Establishment Of Plant Regeneration System And Transformation System Of Ornamental Willow Clone

Salix×aureo-pendula cl."J1010"belongs to Salicaceae Salix, it is one catorgery of hardwood, its leaves are like the shape of needles. Its tress is drooping, yellow in color, elegancy and bright (branchlet is kelly in color in growing season and golden in dormacy season ). The disease-resistance is strong. The male plant has no wadding in spring so that it brings no environmental pollution. S.×aureo-pendula cl."J1010"also grows fast, It is fond of light, humid climate and fertile soil, but it has stong adaptability to soil, is able to endure the damping, and especially grows well near the river. What's more, it can also grow normally in fertile soil and dry hypsography with the best strength of the budding.This thesis studies the tissue culture and regeneration of S.×aureo-pendula cl."J1010"and investigates the cell morphology on the calli of the different doses and hormones by paraffin section, aiming to study different effectcal conditions of the regeneration of the S.×aureo-pendula cl."J1010". Through the study of the genetic transformation of the S.×aureo-pendulacl."J1010"with Agrobacterium tumefaciens, after screening and culturing by the antibiotic, this experiment obtains transgenetic resistant calli. Morever, it conducts primary test to the transgenetic calli by the histochemistry dyeing of GUS.1. Establishment of the tissue culture and regeneration systemAltenative usage of MS+6 -BA0.1mg/L + NAA 0.2mg/L and MS+6-BA 0.5mg/L +NAA0.1 mg/L is the most suitable way for the propagation. Taking the leaves and stem as the explants, MS+BA1 .0mg/L+NAA0.1 mg/L and 6-BA1.0mg/L+2, 4-D0.1 mg/L are preferable for the induction calli. 1/2MS+NAA0.3 mg/L is the most suitable medium for the rooting.2. Establishment of the embryo calliThe result shows that adding the 1.0～2.0mg/L6-BA and 0.1～0.5mg/L NAA to the medium in the proess of inducing the calli may induce embryo calli.3. Establishment of the transformation system of BADH geneWhen taking cefotaxime whose density reaches 500mg/L as the bacteriophates, it is suitable to inhibit the Agrobacterium tumefaciens. Besides, it has litlle affect on the calli and steriled shoot. When the denstity of Kanamycin reaches 50mg/L, the death rate of the calli is up to 97.6%. Using the Agrobacterium tumefaciens, taking the calli as the explants to infect and using 1/2YEB and 1/2MS to activate the Agrobacterium tumefaciens, they will improve the efficiency of the transformation if OD is 0.5, after pre-cultivating for 4 days and co-cultivtaing for 2 days. Transformated calli turns out to gentle bule after being detected by GUS. This result proves that exterior gene has transformated the genome DNA of the plants primarily.4. Observations on the interior strcture of the calli by the paraffin sectionCytology demonstrates the somatic embryo of the S.×aureo-pendula cl."J1010"comes from the surface of the calli and traces back to the thin parietal cell. Whether it traces back to origin of the single or multiple cell need further study.