| Willows (Salix) represente a genus with a broad spectrum of tree and shrub species mainly distributed over the northern continents. Depending on their properties and growth characteristics they are important for ecological stable ecosystems, but also as raw material for pharmaceutical applications of the wood components.This study was started with the intention to establish the regeneration of willows and gene transformation technical system. The results could provide information and technology for breeding program for Salix with gene transfer methods for later research in tissue culture. In this research, the main results were as follows:1. Rapid propagation system of J795(Salix jiangsuensis cv. J795.), J797(S. jiangsuensis cv. J797.) and J799(S. jiangsuensis cv. J799.) were established by using one axillary bud stem as explants which derived from internodal sections of 1-year-old willows. The results as follows: the axillary bud can be initiated on Murashige and Skoog's medium (MS) supplemented with 0.2mg/L benzylaminopurine(6-BA) and 0.1mg/L naphthaleneacetic acid (NAA). On MS containing BAP at 0.1mg/L and NAA at 0.05mg/L or on MS containing BAP at 0.2-0.3mg/L and NAA at 0.1mg/L alternate a 3～5 fold shoot multiplication can be achieved in 4 weeks. The isolation shoots produced in this way, after being rooted on half-strength MS (1/2MS) or MS containing only NAA(0.2mg/L) for 20 days, can be transplanted to pots with over 90% success.2. Callus induction, callus growth, and regeneration were examined using leaf explants of three clones in Salix. Calli were initiated on basal MS medium plus 0.2～0.5mg/L of 2.4-dichlorphenoxyacetic acid (2.4-D) and 0.5～1.0mg/L of NAA or 1.0～2.0mg/L BAP and 0.5～1.0mg/L NAA in a factorial fashion. After 3 weeks of growth, developed calli were subsequently cultured on MS medium supplemented with either1.0 or 2.0mg/L6-BA. Calli exposed to more than 6.0mg/L BAP had deteriorated and were light brown and spongy in appearance after 3 weeks. The effect of thidiazuron (TDZ), zeatin and kinetin (KT) for initiation of callus are not better than 2.4-D, NAA and BAP. But TDZ still have effect after five months. Occasionally shoot can regenerated form petiole directly or callus indirectly. No treatment yielding calli responded with good shoot induction frequencies, and regrettably the shoots can't further differentiation and elongation, after several months, most of them browned and died.3. During inducing the callus of Salix, histological and cytological observations of the callus and embryos were performed with paraffin slice. The results showed that the dedifferentiation of the leaf was initiated in the vascular bundle sheath cells. The embryonic callus began from the appearance of the callus. There were much difference between embryonic callus and non-embryonic callus. The multicelluar embryo was developed from periclinal division primarily. There was lots of starch in the embryonic cell, and the starch gradual decreased accompanying the cell fission. 4. Based on shoot regeneration from stem segments, we selected stem as explants. Explants were inoculated with Agrobacterium tumefaciens stains LBA4404. This vector contains nptII and GUS genes in the T-DNA region, conferring kanamycin resistance andβ-glucuronidase activity. During selective cultivation, shoots were cut into single-node segments and sub-cultured biweekly. Three shoots survived after cultivation on MS medium plus 60mg/L kanamycin for 2 months. But they were negative in the PCR assay. Repeated cultivation indicated that the survived shoots were chimeric and the regeneration from calli for willow require further study.
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