| Experiments focusing on plant growth regulators' concentrations and combination, mineral salt formulations and TDZ pretreatment formations were conducted to optimize in vitro shoot regeneration from leaf and petiole explants of 'Marion' blackberry. Optimum shoot formation was obtained when stock plants were incubated in TDZ pretreatment medium for three weeks before culturing leaf explants on regeneration medium in darkness for 1 week before transfer to light photoperiod at 23°C +/- 2°C for 4 weeks. Under these conditions, about 70% of leaf explants formed approximately forty shoots that could be harvested from each petri dish and rooted to form plantlets.; The expression of an intron-containing uidA gene coding for beta-glucuronidase was used as an indicator of successful T-DNA delivery and GUS gene expression in 'Marion' leaf explants. Using the optimized protocol, about 20% of the leaf explants showed blue staining 7 d after cocultivation. Putative transgenic shoots obtained 3 during the optimization experiments, however, were all GUS negative, indicating the necessity of further evaluation of factors that lead to the recovery of real transgenic plants.; Two transgenic lines of 35S::AtCBF1 increased freezing tolerance 3.1°C and 2.2°C in leaves and stems respectively, when compared to wild type (WT) plants grown under non-acclimation conditions, and a 3.3°C and 3.8°C increase in the freezing tolerance of leaves and stems respectively after 4 weeks of cold acclimation at 2°C. After acclimation, there was no increase in freezing tolerance found in the rd29A::AtCBF1 line compared to the WT. The three 35S::AtCBF2 lines grown under acclimation condition had a decreased freezing tolerance, compared to the WT. All four tested transgenic 35S::AtCBF3 lines gave the most significant increases in freezing tolerance, with an average of 5.3°C increase in leaf tissue tolerance when grown under non-acclimating conditions and 6.1°C after being grown under acclimating conditions, and for stem tissues a 3.0°C and 6.3°C increase in freezing tolerance when grown under non-acclimating and acclimating conditions, respectively. Three of the six rd29A::AtCBF3 lines, after cold acclimation, were significantly more tolerant to freezing than the WT, with an average increase of 2.5°C in leaf tissue tolerance and 3.3°C in stem tissues. Level of cryoprotectant such as soluble sugars (sucrose, fructose, and glucose) and proline in leaf tissue of transgenic lines were increased with a manner along with the increase of freezing tolerance. |
