杉木不同器官愈伤组织和再生芽的诱导效率
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摘要
以杉木未成熟胚、种子发芽的下胚轴、子叶以及组培苗茎段为外植体,研究基本培养基、不同激素组合及浓度对杉木愈伤组织的诱导及再生芽发生的影响。结果表明:不同激素组合及浓度对不同外植体的愈伤组织诱导率有显著影响,对未成熟胚愈伤组织诱导最佳的培养基为MS+2,4-D 2 mg·L~(-1)+6-BA 1 mg·L~(-1)+KT 1 mg·L~(-1),诱导率为92.7%;对子叶、下胚轴愈伤组织诱导最佳的培养基为MS+6-BA 2 mg·L~(-1)+NAA 2 mg·L~(-1)+TDZ 0.01 mg·L~(-1),诱导率分别为97.4%和95.2%;对组培苗茎段愈伤组织诱导最佳的培养基为MS+2,4-D 1 mg·L~(-1)+NAA 2 mg·L~(-1)+TDZ 0.01 mg·L~(-1),诱导率为86.7%;MS+6-BA 0.5 mg·L~(-1)+KT 1.5 mg·L~(-1)是愈伤组织诱导再生芽的最佳培养基,诱导率为80%。质地疏松的愈伤组织其再生芽率最高(65%),单个质地均匀的愈伤组织其再生芽数可达6个;基本培养基对愈伤组织质地形态有较大影响,1/2MS培养基诱导出愈伤组织质地较疏松,MS培养基诱导出的愈伤组织质地均匀且表面可见颗粒状细胞团,DCR培养基诱导出的愈伤组织质地较密。
For the purpose of establishing callus induction and bud regenerate system for Cunninghamia lanceolata( Lamb.) Hook.,and proving an effective receptor procedure for researching into the genetic engineering of somatic hybridization and genetic transformation. Using immature embryo,hypocotyls and cotyledons of seedlings and stems of sterile seedlings as explants,the effect of the following factors including basal medium,different types of hormone concentrations and combinations on callus induction and regeneration buds differentiation were investigated respectively. The results indicated that different hormone combinations on callus induction of different explants have a significant influence. The optimal medium for immature embryo induction callus was MS+2,4-D2 mg·L~(-1)+6-BA 1 mg·L~(-1)+KT 1 mg·L~(-1),the induction rate was 92.7%; the optimal medium for cotyledons and hypocotyls induction callus was MS+6-BA 2 mg·L~(-1)+NAA 2 mg·L~(-1)+TDZ 0.01 mg·L~(-1),the induction rates were 97.4% and 95.2% respectively; the optimal medium for stems from sterile seedlings induction callus was MS+2,4-D 1 mg·L~(-1)+NAA 2 mg·L~(-1)+TDZ 0.01 mg·L~(-1),the induction rate was 86.7%; the optimal medium for callus induction regeneration bud was MS+ 6-BA 0.5 mg·L~(-1)+KT 1.5 mg·L~(-1),the induction rate was 80%. The callus with loose texture has the highest regeneration rate( 65%),but for a single callus with homogeneous texture,its maximum number of regenerated buds was 6. The basic medium has great influence on the texture of callus that the callus induced by 1/2MS basal medium showed loose texture,homogeneous surface with visible granular cell for MS basal medium,and dense callus for DCR basal medium.
For the purpose of establishing callus induction and bud regenerate system for Cunninghamia lanceolata( Lamb.) Hook.,and proving an effective receptor procedure for researching into the genetic engineering of somatic hybridization and genetic transformation. Using immature embryo,hypocotyls and cotyledons of seedlings and stems of sterile seedlings as explants,the effect of the following factors including basal medium,different types of hormone concentrations and combinations on callus induction and regeneration buds differentiation were investigated respectively. The results indicated that different hormone combinations on callus induction of different explants have a significant influence. The optimal medium for immature embryo induction callus was MS+2,4-D2 mg·L~(-1)+6-BA 1 mg·L~(-1)+KT 1 mg·L~(-1),the induction rate was 92.7%; the optimal medium for cotyledons and hypocotyls induction callus was MS+6-BA 2 mg·L~(-1)+NAA 2 mg·L~(-1)+TDZ 0.01 mg·L~(-1),the induction rates were 97.4% and 95.2% respectively; the optimal medium for stems from sterile seedlings induction callus was MS+2,4-D 1 mg·L~(-1)+NAA 2 mg·L~(-1)+TDZ 0.01 mg·L~(-1),the induction rate was 86.7%; the optimal medium for callus induction regeneration bud was MS+ 6-BA 0.5 mg·L~(-1)+KT 1.5 mg·L~(-1),the induction rate was 80%. The callus with loose texture has the highest regeneration rate( 65%),but for a single callus with homogeneous texture,its maximum number of regenerated buds was 6. The basic medium has great influence on the texture of callus that the callus induced by 1/2MS basal medium showed loose texture,homogeneous surface with visible granular cell for MS basal medium,and dense callus for DCR basal medium.
