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

Abstract

The development of transgenic potatoes is imperative to investigate various gene functions as well as to develop robustpotato varieties resistant to different biotic and abiotic stresses. Directing a potato breeding program from cultivated tetraploids to selfcompatible 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 microtubersas the explants. Agrobacterium tumefaciens strain GV2260 harboring pBIN19 expression vector containing gusA gene (interrupted byan intron to induce expression from eukaryotic cells) was used for this purpose. Different inoculation times (10, 20, 30 min) wereapplied in aforementioned explants for gene transfer. After cocultivation, explants were transferred to the media with various hormoneconcentrations [6–benzylaminopurine (BAP), 1–naphthaleneacetic acid (NAA), trans-zeatin, kinetin, and 2, 4–Dichlorophenoxyaceticacid (2,4–D)]. The calli generated were then transferred to shoot generating medium supplemented with thidiazuron (TDZ) andgibberellic acid (GA3). According to histochemical GUS analysis, we found a 20 min inoculation time to be optimal for gene transferand 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 inoculatedexplants (41% callus formation for internodes and 65% for leaf explants). Abundant transcripts levels of gusA gene was found in primarytransformants when subjected to RT-qPCR. GUS fluorometric assay further confirmed the primary transformants at protein level. Thepresent study can serve as a gateway to transfer gene(s) of interest in diploid potatoes.