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BT Cotton – Indian Story

What is Bt-cotton?

Bt cotton is genetically modified cotton crop that expresses an  insecticidal protein whose gene has been derived from a soil  bacterium  called  Bacillus thuringiensis, commonly referred as Bt. Many subspecies of B.thuringiensis are found in soils and are in general known to be toxic to various genera of insects but safe to other living organisms. Bt was first discovered by a Japanese scientist Ishiwata in the year 1901. Bt has been used as an insecticide for control of stored grain pests since 1938 in France and from 1961 as a registered pesticide in the USA and later in many other countries including India as sprays in cotton Integrated Pest Management (IPM) programs to control insects. Bt toxins thus has several decades of proven selective toxicity to insect pests and with established safety record to non-target animals.

Currently there are 67 recognized subspecies of B.thuringiensis most of which produce spores and insecticidal proteins. The B.thuringiensis strains produce three types of insecticidal toxin.

  1. crystal (Cry) toxins
  2. cytolytic (Cyt) toxins
  3. vegetatively expressed insecticidal proteins (vip). These toxins are highly specific to certain insect species.

Thus far until September 2012, a total of 229 cry toxins (Cry1Aa to Cry72Aa), 11 cyt toxins (cyt1Aa to cyt3Aa) and 102 vip toxins (vip1Aa1 to vip4Aa1) amounting to 342 Bt toxin genes are available for research to develop insect resistant GM crops.

The Bt gene cry1Ac was used to develop the first Bt-cotton variety. The gene was transferred into genome of cotton explants tissue pieces using a bacterium called Agrobacterium tumefasciens. The transformed cells were developed into a full GM plant now called Bt-cotton. Cry1Ac toxins are highly specific to insects at species level, and are not known to cause any harm to fish, birds, farm animals, human beings or to any other non-target species.

Currently, Cry1Ac, Cry2Ab and Cry1C have been approved for commercially cultivation in India. Bt cotton hybrids available in India are derived from technologies developed by Monsanto (Cry1Ac and Cry1Ac + Cry2Ab), Metahelix (Cry1C), CAAS, Chinese Academy of Agricultural Sciences, through Nath seeds (modified Cry1Ac called as fusion gene) and JK seeds (Cry1Ac). Dow Agrosciences are conducting field trials

with Cry1Ac + Cry1F and Bayer is introducing Cry1Ab + Cry2Ae. There were 1128 Bt- cotton hybrids in 2012, developed by 40 seed companies, available in the Indian markets.


Why do we need Bt-cotton?

  1.  Cotton is a long duration crop and is attacked by large number of insect pests throughout its growth and development.
  2.  The three bollworms, American bollworm Helicoverpa armigera, Pink bollworm Pectinophora gossypiella and the Spotted bollworms, Earias vittella and Earias insulana are major pests and cause serious threat to the cotton resulting in significant yield losses.
  3. About 9400 M tonnes of insecticides worth Rs 747 crores were used only for bollworm control in 2001
  4. Before the introduction of Bt cotton, insecticide quantity applied on cotton was the highest relative to other cultivated crops.
  5.  Cotton bolls are highly vulnerable to hidden insects such as the American bollworm, pink bollworm and spotted bollworm.
  6. Bollworms, especially the pink and spotted bollworms are hidden feeders and generally do not come into direct contact with insecticide sprays.
  7.  The American bollworm which comes into contact with insecticides, partially, has developed resistance to almost all the insecticides recommended for its control in all regions of the world.
  8. Nearly 90.0% of all insecticides in Pakistan and about 50.0% of all insecticides in India were being unsuccessfully used for cotton pest control, until the year 2001, before Bt cotton was introduced. Of these insecticides about 70.0% was for bollworm control and the rest for sap-sucking insects.
  9. Resistant sources are unavailable in the germplasm and resistance breeding has been unsuccessful.

How does Bt-cotton kill insects?

