Study On Comparison Of Defferent DNA Extraction And Gene Transformation Of Adiantum Capillus-veneris L.

We used the A. capillus-veneris L. as the materials. First, we studied on its whole DNAextraction protocol and examined the modificatory method better than others. Second, we firstestablished the regeneration system of the gametophytes and the sporophytes, and then we discussed thepossibility of the Agrobacterium tumefaciens-mediated genetic transformation and particlebombardment-mediated genetic transformation methods on A. capillus-veneris L. through theregeneration system. Our results include:1. There are large amounts of secondary metabolites such as high polysaccharide and polyphenolcomponents in ferns, so their whole DNA extraction are much difficult than other plants. We investigatedthe advantages and disadvantages of the nine whole DNA extraction protocols using the gametophtye andsprophyte materials of A. capillus-veneris L.. Our results shows that compared to the sporophytes, theDNA purity of the gametophytes was higher but the output was lower. To the gametophytes, the methodsof CTAB、SDS and the Kit were the best ones. To the sporophytes, the whole DNA purity obtained bythe modified CTAB DNA extraction protocol was obviously better than others, the detection PCR alsogot the same results.2. The regeneration system of A. capillus-veneris L. was established. We used young leaves ofsporophyte as materials to induce the callus.The protocol is: the vernations and unfold young leaves arebest materials; the light condition was12h light/12darkness; tight callus would be obtained using0.5mg/L6-BA＋0.5mg/L2,4-D (or1mg/L2,4-D) MS medium and then used the same culture medium tosubculture for3-4generations. We transferred them to the0.5mg/L6-BA or1mg/L6-BA MS forinducing the somatic embryo. After that, we transferred them to the MS medium for inducing thesporophyte seedling. The best spore germination condition was in red light for two days using the Knop’smedium. The life cycle of A. capillus-veneris L. was divided into six phases under laboratory conditions:spore, protonema, prothallium, sex organ phase, embryo, mature plant.3. We attempted the A. tumefaciens-mediated genetic transformation and particlebombardment-mediated genetic transformation methods on A. capillus-veneris L. through theregeneration system. The result indicated that the best material for gene transformation is generativeorgan in both two methods and we could obtain more fluoroscopic signals in this material through GFPtest. Materials were most dead in their regeneration through A. tumefaciens-mediated genetictransformation method because of antibiotics. Materials could not through particlebombardment-mediated genetic transformation because of serious pollution.