培养基成分对黑松体胚发育成熟的影响
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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~(-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~(-1)ABA、125 g·L~(-1)PEG为抗性黑松体胚发育成熟的最佳组合。
【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(30,45,60 g·L~(-1)),abscisic acid(ABA)(0,5,10,20,30,50 mg·L~(-1)),polyethylene glycol(PEG8000)(0,50,75,100,125,150 g·L~(-1)),activated carbon(AC)(1 g·L~(-1),2 g·L~(-1),3 g·L~(-1)),coagulating agent(agar: 6,8,10,12 g·L~(-1); Phytagel 2,2.5,3,3.5 g·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 45 g·L~(-1) medium increased significantly; When the ABA concentration ranged from 5 mg·L~(-1) to 20 mg·L~(-1),the number of somatic embryos increased significantly with ABA concentration,and with abscisic acid(ABA) concentration in 20 mg·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 2 g·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 3 g·L~(-1) was the optimum concentration of somatic embryo maturation. The orthogonal experiment showed that maltose 45 g·L~(-1),ABA 10 mg·L~(-1),and PEG8000 125 g·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 45 g·L~(-1),ABA 10 mg·L~(-1),PEG8000 125 g·L~(-1); from #1537 and #1637,the optimal combination was maltose 30 g·L~(-1),ABA 10 mg·L~(-1),PEG8000125 g·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 45 g·L~(-1),ABA 20-30 mg·L~(-1),PEG8000 125 g·L~(-1),AC 2 g·L~(-1) and 3 g·L~(-1) phytagel promoted somatic embryo maturation. The optimal combination for Japanese black pine somatic embryo maturation is maltose concentration 30 g·L~(-1),ABA 10 mg·L~(-1) and PEG8000 125 g·L~(-1).
【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(30,45,60 g·L~(-1)),abscisic acid(ABA)(0,5,10,20,30,50 mg·L~(-1)),polyethylene glycol(PEG8000)(0,50,75,100,125,150 g·L~(-1)),activated carbon(AC)(1 g·L~(-1),2 g·L~(-1),3 g·L~(-1)),coagulating agent(agar: 6,8,10,12 g·L~(-1); Phytagel 2,2.5,3,3.5 g·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 45 g·L~(-1) medium increased significantly; When the ABA concentration ranged from 5 mg·L~(-1) to 20 mg·L~(-1),the number of somatic embryos increased significantly with ABA concentration,and with abscisic acid(ABA) concentration in 20 mg·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 2 g·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 3 g·L~(-1) was the optimum concentration of somatic embryo maturation. The orthogonal experiment showed that maltose 45 g·L~(-1),ABA 10 mg·L~(-1),and PEG8000 125 g·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 45 g·L~(-1),ABA 10 mg·L~(-1),PEG8000 125 g·L~(-1); from #1537 and #1637,the optimal combination was maltose 30 g·L~(-1),ABA 10 mg·L~(-1),PEG8000125 g·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 45 g·L~(-1),ABA 20-30 mg·L~(-1),PEG8000 125 g·L~(-1),AC 2 g·L~(-1) and 3 g·L~(-1) phytagel promoted somatic embryo maturation. The optimal combination for Japanese black pine somatic embryo maturation is maltose concentration 30 g·L~(-1),ABA 10 mg·L~(-1) and PEG8000 125 g·L~(-1).
