Monsanto and Forage Genetics International Petition 

(12-321-01p) for Determination of Non-regulated 

Status of Event KK179 Alfalfa

OECD Unique Identifier: 

MON-ØØ179-5

Plant Pest Risk Assessment 

August 2013

Agency Contact 

Cindy Eck

Biotechnology Regulatory Services

4700 River Road

USDA, APHIS 

Riverdale, MD 20737 

Fax: (301) 734-8669

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U.S. Department of Agriculture over others not mentioned. USDA neither guarantees nor warrants the standard of any 

product mentioned. Product names are mentioned solely to report factually on available data and to provide specific 

information. 

This publication reports research involving pesticides. All uses of pesticides must be registered by appropriate State 

and/or Federal agencies before they can be recommended. 
TABLE OF CONTENTS

i

A. Introduction................................................................................................................. 1

B. Development of KK179 Alfalfa.................................................................................. 2

C. Expression of the Gene Product and Changes to Plant Metabolism........................... 7

D. Potential Impacts on Disease and Pest Susceptibilities.............................................. 9

E. Potential Impacts on Nontarget Organisms Beneficial to Agriculture ..................... 13

F. Potential for Enhanced Weediness of KK179 Alfalfa .............................................. 14

G. Potential Impacts on the Weediness of Any Other Plants with which It Can 

Interbreed................................................................................................................. 16

H. Potential Changes to Agriculture or Cultivation Practices....................................... 19

I. Potential Impacts from Transfer of Genetic Information to Organism with 

which KK179 Alfalfa Cannot Interbreed................................................................. 19

J. Conclusion ................................................................................................................ 20

K. References................................................................................................................. 21
1

A. Introduction

Monsanto Company (referred hereafter as Monsanto) and Forage Genetics International, 

LLC (hereafter referred to as FGI) have petitioned the Animal and Plant Health 

Inspection Service (APHIS) of the United States Department Agriculture (USDA) for a 

determination that the genetically engineered (GE) ) reduced lignin alfalfa (Medicago 

sativa L.) event KK179 (hereafter referred to as KK179 alfalfa) is unlikely to pose a plant 

pest risk and, therefore, should no longer be a regulated article under the APHIS’ 7 Code 

of Federal Regulations (CFR) part 340. This petition was assigned the number 12-321-

01p-a1, and is hereafter referenced as Monsanto and FGI 2013. APHIS administers 7 

CFR part 340 under the authority of the plant pest provisions of the Plant Protection Act 

(PPA) of 20001(7 U.S.C. 7701 et seq.). This plant pest risk assessment was conducted to 

determine if KK179 alfalfa is unlikely to pose a plant pest risk.

APHIS regulations in 7 CFR Part 340 regulate the introduction (importation, interstate 

movement, or release into the environment) of certain GE organisms and products. A GE 

organism is no longer subject to the plant pest provisions of the PPA or to the regulatory

requirements of 7 CFR part 340 when APHIS determines that it is unlikely to pose a plant 

pest risk. A GE organism is considered a regulated article under 7 CFR part 340 if the 

donor organism, recipient organism, or vector, or vector agent used in engineering the 

organism belongs any genera or taxa designated in 7 CFR § 340.2 and is also considered 

a plant pest. KK179 was produced by Agrobacterium tumefaciens-mediated

transformation and includes introduced genetic sequences derived from plant pest 

organisms listed in 7 CFR § 340.2 (Monsanto and FGI 2013). Monsanto has conducted 

introductions of KK179 as a regulated article under APHIS-authorized notifications since 

2007 (Table A-1, pages 231-235 in Monsanto 2013), in part, to gather information to 

support that it is unlikely to pose a plant pest risk.

Potential impacts in this Plant Pest Risk Assessment are those that pertain to plant pest 

risk associated with KK179 alfalfa and its progeny and their use in the absence of 

confinement. APHIS utilizes data and information submitted by the applicant, in addition 

to current literature, to determine if KK179 alfalfa is unlikely to pose a plant pest risk. 

APHIS regulations in 7 § CFR 340.6(c) specify the information needed for consideration 

in a petition for nonregulated status. APHIS will evaluate information submitted by the 

applicant related to plant pest risk characteristics, expression of the gene product, new 

enzymes, or changes to plant metabolism, weediness of the regulated article, impact on 

the weediness of any other plant with which it can interbreed, changes to agricultural or 

cultivation practices that may impact diseases and pests of plants, effects of the regulated 

 

1

Section 403 (14) of the Plant Protection Act (7USC Sec 7702(14)) defines plant pest as:“Plant Pest - The 

term “plant pest” means any living stage of any of the following that can directly or indirectly injure, cause 

damage to, 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. (G) An infectious agent or other 

pathogen. (H) Any article similar to or allied with any of the articles specified in the preceding 

subparagraphs.”
2

article on nontarget organisms, indirect plant pest effects on other agricultural products, 

and transfer of genetic information to organisms with which it cannot interbreed. 

