In Vitro Agrobacterium-mediated Transformation And Regeneration Of Transgenic Plants With LMV-CP Gene In Chinese Cabbage(Brassica Campestris Ssp.pekinensis)

Chinese cabbage has consistently proven to be one of the most recalcitrant members of the Brassicaceae in tissue culture, and it抯 also refractory to transformation. This dissertation focused on efficient normal shoot regeneration and its application in genetic transformation of heading Chinese cabbage. Results were discussed in these aspects: 3mg/LAgNO3 markedly enhanced shoot regeneration and AgNO3 was an absolute requirement in whatever genotypes and explants tested. Shoot regeneration ability is genotype dependent. Petioles of intact cotyledons as explants, 17 of 20 genotypes tested had shoot regenration ability, moreover shoot regeneration frequency of 10 cultivars in them was above 80%. In No.80~ No.3~ No.10, comparison of the regeneration ability was taken in petioles of intact cotyledons, hypocotyl segments and leaf sections. It was found that petioles of intact cotyledons and leaf sections had higher regeneration ability, which meant that leaf sections could be used as a perfect transformation receptor. Factors affecting the vitrification of regeneration shoots of Chinese cabbage were tested. Results showed that decreasing hormone concentration (BA 2-1 mgL? NAAI-0.5 mgTJ ) or increasing agar content (0.8%-l.4%) facilitated the normal shoot development. But there was no evident effect on inhibiting the occurrence of vitreous shoot by decreasing AgNO3 concentration, increasing sucrose concentration, supplementing AC or changing culture dishes into culture flasks. No.80 and 92-2 Cultivars of Chinese cabbage were selected for genetic transformation. Petioles of intact cotyledons or leaf explants were co-cultivated with Agrobacterium C58 harhouring binary vectors carrying LMV-CP gene and bar gene. Resistant plants were recovered from petioles of intact cotyledons at the frequency of 2-6% in No.80 and 3.7% in 92-2.The frequency was 2.0% in No.80 using leaf 3 sections as explants. According to polymerase chain reaction (PCR) and PCR Southern blot hybridization analyses, 3 of 6 resistant plants, which were randomly selected, were confirmed the introduction of the LMV-CP into the Chinese cabbage genome. Detection of resistant virus is under way.