仙客来离体培养及体细胞无性系变异的研究
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摘要
本实验以仙客来‘浓香’(Cyclamen persicum Mill.)为试材，在MS基本培养基中分别添加不同浓度2，4-D和NAA两种生长素类植物生长调节剂诱导愈伤组织，进而研究了仙客来体细胞无性系建立过程中，由2，4-D或NAA诱导而来的愈伤组织的芽分化、根分化和继代培养问题。本实验初步研究了活性炭(AC)，维生素(Vc)，和聚乙烯吡咯烷酮(PVP)对仙客来外植体抗褐化的作用效果。为了解仙客来体细胞无性系变异的情况，应用秋水仙素为诱变剂对愈伤组织进行诱变，对自然变异株和诱变株进行了形态学及同功酶谱检测。
结果表明：仙客来再生体系建立最佳外植体为幼株叶片。2，4-D和NAA均可诱导愈伤组织；最佳诱导愈伤组织培养基为MS附加2，4-D 0.5mg／L和BA 0.2mg／L。分化培养基为MS附加BA2.0mg／L、2，4-D0.5mg／L和KT 0.2mg／L；生根培养基为1／2MS，附加IBA0.5mg／L和BA0.05mg／L。继代培养中，对于愈伤组织增殖，2，4-D诱导得到的愈伤组织的继代培养基为原诱导培养基；NAA诱导得到的愈伤组织应转入含有NAA1mg／L的继代培养基中；芽分化阶段的继代培养应转入含有BA1mg／L的培养基中。
仙客来适宜的抗褐化剂和使用浓度为Vc使用200mg／L浓度，PVP使用0.04％浓度。
秋水仙素处理时间与浓度对愈伤组织死亡率和不定芽分化率均有影响，但处理时间对仙客来愈伤组织死亡率的影响明显大于处理浓度的效应，在处理后35天观察时处理浓度与处理时间对仙客来的愈伤组织的死亡率存在互作效应。根据秋水仙素处理后愈伤组织死亡率和不定芽分化率衡量，诱变的最佳浓度和时间为100mg／L处理15天。
仙客来体细胞无性系在自然培养条件下变异率很高，变异类型丰富；由2，4-D和NAA诱导得到的再生植株变异率没有明显差别；经过秋水仙素处理后，无论是由2，4-D诱导得到的还是由NAA诱导得到的再生植株变异率对比未经秋水仙素处理得到的植株，变异率提高。
In vitro regeneration of Cyclamen 'dense fragrance' has been attempted in this experiment. Two kinds of auxins were applied respectively to induce the callus. They were 2,4-D and NAA. Then separate experiments and rooting experiments were carried out. The aspect of successive transfer culture was involved too. In the experiment, The question of browning of explants were studied. In order to get variant by chmomorphosis, callus were induced to produce variant by mutagen colchicum. And the variants were detected by methods of morphology observing and electrophoresis of isoenzyme. The results showed:
It was found that the callus from young leaves was easy to induce and grew rapidly. The best inducing medium is MS add 2,4-D0.5mg/L, BA0.2mg/L, the differentiation medium is MS add BA2mg/L, 2,4-D 0.5mg/L and KT 0.2mg/L. Then the explants should be transfer into 1/2MS add IBA0.2mg/L and BA0.02mg/L to root. The differentiation medium were also suitable for the callus induced by NAA. the suitable media for subculture respectively were MS add 2,4-D0.5mg/L and MS add NAA 1mg/L towards the callus induced by 2,4-D or NAA. For adventitious bud multiplication, the caltures with shoot buds should be transfered into MS added BAlmg/L, 2,4-D 0.5mg/L and KT 0.2mg/L.
Appreciate anti-browning agents and concentration were Vc200mg/L, PVP0.04%.
Time and concentration of treatment of colchicum affect mortality and frequency of differentiation of callus. The proper combination of time and concentration of colchicum treatment was 100mg/L and 15 day.
Either the regenerative plant induced by 2,4-D or that of by NAA, The rate of somaclonal variation was great in Cyclamen in vitro regeneration. After the treatment of colchicum, the rate of variation increased. These plantlets often showed deviant phenotypes.
