两种观赏蕨类植物离体快繁体系的建立
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摘要
本文以紫萁和银粉背蕨为材料，采用银粉背蕨孢子、原叶体和紫萁原叶体、根状茎尖作为外植体进行组织培养。通过不同的培养条件、不同的激素种类及其不同浓度的组合试验，成功地诱导出紫萁的GGB(绿色球状体)和愈伤组织、银粉背蕨的GGB，建立了两种蕨的离体快繁体系，为实现其工厂化育苗提供依据。主要研究结果如下：
以紫萁的根状茎尖为外植体诱导GGB的适合培养基是：1／2MS+BA 0.1mg／L+蔗糖30g／L，40天增殖倍数为27.5，GGB诱导率为100％。GGB分化的适合培养基是：1／2MS+蔗糖30g／L，GGB分化率为100％。适合紫萁试管苗生根的培养基是：1／2MS+蔗糖20g／L+NAA 0.1mg／L。将根状茎尖的叶片全部切除后诱导愈伤组织的效果较好，适合诱导愈伤组织的培养基是：1／2MS+2，4-D 3.0mg／L，培养条件为正常的光照培养。愈伤组织在1／2MS+NAA 0.1mg／L的培养基中的分化系数为5.433。生根试管苗炼苗后在腐叶土：珍珠岩=2：1的基质中的移栽成活率为47.5％。
银粉背蕨的孢子用2％NaClO溶液振荡8-10min后过滤，污染率可降低到0，是较适合银粉背蕨孢子的消毒方法。孢子在MS+蔗糖20-40g／L中光照培养，萌发率超过70％。以银粉背蕨原叶体为外植体在MS+BA 1.0-2.0mg／L+NAA 0.3mg／L的培养基中诱导GGB，50天后增殖倍数可达9以上。GGB在MS+KT 0.1mg／L+NAA 0.1mg／L的培养基中的分化率为100％，平均每植株分化的叶片数为6。适合银粉背蕨试管苗生根的培养基是：MS+蔗糖20g／L+IBA 0.5mg／L。生根试管苗炼苗后在腐叶土：珍珠岩=2：1的基质中的移栽成活率为75％。
In vitro culture of Osmunda japonica and Aleuritopteris argentea were studied inthis project. Spores, prothallus and rhizome were used as explants for the culture. TheGGB(green globular bodies) of Aleuritopteris argentea, and the GGB and callus ofOsmunda japonica were successfully induced in the different culture conditions, and thedifferent media with different types of hormone concentrations and the mix of differenttests. In vitro propagation systems of two fems were established, which providedimportant data for the industrialized propagation in macro and exploitation of these ferns.The main results were as follows:
Using rhizome of Osmunda japonica as explant, 1/2MS medium containing 0.1mgl~(-1)BA and sucrose 30gl~(-1) was the appropriate medium for inducing GGB. After 40 daysof culture, the biggest multiplication coefficient was 27.50, and the induction rate of GGBwas 100%. The appropriate medium for GGB differentiation is: 1/2MS with30gl~(-1) sucrose.GGB differentiation rate was 100%. The suitable medium for plantletsrooting was: 1/2MS+sucrose 20gl~(-1)+NAA 0.1mgl~(-1). Rhizomes removed off all leaveswere the best explant for callus induction. The suitable medium for callus induction was:1/2MS+2, 4-D 3.0mgl~(-1). The culture condition of callus induction was the normalillumination(illumination intensity 1200-14001x). The medium of 1/2MS containing0.1mgl~(-1) NAA was optimal for differentiation of callus of Osmunda japonica. Thedifferentiation coefficient was 5.433. The suitable transplanting substrate for plantletswith roots was the mixture of leaf mold and perlite (the ratio of 2:1)and the survival ratiowas 47.5%.
The Aleuritopteris argentea spores were filtrated after oscillating with 2% NaClOsolution for 8-10 minutes, and the contamination rates can be lowered to 0. This was theoptimal disinfection method for Aleuritopteris argentea spores. The suitable cultureconditions for spores germinating were normal illumination(illumination intensity1200-14001x). And the medium was MS together with 20-40gl~(-1) sucrose. The sporesgermination rate was over 70%. Using prothallus as explants, GGB can be induced after50 days of culture. And the optimal medium was MS containing 1.0-2.0mgl~(-1) BA together with 0.3 mgl~(-1) NAA. The multiplication coefficient was up to above 9 after 50 days. Thesuitable medium for GGB differentiation was MS+KT 0.1mgl~(-1)+NAA 0.1mgl~(-1) and thedifferentiation rate was 100%. The average plant leaf of differentiation of was six. Thesuitable medium for plantlets rooting was: MS+sucrose 20gl~(-1)+IBA 0.5mgl~(-1). Thesuitable transplanting substrate for plantlets with roots was the mixture of leaf mold andperlite (the ratio of 2:1) and the survival ratio was 75%.
