乐东拟单性木兰、华木莲、红叶石楠‘红罗宾’的组织培养及快繁技术研究
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摘要
本研究以国家二级保护、濒危树种乐东拟单性木兰Parakmeria lotungensis(Chun et C．Tsoong)Law，江西特有的国家一级保护树种、木兰科单种属华木莲Sinomanglietia glauca Z．X．Yu et Q．Y．Zheng及现行珍稀彩叶观赏树种红叶石楠‘红罗宾’Photinia×fraseri 'Red Robin'为试验材料，利用细胞全能性及其发育调控理论，建立三树种的组织培养再生体系。通过细胞学观察及与生根相关的酶活性分析，探讨红叶石楠的组培再生机理。探索生物防腐剂对组培过程中污染的抑制作用。结合设施园艺栽培技术，有针对的研究林木组培规模化育苗技术体系。本研究取得的主要结果：
1、以成年树嫩枝为外植体建立了乐东拟单性木兰的组培再生系统。10月上旬采取嫩枝作外植体，经4℃冰箱低温处理5～7d，有利于降低褐化和污染率，提高诱导率。试验筛选以MS为基本培养基，较好增殖培养基为：MS+6-BA0.2mg／L(单位下同)+IBA0.05+30g／L糖+7g／L卡拉胶；pH值范围为5.5～6.5；光照强度控制在10001x左右；继代周期以4周为宜，继代9次以后可进入快速、稳定增殖阶段，增殖系数可达3.5以上。1.5～2cm长的不定芽放入1／2MS+NAA3+6-BA0.1的根诱导培养基中暗培养15d后，转入不加任何激素的1／2 MS根形成培养基中，12d左右可出根，生根率达15～25％。
首次将物质E应用于组培苗的根诱导。在以上根诱导培养基上添加物质E，暗培养10d后，转入根形成培养基中，5d左右可出根，生根率达77％，生根率提高了50％以上，生根时间缩短10d左右，表现出对乐东拟单性木兰生根很大的促进作用。
2、初步建立了华木莲组培再生系统。3月份采取即将萌动的休眠芽和10月上旬采集生长旺盛的嫩枝，有利于不定芽的诱导。接种后材料需15d接转一次。试验筛选以M_木为基本培养基，较好增殖培养基为：M_木+6-BA0.5+IBA0.05+30g／L糖+7g／L卡拉胶；pH值调至5.8；光照强度控制在10001x左右；继代周期以10d为宜。分化出的不定芽放入1／2MS+NAA3+6-BA0.1(去卡拉胶，加珍珠岩或蛭石)的根诱导培养基中，暗培养10-12d后，转入不加任何激素的1／2 MS根形成培养基中，12-15d左右可出根。
3、建立了一套经济、高效的红叶石楠组培规模化育苗技术体系，繁育组培商品苗150万株。
以红叶石楠当年生半木质化嫩枝为材料，对红叶石楠进瓶诱导、增殖培养、生根培养、瓶苗移栽的主要影响因子进行了系统的研究。较佳的诱导培养基为：MS+KT1.0+NAA0.2+30g／L糖+7g／L卡拉胶；较好的增殖培养基为：MS+6-BA2.0+KT0-5+IBA0.2+3g／L白糖+7g／L卡拉胶，最高增殖系数可达9.3；适宜的光照为：3-5d的暗培养与1000-1500 1X＼12h／d光照培养相结合，平均每瓶增殖苗中有效芽可达32棵。M_木基本培养基、生长素NAA有利于红叶石楠根的诱导，在M_木+NAA0.3的生根培养基上，20d以后生根率达66％。
将物质E应用于红叶石楠组培苗的根诱导，表现出对红叶石楠生根很大的促
进作用。在M木+NAA 0.3的生根培养基上，添加E物质69/L后，红叶石楠出
根整齐而快、10天内生根率达98.5%，比对照提高了30多个百分点，完成生根
时间缩短10一15d。通过细胞解剖学观察及与生根有关的酶(POD、IAAO、PPO)
活性的测定，在添加E物质后，红叶石楠生根苗的三种酶活性表现为利于生根的
动态变化，而在未添加E物质上的酶活性表现为不利于生根的动态变化。
4、首次将热稳定性强的生物防腐剂702发酵液应用于组培过程中污染的控
制。抑菌试验结果表明对组培过程中常见的14种细菌、真菌、酵母菌的抑菌率
达100%。而对瓶苗的生长尚未发现不良影响。
5、较系统地研究了红叶石楠组培苗移栽管理技术，移栽成活率可达90%。
关键词:乐东拟单性木兰，华木莲，红叶石楠‘红罗宾，组织培养，快繁
The secondary class national preserved and endangered plant Parakmeria lotungensis (Chun et C.Tsoong)Law, The first class national preserval plant Sinomanglietia glauca Z.X.Yu et Q.Y.Zheng endemic in Jiangxi province, and the up-to-date and rare color-leaf plant Photinia fraseri 'Red Robin' were respectively studied to establish tissue culture and rapid propagation system based on plant cell totipotency and its regulation and control theory. Meanwhile, the regeneration mechanism of tissue culture of P. fraseri was discussed by means of anatomy observation and enzyme activity measuration related with rooting. The bio-antisepsis was used in controlling the contamination in plant tissue culture. In addition, by combining with techniques of horticulture, the systematized techniques of rapid micropropagation of woody plant via tissue culture were specially investigated. The results were listed as below:The regeneration system of tissue culture of P. lotungensis was first established by utilizing its twigs from the mature tree as explants. In the first