春石斛兰组培增殖及兰花试管开花研究
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摘要
春石斛兰作为兰花产业中的一个重要的兰花种类,主要靠扦插和高压方法进行繁殖,其繁殖系低、繁殖周期长。近年来春石斛兰在国外市场销量很大,而在国内作为盆花观赏则刚刚崭露头角,市场上较好的春石斛兰品种大多引自引进日本,这限制了我国春石斛兰的深入研究与发展。本研究以从日本引进的23个品种春石斛兰为材料,围绕春石斛兰组培的增殖,对侧芽外植体的建立、不同激素对部分品种增殖的影响和组培苗的继代培养等几个关键技术进行了研究,同时对春石斛兰、密花石斛和建兰‘小桃红’×纹瓣兰的试管开花进行了研究,试验结果如下:
1.对春石斛兰的最佳消毒方案和光照条件进行了研究,初步确定了3个代表品种(004、022和052)春石斛兰的最佳消毒方案,即芽尖外留一层叶鞘,75%酒精消毒15 s和0.1% HgCl2消毒8 min;春石斛兰侧芽外植体的暗培养具有减轻春石斛兰褐化的作用。
2.在相同培养条件下,对日本引进的23个春石斛兰品种进行了启动率、增殖率和增殖系数等方面的研究。结果表明,23个春石斛兰品种间的差异显著,在483个接种的外植体中,51.97%的外植体在诱导培养基中不适宜工厂化繁殖。依据平均增殖系数,以1.0和2.0为标准将23个品种分为高、中和低3个组;6-BA浓度梯度试验得出,适宜3个代表品种(022、021和026)增殖的6-BA浓度分别为3.39、2.07和1.92 mg/L,该浓度满足组内大多品种的组培增殖要求。1/2 MS+NAA 1.0 mg/L+6-BA 4.0 mg/L+卡拉胶7.0 g/L+蔗糖20 g/L+椰子乳100 ml/L+细菌学蛋白胨2.0g/L适合001、002、004和022这4个品种的侧芽组培增殖。
3. 12个春石斛兰品种侧芽增殖系数与3种细胞分裂素6-BA、KT和TDZ的浓度间均存在抛物线关系,对12个品种侧芽平均增殖系数的影响顺序为:KT>6-BA>TDZ。在1/2 MS+NAA 1.0 mg/L+卡拉胶7.0 g/L+蔗糖20 g/L+椰子乳100 ml/L+细菌学蛋白胨2.0g/L基础上,添加3.0 mg/L 6-BA适合004品种的侧芽增殖,添加2.0 mg/L KT适合005和035这2个品种的侧芽增殖,添加3.0 mg/L KT适合015、019和026这3个品种的侧芽增殖。
4.生长素对D.Tubuyaki和D.Shirasagi的增殖率影响显著,6-BA与6-BA×KT对D.Tubuyaki和D.Shirasagi的增殖率和增殖系数影响均显著。0.5mg/L NAA+1.0 mg/L 6-BA+0.5 mg/L KT适合D.Tubuyaki的侧芽增殖;适合D.Shirasagi侧芽增殖的激素组合为0.5 mg/L 2,4-D+1.0 mg/L 6-BA+1.0 mg/L KT。
5. 5个春石斛兰品种侧芽组培苗的继代培养条件存在差异性。1/2 MS +卡拉胶7.0 g/L+蔗糖20g/L+椰子乳100 ml/L+细菌学蛋白胨2.0g/L+6-BA 3.0 mg/L+NAA 0.3 mg/L适合002、004和022的继代培养;通过调整基本培养基配方可改善D.Casiflake和D.Emur侧芽组培苗继代培养时的褐化和生长状况,在上述培养基上添加0.03%活性炭可以有效防止D.Casiflake和D.Emur继代培养褐化。
6.初步建立了兰花试管开花的诱导方案,即在蔗糖40g/L+卡拉胶6g/L+4.0 mg/L 6-BA的基础上,附加低氮高磷和变温。密花石斛无菌播种苗在变温(18℃±1/25±1)下,MS1(1/3 N,9 P)处理中有2株诱导出1个花芽(6.6%),开放畸形的白花;MS4(1/6 N,15 P)处理中有1植株诱导出2个花芽(3.3%),开放正常的黄花。但对建兰杂交种(XTW)无菌播种苗和D.Spot Right侧芽组培苗的离体花芽没有诱导效果。
7.研究了植物材料的成熟程度对兰花离体花芽诱导作用。选取成熟的兰花植株,进行细胞分裂素(6-BA、TDZ)浓度处理,可诱导出花蕾。在1/2MS+蔗糖40g/L+卡拉胶8g/L+活性炭1.0g/L上,附加6-BA 1.0 mg/L+TDZ 0.1mg/L诱导建兰杂交种(XTW)无菌播种苗1株产生花蕾(3.3%);附加6-BA 10 mg/L+TDZ 1.0 mg/L诱导建兰杂交种(XTW)无菌播种苗2株出现花蕾(6.7%);附加6-BA 5.0 mg/L诱导春石斛兰(D.Spot Right)侧芽组培苗6株出现花蕾(20%)。
The Nobile-type Dendrobium, plays an important role in orchid industry, and was mainly propagated in cutting and high layering. But the propagation coefficient of the ways was quite low and reproductive cycle was very long. Recently, the Nobile-type Dendrobium has a large-sale volume in foreign market, but was only used as potted flower in China. Moreover, the better varieties were introduced from Japan, which had restricted the further researching and development of the Nobile-type Dendrobium in China. The twenty-three Nobile-type Dendrobium varieties, which were introduced from Japan, were used as test materials in this study. The main