The Cry1Ac, Cry2Ab, Cry1C, Cry1F etc., belong to the class “Bt-delta- endotoxins” which function as oral toxins. The delta-endotoxins are ingested and the protoxins present in the crystals are proteolytically activated to trypsin-resistant active core δ-endotoxin in the alkaline mid-gut. The active toxin traverses the peritrophic membrane to bind cadherin receptors present on the brush border membrane of the insect midgut. The cadherins process the toxins to form homo-oligomers and bind to specific receptors like alkaline phosphatases and aminopeptidases before causing pores in the epithelial membrane, resulting in osmotic lysis of the cells. This results in cessation of feeding and finally causing mortality.

Amongst the genes that have been deployed in insect resistant transgenic cotton, thus far, Cry1Ac is the most toxic to H. armigera and against a wide range of lepidopteran insect pests that include the other two bollworms the spotted bollworm Earias vittella & the pink bollworm, Pectinophora gossypiella. Bt-cotton incorporated with Cry1Ac is highly toxic to the bollworms and other minor pests such as the cotton semilooper and hairy caterpillar, but not effective on the leaf eating tobacco caterpillar Spodoptera litura. Cry2Ab2 (present in Bollgard-II) is moderately toxic to Helicoverpa armigera, and Spodoptera litura. However the expression levels of Cry2Ab2 are very high (120-350 ppm), as compared to the Cry1Ac (0.5 to 15 ppm) and therefore Bollgard II is highly toxic to H. armigera and toxic to Spodoptera litura.

The Cry1F is not effective on H. armigera but the level of expression in “Widestrike” (Dow Agrosciences) is at 10-40 ppm that confers mild tolerance to H. armigera and fairly good toxicity to Spodoptera litura.

Toxicity to Human Beings and possibility of entering food chain

The Cry toxins Cry1Ac, Cry1Ab, Cry2Ab, Cry1F and Cry1C are considered to be safe to human beings. The stomach of humans, being the first organ of digestion the Bt protein encounters, is acidic and contains proteases like pepsin which degrade the Bt protein. Thus the alkaline conditions needed for pro-toxin solubilization and protease required for toxin activation are absent in the human stomach. More importantly the human intestine lack the specific receptors to which the activated Bt protein binds and initiates the physiological effect. Bt-cotton is being cultivated in at least 12 countries and was cultivated in at least five major countries for more than a decade. Cotton seed oil is a by-product of Bt-cotton and is used in all the cotton growing countries. But, Bt-toxins or Bt-DNA were not detected in refined oil.

The possible routes of Bt-cotton protein entering the food chain are, through human consumption of un-refined cottonseed oil, in which traces of Bt protein may be present with particulate seed residues or through consumption of meat of the animals which fed on Bt cotton seed-cake. However, ELISA tests showed that milk and meat were found to be free of Cry proteins. Thus the chances of Bt proteins entering the human food chain through milk and meat are low.


Toxicity to Cattle

Cry toxins have not been reported to be toxic to higher animals such as goats, sheep and cattle in any part of the world. However, it is only in India that apprehensions were expressed by NGOs regarding sheep mortality at Warangal and Adilabad district of Andhra Pradesh due to grazing in Bt cotton fields. The issue was examined by the State Government, reports received from the Directorate of Animal Husbandry, Hyderabad and the Indian Veterinary Research Institute, Izatnagar, U.P. revealed that the sheep deaths might be due to high content of Nitrates/Nitrites, residues of hydrocyanide (HCN) and organophosphates which are common constituents of pesticides used during cotton cultivation and not due to Bt toxin. Scientific evidence indicates that the possibility of Cry toxins killing goats and sheep is remote. The Cry toxins do not get activated under the acidic conditions of non target animals such as goat, sheep and cattle. Feeding studies did not show any toxicity symptoms that could lead towards extreme toxicity symptoms or mortality.

A field study was carried out at CICR, Nagpur by a team of scientists led by a senior scientist of the Krishi Vigyan Kendra, for two years (2007-2009) by tethering six goats in one hectare of Bt cotton and one hectare of conventional cotton. The goats were fed on the crop continuously for four months and there were no differences in any biological aspects of the two sets of animals. The biochemical and health results clearly showed that Bt cotton was safe to goats.