APHIS may also consider information relevant to reviews conducted by other agencies 

that are part of the ‘Coordinated Framework for the Regulation of Biotechnology’ (51 FR 

23302, June 26, 1986). Under the Coordinated Framework, the oversight of 

biotechnology-derived plants rests with the APHIS, the Food and Drug Administration 

(FDA), and the Office of Pesticide Programs of the U.S. Environmental Protection 

Agency (EPA). Depending on its characteristics, certain biotechnology-derived products 

are subjected to review by one or more of these agencies. The EPA under the Federal 

Insecticide, Fungicide and Rodenticide Act (FIFRA), regulates the distribution, sale, use 

and testing of pesticidal substances produced in plants and microbes, including those 

pesticides that are produced by an organism through techniques of modern 

biotechnology. The EPA also sets tolerance limits for residues of pesticides on and in 

food and animal feed, or establishes an exemption from the requirement for a tolerance, 

under the Federal Food, Drug and Cosmetic Act (FFDCA). The FDA under the FFDCA 

is responsible for ensuring the safety and proper labeling of all plant-derived foods and 

feeds, including those developed through modern biotechnology. 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. 

B. Development of KK179 Alfalfa 

Alfalfa is widely planted throughout the world as a forage legume for hay, pasture and 

silage for its highly nutritious forage and broad adaptability (Li and Brummer 2012). In 

the U.S., alfalfa is the fourth most common crop by both acreage (~24 million acres) and 

value (~$10 million) with approximately 20 to 24 million acres of alfalfa hay harvested 

annually in the United States (USDA-NASS 2012). Alfalfa is grown for forage in all 50 

states (Figure 1, next page) and Canada. In the year 2012, the price per ton was $187 

(USDA-NASS 2012). 

Alfalfa originated in southwestern Asia, and is believed to have been first domesticated 

near present day Caucasus regions and other countries in Asia Minor, and now has a 

worldwide distribution due to its popularity as an agricultural species (California Alfalfa 

and Forage Association 2001). It was introduced into the United States in 1736 in 

Georgia, but it was not until around 1850 that it began to be more widely planted. It is 

naturalized in many areas and distributed in temperate zones of the world, e.g. U.S., 

southern Canada, Europe, China, southern Latin America, and South Africa.

Alfalfa is grown as a perennial forage crop and includes field preparation, planting, stand 

establishment (first year), established stand maintenance (2-8 years), and stand 

termination (Orloff et al., 1997). If a crop is being harvested for hay, it is harvested 

multiple times per growing season, from 2-11 times depending on region. Seed crops can 

be harvested once at the end of each growing season (Putnam et al., 2008). 
3

Figure 1: Alfalfa Hay (Dry) USDA-NASS (2012) 

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

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

product is hay, which is forage that has been dried. Lignin deposition in maturing plants 

has a significant impact on the overall quality of alfalfa (Coors et al., 1986; Marten et al., 

1988). 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., 2008). Improved forage 

quality is associated with lower lignin levels (Guo et al. 2001) 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. 

As described above, alfalfa forage may be harvested multiple times in a season. Alfalfa 

harvested at an immature growth stage (shorter interval between cuttings) gives low yield 

but high forage quality. If alfalfa is cut at a mature growth stage (long intervals between 

cuttings) it results in high yield but low forage quality (Orloff and Putnam 2008). 

Deciding when to harvest forage is a critical decision made by the grower that determines 
4

both forage yield and quality. Growers must obtain a balance between obtaining high 

yield and high quality because the quality of forage declines rapidly as lignin 

accumulates in maturing plants. 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 (Orloff and Putman 2008). 

Alfalfa forage production fields remain economically viable for approximately three to 

five years after initial planting.

Monsanto and FGI have developed biotechnology-derived reduced lignin alfalfa, KK179, 

which has reduced levels of a specific lignin subunit when compared to conventional 

alfalfa at the same stage of growth. This leads to reduced overall accumulation of total 

lignin in alfalfa forage, the principal feed product derived from alfalfa. The levels of 

lignin in KK179 forage are generally similar to those found in conventional forage 

harvested several days earlier under similar production conditions. The reduced lignin 

alfalfa increases forage quality compared to conventional forage of the same age, 

maximizes forage yield by delaying harvest for several days, and gives farmers more 

flexibility in forage harvest timing to: 1) maximize forage quality by producing lower 

lignin levels in the forage; 2) maximize forage yields by allowing farmers to delay

harvest for several days accumulating more forage biomass; and 3) allow more flexibility

in harvest schedules during the alfalfa growth cycle due to untimely weather, labor 

availability, and dairy herd management (Monsanto & FGI 2013).

