Study On The Construction Of Tissue Culture And Transformation System For Hypericum Androsaemum L.

Hypericum androsaemum L.has become much more popular as cut and potted flowers in the market for its valuable ornamental characters. However, the traditional propagation and breeding technology have some disadvantages such as long time to realize the effects, thus difficult to meet market demand. The present research was mainly about the micropropagation, direct regeneration and genetic transformation of Hypericum androsaemum’Hyp3’.The Orthogonal experiment design was used in the micropropagation investigation, this provided a useful method for germplasm conservation and industrial production of the flower. Comparison on the influence of genotypes and hormones on regeneration for establishing a high effective and steady regeneration system of’Hyp3’was done. In addition the genetic transformation of’Hyp3’was studied. The main results were as the follows:1.The establishment of rapid micro-propagation system of Hypericum androsaemum ’Hyp3’ With Hypericum androsaemum’Hyp3’as main materials, the influence of basic media 6-BA and NAA were investigated. The results anlyse the optimal medium for multi-propagation was 1/2MS+6-BA1.0mg/L+NAA0mg/L.2. The establishment of direct regeneration system Using the explant of Hypericum androsaemum’Hyp3’as the main material and’Hyp1’ ’Hyp4’、’Hyp7’as the contrast, the direct regeneration system of Hypericum androsaemum L was investigated. The influence of different genotypes, sugar sources, AgNO3, dark culture and plant hormones on regeneration were discussed. The results showed differences among genotypes.’Hyp3’had the best regeneration rate at 74% after 40d inoculation. The optimal medium was MS+TDZ0.10mg/L+NAA0.01mg/L+AgNO3 1.0mg/L, in dark treatment of 20d. The medium with AgNO3 was better than filter paper and supplied a highly efficient and stable regeneration system.3. Investigation of genetic transformation technique for Hypericum androsaemum L. The leaf explants of Hypericum androsaemum’Hyp3’were used to study the influence of factors of genetic transformation. The Agro-bacterium-mediated genetic transformation method was used. Our results showed that the selective concentration of kanamycin was 20.0 mg/L. We also found that there was only 2.5% GUS transient expression with an infection time 5min,co-culture time 10d, Agro-bacterium OD600= 0.5-0.7 and a very small blue spot visible in the explant. Other treatments all had no transgenic expression. A further research on genetic transformation of Hypericum androsaemum L is required.