Studies On Tissue Culture And Genetic Transformation In 'Huanghua' Pear

Sand pear (Pyrus pyrifolia Nakai) cultivars have serious vitrification and browning when cultured in vitro, and low regeneration efficiency because of the genotype, so it has been difficult to establish a tissue culture system and a regeneration system which is efficient and steady. In this research, the sand pear cv. 'Huanghua' which is the major cultivar in south China was used as material and a series of measures to avoid vitrification and browning of in vitro shoot were described. Based on vigorous in vitro shoots, callus of the leaf section explants was induced. Axillary buds germinated largely after etiolation, then basal segments of etiolated 'Huanghua' shoots were used as explants for genetic transformation. The results were summarized below:1. A series of measures to avoid vitrification and browning of in vitro shoot were studied. The effect of three gelling agents, Phytagel Canageenen and Agar powder on avoiding vitrification was compared. And the result was that Phytagel was better than Agar powder and Canageenen to avoid vitrification. When agar concentration and sucrose concentration respectively were increased, the frequencies of vitrification were both decreased, resultly, Agar powder 0.6% and sucrose 3% were chosen for low frequency of vitrification and high proliferation quotient. The frequency of vitrification was increased as 6-BA concentration increased, so 6-BA 3mg/L was chosen for subculture of in vitro shoots. Low concentration (10μmol/L) of salicylic acid and appropriate concentration (3-5g/L) of activated charcoal can avoid browning efficiently.2. Callus induction from the leaf section explants was studied. Among different combination of plant growth regulator, combination of 2.4-D and 6-BA was optimal owning highest frequency of callus induction (33%). And when 2.4-D concentration fixed 1 mg/L and 6-BA concentration fixed 0.1 mg/L, the frequency of callus induction was highest (85%). Low concentration (100mg/L) of Cef could accelerate the frequency of callus induction. Effect of different accretions on growth of callus was different, and the callus was made obvious proliferation by adding coconut milk to culture.3. Axillary buds from basal segment of in vitro shoots after etiolation could germinate largely. Different soluble protein bands between basal segment of etiolation-shoots and base segment of green-shoots under SDS-PAGE electrophoresis were found. An Agrobacterium-mediated transformation system of the basal segment of etiolation-seedlings explants for 'Huanghua' was established primarily, and GUS activity was showed in buds under this system.