| Lily, a kind of perennial bulb flowers with high ornamental value, has been being popularly used as cut flowers, potted flowers and yard plants. Breeding of lily new cultivars and supplying of commercial bulbs is an urgent task to our country's flower culture industry. The study of lily genetic transformation was started relatively late, but a great progress has been made in the past ten years. Nevertheless, lily cultivars obtained by genetic transformation technology have not yet been reported.The subject of this study includes establishment of lily stable efficient regeneration system and production of transgenic lily plants by Agrobacterium-mediated transforma-tion. The results are as follows:1) One way of lily regeneration is via indirect organogenesis, that is, firstly callus induction from slices of bulblet scales, then plant regeneration from induced calluses, and at last bulblet production and enlargement from regenerated plants. The results indicate that callus induction frequency (68.75%) of slices formed by cutting bulblet scales horizontally is significantly higher than that (25.96%) of the ones vertically. Callus induction frequency (93.33%) of upper slices is highest, and that of bottom ones is lowest. Due to light, calluses induced in the photoperiod of light (16 h)/dark (8h) brown seriously and regenerate buds early, but calluses induced in dark neither brown nor regenerate buds so early.2) In the experiment on Lilium 'Bright Yellow' callus differentiation, the differentiation frequency (90%) on MS medium without any hormone is highest of all. The higher the concentration of BA is, the more serious the browning of calluses is. In order to compare different transformation strategies, we choose MS medium without any hormone as the selection differentiation medium.3) The research in bulblet production from seedlings demonstrates that 0.05 mg/L ABA is the best for bulblet production, as the frequency is 95%. When the concentration of ABA is higher than 0.05 mg/L. bulblet production frequency begins to decrease as ABA concentration increases. Moreover,0.2 mg/L ABA is the best for bulblet enlargement, as the average weight of bulblets is about 166 mg. When ABA concentration is higher than 0.2 mg/L, the average weight of bulblets begins to decrease as ABA concentration increases.4) The experiments on direct bulblet regeneration from scales reveal that 90 g/L sucrose is the best for direct bulblet regeneration and growth.1.5 MS is the best for the enlargement of regenerated bulblets. The combination of 1 mg/L BA and 0.2 mg/L NAA can significantly increase regenerated bulblets per scale. SA inhibits direct bulblet regeneration from scales. Low concentrations of PP333 do not affect bulblet regeneration, but high concentrations inhibit it. The best medium for direct bulblet regeneration from scales is 1.5 MS+1mg/L BA+0.2 mg/LNAA+90 g/L sucrose.5) When L.'Bright Yellow'calluses are used as transformation receptor, Kan selection pressure is determined to be 75 mg/L. The target genes include TA29-Barnase chimeric gene.35S-CBF3 gene and 35S-LsAGRi. L.'Bright Yellow' calluses always fall apart to grains after inoculation and brown to death after co-cultivation. Even if seedlings can be regenerated eventually, they grow rather weakly. Eight Kan-resistant seedlings are obtained after genetic transformation with 35S-CBF3, six of which are proved positive by PCR analysis. Four Kan-resistan seedlings are obtained after genetic transformation with TA29-Barnase. three of which are proved positive by PCR analysis.6) When L. longiflorum bulblet scales are used as transformation receptor, Kan selection pressure is also 75 mg/L. The target genes are TA29-Barnase and 35S-LsAGRi. Nine Kan-resistant seedlings are obtained after genetic transformation with TA29-Barnase. three of which are proved positive by PCR analysis. The ratio of chimeras in resistant seedlings is rather high. |
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