United States Department

of Agriculture

Animal and

Plant Health Inspection 

Service

November 5, 1996

Guide for

Preparing and

Submitting a

Petition for

Genetically Engineered

Plants
Contents

How To Use This Guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . iii

Sample Petition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v

Sample Transmittal Letter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii

Sample Application . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix

Application Cover Page . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xi

Table of Contents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xiii

Abbreviations and Scientific Terms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xv

Required Petition Elements

I. Rationale for Development of the Petitioned Crop . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

II. The Biology of the Petitioned Crop . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

III. Description of Transformation System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

IV. Donor Genes and Regulatory Sequences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

V. Genetic Analysis and Agronomic Performance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

VI. Environmental Consequences of Introduction of Transformed Cultivar . . . . . . . . . . . . . . . . . . . . . 15

VII. Adverse Consequences of New Cultivar Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

VIII. References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
How to use this guide—

When an applicant has field tested a transgenic crop and accumulated enough data to

show that it is free from any risk under 7 CFR 340, the applicant can petition the Animal

and Plant Health Inspection Service (APHIS), U.S. Department of Agriculture that a

transgenic crop should no longer be considered a regulated article. The pages that

follow represent a suggested format for submission of field test data to APHIS for

deregulation of a transgenic crop.

Comments and issues that may need to be addressed by the applicant are in italics in the

left margin adjacent to the portions of the sample permit to which they pertain. 

APHIS took certain liberties in preparing the sample information and data presented in

this publication. With the agronomic performance data and certain of the molecular

biology data, material was condensed to ensure that the guide was a reasonable length

and that the reader would be able to ascertain the kind of information APHIS expects to

be provided in a petition.
Sample Petition
Sample transmittal letter for petition for determination of regulatory status

U.S. Department of Agriculture

Animal and Plant Health Inspection Service

4700 River Road, Unit 147

Riverdale, MD 20782

PETITION FOR DETERMINATION OF REGULATORY STATUS

Enclosed is a copy of a petition for determination on the

regulatory status of Gossypium hirsutum L. cultivar “Banjaran,”

which has been modified to be resistant to the herbicide

glyphosate, which is currently deemed a “regulated article”. 

Based on the data and information contained in the enclosed

petition, we believe that there is no longer “reason to believe”

that the modified cotton plant should be deemed to be a regulated

article. The modified cotton plant does not present a plant pest

risk and is not otherwise deleterious to the environment. The

enclosed petition does not contain confidential business

information. 

The undersigned certifies that, to the best of his/her knowledge

and belief, this petition includes all data, information, and

views relevant to the matter, whether favorable or unfavorable to

the position of the undersigned, which is the subject of the

petition.

/s/Terry Smith

1234 Main Street

American Star, Inc.

Biotechnologies, Ltd.

Detroit, Michigan 46666

313–555–1212 VOICE

313–555–2121 FAX
Sample Application
Application for Determination of Nonregulatory

Status for Banjaran:

The Glyphosate-Tolerant Cotton
Table of Contents

Abbreviations and Scientific Terms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xv

I. Rationale for Development of Banjaran . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

II. The Cotton Family . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

A. Cotton as a Crop . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

B. Taxonomy of Cotton . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

C. Genetics of Cotton . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

D. Pollination of Cotton . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

E. Weediness of Cotton . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

F. Modes of Gene Escape in Cotton . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

G. Characteristics of the Nontransformed Cultivar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

III. Description of Transformation System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

IV. Donor Genes and Regulatory Sequences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

A. 5-Enolpyruvyl-3-Phosphoshikimate Synthase Gene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

B. Neomycin Phosphotransferase Gene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

V. Genetic Analysis and Agronomic Performance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

A. Southern Gel Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

B. Mendelian Inheritance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

C. Expression of Inserted Genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

D. Disease and Pest Resistance Characteristics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

E. Mycotoxins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

F. Gossypol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

G. Characteristics of Glyphosate-Tolerant Cotton . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

VI. Environmental Consequences of Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

A. The Herbicide Glyphosate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

B. Current Uses of Herbicides on Cotton . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

C. Banjaran: The Glyphosate-Tolerant Cotton . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

D. Appearance of Glyphosate-Resistant Weeds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

E. Weediness of Banjaran . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

F. Vertical Transfer of the New Genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

G. Horizontal Transfer of the New Genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

VII. Adverse Consequences of Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

VIII. References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
Abbreviations and Scientific Terms

Cyanazine (herbicide) Bladex®

Dinoseb an insecticide

Diuron (herbicide) Karmex®

DMSA (herbicide) monosodium methylarsonic acid

DSMA (herbicide) disodium methylarsonic acid

EPSP synthase 5-enolpyruvyl-3-phosphoshikimate

synthase 

Fluazifop (herbicide) Fusalide®

Fluometuron (herbicide) Cotoran®

Glyphosate (herbicide) Roundup®

IPM Integrated Pest Management

Linuron (herbicide) Lorox®

NOS nopaline synthase

NPT II neomycin phosphotransferase

Prometryn (herbicide) Caparol®

Sethoxydim (herbicide) Poast®

Ti plasmid tumor-inducing plasmid

Tn5 transposon 5

Triazine a class of herbicides

Trifluralin (herbicide) Treflan®

I. Rationale for Development of Banjaran

American Star Biotechnologies, Inc. has developed genetically

transformed cotton plants that are tolerant to the herbicide

glyphosate. The major weed pests of cotton in the Southern

United States include morning glories, cocklebur, pigweeds,

johnsongrass, nutsedges, prickly sida, and bermuda grass. The

development of glyphosate-tolerant cotton will allow producers

the option of applying glyphosate postemergence in an overthe-top application or replacing preemergence herbicides under

appropriate conditions. Introduction of these plants will offer

producers several advantages: several toxic herbicides,

including arsenic compounds can be replaced with a herbicide

that is more benign to the environment; glyphosate-tolerant

cotton would be compatible with IPM schemes; and glyphosate

does not have carryover problems and restrictions on

application that some currently approved cotton herbicides

have. No increase of the proportion of cotton acreage treated

with herbicide is possible because herbicides are currently used

on 99 percent of the acreage.

II. The Cotton Family

A. Cotton as a Crop

Four species of the genus Gossypium are known as cotton,

which is grown primarily for the seed hairs that are made into

textiles. Cotton is predominant as a textile fiber because the

mature dry hairs twist in such a way that they can be spun into

fine, strong threads. Other products, such as cottonseed oil,

meal, and cotton linters are byproducts of fiber production.

