簸箕柳组培再生体系的建立
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摘要
【目的】建立簸箕柳组培再生体系,为在簸箕柳中开展功能基因组研究奠定基础。【方法】分别以簸箕柳带腋芽茎段和种子为外植体,比较不同消毒方法、基本培养基类型、光照条件及植物生长调节剂等因素对不同外植体脱分化和再分化能力的影响。【结果】簸箕柳组培基本培养基成分以MS+蔗糖(30 g/L)+Phytagel (植物凝胶)(4.4 g/L)为宜。以幼嫩茎段为外植体,最佳消毒方法为70%(体积分数)酒精处理30 s+20%(质量分数)的次氯酸钠处理10 min,适宜诱导腋芽的生长调节剂组合为6-BA(1 mg/L)+NAA (0.01 mg/L),丛生芽继代增殖的适宜生长调节剂配比为6-BA (0.5 mg/L)+NAA (0.05 mg/L),适宜腋芽生根的生长调节剂组合为IBA (0.2 mg/L)+NAA (0.03 mg/L)。以种子为外植体,最佳消毒方法为70%(体积分数)酒精处理10 s+20%(质量分数)的次氯酸钠处理2 min,光照条件下诱导愈伤组织的生长调节剂配比为6-BA (0.5 mg/L)+NAA (0.3 mg/L),并利用6-BA (0.5 mg/L)+NAA (0.5 mg/L)和6-BA (0.5 mg/L)+NAA (0.8 mg/L)两种组合方式交替使用进行愈伤组织的继代培养;愈伤组织分化成芽的生长调节剂配比为6-BA (0.5 mg/L)+TDZ (0.2 mg/L),生根培养基只需添加NAA (0.01 mg/L)。【结论】以茎段和种子作为外植体,通过不同的再生途径,均可以建立簸箕柳组织培养再生体系。
【Objective】In this study, we aimed to establish an efficient plant regeneration protocol for Salix suchowensis through tissue culture, which would serve as a foundation for developing a genetic transformation system for this woody shrub. 【Method】By using young stems with axillary buds and mature seeds as explants, the influences of different factors on plantlet regeneration were systematically studied, including sterilization methods, types of the basic medium, light conditions, and plant growth regulators. 【Result】The optimal basic medium for S. suchowensis was Murashige and Skoog(MS) medium + sucrose(30 g/L) + Phytagel(4.4 g/L). When using young stem nodal segments as explants, the best sterilization method was 70% ethanol for 30 sec and then 20% sodium hypochlorite for 10 min, while the optimal medium for axillary buds induction and multiplication was the basic medium supplemented with 6-BA(1 mg/L) + NAA(0.01 mg/L) and 6-BA(0.5 mg/L) + NAA(0.05 mg/L), respectively, and the appropriate medium for rooting was the basic medium + IBA(0.2 mg/L) + NAA(0.03 mg/L). While for mature seeds, the optimal sterilizing treatment was 70% ethanol for 10 sec and then 20% sodium hypochlorite for 2 min; the appropriate medium for callus indution, proliferation and subsequent shoot differentiation was the basic medium supplemented with 6-BA(0.5 mg/L) +NAA(0.3 mg/L), 6-BA(0.5 mg/L)+ NAA(0.5 or 0.8 mg/L), 6-BA(0.5 mg/L) + TDZ(0.2 mg/L), respectively, and the best rooting medium was the basic medium containing NAA(0.01 mg/L). 【Conclusion】We established a regeneration protocol for S. suchowensis from the axillary buds or callus by using young stem nodal segments and mature seeds as explants respectively.
【Objective】In this study, we aimed to establish an efficient plant regeneration protocol for Salix suchowensis through tissue culture, which would serve as a foundation for developing a genetic transformation system for this woody shrub. 【Method】By using young stems with axillary buds and mature seeds as explants, the influences of different factors on plantlet regeneration were systematically studied, including sterilization methods, types of the basic medium, light conditions, and plant growth regulators. 【Result】The optimal basic medium for S. suchowensis was Murashige and Skoog(MS) medium + sucrose(30 g/L) + Phytagel(4.4 g/L). When using young stem nodal segments as explants, the best sterilization method was 70% ethanol for 30 sec and then 20% sodium hypochlorite for 10 min, while the optimal medium for axillary buds induction and multiplication was the basic medium supplemented with 6-BA(1 mg/L) + NAA(0.01 mg/L) and 6-BA(0.5 mg/L) + NAA(0.05 mg/L), respectively, and the appropriate medium for rooting was the basic medium + IBA(0.2 mg/L) + NAA(0.03 mg/L). While for mature seeds, the optimal sterilizing treatment was 70% ethanol for 10 sec and then 20% sodium hypochlorite for 2 min; the appropriate medium for callus indution, proliferation and subsequent shoot differentiation was the basic medium supplemented with 6-BA(0.5 mg/L) +NAA(0.3 mg/L), 6-BA(0.5 mg/L)+ NAA(0.5 or 0.8 mg/L), 6-BA(0.5 mg/L) + TDZ(0.2 mg/L), respectively, and the best rooting medium was the basic medium containing NAA(0.01 mg/L). 【Conclusion】We established a regeneration protocol for S. suchowensis from the axillary buds or callus by using young stem nodal segments and mature seeds as explants respectively.
