Optimization Of Bromeliaceae Culture In Vitro And Its Cold-tolerant Mutants Induction

In this research, a high-frequency technique for Bromeliaceae rapid propagation in vitrohad been achieved successfully through optimizing the factors which influence its shootregeneration rate during culture in vitro and seedling survival rate during transplantation.Based on the established technique for Bromeliaceae shoot regeneration, the test-tubeseedlings were treated with EMS and screened by HYP to induce the cold-tolerant mutantsof Bromeliaceae. The main results were as follows:1. Optimization of Bromeliaceae Culture in vitroDuring in vitro culture, the explant browning could be alleviated by reducing inorganicmacro elements especially NH₄⁺ concentration, and the 2,4-D concentration in the mediums,combined with dark culture for 7 days at early stage. Supplying 200mg/L Vc in mediumsalso could effectively avoid browning, and furthermore did not produce ill effects on growthof Bromeliaceae explants.It reached 85% for the calli induction rate from explants of young leaf base segments.And there were no obvious differences in the induction and differentiation of callus byusing cane sugar or glucose as carbon resources. MS+2,4-D 4mg/L+NAA 1(or 2)mg/L+sugar 30g/L was proved to be the optimal medium for calli induction, and the differentiationrate had reached 90.6% in 1/2MS with higher concentration of BA and lower concentrationof NAA and IAA. 1/2MS+BA 4mg/L+NAA 1mg/L+IAA 1mg/L+sugar 30g/L was proved tobe the optimal medium for shoot regeneration. The duration was shortened to 40-50 days.Regeneration rate of adventitious shoots reached 85% by supplying cane sugar as carbonresource in 1/2MS. It was proved that 1/2MS+BA 4mg/L+NAA 1mg/L+IAA 1mg/L+canesugar 30g/L was suitable for adventitious buds induction with high frequency.MS was better than 1/2MS for sheet subculture and the optimal hormone combinationwas BA 2mg/L+NAA 0.5mg/L. 50001x light intensity was better than 25001x for plantlets growing with which the plantlet propogation coefficient was 5.5.During Adventitious Root Formation, MS was better than 1/2MS and NAA was betterthan IBA. Lower concentration of sugar gave better effect, and rooting frequency reached100% in MS+NAA 0.2mg/L+sugar 20g/L.2. Transplanting techniques of Bromeliaceae seedlings culture in vitroThe results showed the transplanting survival rate was the up to 100% for test tubeseedlings with more than 3 roots pretreated 5 days with Carbendazin-sulfar dipping. Thetransplanting substrate of coconut coir mixed with sand or moss was good for seedlinggrowth. Fluorescent light and plant growth light had no remarkably different effect on thegrowth of transplanted seedlings.3. Chemical mutagenesis of Bromeliaceae and selection of cold-tolerant mutantsDuring the research choosing Aechmea fasciata as the material for mutagenesis, we'vefound that the ages of test tube buds, treatment duration and EMS concentration hadremarkable effects on their survival rate and differentiation rate. The buds whichsubcultured 20d were suitable materials for mutagenesis and the semi-lethal treatment wastreated 6h with 0.5% EMS.In order to reduce the injury to buds, the concentration of selection agent HYP wasinereased step by step and its final concentration was 8mmol/L. The survival buds wereselected once time again in the medium and then transferred to the normal medium withoutHYP.The average content of free proline in mutant leaves was 1.74 times to that of the control,furthermore there was obviously different in cold resistance between the mutants and thecontrol, the injury degree of cold to the mutants much lower than to the control. In addition,the mutants had some differences from the control in morphlogy.