Establishment Of Different Regeneration System And Genetic Transformation In Rosa Multiflora

Rosa multiflora Thunb., with high ornamental and medicinal value, is a deciduous woody plant in the Rosaceae family. R. multiflora, with characteristics of easy propagation, fast growth and strong heat resistance, can be used as rootstock, as well as breeding parent for Roses. However, many traits still needed to be improved via genetic engineering. In this study, some researches had been done on the regeneration and genetic transformation of R. multiflora. The main research results are as follows:1. Establishment of direct organogenesis system in R. multifloraIn order to determine the optional culture condition and high frequent regeneration genotypes, factors such as genotype、plant growth regulators、and different sources of explants that affect plant regeneration of4rose cultivars (R. multiflora) were studied by using leaves as explants. On the medium1/2MS+0.05mg/L NAA+1.5mg/L TDZ+10mg/L AgNO3+3%Glucose+0.3%GEL, the frequency of adventitious bud regeneration had significant differences among the4R. multiflora. The highest one(78.20%) is from R. multiflora2, the second(61.16%) from R. multiflora3, the third R. multiflora4was13%, and the fourth R. multiflora15.93%. The regeneration rate of leaves harvested directly from field was higher than that from sterile seedling in vitro culture. The results also indicted that genotypes, plant hormones and the source of the explants have great effect on the adventitious shoots induction.2. Establishment of somatic embryogenesis system in R. multifloraThe effects of genotype、additives、plant growth regulators and culture conditions were detailed on the induction of somatic embryos of the4R. multiflora. Furthermore, the effects of plant growth regulators and culture conditions on the proliferation and germination of somatic embryos were also investigated from R. multiflora2and R. multiflora4. Using leaflets as explants, somatic embryogenesis was observed in the four R. multiflora. There are differences among the4R. multiflora:after cultured on the medium MS+5.0mg/L2,4-D+400mg/L L-p+10mg/L AgNO3+3%Glucose+0.3%GEL, the highest rate of somatic embryo induction of R. multiflora1and R. multiflora4was 4.38%and5.01%; after cultured on the medium MS+4.0mg/L2,4-D+400mg/L L-p+10mg/L AgNO3+3%Glucose+0.3%GEL, the highest rate of somatic embryo induction of R. multiflora2and R. multiflora3was4.63%and2.78%. On the medium of MS+1.0mg/L2,4-D+0.01mg/L BA+3%Glucose+0.3%GEL, somatic embryos of R. multiflora2multiplicated greatly, and the multiplication coefficient was4.15. On the medium of MS+0.5mg/L2,4-D+0.005mg/L BA+3%Glucose+0.3%GEL, somatic embryos of R. multiflora4multiplicated greatly, and the multiplication coefficient was3.55. In our present studies, the highest somatic embryo germination of R. multiflora2was91.67%on the medium MS+0.5mg/L BA+l.Omg/L TDZ+3%Glucose+0.3%GEL. The highest somatic embryo germination of R. multiflora4was83.33%on the same medium.3. Primary studies of genetic transformation of R. multifloraThe effects of Km and Hyg on the proliferation of somatic embryos were studied by using somatic embryos of R. multiflora2and R. multiflora4. Results showed that60.18%somatic embryos gradually died under120mg/L Km after one month cultured and93.30%somatic embryos died under80mg/L Hyg in R. multiflora2. Results also showed that66.76%somatic embryo died under70mg/L Hyg in R. multiflora4. Using somatic embryos from R. multiflora2and R. multiflora4as receptors of agrobacterium-mediated genetic transformation, the effect of different infection time and co-culture time on transient expression of GUS gene was studied, the results showed that, after40min infection and3d co-culture, the transient expression rate of GUS gene reached to65.30%in R. multiflora2, while, when transferred to selective medium MS+1.0mg/L2,4-D+300mg/L Cef+50mg/L Hyg+3%Glucose+0.3%GEL for one month,6.52%of those embryos showed the GUS expression. For R. multifora4, after40min infection and5d co-culture, the transient expression rate of GUS gene reached to69.51%, while selective cultured one month on the same medium,7.32%of those embryos showed the GUS expression.