Study On Genetic Transformation Of Dendrobium Candidum Wall.ex Lindl With LEA3 Gene By Particle Bombardment

Orchids, one of important ornamental plants, have a great commercial importance in world industry, which are commercially grown globally for cut flower and potted plants. Dendrobium is the largest genera of Orchidaceae with more than 1600 species, many of which are grown mainly for cut flower. Late embryogenesis abundant (LEA) proteins have strong hydrophilic property and hot stability, which play an important role in plant responses to environmental stresses. To cultivate new material of Orchids with enhanced tolerance to drought and prolonged vase life, protocorm-like bodies (PLBs) from Dendrobiun Candidum Wall.ex Lindl were transformed by particle bombardment with plasmid pBY520 containing late embryogenesis abundant protein gene, lea3, from barely (Hordeum vulgare L.). The main results were obtained as follows:An efficient procedure was developed for PLBs domestication from Dendrobiun Candidum and subsequent subculture for a long time. Subcultured PLBs proliferate rapidly during 3-5 weeks and those cultured for 28d and 25d are suitable for differentiation and transformation study, respectively.To establish an effective regeneration in vitro system for Dendrobiun Candidum, the effect of hormones, spermidine and organic additives was compared. Results showed that PLBs on MS medium supplemented with 0.9 mg/L NAA and 1.0 mg/L 6-BA had the highest differentiation rate at 56.50/g, which was over 27 times as compared with control, and the optimal medium for rooting was MS medium supplemented with 20% banana pulp, on which the rooting rate reached 95% and an average of 5 roots developed from one shoot.Factors that influence percentage of transformation were studied. The highest genetic transformation rate was obtained when 10-day-old PLBs cultured in the light were bombarded one time every sample and then were recovered for 20 days before PPT selection. The suitable concentrations of selective agent PPT for PLBs and for regenerated shoots were 3 mg/L and 20 mg/L, respectively.The plasmid containing lea3 gene and bar gene was delivered to PLBs by particle delivery system. All bombarded PLBs were grown on MS for 20 days then transferred to MS medium with 3 mg/L PPT. After two months' screening, the survival PLBs were transferred to MS medium with 0.9 mg/L NAA and 1.0 mg/L 6-BA to differentiate for 45 days, then the regenerated shoots were selected on MS medium with 20 mg/L PPT. One month later, the resistance shoots were transferred to MS medium with 20% banana pulp for rooting. Finally 35 plantlets were regenerated from the bombarded PLBs, from which 11 detected positive to PPT were obtained. The result of PCR analysis on the transgenic plants with PPT resistance showed 7 lines have obtained lea3 gene.