Development of in vitro regeneration and gene transfer systems for conifer species

In vitro shoot establishment and proliferation of Sequoia sempervirens Endl. stem segments on a Wolter and Skoog (WS) medium supplemented with 15 $\mu$M zeatin was found to be optimum for shoot proliferation. Using in vitro-grown needles of S. sempervirens, shoot organogenesis and somatic embryogenesis were induced. The influence of genotype and various cytokinins (BA, BPA, and TDZ) in combination with 2,4-D were investigated. Among the cytokinins tested, 20 $\mu$M BPA with 5 $\mu$M 2,4-D produced the highest frequency of shoot organogenesis from three genotypes while, 10 $\mu$M TDZ with 5 $\mu$M 2,4-D produced the highest frequency of somatic embryos from two of three genotypes.The effect of adventitious shoot induction on cotyledons of Pinus pinea L. was investigated. Among seven basal, three carbon sources (sucrose, glucose and fructose), and two cytokinins (BA and TDZ) with and without NAA were tested. Cotyledons grown on a 1/2 MS medium containing sucrose and 30 $\mu$M of BA produced the highest frequency of regeneration ($>$90%) and shoot number/explant ($>$40% cotyledons produced over 20 shoots). As for the site of shoot organogenesis along the cotyledonary explant, the highest number of shoots per explant was observed on the apical segment of the cotyledon proximal to the hypocotyl over all cytokinin and auxin treatments tested.The effect of seven basal media (macro and micro elements) and three carbon sources (sucrose, glucose and fructose) on induction of adventitious buds from embryos of Pinus sylvestris L. was also determined.Phlox (Phlox paniculata L.) and Italian stone pine (Pinus pinea L.) were used for serial bombardment with BA-coated particles using biolistic gun. Increasing the concentration of BA from 0.01 to 0.1 mg per $\mu$l increased the mean number of adventitious shoots from phlox leaves (16 shoots per leaf), and elongation of these shoots ($>$1 cm) was observed within a shortened time period of 3 weeks. Bombarding P. pinea cotyledons with BA-coated particles reduced the frequency of regeneration (48%) compared to control explants (90%) grown on a medium containing BA and with no bombardment.In order to study cell competence for regeneration, coltyledon explants of P. pinea were cultured in vitro for three-day intervals on a 1/2 MS medium containing 20 $\mu$M BA, and then transferred to the same medium without growth regulators until day 30. Explants exposed to 6, 9, 12, and 15 days of culture on the regeneration medium showed an increase in shoot regeneration frequency over time reaching an optimum at day 15. As for the site of shoot organogenesis along the cotyledonary explant, the highest number of shoots per explant was observed along the apical portion of the cotyledon proximal to the hypocotyl.In order to optimize conditions for bombardment for stone pine cotyledons, leaf tissues of phlox were used as model for biolistic bombardment. Increasing the vortex time from 2 to 20 min after mixing particles with CaCl\sb2, spermidine, and plasmid DNA (pZA300), resulted in the appearance of blue spots. Increasing the pressure of the rupture disks also enhanced GUS expression of phlox leaves. (Abstract shortened by UMI.)U of I OnlyETDs are only available to UIUC Users without author permissio