researching contents of the lateral bud propagation of the Nobile-type Dendrobium in vitro as follows: the disinfection scheme of their lateral bud explants, the effect of the different hormone on the lateral bud propagation of some varieties and the subculture of their plantlets. Simultaneously, the studies on the in vitro flowering of the D.Spot Right, D.densiflorum and Cymbidium emsifolium (L.)‘XiaoTaoHong’×C.aloifolium (XTW) were implemented. Their experimental results as below:
1. The experimental results of the best disinfection scheme and light condition of the Nobile-type Dendrobium showed that the best disinfection scheme of the three representative varieties (004,022 and 052) was that retained one layer leaf sheath outside the lateral bud tip, the 75% ethanol disinfected for fifteen seconds and the 0.1% HgCl2 disinfected for eight minutes. Besides, the dark culture of the lateral bud explant was beneficial to reducing their browning effect.
2. The germination rate, proliferation rate and propagation ratio of the Nobile-type Dendrobium were studied under the same culture conditions. The results showed: their differences among the twenty-three varieties were significant, 51.97% of the 483 explants were not suitable for large-scale propagation at factories. Took the propagation ratio as basis and their values (1.0 and 2.0) as criterial, the twenty-three varieties were divided into high, central and low groups. The results of the 6-BA concentration gradient experiments suggested that the very 6-BA concentration suitable for proliferating of the three representative varieties (022,021 and 026) were 3.39, 2.07 and 1.92 mg/L, respectively; these 6-BA concentration could meet the proliferation needs of the members within the group. The medium (1/2 MS+NAA 1.0 mg/L +6-BA 4.0 mg/L+ carrageenan 7.0 g/L+sucrose 20 g/L+coconut milk 100 ml/L+ bacteriological peptone 2.0g/L) was fit for the propagation of the four varieties (001,002, 004 and 022).
3. The relationship between the lateral bud propagation ratio of the twelve varieties of the Nobile-type Dendrobium and the concentration of the three kinds cytokinin (6-BA, KT and TDZ) was parabola. The order of the effect on the three kinds of cytokinin for the mean propagation coefficient of the twelve varieties were KT>6-BA>TDZ. Based on the medium (1/2 MS+NAA 1.0 mg/L+carrageenan 7.0 g/L+sucrose 20 g/L+coconut milk 100 ml/L+ bacteriological peptone 2.0g/L), we supplemented 3.0 mg/L 6-BA suitable for the 004 variety propagation, 2.0 mg/L KT fit to the 005 and 035 varieties, and 3.0 mg/L KT for the three varieties, 015,019 and 026.
