Manipulating genome of diploid potato inbred line Solanumchacoense M6 using selectable marker gene

Abstract

The development of transgenic potatoes is imperative to investigate various gene functions as well as to develop robust potato varieties resistant to different biotic and abiotic stresses. Directing a potato breeding program from cultivated tetraploids to self-compatible diploid lines will be highly helpful for potato breeders. However, diploid potatoes are considered recalcitrant to regeneration. We aimed to develop a protocol for the transformation of diploid Solanum chacoense M6 potatoes using leaf, internodes, and microtubers as the explants. Agrobacterium tumefaciens strain GV2260 harboring pBIN19 expression vector containing gusA gene (interrupted by an intron to induce expression from eukaryotic cells) was used for this purpose. Different inoculation times (10, 20, 30 min) were applied in aforementioned explants for gene transfer. After cocultivation, explants were transferred to the media with various hormone concentrations [6-benzylaminopurine (BAP), 1-naphthaleneacetic acid (NAA), trans-zeatin, kinetin, and 2, 4-Dichlorophenoxyacetic acid (2,4-D)]. The calli generated were then transferred to shoot generating medium supplemented with thidiazuron (TDZ) and gibberellic acid (GA3). According to histochemical GUS analysis, we found a 20 min inoculation time to be optimal for gene transfer and the medium containing 2 mg L-1 BAP and 2 mg L-1 NAA was the most suitable medium for callus induction from 20 min inoculated explants (41% callus formation for internodes and 65% for leaf explants). Abundant transcripts levels of gusA gene was found in primary transformants when subjected to RT-qPCR. GUS fluorometric assay further confirmed the primary transformants at protein level. The present study can serve as a gateway to transfer gene(s) of interest in diploid potatoes.