisons could be made in some instances due to lack of variability in the data. 

At cutting #1, two statistically significant differences 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 differences were detected between KK179 and 

the conventional control. KK179 had a higher 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 yield 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 conventional control at IABN (7.0 vs. 8.3 rating) 

and greater regrowth after cutting than the conventional control at PACO (10.0 vs. 9.5 

rating). 

At cutting #4, one statistically significant difference was detected between KK179 and 

the conventional control. KK179 had a lower forage yield for cutting #4 than the 

conventional control at IABN (5.4 vs. 6.0 fresh weight ton/acre). 

At cutting #5, one statistically significant difference was detected between KK179 and 

the conventional control. KK179 had a lower 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 statistically 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 conducted in spring 2012, five significant differences 

were detected between KK179 and the conventional control. KK179 had greater spring 

vigor than the conventional control at PACO (6.5 vs. 5.5 rating) and lower spring vigor 

than the conventional control at WATC (9.3 vs. 10.0 rating). 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 stems/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 differences detected in the individual-site analysis were detected in 

the combined-site analysis, suggesting these differences were not indicative of a 

consistent plant response associated with the trait and were not considered biologically 

meaningful in terms of plant pest/weed potential of KK179 compared to the conventional 

control (Figure VII-1, Step 2, answer “no”). 
Monsanto Company 12-AL-246U 

Table G-6. Individual-Site Phenotypic Comparison of KK179 to the Conventional Control in the First Forage Production 

Cutting #1

Seedling emergence 

(# plants/foot)

Early season vigor 

(1-10 rating) Lodging (0-9 rating) Crop growth stage (stage number 0-6) R

Site KK179 Mean (SE) Control Mean (SE) KK179 Mean (SE) Control Mean (SE) KK179 Mean (SE) Control Mean (SE) KK179 Mean (SE) Control Mean (SE) 

M

CADV 5.4 (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)CANA 28.8 (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) 

IABN 24.7 (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) 

IDNP 13.4 (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) 

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) 

KSLA 12.5 (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) 

NYNR 15.8 (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) † 1

PACO 28.0 (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) 1

WATC 12.9 (1.82) 13.5 (2.33)

–

–

–

–

–

– * 

WIWS 33.0 (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) 
Monsanto Company 12-AL-246U 

Table G-6 (continued). Individual-Site Phenotypic Comparison of KK179 to the Conventional Control in 2011) of Forage Production 

Cutting #2

Lodging (0-9 rating) Crop growth stage (stage number 0-6) Forage yield (fresh weight ton/acre) Re

Site KK179 Mean (SE) Control Mean (SE) KK179 Mean (SE) Control Mean (SE) KK179 Mean (SE) Control Mean (SE) KK

Mea

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 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 

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 

IDNP 7.5 (0.65) 7.3 (1.44) 3.4 (0.28) 3.2 (0.36) 10.2 (0.81) 10.5 (0.86) 8.3 

ILCL 8.5 (0.50) 9.0 (0.00) 3.2 (0.25) 3.2 (0.26) 5.3 (0.49) 4.7 (0.28) 7.3 

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 

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 

PACO 6.0 (0.00) 6.3 (0.25)

–

– 8.9 (0.37) 8.2 (0.49) 9.8 

WATC 8.3 (0.25) 8.0 (0.00) 3.9 (0.10) 3.9 (0.08) * 11.0 (1.10) 8.2 (1.03) 8.8 

WIWS

–

– 3.0 (0.17) 3.1 (0.06) 8.1 (0.38) 8.0 (0.26) 9.5 
Monsanto Company 12-AL-246U 

Table G-6 (continued). Individual-Site Phenotypic Comparison of KK179 to the Conventional Contr(2010-2011) of Forage Production 

Cutting #3

Lodging (0-9 rating) Crop growth stage (stage number 0-6) Forage yield (fresh weight ton/acre) Re

Site KK179 Mean (SE) Control Mean (SE) KK179 Mean (SE) Control Mean (SE) KK179 Mean (SE) Control Mean (SE) KK

Mean 

CADV 8.0 (0.58) 7.8 (0.48) 2.1 (0.12) 2.2 (0.05) 4.7 (0.23) 4.9 (0.38) 7.0 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)

IABN 8.3 (0.48) 8.5 (0.29) 1.7 (0.27) 1.6 (0.25) 3.7 (0.76) 3.2 (0.70)

IDNP 8.5 (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 

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)

WATC 8.5 (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 

Table G-6 (continued). Individual-Site Phenotypic Comparison of KK179 to the Conventional Control in 2011) of Forage Production 

Cutting #4

Lodging (0-9 rating) Crop growth stage (stage number 0-6) Forage yield (fresh weight ton/acre) Fal

Site KK179 Mean (SE) Control Mean (SE) KK179 Mean (SE) Control Mean (SE) KK179 Mean (SE) Control Mean (SE) KK17

Mean (

CADV 5.8 (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.CANA NA NA NA NA NA NA 3.1 (0.

IABN NA NA NA NA NA NA 7.5 (0.

IDNP NA NA NA NA NA NA 4.1 (0.

ILCL NA NA NA NA NA NA 4.1 (0.

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.

NYNR NA NA NA NA NA NA 4.2 (0.

PACO NA NA NA NA NA NA 9.8 (0.

WATC NA NA NA NA NA NA 7.5 (0.

