Research On Culture Condition For Haplocladium Microphyllum And Physcomitrella Patens, And Transformation Condition Of Physcomitrella Patens

Bryophyte is the most primitive higher plant. It is widely distributed. There are abundant resources and many species. Due to environmental pollution, climate change, improper exploitation, many bryophytes are on the verge of extinction, even haved recently. Tissue culture is an important way to study moss germplasm conservation and reproduction. On the other hand, moss tissue culture is also an important premise technology for scientific research of bryophytes. Tissue culture developed slowly and got immature culture system because it is difficult for bryophytes to use conventional culture methods which used for other higher plant because of different organizational structure and habits. Moss tissue culture has a certain academic and practical value. Therefore, it is essential to start tissue culture conditions for different bryophytes and carry out in-depth and detailed comparative studies. In this paper, we established a feasible system of moss tissue culture including drawning, cultivating, testing. And we investigated the effects of pH, nitrogen, phosphorus and potassium on the growth and development of Physcomitrella patens and Haplocladium microphyllum. The results showed that the growth effects of phosphorus and potassium on them are less obvious while the pH and nitrogen are more. Moreover, they have different requirements.The growth of P. patens was the best when pH was 6-7, whereas the growth of H. microphyllum was 7. We found that the gametophore of P. patens appear stem browning, leaf chlorosis and protonema cell become shorter when pH was less than 6, even protonema died at 4. In case of pH greater than 7, there will be more rhizoids from gametophore and protonema differentiates into caulonema easier. Meanwhile, we found that H.microphylluma was live when pH was 3. When pH was 8-9, P. patens have rhizoids but elongation was not significantly inhibited and it is easier for protonema to differentiate into caulonema, however, H. microphyllum generated more rhizoids, elongation growth was significantly inhibited.In a certain, improving the nitrogen concentration is benefit for inducing the formation of protonema and inhibiting elongation of gametophore. When the nitrogen concentration is 0.04M, the function is most obvious. In higher concentrations of nitrogen, caulonema differentiation is suppressed while in favor of the growth of chloronema. However higher concentration of nitrogen will inhibit elongation of gametophore. It can grow normally in lower concentration of nitrogen even nitrogen-free. The results of this study provide a laboratory for H. microphyllum and P. patens and the best tissue culture conditions important guidance, but also for an initial moss tissue culture and propagation provide a good platform.Due to its capacity to undergo efficient mitotic homologous recombination, P. patens has emerged as an important model system in recent years. Here, we preliminary optimized that protoplast preparation and regeneration of protoplasts condition. This method can generate even more than 1.6×106 per ml (standard) protoplasts and regeneration rate has also been improved to 2.8%. PEG mediated transformation does not require specialized equipment, and can be performed in any laboratory. The establishment and stable operation is important for future research related to Molecular Biology.