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V. P. Gupta*Compiled by

DEVELOPMENT OF

BIOTECHNOLOGY

IN INDIA

he Government of India accorded high priority to biotechnology by settingup a Department of Biotechnology (DBT) under the Ministry of Science

& Technology in 1986. The DBT serves as a focal point for planning, promoting

and coordinating biotechnological activities and research programs in the country.This has given a new impetus to the development of the field of biology and

biotechnology. Apart from the DBT, other government departments and agencies

such as the Department of Science & Technology, Council for Scientific andIndustrial Research (CSIR), Indian Council of Agricultural Research (ICAR),

Indian Council of Medical Research (ICMR), and many industries support bio-

technological programs in the country.

In India, not only the application of the knowledge and techniques of molecular

biology is considered as biotechnology but a wider definition given by theOffice of the Technology Assessment of the US Congress in 1984, which

defines biotechnology as ‘A technology that uses living organisms or components

of living organisms to make or modify products and to improve plants or

animals and micro-organisms for specific uses,’ is accepted.

Human resource development, creation and augmentation of germplasm re-

positories, infrastructural facilities and centers of excellence, basic research inidentified areas, product and process development, demonstration and field

trials have been the priorities. Various multi-institutional and multi-disciplinary

projects of national importance have been pursued. Through its multifacetedapproach in furthering advanced research and development in biotechnology,

the DBT has given a definite thrust to the sectors of agriculture, human health,

livestock, environment and industrial development. Some of the salient featuresare briefly presented here.

T PLANT BIOTECHNOLOGY

Development of Transgenic Crops

In the development of transgeniccrops, much emphass is on engi-neering plants for value addition,development of male sterility, tole-rance against various biotic andabiotic stresses, etc. Importantachievements in this field are givenbelow.

The grain Amaranth is a pseudo-cereal with a high protein content —17–19% of dry weight as comparedto traditional crops, which have anaverage of 10% protein. AmA1, aseed albumin protein of Amaranth, isrich in essential amino acids suchas lysine, tryptophan, and sulfurcontaining amino acids, which areusually present in limited quantitiesin cereals and legumes.

In an attempt to improve the nutri-tional quality of potatoes, the codingsequence of AmA1 gene was stablyintroduced into potato plants using asimple and reproducible regenerationprotocol. The protein was expressed

*Director, Department of Biotechnology (DBT), Ministry of Science and Technology, CGO Complex, Lodi Road, New Delhi 110 003,India. Dr. Gupta has been working with the DBT for the past 14 years. At present, as the director, he coordinates and monitorsnational programs in agriculture biotechnology covering various research projects for the development of transgenic crops,containment facility, the National Centre for Plant Genome Research, etc.

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Spodoptera litura larvae (5th instar) showing 100% killing following 48 hours of

feeding on transgenic leaf expressing synthetic Cry gene (left). During the same

time period, the larvae fed on control leaves had ingested eight leaves (right).

in the transgenic tubers and a signi-ficant increase in most of the essentialamino acids was observed. On anaverage, the amount of essentialamino acids in tubers increased bytwo to seven fold.

Oxalate decarboxylase (OXDC)gene has been cloned into tomatoto remove the nutritional stress factor— mainly oxalic acid. The transgenictomato showed stable expression ofthe foreign gene and also accumulatedvery little oxalic acid in comparisonto wild type plants. The transgenicplants were also found to be re-sistant to infestation by Sclerotiniasclerotiorum.

For developing transgenic cottonresistant to bollworm, prolific somaticembryogenesis was achieved in cokercultivar. By using synthetic Cry 1CEgene and np II (kanamycin resistance)genes as selection markers, transfor-mation was achieved in both cokerand agronomically important Indiancultivars. Transgenic tobacco plantshave been developed, taking them asa model system to assess the per-formance of this synthetic gene.

On feeding Spodoptera litura larvae,100% killing of larvae up to the 5thinstar was observed in some of thetransformed tobacco plants.

Molecular methods have beendeveloped for hybrid seed productionusing barnase and barstar genes, andtransformation of Brassica juncea cv.varuna has been achieved with theseconstructs. In order to develop insectresistant transgenic Brassica lines,synthetic CryIAbs gene was mobilizedinto Agrobacterium tumefaciens, andtransgenic cabbage plants wereobtained after selection on kanamycin.For developing transgenic fungalresistant Brassica, cDNA constructsof basic chitinase of tobacco with35 S promoter and NOS terminatorin an Agrobacterium vector was usedsuccessfully for transforming B.juncea. Transgenic brinjal, tomato andpigeonpea with BT gene have beendeveloped and tested in open-fieldswith considerable success.