Description of Genetic Modifications

Monsanto and FGI have developed biotechnology-derived low lignin KK179 alfalfa

designed to have lower quantities of guaiacyl lignin (commonly referred to as G lignin), 

one of the major subunits of total lignin, when 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. 

Lignin provides strength to plants and allows the plant vascular system to transport water 

in the plant without leakage. Lignin increases with advanced maturity in alfalfa 

(Undersander 2010). However, lignin is indigestible and reduces fiber digestibility in 

ruminants. Thus reducing lignin content should increase fiber digestibility at any maturity

stage. Lignin molecule fills the spaces between cellulose, hemi-cellulose and pectins as 

the plant ages and binds with the hemicellulose. Lignin coating the cellulose allows water 

to move up the plant stem without leakage but also reduces digestion of the cellulose in 

the rumen of forage animals (Undersander 2010).

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
5

reduction in G lignin subunit synthesis leads to reduced accumulation of total lignin. 

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).

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, Monsanto and FGI 2013). 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 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.

KK179 alfalfa was developed through Agrobacterium tumefaciens-mediated 

transformation with conventional alfalfa, R2336, with the plasmid vector PVMSPQ12633. The PV-MSPQ12633 plasmid contains two separate transfer DNAs (TDNAs), each delineated by Left and Right Border sequences (See Table 1, next page).

The first T-DNA, designated T-DNA1, contains the caffeoyl CoA O-methyltransferase 

(CCOMT) suppression cassette, the phenylalanine ammonia-lyase (Pal2) promoter and 

the nopaline synthase (nos) 3 ́ untranslated region (UTR) regulatory elements. T-DNA1 

also contains the RNA-based suppression of CCOMT in KK179 is mediated by double 

stranded RNA (dsRNA) molecules transcribed from the suppression cassette, which 

decrease the level of endogenous CCOMT RNA transcripts resulting in reduced levels of 

G lignin. 

The second T-DNA, designated T-DNA II, contains the neomycin phosphotransferarse II 

(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 TDNA II, containing the nptII expression cassette functioned as a marker gene for the in

vitro selection of transformed plantlets (Monsanto & FGI, 2013). This second T-DNA, 

however, was subsequently bred out of the alfalfa line through conventional breeding and 

is not present in KK179 alfalfa. 
6

Table 1. The number of inserts and a brief description of the nature of 

the inserts in KK179 alfalfa

T-DNA I Genetic Components

Genetic Element Function (Reference)

B1-Left Border

Region

DNA region from Agrobacterium tumefaciens containing the Left 

Border sequence used for transfer of the T-DNA (Barker et al., 1983)

P2-Pal2 Promoter of the Pal2 gene from Phaseolus vulgaris

encoding the phenylalanine ammonia-lyase that directs transcription in 

plant cells (Cramer et al., 1989)

CCOMT* 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

CCOMT* 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

T3-nos 3′UTR sequence of the nopaline synthase (nos) gene from 

Agrobacterium tumefaciens pTi encoding NOS that directs 

polyadenylation (Bevan, 1984; Fraley et al., 1983)

B-Right

Border Region

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

Genetic Element Function (Reference)

aadA 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.

OR4-ori-pUC Origin of replication from plasmid pUC for maintenance of plasmid in 

E. coli (Vieira and Messing, 1987)

CS5-rop Coding sequence for repressor of primer protein from the ColE1 

plasmid for maintenance of plasmid copy number in E. coli (Giza and 

Huang, 1989)

OR-oriV Origin of replication from the broad host range plasmid RK2 for 

maintenance of plasmid in Agrobacterium (Stalker et al., 1981)

Description of T-DNA II Genetic Components

Genetic Elements Function 

B-Left Border

Region

DNA region from Agrobacterium tumefaciens

containing the Left Border sequence used for transfer of the T-DNA 

(Barker et al., 1983)

P-35S

Promoter and leader from the 35S RNA of cauliflower mosaic virus 

(CaMV) (Odell et al., 1985) that directs transcription in plant cells

CS-nptII

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)

T-nos

3′ UTR sequence of the nopaline synthase (nos) gene from 

Agrobacterium tumefaciens pTi encoding NOS that directs 

polyadenylation (Bevan, 1984; Fraley et al., 1983)

B-Right

Border Region

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)
7

The transformed plant was crossed with MS 208, an elite, male sterile, conventional 

alfalfa plant, in which the unlinked insertions for T-DNA I and T-DNA II were 

segregated to produce KK179 and confirmed by PCR analysis. The segregation of the 

two markers was 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 

line of KK179 (Monsanto and FGI 2013).

Genetic elements outside 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 the 

plasmid vector backbone. In addition to the above mentioned genetic elements, the 

inserted T-DNA also contains short non-coding intervening DNA sequences. The 

intervening sequences contain restriction enzyme recognition sites and are used for 

cloning purposes.