结果表明：仙客来再生体系建立最佳外植体为幼株叶片。2，4-D和NAA均可诱导愈伤组织；最佳诱导愈伤组织培养基为MS附加2，4-D 0.5mg／L和BA 0.2mg／L。分化培养基为MS附加BA2.0mg／L、2，4-D0.5mg／L和KT 0.2mg／L；生根培养基为1／2MS，附加IBA0.5mg／L和BA0.05mg／L。继代培养中，对于愈伤组织增殖，2，4-D诱导得到的愈伤组织的继代培养基为原诱导培养基；NAA诱导得到的愈伤组织应转入含有NAA1mg／L的继代培养基中；芽分化阶段的继代培养应转入含有BA1mg／L的培养基中。
仙客来适宜的抗褐化剂和使用浓度为Vc使用200mg／L浓度，PVP使用0.04％浓度。
秋水仙素处理时间与浓度对愈伤组织死亡率和不定芽分化率均有影响，但处理时间对仙客来愈伤组织死亡率的影响明显大于处理浓度的效应，在处理后35天观察时处理浓度与处理时间对仙客来的愈伤组织的死亡率存在互作效应。根据秋水仙素处理后愈伤组织死亡率和不定芽分化率衡量，诱变的最佳浓度和时间为100mg／L处理15天。
仙客来体细胞无性系在自然培养条件下变异率很高，变异类型丰富；由2，4-D和NAA诱导得到的再生植株变异率没有明显差别；经过秋水仙素处理后，无论是由2，4-D诱导得到的还是由NAA诱导得到的再生植株变异率对比未经秋水仙素处理得到的植株，变异率提高。
In vitro regeneration of Cyclamen 'dense fragrance' has been attempted in this experiment. Two kinds of auxins were applied respectively to induce the callus. They were 2,4-D and NAA. Then separate experiments and rooting experiments were carried out. The aspect of successive transfer culture was involved too. In the experiment, The question of browning of explants were studied. In order to get variant by chmomorphosis, callus were induced to produce variant by mutagen colchicum. And the variants were detected by methods of morphology observing and electrophoresis of isoenzyme. The results showed:
It was found that the callus from young leaves was easy to induce and grew rapidly. The best inducing medium is MS add 2,4-D0.5mg/L, BA0.2mg/L, the differentiation medium is MS add BA2mg/L, 2,4-D 0.5mg/L and KT 0.2mg/L. Then the explants should be transfer into 1/2MS add IBA0.2mg/L and BA0.02mg/L to root. The differentiation medium were also suitable for the callus induced by NAA. the suitable media for subculture respectively were MS add 2,4-D0.5mg/L and MS add NAA 1mg/L towards the callus induced by 2,4-D or NAA. For adventitious bud multiplication, the caltures with shoot buds should be transfered into MS added BAlmg/L, 2,4-D 0.5mg/L and KT 0.2mg/L.
Appreciate anti-browning agents and concentration were Vc200mg/L, PVP0.04%.
Time and concentration of treatment of colchicum affect mortality and frequency of differentiation of callus. The proper combination of time and concentration of colchicum treatment was 100mg/L and 15 day.
Either the regenerative plant induced by 2,4-D or that of by NAA, The rate of somaclonal variation was great in Cyclamen in vitro regeneration. After the treatment of colchicum, the rate of variation increased. These plantlets often showed deviant phenotypes.
引文
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2．陈杭．国际园艺学会第25届大会简况．长江蔬菜，1999，(2)49
3．陈俊愉主编．中国农业百科全书—观赏园艺卷．农业出版社(北京)．1996：
4．陈受宜，朱立煌，洪建等．水稻抗盐突变体的分子生物学鉴定．植物学报．1991，33(8)：569
5．陈少麟．高等植物细胞突变体地筛选与作物改良．北京工业大学出版社(北京)1988：268
6．程金水主编．园林植物遗传育种学．中国林业出版社(北京)．2001
7．迟惠梅．芦荟抗寒变异系的筛选及其特性的研究，东北农业大学硕士学位论文．2000：4
8．费水章，周维燕．切花菊再生株的形态和细胞学变异的研究．园艺学报．1994，21(2)：193
9．傅桂荣，汪清胤，杨旭升．哈尔滨师范大学学报(自科版)．1994，10(2)：98
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