Establishment Of High Efficient Regeneration System And Study On Genetic Transformation Of Hydrangea Macrophylla

Hydrangea[Hydrangea macrophylla (Thunb.) Ser] is one of the ornamental plants. It has big and colorful flowers and used as a potted flowering plant, cut flower or garden ornamental. Tissue culture was systematically studied in order to provide the technique support for industrial production of Hydrangea. By investigating the factors of different plant growth regulators, incubation conditions, inoculation method and genotypes, we established a high efficient regeneration system and studied on Agrobacterium tumefaciens-mediated genetic transformation of H.macrophylla. The major results were as follows:(1) The establishment of rapid propagation system for HydrangeaThe stems with axillary buds of’Hydl’,’Hyd4’and’Hyd5’were used as explants during the establishment of micropropagation technique system. The optimum medium for proliferation of the three genotypes was B5 supplemented with 0.5 mg/L 6-BA and 0.1 mg/L NAA, which resulted in multipcation coefficient over 3.0. With’Hyd1’terminal buds as explants, the rooting medium with 60.0 mL perlite plus 30.0 mL liqudid medium of 1/2 B5+0.5 mg/L IBA had the best effect on rooting rate (100.0%). The rooted shoots transplanted to the mixed substrates of peat soil and perlite (1:3), and the survival rate reached 100.0% after 30 days.(2) The establishment of regeneration system for HydrangeaLeaves and petioles were used as explants for the direct and indirect regeneratin of Hydrangea. The results showed that there were great differences on the regeneration capacity among different genotypes. On B5 medium supplemented with 2.25mg/l 6-BA, 0.1mg/l IBA and 1.0 mg/L AgNO3, the regeneration rate and average number of shoots per explant were 96.3% and 3.0 from leaf explants of’Hyd1’. With the same culture condition, the regeneration rate of ’Hyd4’ and’Hyd8’leaf explants were 26.7% and 10.0%, respectively. For the indirect regeneration of’Hyd1’ genotype, callus induction rate from leaf and petiole explants was 100.0% with hormone combination of 1.0-1.5 mg/L KT and 1.0 mg/L 2,4-D. Callus could be differentiated into adventitious shoots on the B5 medium puls 1.0 mg/L 6-BA and 0.1 mg/LIBA. The differentiation coefficient was low about 26.7%。(3) Study on the genetic transformation system of HydrangeaAgrobacterium tumefaciens-mediated genetic transformation was studied by using leaf explants of’Hyd1’. The concentration of Kanamycin selection pressures was determined as 20.0 mg/L for shoot regeneration of leaves and 200.0 mg/L for rooting. A. tumefaciens could be inhibited with 200.0 mg/L Cefotaxime. According to the result of GUS transient expression analysis, the leaf explants with 0 d pre-culture, infected for 35.0 min and co-cultured for 3.0 day had the highest GUS transient expression rate of 100.0%. Resistent callus and 10 regenerated plants were obtained under the optimal transformation conditions.