Cotton, a perennial plant cultivated as an annual, is grown in

the United States mostly in areas from Virginia southward and

westward to California in an area often referred to as the

Cotton Belt (McGregor, 1976).

B. Taxonomy of Cotton

The genus Gossypium, a member of the Malvaceae, consists of

39 species, 4 of which are generally cultivated (Fryxell, 1984). 

SAMPLE PETITION—Herbicide-Tolerant Plants 1
The most commonly cultivated species is G. hirsutum L. Other

cultivated species are G. arboreum L., G. barbadense L., and

G. herbaceum L.

Four species of Gossypium occur in the United States (Fryxell,

1979; Kartesz and Kartesz, 1980). G. hirsutum is the primary

cultivated cotton. G. barbadense is also cultivated. The other

two species, G. thurberi Todaro and G. tomentosum Nuttall ex

Seemann, are wild plants of Arizona and Hawaii, respectively. 

G. tomentosum is known from a few isolated locations very

close to the ocean.

C. Genetics of Cotton

At least seven complete sets of genes, designated A, B, C, D, E,

F, and G, are found in the genus (Endrizzi, 1984). Diploid

species (2n=26) are found on all continents, and a few are of

some agricultural importance. The A genome is restricted in

diploids to two species (G. arboreum and G. herbaceum) of

the Old World. The D genome is restricted in diploids to some

species of the New World, such as G. thurberi. 

By far the most important agricultural cottons are G. hirsutum

and G. barbadense. These are both allotetraploids of New

World origin and presumably resulted from an ancient cross

between Old World A genomes and New World D genomes. 

How and when the original crosses occurred are speculative. 

Euploids of these plants have 52 somatic chromosomes and are

frequently designated as AADD. Four additional New World

allotetraploids occur in the genus, including G. tomentosum,

the native of Hawaii. G. tomentosum has been crossed with G.

hirsutum in breeding programs.

The New World allotetraploids are peculiar in the genus

because the species, at least in their wild forms, grow near the

ocean as invaders in the constantly disturbed habitats of strand

and associated environs. It is from these “weedy” or invader

species that the cultivated cottons developed (Fryxell, 1979).

2 SAMPLE PETITION—Herbicide-Tolerant Plants
D. Pollination of Cotton

Gossypium hirsutum is generally self-pollinating, but in the

presence of suitable insect pollinators it can cross-pollinate. 

Bumble bees (Bombus spp.), Melissodes bees, and honey bees

(Apis mellifera) are the primary pollinators (McGregor, 1976). 

Concentration of suitable pollinators varies from location to

location and by season, and is considerably suppressed by

herbicide use. If suitable bee pollinators are present,

distribution of pollen decreases considerably with increasing

distance. McGregor (1976) reported results from an experiment

in which a cotton field was surrounded by a large number of

honeybee colonies, and movement of pollen was traced by

means of fluorescent particles. At 150 to 200 feet from the

source plants, 1.6 percent of the flowers showed the presence

of the particles. The isolation distance for Foundation,

Registered, and Certified seeds in 7 CFR Part 201 are 1,320,

1,320, and 660 feet, respectively.

Unlike G. hirsutum, G. tomentosum seems to be pollinated by

lepidopterans, presumably moths (Fryxell, 1979). The stigma in

G. tomentosum is elongated, so that the plant seems incapable

of self-pollination until acted upon by an insect pollinator. The

flowers are unusual, too, because they stay open at night; most

Gossypium flowers are ephemeral—they open in the morning

and wither at the end of the same day. 

E. Weediness of Cotton

Although the New World allotetraploids show some tendencies

to “weediness” (Fryxell, 1979), the genus shows no aggressive,

weedy tendencies in the South. Cotton is a poor competitor in

most of the southern U.S. cotton-growing regions and is not

allowed to overwinter. In more northerly areas where freezing

conditions occur, the cotton plant cannot overwinter, and there

is essentially no volunteerism from seed.

F. Modes of Gene Escape in Cotton

Genetic material of G. hirsutum may escape from a planting

site by vegetative material, by seed, or by pollen.

SAMPLE PETITION—Herbicide-Tolerant Plants 3
Description of the biology of the nonmodified recipient

organism should include taxonomy, genetics,

pollination, evidence of reported weediness (e.g., noting

whether the crop or sexually compatible species is listed

in the relevant publications of the Weed Society of

America), and discussion of sexual compatibility with

wild and weedy free-living relatives in natural crosses or

crosses with human intervention. The applicant should

provide the source of recipient (cultivar name or

accession number) and the weed status of its sexually

compatible relatives.

Vegetative propagation is not a common mechanism by which

cotton reproduces. Movement of genetic material by pollen is

possible only to those plants with the proper chromosomal

type, in this instance only to those allotetraploids with AADD

genomes. In the United States this group would include only

the cultivated species G. hirsutum, G. barbadense, and the wild

species G. tomentosum. G. thurberi, the native diploid from

Arizona with a DD genome, is not a suitable recipient. 

Movement to G. hirsutum and G. barbadense is possible if

suitable insect pollinators are present and if there is a short

distance from transgenic plants to recipient plants. Physical

barriers, intermediate pollinator-attractive plants, and other

temporal or biological impediments would reduce the potential

for pollen movement.

The applicant should explicitly identify the lines to be

considered in the petition and the cultivars from which

they are derived. If there are multiple lines, each line

must be given a unique identifier and listed in the

application. For virus-resistant plants, applicants should

provide in an additional section the following

information on the nature of the virus that provided the

sequences encoding the resistance phenotype:

i) the taxonomic name of the virus including family,

genus, and strain designation including any synonyms;

ii) the type of nucleic acid contained in the virus;

iii) whether the infection is systemic or tissue specific;

iv) whether the virus is associated with any satellite or

helper viruses; v) the natural host range of the virus; vi)

how the virus is transmitted; vii) if transmitted by a

vector, the identity of the vector including mode of

transmission (e.g., persistent or nonpersistent);

viii) whether any synergistic or transcapsidation

interactions with other viruses under field situations have

been reported in the literature, and situations have been

reported in the literature; and ix) the location and the

name of the host from which the plant the virus was

originally isolated.

The above information can be provided in a table

format (see Table 1). This information can be

supplemented by listing references that report the host

range, insect vectors, etc., for the virus.