引文
[1]欧阳磊,郑仁华,翁玉榛,等.杉木优良无性系组培快繁技术体系的建立[J].南京林业大学学报(自然科学版),2007,31(3):47-51.
[2]朱木兰,王骥,卫志明.杉木再生系统的比较研究[J].分子细胞生物学报,2007,40(4):239-244.
[3]阙国宁.杉木组培嫩梢增殖与复壮的分析[J].林业科学研究,1989(6):546-551.
[4]阙国宁.杉木组织培养的初步研究[J].林业科学,1980,16(S1):137-140.
[5]吴擢溪,李振问,吴大忠.杉木组织培养繁殖体系建立的研究[J].福建林学院学报,1991,11(1):67-74.
[6]TZFIRA T,CITOVSKY V.Agrobacterium-mediated genetic transformation of plants:biology and biotechnology[J].Current Opinion in Biotechnology,2006,17(2):147-154.
[7]ROMMENS C M.All-native DNA transformation:a new approach to plant genetic engineering[J].Trends in Plant Science,2004,9(9):457-464.
[8]王关林.植物基因工程[M].北京:科学出版社,2014.
[9]席梦利,施季森.杉木合子胚愈伤组织的诱导及植株再生[J].南京林业大学学报(自然科学版),2006,30(2):6-10.
[10]陈剑勇.杉木茎尖诱导愈伤组织及植株再生研究[J].西南林业大学学报,2009,29(5):37-41.
[11]MATHUR G,NADGAUDA R.In vitro plantlet regeneration from mature zygotic embryos of Pinus wallichiana A.B.Jacks[J].Plant Cell Reports,1999,19(1):74-80.
[12]黄健秋,卫志明.松属树种的组织培养和原生质体培养[J].植物学报,1994,11(1):34-42.
[13]ROUNT G R,MOHAPATRA A,JAIN S M.Tissue culture of ornamental pot plant:a critical review on present scenario and future prospects[J].Biotechnology Advances,2006,24(6):531-560.
[14]BONGA J M,KLIMASZEWSKA K K,ADERKAS P V.Recalcitrance in clonal propagation,in particular of conifers[J].Plant Cell Tissue&Organ Culture,2009,100(3):241-254.
[15]王祎,赵彤彤,杨魏,马辉,丁令智,梁艳,沈海龙.红松成熟合子胚愈伤组织诱导的适宜培养条件[J].森林工程,2015,10(2):5-7,13.
[16]耿菲菲,肖丰坤,吴涛,等.思茅松成熟胚的胚性愈伤组织诱导与增殖[J].东北林业大学学报,2015,43(5):59-63.
[17]徐刚标,何方,陈良昌.银杏愈伤组织诱导与继代培养的研究[J].中南林业科技大学学报,1999,10(3):32-36.
[18]张建瑛,张海峰,田新华,等.长白落叶松组织培养中愈伤组织的诱导[J].黑龙江生态工程职业学院学报,2012,13(2):15-16.
[19]张丽杰,赵丽蒙,陆秀君,等.水曲柳子叶和下胚轴愈伤组织和体胚的诱导[J].分子植物育种,2015,13(7):1 645-1 652.
[20]唐巍,郭仲琛.火炬松(Pinus teada L.)合子胚愈伤组织的器官发生和植株再生[J].实验生物学报,1998,31(1):87-93.
[21]GLOWACKA K,JEZOWSKI S,KAZMAREK Z.The effects of genotype,inflorescence developmental stage and induction medium on callus induction and plant regeneration in two Miscanthus species[J].Plant Cell Tissue&Organ Culture,2010,102(1):79-86.
[22]顾淑荣,朱至清,赵敬芳,等.白皮松雌配子体愈伤组织的诱导和分化[J].植物学报,1994,11(3):217-221.
[23]杨模华,李志辉,张冬林,等.马尾松嫩茎愈伤组织保持、增殖与不定芽分化培养[J].中国农学通报,2011,27(10):12-17.
[24]张文泉,闫伟.樟子松愈伤诱导及植株再生的初步研究[J].中南林业科技大学学报,2012,32(11):37-41.
