Bt-Brinjal: Production and Its Development | India

After reading this article you will learn about:- 1. Production of Bt-Brinjal 2. Development of Bt Brinjal 3. Mode of Action of Bt To Control FSB 4. Bt Brinjal – The Regulatory Framework In India 5. Chronology of the Development and Approval of Bt Brinjal In India.

Production of Bt-Brinjal:

India is the second largest producer of vegetables and brinjal in the world after China. India’s share of global vegetable production is 9.2% compared to 36.6% of China. India produces 26% of the total 32 million tons of world brinjal production (Table 38.1) whereas China leads with 56%.

In terms of area, India cultivates 512,800 hectares brinjal (550,000 hectares as per NHB) of an estimated 2.04 million hectares worldwide. Other important countries that grow brinjal include Bangladesh, Indonesia, Egypt, Turkey, Iraq, the Philippines, Italy, Japan. Sudan, Thailand. Romania, Pakistan, Syria, Azerbaijan, Algeria, Greece, Kazakhstan, the United States, Venezuela and France.

The major brinjal producing States in India are West Bengal, Orissa, Gujarat, Bihar, Maharashtra, Andhra Pradesh, Chhattisgarh, Madhya Pradesh, Karnataka, Assam, Haryana and Tamil Nadu. The national average productivity of brinjal is around 16.0 tons per hectare which varies substantially from 12.9 to 22.1 tons per hectare.

The National Horticulture Board (NHB) reported the highest productivity 22.1 tons per hectare in Karnataka, followed by 21.0 tons in Andhra Pradesh, 19.2 tons in Bihar, 18.0 tons in West Bengal, 15.7 tons in Gujarat, 15 tons in Maharashtra and the lowest was 12.9 tons per hectare in Haryana. The state-wise distribution of area, production and productivity of brinjal is listed in Table 38.2.

The eggplant growing pockets in major eggplant growing states are as follows:

a. Andhra Pradesh – East and West Godavari, Krishna, Guntur, Nellore, Kurnool, Ananthapur, Srikakulam, Visakhapatnam.

b. Karnataka – Belgaum, Dharwad, Bijapur, Hassan, Mysore

c. West Bengal – Hooghly, 24 – Paraganas, Burdwan

d. Tamil Nadu – Salem, Dindigual, Coimbatore

e. Rajasthan – Alwar, Jaipur, Kota, Tonk, Ajmer, Sriganganagar

f. Maharashtra, Orissa, Uttar Pradesh, Gujarat, Bihar, Haryana and Assam

There has been gradual and steady increase in productivity of brinjal over the last two decades. At the national level, productivity increased from 12.6 tons per hectare in 1987-88 to 15.3 tons per hectare in 1993-94 and to a high of 16.5 tons per hectare in 2005-06 as given in Table 38.3.

However, the productivity gains in India are lowest as compared to other brinjal growing countries of the world. France reported highest productivity of brinjal with 41.2 tons per hectare followed by 31.2 tons per hectare in Japan, 26.4 tons per hectare in Turkey. 23.2 tons per hectare in Egypt and 22.5 tons per hectare in Italy.

Brinjal Fruit and Shoot Borer-The Major Constraint in Increasing Productivity:

It is well recognized farmers often lose a significant share of production due to insect- pests and that fruit and shoot borer (Leucenodes orbonalis) is the most destructive and unmanageable pest of brinjal which accounts for the majority of insecticide usage and yield losses.

On the other hand, consumers have generally no choice but to buy insect-damaged and infested fruits or those with high pesticide residues. Farmers suffer because brinjal is highly susceptible to insect-pests and have no option except to apply insecticides which provide ineffective control of the principal pest, fruit and shoot borer (FSB).

One of the critical factors involved in the control of FSB is that farmers have to time the application of insecticides in such a way as to kill the larvae before they bore into shoots and fruits.

If they are not controlled at this early stage, FSB larvae remain and feed within the shoots and fruits. Infested shoots retard vegetative growth and affect yield while damaged fruits are responsible for direct losses in marketable yield.

It is estimated that FSB causes yield losses of 60-70% even after repeated insecticide sprays. Farmers also have to apply additional insecticides to control other pests including epilachna beetle (hadda), stem borer, red spider mite and jassids.

Collectively these insect-pests cause substantial crop losses leading to heavy economic losses. Application of frequent insecticide sprays results in a high pesticide exposure for fanners and sometimes this can be associated with recurring health problems.

The wilted shoots are readily visible. There may be small darkened holes surrounded with brownish areas on fruit surface and/or fruit stalk. The inside of the fruit is hollow and filled with frass resulting into non-marketable fruits.