Movement of genetic material to G. tomentosum is less well

documented. The plants are chromosomally compatible with

G. hirsutum, but there is some doubt as to the possibility for

pollination. The flowers of G. tomentosum seem to be

pollinated by moths, not bees, and the flowers are receptive at

night, not in the day. Both these factors would seem to

minimize the possibility of cross-pollination. However, Fryxell

(1979) reports that G. tomentosum may be losing its genetic

identity from introgression hybridization of cultivated cottons

by unknown means.

G. Characteristics of Nontransformed Cultivar

G. hirsutum L. cv. “Stoneville 825” is the cultivar that we

genetically transformed. This cultivar is widely grown in the

United States and was specially developed for introduction in

the Mississippi Delta region. American Star Inc. has received a

U.S. patent on the specific herbicide tolerant gene that has

been transformed into this cultivar, and the transformed cultivar

has received additional protection under the Plant Variety

Protection Act of 1970. American Star Inc. intends to introduce

the new traits into other cotton cultivars by traditional breeding

techniques. The cultivar that has been transformed to be

herbicide tolerant is called “Banjaran.”

4 SAMPLE PETITION—Herbicide-Tolerant Plants
For Agrobacterium-based transformation protocol, the

applicant must indicate how Ti plasmid based vector

was disarmed (i.e., all tumorigenic DNA was removed). 

Applicants can provide citations that describes the

transformation procedure. However, any significant

modifications of transformation, strain designation, etc.

should be described.

For other methods of transformation, the applicant can

describe the sources of various components of the

plasmid (or other DNA including possible carrier DNA)

and method of transformation by citation. However,

any significant modifications in transformation, tissue

regeneration, etc. should be described.

III. Description of Transformation System

The vector system (pVST1) used to transfer the genes to cotton

plants is based on the Ti plasmid from Agrobacterium

tumefaciens (Fig. 1). The DNA becomes incorporated into the

plant cell chromosome (Klein et at., 1989). Although some of

the DNA sequences used in the transformation process were

derived from the plant pathogen A. tumefaciens, the genes

which cause crown gall disease were first removed, and

therefore the recipient plant does not have crown gall disease

(Zambryski, 1988; Klee and Rogers, 1989). Once the

introduced genes are inserted into the chromosome of the

recipient, they are maintained in the same manner as any other

genes.

The T-DNA, which includes the glyphosate resistance and nptII

genes, was transferred into the genomes of individual cotton

hypocotyl sections as described by Umbeck et al. (1987). 

Plants were regenerated as described by Trolinder and Goodlin

(1987).

The applicant must provide a detailed restriction map of

the plasmid that is sufficient to be used in the analysis

of Southern data. Description of added restriction sites is

helpful in interpretation of Southern data and should be

provided (see Fig. 1).

BamH1 BamH1 RI RI PstI PstI

↓ ↓ ↓ ↓ ↓ ↓

RB nos5' nptII nos3' CaMV35s aroA nos3' LB amp ori lac

Fig 1—pVST1: A linear schematic illustration of the plasmid vector (7.8kb)

that was conjugated into Agrobacterium tumefaciens (Malik and Wahab, 1993).

Various components of pVST1 have been described in Table 1. Map reflects

the gene order but not the sizes. The plasmid does not have restriction sites

for SpI and NdeI, but has one site for SmaI and NotI.

SAMPLE PETITION—Herbicide-Tolerant Plants 5
Table 1—Summary of DNA components in pVSTI

GENETIC

ELEMENT

SOURCE FUNCTION

RB A. tumefaciens Right border of the Ti plasmid of A.

tumefaciens LB 444 (White, 1992)

nos 5' Ti plasmid of A.

tumefaciens

Promoter for transcription of the

nptII gene (White, 1992)

nptII E. coli Neomycin phosphotransferase type

II enzyme that confers resistance to

kanamycin and allows for selection

in plant cells (Beck et al., 1982)

nos 3' A. tumefaciens A 3' nontranslated region of the

nopaline synthase gene involved in

transcription termination and

polyadenylation (Depicker et al.,

1982)

CaMV35s Cauliflower mosaic virus Promoter that directs transcription

of cryIA(B) gene (Odell et al.,

1982) 

aroA Salmonella typhimurium 5-Enolpyruvyl-3-Phosphoshikimate

Synthase Gene (Pittard, 1987)

nos 3' see above see above

LB A. tumefaciens Left border of the Ti plasmid of A.

tumefaciens (White, 1992)

amp E. coli Resistance to ampicillin expressed

only in E. coli (White, 1992)

Ori Col E1 plasmid of E. coli The origin of replication for the

PUC plasmids that allows for

plasmid replication in E. coli (Vieira

and Messing, 1987)

lac E. coli β-galactosidase of E. coli (White,

1992)

Indicate the functions of the gene(s), promoters,

leader sequences, enhancers, introns, and any other

sequences that are used for gene expression in the

plant and a reference describing from where the

sequences were obtained. Discussion should include

whether the inserted sequences are responsible for

disease or injury to plants or other organisms. The

nucleotide sequence(s) of the plant-expressed gene(s)

should be provided by citation and not submitted

in the application. If there has been a significant

modification to sequences and the modified sequence has not been published, provide the complete

sequence highlighting the modifications. If there

have been minor modifications to the sequence of

the plant-expressed gene, they should be provided. 

For example, if in a chemically synthesized Bt gene

amino acid 23 was changed from methionine to

alanine it should be stated without providing the

complete sequence.

6 SAMPLE PETITION—Herbicide-Tolerant Plants
IV. Donor Genes and Regulatory Sequences

A. 5–Enolpyruvyl Shikimate Phosphate (EPSP) Synthase Gene

The donor organism used to supply the glyphosate tolerance

gene, aroA, was the bacterium Salmonella typhimurium. S.

typhimurium is a well-characterized enteric bacterium with

homology to E. coli (Ochman and Wilson, 1987). Some strains

of S. typhimurium are known to cause a disease in susceptible

mice and humans, but there is no evidence that strains of the

bacterium are plant pests (Le Minor, 198l). Salmonella species

may be associated with vegetation as free-living organisms

whenever these plants have been contaminated with fertilizers

of fecal origin or when they have been irrigated with polluted

water. Salmonella organisms do not seem to multiply

significantly in the natural environment (outside of digestive

tracts), but they can survive several weeks in water and several

years in soil if the conditions of temperature, humidity, and pH

are favorable (Delage, 1960).