WIWS NA NA NA NA NA NA 8.8 (0.
Monsanto Company 12-AL-246U 

Table G-6 (continued). Individual-Site Phenotypic Comparison of KK179 to the Conventional Contr(2010-2011) of Forage Production 

Total forage yield 

(fresh weight ton/acre) Spring vigor (1-10 rating) Spring stand recovery (1-10 rating) S

Site KK179 Mean (SE) Control Mean (SE) KK179 Mean (SE) Control Mean (SE) KK179 Mean (SE) Control Mean (SE) KK1

Mean 

CADV 15.6 (0.78) 14.7 (0.81) 7.8 (0.25) 7.5 (0.50) 7.8 (0.75) 7.3 (0.48) 39.8 (6CANA 2.5 (0.52) 2.9 (0.47) 5.8 (0.25) 5.3 (0.48) † 10.0 (0.00) 10.0 (0.00)

–

IABN 9.7 (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

IDNP 18.1 (0.72) 19.1 (1.20) 8.0 (0.41) 8.0 (0.00) 7.8 (0.63) 8.5 (0.65) 86.7 (1

ILCL 8.2 (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

KSLA 20.1 (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

NYNR 10.2 (0.56) 9.8 (0.61)

–

–

–

– 50.5 (7

PACO 12.5 (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

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

WIWS 10.8 (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

Note: The experimental design was a randomized complete block with four replications. Means based on n=4 for all characteristics exc

growth 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 fcontrol 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 KKcontrol at CADV; for forage yield #4, n=3 for the conventional control at CADV; and for total forage yield, n=3 for the conventional coSE = 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 

Table G-7. Individual-Site Phenotypic Comparison of KK179 to the Conventional Control in the Second Forage Production 

Cutting #1

Lodging (0-9 rating) Crop growth stage (stage number 0-6) Forage yield (fresh weight ton/acre) Reg

Site KK179 Mean (SE) Control Mean (SE) KK179 Mean (SE) Control Mean (SE) KK179 Mean (SE) Control Mean (SE) KK1

Mean 

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 (0CANA 7.0 (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

IABN 6.5 (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

IDNP 4.5 (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

ILCL 8.0 (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

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

NYNR † 9.0 (0.00) 9.0 (0.00)

–

– 5.8 (0.39) 6.4 (0.28) 8.8 (0

PACO

–

–

–

– 13.6 (0.56) 12.5 (0.46) 8.0 (0

WATC 7.0 (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

WIWS 4.0 (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
Monsanto Company 12-AL-246U 

Table G-7 (continued). Individual-Site Phenotypic Comparison of KK179 to the Conventional Control (2011-2012) of Forage Production 

Cutting #2

Lodging (0-9 rating) Crop growth stage (stage number 0-6) Forage yield (fresh weight ton/acre) Reg

Site KK179 Mean (SE) Control Mean (SE) KK179 Mean (SE) Control Mean (SE) KK179 Mean (SE) Control Mean (SE) KK1

Mean 

CADV † 9.0 (0.00) 9.0 (0.00) * 2.0 (0.20) 2.5 (0.04)

–

– 8.5 (0

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

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

IDNP 6.0 (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

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

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

NYNR † 9.0 (0.00) 9.0 (0.00)

–

– 3.4 (0.24) 3.5 (0.22) † 10.0 (0

PACO 5.8 (0.25) 5.5 (0.29)

–

– 12.4 (0.50) 12.1 (0.41) 9.0 (0

WATC 9.0 (0.00) 9.0 (0.00)

–

– 13.1 (0.28) 13.2 (0.66) 9.3 (0

WIWS 5.0 (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
Monsanto Company 12-AL-246U 

Table G-7 (continued). Individual-Site Phenotypic Comparison of KK179 to the Conventional Control (2011-2012) of Forage Production 

Cutting #3

Lodging (0-9 rating) Crop growth stage (stage number 0-6) Forage yield (fresh weight ton/acre) Reg

Site KK179 Mean (SE) Control Mean (SE) KK179 Mean (SE) Control Mean (SE) KK179 Mean (SE) Control Mean (SE) KK1

Mean 

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 (0CANA † 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

IABN 3.3 (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

IDNP 6.5 (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

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

KSLA 5.8 (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

NYNR † 9.0 (0.00) 9.0 (0.00)

–

– 3.7 (0.02) 3.6 (0.23) † 10.0 (0

PACO 7.3 (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

WATC 7.8 (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

WIWS 5.5 (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
Monsanto Company 12-AL-246U 

Table G-7 (continued). Individual-Site Phenotypic Comparison of KK179 to the Conventional Control (2011-2012) of Forage Production 

Cutting #4

Lodging (0-9 rating) Crop growth stage (stage number 0-6) Forage yield (fresh weight ton/acre) Reg

Site KK179 Mean (SE) Control Mean (SE) KK179 Mean (SE) Control Mean (SE) KK179 Mean (SE) Control Mean (SE) KK1

Mean 

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 (0CANA † 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

IABN 8.5 (0.29) 8.5 (0.29) 2.4 (0.07) 2.4 (0.09) * 5.4 (0.07) 6.0 (0.16)

–

IDNP 6.8 (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

NYNR † 9.0 (0.00) 9.0 (0.00)

–

– 3.2 (0.21) 3.3 (0.34)

–

PACO 4.3 (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

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

WIWS 5.3 (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 

Table G-7 (continued). Individual-Site Phenotypic Comparison of KK179 to the Conventional Control (2011-2012) of Forage Production 

Cutting #5

Lodging (0-9 rating) Crop growth stage (stage number 0-6) Forage yield (fresh weight ton/acre) Fal

Site KK179 Mean (SE) Control Mean (SE) KK179 Mean (SE) Control Mean (SE) KK179 Mean (SE) Control Mean (SE) KK1

Mean 

CADV 8.5 (0.29) 8.8 (0.25) 3.3 (0.16) 3.5 (0.09) 6.9 (1.02) 4.9 (0.78) 4.7 (0CANA † 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

IDNP

–

–

–

–

–

– 8.7 (0

ILCL

–

–

–

–

–

– 10.6 (0

KSLA † 9.0 (0.00) 9.0 (0.00)

–

– 5.0 (0.21) 5.4 (0.06) 4.6 (0

NYNR

–

–

–

–

–

–

–

PACO 5.8 (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

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

WIWS

–

–

–

–

–

– 4.3 (0
Monsanto Company 12-AL-246U 

Table G-7 (continued). Individual-Site Phenotypic Comparison of KK179 to the Conventional Control (2011-2012) of Forage Production 

Total forage yield 

(fresh weight ton/acre) Spring vigor (1-10 rating) Spring stand recovery (1-10 rating) Sp

Site KK179 Mean (SE) Control Mean (SE) KK179 Mean (SE) Control Mean (SE) KK179 Mean (SE) Control Mean (SE) KK1

