Comparison of Regeneration Efficiency of the Indian Potato Cultivars during Agrobacterium-Mediated Genetic Transformation

Abstract

The present study was focused to study the effect of various parameters on potato regeneration in some selected Indian potato cultivars namely Kufri Chipsona-1, Kufri Chipsona-2, KufriJyoti along with an exotic cultivar Desiree. These cultivars vary with regard to the maturation period, genetic makeup and adoptability to different agro-climatic zones of the Indian subcontinent.  The germplasms of the cultivars under study were routinely subcultured on MS Basal medium and maintained in our laboratory under controlled conditions.  The Agrobacterium-based Ti-plasmid vectors (pBI121 and pAN-GB01) were mobilized to Agrobacterium tumefaciens LBA4404 through triparental mating. The plasmid DNA was isolated from the Agrobacteriumtransformants and the orientation of the different components of T-DNA of the vectors were further checked by PCR approach.  The Agrobacterium strainharbouring Ti-plasmid based vectors was co-cultivated with the explants of the individual potato cultivars under varying parameters such as, bacterial cell density, age of the explants, time period of co-cultivation, phytohormone formulations to see their effects on overall regeneration efficiency.  The regeneration response of the individual potato cultivars was noted carefully. The salient observations of this study include: At bacterial cell density of OD600= 0.2, the regeneration efficiency was found to be effective for all the potato cultivars under study as compared to higher cell densities as used during co-cultivation. The explants corresponding to the cultivars KufriChipsona I and Desiree showed efficient regeneration on zeatin-based media, whereas the cultivars KufriChipsona 2 and KufriJyoti performed better after passage through callus inducing medium (CIM) for a period of around two weeks (however, to avoid somaclonal variations a minimum intervening callus phase was allowed prior to shoot regeneration). In CIM, it was noted that the explants of 45 days old culture showed more efficient regeneration as compared to the response of explants from 30 days old plantlets. 20 min duration of co-cultivation of the explants with Agrobacterium was found to be optimum in case of CIM formulation, whereas for zeatin-based media the duration of co-cultivation was around 30 min. for optimium regeneration efficiency. All these observations together will provide clues to ensure efficient regeneration of the individual potato cultivars during transgenic approaches.