The aroA gene, which encodes the sequence for the enzyme

EPSP synthase, has no known inherent plant-pest characteristics

nor direct involvement in human and animal disease. EPSP

synthase is one of the enzymes in the biosynthetic pathway

leading to chorismate, an intermediate in the formation of

aromatic amino acids and their derivatives (Pittard, 1987). This

pathway is found in most plants, many single-celled organisms,

and some lower forms of animals. The aroA gene is

constitutively expressed in both E. coli (Tribe et al., 1976) and

S. typhimurium (Gollub et al., 1983). The gene was mutated in

the bacterium to provide glyphosate resistance by the classical

genetic techniques of mutation and selection (Comai et al.,

1983). The description of the modified aroA gene, including

the method of isolation and complete gene sequence, is

contained in two published papers (Comai et al., 1983 and

Stalker et al., 1985). The EPSP synthase gene was fused to the

CaMV35s promoter and NOS termination/polyadenylation

sequences as previously described (White, 1992). Confirmation

that this gene was indeed inserted into the cotton chromosome

was provided by the following: Southern gel analysis, 

Mendelian inheritance, measurement of expression levels of

SAMPLE PETITION—Herbicide-Tolerant Plants 7
gene product, and demonstration that the whole plant is

resistant to applied glyphosate (see subsequent sections).

B. The Selectable Marker Gene: Neomycin Phosphotransferase

In addition to the aroA gene, the nptII gene from transposon

Tn5 of the bacterium E. coli has been introduced in cotton to

be used as a selectable marker. This gene codes for the

enzyme neomycin phosphotransferase which confers resistance

to the common aminoglycoside antibiotic kanamycin (Fraley et

al., 1986). The DNA sequence of the gene has been

determined (Beck et al., 1982). The lack of risk to humans of

the nptII gene can be supported by its use in the first human

gene therapy trials (Anonymous, 1990). The nptII coding

region is under the control of the nos promoter and nos

terminator. 

None of the introduced genes has any inherent plant pest

characteristics or poses a risk to plant health when introduced

into the modified plants.

8 SAMPLE PETITION—Herbicide-Tolerant Plants
V. Genetic Analysis and Agronomic Performance

A. Southern Gel Analysis

In general, it is always prudent to analyze data

statistically when such analysis is possible. When

unpublished information or an opinion has been

supplied by a scientific expert, a letter communicating

the information should be included in the petition. If

the unpublished information provided is data resulting

from scientific research then these data can be provided

as a personal communication either in a letter from the

researcher or in the text of the petition. In either case

the materials and methods, data analysis, and

discussion of the data analysis should be provided in

detail. Unsupported assertions about the results of the

experiment are not acceptable.

Applicants must report any differences noted between

transgenic and nontransgenic plants that are not directly

attributed to the expected phenotype. Differences observed

could include changes in leaf morphology, pollen

viability, seed germination rates, changes in

overwintering capabilities, insect susceptibilities, diseases

resistance, yield, agronomic performance etc. Applicants

must also note the types of characteristics that were

compared between transgenic and nontransgenic plants

and found to be unchanged. 

The applicant should describe whether data submitted

are from inbred or hybrid plants; if hybrid plants, state

which generation.

State whether data with respect to plant performance

were generated in a greenhouse or field environment. 

If from the field, indicate how many sites, states and

number of years the data represents.

Seed germination, seed dormancy, seed production,

growth rate, and other data relating to the plant’s

performance will be required when the nature of the

gene and the biology of the plant (including sexuallycompatible relatives) warrant such data. This type of

data will usually not be required for plants that have

some of the following attributes: are highly domesticated

(e.g., corn), are exclusively self-pollinating (e.g.,

soybean), are male sterile, and have high seed

germination rates (>90%), and whose phenotypes are

unlikely to affect performance with respect to weediness

or fitness (e.g., delayed ripening or oil seed

modification). Phenotypes that might require

performance data (depending on the plant) include but

are not limited to the following: cold tolerance, salt

tolerance and tolerance or resistance to other biotic or

abiotic stresses.

The identity of the genetic material that was integrated into the

genome of the transgenic plant was probed by Southern

hybridization. In order to determine the number of insertion

events, the DNA from the transgenic regulated plant and the

parental Stoneville 825 recipient lines were digested with the

restriction enzyme Nde1, which does not cleave within the

pVST1 DNA. The hybridizations done with three probes

indicated that the foreign DNA integrated at one site to yield

the transgenic plant. This is supported by the presence of one

hybridizing fragment of 20 kb that is present in the transgenic

DNA but is absent from the parent.

The following restriction fragments were labeled and

hybridized to the Southern blots:

1. a 300 bp EcoR 1 fragment of the aroA gene

2. a 400 bp BamH 1 fragment of the nptII gene

3. a 950 bp Pst 1 fragment with the amp and lac genes

that are not expressed in plants and should not be

integrated into the plant

Hybridization analysis of genomic DNA was performed

following the method of Firoozabady et al. (1987). The results

are shown in figure 2 for the aroA, nptII and Amp marker

genes. Both aroA and nptII probes hybridize to the 20Kb

fragment in lane 1. The data support the Mendelian results

(shown below) that only one expressed copy of the aroA gene

is present in the engineered cultivar and that a single copy of

the npt II marker gene is present. No hybridization with the

ampicillin or lac probe was detected.

Southern blot analysis supports the conclusion that the

amp+lac sequences which lie outside the Ti plasmid left and

right borders, were not integrated in to the genome of

Banjaran, while the sequences inside the Ti left and right

borders were.

SAMPLE PETITION—Herbicide-Tolerant Plants 9
Southern analysis should include DNA isolated from

nonmodified recipient, all or selected transformed lines,

and the vector. Parental plasmid DNA (eg PUC 18) not

containing intended donor genes may be labeled and

hybridized to Southern blots to demonstrate that only the

intended sequences have been incorporated in the

genome of the transgenic plant. Restriction enzymes to

be used might include enzymes that do not cut within

the transforming plasmid but will cut the entire insert

into one fragment from the DNA of the transgenic plant.

In the case of an Agrobacterium-based transformation

system, the applicant should determine if genes that

reside outside the LB/RB are inserted in the genome of

the regulated cultivar. If a complete copy of any of

these genes is present, the applicant should determine

whether it is expressed in the plant. For direct

transformation systems, applicants should determine

which sequences are inserted in transgenic plants and

whether they are expressed. PCR analysis may be used

to prove that only the targeted DNA has been

incorporated. Sequencing of the transgene in plant and

adjacent sequences is not required. Determination of

the number of copies of integrated transgenes is not

required, but the number of insertions may be used to

support analysis of inheritance data.

