Tissue Culture Of Bamboo And Analysis Of Gene Expression Of Differentiation In Phyllostachys Edulis

Bamboo is one of the most important forest resources, which has a high commercial value and ecological function. Bamboo is indispensable in manufacture industry and our daily life. However, genetic improvement researches on bamboo are lag now. In recent years, plant tissue culture and suspension cell culture have been the basic technologies on breeding new varieties by the approaches of somatic cells mutation, cell fusion and genetic transformation. The construction of a tissue culture technology system of bamboo on in vitro micropropogation, regeneation, culture of suspension cell and protoplast, and genetic transformation will provide a research platform for the researches on physiology, molecular biology, and genetics and breeding of bamboo. Browning and hard to regenerate are huge problems on tissue culture of bamboo. The scattered bamboos such as Phyllostachy edulis, are especially hard to regenerate.In this study, Phyllostachys edulis, Cephalostachyum fuchsianum and Bambusa multiplex were sued as materials to establish the culture system of callus or suspension cell lines. The gene expressions of Phyllostachys edulis calli with differentiation potentials were detected by cDNA microarray. The research contents and results are as follows:1. The calii of Phyllostachys edulis can proliferation on the medium of MS macro salt +B5 micro salt +B5 organic +MS ferric salt +500 mg/L proline +500 mg/L glutamine +300 mg/L tryptone +0.5-1.0 mg/L 2,4-D+30 g/L sucrose +6 g/L agar. The highly dedifferentiation calli can not survive on the medium without 2,4-D.2. Four types of calli were seemed as calli with differentiation potential. They were purple campact calli, purple friable calli, yellow compact calli and green compact calli. The purple campact calli, purple friable calli and yellow compact calli can survive on the medium without 2,4-D and differentiate adventitious buds or some tissues wiith structure. However, the green compact calli would brown during subculture.3. The purple campact calli, yellow compact calli and green compact calli were made a pair with yellow friable calli respectively to hybridize to the probes in cDNA microarraies. There were 412 differentially expressed genes detected in the microarray of purple compcat calli VS yellow friable calli. There were 412 differentially expressed genes detected in the microarray of yellow compcat calli VS yellow friable calli. And 567 differentially expressed genes were detected in the microarray of green compcat calli VS yellow friable calli. The unigenes of the differentially expressed genes were analyzed by GO. Genes related to cell differentiation, development, somatic embryogenesis, plant hormones and kinases in signal transduction of differentiation were differentially expressed respectively in 3 types of calli comparing to yellow friable calli. And genes stimulated cell death were upregulated in green compact calli.4. Genes related to ethylene synthesis, JA synthesis, stress response, DNA repair and Ascorbate peroxidase were detected differently expressed in the calli cultured on the medium without 2,4-D for 15 d.5. The calii of Bambusa multiplex could proliferation on the medium of MS macro salt +B5 micro salt +B5 organic +MS ferric salt +500 mg/L proline +500 mg/L glutamine +300 mg/L tryptone + 1.0 mg/L 2,4-D+30 g/L sucrose +6 g/L agar. Adventitious buds could differentiate from yellow compact calli. And amonts of albino buds were acquired. Both normal buds and albino buds could proliferation and rooting.6. Fine suspension cell line could be acquired respectively by inoculation the calli of Phyllostachys edulis and Cephalostachyum fuchsianum into the liquid medium of MS+500 mg/L proline +500 mg/L glutamine +300 mg/L tryptone +1.5 mg/L 2,4-D +30 g/L sucrose. The subculture cycle were 9 d. Calli could be acquired after the cells being cultured in the solid medium by the inoculation density of 20 g/L.