三倍体毛白杨优良无性系微体快速繁殖技术研究
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摘要
本文以三倍体毛白杨新无性系BT17、BT18、BL175、BL193为试材，对其微繁技术进行了系统全面的研究。探讨了从无菌培养物建立、初代培养、继代培养、试管苗生根移栽直到定植栽培的整个过程，弄清了微繁的关键因素、存在问题及其解决方法，为苗木工厂化生产奠定了理论基础。提出了优化快速繁殖程序，为工厂化生产提供了行之有效的技术措施。
研究表明，不同无性系外植体处理最佳的消毒处理组合不一。BT17用酒精处理12秒、升汞处理9分钟效果为最好；BT18用酒精处理12秒、升汞处理5分钟成活率可达82%；BL175、BL193最佳的处理时间分别是20秒、7分钟，5秒、9分钟。
研究表明，在芽增殖及嫩梢生长过程中，除各系号遗传特性差异外，繁殖系数及嫩梢长度主要受营养水平和激素水平的影响。试验证明，MS培养基是嫩梢增殖生长的最佳基本培养基，细胞分裂素选用6-BA最好，生长素以NAA为最好。一定范围内，6-BA决定了芽与嫩梢的增殖数目，NAA决定了嫩梢的生长状况。不仅如此，两者的比例也对芽的增殖与嫩梢的生长有着显著的影响。试验条件下各无性系嫩梢增殖的最适培养基分别为：BT17：MS+6-BA0.3mg/L+NAA0.01mg/L，BT18：MS+6-BA0.6mg/L+NAA0.01mg/L，BL175：MS+6-BA0.3mg/L+NAA0.01mg/L，BL193：MS+6-BA0.9mg/L+NAA0.05mg/L；嫩梢生长最适宜的培养基为：BT17：MS+6-BA0.9mg/L+NAA0.5mg/L，BT18：MS+6-BA0.6mg/L+NAA0.5mg/L，BL175：MS+6-BA0.3mg/L+NAA0.01mg/L，BL193：MS+6-BA0.9mg/L+NAA0.05mg/L。
内源激素研究发现，ZRs与IAA含量高，芽的增殖系数大；GA1/3与IAA含量高且ABA含量低时，芽的嫩梢长度长。玻璃化苗与正常苗从形态及繁殖系数上都明显区别于正常苗，内源激素测定结果显示ZRS和IAA的含量明显少于正常苗，GA1/3和ABA含量高于正常苗。
试管苗玻璃化制约三倍体毛白杨微体快繁效果。玻璃化试管苗既不适
于继续继代培养，同时嫩茎也难以生根。从激素种类与配比、琼脂含量等诸方面研究表明：随着大量元素浓度的增大，试管苗玻璃化率增加；琼脂含量、光照强度与玻璃化率呈显著的负相关；KT和BA相比BA更易产生玻璃化苗。KT或BA与NAA配合使用比单独使用KT或者BA玻璃化苗比率高。封口材料中聚乙烯膜和塑料瓶盖两个处理玻璃化率相似且玻璃化率高，硫酸纸次之，棉塞的最低。
试管苗成本高是限制三倍体毛白杨工厂化生产的重要因子。简化培养基是降低成本的关键。研究表明：自来水代替蒸馏水、白砂糖代替蔗糖、玻璃瓶代替三角瓶基本上能满足三倍体毛白杨试管苗芽的增殖与嫩梢生长的需要，从而降低试管苗的成本。
试管内根的诱导，以1/2MS为基本培养基配合IBA与NAA效果好。供试无性系中BT17、BL175、BL193试管苗根诱导最适培养基配方相同以1/2MS+IBA0.1mg/L+NAA0.1mg/L最好；BT18以1/2MS + IBA0.5mg/L + NAA0.05mg/L为好。试验还研究了试管外发根技术，表明：嫩茎在浓度为50mg/L NAA中浸泡20分钟扦插到营养钵中，试管外生根成活率可达93.5%，且生长状况最好。
三倍体毛白杨试管苗最佳移栽时间为春季3-4月份。相对于菜园土、蛭石和沙子（1：1）、沙子、草炭与珍珠岩（1：1）而言，蛭石是最佳移栽基质。
建立在一系列推理与理论的基础上，提出了年产苗量计算公式及预测模型，据公式推算，每芽年产苗量可高达8.8万多株，商品苗产量可达4万多株。
The rapid micropropagation technique of triploid clones of populus tomentosa (BT17,BT18,BL175,BL193)were comprehensively studied. The tissue and organs from white popular can be rapid micropropagated successfully. The factors affecting propagation, problems existing in this technique and their solutions were also described. The optimal procedure for rapid micropropagation was worked out, which provided the theoretical base and technical measures for industrial production of plantlet.