Probed for aroA Probed for nptII Probed for Ampand lac Molecular Weight

markers

Lane

number 1 2 3 1 2 3 1 2 3 4

 20 Kb

 10 Kb

 5 Kb

Fig. 2—Southern blot analysis of DNA: Detection of aroA and nptII genes

in the Banjaran. Line lanes 1 and 2 contain 10 μg DNA of Banjaran and

Stoneville 825 DNA respectively digested with NdeI. Lane 3 is Sal1

digested pUC 18 DNA as a positive control for amp and lac sequences. 

Lane 4 is a HindIII digest of phage lambda DNA, used as molecular weight

markers. All four DNA samples were electrophoresed on three different

1% agarose gels and transferred to nitrocellulose filters. One

nitrocellulose filter was hybridized with 32P-labeled AroA probe, a second

filter with the nptII probe and a third one with the amp+lac probe. A

similar hybridization pattern was obtained when Southern blots were

hybridized with either AroA or nptII probes. The PstI fragment (amp+lac)

hybridized to pUC 18 DNA but not to the plant genomic DNA. This data is

consistent with the presence of a single insertion of the genes between the

LB and RB incorporated in the genome of the transgenic plant.

B. Mendelian Inheritance

The primary transformants that expressed the NPT II marker

gene and the EPSP synthase gene were allowed to selfpollinate, and the seeds were collected. These seeds (T1

) were

planted in a single 25-foot row. Seedlings were thinned to a

density of two plants per foot. Seedlings were sprayed with

one application of glyphosate at a rate of 8 oz/acre. Symptoms

of bleaching or necrosis appeared 8 to 10 days after application

and were compared to the symptoms of nontransformed plants

that received an identical herbicide application. The number of

resistant and sensitive plants in three separate rows was

counted (Table 2). If primary transformants were expressing

the EPSP gene from a single insertion site (genetic loci), the

expected segregation would be 3:1. The segregation ratio

totals for all 3 rows was 110:40, which fits the Mendelian

prediction of 1121⁄2:371⁄2 (χ

2 = 0.22) well. The nontransformed

plants were fully susceptible to glyphosate.

10 SAMPLE PETITION—Herbicide-Tolerant Plants
If the inserted DNA sequence order is complex, as is

often the case for plants engineered via direct

transformation systems (e.g. electroporation, polyethylene

glycol transformation of protoplasts, or particle

bombardment techniques), the applicants should

summarize the data by providing the following

information for the all genes (whether under the

direction of plant or bacterial promoters). Is there a

complete copy of the gene present in regulated article? 

Is the protein expressed in the plant? If multiple

complete copies of a gene are present, applicants do not

have to determine if each copy of the gene is expressed. 

It is very helpful to provide a table, like the one shown

below, that summarizes the results and indicates where

specific data is to be found.

Is a complete Is protein expressed Location of

Gene copy present? in plant? detailed data

ß-lactamase yes no Fig. 3, p. 7

EPSP synthase yes yes Fig. 4, p. 20

nptII yes yes Fig. 8, p. 21

chloramphenicol no — Fig. 9, p. 31

 acetyltransferase

Mendelian inheritance data and Chi square analysis for

at least 2 generations are appropriate to demonstrate

whether the transgene is stably inserted and inherited in

Mendelian fashion. Such data are generally not

necessary for infertile vegetatively propagated crops such

as male-sterile potatoes.

Table 2—Segregation ratios of progeny of the

seeds of the primary transformants

Ratio of Herbicide 

Tolerant to Susceptible Plants

Variety Observed Expected χ

2

Banjaran 36:14 371⁄2:121⁄2 0.24

Banjaran 39:11 371⁄2:121⁄2 0.24

Banjaran 35:15 371⁄2:121⁄2 0.67

Stoneville 825 0:50 0:50 —

SAMPLE PETITION—Herbicide-Tolerant Plants 11
RNA—Northern analysis is generally not required except

for virus-resistant plants. However, such analysis may

be necessary for ribozyme, truncated sense, or antisense

constructs, when protein levels can not be provided.

PROTEINS—Expression levels of gene(s) of interest and

marker genes in various tissues, developmental stages of

plant, and experimental conditions (induced or

noninduced) are required. Assays can be of enzyme

activity. Serology, ELISA, and Western blots may also be

used. Describing the source of the immunogen is

critical for serological analysis.

For virus resistant plants, the amount of viral transgene

RNA produced should be determined and compared to

the amount of the RNA produced by the viral gene in

an infected nontransgenic plant. Applicants should

address whether the transgene RNA (or protein) is

present in the same tissues as are infected during

natural infections. In addition, provide the amount of

both coat proteins (i.e. from the transgene and the

naturally infecting virus) produced in the transgenic

plant singly infected with the widely prevalent viruses in

the U.S. that normally infect the recipient plant (contact

APHIS for the list of these viruses). For comparison,

provide the amount of both coat proteins produced in

the nonengineered plant in mixed infections of the virus

from which the coat protein gene was derived and the

same widely prevalent viruses used in the single

infection study. Provide description of symptoms of

infected plants in all cases

.

C. Expression of Inserted Genes

The production of the desired proteins in Banjaran was

confirmed by immunoassay. Banjaran has been modified by

the insertion of the aroA gene imparting the glyphosate

resistance trait. In addition to glyphosate resistance, Banjaran

expresses the selectable marker protein, nptII. As measured

by immunoassay, the aroA and nptII proteins were expressed

at low and relatively consistent levels in Banjaran across all

field sites (see data reports 94–000–02, 94–000–03). The mean

from all tests reported in 10 states at 50 sites showed that

Banjaran contained in leaf and seed tissue respectively

approximately 12.6 μg and 17.3 μg 5-enolpyruvyl-3-shikimate

phosphate synthase protein/gram fresh weight of tissue (fwt),

and 6.9 and 3.3 μg nptII protein/gram fwt. At the one field site

at which expression was evaluated over time, the EPSP protein

levels varied less than five fold in young leaf tissue collected

over the growing season with the highest levels observed early

in the season (see data report 94-000-05).

