花烛离体培养与快速繁殖技术研究
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摘要
虽然国内外花烛离体培养取得了巨大进展，但尚存在不足：如尚未归纳出完善、实用的离体培养操作程序；生产周期长；对各种生长调节剂的应用看法不一致；生产成本高昂；继代过程中细菌污染的困扰等问题严重制约着花烛离体培养技术的发展。本研究旨在进一步探索花烛离体培养的快捷途径、降低生产成本、减少污染的技术，为建立完善、实用的离体培养操作程序提供依据。
本研究以花烛(Anthurium andraoanum)材料A1为试验对象，研究了无菌培养体系的建立、愈伤组织的增殖培养及不定芽的诱导培养、壮苗生根培养、试管苗移栽、低成本快速繁殖等技术。
研究外植体、基本培养基类型、接种前灭菌处理方法、叶片生理部位、植物生长调节剂、光照条件等因素对愈伤组织诱导的影响，结果表明：叶片的诱导率最高，污染率最低；诱导愈伤组织发生率及发生速度依次为：1／2MS培养基＞MS＞N6＞B5＞white，幼叶＞中龄叶＞老叶，近轴端＞远轴端，不含叶缘叶片＞含叶缘叶片；经HgCl_2灭菌处理的材料污染率较低，生长势最强。愈伤组织在1／2MS+6-BA 1.0mg／L+NAA0.2 mg／L中长势最好；在1／2MS+6-BA 2.0 mg／L+IBA0.1 mg／L中生长旺盛，不定芽健壮，可进一步生根培养；全面光照不利于愈伤组织形成，12小时光照下愈伤组织发生率最高；6mg／L庆大霉素对花烛试管苗细菌处理效果最好。
研究继代次数、基本培养基、生长调节剂等对愈伤组织的增殖培养及不定芽的诱导培养的影响，结果表明：前五次继代培养愈伤组织产生芽的能力较强，不定芽质量较好；6-BA 1.0 mg／L+NAA 0.1mg／L诱导不定芽的分化率较高，生长健壮；MS是该阶段最优培养基。
研究植物生长调节剂、基本培养基、蔗糖、活性炭对于生根培养的影响，结果表明：细胞分裂素和生长素在壮苗过程中缺一不可，6-BA0.3mg／L+NAA0.5mg／L对于壮苗过程最佳；生长素对于生根苗的生长是必需的；1／2MS培养基、2.0％的蔗糖、1／2MS+NAA0.3mg／L生根效果最佳。
研究基质、温度、试管苗质量、季节对试管苗移栽的影响，结果表明：花烛苗在泥炭中生长好，成活率较高；壮苗移栽30d后成活率90％，弱苗只有55％，后来又有陆续死亡现象；3个月后，两种无菌苗株高和最大叶宽差异显著．简单设施条件下，4-9月份炼苗成活率最高，达到86％以上；1-3月、10-12月炼苗成活率较低。
研究充分利用自然光、刻伤处理、不同碳源运用、瓶外生根法以及不定芽增殖与生根同步进行等对生产成本的影响，结果表明：晴天利用自然光，仅阴天照光处理电力消耗比正常情况下低20％；进行刻伤处理的切块产生愈伤组织块数及不定芽分化率明显高于正常切块；采用白砂糖30g、35g愈伤组织诱导、不定芽增殖效果较理想，比使用蔗糖30g节约资金90％左右。对在试管中生根稍差的无菌苗，用2号生根粉5000倍液浸30min处理新根点发生较多，生长势旺盛．
Although the technology of in vitro culture of Anthurium andraeanum progressed greatly, there were many problems in the following: the perfect and practical manipulation procedure of In Vitro culture could not yet be concluded, produce cycle was long, the opinion of using of several types of growth regulator was disaccord, the produce cost was high, was persecuted by bacterium contamination during subculture. The develop of the technology of culture in vitro of Anthurium andraeanum critically restricted by the above problems. The aim of this study was make further explore quick way of culture In Vitro plantlet, and the technology of reducing produce cost and the rate of contamination, supply the building of the perfect and practical manipulation procedure of culture In Vitro plantlet with basis.
The test of this experiment was Anthurium andraeanum material A1, and the technology of establish of sterility culture system, proliferation culture of callus, shoots proliferation, take root culture of strong In Vitro plantlet, transplant of In Vitro plantlet, and rapid propagation of low produce cost were studied.