Monsanto and FGI provided evidence demonstrating that, 

 KK179 contains a single copy of T-DNA I at a single insertion site in the alfalfa 

genome and is devoid of T-DNA II or backbone sequences from PV-MSPQ12633 

(Figures IV-2 through IV-8, Monsanto and FGI 2013).

 KK179 genetic elements were confirmed through PCR and DNA sequence 

analyses of elements within the insert, and determined the 5 ́ and 3 ́ insert to plant 

junctions (Figure IV-9 and Figure IV-10, Monsanto and FGI 2013). 

 Segregation analysis of heritability and generational stability of T-DNA I was 

maintained through four generations of the breeding history (Figure IV-12), and 

establishes that T-DNA I in KK179 is inherited according to Mendelian principles 

of inheritance (Figure IV-13 and Table IV-3, Monsanto and FGI 2013).

C. Expression of the Gene Product and Changes to Plant Metabolism

USDA-APHIS assessed whether changes in plant metabolism in KK179 alfalfa is likely

to alter their plant pest risk relative to the untransformed control. The assessment 

encompasses (1) a consideration of the specific effects on plant metabolism or 

composition of KK179 due to silencing CCOMT expression; and (2) evaluation of 

whether the nutrient and anti-nutrient levels are comparable to those in the respective 

non-transformed alfalfa and its effect on plant metabolism and if this could lead to an 

increase plant pest risk.

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 resistant 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
8

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 unlikely to produce a protein or 

polypeptide.

The dsRNSs 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 (U.S. FDA 1992). Several biotechnology-derived plant products previously

reviewed by the US FDA, and deregulated by USDA-APHIS, were developed using 

RNA-based suppression mechanisms, including improved fatty acid profile soybean 

MON 87705(09-201-01p), plum pox virus-resistant plum trees (04-264-01p), virusresistant papaya (96-051-01p), virus-resistant squash (95-352-01p), and delayed-ripening 

tomatoes(95-324-01p). The hairpin secondary structure of the ds-RNA 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.

Compositional Analysis

Compositional analyses were conducted to assess whether the composition and nutrient 

levels in grain and forage derived from Event KK179 alfalfa were comparable to the 

negative segregant, which has background genetics very similar to Event KK179 alfalfa,

but without the CCOMT suppression gene cassette. Fourteen different conventional 

commercial alfalfa reference varieties were included across the field production sites in 

six U.S. sites: California (CAPR), Iowa (IARL), Illinois (ILCY), Kansas (KSLA), Texas 

(TXCL), and Wisconsin (WIDL) to provide data on the natural variability of 

compositional component analyzed (Appendix E).

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, 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) (Appendix E). Anti-nutrient and secondary metabolites saponins, and 

canavanine (Section I.B.3, Appendix E). In 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 wall associated 

metabolites (Appendix E).

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 the constituents statistically compared (Appendix E). In the case of ash and 

ferulic acid, the relative magnitudes of the differences were under 10%. The mean levels 
9

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, Monsanto & 

FGI 2013).

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. 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 forage growth stage 

(Section I.B.3, Monsanto & FGI 2013) KK179 is compositionally equivalent to 

conventional alfalfa with regard to levels of nutrients, anti-nutrients, and secondary

metabolites. 

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 in different laboratories likely contributed to the variability in total 

lignin (ADL) values between labs. Both methods did confirm a decrease in total lignin in 

KK179, with one lab reporting a significant decrease of 22.15%, and another lab a 

nonsignificant decrease of 4.89%.

Overall, a comprehensive evaluation of KK179 alfalfa and the controls showed no 

biologically meaningful differences for KK179 alfalfa and forage composition for the 

nutrients or key anti-nutrients, and secondary metabolites. The few differences detected 

differences were either small in magnitude or the mean component values of KK179 

alfalfa and the controls were within the 99% tolerance level. Therefore based on the data 

presented by Monsanto and FGI on KK179, APHIS therefore concludes that feed derived 

from KK1179 alfalfa can be considered compositionally and nutritionally equivalent to 

those derived from conventional alfalfa.

Based on all the above noted considerations, APHIS concludes that KK179 alfalfa poses 

no more of a plant pest risk from new gene products, changes to plant metabolism or 

composition than conventional alfalfa varieties.

D. Potential Impacts on Disease and Pest Susceptibilities

USDA-APHIS assessed whether Event KK179 alfalfa is likely to have significantly 

increased disease and pest susceptibility because of the introduced gene silencing 

constructs compared to the non-transformed alfalfa. This assessment encompasses a 

through consideration of the transformation process, introduced genes, and their genetic 

elements, and their expression products to cause interactions with pests and diseases. 

