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Plant Transgenics |
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Dr. D. V. Amla
Scientist G
| Phone no. 91-522-2205831-35 Ext. 954 | dvamla@nbri.res.in |
Dr. Indraneel Sanyal
Scientist C
| Phone no. 91-522-2205831-35 Ext. 955 | i.sanyal@nbri.res.in |
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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.
- 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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