Effect of explants type, basic medium type, sterilizing method before vaccinate, physiology position of leaves, type of plant growth regulate substance, and Status of light on the inducing of callus were studied. Results indicated that callus inducing rate of leaves was the most highest, the lowest contamination percentage. The inducing engender proportion and engendering rate of callus by the type of medium in proper order were 1/2MS medium>MS medium>N6 medium>B5 medium>White medium, by the type of leaves were spire>medium year leaf>old leaf, adaxial surface>distal surface, leaves of with margin>no margin. The contamination percentage of sterilizing by HgCl_2 was the lowest, and the growth force was the most strong. Callus grew well cultured by 1/2MS. medium+6-BA1.0mg/L+NAA0.2mg/L, and grew vigorously and small bud grew robustly cultured by 1/2MS+6-BA2.0+IBA0.1mg/L, furthermore could culture rooting. Full light was not beneficial to induction of callus, and callus inducing rate was the highest in 12h of light. The effect of 6 mg/L gentamicin on bacterium treatment of test-tube seedling was the best.
The effect of times of continue generation culture, basal medium, growth regulator on the proliferation culture of callus and inducing culture of indefinite bud were studied. Resulted showed that engendering bud was very capable at the top five times of continue generation culture, quality of bud was good. The differentiation percentage of indefinite bud was high inducing by 6-BA 1.0mg/L+NAA 0.1mg/L, and it grew strong. MS medium was the optimal one during this period.
The effect of plant growth regulator, basal medium, sucrose, activated charcoal on rooting culture were studied. Results showed that cytokinin and auxin all were necessary and the result of BA0.3mg/L+NAA0.5mg/L was the best during culturing strong seedling. Growth regulator was necessary to rooted young seedling. Effect of 1/2MS medium, 2.0% sucrose and 1/2MS+NAA0.3mg/L were the best.
Effect of medium, temperature, quality of test-tube shoots, season on the transplant of test-tube shoots were studied. Results indicated that young sprout grew well in peat, its survival rate was high. The transplanting survival rate of the strong sprout was 90% after 30d, but the weak one only was 55%, and died n succession in following time. After 3 months, plant height and leaf biggest width of these two kinds of sprout were different significantly. In Status of simple installation, survival rate was the highest in April to September, was>86%, but were lower in January to March, and October to December
Effect of fully using sunlight, cut treatment, using different kinds of carbon resource, rooting method exvitro, and proliferation of transitory bud synchronize with rooting on produce cost were studied. Results showed that the consume of electric power decrease 20% when using sunlight in sunny day only illuminating in cloudy day than normal. The number of callus and differentiation percentage of transitory bud was higher obviously by cutting treatment than normal. Result of inducing callus, proliferation of transitory bud all were good using by 30g、35g white sand sugar,and reduce expenses as 90% than using by 30g sucrose. When the little weak rooting shoots in test-tube were soaked in 5000 times of No.2 strike root power, new root spot occurred much, grew vigorously.
本研究以花烛(Anthurium andraoanum)材料A1为试验对象，研究了无菌培养体系的建立、愈伤组织的增殖培养及不定芽的诱导培养、壮苗生根培养、试管苗移栽、低成本快速繁殖等技术。
研究外植体、基本培养基类型、接种前灭菌处理方法、叶片生理部位、植物生长调节剂、光照条件等因素对愈伤组织诱导的影响，结果表明：叶片的诱导率最高，污染率最低；诱导愈伤组织发生率及发生速度依次为：1／2MS培养基＞MS＞N6＞B5＞white，幼叶＞中龄叶＞老叶，近轴端＞远轴端，不含叶缘叶片＞含叶缘叶片；经HgCl_2灭菌处理的材料污染率较低，生长势最强。愈伤组织在1／2MS+6-BA 1.0mg／L+NAA0.2 mg／L中长势最好；在1／2MS+6-BA 2.0 mg／L+IBA0.1 mg／L中生长旺盛，不定芽健壮，可进一步生根培养；全面光照不利于愈伤组织形成，12小时光照下愈伤组织发生率最高；6mg／L庆大霉素对花烛试管苗细菌处理效果最好。
研究继代次数、基本培养基、生长调节剂等对愈伤组织的增殖培养及不定芽的诱导培养的影响，结果表明：前五次继代培养愈伤组织产生芽的能力较强，不定芽质量较好；6-BA 1.0 mg／L+NAA 0.1mg／L诱导不定芽的分化率较高，生长健壮；MS是该阶段最优培养基。
研究植物生长调节剂、基本培养基、蔗糖、活性炭对于生根培养的影响，结果表明：细胞分裂素和生长素在壮苗过程中缺一不可，6-BA0.3mg／L+NAA0.5mg／L对于壮苗过程最佳；生长素对于生根苗的生长是必需的；1／2MS培养基、2.0％的蔗糖、1／2MS+NAA0.3mg／L生根效果最佳。
研究基质、温度、试管苗质量、季节对试管苗移栽的影响，结果表明：花烛苗在泥炭中生长好，成活率较高；壮苗移栽30d后成活率90％，弱苗只有55％，后来又有陆续死亡现象；3个月后，两种无菌苗株高和最大叶宽差异显著．简单设施条件下，4-9月份炼苗成活率最高，达到86％以上；1-3月、10-12月炼苗成活率较低。
研究充分利用自然光、刻伤处理、不同碳源运用、瓶外生根法以及不定芽增殖与生根同步进行等对生产成本的影响，结果表明：晴天利用自然光，仅阴天照光处理电力消耗比正常情况下低20％；进行刻伤处理的切块产生愈伤组织块数及不定芽分化率明显高于正常切块；采用白砂糖30g、35g愈伤组织诱导、不定芽增殖效果较理想，比使用蔗糖30g节约资金90％左右。对在试管中生根稍差的无菌苗，用2号生根粉5000倍液浸30min处理新根点发生较多，生长势旺盛．
