The Research On Anther Culture In Vitro For Oil Energy Plant Of Swida Wilsoniana

Swida wilsoniana, a member of Cornaceae, is an important ecological and economic oil energy tree of its extensive adaptability and high fruit oil rate. Swida wilsoniana oil, which mainly enriching unsaturated fatty acids, is an ideal raw material for the preparation of high quality biodiesel. Swida wilsoniana breeding basically about natural variation and clonal selection at present, long cycle and low breeding has seriously hampered its seed process. Through anther culture to obtain protoplast, establish ploidy breeding system, it could shorten the breeding cycle, increase mutation rate, lay the solid foundation for large-scale development and utilization of new varieties resources with large fruit, high oil content and high yield.In this paper, by means of single factor experiments, different factors including genotype, flower development period, source concentration, pretreatment time and hormone ratio on in vitro culture of anther explants collected from six superior clones of Swida wilsoniana were studied. Effects of the tested factors on callus induction, proliferation and cell characteristics were investigated,and then anther callus occurrence mechanism was set forth. Meanwhile, the callus induced from anther was used as experimental materials for study different factors on, such as morphological structure, concentration and combination of enzyme, enzymolysis time and mannitol concentration protoplast isolation. An efficient anther callus protoplast separation system was established initially, which would provid technical and theoretical support for polyploid breeding and genetic improvement. The results showed as followings:1. The best time of explant sampling was early May when buds are yellowish green and theirs vertical and horizontal length ratio about3.8, petals not yet open, anthers with pale yellow, most of the spores were at the uninucleate stage.4℃low temperature pretreatment7-11d or32℃high temperature pretreatment1d was helpd to improve callus induction rate.2. The optimum system of callus induction followed the following steps. First, anther soaked with0.1%HgCl2for5minutes.Then inoculated them to the medium of MS+2,4-D1mg/L+TDZ0.5mg/L+3%sucrose. After one week dark pre-culture in28℃and then transferred to24℃was beneficial to anther callus induction.3. MS+ZT1.5mg/L+NAA0.5mg/L was optimal proliferation medium for anther callus.4. By analyzing the effect of genotype, flower development period, carbon concentration, pretreatment time and hormone ratio on anther in vitro culture, the period of anther development had the greatest impact, followed by genotype, hormone ratio again5. The optimum system for protoplast isolation of anther callus was:loose structure callus with light yellow and green color mixed with enzyme liquid of CPW+2%CellulaseR-10+0.3%Snailase+0.25%Pectinase+13%mannitol+0.6g/LMES, and then shaked slowly for10hours in dark could obtain the highest yield of protoplast, up to3.19X106个/g with viability of73.48%.