Study On Regeneration And Genetic Transformation System Of Hydrangea Macrophylla And Hypericum Androsaemum

Hydrangea macrophylla (Thunb.) and Hypericum androsaemum L. are woody ornamental plants belonging to the Hydrangeaceae and Hypericaceae families respectively. Hydrangea macrophylla (Thunb) is used for planting courtyard flower borders, forest fringes, and ornamental garden, and is used in large scale flower decorations and sold as potted flower due to its colorful and large flower and long-lasting florescence. Hypericum androsaemum L. is a relatively new winter courtyard shrub and is used as new cut flower material because of its attractive looking fruits, long evergreen shoots and long lasting fruiting stage. However, both crops are vulnerable to plant diseases during the growing season, namely Hydrangea leaf spot and Hypericum root-knot nematode. Genetic engineering is potentially an efficient way of improving their disease resistance. By investigating the factors such as choice of genotype and explants, incubation conditions, inoculation methods, and different plant growth regulators, we established a highly efficient regeneration system and studied. An agrobacterium tumefaciens-mediated genetic transformation of Hydrangea macrophylla was developed as well. In addition, the pathogen causing leaf spot was isolated and identified as Cercospora hydrangeae. The major results were as follows:1. The establishment of a rapid propagation and regeneration system for HydrangeaSterile shoots of Hydrangea macrophylla were obtained for genotypes Hydl, Hyd4 and Hyd5. The stems with axillary buds of these three genotypes were used as explants for the establishment of a micropropagation system. The optimum medium for proliferation of the three genotypes was B5+0.5 mg/L BA+0.1 mg/L NAA with a resulting multiplication coefficient of 3.0. Hydl terminal buds were used as explants for rooting. The rooting medium with 60 mL perlite plus 30 mL liquid medium of 1/2 B5+ 0.5 mg/L IBA gave the best results for the rooting rate (100%). Rooted shoots transplanted to mixed substrates of peat soil:perlite (1:1) had a survival rate as high as 100% after 30 days.Leaves and petioles of seven genotypes Hyd1, Hyd4, Hyd5, Hyd6, Hyd7, Hyd8 and Hyd10 were used as explants for experiments involving direct and indirect regeneration. The results showed that for leaf explants of Hydl on the B5 medium supplemented with 2.25mg/L BA,0.1 mg/L IBA and 100 mg/L cefotaxime, the regeneration rate was 100% and average number of shoots per explant was 2.7. There were large differences in regeneration capacity among different genotypes. On the same medium, the regeneration rate in leaf explants of Hyd4 and Hyd8 were lower than 30%.Somaclonal variation of Hydrangea macrophylla regenerants were tested by ISSR markers. There were in total 1857 scorable bands. The observed similarity was between 0.980-0.983. No significant somaclonal variation was abserved.2. Study on development of a genetic transformation system of Hydrangea macrophyllaAgrobacterium tumefaciens-mediated genetic transformation was studied by using leaf explants of Hydl. The concentration of Kanamycin selection pressures was determined as 20 mg/L for shoot regeneration of leaves and 200 mg/L for rooting. A. tumefaciens could be inhibited with 200 mg/L cefotaxime. GUS transient expression analysis revealed that the leaf explants with 0 d pre-culture, infected for 35min and co-cultured for 3 day could reach the highest transient GUS expression rate of 100%. Delayed kanamycin selection for seven days benefited the formation of resistant callus and shoots. Resistant callus (100%) and 12 regenerated plants were obtained under the optimal transformation conditions。Resistant callus was difficult to induce for regeneration on kanamycin medium. Further research should focus on callus differentiation.3. Establishment of a rapid micro-propagation and regeneration system of Hypericum androsaemumBasal medium and BA/NAA were tested in order to establish a rapid micro-propagation sytem of genotype Hyp3. The highest shoot induction percentage (100%) and mean number of shoot per explant (3.7) were found on 1/2MS plus BA1.0mg/L.A regeneration system for Hypericum androsaemum was studied using explants of four genotypes Hypl, Hyp3, Hyp4 and Hyp7. A stable and highly efficient regeneration system of Hyp3 leaf explants was established, viz., media:MS+TDZ0.1 mg/L+AgNO3 1.0 mg/L+sucrose 30 mg/L, initial 20d dark culture followed by photoperiod culture. The highest regeneration rate was 83.3%. Genotype was a significant factor in Hypericum androsaemum regeneration. Medium with AgNO3 supplied a highly efficient and stable regeneration system.4. Study on genetic transformation of Hypericum androsaemumThe factors of genetic transformation were tested in Hypericum androsaemum genotype Hyp3. The optimal protocol was as follows. Genotype Hyp3 leaf explants without pre-culture was immerged into EHA105 for 5 min, co-cultured on MS+ TDZ 0.1 mg/L+NAA 0.01 mg/L+AS 40 mg/L+MES 2.0g/L medium for 3d, selected on MS+ TDZ 0.1 mg/L+NAA 0.01 mg/L+AgNO3 1.0 mg/L+cef200 mg/L medium. High transient GUS expression rated (79.2%) was gained.5. Study on hydrangea leaf spotPathogen was isolated from diseased potted hydrangea leaves and was identified as Cercospora hydrangeae by morphological and rDNA-ITS way. The ITS sequence was deposited to Genbank, accessing No. JF495458.1.