| Loquat was used as the material for the in vitro culture of induction and maintenance of embryonic cultures, immature embryo culture, and further gene cloning of the ACS and ACO genes encoding two key enzymes in ethylene synthesis pathway from embryonic cultures, then the transformation of loquat embryoids via ACS antigene mediated by Agrobacterium and the research of in vitro conservation of loquat plantlets were conducted. The main results were as follows:1. Induction and maintenance of embryogenic cultures in loquatAnthers of loquat "jiefangzhong" were used as materials. The orthogonal test was conducted to research the effects of sucrose and plant growth regulators on anther callus induction rate. The MS medium supplemented with 30 g·L-1 sucrose, 2.0 mg·L-1 2,4-D, 0.5 mg·L-1 6-BA was facilitated to callus induction of anthers in loquat. Moreover, this study utilized the embryonic cultures that selected and conserved for 5 years by Shen Qingbin(2003) to conduct alternative subcultures and it was found that the embryonic cultures could be subcultured for a long time.2. Immature embryo culture and obtaining of plantlets in loquatThe immature embryo germination, plantlet proliferation and rooting of 4 loquat cultivars from different locations were investigated respectively. The results were as follows: the discrepancies in size and genotype resulted in the differences of embryonic germination rates; however, different light intensities and upper portion cutting of cotyledon produced little influence on embryonic germination rates. The 1/2MS medium supplemented with 20 g·L-1 white sugar had the highest germination rate, different concentrations of white sugar and MS had the coherent impact on the germination of four loquat cultivars. Take "Puxinben" for example, the immature embryo germination rate decreased along with the declining MS concentration, on the contrary, this rate rose along with the increasing white sugar concentrations. The MS medium supplemented with 1.0 mg·L-1 BA, 0.1 mg·L-1 or 0.2 mg·L-1 NAA was suitable for plantlet proliferation of different loquat cultivars. The 1/2MS medium supplemented with 0.4 mg·L-1NAA was suitable for rooting of different loquat cultivars.3. Cloning of ACS genes from embryonic cultures in loquatEmbryonic cultures of loquat were firstly used as the materials for RNA extraction for further cloning ACS gene. Two ACS gene fragments of 523bp were obtained via homologous cloning, then a pair of primers were designed to clone the ORF of ACS gene and gained two ACS genes simultaneously with the same length of 1464bp which encoded 487 amino acids. The two genes, which were respectively named EjACS-1 and EjACS-2, had a similarity of 92%, and 115 nucleotides discrepancies existed in a total of 1464 nucleotides. The protein encoded by ACS gene were analysized through bioinformatical methods, the results manifested that these two proteins had different theoretical pIs and both appeared hydrophilic; EjACS-1 was not a transmembrane protein, EjACS-2 might form a transmembrane helix with a N-terminal out of the membrane. Neither of the two ACS proteins had signal peptide hence they were probably not the secreted proteins. ACS gene of 2319bp length was also cloned from loquat genome in this study and name gACS subsequently, compared its sequence with that of EjACS-2, Three introns were founded in ACS gene, possessing the length of 89bp, 106bp and 660bp, respectively.4. Cloning of the ACO gene from embryonic cultures in loquatIn this study ACO gene was cloned from loquat embryonic cultures. Homologous cloning and 3'RACE were used to gain the whole length of loquat ACO gene EjACO-1 of 1160 bp, encoding 322 amino acids. Bioinformatical analysis revealed that the protein encoded by ACO gene had a theoretical PI of 5.35, therefore it belonged to acidic protein, the whole polypeptide chain appeared to be hydrophilic and it might be a non-transmembrane protein and a non-secreted protein. The ACO gene gACO which cloned from loquat genome had a length of 1517bp, compared its sequence with that of EjACS-1, Three introns were found in ACO gene with the length of 114bp, 240bp and 194bp, respectively.5. The introduction of antisense ACS gene into loquat embryoidsIn this study antisense ACS gene was introduced into loquat embryoids by genetic transformation mediated by Agrobacterium, and the orthogonal test was conducted to uncover the factors that affected the transformation efficiency. The optimum factors for this experiment were found by analyzing instantaneous expression rates of GUS, that is, inoculated for 45min, co-cultivated for 3d, Agrobacterium suspension containing 0 g·L-1 sucrose, pretreated for 12d and the OD600 value 0.8, the instantaneous expression rate of loquat embryoids achieved the highest point at this time.6. In vitro conservation of loquat plantletsIn vitro preservation methods and the impact of MS and paclobutrazol of various concentrations were investigated on loquat plantlets. The results indicated that the 1/3MS medium supplemented with 4.0 mg·L-1 PP333 was relatively suitable for the in vitro preservation of loquat plantlets. The survival rate of plantlets remained 55.98 % without subculture for 12 months, and 4.0 mg·L-1 PP333 was facilitate to in vitro preservation of loquat plantlets. 22 accessions of loquat germplasm resources from different locations were preserved in vitro using this culture medium, and the growth situation of loquat resourcesthat had been kept by our institution for 2 years was observed as well as preserved successively for a long term.
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