| Camptotheca acuminata Decaisne (Nyssaceae) is a tree species indigenous and unique to China. The secondary compound, camptothecin (CPT) from this species may sever to treat cancers, which made its worldwide fame. It is an important species for timber utilization in the regions of China southern to Changjiang River. But the poor timber quality limits its utilization in paper production. Therefore, gene transfer for improving timber quality of this species is significant since its increasing economic value. However, this kind of studies is limited, and to our knowledge, no reports on the propagation of this species by leaf explants and subsequent gene transfer are available until now. Moreover, the development of root system during the acclimation of rooted plantlet in green house as well as its influences on photosynthetic traits is also poor understood. This may influence the afforestration practices of the rooted plantlet in future. Under this background, present study was carried out. The aims of this study were to establish the high-frequency regeneration system and then to build a successful protocol for UGPase gene transformation system by Agrobacterium-mediated transformation. We found that,1. B5 medium supplemented with 3%(w/v)sucrose, 6%agar, l.Omg/lBA produced thehighest shoot regenerating rate, 95%. The average number of regenerated shoots was 3.8 per axillary bud. After two times of subculture, one axillary bud could successfully proliferate at most 20 shoots.2. The optimum medium for the callus induction and shoot regeneration from leaf derived from in vitro cultured individual was WPM medium supplemented with 1.0mg/l BA, 0.2mg/l NAA, 3%(w/v)sucrose and agar 6g/l. Callus induction rate was up to 96% and shoot regeneration rate was 70.3%.3. The optimal subculture medium for regenerated shoots were WPM or B5 mediumcontaining 2%(w/v)sucrose, 0.2-0.5 mg/1 BA and 6g/l agar .4. The optimum medium for rooting of the regenerated plantlets was WPM supplemented with 0.5mg/l IBA, 2%(w/v) sucrose and agar 6.0g/l. Root frequencies were 98% with high root number, 4 to 5.5. Survival rate of rooted plantlet was above 90%. Comparing to seedlings, root systemof rooted plantlet in soil was poorly developed. The roottips per unit leaf area were marked lower than that of seedlings, which may influence the supply of water. Thisroot status is demonstrated by the lower stomatal conductance and subsequent lower capacity in photosynthesis.6. The efficient system of genetic transformation was established. 20mg/l kanamycin was used in the shoot regeneration medium to select the resistant shoots. 500mg/l cefotaxine was used to kill the Agrobacteria after infection. Explants were pre-cultured for 3 days before infection. The pre-cultured explants were incubated for 5-10min in the bacterial suspension which concentration (OD600) was between 0.1 and 0.5. Length of co-cultivation period was 3 days. The target genes integrated into the genomic DNA of C. acuminata were detected and confirmed by PCR and Southern blotting analysis. Genetic transformation rate was 6%. |
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