Author(s):

Tirthkar Meera Baburao and Dr. Sumangala Bhat

Abstract: Pigeonpea is an important pulse crop which is widely cultivated throughout the tropical and sub-tropical regions. It is the major source of protein for the people with vegetarian diet. Major constraint in pigeonpea is the attack by pod borer. Conventional plant breeding techniques have limitations to develop resistance to the pest due to non-availability of resistance in the available germplasm. Therefore genetic engineering tools can be employed to transfer foreign genes into pigeonpea to impart pod borer resistance. Development of a reliable regeneration system coupled with efficient transformation has been a major limitation in developing transgenic pigeonpea with useful genes. In planta is an alternative approach to obtain transformed plants in short duration without involving tissue culture regeneration. However major limitations with this approach is screen more number of seeds to identify putative transformants which is time consuming and expensive. Therefore the present investigation was carried out to optimize the concentration of kanamycin for quick identification of putative transformants carrying cry1Ac and npt II using sand based assay. Seeds of Asha and BSMR736 were soaked in different concentrations of kanamycin (10-100, 200, 300, 400 and 500 ppm) for 5hrs and later sown in sand based medium. There was no effect of kanamycin on seedling growth up to 60 ppm and decrease in survival percentage in both the genotypes beyond this concentration. There was significant differences in growth of the seedlings in terms of shoot and root length at 100 ppm and beyond. Therefore this concentration was used to screen seeds of 32 primary transformants carrying cry1Ac and npt II and subjected to gene specific PCR analysis to confirm the usability of simple screening on kanamycin based sand medium for the identification of true transgenic plants. However some discrepancies between the growth inhibition and PCR results were observed.

DOI: 10.22271/phyto.2020.v9.i5u.12551

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