ten days of October, the eugonic twigs were excised from mature tree, and pretreated them with low temperature (4℃) for 5-7d in order to decrease brownness and contamination, and increase survival rate of induced propagation. MS was more effective than the other as basic medium in the whole experiment. The best medium of proliferation was MS+6-BA0.2mg/l + IBA0.05mg/l+30g/l Sucrose +7g/l Gelatin, pH5.5-6.5, illumination intensity 1000 Lx, subculture interval was 4 weeks. After subculture for 8 times, the proliferation was fast and stable, and multiplication rate was 3.5. The buds 1.5-2cm long were planted into root-induced medium (1/2MS+NAA3.0 mg/L +BA0.1 mg/L), after 15d under the darkness, and by transferred into rooting medium (1/2MS) without any hormone, rooting was started at about 12days, and the rooting rate was 15-25%.The matter E was first used in tissue culture root induction. The additive matter E makes a full impact on adventitious root formation of P. lotungensis. The rooting rate has reached 77% , and much more 50% have been increased than comparison,The regeneration system of tissue culture of S. glauca was established by utilizing its twigs and dormancy bud as explants. the eugonic twigs excised in the first ten days of October, and dormancy excised in March were fit for induction of bud. M * was more effective than the other as basic medium. The best proliferation medium was MS+6-BA0.5mg/L+IBA0.05mg/L+30g/lSucrose+7g/lGelatin, pHS.8, illumination intensity lOOOLx, subculture interval was 10 days, The buds 1.5-2cm long were planted into root-induced medium 1/2MS+NAA3.0 mg/L +BA0.1 mg/L without Gelatin but with perlite or vermiculite , afterlO-12d under the darkness, and
by transferred into root-formation medium (l/2MS)without any hormone, rooting was started at about 12-15days.This study established an economic and hight effective technology system on rapid propagation of P. fraseri 'Red Robin ' .By comparision of the mainly factors on induction culture proliferation culture rooting culture and transplant culture , the optimal inducing medium was MS+KT1.0mg/l+NAA0.2mg/l; the optimal multiplication medium was MS+6-BA2.0 mg/l+KT0.5mg/l+IBA0.2mg/l+30g/lsugar.+7g/lGelatin, feasible illumination intensity was 3-5d dark culture combined with 1000-15001x light culture 12h/days, the highest multiplication rate was 9.3. On average, there were 32 shoots white can be used for rooting in one bottle.M medium and NAA was effective on rooting of P. fraseri, and the rooting rate was 66% after 20days by incubation on medium M *+ NAA0.3mg/L.The additive matter E makes a full impact on adventitious root formation of P. fraseri. The rooting rate has reached 98.5% and much more 30% have been increased than comparison, and at the same time ,root grow fast and order; It farther demonstrates in theory that appending substance E or not has an straight effect on the rooting rate and time of root emergence through the examinations of anatomy observation and enzyme activity measuratio