引文
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[10] 王蒂.植物组织培养[M].北京:中国农业出版社,2004.WANG D.Tissue culture of plants[M].Beijing:China Agriculture Press,2004.
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[13] 李永文,刘新波,黄海帆,等.植物组织培养技术[M].北京:北京大学出版社,2007.LI Y W,LIU X B,HUANG H F,et al.Tissue culture technology of plants [M].Beijing:Peking University Press,2007.
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[15] 王慧梅.喜树组织培养与遗传转化的研究[D].哈尔滨:东北林业大学,2004.WANG H M.Study on the tissue culture and genetic transformation of Camptotheca acuminate [D].Harbin:Northeast Forestry University,2004.
[16] 陈宗礼,齐向英,张向前,等.TDZ和6-BA对枣树继代培养的影响[J].西北农业学报,2006,15(3):162-165.DOI:10.3969/j.issn.1004-1389.2006.03.039.CHEN Z L,QI X Y,ZHANG X Q,et al.Effect of TDZ and 6-BA on subculture of Zizyphus jujube Mill[J].Acta Agriculturae Boreali-occidentalis Sinica,2006,15(3):162-165.
[17] 张娜,张芸香,郭晋平.文冠果腋芽诱导关键影响因素与培养体系优化[J].山西农业大学学报(自然科学版),2014,34(1):53-58.DOI:10.13842/j.cnki.issn1671-8151.2014.01.016.ZHANG N,ZHANG Y X,GUO J P.Thekey factors of axillary bud induction of Xanthoceras sorbifolium and the optimization system for its cultivation [J].Journal of Shanxi Agriculture University (Nature Science Edition),2014,34(1):53-58.
[18] 王涛.碱茅再生体系建立及其遗传转化的研究[D].哈尔滨:东北林业大学,2011.WANG T.Studies onconstruction of regeneration system and genetic transformation of Puccinellia chinampoensis [D].Harbin:Northeast Forestry University,2011.
[19] STOEHR M U,CAI M,ZSUFFA L.In vitro plant regeneration via callus culture of mature Salix exigua [J].Canadian Journal of Forest Research,2011,19(12):1634-1638.DOI:10.1139/x89-247.
[20] AGRAWAL D C,GEBHARDT K.Rapid micropropagation of hybrid willow (Salix) established by ovary culture [J].Journal of Plant Physiology,1994,143(6):763-765.DOI:10.1016/S0176-1617(11)81173-9.
[21] LYYRA S,LIMA A,MERKLE S A.In vitro regeneration of Salix nigra from adventitious shoots [J].Tree Physiology,2006,26(7):969-975.DOI:org/10.1093/treephys/26.7.969.
[22] VAHALA T,STABEL P,ERIKSSON T.Genetic transformation of willows (Salix spp.) by Agrobacterium tumefaciens [J].Plant Cell Reports,1989,8(2):55-58.DOI:10.1007/BF00716837.
[23] YANG J L,YI J,YANG C P,et al.Agrobacterium tumefaciens-mediated genetic transformation of Salix matsudana Koidz.using mature seeds [J].Tree Physiology,2013,33(6):628-639.DOI:10.1093/treephys/tpt038.
[2] WHETTEN R W,MACKAY J J,SEDEROFF RR.Recent advances in understanding lignin biosynthesis [J].Annu Rev Plant Physicl Plant Mol Biol,1998 (49):407-416.DOI:10.1146/annurev.arplant.49.1.585.
[3] TAYLOR G.Populus:Arabidopsis for forestry.Do we need a model tree?[J].Annals of Botany,2002,90(6):681-689.DOI:org/10.1093/aob/mcf255.
[4] TUSKAN G.The genome of black cottonwood,Populus trichocarpa (Torr.& Gray) [J].Science,2006,313(5793):1596-1604.DOI:10.1126/science.1128691.
[5] PARSONS T J,SINKAR V P,STETTLER R F,et al.Transformation of poplar by Agrobacterium tumefaciens [J].Nature Biotechnology,1986,4(6):533-536.DOI:10.1038/nbt0686-533.
[6] 沈利爽,朱立煌.植物的比较基因组研究和大遗传系统[J].中国生物工程杂志,1995,15(2):23-28.DOI:10.13523/j.cb.19950207.SHEN L S,ZHU L H.Comparative genomic research and large genetic system in plants [J].Progress in Biotechnology,1995,15(2):23-28.
[7] DAI X G,HU Q J,CAI Q L,et al.The willow genome and divergent evolution from poplar after the common genome duplication [J].Cell Research,2014,24(10):1274.DOI:10.1038/cr.2014.83.