Mean 

CADV2 36.7 (1.95) 35.8 (0.91) 7.3 (1.20) 6.5 (0.29) 8.0 (1.15) 8.8 (0.25) 11.4 (

CANA 17.7 (1.55) 18.7 (1.47) 5.8 (0.25) 5.8 (0.25) † 10.0 (0.00) 10.0 (0.00) 72.4 (

IABN 36.3 (1.09) 38.0 (1.00) 7.5 (0.65) 8.0 (1.08) * 7.8 (0.25) 9.5 (0.29) 50.2 (

IDNP 57.9 (2.42) 58.5 (1.12) 7.5 (0.65) 6.8 (0.75) 7.3 (0.25) 6.5 (0.65) * 54.8 (

ILCL 23.6 (0.35) 23.1 (0.96) 9.5 (0.29) 9.0 (0.00) 8.3 (0.25) 8.0 (0.00) 31.0 (

KSLA 37.7 (0.81) 39.4 (0.69) 9.0 (0.00) 7.5 (1.50) 8.3 (0.25) 8.0 (0.00) 105.8 (

NYNR 16.0 (0.16) 16.8 (0.68) 5.0 (0.00) 5.0 (0.00) 7.0 (1.35) 8.3 (0.25) * 59.3 (

PACO 46.4 (1.27) 44.5 (1.59) * 6.5 (0.29) 5.5 (0.29) 8.3 (0.25) 8.3 (0.25) 32.3 (

WATC 56.3 (0.85) 57.5 (1.71) * 9.3 (0.25) 10.0 (0.00) 7.8 (0.48) 8.3 (0.48) 86.2 (

WIWS 40.1 (0.47) 39.6 (0.79) 9.8 (0.25) 9.3 (0.25) 8.8 (0.63) 9.0 (0.41) 32.3 (

Note: The experimental design was a randomized complete block with four replications. Means based on n=4 for all characteristics exc

growth 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 fcontrol at CADV; for crop growth stage #2, n=3 for KK179 at CADV and at KSLA; for crop growth stage #3, n=2 for KK179 at CADV conventional control at CADV and CANA; for forage yield #3, n=3 for KK179 at PACO and n=3 for the conventional control at CADV#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 forat PACO and n=3 for the conventional control at CADV; for spring vigor, spring stand recovery, and spring stand count, n=3 for KK17SE = 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. Abiotic stressors evaluated 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 conventional control. Diseases evaluated 

included Anthracnose, bacterial wilt, black stem, damping-off, downy 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 damage responses to arthropod stressors were 

observed for 96 comparisons between KK179 and the conventional control. Arthropods 

evaluated included alfalfa caterpillar, alfalfa weevil, aphid, armyworm, blister beetle, 

cutworm, grasshopper, meadow spittlebug, plant bug, potato leafhopper, spider mite, and 

thrips (Table G-10). 

In an individual-site analysis for the first year (2010), no statistically significant 

differences were detected between KK179 and the conventional control for alfalfa weevil 

and potato leafhopper damage among all observations (Table G-11). The lack of 

differences in both the individual-site analysis and the combined-site -analysis supports 

the conclusion that the introduction of the trait for reduced G lignin and total lignin is not 

expected to have an effect on the environment 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 pest- and beneficial-arthropod abundance (Tables 

G-12 and G-13). No statistically significant differences in abundance were detected 

between KK179 and the conventional control for the following pest and beneficial 

arthropods: aphid, alfalfa weevil, 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). KK179 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 potato leafhopper in collection #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 differences detected for these taxa were not 

consistently detected across collection times or sites. Thus, the detected differences were 
Monsanto Company 12-AL-246U 364 of 407

not indicative of a consistent response associated with the trait and were not considered 

biologically meaningful in terms of effects on the environment from KK179 compared to 

the conventional control (See Section VII.B.2.). 

Two statistically significant differences in abundance 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 ILCL and at KSLA (0.0 vs. 1.5 

and 0.0 vs. 1.3, respectively). The mean abundance values of KK179 were within the 

respective reference ranges. Thus, the detected differences were not considered 

biologically meaningful in terms 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 stressors were observed for 129 comparisons between KK179 and 

the conventional control. Abiotic stressors evaluated included drought, frost, hail, heat, 

heaving, nutrient deficiency, soil compaction, 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 conventional control. Diseases evaluated 

included Anthracnose, bacterial wilt, black stem, crown rot, downy mildew, Fusarium 

wilt, leaf spots, root rot, Sclerotinia crown and stem rot, and Verticillium wilt (Table 

G-15). 

No differences in the range of plant damage responses to arthropod stressors were 

observed for 129 comparisons between KK179 and the conventional control. Arthropods 

evaluated included 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 thrips (Table G-16). 

In the individual-site analysis for the second year, three statistically significant 

differences were detected between KK179 and the conventional control for alfalfa weevil 

and potato leafhopper damage among all observation 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 leafhopper 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 considered biologically 

meaningful in terms of effects on the environment from KK179 compared to the 

conventional control (Figure VII-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 and 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, armyworm, false chinch bug, blister beetle, green 

cloverworm, Lygus bug, meadow spittlebug, potato leafhopper, thrips, soybean looper, 

spiders, ladybird beetle, lacewing, nabids, and chalcid wasps. 

One significant difference was detected out of 50 pest-arthropod comparisons between 

KK179 and the conventional control (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 considered biologically meaningful in terms of effects on the 

environment from KK179 compared to the conventional control (See Section VII.B.2.). 

