GM Crop Database

Database Product Description

CBH-351 (ACS-ZMØØ4-3)

Host Organism: Zea mays (Maize)

Trade Name: StarLink™

Trait: Resistance to European corn borer (Ostrinia nubilalis); phosphinothricin (PPT) herbicide tolerance, specifically glufosinate ammonium.

Trait Introduction: Microparticle bombardment of plant cells or tissue

Proposed Use: Production for livestock feed and products for industrial applications.

Product Developer: Aventis CropScience

Summary of Regulatory Approvals

Country	Food	Feed	Environment	Notes
United States		1998	1998

Introduction Expand: Maize line CBH-351 (tradename StarLink) was developed through a specific genetic modification to be resistant to attack by European corn borer (ECB; Ostrinia nubilalis), a major insect pest of maize in agriculture. These novel plants contain a modified cry9C gene from Bacillus thuringiensis subsp. tolworthi strain BTS02618A. CBH-351 maize is also genetically modified to express the bar gene cloned from the soil bacterium Streptomyces hygroscopicus, which encodes a phosphinothricin-N-acetyltransferase (PAT) enzyme. The PAT enzyme is useful as a selectable marker enabling identification of transformed plant cells as well as a source of resistance to the herbicide phosphinothricin (also known as glufosinate ammonium, the active ingredient in the herbicides Basta, Rely, Finale, and Liberty). PAT catalyses the acetylation of phosphinothricin and thus detoxifies phosphinothricin, eliminating its herbicidal activity. Delta-endotoxins such as the Cry9C protein expressed by CBH-351, protects the maize plants against feeding damage of larvae of the lepidopteran insect European corn borer by selectively binding to specific sites localized on the brush border midgut epithelium of susceptible insect species. Following binding, cation-specific pores are formed that disrupt midgut ion flow and thereby cause paralysis and death. Cry9C protein is insecticidal only to lepidopteran insects, and its specificity of action is directly attributable to the presence of specific binding sites in the target insects. There are no binding sites for delta-endotoxins of B. thuringiensis on the surface of mammalian intestinal cells, therefore, livestock animals and humans are not susceptible to these proteins.

Summary of Introduced Genetic Elements Expand

Code	Name	Type	Promoter, other	Terminator	Copies	Form
bar	phosphinothricin N-acetyltransferase	HT	CaMV 35S	A. tumefaciens nopaline synthase (nos) 3'-untranslated region	>=4
bla	beta lactamase	SM	bacterial promoter			Not expressed in plant tissues
cry9c	cry9c delta-endotoxin	IR	CaMV 35S ; leader sequence of the cab22L gene of Petunia hybridia (chloroplast transit peptide)	CaMV 35S poly(A) signal	>=1	Truncated N-,C-terminal

Characteristics of Zea mays (Maize) Expand

Center of Origin	Reproduction	Toxins	Allergenicity
Mesoamerican region, now Mexico and Central America	Cross-pollination via wind-borne pollen is limited, pollen viability is about 30 minutes. Hybridization reported with teosinte species and rarely with members of the genus Tripsacum.	No endogenous toxins or significant levels of antinutritional factors.	Although some reported cases of maize allergy, protein(s) responsible have not been identified.

Donor Organism Characteristics Expand

Latin Name	Gene	Pathogenicity
Streptomyces hygroscopicus	bar	S. hygroscopicus is ubiquitous in the soil and there have been no reports of adverse affects on humans, animals, or plants.
Bacillus thuringiensis subsp. tolworthi	cry9c	While target insects are susceptible to oral doses of Bt proteins, no evidence of acute toxicity in laboratory mammals, birds, or non-target beneficial insects has been reported. The Cry9C protein is resistant to heat and proteolytic degradation and may have allergenic potential

Modification Method Expand: CBH-351 maize was produced by biolistic transformation of the backcrossed hybrid maize line (PA91 x H99) x H99 with two pUC19 based plasmids, PRSVA9909 and pDE110. Both plasmids contained the modified cry9C gene and the bar gene, respectively, engineered for enhanced expression in plants. The cry9C and bar genes were fused to noncoding regulatory sequences that enabled them to be expressed at high levels, constitutively throughout most of the plant. Specifically, the expression of the modified cry9C gene was regulated by the promoter and terminator sequences from the 35S transcript of CaMV, along with the leader sequence of the cab22L gene from petunia. The expression of the bar gene was also directed by the 35S CaMV promoter along with the 3' untranslated region from the nopaline synthase (nos) gene from A. tumefaciens which is involved in transcription termination and polyadenylation. Although most of these regulatory regions were derived from plant pathogens, the regulatory sequences cannot cause plant disease by themselves or with the genes that they are designed to regulate. Additional genetic elements present on the transforming plasmids included the ampicillin resistance gene beta-lactamase (bla) and the origin of replication (ori) both from the enteric bacterium, Escherichia coli. Both were introduced into CBH-351 maize, however these elements are nonfunctional in plants. The bla gene was present on the plasmids only as a selectable marker to detect transformed E. coli host bacteria.