Although the technology of in vitro culture of Anthurium andraeanum progressed greatly, there were many problems in the following: the perfect and practical manipulation procedure of In Vitro culture could not yet be concluded, produce cycle was long, the opinion of using of several types of growth regulator was disaccord, the produce cost was high, was persecuted by bacterium contamination during subculture. The develop of the technology of culture in vitro of Anthurium andraeanum critically restricted by the above problems. The aim of this study was make further explore quick way of culture In Vitro plantlet, and the technology of reducing produce cost and the rate of contamination, supply the building of the perfect and practical manipulation procedure of culture In Vitro plantlet with basis.
The test of this experiment was Anthurium andraeanum material A1, and the technology of establish of sterility culture system, proliferation culture of callus, shoots proliferation, take root culture of strong In Vitro plantlet, transplant of In Vitro plantlet, and rapid propagation of low produce cost were studied.
Effect of explants type, basic medium type, sterilizing method before vaccinate, physiology position of leaves, type of plant growth regulate substance, and Status of light on the inducing of callus were studied. Results indicated that callus inducing rate of leaves was the most highest, the lowest contamination percentage. The inducing engender proportion and engendering rate of callus by the type of medium in proper order were 1/2MS medium>MS medium>N6 medium>B5 medium>White medium, by the type of leaves were spire>medium year leaf>old leaf, adaxial surface>distal surface, leaves of with margin>no margin. The contamination percentage of sterilizing by HgCl_2 was the lowest, and the growth force was the most strong. Callus grew well cultured by 1/2MS. medium+6-BA1.0mg/L+NAA0.2mg/L, and grew vigorously and small bud grew robustly cultured by 1/2MS+6-BA2.0+IBA0.1mg/L, furthermore could culture rooting. Full light was not beneficial to induction of callus, and callus inducing rate was the highest in 12h of light. The effect of 6 mg/L gentamicin on bacterium treatment of test-tube seedling was the best.
The effect of times of continue generation culture, basal medium, growth regulator on the proliferation culture of callus and inducing culture of indefinite bud were studied. Resulted showed that engendering bud was very capable at the top five times of continue generation culture, quality of bud was good. The differentiation percentage of indefinite bud was high inducing by 6-BA 1.0mg/L+NAA 0.1mg/L, and it grew strong. MS medium was the optimal one during this period.
The effect of plant growth regulator, basal medium, sucrose, activated charcoal on rooting culture were studied. Results showed that cytokinin and auxin all were necessary and the result of BA0.3mg/L+NAA0.5mg/L was the best during culturing strong seedling. Growth regulator was necessary to rooted young seedling. Effect of 1/2MS medium, 2.0% sucrose and 1/2MS+NAA0.3mg/L were the best.
Effect of medium, temperature, quality of test-tube shoots, season on the transplant of test-tube shoots were studied. Results indicated that young sprout grew well in peat, its survival rate was high. The transplanting survival rate of the strong sprout was 90% after 30d, but the weak one only was 55%, and died n succession in following time. After 3 months, plant height and leaf biggest width of these two kinds of sprout were different significantly. In Status of simple installation, survival rate was the highest in April to September, was>86%, but were lower in January to March, and October to December
Effect of fully using sunlight, cut treatment, using different kinds of carbon resource, rooting method exvitro, and proliferation of transitory bud synchronize with rooting on produce cost were studied. Results showed that the consume of electric power decrease 20% when using sunlight in sunny day only illuminating in cloudy day than normal. The number of callus and differentiation percentage of transitory bud was higher obviously by cutting treatment than normal. Result of inducing callus, proliferation of transitory bud all were good using by 30g、35g white sand sugar,and reduce expenses as 90% than using by 30g sucrose. When the little weak rooting shoots in test-tube were soaked in 5000 times of No.2 strike root power, new root spot occurred much, grew vigorously.
引文
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