| Plant Transgenics |
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| Group Leader |
Dr. D. V. Amla, Sci
'F' |
Contact
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Work
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91-522-2205838
Ext-333, 334 |
| Home |
91-522-2393261 |
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Other Scientists of the Group
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Dr. Indraneel Sanyal, Scientist
'B'
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Group works on
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Development of stable transgenic plants
of chickpea/pigeonpea and tomato expressing BT-endotoxin gene
for insect pest resistance.
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Expression of human alpha-antitrypsin (AAT)
gene in transgenic plants.
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Objectives
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Project activity is aimed to accomplish
development of geneotype independent, rapid and efficient
in vitro regeneration procedures along with DNA mediated complementary
transformation systems in recalcitrant plant species particularly
grain legume chickpea (Cicer arietinum L.), pigeonpea
(Cajanus cajan L.) and tomato (Lycopersicon esculentum)
to raise their stable transgenic lines resistant to pod borer
insect Heliothis armegera through sufficient expression
of insecticidal crystal protein CryIA (native and highly modified)
transgenes of Bacillus thuringiensis (BT). This include
to elucidate following components:
- Characterization and localization of competent regenerative
tissues in recalcitrant plant species and their in vitro
proliferation.
- To introduce insect resistance single gene trait of
Bt-CryIA or pyramiding of these genes;
- Performance and inheritance pattern of transgene analysis.
- Designing, chemical synthesis of modified human α-antitripsin
(AAT) gene for high level expression in dicot plants as bio
reactor for production of AAT protien.
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Achievements
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- Extensive in vitro regeneration studies on different combinations
of growth regulators with various explants we have characterized
the localization of potent competent regenerative cells in different
mature and developing explants of chickpea and pigeonpea, which
on appropriate combinations of growth regulators may be triggered
for proliferation and in vitro regeneration either through direct
organogenesis or somatic embryogenesis. Similarly with various
immature explants excised at different developmental stages,
a clear stchometric localization and characterization of regenerative
tissues has been documented in chickpea, pigeonpea and tomato,
which may precisely be triggered for dedifferentiation and direct
organogenesis on combination of different cytokinins.
- The vexatious problem of establishing hardening of in
vitro grown plantlets of chickpea and pigeonpea, have been
successfully optimized with combination of different physiological,
physical and light dark regimen during the acclimatization period
of 3 weeks before transferring the plantlets to glass house.
- An excellent rapid and genotype independent in vitro
regeneration system in tomato has been optimized with developing
cotyledons and excised leaf disks through initiation of shoot
buds. The success rate of developing roots, hardening and transfer
of in vitro plantlets to glass house ranges from 85-92+ 2.0%.
- Efficient procedure for Agrobacterium tumefaciens
mediated transformation in chickpea and pigeonpea using dissected
nature embryo axes and processed cotyledonay nodes with different
plasmid constructs harbouring either different reporter genes
viz., uidA (ß-glucuronidase), npt II (kanamycin resistance),
hph (hygromycin resistance), gfp (green fluorescence protein)
or truncated crystal protein CryIA(c) native and modified genes
of B.thuringiensis under the control of 35S CaMV promoter
has been achieved and transgene expression in putative transformants
have been characterized at molecular level to establish stable
integration and transgenosis.
- To optimize direct delivery of DNA into competent regenerate
tissues in various explants of recalcitrant plants and to restrict
our dependence on expensive imported Bio-Rad biolistic system,
we designed and developed an indigenous high velocity bombardment
system using N2 gas acceleration of DNA coated micro
carriers for delivery into the desired tissues. The system is
highly cost effective costing less than one lakh compared to
expensive Bio-Rad system costing 10 lakhs and costing less than
1.25 USD per shoot than about 9.5-10.0 USD for consumables per
shoot used in Bio-Rad system.
- A range of putative transformants of these crops have been
generated with highly modified synthetic Cry1Ac gene encoding
for a highly effective larvicidal toxin against pod borer (Heliothis)
and field insect Spodoptera litura are under investigation
to establish the performance of transgene inheritance and expression.
- Expression of heterologous protein in plants as bioreactors
for inexpensive large-scale production of industrially important
proteins is an important area of plant biotechnology but several
physiological, biochemical and genetic constrains seems to play
important role for expression of heterologous genes in plants.
The most significant component is the precise modification and
optimization of encoding region and 5’ and 3’ regulatory
sequences of the native gene(s) for optimum utilization of plant
machinery for maximum expression, targeting and stability of
the expressed foreign proteins. This involves extensive modifications
and designing of the nucleotide sequences to complement the
metabolic and biochemical environment of the plant cell. The
situation became complex when the expressed protein requires
glycosylation, processing and targeted into particular organ
or tissues in transgenic plants. We have analysed, designed
and synthesized the highly modified cDNA sequence of human alpa-1-antitrypsin
(AAT) gene for high-level expression and localization into dicot
plants. Extensive modifications like codon optimization, RNA
stability factors; UTR sequences (5’ and 3’) are
required to design the gene for enhanced expression in plants.
About 236 nucleotide changes were incorporated in 1.182 kb native
cDNA AAT gene to result 44.5% GC. A total of 113 minor codons
of native AAT were replaced with the dicot-preferred codons.
The TA and CG ending codons were removed to the best possible
because of less abundance of the corresponding tRNA in dicot
plants. Various molecular factors like putative polyadenylation
signals and their variants, mRNA instability sequences and variants,
RNA polymerase II termination signals, secondary structures,
self-dimerizing and cryptic splicing sites are completely removed
to check premature termination of the transcript in higher plants.
An optimal translation initiation context of six nucleotides
in front of ATG was introduced for maximum translation. For
optimum possessing of AAT different secretory signal peptides
at 5’ and KDEL retention signal at the 3’ end were
introduced for proper folding, glycosylation and retension into
endoplasmic reticulum, protein vesicles or apoplast of the plant
cell. The PCR based strategy was used to synthesize and assembly
of the full-length designed genes using overlapping oligos of
50-55 mer having specific Tm followed by sequential subcloning
into binary vectors for Agrobacterium - mediated plant transformation.
The sequencing of modified AAT gene has been performed to have
the correct clone and introduction of the modified AAT gene
into plant is in progress.
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National Relevance
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Grain legumes are the important crops
of Indian subcontinent being the major source
of dietary protein. However, their grain productivity has been consistent
over the last 50 years, primarily due to lack of sufficient information
about the genetic improvement in these plants.
Therefore, development of insect resistant and genetically improved
transgenic lines of chickpea, pigeonpea and tomato through direct
DNA transformation is the promising step towards restricting the
grain losses incurred due to pod borer infestation in field and
expenditure on insecticides. In view of these, development of an
efficient and feasible technology for genetic manipulation with
desired trait require characterization of competent regenerative
tissues for DNA-mediated transformation in these marginal and under
exploited important crops of Indian subcontinent. Expression of
modified human AAT gene in dicot plants is highly relevant to pharmaceutical
applications.
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International Relevance
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In international context extremely
limited work has been carried on these two legume crops while information
exists for in vitro generation and genetic transformation in tomato.
Several international labs are largely focused on soyabean, pea
and beans. There is hardly any data on these two crops of Indian
subcontinent. Conventional programme of genetic improvement in these
crops are restricted due to lack of useful traits in available germplasm
and sexual incompatibility amongst their wild relatives. High level
production of biologically active AAT protein in plants is of significant
economical importance to pharma industry.
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