Changes assessed include those which would: (1) affect not only the new GE crop, but 

that would also result in significant introduction or spread of a damaging pest or disease 
10

to other plants; (2) result in the introduction, spread, and/or creation of a new disease; or 

(3) result in a significant exacerbation of a pest or disease for which APHIS has a control 

program. Any increase in pest or disease susceptibility is evaluated with respect to the 

context of currently cultivated varieties, the ability to manage the pest or disease, and the 

potential impact on agriculture.

Plant Protection and Quarantine (PPQ) is an APHIS program that safeguards agriculture 

and natural resources from the entry, establishment, and spread of animal and plant pests 

and noxious weeds into the United States of America; and supports trade and exports of 

U.S. agricultural products. PPQ responds to many new introductions of plant pests to 

eradicate, suppress, or contain them through various programs in cooperation with state 

departments of agriculture and other government agencies. These may be emergency or 

longer term domestic programs that target a specific pest. A variety of insect, plant 

disease, mollusk, or weed programs exist (see http://www.aphis.usda.gov/plant_health/ 

plant_pest_info/index.shtml ); however none specifically target pests of alfalfa.

APHIS considered the potential for the transformation process to cause or aggravate 

disease symptoms in Event KK179 alfalfa or other plants or to cause the production of 

plant pathogens. Wild type A. tumefaciens carries a tumor-inducing (Ti) plasmid that can 

be transferred to broadleaf plants and cause crown gall disease. Agrobacterium 

tumefaciens strain contains a plasmid PV-MSPQ12633 contains a disarmed plasmid that 

is incapable of inducing tumor formation due to the deletion of the phytohormone genes 

originally present (Koncz and Shell 1986). Agrobacterium-mediated plant transformation 

has been used widely for decades, and has not been implicated in causing plant disease, 

and is highly unlikely to pose a plant pest risk. 

The major alfalfa diseases of economic importance in the United States are caused by 

those pathogens that impact the foliar, crown, root, vascular, and seedling health of 

alfalfa plants. Alfalfa diseases are primarily caused by fungi; however, nematodes, 

bacteria, viruses, and other microbes also cause economic losses in alfalfa production 

(Leath et al., 1988). Field tests were visually monitored for incidence of plant disease 

and pests on KK179 and compared with the conventional alfalfa comparators (an

identical breeding path was used for development of KK179 and non- transgenic 

reference lines). Event KK179 low lignin alfalfa was released in field trials from 2007 

through 2012 in 11 states including Wisconsin, Pennsylvania, New York, Texas, 

Oklahoma, Kansas, Iowa, Illinois, Idaho, California and Washington (133 regulated 

releases ) across a diverse range of environmental conditions representative of where 

KK179 is expected to be grown (pp. 231-235 Monsanto & FGI 2013). All plots were 

uniformly treated with pest control measures at each location based on local protocol. 

The experimental design was a randomized complete block with four replications. The 

evaluations for plant pests and insects 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 
11

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. Monsanto and FGE 

used qualitative and quantitative techniques to observe insect and disease damage in the 

field: Anthracnose, Bacterial wilt, Black stem, Crown Rot, Downy mildew, Fusarium 

wilt, Leaf spots, Phytophthora root rot, Sclerotinia crown and stem rot, Verticillium wilt. 

No differences in the range of plant damage responses to disease stressors were observed 

for 129 comparisons between KK179 and the conventional control (Table G-15, 

Monsanto & FGI 2013). No differences were observed between KK179 and the 

conventional control during any observation for damage caused by any of the assessed 

disease stressors.

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 

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.

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 in insects observed (Section VII). Several insect 

orders were considered and observed in the field trial. Alfalfa weevil (Hypera postica) is 

one of the biggest pests in alfalfa, followed by several species of aphid (Aphididae), and 

army worms (Spodoptera spp.). Observations also included the Potato leafhopper 

(Empoasca fabae), grasshoppers and Lygus bug. There were no meaningful differences 

in observations over combined-site qualitative assessments on insect stressor evaluations 

for KK179 and the conventional control in the 2010 and 2011 year observations.
12

The environmental interactions evaluation (Section VII.C, Monsanto & FGI 2013)

included qualitative assessment of plant response to insects and disease and quantitative 

of insect 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, 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 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 Monsanto &FGI 2013). 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, Monsanto & FGI 2013).

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.). 

Based on all the above noted considerations, APHIS concludes that KK179 poses no 

more of a plant pest risk from changes in susceptibility to disease or pests.
13

E. Potential Impacts on Nontarget Organisms Beneficial to Agriculture

APHIS evaluated the potential for Event KK179 alfalfa to have damaging or toxic effects 

directly or indirectly on non-target organisms. The non-target organisms considered were 

representatives of the exposed species in the agricultural environment. 

Alfalfa is predominantly cross-pollinated and the flowers depend entirely on bees for 

cross-pollination. Wind cross-pollination in alfalfa does not occur (Viands et al., 1988). 

