1.Isolation And Culture Of Protoplasts Of Physcomitrium Sphaericum 2.Studies On RNAi System In Tobacco

Gene targeting by homologous recombination can realized targeted integration. At the same time we can knock-out some gene to know its genetic function and create specific mutation to study some kinds of metabolizing mechanism.But gene targeting was mainly applied to such field as bacterium, yeast and some epiphyte at present, seldom used in higher plants because of the lower frequency of homologous recombination. The moss Physcomitrella patens (Funariales Bryophyta) is being developed as a model system for plant biological studies. Its gene targeting efficiency can be compared with that observed in Saccharomyces cerevisiae.In this thesis, Physcomitrium sphaericum growing in the area of Beijing, which belongs to the same section as Physcomitrella patens, is applied as experiment material to explore the condition of the sterile culture. Because of the special biological structure of bryology, it was very difficult to transfer foreign gene into the protonema or gametophyte by Agrobacterium-mediated transformation. Protoplasts as acceptor, using direct DNA transfer methods such as microprojectile bombardment and PEG-mediated transformation is becoming a good way. So how to obtain high output and high energy protoplasts was very important. Here we studied the relationship of various factors and the quality of protoplasts.which maybe could be the basic of moss gene targeting.Results showed:Inoculated the spores onto diferrent kinds of media, such as MS, Benecke and Knop, we found that there was no difference when the spores germinated and differentiated into cauliform soon. It was very difficult to keep protonema longer. The media types had some effect on the state of protonema.The media supplemented with hormone of different types and concentration to induce callus, Only the medium added with 6-BA 0.5 mg/1 + 2,4-D 0.5 mg/1 had good effect on callus formation.Isolation of protoplasts from different stage of protonema and cauliform revealed that 15-17 days protonema was the best material because of the higher protoplasts output. There was less protoplasts when the protonema was over three weeks old. It was much more difficult when we use the cauliform asmaterial.Pretreatment for 30 minutes by 0.02% Tween-80 before digestion can obviously increase output of protoplasts.Using 0.5 % CELLULASE + 0.4% PECTOLYASE Y-23 + 0.5% MACEROZYME +0.3%HEMICELLULASE H-2125 to digest 0.1 g protonema for 2 hours, can abtain 5 +103 protoplasts /ml, which keep more energy.