1、以成年树嫩枝为外植体建立了乐东拟单性木兰的组培再生系统。10月上旬采取嫩枝作外植体，经4℃冰箱低温处理5～7d，有利于降低褐化和污染率，提高诱导率。试验筛选以MS为基本培养基，较好增殖培养基为：MS+6-BA0.2mg／L(单位下同)+IBA0.05+30g／L糖+7g／L卡拉胶；pH值范围为5.5～6.5；光照强度控制在10001x左右；继代周期以4周为宜，继代9次以后可进入快速、稳定增殖阶段，增殖系数可达3.5以上。1.5～2cm长的不定芽放入1／2MS+NAA3+6-BA0.1的根诱导培养基中暗培养15d后，转入不加任何激素的1／2 MS根形成培养基中，12d左右可出根，生根率达15～25％。
首次将物质E应用于组培苗的根诱导。在以上根诱导培养基上添加物质E，暗培养10d后，转入根形成培养基中，5d左右可出根，生根率达77％，生根率提高了50％以上，生根时间缩短10d左右，表现出对乐东拟单性木兰生根很大的促进作用。
2、初步建立了华木莲组培再生系统。3月份采取即将萌动的休眠芽和10月上旬采集生长旺盛的嫩枝，有利于不定芽的诱导。接种后材料需15d接转一次。试验筛选以M_木为基本培养基，较好增殖培养基为：M_木+6-BA0.5+IBA0.05+30g／L糖+7g／L卡拉胶；pH值调至5.8；光照强度控制在10001x左右；继代周期以10d为宜。分化出的不定芽放入1／2MS+NAA3+6-BA0.1(去卡拉胶，加珍珠岩或蛭石)的根诱导培养基中，暗培养10-12d后，转入不加任何激素的1／2 MS根形成培养基中，12-15d左右可出根。
3、建立了一套经济、高效的红叶石楠组培规模化育苗技术体系，繁育组培商品苗150万株。
以红叶石楠当年生半木质化嫩枝为材料，对红叶石楠进瓶诱导、增殖培养、生根培养、瓶苗移栽的主要影响因子进行了系统的研究。较佳的诱导培养基为：MS+KT1.0+NAA0.2+30g／L糖+7g／L卡拉胶；较好的增殖培养基为：MS+6-BA2.0+KT0-5+IBA0.2+3g／L白糖+7g／L卡拉胶，最高增殖系数可达9.3；适宜的光照为：3-5d的暗培养与1000-1500 1X＼12h／d光照培养相结合，平均每瓶增殖苗中有效芽可达32棵。M_木基本培养基、生长素NAA有利于红叶石楠根的诱导，在M_木+NAA0.3的生根培养基上，20d以后生根率达66％。
将物质E应用于红叶石楠组培苗的根诱导，表现出对红叶石楠生根很大的促
进作用。在M木+NAA 0.3的生根培养基上，添加E物质69/L后，红叶石楠出
根整齐而快、10天内生根率达98.5%，比对照提高了30多个百分点，完成生根
时间缩短10一15d。通过细胞解剖学观察及与生根有关的酶(POD、IAAO、PPO)
活性的测定，在添加E物质后，红叶石楠生根苗的三种酶活性表现为利于生根的
动态变化，而在未添加E物质上的酶活性表现为不利于生根的动态变化。
4、首次将热稳定性强的生物防腐剂702发酵液应用于组培过程中污染的控
制。抑菌试验结果表明对组培过程中常见的14种细菌、真菌、酵母菌的抑菌率
达100%。而对瓶苗的生长尚未发现不良影响。
5、较系统地研究了红叶石楠组培苗移栽管理技术，移栽成活率可达90%。
关键词:乐东拟单性木兰，华木莲，红叶石楠‘红罗宾，组织培养，快繁
The secondary class national preserved and endangered plant Parakmeria lotungensis (Chun et C.Tsoong)Law, The first class national preserval plant Sinomanglietia glauca Z.X.Yu et Q.Y.Zheng endemic in Jiangxi province, and the up-to-date and rare color-leaf plant Photinia fraseri 'Red Robin' were respectively studied to establish tissue culture and rapid propagation system based on plant cell totipotency and its regulation and control theory. Meanwhile, the regeneration mechanism of tissue culture of P. fraseri was discussed by means of anatomy observation and enzyme activity measuration related with rooting. The bio-antisepsis was used in controlling the contamination in plant tissue culture. In addition, by combining with techniques of horticulture, the systematized techniques of rapid micropropagation of woody plant via tissue culture were specially investigated. The results were listed as below:The regeneration system of tissue culture of P. lotungensis was first established by utilizing its twigs from the mature tree as explants. In the first ten days of October, the eugonic twigs were excised from mature tree, and pretreated them with low temperature (4℃) for 5-7d in order to decrease brownness and contamination, and increase survival rate of induced propagation. MS