The insect has chewing mouthparts. The young caterpillar is dull white and turns pink as it matures. It is 1.5-1.8 cm long. The adult moth is white with a pink or bluish tinge and brownish wings. The larvae can be found feeding inside the fruit of a wilted plant. Fruit damage is not obvious.

The first indication is a small hole in the fruit stalk or in the fruit itself. This is where the insect has entered. Cutting the fruit near this hole leads to tunneled areas inside the fruit. The tunnel contains frass and insect remains. Continuing to cut around this area may lead to location of live larvae. Creamy white eggs are laid singly or in groups on the underside of leaves, on stems, flower buds or the base of the fruit.

The newly hatched larvae prefers to bore directly into the fruits. When feeding is complete, pupation occurs on stems, dried shoots, or in the fallen dry leaves. Multiple overlapping generations occur in warm climates. This pest feeds on many other solanaceous plants such as tomato and potato.

Biotechnology Offers Solutions to Problem of Brinjal Fruit and Shoot Borer:

The damage caused by FSB is obvious. It has not been possible to breed FSB resistant cultivars/hybrids as the source of resistance to this insect is just not available in eggplant germplasm. Huge application of insecticides has given partial relief but at the cost of environment pollution and human health related problems due to too much residual toxins on fruit surface of eggplant.

Under this scenario, scientists started to look around devising a strategy based on biotechnology where a gene conferring resistance to this insect could be transferred to eggplant from any source beyond the limit of sexual compatibility. This plainly stated amounts to creation of transgenic brinjal on the lines of several commercialized biotech/GM crops globally.

Globally, 134 million hectares of biotech crops were harvested in 2009 showing fastest adopted crop technology, 80-fold increase from 1996 (the first year of biotech crop commercialization) to 2009, reflecting year to year growth of 9 million ha or 7% (Table 38.4).

The trait-wise distribution of these 134 million hectares of biotech crops in 2009 has been as:

Keeping track of these developments and convinced with utility of transgenics several public sector and private sector research organizations started working on GM fruit and vegetable crops and the information is summarized in Table 38.5. Table 38.5 shows brinjal Bt transgenic in forefront and at the verge of commercialization in India.

Biotech crops are also known as genetically modified (GM) or genetically engineered (GE) crops. Phenotypically they look just like their traditional counterparts.

Among biotech crops, Bt crops such as Bt cotton and Bt corn are already prevalent in many countries. Bt crops are incorporated with one or more modified Bt genes sourced originally from naturally occurring soil bacterium, Bacillus ihuringiensis (Bt is its popular abbreviation).

These crops have been developed worldwide to provide alternative methods to control specific insect-pests in agriculture. Rapid adoption of Bt crops in past twelve years, both in developed and developing countries, is a testimony that this technology works effectively to control target insect-pests in a broad array of agricultural mega-environments.

Biotech crops benefit both the farmers and consumers. Farmers gain higher crop yields with less insecticides and consumers have access to crops grown with fewer insecticides, low pesticide residues and with healthier nutritional characteristics.

With the rapid progress in advanced biology, biotech crops have been developed with the help of genetic engineering tools to possess special characteristics (traits) that make them better. The most common traits deployed in biotech crops include insect resistance, herbicide tolerance, virus resistance and improved product quality.

The “stacking” (use of more than one trait in a single crop) of these traits is an important feature that has been used increasingly to tackle multiple constraints in agriculture.

It is expected that development of crops with tolerance to drought and salinity, improved nitrogen use efficiency, enhanced yield, quality and nutritional status coupled with existing traits will make food better and safer. At the national level, it will make agriculture more efficient and competitive to meet the challenges of hunger, poverty, malnutrition and food security in tomorrow’s world.

Over the years, gene exchange between two plants have been attempted to produce offspring that inherited desired traits. This was done by transferring pollens from male flower of one plant to the female flower of another.

Such traditional cross-breeding methods have some limitations. Firstly, the gene exchange is only feasible between the same or very closely related species. Secondly, it usually takes a long time to achieve desired results and characteristics of interest may often not exist, or not be available at the required level in the species or in any related species. Also, there is little or no guarantee of obtaining specific gene combination from millions of crosses generated.

Undesirable genes also get transferred along with desirable genes or while one desirable gene is gained, another may be lost because genes of both parents are mixed together and re- assorted more or less randomly in the offspring.

Biotech crops offer viable options to counter these limitations. Bt. brinjal is a state-of-the-art technology which is considered as one of the most safe, convenient and viable options to control the fruit and shoot borer (FSB), Leucinodes orbonalis (Lepitoptera, Pyralidae).