There are two types of pollen mediated gene flow in alfalfa that may involve pollinators: 

1) hay to hay field gene flow and, 2) hay to seed field gene flow for alfalfa seed 

production.

In alfalfa hay-to-hay field gene flow forage producers cut hay at regular intervals prior to 

or near early bloom for optimum forage quality and yield. Forage quality of the crop 

deteriorates rapidly after this stage of crop maturity. Since flowering is not advantageous 

to the nutritional quality of alfalfa, the crop is usually cut before flowering. Pollinators 

are not usually found in the field before flowering.

For hay to seed alfalfa of the growers in the eleven (11) western U.S. states that are 

eligible to grow alfalfa for seeds, only those in the Desert Southwest States (CA, AZ, 

etc.) rely predominately on commercially managed honey bees as pollinators. In other 

geographies where wild or commercial honey bees may be present (e.g. the Dakotas) 

alfalfa seed increase will either not be licensed and/or seed producers stock non-Apis bee 

species. There are approximately 44,000 acres of alfalfa seed in the southwestern United 

States, of which approximately 30,000 acres (Van Deynze et al., 2008; McCaslin, 2007) 

is produced for export—this is a market sector that is sensitive to biotechnology-derived 

traits and unlikely to adopt GE varieties at this time (McCaslin, 2007; Van Deynze et al., 

2008).

As stated above, although bees serve no direct agronomic (pollination) purpose in hay

production fields, some alfalfa hay growers opt to allow their late-summer hay crop to 

flower extensively, precluding the harvest of maximum hay quality or yield. Working 

with honey bee keepers, these non-irrigated hay growers may allow their hay crop to 

flower and be used as an intentional source of honey bee forage instead of cutting for 

other livestock feeding purposes. In this situation, the bees are not “pollinators” used to 

produce the alfalfa crop per se; rather they are placed there as “foragers” to produce a 

honey crop and bee progeny (not seed). While it is theoretically possible for exposure of 

honey-producing bees to alfalfa flowers found in fields where harvesting was delayed due 

to weather or on unmanaged feral plants to occur intermittently in some geographies, 

because of factors of scale, hay harvest routines and the seed purchase license 

restrictions, the extent and duration of any unintentional exposure is expected to be minor 

in comparison to intentional seed crop pollination use (see Van Deynze et al. 2008; 

Kendrick et al.; 2005) (Monsanto & FGI 2013).

Event KK179 alfalfa is not expected to be toxic or allergenic to plants or animals. The 

RNAi suppression cassette that precludes translation initiation and protein synthesis, thus 
14

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 ds-RNA are unlikely

to produce a protein. 

Data submitted by Monsanto and FGI 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, between alfalfa KK179 populations comparable to non-transgenic 

control populations for disease and insect disease observations. If deregulated KK179 

would require the same management practices for disease and other pests as for 

conventional alfalfa.

F. Potential for Enhanced Weediness of KK179 Alfalfa

APHIS assessed whether KK179 alfalfa are likely to become more weedy (i.e. more 

prevalent, competitive, damaging or difficult-to-control) than the nontransgenic 

progenitors from which they were derived.

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), however the 

author of the SWSS entry for alfalfa has clarified that alfalfa is not an invasive weed and 

does not displace native species but alfalfa does colonize disturbed areas (USDA-APHIS, 

2010, Brett Serviss, Docket No. 04-085-1 #480).

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). 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).

Seed aging, weathering or mechanical scarification makes 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 (Bass et al., 1988). 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). 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-
15

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 

(Monsanto & FGI 2013).

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 (Monsanto 

& FGI 2013).

Grasses controlled early have a low lignin content, and residue breakdown and soil root 

release occurs far more quickly (Victoria Department of Environment and Primary

Industries 2011). Low lignin grasses with higher nutritional values would allow a return 

to taller more competitive varieties that are more competitive with weeds and close 

canopy over them, lowering the amount of herbicide needed to control weeds (Gressel 

2002). 

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. 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 the 

first four cuttings; lodging, crop growth stage, or forage yield for a fifth cutting; 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 Monsanto 

& FGI 2013). 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 weed potential of KK179 compared to the 

conventional control (See Figure VII-1, Monsanto & FGI 2013).

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 weed potential of KK179 compared to conventional alfalfa. Furthermore,
16

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 weed 

potential or to have an effect on the environment for KK179 compared to

conventional alfalfa. Results of these evaluations show that there are no biologically 

significant differences between KK179 and the conventional control for traits potentially

associated with weediness therefore KK179 is unlikely to be more weedy than their 

respective parent varieties.