The above data was based on leaf and seed. However, the

levels of EPSP and nptII proteins in whole plant tissue were

much lower, on a fresh weight basis, than in leaf or seed

tissues. EPSP is present in whole Banjaran plants from 1.1 to

1.7 μg/g fwt of the whole plant respectively; NPTII protein

levels are 14.6 μg/g/fwt for Banjaran plants. These measured

concentrations were used to estimate the amount of EPSP and

NPTII protein that could enter the environment due to postharvest incorporation of 5000 mature Banjaran plants into the

soil. Predicted values are 23.4 g EPSP protein/acre and 183 g

NPTII protein/acre, Nectar and pollen collected from Banjaran

contain very low levels of EPSP protein. The expression of

EPSP protein in pollen collected from Banjaran greenhousegrown plants were 37.8 ng/g fresh wt, respectively. The EPSP

protein levels in nectar were 0.88 ng/g fresh wt collected from

Banjaran plants. Thus, pollen and nectar produced by

Banjaran present a low source of potential EPSP protein

exposure to nontarget organisms.

12 SAMPLE PETITION—Herbicide-Tolerant Plants
For all diseases and pathogens surveyed, names of the

diseases and the scientific names of the pathogens should

be provided. Data from field tests in foreign countries

are acceptable. If the data on diseases and pests were

obtained in the foreign country, the applicant should

submit information about the distribution of those pests,

disease or pathogens in U.S.. Disease and pathogen

susceptibility on wild type and transgenic plants should

be determined preferably from natural infestations. 

However, if applicant must use direct inoculations; i.e.,

with virus resistant transgenic plants, the source and

taxonomic classification of the virus should be provided.

D. Disease and Pest Resistance Characteristics 

The transformed cotton plants were field-tested for 3 years at

ten sites in five States (see data reports 93–1, 94–1, 95–1 in

appendix). Based on field observation at these sites, pathogensusceptibility or resistance characteristics of the transformed

cultivar were unchanged when compared to those of the

nontransformed cultivar. The transformed cultivar remains

resistant or tolerant to bacterial blight (Xanthomonas campestris

pv. malvacearum), Anthracnose boll rot, and Fusarium

wilt–nematode complex rot. The transformed cultivar remains

susceptible to Alternaria leaf spot, Cercospora leaf spot, and

powdery mildew, as was the nontransformed cultivar.

E. Mycotoxins

Aflatoxins are most commonly found in food and feed

commodities contaminated by Aspergillus flavus. Aflatoxins are

the only contaminants of feeds and food routinely monitored. 

Banjaran was not any more susceptible to mold infection than

its parent cultivar and was not going to be a source of

mycotoxins.

F. Gossypol

Gossypol is a yellow pigment that occurs in various parts of the

cotton plant. Cotton seed usually contains 0.4 percent to 1.7

percent gossypol (Abou-Donia, 1976). When present in

untreated cottonseed meal, gossypol is toxic to animals. When

cottonseed meal is processed under heat and moisture, most of

the free gossypol is removed by solvent extraction or detoxified

by the condensation of the aldehyde groups of gossypol with

the free amino groups of proteins to form nonextractable

(bound) gossypol. Flavanoids which are not major constituents

in cotton were also measured because they can be toxic if

eaten in large amounts. The amount of free or bound gossypol

in the meal did not differ significantly between the transformed

and nontransformed cultivars (Table 2).

SAMPLE PETITION—Herbicide-Tolerant Plants 13
Certain plants have minute quantities of known

toxicants which may adversely impact nontarget

organisms and beneficial insects; e.g., tomatine in

tomatoes, cucurbitin in cucurbits, gossypol in cotton etc. 

If such plants are recipients of transgenes, the applicant

should provide information as to whether the level of

toxicants is altered. If the plant produces no known

toxicant, the applicant should state so and provide the

reference to support the claim. Plant toxins can be

assessed by the tests and criteria that plant breeders

traditionally use in the crop. In some instances, this

may be done qualitatively, e.g. taste testing of cucurbits.

Table 2—Mean toxicant content in cottonseed of transformed and

nontransformed cultivars grown at four sites

Quality factors1Banjaran Line 825

Free gossypol 0.75a 0.75a

Total gossypol 1.0a 1.0a

Flavonoids 1.84a 1.76a

Free and total gossypol and flavonoids are given as percent of kernel weight

assayed according to the methods of Cherry (1983) and Hedin (1988)

respectively. Within each column, means followed by the same letter are not

significantly different according to the Newman-Kreuls multiple range test.

G. Characteristics of Glyphosate-Tolerant Cotton

We determined that the minimum level of glyphosate needed

to control morning glory, cotton’s major weed pest, was 8

oz/acre. At this level, the glyphosate-tolerant cotton was

undamaged by the herbicide. This concentration is generally

also adequate for the control of Morningglory, Common

cocklebur, Pigweed, Johnsongrass, Nutsedges and

Bermudagrass which are all important weeds in cotton

cultivation.

Glyphosate-tolerant cotton is still susceptible to two other

broad-spectrum herbicides, sulfonylurea and bromoxynil, as is

its progenitor cultivar. Thus, the transformed cultivar can be

eliminated using herbicides with a different mode of action

from glyphosate if that is desired.

14 SAMPLE PETITION—Herbicide-Tolerant Plants
VI. Environmental Consequences of Introduction of the

Transformed Cultivar

A. The Herbicide Glyphosate

N-(phosphonomethyl)glycerine (glyphosate) is an

extremely effective broad-spectrum herbicide. The

primary mode of action of the herbicide appears to be

competitive inhibition of 5-enolpyruvylshikimic acid-3-

phosphate (ESPS) synthase, an enzyme in the shikimic

acid pathway of aromatic amino acid biosynthesis. 

Glyphosate provides effective control for the majority of

the world’s worst weeds. It is translocated in the plant

via both phloem and xylem. The broad-spectrum

herbicidal activity is evident only when glyphosate is

applied to foliage because there is little penetration of

bark or woody stems (Franz 1983). Glyphosate becomes

nontoxic upon contacting soil. Its degradation appears to

be mainly microbial. Glyphosate is essentially nontoxic

to mammals and birds (Anonymous 1983). Environmental

impact studies indicate that the herbicide has little direct

effect on animal communities (Sullivan and Sullivan 1979,

1981, 1982). However, some bird communities may show

decreased population densities due to destruction of

habitat caused by use of the herbicide (Morrison and

Meslow 1984). Fish and invertebrates are more sensitive

to the herbicide, especially to the commercial

formulations, as a result of the surfactants in the

formulation (Anonymous 1983). Effects of the herbicide

on soil invertebrates in field situations appear to be minor

(Eijsackers 1985). Although there are numerous reports

of the effects of glyphosate on microbial respiration,

nitrogen cycling, and cellulolytic activity in soils, no

significant effects on any of these microbial processes

should be observed at recommended field application

rate of the herbicide (Carlisle and Trevors 1988). There

have been no reports of groundwater contamination

problems (Goldburg et al. 1990).