Characteristics of the Modification Expand

The Introduced DNA

The cry9C gene was synthesized with plant preferred codons before it was stably inserted into maize plants to produce a truncated and modified Cry9C protein. The CBH-351 expressed Cry9C protein was similar to the trypsin-resistant core protein produced by B. thuringiensis, with the exception of a single amino acid substitution in the internal sequence and the addition of two extra amino acids to the N-terminus.

The modifications that have been introduced into the Cry9C protein expressed in CBH-351 maize, as compared to the wild type 129.8 kDa Cry9C protoxin include the following:

C-terminal truncation that removes all the amino acids (aa) following position 666 of the wild type protoxin just after the conserved sequence block 5 shared by certain Lepidoperta-active Bt Cry proteins;
N-terminal truncation that removes the first 43 aa at the point corresponding to the N-terminus of the 68.7 kDa fragment of the wild type toxin produced by initial trypsin-digestion, and the subsequent N-terminal addition of the amino acids methionine and alanine;
Replacement of arginine by lysine at position 123 in the plant encoded protein which reduces the susceptibility of the protein to trypsin cleavage to a non-toxic 55 kDa fragment.

These modifications did not appear to affect the insecticidal activity of the Cry9C protein against the primary targeted insect, European corn borer (ECB), Ostrinia nubilalis.

Genetic Stability of the Introduced Trait

Molecular and biochemical analyses presented for maize line CBH-351 demonstrated that the number of insertion sites, copy number, and expression level were stably inherited.

Environmental Safety Considerations Expand

Field Testing

Field testing has demonstrated that CBH-351 maize plants are well protected from European corn borer and exhibit tolerance to glufosinate ammonium herbicides at concentrations that provide effective weed control. CBH-351 maize has undergone field testing in a wide variety of locations, in 31 States and territories of the United States since 1995, and in Canada, Belgium, France, Chile and Argentina. This field testing was conducted, in part, to confirm that CBH-351 maize exhibits the desired agronomic characteristics and to demonstrate that CBH-351 maize did not pose a plant pest risk. The kernel composition, quality and other characteristics of CBH-351 maize were found to be similar to non-transgenic maize and should have no adverse impact on raw or processed agricultural commodities.

Outcrossing

Since pollen production and viability were unchanged by the genetic modification resulting in CBH-351, pollen dispersal by wind and outcrossing frequency should be no different than for other maize varieties. Gene exchange between CBH-351 maize and other cultivated maize varieties will be similar to that which occurs naturally between cultivated maize varieties at the present time. In North America, where there are few plant species closely-related to maize in the wild, the risk of gene flow to other species appears remote. Cultivated maize, or maize, Zea mays L. subsp. mays, is sexually compatible with other members of the genus Zea, and to a much lesser degree with members of the genus Tripsacum.

Wild diploid and tetraploid members of Zea collectively referred to as teosinte are normally confined to Mexico, Guatemala, and Nicaragua; however, a fairly rare, sparsely dispersed feral population of teosinte has been reported in Florida. All teosinte members can be crossed with cultivated maize to produce fertile F1 hybrids. Nonetheless, in the wild, introgressive hybridization from maize to teosinte is currently limited, in part, by several factors including distribution, genetic incompatibility, differences in flowering time, developmental morphology, dissemination, and dormancy. First-generation hybrids are generally less fit for survival and dissemination in the wild, and show substantially reduced reproductive capacity which acts as a significant constraint on introgression.

In Central America, many of these species occur where maize might be cultivated, however, gene introgression from CBH-351 maize under natural conditions is highly unlikely. It is further unlikely that potential introgression of European corn borer resistance or glufosinate tolerance traits from CBH-351 maize would cause teosinte to become more weedy in the absence of glufosinate herbicide selection.

The genus Tripsacum contains up to 16 recognized species, most of which are native to Mexico, Central and South America, but three of which exist as wild and/or cultivated species in the U.S. Hybrids of Tripsacum species with Zea are difficult to obtain outside of a laboratory and are often sterile or have greatly reduced fertility, and none are able to withstand even the mildest winters. Furthermore, none of the sexually compatible relatives of maize in the U.S. are considered to be weeds in the U.S. therefore it is unlikely that introgression of the bar gene would provide a selective advantage to these populations as they would not be routinely subject to herbicide treatments.

Weediness Potential

No competitive advantage was conferred to CHB-531 maize, other than that conferred by resistance to European corn borer and herbicide tolerance to glufosinate. Maize is not a weed, and resistance to European corn borer or herbicide tolerance will not render maize weedy or invasive of natural habitats since none of the reproductive or growth characteristics were modified. Potential introgression from CBH-351 maize into wild relatives is not likely to increase the weediness potential of any resulting progeny nor adversely effect genetic diversity of related plants any more than would introgression from traditional maize hybrids.

Cultivated maize is unlikely to establish in non-cropped habitats and there have been no reports of maize surviving as a weed. Maize volunteers are not uncommon but are easily controlled by mechanical or by using herbicides that are not based on glufosinate as appropriate. Zea mays is not invasive and is a weak competitor with very limited seed dispersal.