In rice, protoplast culture and plantregeneration methods have beenperfected. Besides this, RFLP andRAPD methods have been used for

tagging genes responsible for blastresistance. In the area of developingtolerance against salinity and drought,capability has been developed togenerate transgenic plants harboringpyroline-5-carboxylase synthase(P5 cs) under constitutive promoter.Work on tagging gene(s) for leaffolder and Brown Plant Hopper(BPH) resistance in rice also yieldedgood results. Shoot apices of japonicaand indica rice were transformedusing an Agrobacterium strain har-boring a binary vector containingsynthetic Cry1A(C) gene.

In a project on wheat, two geneswere tagged successfully for qualitytraits. Full length cDNAs of triticinewere isolated and strong promotersfor major wheat seed storage pro-teins glutenin and gliadins havebeen cloned using PCR methods. Aprotocol has been developed forinduction of somatic embryogenesisin wheat, and the protocol is beingutilized for transformation of wheatemploying co-cultivation with Agro-bacterium and biolistics techniques.

Under the programs of molecularbiology of fruit ripening, near fulllength clone of ACC synthase genefrom banana has been cloned andpartially sequenced. Further charac-terization of ACC synthase transcrip-tion during ripening process of bananafruit is in progress.

In a project on wheat, two

genes were tagged successfully

for quality traits. Full length

cDNAs of triticine were isolated

and strong promoters for major

wheat seed storage

proteins glutenin and gliadins

have been cloned using

PCR methods.

”

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Tissue culture studies in maggar bamboo.

National Centre for PlantGenome Research

This is a new center established in

New Delhi to carry out work on

structural, functional and applicationgenomics of selected crop plants. The

center has already initiated work

on utilization of molecular biologyapproaches along with tissue culture

and genetic engineering techniques to

identify important genes and use themfor developing transgenic plants with

improved agronomic traits and patho-

gen/abiotic stress resistance.

Plant Tissue Culture

A large number of R&D projects are

being pursued in various Indian

institutions for perfecting micropro-pagation protocols for important forest

trees, horticulture and plantation

crops. Two tissue culture pilot plantswere established in the country for

large scale production of elite planting

material with the main objective ofdemonstrating the field performance

of tissue culture raised plants of

forest trees. Each pilot plant has a

production capacity of 1.0 millionplantlets per annum. Recently the

pilot plants were converted into

Micropropagation Technology Parks(MTP) to provide an effective

platform for transfer of proven

technologies to enterpreneurs. TheMTPs have optimized a large number

of technologies and transferred them

to the industry.

Micropropagation technologies for

the following plants have been

perfected for large scale production

and commercialization.

Forest trees — Anogeissus pendula,

Boswellia serrata, bamboos, eucalyp-

tus, neem, poplars, salvadora, teak,and Wrightia arborea.

Horticulture crops — banana,

citrus, potato, strawberry, and sugar-cane.

Plantation crops — black pepper,cardamom, coffee, ginger, tea, and

turmeric.

Two tissue culture

pilot plants for

large scale

production of elite

planting material

have already been

established in the

country.

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Tissue culture raised plants of coffee in the nursery and field.

Field demonstration of biopesticides for ‘green’ cotton production.

Biological Control of Plant Pests

The concept of Integrated PestManagement (IPM) has emergedwith increasing realization of theimportance of sustainable agriculture.Biological control fits very well in

the IPM concept of plant protection.The department is supporting pro-

grams for developing biopesticides

and their application in various crops.

Among the biocontrol agents, the

fungal antagonist Trichoderma viride

has been used as seed treatment to

prevent root pathogens. Bacillus

thuringiensis, Nuclear PolyhedrosisViruses, of Helicoverpa armigera

and Spodoptera litura and fungal

pathogens, Beauveria bassiana andNoruraea rileayi have been used

against sucking pests and bollworms.

Trichogramma chilonis and eggparasitoid of bollworms are produced

in large quantities, and used success-

fully in cotton crop.

Two pilot plants have been set up

to produce biological control agents

catering to the needs of around0.224 million ha area. The efficacy

of different biopesticides has been

successfully demonstrated in an areaSunflower treated with Trichoderma biopecticide for root pathogen.

Central row is untreated.

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Use of Blue-green algal biofertiliser(BGA) in paddy field showing

luxurient growth.

of 60 000 ha covering importantcrops like cotton, rice, chickpea,

sugarcane, groundnut, sunflower,

safflower, black gram, pigeonpea, green gram and other pulses,

cauliflower, tomato and other

vegetables.

Technology for mass production

of candidate biocontrol agents —

baculovirus, parasites, antagonistic

fungi and bacteria for economically

important crops have been transferred

to the industry.

Pheromone

A few pheromone components havebeen synthesized through divergentsynthetic strategy from 10-undecenoicacid, a readily available material fromcastor oil. Large scale synthesis ofpheromones of Chilosacchariphagus

indicus has also been achieved. Asimple slow release dispenser hasbeen developed for attracting themale insects of Chilosacchariphagus

indicus. Field evaluation of thesynthetic product led to the develop-ment of a successful mating disruptiontechnique by trail following method.Application of the pheromones hasbeen demonstrated in about 25 acres.A significant increase in cane yieldhas been observed in all the trial plots.The pheromones of the insects ofH. armigera, Chilo pertellus, Plutella

xylostella, Ducus doraslis, Agrotis

ipsilon and Trichoplusia ni havebeen synthesized, These are usefulfor IPM in a number of crops likegram, potato, sorghum, maize, redgram, cotton, tomato, cabbage,mango and others.