The attempts made so far to control this pest by developing resistant cultivars through traditional plant breeding, indiscriminate use of insecticides and application of integrated pest management (1PM) have met with limited or almost no success.

In this context, it is expected that timely deployment of Bt brinjal will help farmers to effectively control FSB while significantly reducing insecticide sprays. This will also enable farmers to improve yield by saving damage to marketable fruits and consumers to get healthier vegetables devoid of insect damage and pesticide residues.

Development of Bt Brinjal:

Bt. brinjal is a biotech crop developed using Bt technology and transformation process similar to the one exploited in Bt cotton. Like Bt cotton, Bt brinjal carries an additional gene that provides in-built insect protection against fruit and shoot borer (FSB).

The development of Bt brinjal involves introduction of cry 1Ac gene expressing insecticidal protein to confer resistance against FSB. The cry 1Ac gene is sourced from environment friendly and ubiquitous soil bacterium called Bacillus thuringiensis (Bt), which has been frequently used as a biological control measure in granular or powder form to control FSB and other insect-pests for many years.

The Maharashtra Hybrid Seeds Company (Mahyco) – a leading Indian seed company has developed a new DNA construct, which contains a gene sequence encoding insecticidal protein in all parts of brinjal plant throughout its life.

The cry1 Ac gene along with two other supporting genes namely nptll and aad genes are put together in such a way that they work in tandem to produce insecticidal protein that is toxic to the targeted insect, in this case the fruit and shoot borer (FSB).

The cry 1Ac gene is under the transcriptional control of the enhanced CaMV35S promoter (P-E35S), which works as an on’ off switch and regulates when and where cry 1Ac gene should express.

This new strand of DNA is called the ‘gene construct’ and is illustrated as follows:

Summary of Gene:

The gene construct contains the following genes and a brief summary of each gene is given below:

(i) Cry1 Ac gene:

It is isolated from the common soil bacterium Bacillus thuringiensis sub- sp. kurstaki (B.t.k.) strain HD73, and introduced into the plant after suitable modification, it encodes for an insecticidal protein Cry 1Ac.

(ii) Nptll gene:

It is a selectable marker which encodes enzyme neomycin phosphotransferase II (nptll) and is used to identify transformed cells that contains cry 1Ac gene. It has no insecticidal properties. The nptll gene is derived from prokaryotic transposon Tn5.

(iii) CaMV 35S promoter:

The cry 1Ac gene is expressed under the control of the Cauliflower Mosaic Virus 35S promoter.

(iv) aad gene.

It encodes for bacterial selectable marker enzyme 3″ (9)-0- aminoglycoside adenyl transferase (AAD) and allows for selection of bacteria containing the pMON 10518 plasmid on media containing spectinomycin or streptomycin.

The aad gene is under the control of a bacterial promoter and hence not expressed in Bt brinjal. The plasmid vector pMON 10518 containing the cry 1Ac gene expression cassette has been transferred to Agrobacterium tumefaciens strain LBA4404 using methods such as electroporation or tri-parental mating.

A vector containing cry l Ac gene, nptll gem, CAMV 35S promoter and aad gene was used to transform young cotyledons of brinjal plants by co-cultivation with Agrobacterium and cultured on Kanamycin-containing medium using standard tissue culture techniques for plant regeneration. An improved method for Agrobacterium-medlated brinjal transformation has been developed and used at Mahyco.

This is based on a method that was described earlier by Fari (1995). The regenerated plants from the transformed cells were carried forward and analyzed in subsequent generations to identify lines in which transgene segregated in expected Mendelian fashion. Selected lines were also analyzed by southern blot and a single copy elite event was selected and named as event EE-1 (Elite Event-1).

An event refers to a unique DNA recombination event that took place in one plant cell, which was then used to generate entire transgenic plant. Every cell that successfully incorporates the gene of interest represents a unique “event.” Every plant line derived from a transgenic event is considered as biotech crop.

The event names correspond to the identifiers commonly used by regulatory authorities and international organizations, such as the Organization for Economic Cooperation and Development (OECD). Mahyco has also developed a PCR- based event identification system (ID) for this unique event EE-1 in order to track this event in green-house and field.

The event EE-1 was then introduced into the regular breeding program where it was back-crossed with female parents of seven best performing brinjal hybrids and MHB-4 Bt, MHB-9 Bt, MHB-10 Bt, MHB-11 Bt, MHB-39Bt, MHB-80 Bt and MHBJ-99 Bt were developed at Mahyco Research and Life Sciences Centre at Jalna, Maharashtra.