4. The effects of the auxin on the propagation rate of the D.Tubuyaki and D.Shirasagi were significant, and so were the effects of the 6-BA and 6-BA×KT on the D.Tubuyaki and D.Shirasagi. 0.5mg/L NAA+1.0 mg/L 6-BA+0.5 mg/L KT were suitable for the D.Tubuyaki proliferation, and 0.5 mg/L 2,4-D+1.0 mg/L 6-BA+1.0 mg/L KT were for the D.Shirasagi proliferation.
5. The subculture conditions of the five Nobile-type Dendrobium varieties were different. 1/2MS+carrageenan 7.0g/L+sucrose 20g/L+coconut milk 100ml/L+ bacteriological peptone 2.0g/L+6-BA3.0 mg/L+NAA0.3 mg/L were suitable for the subculture of the three varieties, 002, 004 and 022. The browning and growth conditions of the plantlets from the lateral bud in D.Casiflake and D.Emur via tissue culture could be improved through adjustment of the basal medium composition. And on the basis of the above media, the browning of D.Casiflake and D.Emur during their subculture could be effectively prevented by supplementing it with 0.03% active carbon.
6. The preliminary induction formular of in vitro flowering in orchids was established, which was supplementing with low nitrogen(N) and high phosphrous(P) and variable temperature, on basis of the sucrous 40g/L+carrageenan 6g/L+4.0 mg/L 6-BA. Under variable temperature (18℃±1/25℃±1, night/day), two flower buds of two plants from the D.densiflorum were induced, which opened malformation white flower under the treatment MS1 (1/3N,9P), and two flower buds from a plant of the D.densiflorum were induced, which opened normal yellow flowers under the treatment MS4(1/6N,15P). However, these treatments didn’t have the flower bud inducing effect in the aseptic seedings of XTW and the plantlets of the D.Spot Right in vitro.
7. The effect of the mature degree of the plant materials on the in vitro flowering in orchids was studied. The flower bud could be induced from the mature orchid plants under cytokinin(6-BA and TDZ) treatment. On the condition of the medium: 1/2MS+sucrous 40g/L+ carrageenan 8g/L+active carbon1.0g/L, two flower buds of a plant in XTW were induced by supplementing with 6-BA 1.0 mg/L+TDZ 0.1 mg/L, two flower buds of two plants in XTW were induced by supplementing with 6-BA 10 mg/L+TDZ 1.0 mg/L, and 6 flower buds of 6 plants of D.Spot Right were induced by supplementing with 6-BA 5.0 mg/L.
1.对春石斛兰的最佳消毒方案和光照条件进行了研究,初步确定了3个代表品种(004、022和052)春石斛兰的最佳消毒方案,即芽尖外留一层叶鞘,75%酒精消毒15 s和0.1% HgCl2消毒8 min;春石斛兰侧芽外植体的暗培养具有减轻春石斛兰褐化的作用。
2.在相同培养条件下,对日本引进的23个春石斛兰品种进行了启动率、增殖率和增殖系数等方面的研究。结果表明,23个春石斛兰品种间的差异显著,在483个接种的外植体中,51.97%的外植体在诱导培养基中不适宜工厂化繁殖。依据平均增殖系数,以1.0和2.0为标准将23个品种分为高、中和低3个组;6-BA浓度梯度试验得出,适宜3个代表品种(022、021和026)增殖的6-BA浓度分别为3.39、2.07和1.92 mg/L,该浓度满足组内大多品种的组培增殖要求。1/2 MS+NAA 1.0 mg/L+6-BA 4.0 mg/L+卡拉胶7.0 g/L+蔗糖20 g/L+椰子乳100 ml/L+细菌学蛋白胨2.0g/L适合001、002、004和022这4个品种的侧芽组培增殖。