Biofertilizers

The DBT supports a number of

projects on development and appli-

cation of various biofertilizers. Highnitrogen fixing strains of rhizobia

and blue-green algae suitable for

various crops and agro climaticregions have been isolated and theirefficacy has been demonstrated infield conditions. The increase inpaddy yield with application of blue-green algae ranged from 13 to16.67%, whereas in pulses and oilseedcrops, application of rhizobiumresulted in yield increase of about5 to 13.5%. A network program on‘Integrated Use of Biofertilizers inNutrient Management’ for specificcropping systems prevalent in thevarious agroclimatic zones in thecountry has been taken up. Atechnology has been developed formass production of endomycorrhiza,and this has been transferred to acompany for commercialization.

In addition, a full technologypackage has been developed forgreening of fly ash ponds usingmycorrhizal organo-biofertilizers.It has been found that a variety ofplants including those of somecommercial value can be grown on

fly ash rich substrates. With smallquantity of compost and benefitsof mycorrihzal associations, theseplants can grow and establish them-selves on fly ash overburdens. Asthey grow, their roots hold the flyash together making it less prone tobeing airborne. At the same time, themycorrhizae through their mycelialnetwork, accumulate heavy metalsfrom the fly ash and retain themwithin their cells. Thus, mycorrihzaehelp in checking the heavy metalcontamination of groundwater.

Bioprospecting and BiodiversityConservation

A program is being pursued on char-acterization, inventiorization and

conservation of the biodiversity of

different eco-geographical regions andbio-prospecting of genes and bio-

molecules. Remote sensing and

satellite imaging techniques are beingused for this purpose. The bio-

prospecting is being done for the

candidate genes and molecules inorder to identify and characterize

particular genes for potential use in

other groups of plants. Bioassaystudies and chemotyping are being

carried out to identify the active

principles of utility as biocide,antifeedant and as industrial raw

material. Plants of high economic

importance are also being fingerprinted for complete characterization

and cataloging. Efforts are also being

made to conserve important plantsthrough both in-situ and ex-situ con-

servation methods, and regeneration

protocols are being developed forrapid multiplication of the elite

material.

Tissue culture propagation methodshave been developed for three en-

dangered mangrove species found

in southern coastal eco-system of

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the country. Characterization of 22

mangrove species using RFLP and

RAPD methods is in progress.Protocols for collection, evaluation,

propagation and reintroduction of

15 mangrove species have beenstandardized. More than 5000 micro-

propagated plants have been trans-

planted in semi-natural habitatsafter hardening. Low cost vegetative

propagation technique has been

developed for six species.

Three national gene banks of

medicinal and aromatic plants havebeen established in Lucknow, New

Delhi and Thiruvanthapuram, located

in different parts of the country. Thepurpose of the gene banks is to

conserve important, rare and en-

dangered medicinal and aromaticplant species used in traditional

systems of medicine. These gene

banks have collected nearly athousand accessions from different

biogeographic regions of the country

in field gene banks, seeds banks, in

vitro repository, DNA bank and under

cryopreservation.

MEDICALBIOTECHNOLOGY

New advances in medical biotech-

nology for generating greater insightin understanding molecular mecha-

nisms for disease processes at the

tissue, cellular and molecular levelshas been one of the focus of research

in biomedical sciences. The programs

under medical biotechnology havespecifically been designed for deve-

loping new diagnostics, vaccines,

new drug delivery systems andproviding genetic counseling for

genetic disorders. Basically research

programs are related to developingcandidate recombinant DNA vaccines,

genetically modified organisms as

candidate vaccines, recombinant

reagents for diagnostics, the molecular

probes for emerging and re-emerginginfections and non-communicable

disorders, molecular probes for

providing pre-natal diagnosis forgenetic disorders, drug targeting

and developing transgenic animals

for biomedical and pharmaceuticalresearch.

Diagnostics

Several immunodiagnostics have been

developed. These include anti-IgM

ELISA kit for hepatitis A, antibody

detection kit for aspergellosis, PCR

based system for tuberculosis, western

immuno blot assay for HIV-I/II

naked eyed agglutination test system

utilizing recombinant chimeric re-

agents for HIV-I/II, peptide ELISA

for HIV-I/II, ELISA system for

reproductive hormones, specific

ELISA test system for fetal antigen,

direct agglutination test system for

toxoplasmosis, LDH ELISA for

malaria diagnosis, and ELISA for

detecting specific toxins following

snake bite. Several of these test

systems have been transferred to the

industry for commercialization.

