组织培养中秋水仙素诱导葡萄多倍体研究
摘要
葡萄是我国重要的果树作物。人工诱导多倍体是培育大粒、无核葡萄新品种的重要途径。本试验结合葡萄组织培养技术，在葡萄体细胞愈伤组织和试管苗两个水平上采用不同的方法对诱导葡萄多倍体进行了研究。
愈伤组织水平上诱导葡萄多倍体试验结果表明：以茎段和叶片为外植体，在附加不同浓度2，4-D、6-BA及秋水仙素的MS、B_5培养基上，均可诱导出愈伤组织；愈伤组织的形态特征与外植体的类型、秋水仙素的有无和培养基有关。
对颗粒状愈伤组织进行染色体鉴定结果表明：在加有和不加秋水仙素的培养基上诱导的颗粒状愈伤组织均存在广泛的染色体变异，除正常染色体数为2n=38和2n=4x=76细胞外，还有一些非整倍体细胞。其变异的情况因品种、培养基的不同有所差异。在加有20mg／L秋水仙素并附加1.0mg／L 2，4-D和0.5mg／L 6-BA的B_5培养基上可获得加倍率高的愈伤组织；红提较京秀、黑大粒容易获得加倍率高的愈伤组织。
诱导的愈伤组织在MS、B_5培养基上均未分化再生成苗，可能与愈伤组织的染色体变异有关，染色体变异越大，分化频率越低；或与诱导分化成苗的培养基、培养条件等因素有关。
试管苗水平上，采用羊毛脂涂抹腋芽、单芽茎段浸泡、脱脂棉覆盖腋芽和秋水仙素加入培养基4种方法进行诱导葡萄多倍体研究，结果表明：除第一种方法外，其余三种均具有诱导加倍效果，诱导处理后的成苗率、诱导率、诱变株的加倍率存在明显差异。
茎段浸泡法成苗率15.7％，诱导率4.1％，诱变株系加倍率31.8％；脱脂棉覆盖法成苗率54.2％，诱导率5.9％，加倍率30.3％：秋水仙素加入培养基中诱导处理，成苗率19.0％，诱导率9.0％，加倍率53.0％，其变动幅度30.0％-66.1％。从成苗率看，秋培法不及浸泡和覆盖法，但从最终的诱导率和诱变株系的加倍率看，秋水仙素加入培养基诱导较为理想，其中以20mg／L秋水仙素加入附加有0.01mg／L 6-BA的MS培养基中包埋腋芽诱导处理两周，诱导加倍效果较好。
不同的品种对秋水仙素的敏感反应不一，诱导加倍效果不同，京秀、黑大粒未获得加倍率超过50％的诱变株系，以红提为亲本获得加倍率分别为66.1％、64.6％的诱变株系H0120-1和H0120-3。两株系经连续3代的继代培养，经根尖和茎尖倍性鉴定，倍性结构基本稳定；与二倍体红提进行形态特征比较，具有多倍体的部分特征特性。气孔鉴定结果表明：诱变株与二倍体红提气孔平均大小无明显差异，但诱变株存在20.8％的特大气孔。H0120-3移栽大田，正进一步鉴定筛选。
Grapevine is a major traditional fruit tree in the world. It is a significant way to induce tetraploid to breed new grapevine varieties of high production and top quality. We researched on tetraploid grapevine induction by colchicine treatment through tissue culture.
The results of induced calluses had been obtained as follows: the frequency of callus induction was much high in MS or B5 with different concentration of 2,4-D, 6-BA and colchicine; the appearance and characteristics of induced calluses were closely related to type of explants, media and colchicine.
Observed the chromosome of induced pellet differentiated competence calluses, it was found that the wide-ranging chromosome variation existed in pellet calluses induced by different media with colchicine or absence of ones. Except for normal 2n=38 diploid cells and 2n=4x=76 tetraploid cells, aneuploid cells was found in calluses. The difference of chromosome variation was related to different grapevine varieties and media. The higher 4x cells rate calluses had been obtained in B5 medium with colchicine 20mg/l, 2,4-D 1.0mg/L and 6-BA 0.5mg/L. Red globe was easier than Jingxiu and Exotic in inducing double chromosome calluses . Regenerated plants had not been obtained through inducing calluses in MS and B5 media. It was maybe related to the chromosome variation of calluses. The higher the chromosome frequency variated , the lower the callus differention frequency was.