以紫萁的根状茎尖为外植体诱导GGB的适合培养基是：1／2MS+BA 0.1mg／L+蔗糖30g／L，40天增殖倍数为27.5，GGB诱导率为100％。GGB分化的适合培养基是：1／2MS+蔗糖30g／L，GGB分化率为100％。适合紫萁试管苗生根的培养基是：1／2MS+蔗糖20g／L+NAA 0.1mg／L。将根状茎尖的叶片全部切除后诱导愈伤组织的效果较好，适合诱导愈伤组织的培养基是：1／2MS+2，4-D 3.0mg／L，培养条件为正常的光照培养。愈伤组织在1／2MS+NAA 0.1mg／L的培养基中的分化系数为5.433。生根试管苗炼苗后在腐叶土：珍珠岩=2：1的基质中的移栽成活率为47.5％。
银粉背蕨的孢子用2％NaClO溶液振荡8-10min后过滤，污染率可降低到0，是较适合银粉背蕨孢子的消毒方法。孢子在MS+蔗糖20-40g／L中光照培养，萌发率超过70％。以银粉背蕨原叶体为外植体在MS+BA 1.0-2.0mg／L+NAA 0.3mg／L的培养基中诱导GGB，50天后增殖倍数可达9以上。GGB在MS+KT 0.1mg／L+NAA 0.1mg／L的培养基中的分化率为100％，平均每植株分化的叶片数为6。适合银粉背蕨试管苗生根的培养基是：MS+蔗糖20g／L+IBA 0.5mg／L。生根试管苗炼苗后在腐叶土：珍珠岩=2：1的基质中的移栽成活率为75％。
In vitro culture of Osmunda japonica and Aleuritopteris argentea were studied inthis project. Spores, prothallus and rhizome were used as explants for the culture. TheGGB(green globular bodies) of Aleuritopteris argentea, and the GGB and callus ofOsmunda japonica were successfully induced in the different culture conditions, and thedifferent media with different types of hormone concentrations and the mix of differenttests. In vitro propagation systems of two fems were established, which providedimportant data for the industrialized propagation in macro and exploitation of these ferns.The main results were as follows:
Using rhizome of Osmunda japonica as explant, 1/2MS medium containing 0.1mgl~(-1)BA and sucrose 30gl~(-1) was the appropriate medium for inducing GGB. After 40 daysof culture, the biggest multiplication coefficient was 27.50, and the induction rate of GGBwas 100%. The appropriate medium for GGB differentiation is: 1/2MS with30gl~(-1) sucrose.GGB differentiation rate was 100%. The suitable medium for plantletsrooting was: 1/2MS+sucrose 20gl~(-1)+NAA 0.1mgl~(-1). Rhizomes removed off all leaveswere the best explant for callus induction. The suitable medium for callus induction was:1/2MS+2, 4-D 3.0mgl~(-1). The culture condition of callus induction was the normalillumination(illumination intensity 1200-14001x). The medium of 1/2MS containing0.1mgl~(-1) NAA was optimal for differentiation of callus of Osmunda japonica. Thedifferentiation coefficient was 5.433. The suitable transplanting substrate for plantletswith roots was the mixture of leaf mold and perlite (the ratio of 2:1)and the survival ratiowas 47.5%.
The Aleuritopteris argentea spores were filtrated after oscillating with 2% NaClOsolution for 8-10 minutes, and the contamination rates can be lowered to 0. This was theoptimal disinfection method for Aleuritopteris argentea spores. The suitable cultureconditions for spores germinating were normal illumination(illumination intensity1200-14001x). And the medium was MS together with 20-40gl~(-1) sucrose. The sporesgermination rate was over 70%. Using prothallus as explants, GGB can be induced after50 days of culture. And the optimal medium was MS containing 1.0-2.0mgl~(-1) BA together with 0.3 mgl~(-1) NAA. The multiplication coefficient was up to above 9 after 50 days. Thesuitable medium for GGB differentiation was MS+KT 0.1mgl~(-1)+NAA 0.1mgl~(-1) and thedifferentiation rate was 100%. The average plant leaf of differentiation of was six. Thesuitable medium for plantlets rooting was: MS+sucrose 20gl~(-1)+IBA 0.5mgl~(-1). Thesuitable transplanting substrate for plantlets with roots was the mixture of leaf mold andperlite (the ratio of 2:1) and the survival ratio was 75%.
引文
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