Vaccines

Various medical biotechnology pro-

grams in the country have led to the

following vaccines.

Anti-Leprosy Vaccine

This vaccine is based on the use of

the Mycobacterium MW. The vaccine

has been found to be effective asan immuno-therapeutic agent to be

given along with multi-drug therapy

for leprosy patients. The leprosypatients receiving the vaccine clear

the infection faster and develop

immunity against the lepra bacilliand do not suffer from side effects

due to the vaccine. The ‘total drug

regime of the multi-drug therapy’ is

reduced from a period of two yearsto about six months. During the

year 1997-98, the Drugs Controller

General of India (DCGI) had givenpermission to M/s Cadila Phar-

maceuticals Limited to produce

batches of the vaccine for testing ofits moisture content and contamina-

tion by the Central Research Institute

(CRI) in Kasauli. Upon satisfactoryreport from the CRI, the DCGI

permitted Candila Pharmaceuticals to

manufacture and market this vaccine.The product has been marketed under

the trademark name ‘Leprovac.’ Being

a social cause, the manufacturer has

kept the cost at a very low level.

Anti-Cholera Vaccine

The genetically modified live choleraorganism as candidate vaccine has

been developed by the Institute ofMicrobial Technology (IMTECH) in

Chandigarh, along with the National

Institute of Cholera & EntericDiseases (NICED) and the Indian

Institute of Chemical Biology (IICB)

in Calcutta. This candidate vaccinehas been proved very efficacious in

experimental model immunization

and it has led to the right type ofimmunity with no reactogenecity. The

DCGI had given permission for

phase-I human volunteer trialswhich was carried out at the Sanjay

Gandhi Postgraduate Institute of

Medical Education and Research(SGPGIMER) in Lucknow and

NICED in Calcutta. The candidate

vaccine was found to have no sideeffects, or reactogenecity. Upon com-

pletion of phase-I trials and analysis

of the data so generated under thistrials, the candidate vaccine is

expected to undergo the phase-II

clinical trials soon.

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Confluent monolayers of vero cells infected with rabies vaccine strain in microcarrier culture.

Anti-Fertility Vaccine

The anti-fertility vaccine based on

βhCG to control fertility in women

had earlier undergone phase-II as well

as extended phase-II human volunteer

clinical trials. Analysis of the data

indicated that about 60 percent of

the women who received this vaccine

developed specific antibodies at

desired levels. However, the antibody

level started to decline within a short

period. Therefore, a new formulation

needs to be designed so as to obtain

the predictable and sustainable

immune responses in all vaccine

recipients. Efforts in this direction are

being made at the National Institute

of Immunology, New Delhi. Utilizing

the right type of immuno potentiator,

desired degree of immune response

in a more or less predictive fashion

has been achieved in experimental

animals only. More detailed studies

are needed before βhCG could

again be taken up for clinical trials.

The recombinant βhCG has been

produced so that enough material

is available for any future trials.

Similar observations on the limited

phase-I trial for oFSH for controlling

male contraception have been made.

Modification in formulation is being

worked out.

Anti-Rota Viral DiarrhoealVaccine

Two strains of rota virus, which do

not produce disease in infants and

children, have been isolated. Thisstudy has been carried out under the

Indo-US Vaccine Action Program.

A pilot scale production of thiscandidate vaccine strain has been

initiated in laboratories in the US.

It has already successfully undergonephase-I clinical trials in human

volunteers in the US. Negotiations

are being made with two Indian

pharmaceutical companies to take upupscaling/pilot scale production of

these candidate vaccines for phase-I

clinical trials in India. The DCGI hasalready given permission to undertake

such trials for one of the candidate

vaccine strains.

Japanese Encephalitis (JE) Vaccine

The currently used JE vaccine is from

the virus grown in suckling mouse

brain. In this vaccine, despite puri-fication procedures, it is difficult to

remove the contaminated brain tissue,

which is known to lead to many sideeffects. To overcome this, scientists

at the National Institute of Immuno-

logy have attempted to grow the virususing tissue culture techniques in a

way which gives rise to the required

number of virus particles to producethe vaccine. Such a virus strain has

been adopted to grow in vero cell line

utilizing a micro carrier technology.Efforts are now being made to use

this virus to produce the candidate

vaccine as a replacement for themouse brain driven JE vaccine. In

addition, a candidate DNA vaccine

has also been identified. This hasbeen found to produce the requisite

immune response in animals.

Anti-Rabies Vaccine

A DNA sequence has been identifiedfor its potential use as a vaccineinitially for control of rabies inanimals and subsequently in treatmentof human beings. The pre-clinicaltesting, including toxicological assess-ment, are currently being initiated.

Anti-Tuberculosis Vaccine

Since the existing BCG has variabledegrees of protection especially insouthern states of India, a recom-binant BCG carrying a gene forM. tuberculosis has been developed.Its immunogenicity and efficacy inanimals are being assessed.

