Study On Somatic Embryogenesis And Protoplast Culture In Ramie (Bochmeria Nivea L. Guad.)

Ramie (Bochmeria nivea L. Gaud.) or "China grass" is a perennial bast fiber plant, one of the very important export materials in China. Ramie cultivars and breeding materials are heterozygous because of cross-pollination. It is a big problem of ramie genetic improvement that the period of conventional breeding is long. Somatic embryogenesis is an important pathway for the regeneration of plant cell culture systems, which has been a basic technique in shortening breeding period and fastening popularization of fine cultivars. Protoplast isolation, culture and plant regeneration bring a bright prospect to plant mutants selection in vitro, genetic transformation and somatic hybridization. And protoplast can be used to fundamental research such as physiology, genetics and breeding. To improve this fiber crop via biotechnology, it is essential that cell culture systems be developed, and somatic embryogenesis be effectively induced and maintained in as a wide range of cultivars as possible. In order to seek the condition of somatic embryogenesis and protoplasts culture in ramie, the study was carried out and the main results are as follows:1. Effect on callus induction and differentiation of plant growth regulator, organic appendices and inorganic salts.Calli were produced on these explants (cotyledons, hypocotyls, shoot-tips and seeds). The variety and the development stage of explants affect the potential of morphogenesis. Characters of ramie callus were multiplicity, among all of them, light yellow or green-yellow, friable embryogenic calli developed into embryos via somatic embryogenesis after 4 weeks on modified MSB medium supplemented with CH(300 mg l-1) and Gln(200 mg l-1). The result indicated that the conditions of high potassium, nitrate-nitrogen, myo-inositol, thiamine, low calcium and ammonia-nitrogen, coconut milk, casem hydrolysates, Gin were crucial for ramie callus induction and differentiation, while CPA and 2, 4-D are good for callus induction and proliferation, but the concentration must be low. The best combination of plant growth regulators for callusinduction was CPA(0.5 mg l-1) and BR(0.05 mg l-1).2. Effect on callus induction and differentiation of genotypeGenotype is an important factor that influences somatic embryogenesis. Various caili were produced when explants of cultivars were cultured on the same induction medium. The differentiation potential of cultivars varies, under the same condition, the number of somatic embryos of "Guangxi Heipidou" was first, "Enshi Qingma" was second, very few were produced of "Chuannan Hongpi Xiaoma"and "Huangkezao", and the differentiation time of "3147-3" was very long.3. Growth character of cell suspension linesIn the early stage of suspension culture, pH descended and cells divided vigorously, and then, pH was ascendant, cells division slowed, cells growth was visibly restrained by the ascendant pH after 6d.4. Protoplast isolationThe combination of enzymes for cotyledons protoplast isolation was Cellulose R-10(0.5%), pectinase(0.6%), and Cellulase R-10(4%); Pectinase(2%) was suitable to cell suspension lines for protoplast isolation.5. Protoplast cultureCotyledon protoplasts first divided in KM8p medium supplemented with 2, 4-D(0.2 mg l-1), NAA(0.1 mg l-1) and ZT(0.5 mg l-1), but continuous division has not been observed. Protoplasts from cell suspension lines grew well in KM8p medium contained 2, 4-D(0.5 mg l-1) and BA(0.5 mg l-1), after 25d, little cells cluster was produced and visible to naked eyes.