Result indicated that the best time combinations of sterilizing agents are different for each clone. The optimal sterilization condition were as following: BT17:70%alcohol12s+0.1%HgCl2 9min; BT18: 70%alcohol12s +0.1%HgCl2 25min;BL175: 70%alcohol20s+0.1%HgCl2 7min; BL173: 70%alcohol5s+0.1%HgCl2 9min.
The main factors affecting bud multiplication and shoot growth are nutrients and plant growth regulators besides their genetic potential. MS was found to be the most favorable to bud multiplication and shoot growth. Within certain concentration range, the number of buds was determined by BA, and the growth of shoots was determined by NAA. Bud multiplication and shoot growth were controlled by not only the absolute concentration, but also the relatively ratio of BA/NAA. The optimal mediums for bud multiplication of each clone were: BT17:MS+BA0.3mg/L+NAA0.01mg/L; BT18:MS+BA0.1mg/L+ NAA0.01mg/L;BL175:MS+BA0.3mg/L+NAA0.01mg/L;BL193:MS+BA0.9mg/L+NAA0.05mg/L; The most favorable media for shoot growth were: BT17:MS+BA0.9mg/L +NAA0.5mg/L;BT18:MS+BA0.6mg/L+NAA0.5mg/L;BL175:MS+BA0.3mg/L+NAA0.01mg/L; BL193: MS+BA0.9mg/L+NAA0.05mg/L.
Study on endohormones indicated that propagation coefficient was higher
when the content of ZRs and IAA was more. The shoot length was longer when the content of GA1/3 and IAA was higher and ABA was lower. Result showed that the content of ZRs and IAA in the vitrified plantlet was lower than that in the normal plantlet, while the content of GA1/3 and ABA was higher.
The industrial and commercial micropropagation of white popular plantlet was restricted by vitrification. It was difficult for vitrified plantlet to root or to be subcultured. Study indicated that the higher the concentration of macroelement was, the more the rate of vitrified plantlets was. The correlation coefficient between the agar concentration and the vitrification rate was reverse. And the relation between intensity of light and vitrification rate was also reverse. Compared with KT, vitrification percentage caused by BA was higher. Study also found that vitrification rate was higher when combining BA with KT than that when using them along. The vitrification rate caused by cotton plug wrapped in cheese cloth was the lowest among all seal materials, including polyethylene film, plastic cover and sulfuric paper.
The high cost was unfavorable for the industrial commercial production of plantlet. Study showed that the cost would decrease if we substituted tap water for the distilled water, sugar for sucrose, and glass jar for Erlenmeyer flask. And it wouldn't affect the bud multiplication and shoot growth.
1/2MS was the best rooting medium .For BT18, the optimal rooting medium was 1/2MS+IBA0.1mg/L+NAA0.1mg/L, and other clones were all 1/2MS+ IBA0.5mg/L+NAA0.05mg/L. The ex vitro rooting technique of shoot was also studied. The percentage of rooting shoots could reach 93.5 percent if the shoots were soaked in50mg/L NAA solution for 20 minute.
The appropriate period for plantlet transplanting is from March to April in spring. Compared with other matrixes, such as soil, vermiculite VS sand (1:1), peat VS pearlite (1:1), vermiculite was the best. Meanwhile, shade and intermittent mist are also necessary.
