The Preliminary Study On The Tissue Culture And Genetic Transformation Of Pinus Bungeana

Mature embryo and cotyledon of Pinus bungeana as explants, were studied on Pinusbungeana adventitious bud induction, subculture multiplication and rooting. The gene SOS1and NHX1of Arabidopsis thaliana with different salt tolerance mechanisms were cloned andconstructed the overexpress vectors separately. The overexpress vectors were used to studythe P. bungeana genetic transformation, In order to increase the salt resistance alkaline-resisting features.The main results were summarized as follows:1. The best shoot induction medium for cotyledon was MS+NAA0.1mg/L+6-BA3mg/L+TDZ0.01mg/L,the best shoot induction medium for mature embryo was MS+NAA0.1mg/L+6-BA2mg/L.2. The optimum shoot proliferation medium was MS+NAA0.05mg/L, multiplicationcoefficient up to6.3,bud average height as high as1.56cm after culture two times. Budproliferation coefficient is reduced with increasing the concentrations of6-BA.3. With the concentration of activated carbon increasing, multiplication coefficient isreduced, and2cm young shoots proportion increased significantly, it’s no obvious effect forshoot proliferation, but can significantly promote bud elongation. When it more than3g/L,the bud elongation will be inhibited. GA3can effectively promote bud elongation, along withthe GA3from0mg/L to1.5mg/L, buds are growing faster,1mg/L to2mg/L changesslightly but when more than2mg/L, there is a decrease trend of the bud average height.4. To did not handle and vaccination respectively in bud induction medium on2,3,5weeks of coatedpinus P.bungeana cotyledons paraffin section for analysis.Two weeks afterbud induction cells, including epidermal cells or subepidermal a layer of cortical cells throughpericlinal divisions to form original cells group; Training after three weeks, due to the furtherdivision of the original cells group, coupled with a single cell volume increases the formationof circular protrusions in the cotyledon surface;Training after five weeks,original cells groupfurther split have obvious formation Cytological partition of bud primordia,the budprimordia lateral meristematic tissue cells through periclinal divisions formed scale leafprimordia;Scale leaf primordia continued elongation of the formation of scale leaves, whilethe bud primordia lateral meristematic tissue cells formed new scale leaf primordia; P.bungeana cotyledons in vitro shoot originated in the Epidermal cells or subepidermal alayer of cortical cells, so that exogenous way for the main way of adventitious buds.5. P. bungeana cotyledons Kan background resistance experimental results show that7.5mg/L Kan will be able to completely inhibit the differentiation of cotyledon. On this basis,according to the screening concentration generally slightly higher than the concentration cannot differentiation the principle, in this experiment10mg/L Kan was used to select thetransformed cells.6. The different pre-culture time influence the generation of resistant bud results showthat the P. bungeana cotyledons pre-cultured0-1d, do not produce any resistance buds; pre-cultured for3-7d, the frequency of resistant shoots have increased from4%to18%;Takinginto account the pre-culture for too long, the meristem quickly divide and part of the budmeristem develop into leaf primordia, while the antibiotic to pass slower and difficult tospread to within the meristem group, which will lead to increased false-positive ratio;subsequently, transformation experiments pre-cultured time of4-5days.7. Bacteria liquid concentration and infection time on the effects of the transformationshow that: bacterial concentration is higher(OD600nm＝0.8), it is easy to produce toxic effectson the cotyledons, lead to its in the process of cultivation Browning died, and the rapidgrowth of Agrobacterium, in the later screening culture is difficult to completely removal willaffect the explants produce resistant shoots. When the infection time more than30min, thecotyledons of soft rot increased significantly, and greatly reduces the regeneration rate ofsubsequent cultivation process of resistant shoots. Comprehensive consideration, P. bungeanacotyledons transformation, infection in the OD600nm=0.4under30min to get the best effect.8. AS concentration in Bacteria infection fluid and Cocultivation medium for100umol/L, the frequency of the resistant shoots more than the control have increasedobviously.AS too high concentration (more than200umol/L), explant Browning level deepen,iadventitious buds was significantly inhibited.9. By the delayed choice of step-by-step screening resistant shoots. Recovery culturedone week in the bud induction medium without Kan, then in contain3mg/L Kan of inadventitious bud induction medium screening two rounds, each round one week to the nakedeye can see adventitious buds. Then in contain10mg/L Kan of in adventitious bud inductionmedium screening two rounds, each round one week. This can effectively reduce the explantsallergic reactions to antibiotics, the growth of explants situation improved significantly.