SAMPLE PETITION—Herbicide-Tolerant Plants 15
B. Current Uses of Herbicides on Cotton

Glyphosate is generally used as a foliar-applied herbicide. 

It is most effective for the control of perennial weeds. It

is usually applied before planting to kill winter weeds or

used as a spot spray at any time throughout the growing

season. Glyphosate is also used for destroying weeds

growing adjacent to the field.

Herbicides are applied to cotton before planting (to

preplant weed foliage or in soil-incorporated applications), at planting (preemergence applications), or after

seedlings emerge (postemergence directed or over-thetop). Herbicides were used on 99 percent of the cotton

acreage (2.7 million acres) in the Delta region of the

United States in 1990, on average from 3.6 to 4.1

treatments per acre. The severe weed pressure in the

Delta is demonstrated by the large proportion of the

cotton acreage receiving three or more herbicide treatments per season. At least one-quarter of the acreage in

the Delta is treated with arsenic-based herbicides (DMSA

or MSMA), singly or in combination with other herbicides. 

The total amount of arsenic-based herbicides applied to

cotton in 1990 was approximately 3.5 million pounds.

Several postemergence herbicides are registered for use in

cotton. They are usually applied when the plants are 3 to

6 inches high. These herbicides include diuron, fluometuron, prometryn, and cyanazine, to which MSMA or

DMSA is added to broaden the spectrum. One additional

application of the mixture is often made during the

season since there is a limit of two applications of the

arsenical herbicides. Late postemergence herbicides are

sometimes applied at or near the time of the last

cultivation (“layby”). Direct applications are usually

placed between the rows, in order to maintain cotton

seed quality. A few over-the-top herbicides are available. 

The two used are sethoxydim and fluazifop. Both are

specific for control of grass weeds and have little effect

on broadleaf plants. Before the introduction of

sethoxydim and fluazifop, glyphosate was used to control

grasses. Because glyphosate is nonselective, special

16SAMPLE PETITION—Herbicide-Tolerant Plants
application methods were devised.

In the various cotton-growing regions of the country,

cotton producers manage weeds differently. The

following summarizes typical practices for the mid-South

region (Frans and Chandler 1989).

1. Disk twice and broadcast and incorporate trifluralin

before planting.

2. At planting, apply fluometuron preemergence on

bands.

3. Cultivate and postdirect fluometuron plus MSMA

on bands.

4. Cultivate and postdirect prometryn plus MSMA on

bands.

5. Spot spray with fluazifop.

6. Cultivate and postdirect cyanazine on bands.

7. Hand hoe, cultivate, and postdirect dinoseb on

bands.

Recently yields of cotton lint have declined, and

continued herbicide use is strongly implicated, especially

where cotton is grown continuously and the same

herbicides are applied yearly (Frans and Chandler, 1989). 

Rogers et al. (1985, 1986) summarized results from a longterm experiment in which herbicides were applied to

cotton at different levels for 6 to 7 years. No reduction in

cotton yield occurred following continuous use of a

minimum set of herbicide practices. When intensive

practices were used (trifluralin preplant incorporated,

fluometuron preemergence, two postemergence directed

applications of fluometuron plus MSMA, and linuron

applied at last cultivation), yields dropped on average up

to 8 percent. Of the rotation crops planted on these

areas, corn and sorghum suffered the least damage while

soybeans and rice were severely injured.

Herbicide residues in the cotton crop have also been a

concern, especially those of organic arsenicals. Both

DMSA and MSMA are used postemergence for control of

SAMPLE PETITION—Herbicide-Tolerant Plants 17
grasses. Although most producers apply arsenicals in a

directed manner, some apply them over the top. In the

latter case, there is the possibility of high residue levels

occurring in cotton, especially if applications are made

during the early reproductive stage of cotton growth and

if there are multiple applications (Frans and Chandler

1989).

C. Banjaran: The Glyphosate-Tolerant Cotton

Environmentally desirable feature of the introduction of

glyphosate-tolerant cotton include

It offers producers the option of replacing with

glyphosate several herbicide combinations that

include arsenical compounds.

Glyphosate is less likely to lead to the

development of resistant weeds than many other

herbicides (Benbrook 1991).

The introduction of glyphosate-tolerant cotton is

compatible with IPM. Producers could apply the

herbicide only if needed, thus reducing the use of

preemergence herbicides.

The most damaging components of glyphosate are

its inert components (Goldburg et al., 1990). 

Recently, the Environmental Protection Agency has

given glyphosate an “E” carcinogenicity

(noncarcinogen) rating.

Glyphosate does not have carryover problems.

The introduction of glyphosate-tolerant cotton

could aid in the development of minimum-till

practices that would result in reduced soil erosion.

 

Glyphosate-tolerant cotton could enable producers to

apply herbicide on an as-needed basis, a key principle of

all IPM systems. If a farmer planted a field with a

herbicide-tolerant variety, the farmer could cut back the

initial herbicide application or try to control weeds with

mechanical cultivation. If chemical weed control became

necessary, herbicide could be applied over the top to the

entire field or by spot application in the areas of field

where weeds were threatening.

18SAMPLE PETITION—Herbicide-Tolerant Plants
D. Appearance of Glyphosate-Resistant Weeds

A decade ago, herbicide-resistant weeds were virtually

unknown. Today there are some 109 herbicide-resistant

weed biotypes and more than half of them are resistant to

triazine (LeBaron, 1991). Certain herbicide characteristics

and application regimes favor the development of resistant weeds: a single target site and a specific mode of

action, broad spectrum of activity, long residual activity

the capacity to control weeds through out the year, and

frequent applications without rotation to other herbicides

or cultural control practices. Current application data suggest that glyphosate is unlikely to engender the development of resistance in target vegetation (Benbrook 1991).

E. Weediness of Banjaran

Will the introduction of the herbicide-tolerance genes to a

plant increase its weediness? Exactly which characters

define a weed is debatable, but a general consensus of

the traits shared by many weeds was developed by Baker

(1974). They include (1) the ability to germinate in many

different environments; (2) discontinuous germination and

great longevity of seed; (3) rapid growth through vegetative phase to flowering; (4) continuous seed production

for as long as growing conditions permit; (5) selfcompatibility but partially autogamous and apometic;

(6) ability to be cross-pollinated by unspecialized visitors

or wind-pollinated; (7) high seed output in favorable

environments and some seed production in a wide range

of environments; (8) adaption for short and long-distance

dispersal; (9) vegetative production or regeneration from

fragments and brittleness (hard to remove from the

ground); and (10) ability to compete interspecifically by

special means (e.g., rosette formation and presence of

allelochemicals). Weeds need not have all these characteristics to be successful. G. hirsutum cv. Stoneville 825

that was genetically transformed is not considered a weed

and has few of the 10 weedy traits. Introduction of the

herbicide tolerance gene into this cultivar did not significantly change its weedy characteristics. No change was

noted with transformed cultivar in the number of seeds

SAMPLE PETITION—Herbicide-Tolerant Plants 19
produced, germination and overwintering characteristics

of seeds, or the number of days from planting until first

boll production or flowering. The herbicide-tolerant

cultivar’s sensitivity to all commonly used, registered

cotton herbicides was not altered except for glyphosate

tolerance.

