Tissue Culture And Polyploid Induction In Akebia Trifoliate

In our research wild Akebia trifoliate was used as experimental material. By tissue culture we established rapid propagation of Akebia trifoliate. Also, colchicine was used for polyploid induction of cluster buds. Then polyploid identification was carried out. These laid the foundation of rapid propagation of Akebia trifoliate. And provided technical support for new varieties cultivation. The main results are as follows:1. Selection and treatment of explant. Seeds removed from fruit were the best explant in the study of combinative sterilization of alcohol and mercuric chloride to different explants. 75%alcohol disinfect 15 seconds and then 0.1% mercuric chloride sterilize 7 minutes was an optimal scheme. The optimum condition of seedling emergence of Akebia trifoliate was cutting seeds in half lengthwise and inoculating into MS + 2Ca2+ + 6-BA 1.0 mg/L + NAA 0.3 mg/L + activated carbon 0.1 g/L medium after that. The inoculated seeds were cultivated on the condition of lighting 12 hours per day under 25 ? with 1000 lx light intensity.2. Induction and differentiation of seed callus. The optimized inducing medium for seed callus was MS + 2,4-D 1.0 mg/L + 6-BA 0.3 mg/L medium. On this condition the induction rate was 32.5%. Embryogenic callus can be obtained after seed callus subcultured on MS + 2,4-D 1.5mg/L + 6-BA 0.1 mg/L medium. Embryogenic callus differentiation occurred on MS + 6-BA 2.0mg/L + NAA 0.3 mg/L + 75 g/L banana puree medium. Differentiation rate of embryogenic callus was 11.7%.3. Induction and rooting culture of cluster buds. The suitable medium for axillary buds induction was MS+6-BA 2.0 mg/L medium. Stem with axillary buds can be induced to grow average buds number up to 8 buds on MS + 6-BA 2.0 mg/L + 2,4-D 0.2 mg/L medium, however,we only got main bud and other buds which could not differentiation into axillary buds. Cluster buds induced by single axillary bud also failed in this study. On the MS + 6-BA 2.0 mg/L + NAA0.3 mg/L medium seedling average proliferation number was the highest and then this mediumwas selected as the cluster buds induction medium of seedling. Single axillary bud can not root under different hormone combinations, while multiple bud clumps can root under different hormone combinations.4. Induction and identification of polyploidy. By taking multiple shoots as explant and using tissue culture technology, the influence of colchicine concentrations and time to Akebia trifoliate polyploid induction was investigated. The results showed that a good inductive effect of polyploidy can be obtained under the situation, which 0.3% colchicine acted on multiple shoots 24 hours by shaker vibrating treatment. Chromosome counting method revealed that the chromosome number of untreated multiple shoots were 2n = 2x = 16 and confirmed as diploid. The chromosome number of polyploid multiple shoots which were got by 0.3% colchicine acted on multiple shoots 24 hours with shaker vibrating treatment were 2n = 4x = 32. The polyploid multiple shoots confirmed as tetraploid. And the induction rate under this treatment was 12%.In general, by tissue culture and chromosome doubling induction of Akebia trifoliate, we gained theoretical and technology support for excellent germplasm preservation and polyploid breeding of wild Akebia trifoliate material.