| Chinese fir?Cunninghamia lanceolata?Lamb.?Hook?is a evergreen coniferous tall trees of Taxodiaceae.It is the main afforestation species and commercial timber species in South China which growing fast with high yield,soft material,beautiful texture,corrosion resistance,easy processing and long fiber.In recent years,with the area of Chinese fir plantation of Chinese fir seed seedling growing,increasing demand,how high generation of fine materials and meet the needs of the rapid expansion of Chinese fir afforestation on high generation seed is very urgent.With shorten the breeding process,space saving,annual production many advantages of tissue culture,It is one of the important ways to cultivate Chinese fir which can maintain the female excellent traits of Chinese fir clones and facilitate the large-scale production,to meet the huge market demand for large-scale quality of Chinese fir seedlings.Although at the end of 1980s China began the research on Chinese fir tissue culture,has use of mature and immature zygotic embryos,cotyledons,hypocotyls,stems,shoot tips and other explants in regeneration system of Chinese fir,and achieved certain results.But at present there are still many problems from Chinese fir tissue culture regeneration process,mainly in the sterilization of explants,adventitious bud induction,adventitious root induction and transplanting were carried out step by step,explant and adventitious root induction is difficult,Lack of complete technical system of tissue culture in vitro,the cost of tissue culture seedlings is high,greatly limits the large-scale application of Chinese fir tissue culture technology.The genetic improvement of Chinese fir was greatly limited because of the lack of mature regeneration system in particular.Therefore,it is great practical significance to carry out the research on regeneration system of Chinese fir callusIn view of this,this paper is aimed at the problems existing in the research of callus induction and regeneration system.The cotyledon and hypocotyl,immature embryo and stem segment of Chinese fir seeds were selected as explants.Discussed the difference on explant disinfection,subculture proliferation,callus induction rate,callus proliferation rate,callus differentiation rate,rooting rate,ways of regenerated buds originated.Screened out the most suitable culture medium for induction and establishment of plant regeneration system of different organs of Chinese fir callus formation and differentiation analysis for the origin and process of Chinese fir callus,explore the establishment of regeneration system of Chinese fir callus,provide receptor materials and technical support for regeneration system and genetic transformation of Chinese fir.The main results are as follows:1.There were significant differences in surface sterilization of explants of different Chinese fir:the optimal combination of reagent and its disinfection time was 0.1%Hgcl2 treating 10min.2.Different Chinese fir seedling propagation effect of different culture medium:suitable proliferative mediums were screened out:MS+6-BA2.0 mg·L-1+TDZ0.04 mg·L-1+NAA1.0 mg·L-1 is optimum medium which average number of adventitious bud differentiation was 28;MS+6-BA3.0 mg·L-1+TDZ0.06 mg-L-1+NAA0.3 mg-L-1 is optimum medium which average growth of seedling height was 15.82mm.Different light intensity significantly on Chinese fir seedlings subculture proliferation difference,3200Lux culture condition is most beneficial to the subculture of tissue culture plantlets.3.There were significant differences in callus induction of different explants.:The optimal medium for cotyledon to induce callus was 2,4-D1.0 mg·L-1+NAA2.0 mg·L-1+KT0.5 mg·L-1,the induction rates was 100%;The optimal medium for hypocotyls to induce callus was NAA2.0mg·L-1+6-BA2.0mg·L-1+TDZ0.01 mg·L-1,the induction rates was 95.8%;The optimal medium for Immature embryo to induce callus was 2,4-D1.0 mg·L-1+6-BA2.0 mg·L-1+KT1.0 mg·L-1,the induction rates was 91.9%;The optimal medium for tissue culture seedling stems to induce callus was 2,4-D 1.0 mg·L-1+6-BA2.0 mg·L-1+KTO.5 mg·L-1,the induction rates was 87.98%?The callus induction rate of cotyledon and hypocotyl was the highest at Germination 15d,and the induction rate was 97.5%and 95%respectively?2,4-D+NAA+KT is the optimum hormone combination for callus induction of four explants.There are significant differences in the basic medium of callus induction of texture,The quality of callus induced by MS basic medium was the best,which could be used for late re differentiation,the highest induction rate was 69.46%;The callus induced by DCR basic medium was dense and could be used for later subculture proliferation.The induction rate was 65.73%.4.The effects of different media on callus subculture of Chinese fir were different:1/2MS and MS can be used as subculture medium for callus regeneration.DCR can be used as the basic medium for callus long-term subculture.The optimum medium for callus to subculture were 6-BA2.0 mg·L-1+KT1.5 mg·L-1+NAA1.5 mg·L-1 and 6-BA1.5 mg·L-1+KKT1.0 mg·L-1+NAA1.0 mg·L-1.2,4-D is beneficial to reduce the browning rate of callus and NAA is beneficial to callus subculture,When 2,4-D concentration was 2 mg·L-1,Callus were most suitable for transfer to new medium after subculture of 30-45d;When NAA concentration was 1.5 mg·L-1,Callus should be transferred to new medium after subculture of 30d.The subculture of callus was suitable for dark culture and low intensity light culture.The callus diameter was 11-13mm is beneficial to its long-term subculture.5.Effects of different media on callus differentiation of Chinese fir:MS+6-BA0.5mg·L-1+KT1.5mg·L-1 was optimum medium for callus re-differentiation.The homogeneous callus differentiation rate was 89.57%and the browning rate was about 10.43%.Callus had a high differentiation rate after non-subculture and one-subculture,the differentiation rates were 83.57%and 82.37%respectively.6.Rooting culture:MS+NAA0.5 mg·L-1+IBA1.O mg·L-1 was optimum medium for regeneration seedling rooting which separated from callus.7.Cytological observation on different stages of callus formation and differentiation of Chinese fir:The callus formation of stem segments under different hormone combinations is different in cell structure and physical properties.Some have activity can re-differentiation,some lose activity can not complete normal development.There were two types of embryogenic and non embryogenic callus in the same callus at some time,indicating that the Chinese fir belonged to the heterogeneous tree species of callus development.There were two ways in which adventitious buds originate.The first way of originate was directly from explants;the other was from callus formation.Both of the two ways were "external origin ".The embryogenic callus was homogeneous with pale yellow or rice green. |
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