F. Vertical Transfer of the New Genes

It is apparent from the data that outcrossing from the

transformed cultivar to other domestic cottons does and

will occur. Of course, this kind of gene transfer happens

in nature constantly. Because cotton producers purchase

new seed every year, the cross-pollination phenomenon

does not have a significant impact on the quality or

nature of seed produced in a field where cross pollination

has occurred. Seeds from all transgenic cotton cultivars

will still have to meet existing certification requirements

for cotton seed production.

The noncultivated Gossypium spp. found in the 

Southwestern United States and Hawaii are not

considered weeds, and introgression of the new genes

into these species would not significantly increase any of

the 10 characteristics of weeds unless selection pressure

favors these characteristics. With regard to glyphosatetolerant cotton, we believe that introgression of this trait

into noncultivated Gossypium spp. would not be highly

favored in the absence of herbicide application. 

Herbicide application is likely only in agricultural settings,

not in wild stands. In addition, in Hawaii, where G.

tomentosum is found, cotton is not commercially

produced. The great majority of cotton grown in that

State is in experimental plots where cotton breeding

programs operate. Cotton breeders generally bag or clip

the flowers when performing crosses between plants. 

This practice significantly reduces the chance that flowers

will be visited by pollinating insects and thus reduces the

likelihood of gene movement. 

20 SAMPLE PETITION—Herbicide-Tolerant Plants
G. Horizontal Transfer of the New Genes

Nonsexual, horizontal transfer of transgenes from

genetically engineered plants into other organisms is not

well documented and is difficult to measure (Harding,

1995). Horizontal gene transfer of transgenes from higher

transgenic plants via the soil to a soil microorganism (the

filamentous fungus Aspergillus niger), however, has been

reported (Hoffmann et al., 1994). Genetic transfer across

taxa of eukaryotes is suggested in only a few cases

(Lewin, 1982; Houck et al., 1991), and of these the only

one suggesting a transfer, even over evolutionary time

scale (excepting Agrobacterium) from unrelated taxa to

higher plants is with the case of vertebrate hemoglobin

and legume hemoglobin (Wiborg et al., 1983). During

the field testing of these plants, there was no evidence of

horizontal transfer of the transformed genes to adjacent

nonsexually compatible plants. This observation is based

on sensitivity to glyphosate of weeds in the nearby fields.

Assuming that the levels of known toxicants in the

regulated article reported in Section V are in acceptable

range; that there were no notable differences reported in

Section V between transgenic and nontransgenic plant;

and that the gene(s) engineered into the recipient plant

have no known reported toxic properties; then,

toxicological data on effects of the plant on nontarget

organisms and threatened and endangered species will

usually not be required

.

VII. Adverse Consequences: New Cultivar Introduction

With respect to the herbicide-tolerant cotton, the use of

glyphosate may increase if the transgenic cultivar is widely

accepted by farmers. The increased use of glyphosate will be

offset by the decreased use of organic arsenate-based

herbicides used in conjunction with other herbicides.

The example given does not refer to a plant with a new

pesticidal phenotype. For such plants, however applicants

should indicate to APHIS whether they have applied for or

been granted registration of the pesticide with the

Environmental Protection Agency.

Applicants should also consult with APHIS on data the agency

would deem to be appropriate and sufficient to demonstrate no

significant impact on threatened and endangered species and

beneficial nontarget organisms. This data should be submitted

in the appendix of the petition application. However, brief

summaries of data should appear in the petition application. 

SAMPLE PETITION—Herbicide-Tolerant Plants 21
Below is an example of an acceptable summary of a test of BT

cotton pollen on a beneficial nontarget organism.

A separate petition should be submitted for each

category/phenotype combination. For example, a

petition for Coleopteran insect-resistant potatoes or PVYresistant potatoes should be submitted separately. 

However, when a single plant contains more than one

phenotype modification, submit only one petition. For

example, one petition should be submitted for potatoes

that are both PVY and PVX resistant.

Springborn Laboratories, Inc. (Wareham, MA) conducted a 48-

hour static-renewal test with Bt cotton pollen (homozygous for

the cryIA(b) gene) and isogenic pollen on Daphnia magna,

according to EPA Guideline No. 72-2. Details of these studies

have been submitted in the registration package to the USEPA

(see appendix). Daphnids were <24 hours old at the time of

study initiation. For the definitive test, dose levels of 19, 32,

54, 90, and 150 mg pollen/L (containing 5.87 mg CryIA(b)/g

pollen) were employed. These levels are a hundredfold higher

than the LC50 for target insects. In addition, isogenic controls at

the same pollen concentrations as the treatment group were

tested along with a negative control group. Each test or control

concentration consisted of two replicates of 10 daphnids each

for a total of 20 daphnids/ concentration or control group. 

Daphnids were exposed for 48 hours with complete renewal of

the test solutions after 24 hours.

Mean survival was 100 percent for each of the transgenic,

isogenic, and negative control groups. All daphnids in the

transgenic, isogenic, and negative control groups appeared

normal during the study. No immobilization or sublethal signs

of toxicity were observed. The only effect noted was a

decrease in dissolved oxygen in the higher test concentrations

of both pollen groups. Dissolved oxygen concentrations were

inversely related to the concentration of pollen tested and were

similar in equivalent concentrations of the transgenic and

isogenic groups. The decrease in dissolved oxygen had no

effect on the survival of the daphnids. Higher concentrations

for both types of pollen were cloudy and some daphnids were

observed to be coated with pollen. At 48 hours, the EC50 based

on immobilization was >150 mg pollen/L for both the

transgenic and isogenic groups. Based on these results, the

NOEC was 150 mg transgenic or isogenic pollen/L (the highest

concentration tested).

22 SAMPLE PETITION—Herbicide-Tolerant Plants
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