Study On Tissue Culture Of Four Wild Medicinal Ferns

Medicinal ferns were used as traditional herbal by now. With new technologies and methods used on application of segregation and analysis in herbal components,more important medicinal ingredients of pteridophytes are found, and demand of medicinal pteridophytes is also increasing.Many wild medicinal ferns are in danger of extinction for wantonly exploitation and the ecology distribution,even difficult to meet the large market demand.Tissue culture provide the necessary measure for the study, protection or development and utilization of wild medicinal ferns.In this paper, from mature spores of four medicinal Pteris cultured in vitro,by means of tissue culture,microscope camera,section technology and etc.,investigation about ferns gametophyte development, proliferation of gametophytes,callus induction by gametophyte,and thereinto,study on cytohistology and morphogenesis of embryoid inducement from gametophyte callus that form on plant-hormone free medium of Pteris dissitifolia Bak. and Pteris fauriei Hieron. is of about ferns embryoid for the first time in China,as follows:(1)Pteris dissitifolia Bak.Spore of Pteris dissitifolia Bak. germinate and form sporophyte on both solid medium of 1/2-strength MS with sucrose 1% and 1/2-strength mass element of MS with sucrose 1%.The germination is of Vittaria-type and the gametophyte development is of Ceratopteris-type on medium of 1/2-strength mass element of MS medium with sucrose 1%.With gametophyte, viscidity and wet callus in yellow-green color is induced on hormone medium.The callus can't differentiate to sporophyte on 1/2-strength mass element of MS medium supplement with GA3 0.1mg·L-1, sucrose 3%.On 1/2-strength mass element of MS medium ,gametophyte forme about in 11th weeks after spores were cultured, then compact and pale yellowish-white in color callus form on some gametophyte.The callus differentiate into embryoid and plantlets with shoots and roots on 1/2-strength mass element of MS medium supplement with GA3 0.1mg·L-1 and sucrose 3%. Cytohistology observation show the morphogenetic of the embryoid similar to of zygotic embryo,for example four-cell stage, globular embryo, heart-shaped embryos, torpedo-shaped embryos, mature embryos;polarity and tracheary elements appear in the early stage of the embryoids;different developmental stages embryoids can be found on the same medium.(2)Pteris fauriei Hieron.On 1/2-strength mass element of MS medium, the germination is of Vittaria-type, the gametophyte development is a kind of similar to the Aspidium-type; a protuberance composed by a number of granular cells , grow many multicellular trichomes close to the meristematic of notch on ventral aspect of nearly mature gametophyte.One or two archegonia are found at the protuberance on few gametophyte.Spore and gametophytes of Pteris fauriei Hieron. could bear great changes in acidic environment.Sporophyte formation relate to the above-mentioned protuberance for Pteris fauriei Hieron closely. Not only sporophyte but also callus form at the protuberance,and the callus increase with sucrose concentration(0～3%) of the medium.The callus growth slow down and become hard in texture and ivory-white in color by setting of difference in temperature(12h 15℃,12h 25℃);the callus can differentiate into a lot of embryoids and plantlets on 1/2-strength mass element of MS medium supplement with GA3 0.1mg·L-1 and sucrose 3%. Cytohistology observation show the morphogenetic of the embryoids similar to of zygotic embryo.First the callus proliferate and only many adventitious roots form finally on 1/2-strength mass element of MS medium supplement with 6-BA 0.5 mg·L-1+NAA 0.5 mg·L-1+sucrose 3%, then transferred to 1/2-strength mass element of MS medium supplement with GA3 0.1mg·L-1 more adventitious bud and adventitious root form in the upshot.(3)Pteris Muldifida Poir.When culture on the following solid sterile medium, mature spores of Pteris Muldifida Poir. germinate then develop to prothallus and proliferate to hydrangea shape. On 1/2-strength mass element of MS medium with sucrose 1% ,prothallus are found by naked eye about in the 8th week and in low germination rate, then sporophyte form about in the fourth month;on 1/2-strength MS medium with sucrose 3% ,prothallus about in the 6th week and in very low germination rate, no sporophyte form; on 1/2-strength MS medium without sucrose, prothallus about in the 5th week and in hight germination rate, sporophyte form about in the fifth month;on MS medium with sucrose 3%,no sporophyte form ,but dark yellow and hyaline callus form on some gametophyte.The gametophyte of Pteris Muldifida Poir. could bear great changes in acidic environment during the germination and development. Liquid medium supplement with NAA 0.5～4.0 mg·L-1 +6-BA 0.5～2.0 mg·L-1 promote the gametophyte proliferation,and the best ratio is NAA 4.0 mg·L-1 +6-BA 2.0 mg·L-1. Those gametophyte form sporophytes if they are moved to 1/2-strength MS solid medium, but not sporophyte form on MS solid medium.Callus that induce from those gametophyte on hormone medium can't differentiate into sporophyte on 1/2-strength mass element of MS medium supplement with GA3 0.1mg·L-1 and sucrose 3%.(4)Pteris deltodon Bak.On 1/2-strength MS medium with sucrose 1%, germination began in the second week.The germination is of Vittaria-type and the gametophyte development is of Ceratopteris-type,the gametophytes became mature about in the 12th week and sporophytes formed in the 16th week after inoculation. 1/2-strength MS sucrose free medium is suitable for gametophyte development and sporophytes formation for Pteris deltodon Bak.