Optimazation Of In Vitro Regeneration And Genetic Transformation From ’Mao Tao’(Prunus Persica)

For providing a stable and efficient in vitro regeneration system to peach rootstock (Prunus persica) transgenic research, this experiment was carried out on the basis of previous research in our own laboratory. The aseptic leaves of’mao tao’were used as explants for studying the effects of different content of silver thiosulfate, adenine, zeatin and so on on adventitious shoot regeneration, hoping to largely improve the regeneration rate to meet the need of genetic transformation. Meanwhile using the callus derived from the aseptic leaves of’mao tao’as materials, we have preliminary explored genetic transformation of callus from’mao tao’through the agrobacterium-mediated method, wishing to achieve kanamycin-resistant callus in the selection medium and establish a genetically modified system of callus from’mao tao’. The main results are as follows:1. The optimal subculture medium for’mao tao’in vitro was LP+CPPU0.05mg/L+IBA0.3mg/L+adenine3.0mg/L+sucrose30.0g/L+agar6.8g/L, pH5.85-6.00. The plants were subcultured for every28d.2. The aseptic leaves and petioles from’mao tao’ were cultured on the basis of LP+CPPU0.5mg/L+NAA0.1mg/L+sucrose30.0g/L+agar6.0g/L, and the addition of riboflavin, biotin and calcium pantothenate cannot induce the adventitious shoot regeneration from leaves and petioles of’mao tao’; And the regeneration rate of leaves and petioles from ’mao tao’were15.00%and4.00%respectively, when60μM silver thiosulfate was added; And the regeneration rate of leaves from ’mao tao’ was8.89%when1.5mg/L ZT and0.3mg/L NAA were replaced by0.5mg/L CPPU and0.1mg/L NAA accordingly; And the maximal regeneration rate of leaves from’mao tao’ was18.33%when3.0mg/L Ad was added.3. The optimal medium for preconditioned shoot of ’mao tao’was LP+CPPU0.1mg/L+IBA0.3mg/L+Ad3.0mg/L+sucrose30.0g/L+agar6.0g/L, pH5.85-6.00, dark cultivation21d back to the light.4. The regeneration rate of preconditioned leaves of ’mao tao’was4.44%when the preconditioned leaves of ’mao tao’ were cultured on LP+CPPU0.5mg/L+NAA0.2mg/L+sucrose30.0g/L+agar6.0g/L, pH5.85-6.00. When2.0mg/L Ad was added to the medium, the regeneration rate of preconditioned leaves of’mao tao’ increased to4.92%。5. The effects of LP basic medium on callus induction and adventitious shoot regeneration of leaves from ’mao tao’ were better than AP basic medium; Conservation of callus from ’mao tao’needs PGR. Otherwise, the proliferation times of callus from ’mao tao’ decreas and the callus fail to grow well. Callus from ’mao tao’were cultured in suspension medium of LP+CPPU0.5mg/L+IBA/NAA0.1mg/L+sucrose30.0g/L, and no regeneration occurred.6. The optimal medium for rooting of ’mao tao’ in vitro was1/2LP+IBA1.0mg/L+NAA0.1mg/L+PG40mg/L+sucrose30.0g/L+agar6.8g/L, pH5.85-6.00. The rooting percentage was up to79.23%, the mean of root number of every plantlet was7.66and the average length was5.38cm.7. In the genetic transformation of callus from ’mao tao’, we choose30mg/L km as selection pressure to screen the kanamycin-resistant callus in the selection medium, and choose400mg/L cef as additive amount in the co-culture medium.