Studies On Tissue Culture And Genetic Transformation System Of Three Ferns

In this paper, the gametophyte development and sex organ differentiation of Adiantum flabellulatum were studied in the first. Spores were used as explants for the study. The influence factors were different exogenous substances, including BA, IAA, GA3, GA4+7 and Ca2+. Secondly, the young leaves were used to study the tissue culture of Platycerium wallichii. Three archusias and three cytokinins were added to MS medium. They were IBA, NAA,2,4-D, BA, ZT and KT. The growth and differentiation situations of young leaves were observed to determine the effects of the hormones. Finally, the genetic transformation systems of Adiantum reniforme var. sinense and Platycerium wallichii were studied. The GGB and prothallus with buds were used as transformation receptor materials for Adiantum reniforme var. sinense as the GGB were used for Platycerium wallichii. The main results were as follows:1.1/4 strength MS medium containing IAA 1.0 mg/L was the appropriate medium for germination of spores of Adiantum flabellulatum.0.1 mg/L BA, IAA, GA4+7 or 3.0 mg/L GA4+7 inhibited the germination of spores; GA3, Ca2+ and 0.5 mg/L BA made the gametophytes of Adiantum flabellulatum develop to heart shaped gametophytes effectively, and there was no significant difference in these deals. Most of the gametophytes cultured in mediums with IAA, GA4+7, or 0.1,1.0 mg/L BA were filamentous gametophytes; IAA, GA3, Ca2+ and 0.5 mg/L BA promoted the meristem region formation and archegonium differentiation of Adiantum flabellulatum in different levels. Medium containing GA31.0 mg/L was the most effective medium. GA4+7 had no significant effect on the formation of meristem region, but it promoted the differentiation of archegonium.2. MS medium supplemented with 0.5 mg/L BA was the most effective medium for the induction of GGB of Platycerium wallichii. NAA and 2,4-D had no effect on the generation of GGB. Appropriate amounts of ZT, KT or IBA can extent the formation of GGB. The differentiation rate of buds of Platycerium wallichii under the concentration of 0.5 mg/L NAA was the highest, which was 89.58%.0.5 mg/L ZT followed, and there was no significant difference between the two deals. Proper amounts of KT, IBA or 2, 4-D can improve the differentiation of buds. BA inhibited the differentiation of buds seriously. Only IBA or 2,4-D can induce differentiation of callus of Platycerium wallichii. The induction rate of callus under the concentration of 5.0 mg/L IBA was the highest, which was higher than 2,4-D obviously. However, the activity of callus formatted can only be maintained for a period of time, and they all became brown and went to death eventually.3. The transformation system for Adiantum reniforme var. sinense was established preliminarily. The prothallus with buds and GGB were pre-cultured respectively in MS medium containing Ca2+ 0.2 mg/L or ZT 1.0 mg/L for 3 days. The suitable concentration of Agrobacterium tumefaciens and the time for transforming was OD6oo=0.5-0.8 and 10 minutes. Explants were co-cultivated in dark for 3 days after infection. Delayed cultivation in medium containing Cef 500 mg/L for 15 days was suitable for transformation of prothallus with buds and 30 days were suitable for GGB. Subsequently, explants were selected under the critical concentration of 100 mg/L Kan and 500 mg/L Cef.4. The transformation system for Platycerium wallichii was established preliminarily. The GGB were pre-cultured in MS medium supplemented with 0.5 mg/L ZT for 3 days. The suitable concentration of Agrobacterium tumefaciens and the time for transforming was OD6oo=0.5-0.8 and 10 minutes. GGB were co-cultivated in dark for 3 days after infection. Delayed cultivation in medium containing Cef 200 mg/L for 30 days was suitable for transformation. Subsequently, GGB were selected under the critical concentration of 300 mg/L Kan and 200 mg/L Cef.