[8] 梁春秀,严华兵,黄京华,等.激素对罗汉果增殖、生根及愈伤组织大小影响研究[J].南方农业学报,2009,40(7):807-809.DOI:10.3969/j.issn.2095-1191.2009.07.003.LIANG C X,YAN H B,HUANG J H,et al.Effects of hormones on subculture,rooting and size of callus of grosvenor momordica fruit (Siratia grasvenorii Swingle) [J].Guangxi Agricultural Sciences,2009,40(7):807-809.
[9] 祝晨辰,徐进,欧阳磊,等.柳杉优良无性系离体培养再生体系研究[J].南京林业大学学报(自然科学版),2015,39(3):55-58.DOI:10.3969/j.issn.1000-2006.2015.03.011ZHU C C,XU J,OUYANG L,et al.Study on regeneration system of Cryptomeria clones in vitro culture[J].Journal of Nanjing Forestry University (Natural Sciences Edition),2015,39(3):55-58.
[10] 王蒂.植物组织培养[M].北京:中国农业出版社,2004.WANG D.Tissue culture of plants[M].Beijing:China Agriculture Press,2004.
[11] 李洁.雪柳的组织培养初探[J].山西农业大学学报(自然科学版),2003,23(3):292-294.DOI:10.3969/j.issn.1671-8151.2003.03.028.LI J.Theinitial study of Fontaresia fortunei Carr' s organize culture [J].Journal of Shanxi Agriculture University (Nature Science Edition),2003,23(3):292-294.
[12] 关亚丽,陈英,黄敏仁.簸箕柳组织培养初步研究[J].海南师范大学学报(自然科学版),2009,22(2):204-208.DOI:10.3969/j.issn.1674-4942.2009.02.023.GUAN Y L,CHEN Y,HUANG M R.Thepreliminary study on tissue culture of Salix suchowensis [J].Journal of Hainan Normal University (Natural Science),2009,22(2):204-208.
[13] 李永文,刘新波,黄海帆,等.植物组织培养技术[M].北京:北京大学出版社,2007.LI Y W,LIU X B,HUANG H F,et al.Tissue culture technology of plants [M].Beijing:Peking University Press,2007.
[14] 潘学峰,李绍良.生姜茎尖离体培养的研究[J].海南大学学报(自然科学版),1999,17(1):59-63.DOI:10.3969/j.issn.1004-1729.1999.01.011.PAN X F,LI S L.Study on the shoot tip culture of common ginger in vitro [J].Natural Science Journal of Hainan University,1999,17(1):59-63.
[15] 王慧梅.喜树组织培养与遗传转化的研究[D].哈尔滨:东北林业大学,2004.WANG H M.Study on the tissue culture and genetic transformation of Camptotheca acuminate [D].Harbin:Northeast Forestry University,2004.
[16] 陈宗礼,齐向英,张向前,等.TDZ和6-BA对枣树继代培养的影响[J].西北农业学报,2006,15(3):162-165.DOI:10.3969/j.issn.1004-1389.2006.03.039.CHEN Z L,QI X Y,ZHANG X Q,et al.Effect of TDZ and 6-BA on subculture of Zizyphus jujube Mill[J].Acta Agriculturae Boreali-occidentalis Sinica,2006,15(3):162-165.
[17] 张娜,张芸香,郭晋平.文冠果腋芽诱导关键影响因素与培养体系优化[J].山西农业大学学报(自然科学版),2014,34(1):53-58.DOI:10.13842/j.cnki.issn1671-8151.2014.01.016.ZHANG N,ZHANG Y X,GUO J P.Thekey factors of axillary bud induction of Xanthoceras sorbifolium and the optimization system for its cultivation [J].Journal of Shanxi Agriculture University (Nature Science Edition),2014,34(1):53-58.
[18] 王涛.碱茅再生体系建立及其遗传转化的研究[D].哈尔滨:东北林业大学,2011.WANG T.Studies onconstruction of regeneration system and genetic transformation of Puccinellia chinampoensis [D].Harbin:Northeast Forestry University,2011.
[19] STOEHR M U,CAI M,ZSUFFA L.In vitro plant regeneration via callus culture of mature Salix exigua [J].Canadian Journal of Forest Research,2011,19(12):1634-1638.DOI:10.1139/x89-247.
[20] AGRAWAL D C,GEBHARDT K.Rapid micropropagation of hybrid willow (Salix) established by ovary culture [J].Journal of Plant Physiology,1994,143(6):763-765.DOI:10.1016/S0176-1617(11)81173-9.
[21] LYYRA S,LIMA A,MERKLE S A.In vitro regeneration of Salix nigra from adventitious shoots [J].Tree Physiology,2006,26(7):969-975.DOI:org/10.1093/treephys/26.7.969.
[22] VAHALA T,STABEL P,ERIKSSON T.Genetic transformation of willows (Salix spp.) by Agrobacterium tumefaciens [J].Plant Cell Reports,1989,8(2):55-58.DOI:10.1007/BF00716837.
[23] YANG J L,YI J,YANG C P,et al.Agrobacterium tumefaciens-mediated genetic transformation of Salix matsudana Koidz.using mature seeds [J].Tree Physiology,2013,33(6):628-639.DOI:10.1093/treephys/tpt038.