Studies On Regeneration System And Agrobacterium Tumefaciens-mediated Genetic Transformation In Casuarina Cunninghamiana Miq.

Casuarina cunninghamiana Miq. is an environmentally and economically important tree species of the Casuarinaceae family for coastal protection in China. It is characterized by drought tolerance, low soil fertility tolerance, saline alkali tolerance, fast-growing, windbreak, sand fixation, nitrogen fixation and other things. So, it has been widely used in coastal sandy and degraded sites for establishment of agroforestry systems in tropical and subtropical regions. It is a multi-purpose tree species for afforestation, as well as fuel wood and pulp. In recent years, due to over-pursuiting econmic value based on casuarina's outstanding rooting ability, very few casuarina clones are for long-term use. This have resulted in narrowing of genetic diversity and decreasing of shelterbelt protective functions. Meanwhile, as a consequence of desertification and salinization in the coastal sandy lands that resulted from zirconium titanium mining and aquaculture activities, remarkable recession of coastal casuarina plantations was found, which would threaten the economic development and ecological security of coastal areas. Therefore, selecting and creating new casuarina varieties or clones which are suitable for changed enviroments to update or replace the existing casuarina few cultivats and varieties have become a major task of current casuarina research.C. cunninghamiana explants were used as research materials, and then protocols of plant regeneration via indirect organogenesis and Agrobacterium tumefaciens-mediated genetic transformation were established. At the same time, three clones of C. cunninghamiana were selected and obtained. Then these clonal materials were used for genetic transformation. This study aims to establish a casuarina genetic engineering technology platform by developing the gene transformation protocol of casuarina, which would become a tool for gene function and expression analyses of C. cunninghamiana tree. The main conclusions are as follows:1) Seedling epicotyls of C. cunninghamiana were used (Pintan No. 3) as the initial explants, effects of plant growth regulators, sucrose, pH and AgNO₃ on callus induction and growth, adventitious bud differentiation and shoot development were studied. Results indicated that the optimal medium for callus induction of C. cunninghamiana was MSC + 0.54μmol/L NAA + 3.30μmol/L BAP + 30 g/L sucrose + pH 5.9. After being cultured one month in this medium, more than 94% induction rate of callus and 4.42 mm callus size could be achieved. When callus were cultured in the same medium for two months, the concentrations of NAA and AgNO₃ in the medium were adjusted to 0.27μmol/L and 1 mg/L respectively for two more months culturing, then 47.5% of adventitious bud differentiation, 27.38 bud number per callus and 4.75 shoots (≥2 cm bud) were obtained. Further more, When regeneration shoots were treated with liguid MSC medium containing 25μmol/L NAA for three days, whereafter, were cultured one month for root induction, 92% shoots could be observed one to three new-developed roots. After acclimatisation process, around 80% plantlets survived and showed no sign of desiccation under constant humidity for four weeks. Observation of adventitious bud differentiation from callus by scanning electron microscope and paraffin slice showed that: the plant regeneration protocol of C. cunninghamiana via indirect organogenesis was achieved by somatic embryo, which was developed from embryogenic callus.2) Seedling epicotyls of C. cunninghamiana (Pintan No. 3) were used as the research materials, effects of factors including work concentration of kanamycin and cefotaxime, co-cultivation time, OD600nm and acetosyringone on genetic transformation were studied. The aim was to establish the protocol of Agrobacterium tumefaciens-mediated genetic transformation for C. cunninghamiana. The results showed that the optimal kanamycin work concentrations were 20 mg/L for selection of callus induction and 50 mg/L for selection of callus growth and adventitious bud differentiation. The optimal cefotaxime work concentration is 200 mg/L for inhibiting growth of Agrobacterium. Moreover, five days of co-cultivation, OD 600nm 0.2 and 50μmol/L acetosyringone could lead to 88.89% of the genetic transformation rate and 40.66% gus gene expression.3) Genetic transformation experiments were conducted on seedling explants of C. cunninghamiana by Agrobacterium tumefaciens-mediated method (strain: C58C1, plasmid: pBIN35GUSINT). After several stages of antibiotic selection culture, some transgenic plants were obtained and five samples were selected for molecular biological detection. PCR analysis results showed: the gus gene was successfully transformed into the five plants. And by sequencing the PCR extraction, the study indicated the gene which was transformed into the plants was the target gene. Further analyzing by Southern blot hybridization, it also indicated that the target gene has been integrated into genomic DNA of C. cunninghamiana transgenic plants. Interestingly, we found that the transgenic plants inoculated with Frankia could also produce N-fixation nodulation. it implied that genetic transformation of C. cunninghamiana had not affect the symbiotic properties between transgenic plants and Frankia.4) Three clones (PT3-1, PT3-2, PT3-3) with high growth advantage from C. cunninghamiana seedling (Pintan No. 3) were obtained by seedling selection, in which the adventitious bud differentiation rate were 48.3%, 43.7% and 51.6%, respectively. Genetic transformation for these three clones by Agrobacterium tumefaciens-mediated (strain EHA105, plasmid pLR, LEA gene) method was successful conducted in terms of PCR detection on the antibiotic resistant callus, and RT-PCR analyses on three transgenic plants from PT3-3. The results showed that there were two transgenic plants that had integrated the LEA gene in genomic DNA, and expressed in the RNA level.