Establishment Of Plant Regeneration System And Preliminary Studies On Genetic Transformation Of Rosa Laevigata Michx.

Rosa laevigata Michx.is an evergreen climbing shrub of the Rosa in Rosaceae,generally grows on the sunny hillside with an altitude of 100-1600m.It is widely distributed in China,mainly in the south and southwest of the Huaihe River and Qinling Mountains.It has extremely high edible value and medicinal value.In this experiment,in vitro rapid propagation system was established using stem of Rosa laevigata Michx.as explant,and the direct and indirect organogenesis pathway from detached leaves and genetic transformation system were preliminarily studied.The main findings are listed below:1.The stem with segments from wild seedlings of Rosa laevigata Michx.was used as explants and in vitro rapid propagation system was established.The conditions affecting primary culture,axillary bud proliferation and rooting culture of Rosa laevigata Michx.were studied.The results showed that the suitable medium for primary culture of Rosa laevigata Michx.was MS+NAA0.1 mg/L+6-BA0.5 mg/L+GA3 4 mg/L+sucrose30g/L+agar7.5 g/L.After 3 weeks of culture,axillary buds could germinate and grow well.The suitable medium for proliferation and subculture was MS+NAA0.1 mg/L+6-BA1 mg/L+sucrose30 g/L+agar 7.5 g/L,after culture for 3 weeks,the proliferation coefficient was 4.5;the best rooting medium was MS+NAA0.1 mg/L+sucrose 30 g/L+agar7.5 g/L,the rooting rate can reach 100%after culture for 3 weeks.2.The young leaves with scars from seedlings in vitro of Rosa laevigata Michx.were used as explants,and the effects of different plant growth regulator combinations,dark culture time,additive L-proline and different parts of leaves on the direct regeneration of adventitious buds were studied.The results showed that the optimal hormone combination was 1.5 mg/L of TDZ and 0.0005 mg/L of NAA for the direct induction of adventitious buds with the highest rate of 9.5%.There were significant differences in the induction rates of adventitious buds under different dark culture time.When the dark culture time was 10d,the direct induction rate of adventitious buds was 10.5%.After adding different concentrations of L-proline,the induction rate of adventitious buds did not increase.In the comparison of the regeneration rates among different leaf parts,only the leaf with petiole could generate adventitious buds.In summary,the culture medium for directly regenerating adventitious buds from leaves with petioles was 1/2MS+TDA1.5 mg/L+NAA0.0005 mg/L+CH100 mg/L+AgNO310 mg/L+sucrose30 g/L+agar7.5 g/L.The suitable dark culture time was 10-day before transferring to normal photoperiod for about 4-week culture,the final adventitious bud induction rate was 10.5%.3.The young leaves with scar of Rosa laevigata Michx.seedlings in vitro were used as explants,and the effects of different plant growth regulator combinations,dark culture time,additive L-proline and different regions of explants on indirect regeneration of adventitious buds were examined.The results showed that the combination of 1.5 mg/L of TDZ and 0.0005 mg/L of NAA had the highest indirect induction rate of adventitious buds,which was 12%.There were significant differences in the induction rate of adventitious buds under different dark culture time.The indirect induction rate of adventitious buds was the highest under 12-day of dark culture,which was 11.5%.There were significant differences in the induction rate of adventitious buds among different concentrations of L-proline.The optimal concentration of L-proline was 600 mg/L for the indirect adventitious bud induction with the highest rate of 10.5%.The leaf disc and the leaf with petiole both could generate adventitious buds.In summary,the culture medium for indirect regeneration of adventitious buds from leaf with petiole or leaf discs was MS+TDA1.5 mg/L+NAA0.0005 mg/L+L-Proline600 mg/L+CH100 mg/L+AgNO310 mg/L+sucrose30 g/L+agar7.5 g/L,the suitable dark culture time was 12-day,after about 4-week culture under normal photoperiod the final adventitious bud induction rate was 12%.4.The sterile leaflets of Rosa laevigata Michx.were used for gene transforming,and the effects of antibiotics concentration,infection methods and co-cultivation time on the Agrobacterium-mediated gene transformation efficiency were studied.The results showed that the sensitivity of leaves and callus of Rosa laevigata Michx.to Basta was consistent.The optimum selective concentration of Basta was 0.8 mg/L.Termetin had strong inhibitory effect on Agrobacterium tumefaciens.In this experiment,the optimum selective concentration of Termetin was 500 mg/L.At this concentration,explants grew well and Agrobacterium tumefaciens were completely inhibited.The optimum transformation method was vacuum infiltration with 3-day co-culture.The highest activity of luciferase from callus reflecting transformation rate was obtained after 3-week subculture.