3. 12个春石斛兰品种侧芽增殖系数与3种细胞分裂素6-BA、KT和TDZ的浓度间均存在抛物线关系,对12个品种侧芽平均增殖系数的影响顺序为:KT>6-BA>TDZ。在1/2 MS+NAA 1.0 mg/L+卡拉胶7.0 g/L+蔗糖20 g/L+椰子乳100 ml/L+细菌学蛋白胨2.0g/L基础上,添加3.0 mg/L 6-BA适合004品种的侧芽增殖,添加2.0 mg/L KT适合005和035这2个品种的侧芽增殖,添加3.0 mg/L KT适合015、019和026这3个品种的侧芽增殖。
4.生长素对D.Tubuyaki和D.Shirasagi的增殖率影响显著,6-BA与6-BA×KT对D.Tubuyaki和D.Shirasagi的增殖率和增殖系数影响均显著。0.5mg/L NAA+1.0 mg/L 6-BA+0.5 mg/L KT适合D.Tubuyaki的侧芽增殖;适合D.Shirasagi侧芽增殖的激素组合为0.5 mg/L 2,4-D+1.0 mg/L 6-BA+1.0 mg/L KT。
5. 5个春石斛兰品种侧芽组培苗的继代培养条件存在差异性。1/2 MS +卡拉胶7.0 g/L+蔗糖20g/L+椰子乳100 ml/L+细菌学蛋白胨2.0g/L+6-BA 3.0 mg/L+NAA 0.3 mg/L适合002、004和022的继代培养;通过调整基本培养基配方可改善D.Casiflake和D.Emur侧芽组培苗继代培养时的褐化和生长状况,在上述培养基上添加0.03%活性炭可以有效防止D.Casiflake和D.Emur继代培养褐化。
6.初步建立了兰花试管开花的诱导方案,即在蔗糖40g/L+卡拉胶6g/L+4.0 mg/L 6-BA的基础上,附加低氮高磷和变温。密花石斛无菌播种苗在变温(18℃±1/25±1)下,MS1(1/3 N,9 P)处理中有2株诱导出1个花芽(6.6%),开放畸形的白花;MS4(1/6 N,15 P)处理中有1植株诱导出2个花芽(3.3%),开放正常的黄花。但对建兰杂交种(XTW)无菌播种苗和D.Spot Right侧芽组培苗的离体花芽没有诱导效果。
7.研究了植物材料的成熟程度对兰花离体花芽诱导作用。选取成熟的兰花植株,进行细胞分裂素(6-BA、TDZ)浓度处理,可诱导出花蕾。在1/2MS+蔗糖40g/L+卡拉胶8g/L+活性炭1.0g/L上,附加6-BA 1.0 mg/L+TDZ 0.1mg/L诱导建兰杂交种(XTW)无菌播种苗1株产生花蕾(3.3%);附加6-BA 10 mg/L+TDZ 1.0 mg/L诱导建兰杂交种(XTW)无菌播种苗2株出现花蕾(6.7%);附加6-BA 5.0 mg/L诱导春石斛兰(D.Spot Right)侧芽组培苗6株出现花蕾(20%)。
The Nobile-type Dendrobium, plays an important role in orchid industry, and was mainly propagated in cutting and high layering. But the propagation coefficient of the ways was quite low and reproductive cycle was very long. Recently, the Nobile-type Dendrobium has a large-sale volume in foreign market, but was only used as potted flower in China. Moreover, the better varieties were introduced from Japan, which had restricted the further researching and development of the Nobile-type Dendrobium in China. The twenty-three Nobile-type Dendrobium varieties, which were introduced from Japan, were used as test materials in this study. The main researching contents of the lateral bud propagation of the Nobile-type Dendrobium in vitro as follows: the disinfection scheme of their lateral bud explants, the effect of the different hormone on the lateral bud propagation of some varieties and the subculture of their plantlets. Simultaneously, the studies on the in vitro flowering of the D.Spot Right, D.densiflorum and Cymbidium emsifolium (L.)‘XiaoTaoHong’×C.aloifolium (XTW) were implemented. Their experimental results as below:
1. The experimental results of the best disinfection scheme and light condition of the Nobile-type Dendrobium showed that the best disinfection scheme of the three representative varieties (004,022 and 052) was that retained one layer leaf sheath outside the lateral bud tip, the 75% ethanol disinfected for fifteen seconds and the 0.1% HgCl2 disinfected for eight minutes. Besides, the dark culture of the lateral bud explant was beneficial to reducing their browning effect.
