Genetic transformation is a key technique for plant molecular breeding to introgress desirable characteristics into the existing genome while preserving genetic identity of plants. Agrobacterium-mediated genetic transformation has become the first choice for basic plant research and many agronomically and horticulturally important species are routinely transformed using this method. Agrobacterium-mediated transformation is a highly efficient process of gene transfer in dicotyledonous plants (dicots), and predominantly results in the integration of transgenes at a single locus.

1. Surender Khatodia

2. Mechanism of Agrobacterium mediated
transformation in plants

3. Need of in planta transformation in chickpea
Till date, transformed chickpea plants resistant to pod
borer have been produced by Agrobacterium-mediated
genetic transformation by several groups by transferring
different versions of cry genes.
But chickpea transformation is quite difficult and the rate
of transformation frequency is very low
Chickpea considered to be highly recalcitrant due to lack
of in vitro induction, development of strong root system
and establishment of in vitro raised plantlets in pots.

4. In planta Agrobacterium-mediated
transformation
Tissue culture independent
High throughput transformation
Minimizes labor, expense and expertise
Reduces rate of unintended mutagenesis
Avoids somaclonal variation
Limitations using intact and differentiated tissue explant
Chimeric plants
Low transformation frequency

5. Direct seed transformation strategy
Taking seed as target for in planta transformation
Non-selective/PCR selection
relatively high transformation efficiency
time efficient,
cost-effective
genotype independent

6. Bt chickpea development
Transformation with cry1Ac gene following the non-
selective/PCR detection system using direct plant PCR screening
indicated the putative transgenic nature of plants.
Transformation frequency of up to 41% with LBA4404 strain
carrying pBinAR plasmid.
The putative transgenic chickpea plants were analyzed adopting
multiple evaluation strategies, such as PCR, ELISA and southern
blotting, for selection of plants for further advancement.

7. Direct PCR screening of putative chickpea transformants
carrying cry1Ac gene
M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 NT P NC
M 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 1068 bp

8. Southern analysis of T2 transgenic chickpea plants
carrying Cry1Ac gene
1 2 3 C P
- 23kb
- 9.4kb
- 6.5kb
- 4.3kb
- 2.3kb
- 2kb
- 0.5kb
Lanes:
1- TH2-44 (cv. HC-1),
2- TH2-166(cv. HC-1),
3- TC2-33(cv. C-235),
C-Control plant,
B- Blank
P- Positive control

9. Future prospects
Super Chickpea plants with novel traits
Marker free transgenic events using binary vectors that
are devoid of the marker gene upfront, allows stacking
of multiple genes
Applicability across different genotypes/cultivars
Other recalcitrant grain legume crops like pigeonpea
Other plant species

10. Plant mediated RNAi of Heliocverpa for insect pest
resistance in cotton and chickpea using direct seed
transformation
Helicoverpa armigera - the most devastating insect pest of cotton and
chickpea
Sustainability of Bt transgenic crops is threatened by the accelerated
emergence of insect resistance.
RNAi induced in insects after ingestion of plant-expressed hairpin
RNA offers promise for managing devastating crop pests.
The lethal or highly detrimental effect of down-regulating three crucial
target genes of Helicoverpa by plant mediated RNAi for resistance in
cotton and chickpea is to be studied.

11. Plant mediated RNAi

12. RNAi for Helicoverpa
CytP450 (involved in detoxification of allelochemicals),
HaHR3 (molt-regulating transcription factor gene),
Chymotripsin (involved in digestion of proteins).
In silico designing of common potential insecticidal siRNAs for
the selected target genes from various populations of H.
armigera will be done.
which can be applied to any species for RNAi-mediated, off-
target minimized, effective gene silencing.

13. Thank you