The Bt technology available with M/s Mahyco has been transferred (free of cost) to TNAU, Coimbatore, UAS, Dharwad and IIVR, Varanasi. Mahyco has transferred this technology to institutions in Bangladesh and Philippines also.

The transfer of this technology to the Institute of Plant Breeding of Philippines in Philippines and Bangladesh Agric. Res. Inst. (BARI) and East West Seeds Ltd in Bangladesh materialized under the aegis of Agricultural Biotechnology Support Project (ABSP II) of Cornell University which is supported by USAID.

Many small and medium private seed and biotech companies have invested heavily in expanding research base, developing state- of-the-art biotech laboratories and in establishing high quality and high quantity seed processing plants.

Recently, there has been emphasis on public-private partnership, particularly in agriculture sector and many such collaborative projects have been successfully materialized. Another important milestone in the development is the transfer of Bt brinjal technology developed using cry1Fal gene by the National Research Centre of Plant Biotechnology (NRCPB) to Bejo Sheetal Seeds Pvt. Ltd. Krishidhan Seeds Pvt. Ltd., Nath Seeds Pvt. Ltd. and Vibha Agro-tech Pvt. Ltd. These companies have made considerable progress and Bt brinjal hybrids expressing cry1Fal gene are at the confined limited and multi-location research trial stages.

Mode of Action of Bt To Control FSB:

Bt brinjal hybrids containing cry1Ac gene express Bt protein in all parts of the plant (i.e. constitutive expression) throughout its life cycle. To get activated and exhibit insecticidal property, Bt protein must be ingested by FSB. When FSB larvae feed on Bt brinjal plants, they ingest Bt protein along with plant tissue. In the insect gut, it is solubilized and activated by gut proteases generating a toxic fragment.

The activated insecticidal protein then binds to two different receptors in a sequential manner. The first contact of the insecticidal protein is with the cadherin receptor, triggering the formation of oligomer structure. The oligomer then has increased affinity to a second receptor, amino-peptidase-N (APN).

The APN facilitates insertion of the oligomer into membrane causing ion pores. These events disrupt digestive processes such as loss of trans­-membrane potential, cell lysis, leakage of the mid-gut contents and paralysis that in turn cause the death of fruit and shoot borer.

This exemplifies how Bt technology can work as a safe and viable strategy for insect-pest management in brinjal and other potential vegetable crops like cauliflower, cabbage, okra and chilli. Bt proteins require certain specific conditions to be active.

These include:

1. Bt protein has to be ingested by the target insect. In the case of Bt brinjal, FSB larvae ingest Bt protein when the larvae feed on plant tissues.

2. Bt protein requires an alkaline gut with a suitable pH (9.5 and above) for its activation.

3. Presence of specific receptors particularly cadherin and APN, in the insect mid-gut epithelial cells is required for protein binding.

All these specific conditions are met by FSB larvae and therefore they succumb when they feed on Bt brinjal. Bt brinjal does not harm or pose any threat to higher order organisms and non-target organisms as they lack specific receptors and conditions for activation of Bt protein in their gut.

So, Bt brinjal is safe for consumption by all non-lepidopteran insects, birds, fish, animals and human-beings. Owing to its in-built insect resistance and specificity, Bt technology is regarded as a superior technology for control of target pests, in this case FSB, either alone or as one of the important components of integrated pest management (IPM).

Bt Brinjal – The Regulatory Framework In India:

The research and product development in rDNA technology or biotechnology are strictly regulated in India. The regulatory framework is well established and functional for the past two decades.

The Ministry of Environment and Forest (MoEF) notified the EPA Rules ‘Manufacture /Use/Import/Export and Storage of Hazardous Microorganisms, Genetically Engineered Organisms or Cells way back in 1989 under the provisions of the Environment Protection Act (EPA), 1986.

The EPA Rules 1989 clearly define the composition, function and roles of competent authorities. These are responsible for regulating various biosafety, food safety, agronomic evaluation and environmental concerns of biotech crops as shown in Table 38.6.

The Ministry of Environment and Forest (MoEF) and the Department of Biotechnology (DBT) coordinate the implementation of various provision of the Rules 1989 which are assigned to relevant ministries, state governments and public sector institutions.

The competent authorities under the Rules 1989 have framed guidelines, protocols and procedures for evaluating biosafety, toxicity, allergenicity, field trials, food and feed safety, production processes, large-scale use of genetically modified organism (GMOs) and products thereof and their release into the environment.