No statistically significant differences in abundance were detected between KK179 and 

the conventional control out of 33 beneficial-arthropod comparisons (Table G-19). The 

lack of differences supports the conclusion that 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 Assessment: Abiotic Stressor Evaluations 

Using an Observational Severity Scale for 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 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 2010 

Number of observations with no 

differences between KK179 and 

the conventional control across all 

sites in 2010 

Total 93 93 

Drought 16 16 

Flood1 15 15 

Frost damage 1 1 

Hail damage 7 7 

Heat 22 22 

Nutrient deficiency 11 11 

Soil compaction 5 5 

Wind damage 16 16 
Monsanto Company 12-AL-246U 367 of 407

Table G-9. Combined-Site Qualitative Assessment: Disease Damage Evaluations 

Using an Observational Severity Scale for 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 rot1

 6 6 

Sclerotinia crown and stem rot 4 4 

Stem nematode 4 4 

Verticillium wilt 10 10 

 
Monsanto Company 12-AL-246U 368 of 407

Table G-10. Combined-Site Qualitative Assessment: Insect Stressor Evaluations 

Using an Observational Severity Scale for 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 13 
Monsanto 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 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 

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

Pest Arthropod

Aphid (Aphididae) Alfalfa weevil (Hypera postica) Alfalfa looper (A

Site KK179 Mean (SE) Control Mean (SE) Reference Range1 KK179 Mean (SE) Control Mean (SE) Reference Range1 KK179 Mean (SE) 

M

Collection #1

ILCL

–

–

–

–

–

–

–

KSLA 22.3 (3.20) 26.0 (4.56) 17.5 – 27.3 0.8 (0.48) 0.0 (0.00) 0.0 – 0.5

–

NYNR 22.8 (4.77) 15.8 (3.99) 17.0 – 24.5

–

–

–

–

Collection #2

ILCL 12.8 (3.77) 13.8 (3.71) 13.8 – 22.8

–

–

–

–

KSLA 167.8 (35.73) 181.3 (25.38) 114.0 – 294.5

–

–

–

–

NYNR 156.5 (19.75) 118.3 (6.16) 116.8 – 159.5

–

–

–

–

Collection #3

ILCL 44.5 (17.33) 41.5 (8.21) 28.0 – 44.3

–

–

–

–

KSLA 510.3 (62.90) 509.0 (172.02) 404.0 – 813.5

–

–

– 0.8 (0.25) 0.

NYNR 8.3 (0.85) 12.3 (2.66) 7.0 – 11.5

–

–

–

–

Collection #4

KSLA 5.8 (1.89) 9.0 (1.78) 5.8 – 9.0

–

–

–

–
Monsanto Company 12-AL-246U 

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

Pest Arthropod

False chinch bug (Nysius raphanus) Green cloverworm (Hypena scabra) Garden webwo

Site KK179 Mean (SE) Control Mean (SE) Reference Range KK179 Mean (SE) Control Mean (SE) Reference Range KK179 Mean (SE) 

M

Collection #1

ILCL 4.5 (1.32) 10.0 (2.86) 7.0 – 11.3 1.3 (0.25) 1.8 (1.11) 1.3 – 4.3

–

KSLA 9.0 (1.96) 7.5 (0.50) 6.3 – 13.8

–

–

–

–

NYNR

–

–

–

–

–

–

–

Collection #2

ILCL 7.3 (1.11) 9.5 (1.94) 10.0 – 13.3

–

–

– 2.3 (0.63) 2

KSLA 1.0 (0.41) 1.5 (1.50) 0.8 – 1.3

–

–

–

–

NYNR 2.0 (0.71) 1.8 (0.75) 0.8 – 1.5

–

–

–

–

Collection #3

ILCL 6.8 (1.93) 7.5 (2.02) 3.8 – 10.3

–

–

–

–

KSLA 10.5 (3.01) 10.3 (0.85) 9.0 – 10.5

–

–

– 3.3 (0.75) 3

NYNR

–

–

–

–

–

–

–

Collection #4

KSLA 6.0 (1.58) 3.0 (1.08) 3.8 – 6.8

–

–

–

–
Monsanto Company 12-AL-246U 

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

Pest Arthropod

Lygus bug (Miridae) Potato leafhopper (Empoasca fabae) Thrips 

Site KK179 Mean (SE) Control Mean (SE) Reference Range KK179 Mean (SE) Control Mean (SE) Reference Range KK179 Mean (SE) 

Collection #1

ILCL 7.8 (1.49) 8.8 (3.07) 6.8 – 11.0 *152.3 (30.90) 329.0 (90.25) 178.8 – 256.8

–

KSLA 0.3 (0.25) 1.5 (0.96) 0.7 – 1.3 6.5 (1.66) 11.0 (1.78) 6.7 – 13.3

–

NYNR 1.5 (0.50) 1.0 (0.71) 1.5 – 2.3 12.0 (3.24) 18.5 (3.07) 12.3 – 14.3

–

Collection #2

ILCL

–

–

– 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) 1

NYNR 11.8 (0.95) 11.8 (3.71) 5.3 – 11.0

–

–

–

–

Collection #3 

ILCL

–

–

– 9.0 (1.68) 1

KSLA 0.3 (0.25) 0.8 (0.48) 0.0 – 0.5

–

–

–

–

NYNR 1.8 (0.63) 4.3 (2.36) 1.8 – 4.8 25.5 (1.04) 19.8 (1.49) 14.8 – 21.8 12.0 (3.56) 1

Collection #4

KSLA

–

–

– 1.5 (0.29) 1.5 (0.29) 0.3 – 1.3 27.0 (3.76) 2

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 values among the conventional commercial reference varieties.
Monsanto Company 12-AL-246U 

Table G-13. Abundance of Beneficial Arthropods in Samples Collected from KK179, the Convention

Conventional Commercial Reference Varieties in the First Year (2010) 

Beneficial Arthropod

Spiders (Araneae) Damsel bug (Nabis spp.) Ladybird beetl

Site KK179 Mean (SE) Control Mean (SE) Reference Range1 KK179 Mean (SE) Control Mean (SE) Reference Range1 KK179 Mean (SE) C

