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Event-1 Host Organism Gossypium hirsutum L. L. (Cotton) Trait Resistance to lepidopteran pests. Trait Introduction Microparticle bombardment of plant cells or tissue Proposed Use Production of cotton for fibre, cottonseed and cottonseed meal for livestock feed, and cottonseed oil for human consumption. Company Information JK Agri Genetics Ltd (India) 1-10-177, 4th Floor, Varun Towers Begumpet Hyderabad 500 016 Andhra Pradesh India

Summary of Regulatory Approvals

Country
India

Environment
2006

Food and/or Feed

Food Feed

Marketing

Click on the country name for country-specific contact and regulatory information.

Introduction
Transgenic cotton Event-1 was developed through specific genetic modifications to be resistant to major caterpillar pests of cotton, including Helicoverpa armigera (American Bollworm), Pectinophora gossypiella (Pink Bollworm) and Earis vittella (Spotted Bollworm). Event-1 express a modified gene (cry1Ac) that encodes an insecticidal crystalline Cry1Ac delta-endotoxin protein, derived from the soil bacterium Bacillus thuringiensis subsp. kurstakistrain HD73. Insecticidal activity is caused by the selective binding of Cry1Ac protein 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 gut paralysis and eventual death due to bacterial sepsis. Delta-endotoxins, such as the Cry1Ac protein expressed in cotton Event-1, exhibit highly selective insecticidal activity against a narrow range of lepidopteran insects. The specificity of action is directly attributable to the presence of specific receptors in the target insects. There are no receptors for deltaendotoxins of B. thuringiensis on the surface of non-lepidopteran insect guts or mammalian intestinal cells, therefore, livestock animals and humans are not susceptible to these proteins. An antibiotic resistance marker gene (nptII) encoding the enzyme neomycin phosphotransferase II (NPTII), which inactivates aminoglycoside antibiotics such as kanamycin and neomycin, was also introduced into the genome of Event-1. This gene was derived from a bacterial transposon (Tn5 transposable element from Escherichia coli) and

was included as a selectable marker to identify transformed plants during tissue culture regeneration and multiplication. The expression of the nptII gene in these plants has no agronomic significance and the safety of the NPTII enzyme as a food additive was evaluated by the United States Food and Drug Administration in 1994.

Summary of Introduced Genetic Elements

Code
cry1Ac

Name

Type
IR

Promoter, other
double enhanced CaMV 35S NULL CaMV 35S NULL

Terminator
A. tumefaciens nopaline synthase (nos) 3'untranslated region A. tumefaciens nopaline synthase (nos) 3'untranslated region

Copies Form
1 Truncated Cry1Ac protein Native

Cry1Ac deltaendotoxin (Bacillus thuringiensis subsp. kurstaki (Btk)) nptII neomycin phosphotransferase II (Escherichia coli)

SM

Characteristics of Gossypium hirsutum L. (Cotton)

Center of Origin
Believed to originate in Meso-America (Peruvian-EcuadorianBolivian region).

Reproduction
Generally self-pollinating, but can be cross-pollinating in the presence of suitable insect pollinators (bees). In the U.S., compatible species include G. hirsutum, G. barbadense, and G. tomentosum.

Toxins Allergenicity
Gossypol in cottonseed meal.

Donor Organism Characteristics

Latin Name

Gene

Pathogenicity

Bacillus cry1Ac Although target insects are susceptible to oral doses of Bt proteins, there is no thuringiensissubsp.kurstaki evidence of toxic effects in laboratory mammals or birds given up to 10 g protein / g body wt. There are no significant mammalian toxins or allergens associated with the host organism.

Modification Method
The transgenic Bt cotton was developed by using the biolistic method of transformation system. The DNA transferred from the plasmid to the genome of individual cotton cells (T-DNA), included the cry1Ac and nptII genes. The transformed cotton shoots containing thenptII gene were selected on medium supplemented with kanamycin. A procedure for biolistics method of transformation of cotton is novel and performed using shoot meristem. Plants were regenerated and ultimately plantlets were grown in soil and assayed for insect resistance.