A science-based regulatory system for GM crops has been developed in India with the efforts of MoEF and DBT. The Indian regulatory system is considered by many national and international experts as one of the most robust regulatory systems in the world.

Some of the salient features of the Indian regulatory system are the following:

a. Provision of revision and improvement of regulatory guidelines from time to time, keeping pace with the rapid biotechnological advances taking place in other parts of the world.

b. Seven layers of regulatory committees to keep track of product development.

c. ICAR as a parallel system to check and validate data submitted by the technology developers.

d. Periodic revision of biosafety and food safety guidelines and also field trial protocols to ensure foolproof system.

e. Time-bound approval system to track progress and take corrective action, if any, and,

f. Post-commercial monitoring system to review safety and evaluate performance.

Chronology of the Development and Approval of Bt Brinjal In India:

2000:

Transformation and greenhouse breeding for integration of cry1Ac gene into brinjal hybrids and seed purification.

2001- 2002:

Preliminary greenhouse evaluation to study growth, development and efficacy of Bt brinjal.

2002- 2004:

Confined field trials to study pollen flow, germination, aggressiveness and weediness, biochemical, toxicity and allgergenicity studies and backcrossing into the regular breeding programme.

2004:

RCGM approves conducting multi-location research trials of seven Bt brinjal hybrids.

2005:

Through a MoU under the aegis of Agri-biotechnology Support Programme II (ABSP II) of USAID Mahyco shares the technology with TNAU, DAU and IIVR to develop open-pollinated verities of Bt brinjal, back-crossing and integration of EE 1 into 4 verities of TNAU, Coimbatore and 6 verities of UAS, Dharwad is done.

2004-05:

Biosafety data on the effects of Bt brinjal on soil micro-flora, efficacy against fruit-shoot borer, pollen flow, germination, aggressiveness and weediness; toxicity and allergenicity studies, chemical composition etc. submitted to the Review Committee on Genetic Modification (RCGM). RCGM recommends large scale trials to the GEAC.

2006:

1. Mahyco submits bio-safety data to Genetic Engineering Approval Committee (GEAC) and seeks permission for large scale trials.

2. GEAC posts the biosafety data on Bt brinjal on GEAC website.

3. GEAC constitutes a subcommittee to look into the concerns raised by civil society.

4. Supreme Court stops ongoing field trails of GM crops due to a PIL filed by civil society representatives.

2007:

a. The subcommittee [Expert Committee 1] submits its report, recommends that 7 more studies on bio-safety be repeated for reconfirmation of data generated during confined multi-location trials but gives a green signal for large scale trials.

b. Supreme Court lifts ban on GM crop field trials subject to conditions such as isloation distance etc.

c. GEAC approves large scale trial.

d. As per GEAC direction, Indian Institute of Vegetable Research [IIVR] takes up the responsibility of large scale trials of Mahyco’s Bt brinjal trials at 10 research institutions across the country in 2007 and 11 in 2008.

2009:

January- IIVR submits the results of the large scale trials. Due to concerns raised by several stakeholders including some national and international experts, GEAC constitutes a 2nd sub-committee [Expert Committee 2 or EC2] to look into adequacy of biosafety data generated as well as the concerns raised by all stake holders.

2009 Oct. 14^th:

The Subcommittee submits its report based on which GEAC approves the environmental release of Bt brinjal containing the event EE1. However, GEAC in its 97th meeting held on 14 Oct, 2009 observed that “……………… as this decision

of the GEAC has very important policy implication at the national level, the GEAC decided its recommendation for environmental release may be put up to the Government for taking final view on the matter. The GEAC, being located in the Ministry of Environments and Forests, sent its recommendation to the Union Minister of State for Environment and Forests (Hon. Sri Jairam Ramesh).

2009 Oct. 16^th:

Responding to strong view expressed both for and against the release of the Bt brinjal, the Minister of State for Environment and Forests (I/C) (to whom the GEAC reports) announces a nationwide consultation in January and February of 2010 (13-1-10 to 6-2-10) pending a final decision of this issue.

2010 Feb.9:

Union Minister of State for Environment and Forests after considering all the views of the proponents and the opponents, adopted a cautious, precautionary principle-based approach and imposed a moratorium on release of Bt-brinjal till such time independent scientific studies establish, to the satisfaction of both public and professionals, the safety of the product from the point of view of its long term impact on the human health and environment, including the rich genetic wealth existing in brinjal in our country.

2010 Sep:

Six academies submit a joint report stating that Bt brinjal should be released. However, Honble’ Minister of Environment and Forests refused to lift the moratorium.