Study On Tissue Culture Of P.sylvestris Var Mongolica

P.sylvestris Var Mongolica is a kind of conifer with more root, cold-resistant, drought-resistant, liking sun, sandy soil, good wood, fast-growing, which is widely used to windbreak and sandy-fixing and wood-use.Owing to breed sapling difficultly, produce seed time long and seed output low, the supply of seed from orchard cannot meet the demand in the production. Nowadays, it is an effective way to enlarge propagation by tissue culture. so to carry out in vitro and micro propagation is important in theory and practice. Although study about it have been done in our country and abroad, it is not much and preliminary. The subject via to tissue culture and micro propagation study of P.sylvestris Var Mongolica, in vitro condition producing plantlet regeneration from mature embryo of P.sylvestris Var Mongolica. Tentatively exploring the effective way of micro propagation of P.sylvestris Var Mongolica, which provid some tentative and theoretical bases for plant breeding. Meanwhile, it provid possibility to obtain engineering pine. The main results as the following. (一)Study on inducing adventitious buds ⅠThe mature stratified embryo is the optimal explants of inducing adventitious buds. ⅡSeed is soaked in 700g.Kg-1 alcohol 40s, then transferred into 1 g.Kg-1Hgcl2 5-6min to sterilize, and used antisepsising water to wash 4 times. Under disinfection condition using little dissection knife to strip endosperm and inoculate mature embryo to adventitions medium. Embryo is rarely polluted. ⅢThe basal medium is key to form adventitious buds of P.sylvestris Var Mongolica mature embryo in vitro.1/2MS is the best basal medium. ⅣThe location of explants on the medium is divided into three kinds. They are level, erect and declivous. When the embryo is inoculated in terms of level location, much adventitious buds can be induced on cotyledon and hypocotyls. (二)Study　on　buds　proliferation (a) Buds proliferation on embryo Ⅰ0.05~0.1mg.L-1IBA and0.1~0.2mg.L-1NAA and 1.0~2.0mg.L-16-BA have significant promotion on inducing buds, ⅡThe 1/2MS medium supplemented with 6-BA1.0 mg.L-1and NAA0.2 mg.L-1 and 3%蔗糖was the best for inducing adventitious buds. It mainly induced adventitious buds from the tips of cotylendous. ⅢThe 1/2MS medium supplemented with 6-BA1.0 mg.L-1and IBA0.1 mg.L-1 and 3%蔗糖was the best for inducing adventitious buds. It mainly induced adventitious buds from the bases of cotylendous. (b) Study on axillary buds proliferation Ⅰ1/2MS supplemented with 6-BA0.5 mg.L-1 and 2% 蔗糖is better for axillary buds proliferation of P.sylvestris Var Mongolica. Ⅱ1/2MS supplemented with 6-BA0.5 mg.L-1 and 2%蔗糖and NAA is not good for axillary buds proliferation of P.sylvestris Var Mongolica. Callus can be induced in the bases of buds. ⅢThe experiment of different activated charcoal concentration on inducing axillary buds showed that it has no prominent discrepancy influence . (三)Study on buds growth and elongation ⅠThe best medium for increasing the height of cluster buds is 1/2MS +0.1%活性炭+2%蔗糖. ⅡGA3 has no prominent discrepancy influence on buds growth and elongation . ⅢDuring tissue culture of mature embryo, the buds are always browning and withered. In order to avoid this phenomenon, different periods of culture are chosen. The results showed to subculture every other 25days can inhibit this phenomenon effectively. ⅣDuring buds growth and elongation, terminal buds will be found after 40 days. (四) Study on rooting culture ⅠUsing singly auxin cannot induce root formation. Ⅱthe basic medium played a determinative role in inducing the adventitious roots . Decrease the concentration of large elementary (1/2MS to 1/4MS) and source (2% to 1%) can induce adventitious roots. ⅢThe induction of roots of P.sylvestris Var Mongolica have two periods. Form period the buds are cultured in 1/4MS+0.5mg.L-1NAA+0.2mg.L-1IBA+1%蔗糖for 5 days. Follow period, they are subcultured in 1/4MS+1%蔗糖. Such can promote the formation of root . ⅣIt is very important that buds should be subcultured in 1/4MS+1%蔗糖in following period. ⅤThe buds are derectly cultured in 1/4MS+1%蔗糖, the buds with height over 2cm can be induced effective root, the buds with height below 2cm and having callus in base cannot form effective root. ⅥWhen the height of buds in vitro are over 2cm,it is beneficial for forming effective root .