Anti-Malaria Vaccine

Two significant candidate vaccinesare being followed up. One of themis based on stitching nine differentgenes from different stages ofPlasmodium falciparum which hasbeen expressed in a Baculovirus ex-pression system. The recombinantproteins have been shown to producethe immune response in animals. Theefficacy trials in animals have beenplanned. The second candidatevaccine — a peptide which is com-mon to P. falciparum and P. vivex —

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has been identified and found toeliciate appropriate immune responsein animals. This peptide has beenfound to have no side effects. Thetoxicity analysis of this peptide hasalso been made. The DCGI has beenapproached to clear this candidatevaccine for the phase-I humanvolunteer trial.

Anti-HIV Vaccine

A group of scientists has been giventhe challenge to design and developanti-HIV sub-type C vaccine for thecountry. Efforts in this direction havejust been initiated.

Program on Infectious Diseases

A multi-centric program to under-stand the genotype of HIV-I/II incirculation in different geographic

regions of the country led to identi-

fication of HIV subtype C to be pre-determined HIV-I virus, although

other subtypes like B and E have

also been identified. In addition,recombinant HIV-1 virus has also

been identified. This information is

important for designing the righttype of HIV vaccine for the country.

An assessment on cytokine analysis

of sero-positive and sero-negativesubjects is underway. Studies have

been initiated on co-receptors and

their role in genetic resistance to HIVinfection.

Several programs at cutting edgescience in relation to tuberculosis,typhoid, Helicobacter pylori, leish-maniasis, chikungunya, JapaneseEncephalitis, etc., have led to signi-ficant understanding of diseasesprocesses and identifying a suitablecandidate vaccine to control theseinfections. Efforts to develop vaccinesalternative to BCG is also in pro-gress. Total genomic mappingof Leishmania donovani, Shigella

dysentery and Plasmodium vivex hasyielded significant lead in terms ofidentifying new drug targets. Effortsare also being made to developedible vaccines for use by children.Vibrio cholerae gene in tomato/banana has yielded appropriate levelof expression. More in-depth studiesin designing edible vaccines arebeing initiated.

Program on Non-CommunicableDiseases

The focus has been to develop dia-gnostics for cancer especially oral/cervical and breast cancers. Molecularregulation in these cancers is alsobeing studied.

Human Genetics and GenomeAnalysis

Fourteen genetic counseling clinicshave been established for providingmolecular diagnosis, counseling forcommon genetic disorders such asthalassemia, duchenne musculardystrophy and other haemopathies.A major program on functionalgenomics in relation to Schizophreniaand other neurogenetic disorders has

provided insight in understandingthese neurogenetic disorders. Anothermajor initiative is the study of humangenome diversity in different popula-tions of India in order to understandthe genes responsible for suscepti-bility or refractiveness to diseasesand disorders. Once these programshave been completed, more effectiveand new diagnostics, drug targets andeffective vaccines are expected to beavailable for the healthcare system ofthe country.

ANIMAL BIOTECHNOLOGY

Overall efforts in animal biotechno-logy are aimed at production andproductivity enhancement throughprojects on embryo transfer, health,nutrition, etc. Some of the achieve-ments in this field are given below.

Embryo Transfer Technology(ETT)

ETT in cattle and buffaloes has beensuccessfully disseminated to farmersresulting in birth of about 1000 calves.ETT cows produce 3000–6000 litersmilk per lactation. DNA markers forsex determination of embryos duringembryo transfer were developed andfield tested. This technology is nowa customized service.

To alleviate diseases in animals,major projects for development ofvaccines and diagnostics have beenundertaken. All validation trialshave been completed for tissueculture based vaccine for New Castle(Raniket) disease in poultry andinfectious bovine rhinotracheitis(IBR) disease in cattle. The IBRvaccine technology has been trans-ferred to the industry.

ETT in camel was standardized anda new protocol for camel super-ovulation was developed for the first”

“A major program on

functional genomics in

relation to Schizophrenia

and other neurogenetic

disorders has provided

insight in understanding

these neurogenetic

disorders.

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time. Expression of bovine growth

hormone gene in E.coli bacteria and

preparation of reconstituted collagen

sheet of bovine origin for wound

healing applications are other high-

lights.

Transgenic mice carrying hepatitis

B virus X gene have been developed

in order to study the mechanism of

hepatocellular carcinoma. Transgenic

animals carrying fragile X gene have

also been developed to observe the

mechanism of X fragile syndrome,

which leads to mental retardation.

Transgenic mice have been produced

by injecting 1.9 kb linear construct

of neomycin resistant gene under the

control of pPGK promoter. These

may be used as a primary source of

feeder cells for growing embryonic

stem cells in order to maintain their

totipotency and to observe the effect

of neomycin resistant gene widely

used for human gene therapy.

Vaccines

Rabies vaccine for animals using

laboratory rabies strain PV-11 seed

virus has been produced. The potency

of the bulk vaccine was found to be

3.6 IU by NIH test approved by the

World Health Organization (WHO).