was more effective than the other as basic medium in the whole experiment. The best medium of proliferation was MS+6-BA0.2mg/l + IBA0.05mg/l+30g/l Sucrose +7g/l Gelatin, pH5.5-6.5, illumination intensity 1000 Lx, subculture interval was 4 weeks. After subculture for 8 times, the proliferation was fast and stable, and multiplication rate was 3.5. The buds 1.5-2cm long were planted into root-induced medium (1/2MS+NAA3.0 mg/L +BA0.1 mg/L), after 15d under the darkness, and by transferred into rooting medium (1/2MS) without any hormone, rooting was started at about 12days, and the rooting rate was 15-25%.The matter E was first used in tissue culture root induction. The additive matter E makes a full impact on adventitious root formation of P. lotungensis. The rooting rate has reached 77% , and much more 50% have been increased than comparison,The regeneration system of tissue culture of S. glauca was established by utilizing its twigs and dormancy bud as explants. the eugonic twigs excised in the first ten days of October, and dormancy excised in March were fit for induction of bud. M * was more effective than the other as basic medium. The best proliferation medium was MS+6-BA0.5mg/L+IBA0.05mg/L+30g/lSucrose+7g/lGelatin, pHS.8, illumination intensity lOOOLx, subculture interval was 10 days, The buds 1.5-2cm long were planted into root-induced medium 1/2MS+NAA3.0 mg/L +BA0.1 mg/L without Gelatin but with perlite or vermiculite , afterlO-12d under the darkness, and
by transferred into root-formation medium (l/2MS)without any hormone, rooting was started at about 12-15days.This study established an economic and hight effective technology system on rapid propagation of P. fraseri 'Red Robin ' .By comparision of the mainly factors on induction culture proliferation culture rooting culture and transplant culture , the optimal inducing medium was MS+KT1.0mg/l+NAA0.2mg/l; the optimal multiplication medium was MS+6-BA2.0 mg/l+KT0.5mg/l+IBA0.2mg/l+30g/lsugar.+7g/lGelatin, feasible illumination intensity was 3-5d dark culture combined with 1000-15001x light culture 12h/days, the highest multiplication rate was 9.3. On average, there were 32 shoots white can be used for rooting in one bottle.M medium and NAA was effective on rooting of P. fraseri, and the rooting rate was 66% after 20days by incubation on medium M *+ NAA0.3mg/L.The additive matter E makes a full impact on adventitious root formation of P. fraseri. The rooting rate has reached 98.5% and much more 30% have been increased than comparison, and at the same time ,root grow fast and order; It farther demonstrates in theory that appending substance E or not has an straight effect on the rooting rate and time of root emergence through the examinations of anatomy observation and enzyme activity measuratio
引文
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