Me

Collection #1

ILCL 1.5 (0.29) 1.5 (0.50) 0.8 – 1.5 1.8 (0.85) 1.0 (0.41) 0.8 – 2.0 2.0 (0.91) 2.3 

KSLA

–

–

– 1.0 (0.58) 0.8 (0.25) 0.5 – 2.0 1.0 (0.71) 0.3 

NYNR

–

–

– 0.0 (0.00) 0.0 (0.00) 0.0 – 0.3 0.0 (0.00) 0.3 

Collection #2

ILCL * 0.0 (0.00) 1.5 (0.65) 0.0 – 1.8 0.5 (0.29) 0.5 (0.29) 0.3 – 2.0 1.5 (0.65) 1.8 

KSLA * 0.0 (0.00) 1.3 (0.48) 0.0 – 0.5

–

–

– 0.3 (0.25) 0.0 

NYNR

–

–

– 1.5 (0.65) 0.5 (0.50) 0.3 – 1.5 1.8 (0.85) 1.3 

Collection #3

ILCL 0.0 (0.00) 0.5 (0.29) 0.0 – 0.5 0.0 (0.00) 0.5 (0.29) 0.0 – 0.5

–

KSLA 2.5 (0.65) 1.5 (0.87) 1.3 – 2.5

–

–

– 5.5 (1.04) 2.8 

NYNR

–

–

– 0.5 (0.29) 0.8 (0.48) 0.3 – 1.0 0.3 (0.25) 0.5 

Collection #4

KSLA 1.3 (0.48) 1.0 (0.41) 0.8 – 2.3 0.5 (0.29) 0.3 (0.25) 0.0 – 0.5

–
Monsanto Company 12-AL-246U 

Table G-13 (continued). Abundance of Beneficial Arthropods in Samples Collected from KK179, the Cand the Conventional Commercial Reference Varieties in the First Year (2010) 

Beneficial Arthropod

Parasitic wasps (Hymenoptera)2 Lacewing (Chrysopid

Site KK179 Mean (SE) Control Mean (SE) Reference Range KK179 Mean (SE) Control Mean (SE) 

Collection #1

ILCL

–

–

–

–

–

KSLA 5.0 (1.47) 9.8 (1.18) 5.0 – 12.3

–

–

NYNR 0.0 (0.00) 0.5 (0.50) 0.0 – 0.5

–

–

Collection #2

ILCL

–

–

–

–

–

KSLA 0.8 (0.25) 2.0 (0.91) 0.8 – 1.5

–

–

NYNR 1.0 (1.00) 1.0 (0.71) 0.5 – 1.5

–

–

Collection #3

ILCL

–

–

– 0.0 (0.00) 0.0 (0.00) 

KSLA 3.0 (1.08) 1.8 (0.85) 1.8 – 3.0

–

–

NYNR 2.8 (0.75) 1.5 (0.65) 0.5 – 3.8

–

–

Collection #4

KSLA 0.5 (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 values among 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 Assessment: Abiotic Stressor Evaluations 

Using an Observational Severity Scale for 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 soil1

 22 22 

Wind damage 30 30 

Winter injury kill 8 8 

 
Monsanto Company 12-AL-246U 376 of 407

Table G-15. Combined-Site Qualitative Assessment: Disease Damage Evaluations 

Using an Observational Severity Scale for 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 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 stem1

 12 12 

Crown Rot 3 3 

Downy mildew 28 28 

Fusarium wilt 4 4 

Leaf spots2

 36 36 

Root rot3

 6 6 

Sclerotinia crown and stem rot 4 4 

Verticillium wilt 19 19 

 
Monsanto Company 12-AL-246U 377 of 407

Table G-16. Combined-Site Qualitative Assessment: Insect Stressor Evaluations 

Using an Observational Severity Scale for 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 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 

Aphid1

 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 17 

 
Monsanto 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 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. 
Monsanto Company 12-AL-246U 

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

Pest Arthropod

Aphid (Aphididae) Alfalfa weevil (Hypera postica) Armyworm 

Site KK179 Mean (SE) Control Mean (SE) Reference Range1 KK179 Mean (SE) Control Mean (SE) Reference Range1 KK179 Mean (SE) 

M

Collection #1ILCL 52.8 (2.14) 53.8 (16.04) 60.0 - 100.8 * 0.3 (0.25) 7.0 (5.05) 0.0 – 1.0

–

KSLA 81.5 (11.38) 75.3 (10.95) 64.3 – 97.0 25.0 (7.69) 29.8 (5.12) 22.0 – 43.3

–

NYNR 185.8 (19.26) 181.5 (38.49) 220.0 – 252.0 44.3 (10.26) 48.5 (6.74) 35.5 – 64.3

–

Collection #2ILCL

–

–

–

–

–

–

–

KSLA 173.0 (24.19) 177.0 (25.26) 158.3 – 281.3

–

–

–

–

NYNR 52.5 (5.07) 52.3 (7.09) 41.0 – 58.0

–

–

–

–

Collection #3ILCL 73.5 (14.39) 54.3 (7.38) 76.3 – 87.8

–

–

–

–

KSLA 186.5 (25.31) 189.3 (31.98) 134.3 – 236.0

–

–

–

–

NYNR 114.0 (17.97) 96.3 (8.40) 62.3 – 112.0

–

–

–

–

Collection #4ILCL 141.5 (29.22) 112.8 (18.14) 99.3 – 134.3

–

–

–

–

KSLA 167.8 (35.46) 232.8 (24.54) 189.3 – 276.5

–

–

–

–

NYNR 3.3 (1.60) 4.8 (1.11) 4.8 – 6.8

–

–

– 0.3 (0.25) 

Collection #5KSLA 11.0 (2.35) 8.3 (1.31) 6.5 – 8.0

–

–

– 5.3 (2.21) 
Monsanto Company 12-AL-246U 

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

Pest Arthropod

False chinch bug (Nysius raphanus) Blister beetle (Meloidae) Green cloverworm 

Site KK179 Mean (SE) Control Mean (SE) Reference Ran

ge

KK179

Mean (SE)

Control

Mean (SE)

Reference 

Ran

ge

KK179

Mean (SE)

M

Collection #1

ILCL

–

–

–

–

–

–

–

KSLA 1.5 (0.50) 2.3 (1.03) 2.0 – 4.8

–

–

–

–

NYNR

–

–

–

–

–

–

–

Collection #2

ILCL

–

–

–

–

–

–

–

KSLA 21.3 (5.22) 20.3 (2.69) 19.0 – 29.5 0.3 (0.25) 0.5 (0.50) 0.0 – 1.0

NYNR

–

–

–

–

–

–

–

Collection #3

ILCL

–

–

–

–

–

–

–

KSLA 18.5 (7.92) 17.3 (2.29) 7.0 – 20.0

–

–

–

–

NYNR

–

–

–

–

–

– 1.3 (0.63) 