Secondary and Non-Target Adverse Effects

The history of use and literature suggest that the bacterial Bt protein is not toxic to humans, other vertebrates, and beneficial insects. This protein is active only against specific lepidopteran insects and no lepidopteran species are listed as threatened or endangered species in North America. The Cry9C toxin is also toxic against other Lepidoptera, including members of the families Pyralidae, Plutellidae, Sphingidae, and Noctuidae.

Studies were conductive on several nontarget organisms to determine the potential toxic effects of Cry9C protein on test organisms including adult honeybees, a predator ladybird beetle (H. convergens), juveniles of the soil-dwelling invertebrate Collembola (springtails) (Folsomia candida), earthworms, juveniles of the freshwater invertebrate Daphnia magna, Northern bobwhite chicks, and mice. The Cry9C was expressed in either whole plant powder or pollen derived from CBH-351 maize plants; or as purified from a Cry-minus B.t. bacterial strain engineered to express the protein toxin. Control maize plant powder and pollen test substances lacking insecticidal activity (as assayed against the European corn borer) were used in these studies to determine whether effects were specific to the CBH-351 transformation event. No effects on these organisms were detected during any of these studies that could be related to the presence of the insecticidal Cry9C protein in CBH-351 maize.

Small scale field studies conducted in 1996 in Johnston, Iowa demonstrated that the number of predators observed on plots planted to either CBH-351 maize or a non-transformed counterpart did not show a significantly different pattern among plots. In addition, the diversity of predators observed in both types of plots was equal.

In summary, it was concluded that cultivation of CBH-351 maize should not have a significant potential to harm nontarget and beneficial organisms common to agricultural ecosystems, nor should it significantly impact species recognized as threatened or endangered.

Impact on Biodiversity

CBH-351 maize has no novel phenotypic characteristics that would extend its use beyond the current geographic range of maize production. Since the risk of outcrossing with wild relatives in North America is remote, it was determined that the risk of transferring genetic traits from CBH-351 maize to species in unmanaged environments was insignificant.

CBH-351 maize plants are not likely to eliminate the use of chemical insecticides which are traditionally applied to about 25 to 35% of the total maize acreage planted, since the primary target for most of these applications has been the coleopteran, corn rootworm. CBH-351 maize may positively impact current agricultural practices used for insect control by 1) offering an alternative method for control of European corn borer (and potentially other Cry9C-susceptible pests of maize); 2) reducing the use of insecticides to control European corn borer and the resulting potential adverse effects of such insecticides on beneficial insects, farm worker safety, and ground water contamination; and 3) offering a new tool for managing insects that have become resistant to other insecticides currently used or expressed in maize, including other Bt-based insecticides.

CBH-351 maize exhibits tolerance to glufosinate ammonium herbicides at concentrations that provide effective weed control and excellent crop safety. CBH-351 maize, along with glufosinate ammonium herbicides, is expected to positively impact current agricultural practices used for weed control by 1) offering growers a broad spectrum, post-emergent weed control system; 2) providing the opportunity to continue to move away from pre-emergent and residually active herbicides; 3) providing a new herbicidal mode of action in maize that allows for improved management of weeds which have developed resistance to herbicides with different modes of action; and 4) decreasing cultivation needs and increasing the amount of no-till acres. Volunteers of CBH-351 can be easily controlled by selective mechanical or manual weed removal or by the use of herbicides with active ingredients other than glufosinate ammonium.

Other Considerations

In order to prolong the effectiveness of plant-expressed Bt toxins, and the microbial spray formulations of these same toxins, regulatory authorities in Canada and U.S. have required developers to implement specific Insect Resistant Management (IRM) Programs. These programs are mandatory for all transgenic Bt-expressing plants, including CBH-351 maize, and require that growers plant a certain percentage of their acreage to non-transgenic varieties in order to reduce the potential for selecting Bt-resistant insect populations. Details on the specific design and requirements of individual IRM programs are published by the relevant regulatory authority.

Food and/or Feed Safety Considerations Expand

Maize line CBH-351 has only been approved in the USA for livestock feed use. The safety for use in animal feed was supported by the lack of toxicity of bacterial-expressed Cry9C protein in high oral dose feeding studies with laboratory animals, the low potential for toxicity of plant-expressed Cry9C protein demonstrated by a lack of amino acid sequence homology with known protein toxins and rapid digestion in simulated gastric juices.

Nutritional Data

Kernel composition, quality and other characteristics of CBH-351 maize were not significantly different from non-transgenic maize.

Toxicity

The low potential for toxicity of plant-expressed Cry9C protein was demonstrated by a lack of amino acid sequence homology with known protein toxins and the lack of toxicity in high oral dose feeding studies with laboratory animals.
A high-dose acute oral toxicity study was done to assess the potential mammalian toxicity to Cry9C protein present in CBH-351. Bacterial expressed protein was used in these studies because insufficient amounts could be purified from plant tissue. Data demonstrating the molecular equivalence of bacterial and plant-expressed Cry9C protein were provided.