G. Potential Impacts on the Weediness of Any Other Plants with which 

It Can Interbreed

Alfalfa is self-incompatible and predominately outcrossing plant and is not capable of 

natural hybridization with any other species found in North America (USDA-APHIS 

2010). Alfalfa belongs to the genus Medicago and the tribe Trifolieae, which includes 

genera Trifolium (true clovers), Melilotus (sweetclover), and Trigonella (fenugreek). 

Medicago species do not hybridize (interbreed to form hybrid offspring) with these or 

other genera. The M. sativa complex has been successfully hybridized with 12 other 

perennial Medicago species (McCoy and Bingham, 1988). However, many of these 

interspecific hybrids have been successful only by using embryo culture of the hybrid in 

the greenhouse or laboratory (McCoy and Smith, 1986), making them highly unlikely to 

occur in nature. Moreover, no perennial Medicago species occur naturally in the U.S. 

(Xu et al., 2004). There are no sexually compatible relatives of Medicaga sativa in North 

America, thus, the risk for cross pollination and subsequent transgene introgression is 

limited to cultivated and feral alfalfa populations growing in North America (USDAAPHIS 2010).

Gene introgression is a process whereby one or more genes successfully incorporate into 

the genome of a recipient plant. 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. Gene flow is a measure of the exchange of genes between populations, and it 

occurs naturally between alfalfa populations. Review of information previously provided 

by Monsanto and FGI, information in the (USDA APHIS 2004, Petition 04-110-01p), as 

well as in the technical report, The Potential for Gene Flow from Glyphosate-Tolerant 

Alfalfa (Medicago sativa L.) to Related Species (appendix I) concludes that alfalfa does 

not naturally hybridize with any wild relatives in North America (USDA-APHIS 2010).

Gene Flow among Alfalfa Populations

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 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 
17

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).

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).

Gene flow to feral alfalfa plants from large-scale seed or hay 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) 

The data presented in this petition strongly suggest that the trait in KK179 would not 

confer a selective advantage to a feral alfalfa plant. 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.

Hybridization with Annual Species of 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

unsuccessful (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 alfalfas 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 (USDAAPHIS, 2010). Recent research further supports that M. lupulina and M. sativa 

hybridization is unlikely (Chandra et al., 2011; Steele et al., 2010).
18

Hybridization with the Perennial Species of Medicago

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). The lack of differences between the KK179 and conventional 

control with respect to plant pest potential, described in Section VII.C. (Monsanto & FGI 

2013) includes aspects related to gene flow and introgression such as pollen, flower, and 

seed characteristics.

No perennial Medicago species are present naturally in the Americas, Australia, New 

Zealand, or South Africa (Quiros and Bauchan, 1988; USDA APHIS, 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).

APHIS evaluated the potential for hybridization and gene introgression to sexually

compatible wild (free-living) relatives, and considered whether such introgression would 

result in increased weediness. Alfalfa is sexually compatible with several subspecies 

within the M. sativa complex (Small and Jomphe, 1989). The center of origin for the 

genus Medicago is generally believed to be in the Caucasus, northwestern Iran and 

northeastern Turkey; the genus is not native in North America. An additional 18 

Medicago species are known to be naturalized (free-living) or possibly so within the 

United States, of which only M. lupulina (black medic) is widely naturalized throughout 

the United States. None of these species are native to the United States, and none are 

sexually compatible with M. sativa. The M. sativa complex, which was introduced into 

North America early by Europeans for forage and includes all the commercial alfalfa 

varieties, is a group of closely related subspecies, including the cultivated M. sativa ssp. 

sativa and M. sativa ssp. falcata (synonym M. falcata) (Small and Jomphe, 1989). 

In addition to the M. sativa complex within which all of the subspecies are sexually

compatible to some degree, an additional 17 and possibly 18 Medicago species have been 

recognized as being naturalized (free-living) or possibly so in the United States (USDANRCS, 2004; Kartesz, 2004). All of these 18 species are annual species, except for the 

species M. hybrida (in Medicago section Medicago) hybrids of which have only been 

produced experimentally by embryo culture. No annual species are known to hybridize 

with M. sativa (Quiros and Bauchan, 1988; McCoy and Bingham, 1988; (USDA APHIS 

2010). 

Medicago lupulina (black medic) is the species that might be of most concern within this 

list of 18 species. It is considered a weed in lawns and waste places and in forages since 

its seeds frequently contaminate forage legume seed crops. Black medic is an annual 

(possibly sometimes short-lived perennial) self-pollinating species and is known to occur 

throughout the United States. Successful hybridizations between M. sativa and M. 

lupulina have been reported (Southworth, 1928; Fryer, 1930; Shrock, 1943). However, 

because of the lack of hybrids after many subsequent experiments, there is general 
19

agreement that these putative “hybrids” were most likely not hybrids but due to selffertilization (Lesins and Gillies, 1972; Fridriksson and Bolton, 1963; Valizadeh et al.,

1996). APHIS’ opinion is that hybridization between Medicago sativa and M. lupulina

has an extremely low to non-existent probability of occurring in a non-experimental or 

even in an experimental setting.