2. The germination rate, proliferation rate and propagation ratio of the Nobile-type Dendrobium were studied under the same culture conditions. The results showed: their differences among the twenty-three varieties were significant, 51.97% of the 483 explants were not suitable for large-scale propagation at factories. Took the propagation ratio as basis and their values (1.0 and 2.0) as criterial, the twenty-three varieties were divided into high, central and low groups. The results of the 6-BA concentration gradient experiments suggested that the very 6-BA concentration suitable for proliferating of the three representative varieties (022,021 and 026) were 3.39, 2.07 and 1.92 mg/L, respectively; these 6-BA concentration could meet the proliferation needs of the members within the group. The medium (1/2 MS+NAA 1.0 mg/L +6-BA 4.0 mg/L+ carrageenan 7.0 g/L+sucrose 20 g/L+coconut milk 100 ml/L+ bacteriological peptone 2.0g/L) was fit for the propagation of the four varieties (001,002, 004 and 022).
3. The relationship between the lateral bud propagation ratio of the twelve varieties of the Nobile-type Dendrobium and the concentration of the three kinds cytokinin (6-BA, KT and TDZ) was parabola. The order of the effect on the three kinds of cytokinin for the mean propagation coefficient of the twelve varieties were KT>6-BA>TDZ. Based on the medium (1/2 MS+NAA 1.0 mg/L+carrageenan 7.0 g/L+sucrose 20 g/L+coconut milk 100 ml/L+ bacteriological peptone 2.0g/L), we supplemented 3.0 mg/L 6-BA suitable for the 004 variety propagation, 2.0 mg/L KT fit to the 005 and 035 varieties, and 3.0 mg/L KT for the three varieties, 015,019 and 026.
4. The effects of the auxin on the propagation rate of the D.Tubuyaki and D.Shirasagi were significant, and so were the effects of the 6-BA and 6-BA×KT on the D.Tubuyaki and D.Shirasagi. 0.5mg/L NAA+1.0 mg/L 6-BA+0.5 mg/L KT were suitable for the D.Tubuyaki proliferation, and 0.5 mg/L 2,4-D+1.0 mg/L 6-BA+1.0 mg/L KT were for the D.Shirasagi proliferation.
5. The subculture conditions of the five Nobile-type Dendrobium varieties were different. 1/2MS+carrageenan 7.0g/L+sucrose 20g/L+coconut milk 100ml/L+ bacteriological peptone 2.0g/L+6-BA3.0 mg/L+NAA0.3 mg/L were suitable for the subculture of the three varieties, 002, 004 and 022. The browning and growth conditions of the plantlets from the lateral bud in D.Casiflake and D.Emur via tissue culture could be improved through adjustment of the basal medium composition. And on the basis of the above media, the browning of D.Casiflake and D.Emur during their subculture could be effectively prevented by supplementing it with 0.03% active carbon.
6. The preliminary induction formular of in vitro flowering in orchids was established, which was supplementing with low nitrogen(N) and high phosphrous(P) and variable temperature, on basis of the sucrous 40g/L+carrageenan 6g/L+4.0 mg/L 6-BA. Under variable temperature (18℃±1/25℃±1, night/day), two flower buds of two plants from the D.densiflorum were induced, which opened malformation white flower under the treatment MS1 (1/3N,9P), and two flower buds from a plant of the D.densiflorum were induced, which opened normal yellow flowers under the treatment MS4(1/6N,15P). However, these treatments didn’t have the flower bud inducing effect in the aseptic seedings of XTW and the plantlets of the D.Spot Right in vitro.
7. The effect of the mature degree of the plant materials on the in vitro flowering in orchids was studied. The flower bud could be induced from the mature orchid plants under cytokinin(6-BA and TDZ) treatment. On the condition of the medium: 1/2MS+sucrous 40g/L+ carrageenan 8g/L+active carbon1.0g/L, two flower buds of a plant in XTW were induced by supplementing with 6-BA 1.0 mg/L+TDZ 0.1 mg/L, two flower buds of two plants in XTW were induced by supplementing with 6-BA 10 mg/L+TDZ 1.0 mg/L, and 6 flower buds of 6 plants of D.Spot Right were induced by supplementing with 6-BA 5.0 mg/L.
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