Based on a series of scientific theories and assumptions, the ratio of
multiplication was estimated and the formula for calculating plantlets yield was figured out. The result of calculation by formula indicated that 88 thousand plantlets and 40 thousand marketable plantlets could be produced yearly for
研究表明，不同无性系外植体处理最佳的消毒处理组合不一。BT17用酒精处理12秒、升汞处理9分钟效果为最好；BT18用酒精处理12秒、升汞处理5分钟成活率可达82%；BL175、BL193最佳的处理时间分别是20秒、7分钟，5秒、9分钟。
研究表明，在芽增殖及嫩梢生长过程中，除各系号遗传特性差异外，繁殖系数及嫩梢长度主要受营养水平和激素水平的影响。试验证明，MS培养基是嫩梢增殖生长的最佳基本培养基，细胞分裂素选用6-BA最好，生长素以NAA为最好。一定范围内，6-BA决定了芽与嫩梢的增殖数目，NAA决定了嫩梢的生长状况。不仅如此，两者的比例也对芽的增殖与嫩梢的生长有着显著的影响。试验条件下各无性系嫩梢增殖的最适培养基分别为：BT17：MS+6-BA0.3mg/L+NAA0.01mg/L，BT18：MS+6-BA0.6mg/L+NAA0.01mg/L，BL175：MS+6-BA0.3mg/L+NAA0.01mg/L，BL193：MS+6-BA0.9mg/L+NAA0.05mg/L；嫩梢生长最适宜的培养基为：BT17：MS+6-BA0.9mg/L+NAA0.5mg/L，BT18：MS+6-BA0.6mg/L+NAA0.5mg/L，BL175：MS+6-BA0.3mg/L+NAA0.01mg/L，BL193：MS+6-BA0.9mg/L+NAA0.05mg/L。
内源激素研究发现，ZRs与IAA含量高，芽的增殖系数大；GA1/3与IAA含量高且ABA含量低时，芽的嫩梢长度长。玻璃化苗与正常苗从形态及繁殖系数上都明显区别于正常苗，内源激素测定结果显示ZRS和IAA的含量明显少于正常苗，GA1/3和ABA含量高于正常苗。
试管苗玻璃化制约三倍体毛白杨微体快繁效果。玻璃化试管苗既不适
于继续继代培养，同时嫩茎也难以生根。从激素种类与配比、琼脂含量等诸方面研究表明：随着大量元素浓度的增大，试管苗玻璃化率增加；琼脂含量、光照强度与玻璃化率呈显著的负相关；KT和BA相比BA更易产生玻璃化苗。KT或BA与NAA配合使用比单独使用KT或者BA玻璃化苗比率高。封口材料中聚乙烯膜和塑料瓶盖两个处理玻璃化率相似且玻璃化率高，硫酸纸次之，棉塞的最低。
试管苗成本高是限制三倍体毛白杨工厂化生产的重要因子。简化培养基是降低成本的关键。研究表明：自来水代替蒸馏水、白砂糖代替蔗糖、玻璃瓶代替三角瓶基本上能满足三倍体毛白杨试管苗芽的增殖与嫩梢生长的需要，从而降低试管苗的成本。
试管内根的诱导，以1/2MS为基本培养基配合IBA与NAA效果好。供试无性系中BT17、BL175、BL193试管苗根诱导最适培养基配方相同以1/2MS+IBA0.1mg/L+NAA0.1mg/L最好；BT18以1/2MS + IBA0.5mg/L + NAA0.05mg/L为好。试验还研究了试管外发根技术，表明：嫩茎在浓度为50mg/L NAA中浸泡20分钟扦插到营养钵中，试管外生根成活率可达93.5%，且生长状况最好。
三倍体毛白杨试管苗最佳移栽时间为春季3-4月份。相对于菜园土、蛭石和沙子（1：1）、沙子、草炭与珍珠岩（1：1）而言，蛭石是最佳移栽基质。
建立在一系列推理与理论的基础上，提出了年产苗量计算公式及预测模型，据公式推算，每芽年产苗量可高达8.8万多株，商品苗产量可达4万多株。
The rapid micropropagation technique of triploid clones of populus tomentosa (BT17,BT18,BL175,BL193)were comprehensively studied. The tissue and organs from white popular can be rapid micropropagated successfully. The factors affecting propagation, problems existing in this technique and their solutions were also described. The optimal procedure for rapid micropropagation was worked out, which provided the theoretical base and technical measures for industrial production of plantlet.
