BIOTECHNOLOGY FOR THE COTTON FARMER PROBLEMS AND PROSPECTS

Cotton or white gold as it is aptly called is grown for its lint and seed which yield cotton fibre and seed oil, respectively. This crop occupies 32-33 mha of world area with a production of 20-25 metric tonnes. In India its area spans over 7.5 mha with an average yield of 290 kg/ha of lint and 870 kg/ha of seed cottom. To meet the challenges of 2000AD with a population of more that 960 million, a total productiong of 19 million bales is required as against the 13-14 million bales of today. This can be achieved by the use of improved crop production practices coupled ith appropriate pest management tacticas. In addition, generation of novel. transgenics may help acheive the near impossible. Genes that have been identified as potentially profitable, if engineered into acceptale cultivars can be used to generate such transgenics. Among these are genes imparting resistance to herbicides, insects, pathogens and abiotic stresses. It is also widely accepted now that a number of other qualitative characters can be improved, such as fibre strength, fineness, colour and thermal adaptability of the fibre.

Thansgenic plants have become realistic components of stress management world over. Bollworm and herbicide resistant transgenic cotton have received the approval of the Environmental Protection Agency and have been commercially released in the US for cultivation. Considering the fact that numerous biotic and abiotic stresses limit cotton production, it is likely that future strategies might orient towards the development of a multiadversity resistant high yielding transgenic cotton variety with superior fibre qualities.

The most important aspects in the development of transgenic planst are

* Identification, isolation and clining of the desirable genes

* Transffirmation of plant cells or tissues with a suitable vector

* Regeneration of the transformed cells or tissue

* Confirmation of the gene integration and expression

* Hardening and field establishment of the transgenics

Major achievements worldover in the field of cotton transgenics

1. GENES

Genes for Insect resistance used

* Toxin genes from Bacillus thuringiensis

* Protease inhibotor genes from plants & the horn worm

Genes used for herbicide resistance

* Nitrilase gene ftom a bacteria Klebsielia spp. for Bromoxynil resistance

* Mono-oxygenase gene from a bacteria Alcaligenes eutrophus for 2,4-D resistance

* Acstolactate synthase gene for resistance against sufonyl urea & imidazoline group

* ERPSP synthase gene for glyphosate resistance.

Genes used to Improve fibre qualities

* Polyhydroxybutyrate & polyhydroxyacetate from bacteria

* Expression of indigo pigment in fibres

Genes to be used

* Cholesterol oxidase genes against insects

* Heliothis stunt virus genes against Heliothis

* Genes encoding chitinases glucanases, attacins cecropins vital coat proteins etc. against diseases.

2.TRANSFORMATION

* Agrobacterium medicated transformation

* Particle acceleration gene delivery systems have been successfully used all over the world.

3.REGENERATION

* Callus fissue regeneration - mostly genotype specific

* Protoplasts were regenerated successfully

Meristematic tissues were successfully regenerated

4. EXPRESSION

* Enhanced expression was achieved using truncated forms of the full length genes, repeat copies of the propoter, modification of the bacterial codons to plant prefered type.

5. COMMERCIAL RELEASE

* Bollworm resistant transgenics - NUCOTN 33M & NUCOTN 35B were released in the US and cotton transgenics - are in now under field cultivation in Australia, South Africa and China.

* 8 lakh hectares were planted with the transgenic cottons in 1996 and 1997

* More than 8,000 hectares were damaged by bollworms.

* Transgenic cotton plants in Australia were also found to be attacked by the bollworms.

* Herbicide (Bromoxynil) resistant transgenics BXN 57 and BXN 53 were released in the US in 1996.

6. PROBLEMS ENCOUNTERED / EXPECTED

* Gene silencing and expression instability

* Resistance in insects to the toxins used. Studies on baseline toxicity indicated that there is a natural variation in the ability of Helicoverpa armigera in tolerating the CrylAc toxins. Some strains are capable of surviving the totoxins by virtue of an in built capacity to tolerate the toxins, while others are not. While development of Helicoverpa resistance to the CrylAc toxins itself may not be of immediate concern, a slight shift in the tolerance levels resulting from continuous exposure to the transgenics, can cause control difficultes.