Environmental Safety Considerations

Gene Flow The pollen flow / out crossing study conducted in 2003 using Bt Event-1 revealed that out-crossing occurred only upto 2 meters, and only 1% of the pollen reached a distance of 2 meters and beyond 2mt distance no plants were out crossed with transgenic pollen. As the cotton pollen is heavy and sticky, the range of pollen transfer is limited because it was observed that the pollen out crossing taken place upto the distance of 1 mt in East and North direction and 2mt in west direction of experimental site. Since the Bt gene is incorporated into tetraploid species and the native Indian cottons are of diploids and are not crossable to tetroploids, there is no chance that the Bt gene will transfer from cultivated tetraploid species to near relatives such as traditionally cultivated diploid species. Potential Weediness The potential weediness of JK Bt Cotton Event-1 was assessed in comparison with non transgenic line of same by testing its vigor and the rate of germination in laboratory test as well in soil. The results demonstrated that there are no significant differences observed between Bt and non-Bt cotton for germination and vigor. This indicates that the Bt and non-Bt cotton are not different in regard to their weediness potential. In general the cotton plants are not having the potential weediness characteristics such as seed dormancy, soil persistence, germination under diverse environmental conditions, rapid vegetative growth, short life cycle or high seed out put and dispersal.

Potential Impacts on Non-Target Organisms The studies conducted on non-target organisms and beneficial insects during the multi-location and large scale field trials of JK- Bt Cotton Event ?1 revealed that the Bt cotton hybrids do not have any toxic effects on the nontarget species, namely sucking pests (aphids, jassids, white fly and mites). The population of secondary lepidopteran pests (Spodoptera litura) remained negligible during the study period in both Bt and non-Bt hybrids. The movement of beneficial insects populations (Chrysopa, Coccinellids, Syrphids and Spider) are equal and remained active over both Bt and non Bt cotton plots. Environmental Fate Studies were conducted to assess the possible risk of accumulation of Bt protein in the soil as exudates from the roots of Bt Cotton. The soil samples from the Bt plots of different locations were subjected to quantify the Bt protein and the results showed that it was not detected in soil samples. This indicates that Bt protein is rapidly degraded in the soil on which Bt cotton is grown. This study showed that the Cry 1AC protein was rapidly degraded in the soil in both the purified form of the protein and as part of the cotton plant tissue. The half-life for the purified protein was less than 20 days. The half-life of the Cry 1AC protein in plant tissue was calculated to 41 days which is comparable to the degradation rates reported for microbial formulations of Bt. Potential Impacts on Soil Microflora This Study was conducted in 2004 and 2005 to evaluate any impact of Bt protein exudated by roots of Bt cotton on the soil micro-flora. There was no significant difference in population of microbes and soil invertebrates like earthworm and Gllembola between Bt and non-Bt plant rhizosphere soil samples.