The tests of tetraploid induction in micro propagated buds was done through daubing axillary buds with grease colchicine, immersing ones in colchicine solution, covering ones with colchicine absorbent cotton, and inducing ones on media with colchicine. The results showed that the last three methods was more effective in inducing tetraploid than the first one. But the seedling rate, mutation rate, induction frequency of treated materials was obviously different.
As far as plants induced by immersing axillary buds were concerned, seedling rate was up to 15,1%, aberrant buds rate 4.1%, 4x cells rate 31.8%; seedling rate 54.2%, aberrant buds rate 5.9%, 4x cells rate 30.3% for ones induced by covering buds; seedling rate is 19.0%, aberrant buds rate 9.0%, 4x cells rate 53.3%, and its rang of change 30.3%-66.1% for ones induced by media with colchicine.
Induction effect through media with colchicine was inferior to that through covering axillary buds in seedling rate, but it was better in induction frequency and 4x cells rate of induced plant. To obtaining higher 4x cell rate of induced plant, the optimal way was to induce axillary buds on MS with colchicine 20mg/L and 6-BA 0.01mg/L for 2 weeks. The effect of induction was related to different varieties. The sports whose 4x cells rate were up to 50% had not been obtained by inducing Jingxiu and Exotic. The two induced plant H120-1 and H120-3 whose 4x cells rate was 66.1%, 64.6% respectively had been obtained by inducing diploid Red globe through media with colchicine.
The results of observing chromosomes variation in 3 progenies showed that the ploidy of H120-3 was stable. Compared with maternal plant, the lines exhibited typical polyploidy characteristics: leaf largen, stalk bulkyen, top and axillary bud fullen plump, growth strongen, and its propagation multiple obviously increased. The elite lines were being appraised for further selection through cutting axillary buds.
愈伤组织水平上诱导葡萄多倍体试验结果表明：以茎段和叶片为外植体，在附加不同浓度2，4-D、6-BA及秋水仙素的MS、B_5培养基上，均可诱导出愈伤组织；愈伤组织的形态特征与外植体的类型、秋水仙素的有无和培养基有关。
对颗粒状愈伤组织进行染色体鉴定结果表明：在加有和不加秋水仙素的培养基上诱导的颗粒状愈伤组织均存在广泛的染色体变异，除正常染色体数为2n=38和2n=4x=76细胞外，还有一些非整倍体细胞。其变异的情况因品种、培养基的不同有所差异。在加有20mg／L秋水仙素并附加1.0mg／L 2，4-D和0.5mg／L 6-BA的B_5培养基上可获得加倍率高的愈伤组织；红提较京秀、黑大粒容易获得加倍率高的愈伤组织。
诱导的愈伤组织在MS、B_5培养基上均未分化再生成苗，可能与愈伤组织的染色体变异有关，染色体变异越大，分化频率越低；或与诱导分化成苗的培养基、培养条件等因素有关。
试管苗水平上，采用羊毛脂涂抹腋芽、单芽茎段浸泡、脱脂棉覆盖腋芽和秋水仙素加入培养基4种方法进行诱导葡萄多倍体研究，结果表明：除第一种方法外，其余三种均具有诱导加倍效果，诱导处理后的成苗率、诱导率、诱变株的加倍率存在明显差异。
茎段浸泡法成苗率15.7％，诱导率4.1％，诱变株系加倍率31.8％；脱脂棉覆盖法成苗率54.2％，诱导率5.9％，加倍率30.3％：秋水仙素加入培养基中诱导处理，成苗率19.0％，诱导率9.0％，加倍率53.0％，其变动幅度30.0％-66.1％。从成苗率看，秋培法不及浸泡和覆盖法，但从最终的诱导率和诱变株系的加倍率看，秋水仙素加入培养基诱导较为理想，其中以20mg／L秋水仙素加入附加有0.01mg／L 6-BA的MS培养基中包埋腋芽诱导处理两周，诱导加倍效果较好。
不同的品种对秋水仙素的敏感反应不一，诱导加倍效果不同，京秀、黑大粒未获得加倍率超过50％的诱变株系，以红提为亲本获得加倍率分别为66.1％、64.6％的诱变株系H0120-1和H0120-3。两株系经连续3代的继代培养，经根尖和茎尖倍性鉴定，倍性结构基本稳定；与二倍体红提进行形态特征比较，具有多倍体的部分特征特性。气孔鉴定结果表明：诱变株与二倍体红提气孔平均大小无明显差异，但诱变株存在20.8％的特大气孔。H0120-3移栽大田，正进一步鉴定筛选。
Grapevine is a major traditional fruit tree in the world. It is a significant way to induce tetraploid to breed new grapevine varieties of high production and top quality. We researched on tetraploid grapevine induction by colchicine treatment through tissue culture.