* High cost of transgenic seed. v* Microecological changes resulting in pest shifts. CrylAc is a broad spectrum lepidopteran toxins that can cause significant changes in almost all populations of lepidopteran insects occuring on the cotton crop. Some of these insects, which cause negligible economic damage to the crop, harbour parasitoids which have the potential to keep the bollworm populations under check. Consequently, in the absence or low populaions of natural enemies, the bollworms can emerge as stronger pests that before. Insects pests such as Spodoptera litura which are less affected by the CrylAc toxins and which have been under check due to the use of pyrethroids can resurgace as major pests as pyrethroid use is likely to be reduced on transgenic Bt cottons.

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7. SOME RECENT DEVELOPMENT IN COTTON TRANSGENICS

* Three Bt transgenic lines, 95-1, RIOI and R-19 have been indigenously developed in China through Agrobacterium medicated (Somatic embryo) and pollen tube pathway transformation. Thestability of Bt gene expression and field efficacy have been confirmed.

* Microprojectile bombardment parameters for pollen mediated transformation and genetype independent protocols have been develop in China.

* A new methos of transformation through injection of Agrobacterium into developing embryos, has been reported from South Africa.

* Stable transformation and regermation has been reported for Bt transgenics in Uzbekistan, China, Egypt and Australia. Pakistan reports development of cotton transgenic plants resistant torearcurl virus.

* An antisense DNA of CLCUV DNA-A bome ACI gone along with the antesense DNA of the AC2 and AC3 gene was used for the vector construction and transgenic cotton resistant to the CLCUV was developed in Pakistan.

* In Russia, A glucanase gene has been isolated from thermo philic bacteria and is being used as a new reporter system fortransgenic cotton development.

* Genetically engineered Baculovirus ACMNPV with genes from straw itch mite has been developed been developed by Monsanto and Zeneca with researchers from Madison, USA.

* Bt rransgenic cotton currently occupies 20% (85,000 ha ) of cotton area in Australia. Isecticide use has been reduced by about 60% The efficacy has been moderate. The transgenic plants were found to express Bt proteimns only till the 95th day after which fluctuaions were observed. The reduction in expression was primarily due to down regulation which post transcriptional changes of the unstable RNA It was also reported that cotton tannins whoch increase with growth phase, act as antagonistswith Bt toxins.

* After two years of field cuftivation, true resistance to Bt has not yet been detected in Australia.

* Reslistancr to Bt in field populations of Helicoverpa armigera is reported form China

* Annually 100 million dollars are now being spent in the US, only in search of new insecticidal genes.

* In the US, 45% of cotton area this year will be under transgenic cotton ( herbicide and insect resistant). However, the area under Bt cotton seems to on the decline.

* Transgenic cotton developed in Australia using the Stunt virus genes, is not performing well.

* Transgenic cotton developed in the US using lectin genes, is not performing well against Heliothis virscens.

* The Australian report that Helicoverpa armigera is at least 100 times less susceptible to Cry toxins as cinoared to Heliothis virescens.

* The Window strategy for insecticide resistance management in Australia is still important for transgenic cotton.

* Cotton Pest spetrum in the US is getting altered after the introduction of Bollgard. Unsprayed Bt cotton sustained 4 times more attack of tarnished bugs, 2.4 times more with boll weevil, 2.8 times more with stink bugs and Spodoptera.

* Due to these changes in pest complex farmers had to spray 3-5 times on boligard as compared to 6-8 times on non-Bt cottons.

Prospects and potential

The application of biotechnonogy in cotton farming can be either in the form of production of fermentation products or novel recombinant products for use ( example, recombinant Pseudomonas expressing CrylAc ) or as transgenic plant with in buitt resistance to biotic and abiotic stresses. Transgenic crops with in built resistance to insect pests and diseases can be extremely useful as this would result in the reduction of insecticide use apart from making pest management simple for the farmer. Introduction of the bollworm resistant transgenic cotton is expected to reduce the use of chemicals used to control bollworm especially Helicoverpa importantly at a time when resistance to most insecticides including pyrethrolids is on the increase all over the world. Even if the introduction of the Bt cotton in India could result in a 25-30% reduction in insecticide use on cotton, this would mean a benefit of about of about Rs 300 crores, apart from the f-avourable impact on the environment so far, transgenic plants have been produced in about 60 plant species. Cotton has received special attention of the biotechnological companies in the developed countries who were attracted by the profit motives associated with the high value added to the transgenic seeds.