Technology development and the availability of commercial BT-Cotton

Six Bt cotton events have been approved thus far in India for commercial cultivation. There are four Bt Cotton events expressing Cry1Ac, one event with Cry1C, and one event with Cry2Ab2. The various technology developers are:

  1. Monsanto: MON531 (Cry1Ac) event Bollgard
  2. Monsanto: Mon15985 (Cry2Ab2) event in Bollgard-II
  3. JK seeds, India: JK Event-1 (Cry1Ac);
  4. Chinese Academy of Agricultural Sciences, China: GFM Cry1A (Cry1Ac), introduced by Nath seeds India;
  5. NRCPB, New Delhi and UAS Dharwad, India: BNLA601 (Cry1Ac) event; Commercialized by CICR, Nagpur:
  6. Metahelix, India: Event 9124 (Cry1C) event

There are four variants of cry1Ac genes apart from one each of the cry2Ab and cry1C genes. All the Cry1Ac genes present in the four events released in India are chimeric fusion genes.

The Cry1Ac gene in the Bollgard event 531 is a chimeric gene of 3534 bp size, with the first 1398 nucleotides (corresponding to the first 466 amino acids) of Cry1Ab gene and rest of the 1399-3534 nucleotides (corresponding to the 467-1178 amino acids) from the Cry1Ac gene. Except for one amino acid at 766 position, the Cry1Ac amino acid sequences are identical to that of the wild type Cry1Ac gene. The chimeric gene produces a protein that is 99.4% identical to that of the wild type Cry1Ac.

The Cry1Ac genes in JK and BNLA106 are chimeric fusion genes of 1842 bp with the first 1398 nucleotides (corresponding to the first 466 amino acids) of Cry1Ab gene and rest of the 453 nucleotides (corresponding to 151 amino acids at 467-671 position) from the Cry1Ac gene.

The Cry1Ac in Nath seeds is >99% identical to the Cry1Ac used in JK and BNLA106 events except that the size is smaller at 1824 bp with the first 1377 nucleotides (corresponding to the first 459 amino acids) of Cry1Ab gene and rest of the 453 nucleotides (corresponding to 151 amino acids at 460-664 position) from the Cry1Ac gene.

Trend of Bt cotton in the various states

The uptake of Bt-cotton was rapid in Punjab, Haryana, MP, AP and Maharashtra. Desi cotton, Gossypium herbaceum is still cultivated in about 4.0 to 5.0 lakh hectares of “Wagad” soils or saline belt in Gujarat and an estimated 2.0 to 3.0 lakh hectares are still under illegal Bt-hybrids. Thus the area under GEAC approved Bt-cotton hybrids is about 74.0%. The uptake in Rajasthan was slow on account of the unsuitability of several Bt- hybrids because of their susceptibility to the Cotton leaf curl virus disease. Some regions in Karnataka are still under DCH 32 and the Desi species Gossypium herbaceum.

Refugia

 Refugia is a method in which the non-Bt version of the crop is planted in the vicinity of the Bt-crop so as to ensure the survival and maintenance of susceptible insect populations on the non- Bt crop. The strategy is based on the fact that if a small defined area of non- trangenic plants are cultivated in close vicinity of the toxin expressing transgenic plants, they serve as hosts of the target Bt-

 susceptible insect pests to multiply. These would then serve as reservoirs of the susceptible alleles and when mated with the rare resistant survivors from transgenic plants would result in heterozygous progeny which would express susceptibility, especially if the resistant alleles are recessive in nature. The  probability of  the  susceptible alleles

mating with the resistant insects from Bt plants would be high because of the large population of susceptible insects from the non-Bt refuge. Hence having a refuge in close proximity helps in the effectiveness of the refuge.

In India the Genetic Engineering Approval Committee (GEAC) has recommended refuge of non-Bt (5 border rows) with Bt-cotton per acre or an area of 20% Bt cotton that can be subjected to insecticide sprays. Recently, in 2009, pigeonpea has also been approved as refugia to be cultivated as border rows around Bt cotton.

Conclusion

Bt technology in cotton is a golden opportunity for the Indian cotton farmers to shift from the conventional desi cotton cultivation to the technology based cultivation. It is sure to improve the production of cotton in India and make India the world leader in cotton cultivation provided farmers follow the recommendations of the technologists.