The vaccine is currently being sub-

jected to sterility, identity, toxicity and

potency tests in laboratory mice,

rabbits and guinea pigs. Recom-

binant protective antigen of anthrax

(rPA) was purified and found to be

biologically as well as functionally

active and on par with the native PA

purified from B. anthracis. This may

be used to develop an effective

recombinant vaccine against anthrax.

Recombinant dog zona pellucida

peptides have also been developed

and a process to grow these in a batch

fermenter has been optimized. This

will be helpful in controlling the straydog population.

Diagnostics

Immunodiagnostic tests for trypano-

somiasis including double immuno-

diffusion test, Card agglutination

test (CAT) and ELISA have been

standardized and evaluated using

serum from experimental as well as

natural cases of trypanosomiasis in

camel, buffalo and cattle. The Ag-

ELISA test developed in the project

was also evaluated using serum

samples obtained from various re-

search institutes. The efficacy of Ag-

ELISA test was between 75-80%

under field conditions.

Production of monoclonal anti-

bodies against PPR (a disease in

cattle) has been undertaken and the

virus is being propagated in a vero

cell line. A PCR method has been

standardized by amplifying the DNA

from live and inactivated tissue

culture viruses. PCR based detection

of Equrine Herpes Virus-1 (EHV -1)

was also standardized using Pneuma-

bort ‘K’ vaccine as a positive control.

EHV-1 virus is being propagated in

BHK-21 cell line and this has been

confirmed by PCR test.

AQUACULTURE

This is a major food producing

sector with emphasis on enhancing

production and productivity through

established culture systems and

culture of the non-traditional species.

Biotechnological applications in the

areas of health, nutrition, feed, and

genetics, among others, have received

much attention. Some of the signi-

ficant achievements are given below.

A production of about 18 tonnes

per hectare of carp per year has been

achieved through intensive carp

farming and similarly, tiger prawn

production of more than 10 t/ha was

demonstrated through semi-intensive

farming. A hatchery with 10 million

capacity has also been set up for

freshwater prawn at the Central

Institute for Freshwater Aquaculture

(CIFA) in Bhubaneshwar, along

with a feed mill which is now in

production.

Transgenic mice harbouring neomycin resistant gene.

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Genomic libraries of Catla catla,

Labeo rohita and Clarias batrachus

have been constructed. Rainbow trout

growth hormone gene has been

integrated in catfish. Presumptive

transgenic catfish on analysis with

southern blot analysis confirmed

genetic integration. Molecular cloning

of fish growth hormone cDNA has

been achieved and recombinant

clones were developed in Indian

major carp, rohu (Labeo rohita), and

catfish (Heteropneustes fossilis).

A vibrio based immuno-stimulanthas been developed for use againstwhite spot disease in prawn. Anindigenous prawn feed technologywas developed using locally availableraw materials. Three grades of feedsnamely starter, grower and finisher,with protein levels of 42%, 38% and35% respectively were produced forPenaeus monodon and P. indicus. Thefeeds were tested in yard experimentsas well as in farmer’s growout ponds.Both tiger prawn and white prawnshowed excellent growth with anaverage Feed Conversion Ratio (FCR)ranging from 1.6 to 2.0, and this feedis comparable with commercial feeds.The pilot scale production of the feedhas been successfully carried outusing a commercial pellet mill. Thetechnology package currently is beingtransferred to enterpreneurs. The feedis suitable for semi-intensive andextensive culture of tiger prawn andwhite prawn. The cost is about Rs.30/kg. Development of a technologyfor effective utilization of silkwormpupae for aquaculture feeds isunderway.

Other leads obtained are on cryo-

preservation of prawn embryo, setting

up of hatcheries for mussels andclams, development of culture techno-

logy for freshwater pearls, develop-

ment of fish spawning agents, etc.

Important results have been generatedfor the development of bioreactor

systems for prawn hatchery water

treatment and towards developmentof transgenic fish using indigenous

constructs.

SERI-BIOTECHNOLOGY

The DBT has identified thrust areas

in sericulture (both mulberry and non-

mulberry) in which biotechnology

can play a vital role in increasing

productivity, enhancing silk quality

and improving host plants.

A method has been developed

for gene delivery into the silkworm

(Bombyx mori) using the sperm as a

vector. Expression of green fluore-

scent protein (GFP) has been achieved

in cell lines/larval caterpillars of

Bombyx mori after infecting them

with recombinant Bm NPV harboring

reporter gene (GFP). Studies have

been initiated for expression of

hepatitis E viral antigen using this

system.

Degree of polymorphism has been

assessed in 13 strains of silkworm

(Bombyx mori) using cloned micro

and mini-satellites as probes coupled

with PCR. A dendrogram has been

generated using different molecular

assays for 13 silkworm strains. The

genetic analysis of recombinant lines

using micro and mini satellite markers

showed that the diapause and non-

diapause traits of silkworm are linked

to some of the quantitative trait loci

(QTL) associated with cocoon shell

weight and fecundity. The studies will

provide important contributions to the

Silkworm Genome Project.