An oral dose of the tryptic core Cry9C protein of at least 3,760 mg/kg was administered to 10 animals without mortality, demonstrating a high degree of safety for the protein. Transient weight losses were seen in three female treated rodents, with one not recovering her pre-dosing, pre-fast weight at 14 days after dose administration. The treated males showed no weight losses. Transient weight loss has been observed in similar studies conducted on other purified Cry proteins as well as microbial pesticides containing Cry proteins and is not considered a significant adverse effect.

As there was no indication of mammalian toxicity to the Cry9C protein from the studies submitted, there was no reason to believe there would be cumulative toxic effects on adults as well as on infants and children from residues of Cry9C and other substances with a common mechanism of toxicity.

Allergenicity

The Cry9C protein was evaluated for potential allergenicity by examining the amino acid sequence homology to known protein allergens, digestibility, and the history of safe use of microbial insecticides containing this protein.

A search for amino acid homology between the Cry9C protein and the amino acid sequences of known toxins or allergens, using the databases PIR, Swiss-Prot and HIV AA, did not reveal any significant homology matches.

The digestibility of Cry9C protein was determined experimentally using an in vitro digestibility study. Results showed the Cry9C protein to be stable to pepsin digestion at pH 2.0 for 4 hours. The Cry9C protein was also found to be heat stable, not being affected by incubation at 90C for 10 minutes. The Cry9C protein contains a trypsin resistant core and is therefore stable to tryptic digest. Digestibility study showed the Cry9C protein to be stable to pepsin at pH 2.0.

The conclusion of the FIFRA Scientific Advisory Panel Report (December 2000) was that there is a medium likelihood that the Cry9C protein is a potential allergen based on the biochemical properties of Cry9C protein itself – not its levels in the food supply. Relative to the characteristics of known food allergens, there is no evidence disproving the potential allergenicity of Cry9C.

Abstract Collapse

Maize (Zea mays L.), or corn, is grown primarily for its kernel, which is largely refined into products used in a wide range of food, medical, and industrial goods.

Only a small amount of whole maize kernel is consumed by humans. Maize oil is extracted from the germ of the maize kernel and maize is also a raw material in the manufacture of starch. A complex refining process converts the majority of this starch into sweeteners, syrups and fermentation products, including ethanol. Refined maize products, sweeteners, starch, and oil are abundant in processed foods such as breakfast cereals, dairy goods, and chewing gum.
In the United States and Canada maize is typically used as animal feed, with roughly 70% of the crop fed to livestock, although an increasing amount is being used for the production of ethanol. The entire maize plant, the kernels, and several refined products such as glutens and steep liquor, are used in animal feeds. Silage made from the whole maize plant makes up 10-12% of the annual corn acreage, and is a major ruminant feedstuff. Livestock that feed on maize include cattle, pigs, poultry, sheep, goats, fish and companion animals.

Industrial uses for maize products include recycled paper, paints, cosmetics, car parts and pharmaceuticals.

The European corn borer (ECB), Ostrinia nubilalis, is the most damaging insect pest of maize in the United States and Canada; losses resulting from ECB damage and control costs exceed $1 billion each year. An average of one ECB cavity per maize stalk across an entire field can reduce yield by as much as 5% when caused by first generation larvae, and 2.5% when caused by second generation larvae, with annual yield losses estimated at 5 to 10 %.

Despite consistent losses to ECB, chemical insecticides are utilized on a relatively small acreage (less than 20%). Historically, this reluctance stems from the difficulties in identifying and managing ECB in maize crops: ECB larval damage is hidden, heavy infestations are unpredictable, insecticides are costly, timing of insecticide application is difficult and multiple applications may be required to guarantee ECB control.

Weeds are also a major production problem in maize cultivation. Even a light infestation of weeds can reduce yields by 10 to 15%; severe infestations can reduce yields by 50% or more. Typically, weeds are managed using a combination of cultural (e.g., seed bed preparation, clean seed, variety selection) and chemical controls. Depending on the production area and the prevalent weed species, herbicides may be incorporated into the soil before planting (pre-plant), applied after planting but before emergence (pre-emergence), or applied after the maize plants emerge (post-emergence). Ideally, for maize production, weeds should be controlled for the full season. However, the most critical period for weed control is usually about six to eight weeks after crop emergence, during the 4th to 10th leaf stages. This critical period in the life cycle of maize must be kept weed free in order to prevent yield loss.

Maize line CBH-351 was genetically modified to contain two novel genes, cry9C and bar, for insect and herbicide tolerance respectively. Both genes were introduced into a maize line by particle acceleration (biolistic) transformation.
The transgenic maize line CBH-351 was developed to resist ECB by producing its own insecticide. This event was genetically engineered by introducing the cry9C gene, isolated from the common soil bacterium Bacillus thuringiensis (Bt), into a maize line by particle acceleration (biolistic) transformation. The cry9C gene produces the insect control protein Cry9C, a delta-endotoxin. Cry proteins, of which Cry9C is only one, act by selectively binding to specific sites localized on the lining of the midgut of susceptible insect species. Following binding, pores are formed that disrupt midgut ion flow, causing gut paralysis and eventual death due to bacterial sepsis. Cry9C is lethal only when eaten by the larvae of lepidopteran insects (moths and butterflies), and its specificity of action is directly attributable to the presence of specific binding sites in the target insects. There are no binding sites for the delta-endotoxins of B. thuringiensis on the surface of mammalian intestinal cells, therefore, livestock animals and humans are not susceptible to these proteins.

