| 培养基成分对黑松体胚发育成熟的影响 |
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| 引用本文： | 孙婷玉,王艳丽,沈李元,吴小芹,朱丽华,叶建仁.培养基成分对黑松体胚发育成熟的影响[J].林业科学,2019(4):178-186. |
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| 作者姓名： | 孙婷玉 王艳丽 沈李元 吴小芹 朱丽华 叶建仁 |
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| 作者单位： | 南京林业大学林学院;南方现代林业协同创新中心 |
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| 基金项目： | 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) |
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| 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-1PEG8000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的变化趋势:胚性细胞系#1337体胚发育成熟的最佳组合为麦芽糖45 g·L-1+ABA 10 mg·L-1+PEG 125 g·L-1;#1537和#1637体胚发育成熟的最佳组合为麦芽糖30 g·L-1+ABA 10 mg·L-1+PEG 125 g·L-1;在3个无性系中PEG的极差最大,对抗性黑松体细胞胚发育成熟的影响最大。【结论】抗性黑松体胚发育成熟过程中,麦芽糖45 g·L-1、ABA20～30 mg·L-1、PEG8000 125 g·L-1、AC 2 g·L-1和植物凝胶3 g·L-1对体细胞胚成熟都具有促进作用。30 g·L-1麦芽糖、10 mg·L-1ABA、125 g·L-1PEG为抗性黑松体胚发育成熟的最佳组合。
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| 关 键 词： | 黑松 松材线虫病 体细胞胚 聚乙二醇(PEG8000) |
Impact of Medium Components on Somatic Embryo Maturation in Pinus thunbergii |
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| Sun Tingyu,Wang Yanli,Shen Liyuan,Wu Xiaoqin,Zhu Lihua,Ye Jianren.Impact of Medium Components on Somatic Embryo Maturation in Pinus thunbergii[J].Scientia Silvae Sinicae,2019(4):178-186. |
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| Authors: | Sun Tingyu Wang Yanli Shen Liyuan Wu Xiaoqin Zhu Lihua Ye Jianren |
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| Institution: | (College of Forestry, Nanjing Forestry University Nanjing 210037;Collaborative Innovation Center of Sustainable Forestry in Southern Nanjing 210037) |
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| Abstract: | 【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 the mature medium that could not solidify;phytagel was more suitable for solidifying mature medium.phytagel of 3g·L^-1 was the optimum concentration of somatic embryo maturation.The orthogonal experiment showed that maltose 45g·L^-1 , ABA 10mg·L^-1 , and PEG8000 125g·L^-1 were the media on which the embryogenic cells (cell line #1337,#1537,#1637) produced the most somatic embryos among the nine maturation medium combinations.Among different combinations of maltose, ABA and PEG8000, the productivity of somatic embryos produced from different clones showed different trends.The optimal combination for somatic embryo development and maturation from embryogenic cell #1337 was maltose 45g·L^-1 , ABA 10mg·L^-1 , PEG8000 125g·L^-1;from #1537 and #1637, the optimal combination was maltose 30g·L^-1 , ABA 10mg·L^-1 , PEG8000 125g·L^-1;PEG8000 had the biggest range in the three clones, and thus, PEG8000 had the most important effect on somatic embryos development and maturation of Pinus thunbergii .【Conclusion】In process of somatic embryo maturation (#1337), maltose 45g·L^-1 , ABA 20-30mg·L^-1 , PEG8000 125g·L^-1 , AC 2g·L^-1 and 3g·L^-1 phytagel promoted somatic embryo maturation.The optimal combination for Japanese black pine somatic embryo maturation is maltose concentration 30g·L^-1 , ABA 10mg·L^-1 and PEG8000 125g·L^-1 . |
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| Keywords: | Pinus thunbergii pine wilt disease somatic embryo polyethylene glycol (PEG8000) |
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