Collection #4

ILCL

–

–

–

–

–

–

–

KSLA 3.0 (0.41) 5.8 (2.10) 6.0 – 9.0

–

–

–

–

NYNR

–

–

–

–

–

–

–

Collection #5

KSLA 4.0 (1.73) 3.3 (1.11) 4.0 – 5.8

–

–

–

–
Monsanto Company 12-AL-246U 

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

Pest Arthropod

Lygus bug (Lygus hesperus) Meadow spittlebug (Philaenus spumarius) Potato leafhoppe

Site KK179 Mean (SE) Control Mean (SE) Reference Range KK179 Mean (SE) Control Mean (SE) Reference Range KK179 Mean (SE) Co

Mea

Collection #1

ILCL 3.0 (1.08) 2.5 (1.50) 1.0 – 6.3

–

–

–

–

KSLA

–

–

–

–

–

–

–

NYNR 9.8 (0.85) 9.0 (4.83) 6.3 – 11.3 2.0 (1.00) 0.8 (0.75) 0.3 – 2.5 3.0 (1.08) 5.0 

Collection #2

ILCL 14.5 (1.32) 16.8 (5.53) 9.5 – 16.5

–

–

– 74.5 (11.53) 69.0 

KSLA

–

–

–

–

–

–

–

NYNR 8.3 (2.93) 11.8 (1.75) 8.3 – 14.0

–

–

– 49.3 (14.87) 65.0 

Collection #3

ILCL 6.8 (0.85) 12.0 (3.72) 5.8 – 12.0

–

–

– 109.8 (21.26) 129.5 

KSLA

–

–

–

–

–

– 3.8 (1.49) 2.5 

NYNR 21.5 (5.20) 19.8 (3.12) 13.3 – 16.3

–

–

– 215.5 (83.92) 169.3 

Collection #4

ILCL

–

–

–

–

–

– 22.0 (6.65) 14.3 

KSLA

–

–

–

–

–

–

–

NYNR 2.0 (1.68) 1.8 (0.25) 2.3 – 3.8

–

–

– 6.5 (1.04) 3.5 

Collection #5

KSLA

–

–

–

–

–

–

–
Monsanto Company 12-AL-246U 

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

Pest Arthropod

Thrips (Thysanoptera) Soybean looper (Pseudoplusi

Site KK179 Mean (SE) Control Mean (SE) Reference Range KK179 Mean (SE) Control Mean (S

Collection #1

ILCL 32.0 (1.08) 33.0 (12.73) 27.8 – 48.3

–

–

KSLA 25.3 (2.50) 37.5 (9.95) 37.8 – 46.8

–

–

NYNR

–

–

–

Collection #2

ILCL 181.5 (33.26) 177.0 (10.23) 87.5 – 166.5

–

–

KSLA 739.3 (174.43) 976.3 (118.06) 796.5 – 1190.5

–

–

NYNR

–

–

–

–

–

Collection #3

ILCL

–

–

–

–

–

KSLA 138.5 (39.86) 172.8 (43.35) 132.3 – 213.8

–

–

NYNR

–

–

–

–

–

Collection #4

ILCL 52.5 (4.56) 49.3 (5.62) 36.3 – 58.0

–

–

KSLA 76.3 (7.04) 137.3 (18.20) 115.3 – 160.3 2.5 (0.96) 2.5 (1.04) 

NYNR 12.0 (2.38) 10.3 (2.46) 8.0 – 14.0

–

–

Collection #5

KSLA 174.0 (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 values among the conventional commercial reference varieties.
Monsanto Company 12-AL-246U 

Table G-19. Abundance of Beneficial Arthropods in Samples Collected from KK179, the Convention

Conventional Commercial Reference Varieties in the Second Year (2011) 

Beneficial Arthropod

Spiders (Araneae) Ladybird beetle (Coccinellidae) Lacewing (

Site KK179 Mean (SE) Control Mean (SE) Reference Range1 KK179 Mean (SE) Control Mean (SE) Reference Range1 KK179 Mean (SE) C

Me

Collection #1

ILCL 0.0 (0.00) 0.3 (0.25) 0.0 – 0.5 1.5 (0.29) 3.0 (1.73) 1.8 – 2.5 0.3 (0.25) 0.0 

KSLA 0.5 (0.29) 0.3 (0.25) 0.3 – 0.5 1.0 (0.71) 1.0 (0.71) 0.8 – 2.3

–

NYNR

–

–

–

–

–

–

–

Collection #2

ILCL

–

–

–

–

–

–

–

KSLA 0.8 (0.25) 1.0 (0.71) 0.8 – 1.5 2.3 (1.31) 1.5 (0.65) 1.5 – 2.0

–

NYNR 0.0 (0.00) 0.3 (0.25) 0.0 – 0.3

–

–

–

–

Collection #3

ILCL 0.8 (0.25) 1.8 (0.25) 1.0 – 2.3

–

–

– 0.3 (0.25) 0.3 

KSLA 2.8 (0.75) 4.5 (1.44) 3.3 – 4.0 1.3 (0.48) 0.8 (0.25) 0.5 – 2.8 0.5 (0.29) 0.5 

NYNR 1.3 (0.25) 0.8 (0.25) 1.0 – 1.8 7.5 (1.66) 6.0 (1.08) 3.3 – 5.5 1.0 (0.41) 0.8 

Collection #4

ILCL 4.0 (1.47) 1.3 (0.95) 1.3 – 2.8 0.3 (0.25) 0.5 (0.29) 0.0 – 1.3

–

KSLA 2.0 (0.58) 4.3 (0.48) 2.8 – 4.8

–

–

– 0.5 (0.29) 0.8 

NYNR 0.0 (0.00) 0.3 (0.25) 0.3 – 0.8 0.0 (0.00) 0.0 (0.00) 0.0 – 0.3

–

Collection #5

KSLA 3.0 (0.71) 4.0 (1.08) 3.3 – 6.3 0.3 (0.25) 0.3 (0.25) 0.0 – 0.5 1.5 (0.29) 0.8 
Monsanto Company 12-AL-246U 