In addition to the cry9c gene, CBH-351 was developed to allow for the use of glufosinate ammonium, the active ingredient in phosphinothricin herbicides (Basta®, Rely®, Liberty®, and Finale®), as a weed control option, and as a breeding tool for selecting plants containing the cry9c gene. CBH-351 contains the bar gene isolated from a common soil actinomycete, Streptomyces hygroscopicus. This gene allows for the production of the enzyme phosphinothricin N-acetyltransferase (PAT) which confers tolerance to glufosinate.

The PAT enzyme in maize line CBH-351 converts L-phosphinothricin (PPT), the active ingredient in glufosinate ammonium, to an inactive form, thereby conferring resistance to the herbicide. In the absence of PAT, application of glufosinate leads to reduced production of the amino acid glutamine and increased ammonia levels in the plant tissues, resulting in the death of the plant. The PAT enzyme is not known to have any toxic properties.

Maize line CBH-351 was tested in field trials in the United States and Canada, beginning in 1995. Data collected from these trials demonstrated that CBH-351 was not different from conventional maize varieties. Agronomic characteristics and kernel traits were within the expected range of expression reported for commercial maize hybrids. Maize line CBH-351 was comparable to conventional maize lines and did not exhibit weedy characteristics, or negatively affect beneficial or nontarget organisms. CBH-351 was not expected to impact on threatened or endangered species.

Maize does not have any closely related species growing in the wild in the continental United States or Canada. Cultivated maize can naturally cross with annual teosinte (Zea mays ssp. mexicana) when grown in close proximity, however, these wild maize relatives are native to Central America and are not naturalized in North America. Additionally, reproductive and growth characteristics were unchanged in maize line CBH-351. Gene exchange between CBH-351 and maize relatives was determined to be negligible in managed ecosystems, with no potential for transfer to wild species in Canada and the United States.

Regulatory authorities in Canada and the United States have mandatory requirements for developers of Bt maize to implement specific Insect Resistant Management (IRM) Programs. The potential for ECB populations to develop tolerance or become resistant to the Bt toxin is expected to increase as more maize acreage is planted with Bt hybrids. These IRM programs are designed to reduce the potential development of Bt-resistant insect populations, as well as prolonging the effectiveness of plant-expressed Bt toxins, and the microbial Bt spray formulations of these same toxins.

Kernel composition, quality and other characteristics of CBH-351 maize were not significantly different from non-transgenic maize.

CBH-351 maize has been approved in the United States only for livestock feed use. The safety for use in animal feed was supported by the lack of toxicity of bacterial-expressed Cry9C protein in high oral dose feeding studies with laboratory animals and the low potential for toxicity of plant-expressed Cry9C protein demonstrated by a lack of amino acid sequence homology with known protein toxins.

The allergenic potential of the Cry9C protein was assessed by examining the amino acid sequence homology between the Cry9C protein and known protein allergens. A database search determined that there were no significant similarities in the amino acid sequences for the Cry9C protein and known protein allergens. A pepsin digestibility study determined that the Cry9C protein was stable to digestion for up to 4 hours and was heat stable when incubated at 90C for 10 minutes.

The Environmental Protection Agency (EPA) ruled, in the pesticide assessment of Cry9C in maize line CBH-351, that the use of maize line CBH-351 be restricted to animal feed only. The EPA also established that it was acceptable to find the Cry9C protein in meat, poultry, milk, or eggs derived from animals fed CBH-351 maize feed. Following authorization of CBH-351 for use in animal feed, the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA) Scientific Advisory Panel (SAP) was tasked with reviewing scientific information concerning the allergenic potential of the Cry9C protein. In the SAP Report (December 2000) it was concluded that there was a medium likelihood that the Cry9C protein was a potential allergen.

Links to Further Information Expand

Aventis CropScience USA

Updated safety information on Cry9C protein. Submitted to the U.S. Environmental Protection Agency on 24 October 2000.
[PDF Size: 604.58K bytes]

FIFRA Scientific Advisory Panel: Assessment of Scientific Information Concerning StarLink™ Corn

Report of the FIFRA Scientific Advisory Panel Meeting, November 28, 2000, held at the Holiday Inn Rosslyn Hotel. A Set of Scientific Issues Being Considered by the US Environmental Protection Agency Regarding the Scientific Safety Assessment of StarLink Corn.
[PDF Size: 80.62K bytes]

Impact of Bt corn pollen on monarch butterfly populations: A risk assessment

Mark K. Sears, Richard L. Hellmich, Diane E. Stanley-Horn, Karen S. Oberhauser, John M. Pleasants, Heather R. Mattila, Blair D. Siegfried, and Galen P. Dively (2001). Proc. Natl. Acad. Sci. USA Early Edition
[PDF Size: 162.67K bytes]

