Studies On Cell Culture And Somatic Embryogenesis In Acacia Spp.

The calli from Acacia confusa, Acacia concinnata and Acacia melanoxylon were used as initial materials in this experiment,which included the effects of different culture conditions on maintaining and regulating the physiological conditions of calli, the comparison of the differences among the three kinds of Acacia in the establishment and maintenance of cell suspension culture, and the separation of single cell of the three species of Acacia from suspension cell culture, and establishment of the cloning system of single cells. Meanwhile, the calli of Acacia melanoxylon were used to establish the system of somatic embryogenesis of Acacia melanoxylon, and cytological observation during somatic embryogenesis and comparison of the changes of peroxidase activities and protein contents during somatic embryogenesis were also carried out. The main results were as follows:1. Establishment of maintenance system for the callus. The different effects such as plant growth regulators, light conditions, and genotypes on the maintenance of the three species of Acacia were compared in the experiment, and the results showed that MS medium supplemented with 0.2 mg·L^-1 2,4-D, 0.5 mg·L^-1 BA, and 0.2 mg·L^-1 NAA was beneficial to the maintenance of calli, and the superior inoculation amount was 0.3 g calli in each bottle.2.Status regulating and screening of Acacia callus. The effects of different 2, 4-D concentrations on the status of Acacia calli were analyzed in this experiment, and the results showed that the calli could be well maintained on the medium supplemented with 0.2-1 mg·L^-1 2,4-D, and most of the calli were fresh yellow, belonging to the loose and soft calliâ… andâ…¡, which were suitable for cell suspension culture. Moreover, the calli could be kept loose and soft in the weak light by comparing the effects of different light conditions.3.The maintenance of cell suspension systems for three species of Acacia spp. The effects of different genotypes, callus types, concentrations of 2, 4-D, sucrose concentrations, and inoculation amount on the cell suspension culture were analyzed in the experimént, and the results showed that the best initial material of the Acacia spp. for cell suspension culture was the fresh yellow, loose and soft callusâ… andâ…¡, which were propitious to establish the suspension cell culture system quickly. In addition, the cells of the Acacia melanoxylon were small, of strong embryogenic characteristics and fine dispersion, which were easy to establish the cell suspension system. The cell suspension could be maintained well under the conditions that the sucrose concentration was 30 g/L and the 2, 4-D concentration was 0.2 mg/L. And the initial inoculation amount also influenced the culture of cell suspension, of which the best inoculation amount was 0.3 g calli in each bottle. The suitable subculture cycle of the cell suspension culture was 7d in Acacia confusa, while 8-10 d in Acacia concinnata. 4. In vitro culture and differentiation of single cells for Acacia spp. The conditions of in vitro culture and differentiation of single cells for Acacia melanoxylon, Acacia cincinata, and Acacia confusa were analyzed in the experiment, including different initial densities, methods of cell culture, additive agents, and subculture times, etc. The results showed that the best plating density for cell culture of both Acacia cincinata and Acacia confusa was 5×10⁴～5×10⁵ cells/mL, and 1×10⁵～1×10⁶ cells/mL for Acacia melanoxylon. Embryogenic calli were induced from Acacia melanoxylon by plate culture, suspension culture and nursing culture, with the occurrence of embryoids. And plate culture of single cell was more effective for callus differentiation in Acacia cincinata and Acacia confusa. Moreover, some hydrolysis casein added in the media was benefit to single cell culture of the three species of Acacia spp..5. Somatic embryogenesis of Acacia melanoxylon. The effects of plant growth regulators on the induction of somatic embryogenesis in Acacia melanoxylon calli were studied in the experiment, the results showed that green embryoids could be induced in Acacia melanoxylon on the medium containing some concentrations of BA, of which the differentiation rate of somatic embryos was up to 94 % even more on the MS medium supplemented with 3 mg·L^-1 BA, 0.5 mg·L^-1 IAA and 0.5 mg·L^-1 NAA, while most of the somatic embryos were abnormal and their plantlet regeneration rate was low.6. The cytohistological observation as well as physiological and biochemical changes during somatic embryogenesis in Acacia melanoxylon. The cytohistological observation of somatic embryogenesis in Acacia melanoxylon was performed by paraffin section technique. The results showed that the origination of somatic embryogenesis of Acacia melanoxylon was classified into two ways, i.e., outer origination and inner one, developing through the stages of globular embryoids, heart-shaped embryoids and torpedo-shaped embryoids and cotyledonary embryoids. The peroxidase activities and protein contents experienced remarkable changes when the embryoid was formed from embryonic calli, and two peaks emerged from peroxidase activities, one in the early-stage embryoids cultured for 10 days, and the other in mature-stage embryoids cultured for 40 days.