APHIS concludes that the potential of KK179 low lignin trait moving to other sexually

compatible Medicago species in the United States is essentially non-existent. 

H. Potential Changes to Agriculture or Cultivation Practices

The potential of KK179 alfalfa to impact current alfalfa cultivation practices and 

management are described in Section VIII of Monsanto and FGI 2013. Implementing the 

first production strategy, the cutting schedules and the timing of harvest remains the same 

as those used with conventional alfalfa. 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. A delayed harvest 

schedule with its longer cutting intervals could potentially lower production costs over 

the life of the KK179 alfalfa stand. 

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.

 

I. Potential Impacts from Transfer of Genetic Information to 

Organism with which KK179 Alfalfa Cannot Interbreed

APHIS examined the potential for the new genetic material inserted into KK179 alfalfa to 

be transferred to other organisms without sexual reproduction (horizontal gene transfer) 

and whether such an event could lead directly or indirectly to disease, damage, injury or 

harm to plants. KK 179 alfalfa does not contain any coding sequence from plant 

pathogenic organisms. 

The horizontal gene transfer between unrelated organisms is one of the most intensively

studied fields of science. Horizontal gene transfer and expression of DNA from a plant 

species to bacteria or animal species is unlikely to occur. A number of points support this 

conclusion: 

 Many genomes (or parts thereof) from bacteria that are closely associated with 

plants have been sequenced including Agrobacterium and Rhizobium (Kaneko et 

al. 2000; Wood et al. 2001; Kaneko et al. 2002). There is no evidence that these 

organisms contain genes derived from plants. Also, Agrobacterium tumefaciens or 
20

Arthrobacter globiformis species are generally common in soil and therefore 

various epsps genes have been available for long periods of time for horizontal 

transfer from Agrobacterium tumefaciens or Arthrobacter globiformis to plants or 

other soil microorganisms and decaying plant material. Therefore the likelihood of 

any impact or new horizontal gene transfer that is not already capable of taking place 

in the soil is extremely unlikely.

 No evidence has been identified for any mechanism by which alfalfa genes could be 

transferred to humans or animals, or any evidence that such gene transfer has 

occurred for any plant species during evolutionary history, despite animals and 

humans eating large quantities of plant DNA. In cases where review of sequence data 

implied that horizontal gene transfer occurred, these events are inferred to occur on 

an evolutionary time scale on the order of millions of years (Koonin et al. 2001; 

Brown 2003). 

 Transgene DNA promoters and coding sequences are optimized for plant expression, 

not prokaryotic bacterial expression. Thus, even if horizontal gene transfer occurred, 

proteins corresponding to the transgenes are not likely to be produced. 

 FDA has evaluated horizontal gene transfer from the use of antibiotic resistance 

marker genes, and concluded that the likelihood of transfer of antibiotic resistance 

genes from plant genomes to microorganisms in the gastrointestinal tract of humans 

or animals, or in the environment, is extremely unlikely (US-FDA 1998). 

 APHIS also considered whether horizontal transfer of DNA from KK179 alfalfa to 

plant viruses was likely to occur and would lead to the creation or selection of a more 

virulent plant pathogen through recombination with other plant viruses. This issue 

has been considered before by other science review panels and government 

regulatory bodies (Keese 2008). Although sequences of the Cauliflower Mosaic Virus 

are contained within KK179 alfalfa, those sequences are limited to the regulatory 

elements. Regulatory elements such as promoters and terminators have not been 

implicated in viral recombination. 

 Finally, under natural conditions, no transfer of an intact functional gene has been 

demonstrated to date (Miki and McHugh, 2004).

Therefore, APHIS concludes that horizontal gene transfer is unlikely to occur and thus poses 

no plant pest risk.

J. Conclusion

APHIS has prepared the plant pest risk assessment in order to determine if Event KK179 

alfalfa is unlikely to pose a pest risk. Based on the information provided by the applicant 

and the from the inserted genetic material, weedy characteristics, responses to disease, 

insects or plant pests in the field, effects on non-targets or beneficial organisms in the 

agro-ecosystem, and horizontal gene transfer, APHIS has concluded that Event KK179 

low lignin alfalfa is highly unlikely to pose a plant pest risk. 
21

Based on all the above noted considerations, APHIS concludes that KK179 alfalfa poses 

no more of a plant pest risk from new gene products, changes to plant metabolism or 

composition than conventional alfalfa.

K. References

Alfalfa, Wildlife and the Environment. California Alfalfa and Forage Association (2001) 

24pgs. http://alfalfa.ucdavis.edu/-files/pdf/Alf_Wild_Env_BrochureFINAL.pdf

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