U.S.Department of Agriculture, Animal and Plant Health Inspection Service

AgrEvo USA Company Petition for Determination of Nonregulated Status: BtCry9C Insect Resistant, Glufosinate Tolerant Corn Transformation Event CBH-351
[PDF Size: 10.14M bytes]

USDA-APHIS Environmental Assessment

Determination of Nonregulated Status for Bt Cry9C Insect Resistant and Glufosinate Tolerant Corn Transformation Event CBH-351
[PDF Size: 2.90M bytes]

This record was last modified on Friday, March 26, 2010

Product Related Info

Biology Documents

Maize biology document
Biology of Maize L.
General Description

Maize is a tall, monoecious, annual grass varying in height from 1 to 4 meters (Watson & Dallwitz 1992). The main stem is made up of clearly defined nodes and internodes. Internodes are wide at the base and gradually taper to the terminal inflorescence at the top of the plant. Leaf blades are found in an alternating pattern along the stem. Maize is a unique grass as both male and female flowers are borne on the same plant but are located separately. The tassels or staminate (male) inflorescence form large spreading terminal panicles that resemble spike-like racemes. Pollen is shed from the tassel and is viable for approximately 10 to 30 minutes as it is rapidly desiccated in the air (Kiesselbach 1980). Maize plants shed pollen for up to 14 days. The reproductive phase begins when one or two auxiliary buds, present in the leaf axils, develop and form the pistillate inflorescence or female flower (Purseglove 1972). The auxiliary bud starts the transformation to form a long ‘cob’ on which the flowers will be borne. From each flower a style begins to elongate towards the tip of the cob in preparation for fertilization. These styles form long threads, known as silks. The base of the silk is unique, as it elongates continuously until fertilization occurs (Purseglove 1972). Styles may reach a length of 30 cm, the longest known in the plant kingdom. Individual maize kernels, or fruit, are unique in that mature seed is not covered by floral bracts (glumes, lemmas, and paleas) as in most other grasses, but rather the entire structure is enclosed and protected by large modified leaf bracts, collectively referred to as the ear (Hitchcock and Chase 1951). The mature female flowers will remain ready for fertilization for up to two weeks, at which point if fertilization has not occurred, the nucleus will de-organize and fertilization will no longer be possible (Hitchcock & Chase 1951). The pollen of maize, a protandrous plant, matures before the female flower is receptive (Purseglove 1972). This may have been an ancient mechanism to ensure cross-pollination, but is no longer considered conducive to modern agricultural practices. However, decades of conventional selection and improvement have produced many maize varieties with similar maturities for both male and female flowers, to ensure seed set for agricultural proposes.

Reproduction

Under natural conditions, maize reproduces only by seed production. Pollination occurs with the transfer of pollen from the tassels to the silks of the ear. About 95% of the ovules are cross-pollinated and about 5% are self-pollinated (Poehlman 1959), although plants are completely self-compatible. Maize, as a thoroughly domesticated plant, has lost all ability to disseminate its seeds and relies entirely on the aid of man for its distribution (Stoskopf 1985). The kernels are tightly held on the cobs and if ears fall to the ground, so many competing seedlings emerge that the likelihood that any will grow to maturity is extremely low. Maize cannot reproduce asexually by natural means. It is possible to reproduce maize using tissue culture techniques, however, it has proven extremely difficult with a low rate of success (Hoisington et al. 1998).

Classification and Phylogeny of Maize

Zea is a genus belonging to the grass family, Poaceae, in the Andropogoneae tribe, but is commonly cited in literature to belong to the tribe Maydeae. Zea (zeia) was derived from an old Greek name for a food grass. The genus Zea consists of four species of which only Zea mays ssp. mays L. is economically important. The other Zea species, referred to as teosintes, are largely wild grasses native to Mexico and Central America (Doebley 1990). The number of chromosomes in Zea mays is 2n=20, 21, 22, 24 (FAO 2000c). The tribe Maydeae consists of seven genera: two of American origin, Zea and Tripsacum; and five of Eastern origin which extend from India through Southeastern Asia to Australia and include Coix, Sclerachne, Polytoca, Chionachne and Trilobachne (Watson & Dallwitz 1992). It is generally agreed that maize phylogeny was largely determined by the American genera Zea and Tripsacum, however it is accepted that the genus Coix contributed to the phylogenetic development of the species Z. mays (Radu et al. 1997). The genus Manisuris, from the tribe Andropogoneae, may also have contributed to the evolution of Zea mays (Eubanks 1997a; Radu et al. 1997).

Center of Origin and Progenitors of Maize

The center of origin for Zea mays ssp. mays has been established as the Mesoamerican region, now Mexico and Central America (Watson & Dallwitz 1992). The exact period of domestication and the ancestors from which maize arose are unclear. Archaeological records suggest that domestication of maize began at least 6000 years ago, occurring independently in regions of the southwestern United States, Mexico, and Central America (Mangelsdorf 1974). The origins of domesticated maize have been difficult to trace as hybridization events in its evolution are thought to have involved a now extinct wild maize ancestor of which little evidence has been found (Eubanks 1997a).