The results of induced calluses had been obtained as follows: the frequency of callus induction was much high in MS or B5 with different concentration of 2,4-D, 6-BA and colchicine; the appearance and characteristics of induced calluses were closely related to type of explants, media and colchicine.
Observed the chromosome of induced pellet differentiated competence calluses, it was found that the wide-ranging chromosome variation existed in pellet calluses induced by different media with colchicine or absence of ones. Except for normal 2n=38 diploid cells and 2n=4x=76 tetraploid cells, aneuploid cells was found in calluses. The difference of chromosome variation was related to different grapevine varieties and media. The higher 4x cells rate calluses had been obtained in B5 medium with colchicine 20mg/l, 2,4-D 1.0mg/L and 6-BA 0.5mg/L. Red globe was easier than Jingxiu and Exotic in inducing double chromosome calluses . Regenerated plants had not been obtained through inducing calluses in MS and B5 media. It was maybe related to the chromosome variation of calluses. The higher the chromosome frequency variated , the lower the callus differention frequency was.
The tests of tetraploid induction in micro propagated buds was done through daubing axillary buds with grease colchicine, immersing ones in colchicine solution, covering ones with colchicine absorbent cotton, and inducing ones on media with colchicine. The results showed that the last three methods was more effective in inducing tetraploid than the first one. But the seedling rate, mutation rate, induction frequency of treated materials was obviously different.
As far as plants induced by immersing axillary buds were concerned, seedling rate was up to 15,1%, aberrant buds rate 4.1%, 4x cells rate 31.8%; seedling rate 54.2%, aberrant buds rate 5.9%, 4x cells rate 30.3% for ones induced by covering buds; seedling rate is 19.0%, aberrant buds rate 9.0%, 4x cells rate 53.3%, and its rang of change 30.3%-66.1% for ones induced by media with colchicine.
Induction effect through media with colchicine was inferior to that through covering axillary buds in seedling rate, but it was better in induction frequency and 4x cells rate of induced plant. To obtaining higher 4x cell rate of induced plant, the optimal way was to induce axillary buds on MS with colchicine 20mg/L and 6-BA 0.01mg/L for 2 weeks. The effect of induction was related to different varieties. The sports whose 4x cells rate were up to 50% had not been obtained by inducing Jingxiu and Exotic. The two induced plant H120-1 and H120-3 whose 4x cells rate was 66.1%, 64.6% respectively had been obtained by inducing diploid Red globe through media with colchicine.
The results of observing chromosomes variation in 3 progenies showed that the ploidy of H120-3 was stable. Compared with maternal plant, the lines exhibited typical polyploidy characteristics: leaf largen, stalk bulkyen, top and axillary bud fullen plump, growth strongen, and its propagation multiple obviously increased. The elite lines were being appraised for further selection through cutting axillary buds.
引文
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