DEVELOPMENT OF BIOTECHNOLOGY IN INDIA · DEVELOPMENT OF BIOTECHNOLOGY IN INDIA ... basic research...
Transcript of DEVELOPMENT OF BIOTECHNOLOGY IN INDIA · DEVELOPMENT OF BIOTECHNOLOGY IN INDIA ... basic research...
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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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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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