Germplasm Diversity

A study in 1992 concluded that maize landraces occupied 42% of the land under production in less developed countries (México 1994). Mexico and Central America are the only regions where ancient maize lines, such as pod corn, are found (Mink & Dorosh 1985). The survival of maize landraces has been attributed to the integral role maize sustains in small communities where it has very specific uses for food and other special functions. These landraces have been well characterized in Latin America, and germplasm not yet evaluated is known to be present in the region. There is a growing trend in developing countries to adopt improved maize varieties, primarily to meet market demand. In Mexico, only 20% of the corn varieties grown 50 years ago remain in cultivation (World Watch Institute 2000). The narrowing of genetic diversity in modern maize varieties emphasizes the importance of conserving genetic traits for future plant breeding. Leading the way in preserving maize germplasm is CIMMYT (International Maize and Wheat Improvement Centre), which has the world’s largest collection of maize accessions, with over 17,000 lines (CIMMYT 2000). Collections of distant maize relatives native to Asia have been recommended by conservation experts to ensure their preservation for future research (Sharma 1998).

Relatives of Maize

The closest wild relatives of maize are the teosintes which all belong to the genus Zea. The teosintes are wild grasses native to Mexico and Central America and have limited distribution (Mangelsdorf et al. 1981). Teosinte species show little tendency to spread beyond their natural range and distribution is restricted to North, Central and South America (Watson & Dallwitz 1992). The nearest teosinte relative to Z. mays ssp. mays is Z. mays ssp. mexicana (Schrader) Iltis (previously classified as Euchlaena mexicana, Zea mexicana) (2n = 20). This Central Mexican annual teosinte is a large flowered, mostly weedy grass with a broad distribution across the central highlands of Mexico. It does not spread readily. It has limited use as a forage and green fodder crop, but can be problematic due to weedy tendencies (Doebley 1990, Watson & Dallwitz 1992). The closest wild relatives to maize outside the Zea genus are from the genus Tripsacum. The genus Tripsacum is comprised of about 12 species that are mostly native to Mexico and Guatemala but are widely distributed throughout warm regions in the USA and South America, with some species present in Asia and Southeast Asia (Watson & Dallwitz 1992). All species are perennial, warm season grasses.

Outcrossing

Gene flow from maize can occur by two means: pollen transfer and seed dispersal. Seed dispersal can be readily controlled in maize as domestication has all but eliminated any seed dispersal mechanisms that ancestral maize may have previously used (Purseglove 1972). The kernels are held tightly on the cobs and if the ear falls to the ground, competing seedlings limit growth to maturity (Gould 1968). Pollen movement is the only effective means of gene escape from maize plants. Pollen is released from the tassels at the top of the plant and transported by wind to the female flowers located on the stalk. Pollen shed occurs over a 14 day period, with a peak during the first 5 days of shed (Sears 2000). The stigmas are receptive for a large part of this two week interval (Kiesselbach 1980). Insects, such as bees, have been observed to collect pollen from maize tassels, but they do not play a significant role in cross-pollination as there is no incentive to visit the female flowers (Rayor et al. 1972).

Wind speed and direction affect pollen distribution

Differences in flowering dates among commercial maize varieties are small. Cross-pollination between varieties may occur if grown in adjacent fields. The limited viability of maize pollen reduces the risk of cross-pollination, as a receptive host must be found within the 30 minutes that the pollen remains biologically active. Cross-pollination is also affected by the concentration of maize pollen released; pollen produced by a maize crop will successfully compete with foreign pollen sources when present in higher concentrations (Rayor et al. 1972). The isolation of crops using separation distances and physical barriers are common techniques for restricting gene flow and ensuring seed purity for maize seed production. Cross-pollination is controlled in seed lots by separating different lines. The OECD standards require a minimum 200 m (660 ft) isolation distance for maize seed production. This distance has been estimated to maintain inbred lines at 99.9% purity. Physical barriers, such as trees or barrier rows, are also effective in reducing cross-pollination. Detasseling or bagging the tassel effectively prevents pollen movement and limits gene flow.

Intraspecific hybidization

All forms of Zea mays spp mays, such as dent, sweet, and pop corn, freely cross pollinate forming fertile hybrids (Pursglove 1972).

Interspecific hybridization

All teosintes can be crossed to maize and form fertile hybrids, except for the tetraploid Z. perennis. Maize-teosinte hybrids exhibit low fitness and have little impact on gene introgression in subsequent generations (Galinat 1988). The tendency to form natural hybrids differs among teosintes: Z. luxurians rarely hybridizes with maize, whereas Z. mays ssp. mexicana frequently forms hybrids (Wilkes 1977). Molecular data confirms that there is gene flow between maize and teosinte and suggests that introgression of maize and teosinte occurs in both directions but at low levels (Doebley 1990).