An efficient protocol has been

developed for regeneration of mul-

berry from axillary buds and a proto-

col has also been developed for

production of gynogenic haploids

and triploids in mulberry. Fourteen

triploid plants of CV S-36 have been

transferred for field evaluation.

Under a demonstration project

for controlling uzifly, the increase

in cocoon yield by integrating chemi-

cal and biological methods using

Nesolynx thymus has been recorded

to be about 25 kg/100 DFLs over

controls in tribal villages of Tamil

Nadu state.

Expression of Green Fluorescent Protein (GFP) in Bombyx mori cell line infectedwith recombinant Bm NPV harbouring GFP reporter gene (A—in normal light,B—in fluorescent light).

BA

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Oyster mushroom on coffee pulp.

A process has been developed fordegumming of silk with fungal pro-teases as a viable alternative to theexisting methods of degumming. Apatent application has been filed forthis process.

The silk proteins (fibroin andsericin) from non-mulberry silk-worms — muga (Antheraea assama),tropical tasar (A.mylitta) and erisilkworm (Philosamia ricini) havebeen purified and characterized.

Under the Silkworm GenomeProgram, about 900 clones (moderateto highly repetitive) were selectedfrom the genome library constructedin PUC 18 to convert them assequenced tagged sites.

Out of 24 genotypes of castorevaluated, RG-323 genotype fed tothe eri silkworm recorded high larvalweight, effective rate of rearing (ERR)and fecundity as compared to localcultivar (Aruna).

FOOD BIOTECHNOLOGY

Development of bioprocess techno-logies, value addition to foods, andfood safety aspects have been empha-sized under the program. Some of theimportant achievements have beensummarized here.

A microbial process for productionof polysaccharide Xanthan gum hasbeen developed, in which the ex-pensive organic nitrogen sourcehas been replaced with inexpensiveammonium nitrate and corn steepliquor. The xanthan obtained hasbeen used in selected foods such asfruit squashes, extruded products,traditional foods like idli (steamedrice cake), etc. The toxicity studiesshowed no adverse effects.

Complete process has been deve-loped for (a) maintenance of culture

and production of spawns of oyster

mushroom, (b) commercial produc-

tion of oyster mushroom — rural

model (production 50kg/batch) and

urban model (production 100 kg/

batch), and (c) dehydration of oyster

mushroom. In addition, various

mushroom delicacies like mushroom

pickle, sweet chutney, instant soup

mix, mushroom wafers and dry

mushroom powder have been deve-

loped to optimize its use.

Protocols have been standardized

for detection of food toxicants,

pesticide residues, and colors leaching

from colored plastic containers. Also

a network research program has been

launched for development of rapid

diagnostic kits using PCR technology

for detection of various food borne

pathogens.

Production of low cholesterol egg

— Super critical carbon dioxide

extraction technique has been deve-

loped to reduce the cholesterol

in liquid egg and spray dried egg

powder. The recovery of cholesterol

was high (90%) in spray dried whole

Multiple shoots from leaf explants of Morus indica var. K2 after

10 days of culture.

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64 APBN • Vol. 4 • No. 4 • 2000

egg powder. Phospholipids were notfound to be affected.

Biodegradable eco-friendly packag-

ing films for preservation of fresh

fruits and vegetables and processed

foods have been developed and tested

for their performance. These films

extend the shelf life of fruits and

vegetables by 21 days at ambient

temperature and over 40 days at

cold storage temperature.

Development of low cost nutri-

tious food supplements for mal-

nourished children — Efforts have

been made to develop and formulate

low cost food supplements for

school going children in the age

group of 6–14 years so as to fill up

the gap in their energy requirements.

Through biotechnological endeavor,

four formulations have been deve-

loped which contain more than 15%

protein, 25% fat, 35% carbohydrates

and have more than 400 K.Cal. in

100 gm. These supplements would be

in the form of ready to eat food and

shall cater to regional tastes and

preferences.

ENVIRONMENTALBIOTECHNOLOGY

A biosensor based on the enzymeacetylcholine esterase for detectionand estimation of organo-phosphorouspesticide residues in water has beendeveloped. The biosensor has beensimplified to the form of a dipstickand this technology has been trans-ferred for large scale use.

Technologies based on DNAprobes and ELISA technique forthe detection of Entamoeba andpathogenic viruses in drinking waterhave been developed.

A technology for precombustionbio-benefication of coal containinghigh pyritic sulfur and ash andprecombustion desulfurization ofgaseous fuels and emissions con-taining hydrogen sulphide withconcomitant recovery of elementalsulfur has been developed.

A two-step approach for remedia-tion of crude oil spills has also beendeveloped. The process includesapplication of cost efficient adsorbent,

alkali treated sawdust, as the first stepto recover most of the spilled oil. Thesecond step involves biodegradationof remaining oil by using a bacterialconsortium for removing xenobio-tics. The degradation process is thuscost effective and environmentallybenign.