The firs Bt transgenic cotton has already been released, as Bollgard. The Delta and Pineland using DP 5410 and DP 5690 as recurrent parents. The D and PL brand BT transgenic were labelled as Nucotn 33B Two million acres were planted in 1996 in mid-south region of USA,mainly in regions where Heliothis virescens was problematic. Nearly 1.2 M acres were under Nucotn 33 B and the rest under Nucotn 35B.

The Bollgard brand Bt cotton seed was sold at US $34-36 as compared to US $8-9 per hectare for non-engineered cotton seed. The average cost of control of cotton ilnsect pests in the US was approximately US $150 in the early 1990s. Hence the prices were still found to be attractive. The transgenics were found to be more effective against Helothis virescens as compared to pectinophora gossypiella and Heliothis zea.

Farmers growing transgenics had to sign'an agreement with D & PL stating that they would not keep seed for planting next year. Quick ELISA tests have been devised to test for Bt toxins in plant parts, to check the illegal spread of transgenics.

Two more transgenic cotton varieties tolerant to herbicide Bromoxynil, trademarked as BXN 57 and BXN 53 were developed from the recurrent paren Coker 315 BYTHE Stonevuille pedigree company in collaboration with Calgene Inc.This seed was priced nearly 1.7 times the straight varietes.

In Australia the CSIRO and Narrabri research centre, in collaboration with Monsanto have developed transgenic cotton plarts that were commercial released in 1997. This introduction resulted in 50-60 % reduction in the US $ 93 milfion spent by fafrmers each year on insecticides. Pakistan which uses 90% of its total insecticides on cottonalone and China which is one of the major consumers of insecticides on cotton are reportedly strongly considering the prospects of introduction of Bt transgenic cottons.

Cotton acreage in India is only 5% of the total cropped area, yet it consumes more than 50% of the total insecticides used in the country. The pattern of usage, however , is not uniformly, spread, for instance in Andhra Pradesh alone, where cotton cultivation is only 0.3% of the total cropped area of the country, accounts for 17% of the pesticide use on cotton in India. Again within the average picture of thestate, coastal AP uses 30% more pesticide than the state average. The approximate estimates of insecticide sales in Guntur district alone, where cotton is grown in in 0.15 mHa, is about US $ M. Cleraly areas with maximum pecticides use per hectare, necessitated mostly due to bollworms, are likely targeted market niches for the bollworm resistant transgenic cottons. Moreover, since the price of the transgneic seed in the US is nearly four times the high input cotton cultivators of the irrigated belt Growing Bt transgenics and herbicide tolerant transgenics on marginal land is not recommended by the company as these would not show any impact in the absence of strong insect pressure and broad leaved weeds. In most areas of rainfed cottons the use of insecticides and pest pressures are low hence, it may not be economical in such belts, to encourage the use of transgenics.

Now transgenic production technology for many crops has become an established routine procedure in many countries including India. The research on transgenic cotton in government funded research labs in India is almost in the final stages. Meawhuile Mahyco-Monsanto biotech is ready to market Bt transgenic cotton (bollgard) in India. The introduction of transgenics thorugh sthese companies are to be in the form of FL seeds wherein Bt genes are inherited from the transgenic exotic germplasm. Such seeds may also be priced at a premium and it remains to be seen how enthusiastic the response of Indian farmers would be to the expensive input. Transgenic releases from government organisations would definitly enforce a competitve pricing to restrict the monopoly of the private companies.

The transgenic technology is also likely to be extremely beneficial to the private sector. For instance, a transgenic with resistance to a specific herbicide marginalises the use of other herbicides for chemical weed control and the chemical company holding the patent on the herbicide might be prove to be beneficial to the resource rich farmers. Clearly, here the targeted market is primarily high input farmers. The monipoly of transgenics due to patenting rights held by the biotech companies may also lead to high pricing of the seeds.

A Signfficant socioeconomic issue that arises from the introduction of transgenics into the Indian farming system is that the high priced seeds may benefits the prosperous and large farmers thus providing a negafive eaxternality on small and marginal farmers. On the other had it is also

argued that the developments from the application of biotechnology would be beneficial to low input farming practises wherein the cost of chemical inputs can be minimised. It is now only a matter of time before we experience the full social. economic and environmental impact of transgenics in our country.