Characteristics Of Nuclear/cytoplasmic Genome Of Somatic Hybrids Between G.paludosa And B.Willd,S,Musstii And D.carota And Chemical Composition Study

For plant somatic hybridization, it is hard to obatain regenerated plants. Before this assignment, few achievements for obtaining regenerated plants were reported. The study on how to increase the content of efficacious component in Chinese medicinal herbs by the method of somatic hybridisation is only a start. For example, it is only reported in the hybrids between P.quinquefolia L. and D.carota. The characteristics of nuclear/cytoplasmic genome of somatic hybrids between medical plants and their interaction have not been reported.In this work, protoplasts of G paludosa as a donor were irradiated by UV at the intensity of 380uW/cm2 for Os, 30s and lmin respectively and were fused with those of B. Willd as a recipient by PEG method. Great amount of regenerated clones capable of subsequent grwoth were obtained after cultures of two months without any light, some of which were identified as symmetric/asymmetric somatic hybrids by morphological , chromosome observation, isozyme patterns, 5SrDNA space sequence analysis and RAPD analysis. The above analysis showed that the nuclear genome of hybrids was mainly composed of that of B. Willd and recombination was also found. With mitochondria! and chloroplast specific probes, RFLP analysis revealed that the chloroplast genome of hybrids separated randomly. With chloroplast specific primers, no patterns of G paludosa were observed, which demonstrated that the chloroplast genome is mainly composed of that of B. Willd. By HPLC analysis, oleanolic Acid was identified in both parents and swertiamarin was only found in G paludosa. Meanwhile, both efficacious component were found in hybrid clone of 1-21,11-21 and III-4. Moreover, we make an further study on metabolism gram by GC-MS analysis, which shows that most of chemical substances of hybrids were identical with B. Willd. Several of those were identical with G paludosa and some novel compounds were also found in hybrids which manifested that the related genes frommetabolism of G paludosa were transformed to the hybrids.For another, protoplasts of S.musstii as a donor were irradiated by UV at the intensity of 380uW/cm2 for Os, 30s and lmin respectively and were fused with those of D. carota as a recipient by PEG method. Regenerated clones with UV intensity of 30s and lmin , capable of subsequent growth, were obtained after cultures of about 45 days without any light. By chromosome observation, isozyme pattern, 5SrDNA space sequence analysis and RAPD analysis, asymmetric somatic hybrids were selected, which shows that the nuclear genome of hybrids was mainly composed of that of D. carota and recombination was also found. With chloroplast specific primer, SSR analysis reveals that cp genome of the hybrid clones was both-parents type and some novel bands and deletion were observed meanwhile.In this dissertation, the work on somatic hybridization of medical plants with long genetic distance was carried out and the technology system of regenerated plants was established. The relationship and UV dosage were analyzed with the effect of chromosome diminishing and fragmentation. Furthermore, the characteristics of nuclear/cytoplasmic genome of somatic hybridization were also discussed. Finally, we investigated the difference of the metabolism grams of the activate principles and chemical gradients of G paludosa in different hybrid clones.