Abstract
Studies were conducted to develop an efficient and reliable protocol for in vitro direct
regeneration and Agrobacterium-mediated genetic transformation of potato. Nine experiments
were conducted. Six different concentrations of Mercuric chloride (HgCl2) and Sodium
hypochlorite (NaOCI) with five sterilization periods were tested separately. 0.05% HgCl2 with
15 minutes sterilization or 1% NaOCI with 10 minutes sterilization was the best to
decontaminate potato shoot tip. Among the tested five media, MS medium + 0.5 mg l¹ IВA +
3% sugar having pH 5.7 supported the best growth. Among the 22 genotypes, Diamant,
Cardinal, Asterix, Felsina, Processor, Provento, Dura, Heera, Innovator and Granola had better
in vitro regeneration both in solid and liquid medium. Healthy and vigorous shoots were also
obtained earlier in liquid medium. Among the different concentrations of BAP, TDZ and ZR,
MS medium supplemented with 5 mg I¹ ZR showed the best performance for in vitro direct
regeneration of potato from internode and leaf explants. But MS medium supplemented with
BAP and TDZ did not produce any shoots from leaf explants. Among the different
concentrations (0.0, 0.1, 0.5, 1.0, 1.5 and 2.0 mg I¹) of IBA, ½MS medium supplemented with
0.5 mg I IBA resulted the best performance as to the rooting from ZR treated regenerated
shoots. Among the five varieties (Diamant, Asterix, Felsina, Processor and Dura), 3 levels of
ZR and 2 types of explants tested for in vitro direct regeneration, internode explants of Asterix
variety with 5 mg I¹ ZR exhibited the best performance. Moreover, internode explants of 13-16
days and 19-22 days old from liquid and solid media, respectively showed the excellent results
on MS medium supplemented with 5 mg l ZR for in vitro direct regeneration. Between the
varieties of Diamant and Asterix for the standardization of Agrobacterium-mediated genetic
transformation protocol, Asterix variety performed better followed by Diamant. Histochemical
assay revealed the stable expression of the GUS gene within the various tissues of transformed
plantlets. Internode explants infected for 30-minute and 3-day co-culture confirmed nptII
positive through PCR amplification resulted transformation rate of 28.97% and 24.69% in
Asterix and Diamant variety, respectively. Kanamycin at a concentration of 100 mg I¹ was
found optimum for selection of transformed shoots. Besides this, Agrobacterium-mediated
transformation of PsCIPK gene for salinity tolerance, internode explant of Asterix variety with
30-minute infection and 3-day co-culture also confirmed nptII and GUS positive. Stable
integration of nptlI gene was confirmed by PCR analysis of genomic DNA isolated from the
transformed shoots which showed the good performance up to the 120 mM NaCl at in vitro
node cutting and root tip bioassay.