Regeneration and genetic transformation of rose and evergreen azalea

The overall goals of this research are to establish tissue culture systems for rose (Rosa hybrida L. and R. chinensis minima) and evergreen azalea (Rhododendron spp.), develop efficient and highly reliable regeneration systems via organogenesis and/or somatic embryogenesis, and develop gene transfer systems for these two important floral crops.Shoot organogenesis was observed on callus tissue upon transfer of rhizogenic explants pre-incubated with 2,4-D to a regeneration medium containing 22.7 $\mu$M TDZ and 2.9 $\mu$M GA$\sb3.$ Secondary embryogenesis was observed, and increased numbers of somatic embryos were obtained following transfer of embryogenic calli to a growth regulator-free medium. For 'Carefree Beauty', glucose at 111 mM promoted higher organogenesis and somatic embryogenesis than sucrose at either 59 or 117 mM concentrations; however, for 'Baby Katie', no differences were observed between glucose and sucrose.The best growth regulator combination for adventitious shoot regeneration of azalea 'Fuchsia' and 'Hino Crimson' was 22.74 $\mu$M TDZ and 22.8 $\mu$M IAA. In general, incubating leaf explants in the dark for at least 1 week followed by low-light or high-light intensity was enhanced regeneration frequency. Continuous culture under high-light intensity suppresses shoot regeneration; however, a 2-week dark pretreatment promotes shoot organogenesis even when explants were grown under high-light intensity.Several factors for optimizing microprojectile-mediated gene transfer methods for rose and azalea were investigated. Transformation efficiency was increased by elevating the osmotic level of the medium during bombardment. Transformation efficiency was enhanced by combining osmotic treatment, cotyledonary-stage of embryogenic calli, and higher accelerating pressure settings. Comparing GUS ($\beta$-glucuronidase) transient expression of two particle guns, the PIG (particle inflow gun) and the biolistic PDS-1000/He gene gun, the PIG device resulted in higher GUS expression than the biolistic gene gun. A 10% GUS transient expression was obtained at 1100 psi helium pressure with 6 cm distance from stopping screen-to-leaf sections of azalea using the biolistic gun. A 22.2% GUS transient expression was obtained on shoot tip-derived calli of azalea using the PIG device at 60 psi with an open-chamber accelerating setting.Several factors for optimizing Agrobacterium-mediated gene transfer methods for rose and azalea were also investigated. Highly proliferating tissues, rose somatic embryogenic calli and azalea shoot-tips, have been found to be amenable to Agrobacterium-mediated transformation. The cotyledonary-stage of rose somatic embryos was found to be most amenable for gene transfer. Regeneration and transformation of azalea shoot-tips were better than stem segments or leaf sections. Kanamycin was effective for inhibiting regeneration of azalea leaf sections; however, it was less efficient for selection of rose embryogenic calli. Wounding manipulations were not necessary for rose embryogenic calli; however, for certain Agrobacterium strains, a wounding treatment might increase regeneration and/or transformation frequency. Based on GUS expression, bombardment and fresh cut treatments showed a higher blue color intensity. Adding acetosyringone or a nurse culture of minced tobacco leaf nurse culture to the cocultivation medium enhanced the number and intensity of blue spots; moreover, adding acetosyringone to the bacterial culture probably increased stable transformation in azalea. The susceptibility of various tissue types to different Agrobacterium strains was observed.U of I OnlyETDs are only available to UIUC Users without author permissio