水曲柳离体再生系统的建立与完善
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摘要
取水曲柳成熟种子的胚轴作为外植体，对其组织培养进行了系统的研究，获得了不定芽增生和植株再生。
初步试验结果显示：对于不定芽的诱导，TDZ比BA更为有效。以MS作为基本培养基添加1～2mg/L的TDZ进行初代培养，2周后不定芽增生达到最大值。继代培养后极小部分的不定芽伸长，转移到生根培养基后可诱导生根成为完整植株。然而存在着两大问题：1)外植体大量死亡；2)不定芽难以伸长。
由此，在初步实验结果的基础上，我们对不定芽死亡率高和不定芽难伸长的问题进行了进一步的研究，寻求解决途径。结果表明，高盐浓度的MS培养基对不定芽的诱导有促进作用，但不定芽死亡率高；低盐浓度的WPM培养基尽管对不定芽诱导没有优势，但可维持再生不定芽的正常生长。TDZ与BA配合使用对不定芽诱导的促进效果，比单独使用其中的任何一种激素的效果都要大。而且，这两种细胞分裂素的配合使用，对降低外植体的死亡率有很大的作用。最佳的诱导培养基为MS+2mg/L TDZ+0.01mg/LBA，诱导率可达83.3％。WPM，以其低盐浓度、“温和”的特性，被用于进行不定芽的继代培养。BA与IBA配合使用，对不定芽的伸长生长有很好的促进效果。最佳的配比为2mg/L BA+0.01mg／L IBA。
综合研究结果，我们认为，培养基、细胞分裂素和生长素的“协同作用”对水曲柳胚轴外植体的不定芽诱导以及再生不定芽的伸长生长起着至关重要的作用，尤其是激素之间的平衡互作是一个很重要的影响因素。
Embryo hypocotyls of mature seeds of Mandshurican ash (Fraxinus Mandshurica Rupr.) were used as explants. Then in vitro culture of this species was investigated.
Adventitious buds were obtained from embryo hypocotyls explants. Maximum regeneration occurred after 2 weeks of culture. TDZ showed its advantages over BA on the initiation of adventitious buds. The results showed that MS medium supplemented with 1?mg/L TDZ could induced maximum adventitious buds. A few of buds elongated in subculture, and then rooted in rooting medium. Thus rooting seedlings of Mandshurican ash were obtained. However, mortality of explants and short buds were two big problems.
On the basis of the above studies, factors affecting the mortality of adventitious buds of Mandshurican ash and ways to stimulate the elongation of buds were studied. High-salt medium (MS) was effective to the regeneration of adventitious buds, but resulted in higher mortality; Low-salt medium (WPM), while had no advantages over other media on the induction of adventitious buds, supported the continuous growth of adventitious buds of Mandshurican ash.. The combination of TDZ and BA was more effective than used alone on adventitious bud regeneration. Additionally, this combination could reduce the mortality of the adventitious buds. MS medium supplemented with TDZ 2mg/L and BA 0. Olmg/L resulted in best regeneration (83. 3%). In subculture, WPM medium was used, because it was more friendly than other media for the growth of explants. BA, when combined with IBA, showed its effectiveness on the growth and elongation of the adventitious buds. Best result was obtained on medium supplemented with BA 2mg/L and IBA 0.01mg/L.
The results indicated that the "co-effects" of media, cytokinins and auxins , especially the balance of hormones played important role in the regeneration of Mandshurican ash and development of regenerated buds.
初步试验结果显示：对于不定芽的诱导，TDZ比BA更为有效。以MS作为基本培养基添加1～2mg/L的TDZ进行初代培养，2周后不定芽增生达到最大值。继代培养后极小部分的不定芽伸长，转移到生根培养基后可诱导生根成为完整植株。然而存在着两大问题：1)外植体大量死亡；2)不定芽难以伸长。
由此，在初步实验结果的基础上，我们对不定芽死亡率高和不定芽难伸长的问题进行了进一步的研究，寻求解决途径。结果表明，高盐浓度的MS培养基对不定芽的诱导有促进作用，但不定芽死亡率高；低盐浓度的WPM培养基尽管对不定芽诱导没有优势，但可维持再生不定芽的正常生长。TDZ与BA配合使用对不定芽诱导的促进效果，比单独使用其中的任何一种激素的效果都要大。而且，这两种细胞分裂素的配合使用，对降低外植体的死亡率有很大的作用。最佳的诱导培养基为MS+2mg/L TDZ+0.01mg/LBA，诱导率可达83.3％。WPM，以其低盐浓度、“温和”的特性，被用于进行不定芽的继代培养。BA与IBA配合使用，对不定芽的伸长生长有很好的促进效果。最佳的配比为2mg/L BA+0.01mg／L IBA。
综合研究结果，我们认为，培养基、细胞分裂素和生长素的“协同作用”对水曲柳胚轴外植体的不定芽诱导以及再生不定芽的伸长生长起着至关重要的作用，尤其是激素之间的平衡互作是一个很重要的影响因素。
Embryo hypocotyls of mature seeds of Mandshurican ash (Fraxinus Mandshurica Rupr.) were used as explants. Then in vitro culture of this species was investigated.
Adventitious buds were obtained from embryo hypocotyls explants. Maximum regeneration occurred after 2 weeks of culture. TDZ showed its advantages over BA on the initiation of adventitious buds. The results showed that MS medium supplemented with 1?mg/L TDZ could induced maximum adventitious buds. A few of buds elongated in subculture, and then rooted in rooting medium. Thus rooting seedlings of Mandshurican ash were obtained. However, mortality of explants and short buds were two big problems.
On the basis of the above studies, factors affecting the mortality of adventitious buds of Mandshurican ash and ways to stimulate the elongation of buds were studied. High-salt medium (MS) was effective to the regeneration of adventitious buds, but resulted in higher mortality; Low-salt medium (WPM), while had no advantages over other media on the induction of adventitious buds, supported the continuous growth of adventitious buds of Mandshurican ash.. The combination of TDZ and BA was more effective than used alone on adventitious bud regeneration. Additionally, this combination could reduce the mortality of the adventitious buds. MS medium supplemented with TDZ 2mg/L and BA 0. Olmg/L resulted in best regeneration (83. 3%). In subculture, WPM medium was used, because it was more friendly than other media for the growth of explants. BA, when combined with IBA, showed its effectiveness on the growth and elongation of the adventitious buds. Best result was obtained on medium supplemented with BA 2mg/L and IBA 0.01mg/L.
The results indicated that the "co-effects" of media, cytokinins and auxins , especially the balance of hormones played important role in the regeneration of Mandshurican ash and development of regenerated buds.
引文
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6．孔冬梅，谭燕双，沈海龙．白蜡树属植物组织培养和体细胞胚发生．植物生理学通讯，2003；39(6)：677—680
7．梁预，姚敦义．关于体细胞的全能性问题植物学通报，1998：15(4)：41-44
8．刘长莉，沈海龙，张羽，黄剑．新疆杨组织培养技术．东北林业大学学报，2004；32(1)：5～7
9．潘瑞炽，董愚得．植物生理学(第四版)．高等教育出版社．北京．2001
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11．沈海龙，邢朝斌，黄剑，胡立江，刘华英．水曲柳扦插繁殖影响因子的分析．东北林业大学学报，2004(已接收)
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