金钗石斛试管开花研究
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摘要
利用组织培养的方法，研究金钗石斛(Dendrobium nobile Lindl)的试管开花，缩短营养生长向生殖生长转变的时间，探讨花芽分化规律在理论上和应用上都具有重要的意义。
本文研究了PP_(333)、ABA、TDZ、BA、含氮量和糖浓度等因素对金钗石斛组培苗花芽发生和花发育的影响。研究发现，BA诱导金钗石斛的花芽形成率较低。单独使用TDZ处理，花芽形成率最高仅达34.3％。减少培养基中氮的含量(1／10氮)，增加磷含量(5倍磷)，同时将糖浓度增加到40g／t,，可使TDZ诱导的花芽形成率明显提高，最高约76.3％。单独使用PP_(333)或ABA对材料进行预处理，对TDZ诱导花芽形成的影响效果均不明显；若将PP_(333)和ABA联合使用，可使花芽数量明显增加，但所形成的花大多数不能正常开放。通过筛选，诱导金钗石斛花芽形成效果最好的培养方法是：以1／2MS附加蔗糖30g／L，琼脂9g／L，活性炭0.5g／L为基本培养基(pH5.6～5.8)，将组培苗在加有PP_(333)0.5mg／L+ABA0.5mg／L的培养基上预培养35d后，再转入到附加TDZ0.1mg／L+PP_(333)1.0mg／L的培养基中。150d内统计的花芽形成率高达62.2％。根的修剪对花芽形成有显著影响，这种诱导提早开花的现象通常发生在进行了根修剪的情况中，开花的植株多是接种的小苗基部萌发的幼芽形成的新植株，在其顶端形成单花或具有2～4朵小花的花序。以MS+NAA0.5mg／L为基本培养基，附加不同浓度的PP_(333)，可促使金钗石斛幼苗茁壮生长。在壮苗培养过程中也有花芽形成，但形成率较低。用经过壮苗培养的材料进行处理，在预处理条件及成花诱导条件不变的情况下，花芽形成率和正常花的比率均明显增加，分别达到80.0％和58.3％。
以诱导花芽形成效果最佳的配方处理的金钗石斛小苗为材料，通过石蜡切片，观察了金钗石斛花芽分化初期形态发育过程，整个过程大致分为8个时期。处理20d时花芽仍未分化，30d时将分化，40d左右逐渐分化出花原基，50～80d可观察到萼片原基、花瓣原基、合蕊柱。
以诱导花芽形成效果最佳的培养基作为处理组，同时设置一组对照。通过SDS-聚丙烯酰胺凝胶电泳进行蛋白组分的分析。30d的对照组中出现了两个特异性蛋白，分子量为36KD左右和29KD左右。75d的对照组中出现了一个分子量
约12KD的特异性蛋白。90d对照组中的特异性蛋白分子量约41KD;处理组中三
个特异性蛋白分子量分别为54KD左右、13KD左右和12KD左右。另外，105d
的处理组中也出现了三个特异性蛋白，分子量分别约为82KD、42KD和28KD。
Wild Dendrobium nobile Iindl take several years to flower. In vitro flowering by plantlets produced through tissue culture can decrease the juvenile phases from a vegetative to a reproductive stage. Studing molecular mechanisms of D. nobile Lindl floral induction, floral meristem formation, and floral organ development are important not only in theory but also in application.
The objective of this research is to study the effects of PP333, ABA, TDZ, BA, nitrogen concentrations and sucrose concentrations on flowering in vitro of D. nobile Lindl. The results of our research show that BA had an indistinctive effect on in vitro flowering of D. nobile lindl. The highest frequency of floral bud induction was about 34.3% if induced by TDZ only. 1/2MS medium with less nitrogen sources (1/10N) and more phosphor concentration (5P) and supplemented with 40g/L sucrose could make the frequency of floral buds induction increased greatly reaching 76.3% under the induction of TDZ. Effects of PP333 or ABA pretreatment on in vitro flowering of D. nobile plantlets induced by TDZ were not obvious. The number of floral buds was increased if pretreated by "PP333+ABA". The optimal way for inducing floral buds is to preculture the plantets on 1/2MS medium supplemented with 0.5mg/L ABA and 0.5mg/L PP333 for 35 days, then transferred them onto 1/2MS medium supplemented with 1.0mg/L PP333 and 0.1mg/L TDZ.
