Proceedings Volume 2010 (format .pdf) - SimpBTH

transferred OCI and OCII genes were confirmed by PCR technique using specificpin:OCI primers (5’- GGC TCC TCC GTC CAA TTA TA -3’and 5’-ATC GACAGG CTT GAA CTC CT-3’) and pin:OCII primers (5’-GGC TCC TCC GTCCAA TTA TA-3’and 5’-GGT GGC GTC GTC GAG GGG-3’). Reactions werecarried out for 35 cycles of 95ºC for 1 min, 55°C for 1 min, and 72°C for 2 min(OCI) and 35 cycles of 95ºC for 1 min, 53°C for 1 min, and 72°C for 2 min (OCII).PCR amplified gene products were analyzed by electrophoresis on 1.2% agarosegels.3. RESULTS AND DISCUSSIONSimple and rapid transformation protocol employed in this work was basedon previously established potato transformation procedure (Cingel et al., 2010).MS medium supplemented with BAP and NAA for callus induction and with BAPand GA 3 for shoot formation appeared to be suitable for plant regeneration fromleaf explants. These results correspond to a number of published papers(Benchekroun et al., 1995; Visser et al., 1989; Wenzler et al., 1989; Webb et al.,1983).Within 7 days after bacterial infection average 86% leaf explants cultivatedon selective CIM manifested callus proliferation along the cut edge. Shoot budregeneration was observed 10 days after calli were transferred on selective SIM.Within 3 weeks on SIM 74% calli produced an average 7.6 buds per explant (Table1). After 10 days of culture on selective RIM 96% shoots developed roots. Therewere no apparent differences between shoot buds regeneration from control andtransformed explants and all rooted plants exhibits normal phenotype.Table 1: Co-transformation frequency and shoot bud regeneration efficiencyof leaf explants.Number of explants thatdeveloped calli (%)*Number of calli thatdeveloped buds (%) **Number ofbuds/explantscontrol 95.0 ± 0.3 88.0 ± 0.6 8.5 ± 0.6OCI/OCII 86.0 ± 0.4 74.0 ± 0.9 7.6 ± 0.4Results are expressed as mean ± S.E. (No. of explants =100)* On CIM, mean was calculated as No. of explants per petri dish with calli/No. of explants** On SIM, mean was calculated as No. of explants per petri dish with developed buds/No. ofexplants.PCR detection for two target genes showed that 100% of putative rootedtransgenic plants carry either or both transgenes. Transformed plants producedabout 800 bp or/and 1000 bp amplification products that were not detected innontransformed control plants (Figure 1). Apparently, rooting on selective RIMwith increased kanamicyn concentration to 75 mg l −1 was a good indicator of27