培养基成分对黑松体胚发育成熟的影响  

Impact of Medium Components on Somatic Embryo Maturation in Pinus thunbergii

作　　者：孙婷玉 王艳丽 沈李元 吴小芹 朱丽华 叶建仁 Sun Tingyu;Wang Yanli;Shen Liyuan;Wu Xiaoqin;Zhu Lihua;Ye Jianren(College of Forestry, Nanjing Forestry University Nanjing 210037;Collaborative Innovation Center of Sustainable Forestry in Southern Nanjing 210037)

机构地区：[1]南京林业大学林学院,南京210037 [2]南方现代林业协同创新中心,南京210037

出　　处：《林业科学》2019年第4期178-186,共9页Scientia Silvae Sinicae

基　　金：江苏省林业三新工程项目"抗松材线虫病马尾松体胚再生植株高效繁育技术"(LYSX[2016]47);国家重点研发计划(2017YFD0600104);江苏高校优势学科建设工程项目(PAPD)

摘　　要：【目的】对日本抗性黑松体细胞胚成熟的影响因子进行研究,探究各因素在体细胞胚发生过程中的作用,为进一步提高体细胞胚发生的质量和数量提供理论依据。【方法】胚性细胞团在固体增殖培养基上继代生长10天,使用灭菌(75%乙醇擦拭、紫外灭菌30 min)的电子天平称量1 g胚性细胞团,采用50 mL无菌量筒(紫外灭菌30 min)量取30 mL培养液中于100 mL的三角瓶中,将1 g胚性细胞团转至100 mL的三角瓶中,手动搅拌至细胞团分散,置于90 r·min^(-1)摇床上,25℃黑暗培养7～8天。随后,取3 mL沉淀悬浮细胞进行继代增殖,每星期继代增殖一次至胚性细胞全部均匀散落于培养液。将继代4次的胚性细胞充分摇匀,取2 mL(鲜质量约200 mg)胚性悬浮细胞喷洒在不同组分麦芽糖(30、45、60 g·L^(-1))、脱落酸(ABA)(0、5、10、20、30、50 mg·L^(-1))、聚乙二醇(PEG8000)(0、50、75、100、125、150 g·L^(-1))、活性炭(AC)(1、2、3 g·L^(-1))、琼脂(琼脂6、8、10、12 g·L^(-1),植物凝胶2、2. 5、3、3. 5 g·L^(-1))以及麦芽糖、ABA、PEG 3因素的组合固体成熟培养基上。【结果】以#1337为材料的体细胞胚成熟发育过程中,麦芽糖45 g·L^(-1),获得的体细胞胚数量显著增多。ABA浓度为5～20 mg·L^(-1),体细胞胚数量呈显著上升趋势,ABA为20 mg·L^(-1)时,获得的体细胞胚最多。125 g·L^(-1)PEG8000为体胚发生最佳浓度; AC 2 g·L^(-1)时,胚性细胞形成结构完整、数量较多的成熟体胚;成熟培养基中添加琼脂粉,培养基不能凝固,植物凝胶更适合作为成熟培养基凝固剂。3 g·L^(-1)植物凝胶为抗性黑松体胚发育成熟的最佳浓度。在设计的9种成熟培养基组合中(胚性细胞#1337、#1537、#1637),胚性细胞产生体细胞胚最多的成熟培养基组合为麦芽糖45 g·L^(-1)、ABA 10 mg·L^(-1)、PEG8000 125 g·L^(-1)。在不同的麦芽糖、ABA和PEG组合中,不同无性系生产体细胞胚数量呈现出不同�【Objective】In this study, we investigated the role of medium components in somatic embryo maturation, in order to further improve the quality and quantity of somatic embryos.【Method】The embryogenic cells were incubated for 10 days in the solid proliferation medium.Sterile electronic scales (75% ethanol wipe, ultraviolet sterilize 30 min) were used to weigh 1 g embryonic cells, a 50 mL sterile measuring cylinder (ultraviolet sterilization 30 min) was used to measure 30 mL liquid medium which was poured into a 100 mL conical flask.Then, 1 gram embryonic cells were transferred into a 100 mL conical flask which was transferred into a constant temperature incubator shaker.The culture was incubated 7-8 days in darkness at 25 ℃, 90 r·min -1 .Subsequently, suspension cells (precipitated volume 3 mL) were taken for the subculture.The subculture was proliferated once a week until all embryogenic cells were evenly dispersed in the culture medium.The suspension cells of 2 mL (fresh mass 200 mg) from the forth culture were sprayed on solid mature media which contained various components of maltose (30,45,60g·L^-1 ), abscisic acid (ABA)(0,5,10,20,30, 50mg·L^-1 ), polyethylene glycol (PEG8000)(0,50,75,100,125,150g·L^-1 ), activated carbon (AC)(1g·L^-1 , 2g·L^-1 , 3g·L^-1 ), coagulating agent (agar: 6, 8, 10, 12g·L^-1;Phytagel 2, 2.5, 3, 3.5g·L^-1 ) and the combination of maltose, ABA and PEG8000.【Result】During the development and maturation of somatic embryos (cell line #1337 ), the number of somatic embryos obtained from maltose 45g·L^-1 medium increased significantly;When the ABA concentration ranged from 5mg·L^-1 to 20mg·L^-1 , the number of somatic embryos increased significantly with ABA concentration, and with abscisic acid (ABA) concentration in 20mg·L^-1 , the maximum quantity of somatic embryos was obtained;The optimal concentration of polyethylene glycol (PEG8000) and activated carbon (AC) for somatic embryogenesis was 125 and 2g·L^-1 , respectively;Agar powder, as coagulant, was added to t

关 键 词：黑松 松材线虫病 体细胞胚 聚乙二醇(PEG8000) 

分 类 号：S722.3[农业科学—林木遗传育种] S763.18[农业科学—林学]