| In this study, Chinese narcissus (Narcissus tazetta var. chinensis) and antisense ACS transgenic bulblets of Chinese narcissus were used for materials for investigations as follows: homology cloning of ACS from leaf; improvement of conditions of callus culture; subculture and transplanting of the first transgenic bulblets. The main results were as follows:1 Cloning of ACS gene from leafRNA kit (Solarbio) coupled with TRIZOL was used for extracting of total RNA. The results showed that integrity of total RNA extracted by this method was excellent, high-yield, high-purity, polysaccharides and polyphenols could be removed efficiently. This method was effective for extracting of total RNA from leaf of Chinese narcissus. cDNA of ACS from leaf of Chinese narcissus was cloned by homology cloning coupled with RACE. Its full-length was 1427bp, and contained 17 poly (A) at the 3'-end. It was highly similar with ACS from other plants in GenBank. Sequence analysis showed it contained a 1200bp ORF, encoding 400 amino acid, TAA was the stop condon. It was registered in GenBank, and the registered NO. was GU182503.2 Improvement of conditions of callus cultureSlivers of Chinese narcissus bulbs were used for induction of callus, and culture conditions of callus of Chinese narcissus was investigated, and conditions of sunbculture were modified. Slivers of Chinese narcissus bulbs were subcultured on MS medium containing 1.0mg/L2,4-D, 0.1 mg/L KT, 0.5 mg/L BA and 30g/L sugar and cultured in darkness was efficient.3 Subculture and transplanting of the first transgenic bulbletsThe highest proliferation rate was obtained when first transgenic bulblets were subcultured on 1/2MS medium containing 2.0mg/L6-BA, 0.1mg/L 2,4-D and 30g/L sugar. MS medium containing 0.5g/L NAA and 0.5g/L active carbon was effective for seedling growth and rooting of antisense ACS transgenic bulblets of Chinese narcissus. The best medium for transplantation was natural soil: peatsoil 1:1, after pretreatment and autoclaving twice. |
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