引文
陈金慧,施季森,诸葛强,等.2003.杂交鹅掌楸体细胞胚胎发生研究.林业科学,39(4):49-53.(Chen J H,Shi J S,Zhuge Q,et al.2003.Studies on the somatic embryogenesis of liriodendron hybrids(L.chinense×L.tulipifera).Scientia Silvae Sinicae,39(4):49-53.[in Chinese])
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崔凯荣,邢更生,周功克,等.2000.植物激素对体细胞胚胎发生的诱导与调节.遗传,22(5):349-354.(Cui K R,Xin G S,Zhou G K,et al.2000.The induced and regulatory effects of plant hormones in somaitc embryogenesis.Hereditas,22(5):349-354.[in Chinese])
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梁素婷.2013.抗病马尾松器官发生植株再生与体胚发生研究.南京:南京林业大学硕士学位论文.(Liang S T.2013.Study on plant regeneration by organogenesis and somatic embryogenesis in resistance masson pine.Nanjing:MSthesis of Nanjing Forestry University.[in Chinese])
林丽,周蕾,潘珺,等.2017.无菌和带菌松材线虫对赤松的致病性.林业科学,53(5):82-87.(Lin L,Zhou L,Pan J,et al.2017.Pathogenicity of aseptic and germcarrying Bursaphelenchus xylophilus on Pinus densiflora.Scientia Silvae Sinicae,53(5):82-87.[in Chinese])
齐力旺,韩一凡,韩素英,等.2004.麦芽糖、NAA及ABA对华北落叶松体细胞胚成熟及生根的影响.林业科学,40(1):52-58.(Qi L W,Han Y F,Han S Y,et al.2004.Effects on maltose,NAA and ABA on somatic maturation and radicle rooting of Larix principisrupprechtii.Scientia Silvae Sinicae,40(1):52-58.[in Chinese])
孙婷玉.2014.抗性黑松体胚发生及低温保存技术.南京:南京林业大学硕士学位论文.(Sun T Y.2014.Somatic embryogenesis and cryopreservation method of Pinus thunbergii resistant to Bursaphelenchus xylophilus.Nanjing:MS thesis of Nanjing Forestry University.[in Chinese])
孙婷玉,叶建仁,吴小芹,等.2015.抗松材线虫病黑松胚性愈伤组织诱导.东北林业大学学报,(9):96-99.(Sun T Y,Ye J R,Wu X Q,et al.2015.Induction of embryonic callus from Pinus thunbergii resistance to pine wilt disease.Journal of Northeast Forestry University,(9):96-99.[in Chinese])
唐巍,欧阳藩,郭仲琛.1988.火炬松合子胚愈伤组织的器官发生和植株再生.实验生物学报,31(1):87-93.(Tang W,Ouyang F,Guo Z C.1988.Organogenesis and plant regeneration of zygotic embryo in loblolly pine(Pinus taeda L.).Acta Biologiae Experimentalis Sinica,31(1):87-93.[in Chinese])
唐巍,欧阳藩,郭仲琛.1997.湿地松体细胞胚胎发生和植株再生.植物资源与环境,6(2):8-11.(Tang W,Ouyang F,Guo Z C.1997.Plantlet regeneration via somatic embryogenesis in Slash pine.Journal of Plant Resources and Environment,6(2):8-11.[in Chinese])
王贺飞,柴明良.2006.4种固化剂对若干草坪草愈伤组织诱导的影响.园艺学报,3(2):437-440.(Wang H F,Chai M L.2006.Effect of four brands of gelling agents on callus induction from mature seeds of several turfgrass species.Acta Horticulturae Sinica,3(2):437-440.[in Chinese])
吴丽君.2009.湿地松合子胚发育与体胚诱导的研究.福建林学院学报,29(3):243-246.(Wu L J.2009.Study on development course of zygotic embryos and initiation of somatic embryos of Slash pine.Journal of Fujian College of Forestry,29(3):243-246.[in Chinese])
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叶建仁.2010.松材线虫病诊断与防治技术.北京:中国林业出版社.(Ye J R.2010.Techlonogy of diagnosis and treatment of pine wilt disease.Beijing:China Forestry Publishing House.[in Chinese])
张巧,汪静儿,林君,等.2010.不同凝固剂对陆地棉体细胞胚胎发生和植株再生的影响.棉花学报,22(1):3-9.(Zhang Q,Wang J E,Lin J,et al.2010.Effects of different gelling agents on the somatic embryogenesis and plant regeneration in upland cotton.Cotton Science,22(1):3-9.[in Chinese])
赵捷,韩骁,石娟.2017.低温条件下松材线虫在中国的风险分布区预测.生物安全学报,26(3):191-198.(Zhao J,Han X,Shi J.2017.Potential distribution of Bursaphelenchus xylophilus in China due to adaptation cold conditions.Journal of Biosafety,26(3):191-198.[in Chinese])
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Bozhkov M R,Nagmani R.2002.Initiation of embryogenic culture and somatic embryo development in loblolly pine.Canadian Journal of Forestry Research,20(6):810-817.