Intergeneric hybridization Tripsacum species (T. dactyloides, T. floridanum, T. lanceolatum, and T. pilosum) have been successfully hand crossed with maize to form hybrids. The hybrids generally have 28 chromosomes, 10 from maize and 18 from Tripsacum, and are pollen sterile with limited female fertility (Mangelsdorf & Reeves 1939, Mangelsdorf 1974). This infertility is common in such wide crosses because of differences in chromosome number and lack of pairing between chromosomes (Eubanks 1997b). Maize readily crosses with hexaploid wheat (Triticum aestivum) with high frequencies of fertilization and embryo formation (plant breeders use maize pollen to develop double haploids of hexaploid wheat). However, maize chromosomes are eliminated from the genome during the initial stages of meiosis and result in haploid embryos. There is little evidence to suggest fertile hybrids between maize and hexaploid wheat could be produced in nature. There have been unsubstantiated reports of hybridization between maize and sugar cane (Saccaharum sp.).

Biology References
1. CIMMYT (International Maize and Wheat Improvement Centre) (2000). CGIAR Research, Areas of Research: Maize (Zea mays L.) http://www.cgiar.org/areas/maize.htm
2. Doebley, J. F. (1990). Molecular Evidence for Gene Flow among Zea species. BioScience 40, 443- 448.
3. Eubanks, M.W. (1997a). Further evidence for two progenitors in the origin of maize. Maize Newsletter 71, 35-35.
4. FAO (2000c). Species Description. Zea mays L. http://www.fao.org/WAICENT/faoinfo/agricult/agp/agpc/doc/gbase/data/pf000342.htm
5. Galinat, W.C. (1988). The origin of corn. In: Corn and Corn Improvement. Agronomy Monographs No.18. American Society of Agronomy, G.F Sprague and J.W. Dudley, (eds.). Madison, Wisconsin, pp. 1-31.
6. Gould, F.W. (1968). Grass Systematics. McGraw Hill, N.Y., USA.
7. Hitchcock, A.S. & A. Chase. (1951). Manual of the Grasses of the United States (Volume 2). Dover Publications: N.Y. p. 790-796.
8. Hoisington, D., Listman, G.M. & Morris, M.L. (1998). Varietal development: applied biotechnology. In: Maize Seed Industries in Developing Countries. M. L. Morris (ed). Lynne Rienner Publishers, Inc. pp. 77-102.
9. Kiesselbach, T.A. (1980). The structure and reproduction of corn. Reprint of: Research Bulletin No. 161. 1949. Agricultural Experiment Station, Lincoln, Nebraska. University of Nebraska Press. p. 93.
10. Laurie, D.A. & Bennett, M.D. (1986). Wheat x maize hybridization. Can. J. Genet. Cytol. 28, 313-316.
11. Mangelsdorf, P. C. & Reeves, R.G. (1939). The origin of Indian corn and its relatives. Texas Agric. Expt. Sta. Bull. 574.
12. Mangelsdorf, P. C. (1974). Corn: its Origin, Evolution and Improvement. Harvard Univ. Press, Cambridge, Mass.
13. Mangelsdorf, P. C., Roberts, L.M. & Rogers, J.S. (1981). The probable origin of annual teosintes. Bussey Inst., Harvard Univ. Publ. 10, 1- 69.
14. México, D.F. (1994). Maize seed industries revisited: emerging roles of the public and private sectors. World Maize Facts and Trends1993/94. CIMMYT.
15. Mink, S. D. & Dorosh, P.A. (1987). An overview of corn production. In: The Corn Economy of Indonesia. Cornell University Press, London.
16. Poehlman, J.M. (1959). Breeding Field Crops. Holt, New York, USA.
17. Purseglove, J.W. (1972). Tropical Crops: Monocotyledons 1. Longman Group Limited., London.
18. Radu, A, Urechean, V., Naidin, C. & Motorga, V. (1997). The theoretical significance of a mutant in the phylogeny of the species Zea mays L. Maize Newsletter 71, 77-78.
19. Rayor, G.S., Ogden, E.C. & Hayes, J.V. (1972). Dispersion and deposition of corn pollen from experimental sources. Agronomy Journal 64, 420-427.
20. Sears, M.K., Stanley-Horn, D.E. & Matilla, H.R. (2000). Ecological impact of Bt corn pollen on Monarch butterfly in Ontario. Canadian Food Inspection Agency (http://www.cfia-acia.agr.ca/english/ plaveg/pbo/btmone.shtml)
21. Sharma, A.B. (1998). Experts for conservation of wild crop varieties. The Financial Express. Indian Express Group.
22. Stoskopf, N.C. (1985). Cereal Grain Crops. Reston, Virginia: Reston Publishing Company, Inc., Prentice-Hall Company.
23. Watson, L. & Dallwitz, M.J. (1992). Grass Genera of the World: Descriptions, Illustrations, Identification, and Information Retrieval; including Synonyms, Morphology, Anatomy, Physiology, Phytochemistry, Cytology, Classification, Pathogens, World and Local Distribution, and References. Version:18th August 1999. http://biodiversity.uno.edu/delta
24. World Watch Institute (2000). Crop gene diversity declining. Trends in Plant Science 5, 7.
25. Wilkes, H.G. (1977). Hybridization of maize and teosinte, in Mexico and Guatemala and the improvement of maize. Economic Botany 31, 254-293.
LAST UPDATED: SEPTEMBER 2002

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