An integrated biotechnologicalapproach has been developed forbioremediation of mine spoil dumps.The process leads to the develop-ment of supportive and nutritiverhizosphere in manganese and coalmine spoil dumps through appropriateblending of spoil and organic wastesfor the establishment of plant-microflora and inoculation of plantswith specialized cultures of nitrogenfixing microorganisms. Strains ofendomycorrhizal fungi are used forprofuse root development and stresstolerance in plants.

A complete process for treatingazo-dyes wastewater has been deve-loped on a pilot scale. The processmakes use of phragmites and typhawetlands and involves a consortiumof microbes. Trials of dyes wastewater

Bioremediation of hydrocarbon contaminated land at an oil refinery with oilzapper: live culture of oil degrading bacteria.

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treatment were conducted, andoptimal conditions for dyesdegradation in the system wereidentified. The treated waste waterhad no adverse effects on the growthof most susceptible aquatic plantspecies such as Azolla, Hydrilla, andSpirodela, as well as cultivated onessuch as Cymopsis. The process hasbeen validated by the Association ofDye Manufacturers.

A marine strain of Yarrowia lipo-

lytica, which can degrade 58% of

the oil present in treated palm oilmill effluent within 5 days, has

been isolated. The process is being

upscaled on a pilot level at the siteof a palm oil mill.

BIOINFORMATICS

The Biotechnology Information Sys-

tem (BTIS) Program of the DBT hasbeen launched to harness the scientific

knowledge in various interdisciplinary

sciences of biotechnology. The pro-gram is a satellite based network

system namely the BTISnet which

covers 48 centers including 10 Dis-tributed Information Centres (DICs)

and 38 sub-DICs spread all over the

country covering most of the states.Besides providing communication

facilities among scientists and

institutions engaged in biotechnogicalresearch and applications, the program

facilitates in data analysis, modeling

of biological macro-molecules andtheir complexes, metabolic path-

ways, designing of new molecules

such as drugs, peptides, vaccines,proteins, etc.

BIOSAFETY

The DBT has already formulated a

detailed ‘Recombinant DNA Safety

Guidelines’ in respect of measures for

the research activities, large scale use

and also the environmental impact

during field application of genetically

altered material and their products.

The level of risk and the classi-

fication of the organisms within these

levels based on pathogenicity and

local prevalence of diseases and on

epidemic causing strains in India are

defined in these guidelines. Some

of the microorganisms not native to

the country have been assigned to a

special category requiring highest

degree of safety. These include

lassa virus, yellow fever virus, etc.

Appropriate practices, equipment

and facilities are recommended for

necessary, safeguards in handling

organisms, plants, and animals in

various risk groups. The guidelines

employ the concept of physical and

biological containment and are also

based upon the principle of good

laboratory practices.

The concern does not diminish

when it comes to the use of re-

combinant organisms on large fer-

mentation operations or applications

of it in the environment. As such,

the guidelines prescribe for good large

scale practices for using recombinant

organisms which include measures

such as proper engineering for con-

tainment, quality control, personnel

protection, medical surveillance, etc.

Application and release of engi-

neered organisms into the environ-

ment could lead to ecological con-

sequences and potential risks unless

necessary safeguards are taken

into account. The guidelines outline

a criteria for assessment of the

ecological aspects on a case by

case basis for planned introduction

of rDNA organisms into the

environment.

The guidelines also suggest re-gulatory measures to ensure safetyfor import of genetically engineeredmaterials, plants and animals. Therecommendations also cover thevarious quality control methodsneeded to establish the safety, purityand efficacy of rDNA products.

All institutions involved in such re-search have to constitute an Insti-tutional Biosafety Committee (IBSC)to monitor their research work forstrict compliance of the rDNA safetyguidelines. The Review Committeeon Genetic Manipulation (RCGM)functions under the DBT, whichissues clearance for import/export ofvectors, germplasms, organelle, etc.,needed for experimentation. It alsoevaluates research data beforeupscaling the laboratory data. Re-garding category III experiment, priorapproval of RCGM is required. TheGenetic Engineering Approval Com-mittee (GEAC) functions under theMinistry of Environment & Forestswhich examines and issues clearanceafter looking into environmentalsafety issues. These guidelines havebeen notified under the EnvironmentalProtection Act 1986 and the DBT isactively implementing the rules andprocedures to monitor the rDNAresearch work in India.

The DBT has been continuing itsefforts in creating research facilities,development of human resource,basic research, product, process andtechnology development in areas ofbiotechnology. Efforts are being madeto complement and supplement in thegap areas where biotechnologicalinputs could enhance the productivityand efficiency of the concernedsectors, provide easy to use, low costand environmentally sound solutionsaiming at sustainable developmentand overall socio-economic progressof the nation.

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