Table G-19 (continued). Abundance of Beneficial Arthropods in Samples Collected from KK179, the Cand the Conventional Commercial Reference Varieties in the Second Year (2011) 

Beneficial Arthropod

Nabids (Nabidae) Chalcid wasps (Chalcid

Site KK179 Mean (SE) Control Mean (SE) Reference Range KK179 Mean (SE) Control Mean (S

Collection #1

ILCL

–

–

–

–

–

KSLA

–

–

–

–

–

NYNR

–

–

–

–

–

Collection #2

ILCL

–

–

–

–

–

KSLA 1.0 (0.71) 2.3 (0.48) 0.8 – 1.8

–

–

NYNR 0.5 (0.29) 0.5 (0.29) 0.0 – 0.8

–

–

Collection #3

ILCL 0.8 (0.48) 0.3 (0.25) 0.0 – 0.5

–

–

KSLA 1.8 (1.44) 0.3 (0.25) 0.0 – 0.5

–

–

NYNR 0.8 (0.48) 1.3 (0.25) 0.0 – 1.5

–

–

Collection #4

ILCL

–

–

–

–

–

KSLA 0.0 (0.00) 0.5 (0.29) 0.0 – 0.8

–

–

NYNR

–

–

–

–

–

Collection #5

KSLA 0.8 (0.48) 0.8 (0.48) 0.8 – 1.5 9.5 (2.99) 4.5 (0.65) 

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 values among 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 establishment. 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 Methods from Phenotypic and Agronomic 

Assessment of KK179 under Field Conditions during Seed Production 

H.1. Materials 

Agronomic and phenotypic characteristics were assessed for KK179, the conventional 

control (C0-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 the starting seed of KK179, the conventional 

control, and three of the conventional commercial reference varieties, which were 

produced in the same production with KK179 and 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 location in Canyon County, 

ID. Seed of KK179, the conventional control, seven conventional commercial reference 

varieties, and one other material was planted in a randomized 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 cropping history of the study area are listed in 

Table H-2. Prior to planting, the Principal Investigator at the site prepared a proper seed 

bed according to local agronomic practices, including tillage, fertilization and pH 

adjustment, and pest management. After planting, all plots were thinned to a uniform 

density of 200 plants per plot. During the growing season, the field site 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 plot area. 

H.5. Study Management 

The field trial was uniformly managed under the local agronomic practices for seed 

production. Plots were not harvested 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 control for seedling establishment, seedling 

vigor, lodging, seed weight, seeds per pod, split pods, and seed yield. A description of 

each phenotypic characteristic assessed is listed in Table VII-1. 
Monsanto Company 12-AL-246U 387 of 407

H.7. Data Assessment 

Experienced scientists familiar with the experimental design and evaluation criteria were 

involved in all components of data collection, summarization, and analysis. Study 

personnel assessed that measurements were taken properly, data were consistent with 

expectations based on experience 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 during the study that would impact the study 

objectives were noted. Data were then subjected to statistical analysis as indicated 

below. 

H.8. Statistical Analysis 

An analysis of variance was conducted according 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 significance was predetermined to be 5% (α=0.05). KK179 

and the conventional control were not statistically compared to the conventional 

commercial reference varieties. Minimum and maximum mean values were calculated 

for each characteristic from the seven conventional 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

C0-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 

Table H-2. Field and Planting Information Plot area (ft2

) Rows (#/plot) Planting date Planting density1 (lb/A) Soil Series OM2 (%) 

73.3

2 4/20/2010 2.6 Sandy Loam 1.5 

1All plots were thinned to a uniform density of 200 plants per plot after seedling establishment and seedling vigor data wer2OM =Organic Matter 
Monsanto 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 (C0-Syn1), and four conventional commercial reference 

varieties were grown under similar agronomic conditions in a greenhouse (Table I-1). 

Approximately 1-2 weeks after planting, seedlings 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 container and stored on wet ice for transport and 

storage in the microscope laboratory. A minimum of 15 mature flowers were removed 

from the collected racemes of each entry within a replication. 

I.3. Pollen Sample Preparation 

From the collected flowers, five individual sub-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 stain diluted 1:5 (Alexander, 1980). 

I.4. Data Collection 

Pollen characteristics were assessed by viewing samples under an Olympus BX51 light 

microscope equipped with an Olympus DP70 digital color camera. The microscope and 

camera were connected to a computer running 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. Non-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 random 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 two perpendicular axes for 10 representative 

pollen grains per replication. Mean pollen diameter 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 conventional control, and the conventional 

commercial reference varieties was observed as shown in Figure I-1. 

I.5. Statistical Analysis 

An analysis of variance was conducted according 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 conventional control for percent viable pollen 

and pollen grain diameter. KK179 and the conventional control were not statistically 

compared to the conventional commercial reference varieties. Minimum and maximum 

mean values were calculated for each characteristic from the four conventional 

commercial reference varieties. General pollen morphology was qualitative; therefore, 

no statistical analysis was conducted 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

C0-Syn1 Conventional; Syn1 population 11265474

Pioneer 54H11 Conventional 11265478

WL 319HQ Conventional 11265484

DKA50-18 Conventional 11265481

Dow/Dairyland HybriForce 400 Conventional 11265485

 

 
Monsanto Company 12-AL-246U 394 of 407

Figure I-1. General Morphology of Pollen from KK179, the Control, and 

Conventional Commerical Reference Varieties under 200X Magnification 

The alfalfa pollen samples were stained with Alexander’s stain diluted 1:5. Viable pollen 

grains stained red to purple, while non-viable pollen grains stained blue to green and may 

appear round to collapsed depending on the degree of hydration. No visual differences 

were observed between KK179 and the conventional control or conventional commercial 

reference alfalfa varieties. 
Monsanto Company 12-AL-246U 395 of 407

References for Appendix I 

Alexander, M.P. 1980. A versatile 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 (C0-Syn1), and four conventional 

commercial reference varieties were grown under similar agronomic conditions in a field 

trial in 2010 in Walworth County, WI (WIDL). Prior to transplantation, seedlings of 

