A high-efficiency regeneration/transformation system was developed for three elite aspen hybrids (Populus tremuloides × P.tremula, P.tremuloides × P.davidiana, and P. × canescens × P. grandidentata). On both modified MS medium for aspen (MSA) and Woody Plant Medium (WPM) supplemented with zeatin (2.0 mg/L) and NAA (1.0 mg/L), nearly 100% leaf explants formed calli, of which 80% to 100% regenerated into shoots on both media with 2.0mg/L zeatin and 0.01 mg/L TDZ. Bacterial concentration, pH value of the co-cultivation medium, and acetosyringone were evaluated for enhancing transformation efficiency. Agrobacterial concentration at 1.0 Absorbance at OD600 was better than at 0.1, 0.5 Abs, yielding 80% and 75% of callus induction rates from agrobacterium harboring CaMV35s and Heat shock promoter constructs, respectively. The pH of co-cultivation medium, ranging 5.0 to 5.9, did not have any effect on transformation frequency. Acetosyringone was added to the co-cultivation medium and/or to the callus induction medium, the induction of kanamycin-resistant callus increased from 70% to 80% to 90% to 100%, and the size of callus also increased. Acetosyringone had no effect on shoot regeneration from kanamycin-resistant calli. Regenerated aspen shoots were screened on the kanamycin-containing medium, and confirmed by GUS histochemical assay. The GUS-positive plants were further confirmed by polymerase chain reaction, showing that the nptII, uidA, and rolB genes were integrated into the aspen genomes.
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