[25]庞晓峰,王伟达,李成浩,等.长白落叶松愈伤组织诱导与不定芽分化[J].东北林业大学学报,2008,36(2):5-7.
[26]陈发菊,赵德修,杨映根,等.水母雪莲愈伤组织的多态性及其再分化条件的研究[J].华中师范大学学报(自然科学版),2000,34(3):331-335.
[2]朱木兰,王骥,卫志明.杉木再生系统的比较研究[J].分子细胞生物学报,2007,40(4):239-244.
[3]阙国宁.杉木组培嫩梢增殖与复壮的分析[J].林业科学研究,1989(6):546-551.
[4]阙国宁.杉木组织培养的初步研究[J].林业科学,1980,16(S1):137-140.
[5]吴擢溪,李振问,吴大忠.杉木组织培养繁殖体系建立的研究[J].福建林学院学报,1991,11(1):67-74.
[6]TZFIRA T,CITOVSKY V.Agrobacterium-mediated genetic transformation of plants:biology and biotechnology[J].Current Opinion in Biotechnology,2006,17(2):147-154.
[7]ROMMENS C M.All-native DNA transformation:a new approach to plant genetic engineering[J].Trends in Plant Science,2004,9(9):457-464.
[8]王关林.植物基因工程[M].北京:科学出版社,2014.
[9]席梦利,施季森.杉木合子胚愈伤组织的诱导及植株再生[J].南京林业大学学报(自然科学版),2006,30(2):6-10.
[10]陈剑勇.杉木茎尖诱导愈伤组织及植株再生研究[J].西南林业大学学报,2009,29(5):37-41.
[11]MATHUR G,NADGAUDA R.In vitro plantlet regeneration from mature zygotic embryos of Pinus wallichiana A.B.Jacks[J].Plant Cell Reports,1999,19(1):74-80.
[12]黄健秋,卫志明.松属树种的组织培养和原生质体培养[J].植物学报,1994,11(1):34-42.
[13]ROUNT G R,MOHAPATRA A,JAIN S M.Tissue culture of ornamental pot plant:a critical review on present scenario and future prospects[J].Biotechnology Advances,2006,24(6):531-560.
[14]BONGA J M,KLIMASZEWSKA K K,ADERKAS P V.Recalcitrance in clonal propagation,in particular of conifers[J].Plant Cell Tissue&Organ Culture,2009,100(3):241-254.
[15]王祎,赵彤彤,杨魏,马辉,丁令智,梁艳,沈海龙.红松成熟合子胚愈伤组织诱导的适宜培养条件[J].森林工程,2015,10(2):5-7,13.
[16]耿菲菲,肖丰坤,吴涛,等.思茅松成熟胚的胚性愈伤组织诱导与增殖[J].东北林业大学学报,2015,43(5):59-63.
[17]徐刚标,何方,陈良昌.银杏愈伤组织诱导与继代培养的研究[J].中南林业科技大学学报,1999,10(3):32-36.
[18]张建瑛,张海峰,田新华,等.长白落叶松组织培养中愈伤组织的诱导[J].黑龙江生态工程职业学院学报,2012,13(2):15-16.
[19]张丽杰,赵丽蒙,陆秀君,等.水曲柳子叶和下胚轴愈伤组织和体胚的诱导[J].分子植物育种,2015,13(7):1 645-1 652.
[20]唐巍,郭仲琛.火炬松(Pinus teada L.)合子胚愈伤组织的器官发生和植株再生[J].实验生物学报,1998,31(1):87-93.
[21]GLOWACKA K,JEZOWSKI S,KAZMAREK Z.The effects of genotype,inflorescence developmental stage and induction medium on callus induction and plant regeneration in two Miscanthus species[J].Plant Cell Tissue&Organ Culture,2010,102(1):79-86.
[22]顾淑荣,朱至清,赵敬芳,等.白皮松雌配子体愈伤组织的诱导和分化[J].植物学报,1994,11(3):217-221.
[23]杨模华,李志辉,张冬林,等.马尾松嫩茎愈伤组织保持、增殖与不定芽分化培养[J].中国农学通报,2011,27(10):12-17.
[24]张文泉,闫伟.樟子松愈伤诱导及植株再生的初步研究[J].中南林业科技大学学报,2012,32(11):37-41.
[25]庞晓峰,王伟达,李成浩,等.长白落叶松愈伤组织诱导与不定芽分化[J].东北林业大学学报,2008,36(2):5-7.
[26]陈发菊,赵德修,杨映根,等.水母雪莲愈伤组织的多态性及其再分化条件的研究[J].华中师范大学学报(自然科学版),2000,34(3):331-335.