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:

  1. 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;
  2.  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;
  3. 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


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