Food and/or Feed Safety Considerations

The composition of cottonseed and oil from Bt cotton hybrids (Event-1) was compared to that of non-Bt cotton hybrids and other conventional cotton varieties. The nutrients measured in the cottonseed included protein, fat, fiber, moisture, ash, carbohydrate, and the anti-nutrients gossypol, fatty acids and aflatoxins. All these analysis revealed that Bt cotton hybrids are equivalent and as safe and nutritious as non-Bt hybrids and other conventional cotton varieties. Also the nutritional studies in cows revealed that no difference on feed intake, milk yield and composition between Bt and non-Bt cotton seed feed groups of cow as well the safety studies confirmed the food and feed safety of Bt cotton to cows Feeding experiments conducted with Bt cotton seed meal on fish chicken, goat and cows indicated that Bt cotton seed meal is nutritionally equivalent and safe as the non-Bt cottonseed meal. The feeding experiments were conducted from different institutes in India in which Central Avian Research Institute (CARI), Izatnagar (UP) ICAR conducted studies on Bird; IVRI, Izatnagar on goat and cow; Central Institute of Fisheries Education (Deemed University, ICAR), Mumbai on fish. The mammalian acute oral toxicity of Bt protein was studied in rat and the specificity and safety of the Cry1Ac protein was recorded. No toxic effects were observed even at extremely high dose levels (5000 mg/kg of body weight), when the Cry1Ac protein was administered orally to rats. Also the sub-chronic oral toxicity study on rat revealed the Cry1Ac protein found safe to animal health. The Cry1Ac protein expressed in the cotton plant is not expected to present a risk of dermal or inhalation toxicity. The proteins that are non-toxic by the oral route are not expected to be toxic by the dermal or pulmonary route. This was confirmed by the study conducted on irritation test to skin and mucous membrane in rabbits and dermal sensitization study in guinea pigs. All these oral toxicity and irritation tests were conducted at Shriram Institute for Industrial Research, New Delhi Feeding studies on ruminants (goat) showed that Bt cottonseed when fed, it was as nutritious as non Bt cotton seeds and did not cause any deleterious effect on the ruminants in comparision to non-Bt cotton seeds. Nutritional studies on lactating cows conducted in IVRI, Izatnagar reconfirm these conclusions. The toxicological studies on chicken and fish under Indian conditions confirmed the safety of cottonseed meal derived from Bt cotton. Studies conducted to determine the presence of Cry1Ac protein in refined oil and lint obtained from Bt cotton showed absence of Cry1Ac protein at the detection standard of the experiment. Similarly studies conducted to determine the presence of Cry1Ac gene in cottonseed oil obtained from Bt cotton by using forward and reverse primers specific for internal sequence of the Cry1Ac gene, reveal no DNA amplification in the oil samples. Source: India Biosafety Clearing (http://www.indbch.nic.in/risk_assessment_jk.asp). Last accessed 26 February 2009. House

References
Acute Oral Toxicity Study in Rats. Shriram Institute for Industrial Research (2003). Annexure to report no. 000066702. Dermal sensitization study in guinea pigs (Buehler method). Shriram Institute for Industrial Research (2003). Annexure to report no. 000066702. Irritation to mucous membrane in rabbits. Shriram Institute for Industrial Research (2003). Annexure to report no. 000066702. Jain, R.K. and Ghai, S. Studies on soil microflora of Bt and non-Bt cotton fields: a comparative evaluation. Institute of Microbial Technology, Chandigarh, India. Mehra, U.R., Singh, N., Sharma, M.C., Somvanshi, R., Verma, A.K. and Singh, P. (2005). Subchronic oral toxicity in goats -- 90 days study for genetically engineered Bt cotton seeds. Indian Veterinary Research Institute, Izatnagar, India. Mukherjee, S.C., Srivastava, P.P. and Jain, K.K. (2005). Transgenic Bt cotton seed (JKC738 Bt., Event 1) containing Bacillus thuriengensis cry1Ac (truncated) gene feeding studies in Indian major carp, Cirrhinus mrigala. Central Institute of Fisheries Education, Mumbai, India. Study no. ALNUBI-CR-2005-05. Primary skin irritation test on rabbits. Shriram Institute for Industrial Research (2003). Annexure to report no. 000066702. Report on pollen flow trial for Cry1Ac Event-1 (Kharif 2003). Sharma, M.C., Yadav, M.P., Singh, N., Pandey, H.N., Dimri, U., Tomar, A.K.S. and Chaturvedi, V.B. (2006). Feeding studies of transgenic Bt cotton seed of JKAL Event 1 containing Cry1Ac gene in lactating cross bred dairy cows. Indian Veterinary Research Institute, Izatnagar, India. Shrivastav, A.K., Tyagi, P.K., Tyagi, P. and Elangovan, A.V. (2004). Broiler chicken performance as influence by feeding diest containing transgenic Bt cottonseed meal. Central Avian Research Institute, Izatnagar, India. Subchronic oral toxicity study in rats (90-day feeding). Shriram Institute for Industrial Research (2004). Annexure to report no. 000070354.

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THIS RECORD WAS LAST MODIFIED ON WEDNESDAY, FEBRUARY 25, 2009

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