KK179 were sampled and tested using event-specific PCR analyses. Plants not 

containing KK179 were not used in the field trial. The trial was arranged in a 

randomized complete block design with four replications. 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 inner 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 that was pre-labeled with entry number, entry 

name, and plot number from which the sample originated. Each raceme had 

approximately 5-40 flowers, with not less than half of the individual flowers fully 

opened. The containers were kept on wet ice 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 detailed 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 control for flowers per raceme, standard petal 

length, keel petal length, calyx tube diameter, sexual column length, wing petal length, 

flower color class (USDA-ARS, 1972), gross raceme morphology, and gross flower 

morphology (Table VII-15). Flower characteristics were assessed by viewing samples 

with an Olympus SZ60 stereo-zoom microscope 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 characteristic 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 flowers of KK179, the conventional control, and 

the conventional commercial reference varieties (Figures J-2 and J-3). 

J.6. Statistical Analysis 

An analysis of variance was conducted according 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 petal length, calyx tube 

diameter, sexual column length, and wing petal length. The level of statistical 

significance was predetermined to be 5% (α=0.05). KK179 and the conventional control 

were not statistically compared to the conventional commercial reference varieties. 

Minimum and maximum mean values were calculated for each characteristic from the 

four conventional commercial reference varieties. Analysis of variance and the 

calculation for quantitative descriptive statistics was not appropriate for flower color 

class, gross raceme morphology, and gross flower morphology due to the nominal nature 

of the responses. For these characteristics, the rating observed most often is reported for 

KK179 and the conventional control, 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 Untripped (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 sexual 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 column 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 

Table J-1. Starting Plants for Flower Morphology Assessment Material Name Genotype / Phenotype Monsanto Seed Lot Number Monsanto TranKK179 Reduced Lignin; Syn1 population 11265471 11266289C0-Syn1 Conventional; Syn1 population 11265474 11266292Pioneer 54V54 Conventional 11265475 11266293 

Croplan LegenDairy 5.0 Conventional 11265476 11266294 Dow/Dairyland HybriForce 400 Conventional 11265485 11266303 Vernal Conventional 11265487 11266305 

Note: The field production source of flowers evaluated also included two additional materials that are outside of the scope of the object

KK179. 

1Transplants were produced in a greenhouse and were analyzed by PCR to verify identity.
Monsanto Company 12-AL-246U 

Figure J-2. General Morphology of Whole Racemes from KK179, the Conventional Control, anCommercial Reference Varieties No visual differences were observed between KK179 and the conventional control or the conventional commerc

KK179 Control

Reference

Reference ReferenceReference
Monsanto Company 12-AL-246U 401 of 407

Figure J-3. General Morphology of Tripped Flowers from KK179, the 

Conventional Control, and the Conventional Commercial Reference Varieties 

No visual differences were observed between KK179 and the conventional control or the 

conventional commercial reference varieties. 

KK179 Control

Reference Reference

Reference Reference
Monsanto 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 classifying alfalfa flower color. Agriculture 

Handbook No. 424. U.S. Department of Agriculture, 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 plants and fix a substantial 

portion of the world’s supply of organic nitrogen (Gage, 2004). The nitrogen-fixing 

plant-microbe symbiosis results in the formation 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, 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 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 nitrogen status are commonly 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 (C0-Syn1), and six conventional commercial reference 

varieties were used in a symbiont interaction assessment (Table K-1). Nodules, root 

tissue, and shoot tissue collected from KK179, the conventional control, and the 

conventional commercial reference 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. Approximately 1-2 weeks after planting, seedlings 

of KK179 were sampled and tested using event-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 bag each of Premier Horticulture Promix PGX 

and Promix BX, 1⁄4 cup lime and 2 cups sand). Plants were grown 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 for a minimum of four hours. Following this, 

approximately 500 ml of nitrogen-free nutrient 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 reference varieties. At planting, each seed 

was inoculated with a dry, clay-based inoculant containing a minimum of 7 × 107

 cells of 

S. meliloti (Dormal, Becker Underwood, Ames, IA). Pots were arranged in ten replicated 
Monsanto 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 (target 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 harvested 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 commercial reference varieties 

with the following exceptions: four pots had an extra plant emerge after thinning, which 

was not noticed until harvest, and one pot contained 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 excised at the surface of the potting medium and 

shoot and root plus nodule material were removed from the pots. 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 washing with water. Excess moisture was 

removed using absorbent paper towels, and the roots plus nodules were placed in labeled 

bags. The same day that plants were harvested, nodules were removed by hand from the 

roots of each plant and weighed to determine the fresh weight (fwt) of the nodules. 

Nodules were not enumerated as the alfalfa nodules were branched 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 plant. Nodules as well as root and shoot 

material were placed in a drying oven on the same 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 values. Any remaining seeds and 

plants were devitalized per USDA-APHIS requirements. 

 
Monsanto Company 12-AL-246U 405 of 407

K.6. Statistical Analysis 

The data analyzed consisted of five measurements 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) (Table VII-16). 

An analysis of variance was conducted using a randomized complete block design with 

ten replications for each of KK179, the conventional control, and the six conventional 

commercial reference varieties. Data were analyzed using SAS (SAS, 2008) with the 

level of statistical significance 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 for 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, Conventional 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

C0-Syn1 Conventional; Syn1 population 11265474

DKA50-18 Conventional 11265481

FG78T803 Conventional 11293630

FG98M817 Conventional 11293629

Ranger Conventional 11265488

Vernal Conventional 11265487

WL 319HQ Conventional 11265484

 
Monsanto 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. Nutrition and fertilizer use. Pages 333-372 in 

Alfalfa and Alfalfa Improvement. A.A. Hanson, D.K. Barnes, and R.R. Hill, Jr. (eds.). 

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.