MOLECULAR AND GENETIC ANALYSIS OF PUTATIVE TRANSFORMANTS IN TRANSGENIC PIGEONPEA.
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Date
2017-12-05
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Dr. Panjabrao Deshmukh Krishi Vidyapeeth, Akola, Maharashtra.
Abstract
The gram pod borer (Helicoverpa armigera Hubner) is the most serious insect pest of pigeonpea. It is highly susceptible to the insecticidal proteins of Bacillus thuringiensis (Bt). A codon-optimized chimeric Cry1Aabcgene of Bt driven by a constitutive promoter was introduced pigeonpea (PKV Tara). Molecular analyses were carried out by most of worker in T1 and T2 generation.
From 7 plants of T3 generation total 57 progenies were tested for PCR from which 47 plants were seen to be positive through PCR analysis. 38 plants of T1 generation from DK+ material were tested for PCR analysis through gene specific primers from which 22 plants were seen to be positive.
Kamble in year 2014-15 attempted 130 embryo infections with Agrobacterium strain EHA105 harboring Cry1Aabc and NptII gene out of which 73 were survived but only two plants were confirmed positive through PCR. T1 and T2 generation of these two plants namely A and B were sown. Three plants from eachwere selected for PCR confirmation. From two positive plants total 6 progenies from T1 generation and 3 plants from T2 generation were tested for PCR analysis all the 6 plants from T1 generation were seen to be positive while from T2 generation out 3 only two plants were positive. Sample population size of T1 and T2 plants was too less to study inheritance pattern.
In order to study Mendalian inheritance pattern in T3 progeny PCR analysis for the amplification of the cry1Aabc gene and NptII gene was carried out. The plants from same parents plant was consider together for the inheritance study. For undertaking the Chi-square test for segregation the minimum population size of 6 was taken.
Calculated x2 value is less than table value in all the plant progenies. Thus plants for the amplification of the Cry1Aabc gene and NptII gene and progeny of each clone shows 3:1 (presence: absence) segregation pattern.
For detached leaf bioassay 19 positive plants and one negative plant which were confirmed by DNA extraction and PCR amplification with NptII and Cry1Aabc gene specific primers were tested using leaf feeding assay with neonate larvae of Helicoverpa armigera. 20 putative transformants of pigeonpea were subjected to insect (Helicoverpa armigera) bioassay using neonates of Helicoverpa armigera and young trifoliate leaves and WT (non-transformed) PKV Tara was used as control.
Thus the result revealed that the level of Cry protein expressed in positive plants were able to reduce the leaf damage. Thus, it was concluded from above results that like positive plants selected for bioassay study having potential for providing tolerance against Helicoverpa armigera. The further confirmation in subsequent generation is required to come to more robust conclusion.
Description
Molecular characterization of transgenic plants as the number of transgene copies influence the expression level and genetic stability of the transgene. Transformation technologies have been advanced to the point of commercialization of transgenic crops.
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Citation
KADAM, KARTIKI DADASAHEB. (2017). Molecular and genetic analysis of putative transformants of pigeonpea. Department of Agricultural Botany. Dr. Panjabrao Deshmukh Krishi Vidyapeeth, Akola. M. Sc. 2017. Print.xi, 60p.