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崔凯荣,邢更生,周功克,等.2000.植物激素对体细胞胚胎发生的诱导与调节.遗传,22(5):349-354.(Cui K R,Xin G S,Zhou G K,et al.2000.The induced and regulatory effects of plant hormones in somaitc embryogenesis.Hereditas,22(5):349-354.[in Chinese])
黄健秋,卫志明,许智宏.1995.马尾松成熟合子胚的体细胞胚胎发生和植株再生,植物学报,37(4):289-294.(Huang J Q,Wei Z M,Xu Z H,1995.Somatic embryogenesis and plantlet regeneration from callus of mature zygotic embryos of masson pine.Acta Botanica Sinica,37(4):289-294.[in Chinese])
梁素婷.2013.抗病马尾松器官发生植株再生与体胚发生研究.南京:南京林业大学硕士学位论文.(Liang S T.2013.Study on plant regeneration by organogenesis and somatic embryogenesis in resistance masson pine.Nanjing:MSthesis of Nanjing Forestry University.[in Chinese])
林丽,周蕾,潘珺,等.2017.无菌和带菌松材线虫对赤松的致病性.林业科学,53(5):82-87.(Lin L,Zhou L,Pan J,et al.2017.Pathogenicity of aseptic and germcarrying Bursaphelenchus xylophilus on Pinus densiflora.Scientia Silvae Sinicae,53(5):82-87.[in Chinese])
齐力旺,韩一凡,韩素英,等.2004.麦芽糖、NAA及ABA对华北落叶松体细胞胚成熟及生根的影响.林业科学,40(1):52-58.(Qi L W,Han Y F,Han S Y,et al.2004.Effects on maltose,NAA and ABA on somatic maturation and radicle rooting of Larix principisrupprechtii.Scientia Silvae Sinicae,40(1):52-58.[in Chinese])
孙婷玉.2014.抗性黑松体胚发生及低温保存技术.南京:南京林业大学硕士学位论文.(Sun T Y.2014.Somatic embryogenesis and cryopreservation method of Pinus thunbergii resistant to Bursaphelenchus xylophilus.Nanjing:MS thesis of Nanjing Forestry University.[in Chinese])
孙婷玉,叶建仁,吴小芹,等.2015.抗松材线虫病黑松胚性愈伤组织诱导.东北林业大学学报,(9):96-99.(Sun T Y,Ye J R,Wu X Q,et al.2015.Induction of embryonic callus from Pinus thunbergii resistance to pine wilt disease.Journal of Northeast Forestry University,(9):96-99.[in Chinese])
唐巍,欧阳藩,郭仲琛.1988.火炬松合子胚愈伤组织的器官发生和植株再生.实验生物学报,31(1):87-93.(Tang W,Ouyang F,Guo Z C.1988.Organogenesis and plant regeneration of zygotic embryo in loblolly pine(Pinus taeda L.).Acta Biologiae Experimentalis Sinica,31(1):87-93.[in Chinese])
唐巍,欧阳藩,郭仲琛.1997.湿地松体细胞胚胎发生和植株再生.植物资源与环境,6(2):8-11.(Tang W,Ouyang F,Guo Z C.1997.Plantlet regeneration via somatic embryogenesis in Slash pine.Journal of Plant Resources and Environment,6(2):8-11.[in Chinese])
王贺飞,柴明良.2006.4种固化剂对若干草坪草愈伤组织诱导的影响.园艺学报,3(2):437-440.(Wang H F,Chai M L.2006.Effect of four brands of gelling agents on callus induction from mature seeds of several turfgrass species.Acta Horticulturae Sinica,3(2):437-440.[in Chinese])
吴丽君.2009.湿地松合子胚发育与体胚诱导的研究.福建林学院学报,29(3):243-246.(Wu L J.2009.Study on development course of zygotic embryos and initiation of somatic embryos of Slash pine.Journal of Fujian College of Forestry,29(3):243-246.[in Chinese])
吴小芹,张艺,谢小桃,等.2009.外生菌根菌对抗性抗性黑松和赤松不同家系生长影响.林业工程学报,23(1):19-22.(Wu X Q,Zhang Y,Xie X T,et al.2009.Effect of ectomycorrhizal fungi(EMF)on growth of the Pinus densiflora and P.thunbergii family trees resistant-Bursaphelenchus xylophilus.Journal of Forestry Engineering,23(1):19-22.[in Chinese])
叶建仁.2010.松材线虫病诊断与防治技术.北京:中国林业出版社.(Ye J R.2010.Techlonogy of diagnosis and treatment of pine wilt disease.Beijing:China Forestry Publishing House.[in Chinese])
张巧,汪静儿,林君,等.2010.不同凝固剂对陆地棉体细胞胚胎发生和植株再生的影响.棉花学报,22(1):3-9.(Zhang Q,Wang J E,Lin J,et al.2010.Effects of different gelling agents on the somatic embryogenesis and plant regeneration in upland cotton.Cotton Science,22(1):3-9.[in Chinese])
赵捷,韩骁,石娟.2017.低温条件下松材线虫在中国的风险分布区预测.生物安全学报,26(3):191-198.(Zhao J,Han X,Shi J.2017.Potential distribution of Bursaphelenchus xylophilus in China due to adaptation cold conditions.Journal of Biosafety,26(3):191-198.[in Chinese])
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