Result indicated that the best time combinations of sterilizing agents are different for each clone. The optimal sterilization condition were as following: BT17:70%alcohol12s+0.1%HgCl2 9min; BT18: 70%alcohol12s +0.1%HgCl2 25min;BL175: 70%alcohol20s+0.1%HgCl2 7min; BL173: 70%alcohol5s+0.1%HgCl2 9min.
The main factors affecting bud multiplication and shoot growth are nutrients and plant growth regulators besides their genetic potential. MS was found to be the most favorable to bud multiplication and shoot growth. Within certain concentration range, the number of buds was determined by BA, and the growth of shoots was determined by NAA. Bud multiplication and shoot growth were controlled by not only the absolute concentration, but also the relatively ratio of BA/NAA. The optimal mediums for bud multiplication of each clone were: BT17:MS+BA0.3mg/L+NAA0.01mg/L; BT18:MS+BA0.1mg/L+ NAA0.01mg/L;BL175:MS+BA0.3mg/L+NAA0.01mg/L;BL193:MS+BA0.9mg/L+NAA0.05mg/L; The most favorable media for shoot growth were: BT17:MS+BA0.9mg/L +NAA0.5mg/L;BT18:MS+BA0.6mg/L+NAA0.5mg/L;BL175:MS+BA0.3mg/L+NAA0.01mg/L; BL193: MS+BA0.9mg/L+NAA0.05mg/L.
Study on endohormones indicated that propagation coefficient was higher
when the content of ZRs and IAA was more. The shoot length was longer when the content of GA1/3 and IAA was higher and ABA was lower. Result showed that the content of ZRs and IAA in the vitrified plantlet was lower than that in the normal plantlet, while the content of GA1/3 and ABA was higher.
The industrial and commercial micropropagation of white popular plantlet was restricted by vitrification. It was difficult for vitrified plantlet to root or to be subcultured. Study indicated that the higher the concentration of macroelement was, the more the rate of vitrified plantlets was. The correlation coefficient between the agar concentration and the vitrification rate was reverse. And the relation between intensity of light and vitrification rate was also reverse. Compared with KT, vitrification percentage caused by BA was higher. Study also found that vitrification rate was higher when combining BA with KT than that when using them along. The vitrification rate caused by cotton plug wrapped in cheese cloth was the lowest among all seal materials, including polyethylene film, plastic cover and sulfuric paper.
The high cost was unfavorable for the industrial commercial production of plantlet. Study showed that the cost would decrease if we substituted tap water for the distilled water, sugar for sucrose, and glass jar for Erlenmeyer flask. And it wouldn't affect the bud multiplication and shoot growth.
1/2MS was the best rooting medium .For BT18, the optimal rooting medium was 1/2MS+IBA0.1mg/L+NAA0.1mg/L, and other clones were all 1/2MS+ IBA0.5mg/L+NAA0.05mg/L. The ex vitro rooting technique of shoot was also studied. The percentage of rooting shoots could reach 93.5 percent if the shoots were soaked in50mg/L NAA solution for 20 minute.
The appropriate period for plantlet transplanting is from March to April in spring. Compared with other matrixes, such as soil, vermiculite VS sand (1:1), peat VS pearlite (1:1), vermiculite was the best. Meanwhile, shade and intermittent mist are also necessary.
Based on a series of scientific theories and assumptions, the ratio of
multiplication was estimated and the formula for calculating plantlets yield was figured out. The result of calculation by formula indicated that 88 thousand plantlets and 40 thousand marketable plantlets could be produced yearly for
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