The frequency of floral buds induction increased sharply reaching 62.2% during a period of 150 days. All the mediums were added to 30g/L sucrose and 9g/Lagar. The pH was adjusted to 5.6-5.8 with IN NaOH. In addition, the frequency of floral bud induction was also affected by root excision. Most of the plantlets able to form floral buds were not the original ones but those grew up from new stems. On the top of them, a single flower or a inflorescence including 2~3 small flowers could be observed. MS medium supplemented with 0.5mg/L NAA and different concentrations of PP333 could make D. nobile plantlets thrived. Floral buds were also formed during the process of thriving treatment. If thriving the plantlets before precultured, the frequency and the sum of normal flowers were sharply raised at
the same conditions of pretreatment and induction.
After having screened the optimal medium for inducing flower buds initiation on D. nobile plantlets, we observed the process of early stage of floral bud differentiation. The whole process was divided into 8 stages. After 20 days of culture, the floral bud was still undifferentiated and it would differentiate in 30 days. Floral anlage was formed after 40 days of culture. After that, sepal anlage, petal anlage and habenaria reniformis appeared gradually after 50-80 days of culture.
The components of the proteins were analyzed by SDS-PAGE. When plantlets were cultured in comparison mediums, two special proteins about 36KD and 29KD appeared in 30 days, and a special protein about 12KD appeared in 75 days. In addition, three special proteins about 54KD, 13KD and 12KD appeared when plantlets were cultured in the optimal medium for 90 days, and another three special proteins about 82KD, 42KD and 28KD could be observed after cultured in the same medium for 105 days.
本文研究了PP_(333)、ABA、TDZ、BA、含氮量和糖浓度等因素对金钗石斛组培苗花芽发生和花发育的影响。研究发现，BA诱导金钗石斛的花芽形成率较低。单独使用TDZ处理，花芽形成率最高仅达34.3％。减少培养基中氮的含量(1／10氮)，增加磷含量(5倍磷)，同时将糖浓度增加到40g／t,，可使TDZ诱导的花芽形成率明显提高，最高约76.3％。单独使用PP_(333)或ABA对材料进行预处理，对TDZ诱导花芽形成的影响效果均不明显；若将PP_(333)和ABA联合使用，可使花芽数量明显增加，但所形成的花大多数不能正常开放。通过筛选，诱导金钗石斛花芽形成效果最好的培养方法是：以1／2MS附加蔗糖30g／L，琼脂9g／L，活性炭0.5g／L为基本培养基(pH5.6～5.8)，将组培苗在加有PP_(333)0.5mg／L+ABA0.5mg／L的培养基上预培养35d后，再转入到附加TDZ0.1mg／L+PP_(333)1.0mg／L的培养基中。150d内统计的花芽形成率高达62.2％。根的修剪对花芽形成有显著影响，这种诱导提早开花的现象通常发生在进行了根修剪的情况中，开花的植株多是接种的小苗基部萌发的幼芽形成的新植株，在其顶端形成单花或具有2～4朵小花的花序。以MS+NAA0.5mg／L为基本培养基，附加不同浓度的PP_(333)，可促使金钗石斛幼苗茁壮生长。在壮苗培养过程中也有花芽形成，但形成率较低。用经过壮苗培养的材料进行处理，在预处理条件及成花诱导条件不变的情况下，花芽形成率和正常花的比率均明显增加，分别达到80.0％和58.3％。
以诱导花芽形成效果最佳的配方处理的金钗石斛小苗为材料，通过石蜡切片，观察了金钗石斛花芽分化初期形态发育过程，整个过程大致分为8个时期。处理20d时花芽仍未分化，30d时将分化，40d左右逐渐分化出花原基，50～80d可观察到萼片原基、花瓣原基、合蕊柱。
以诱导花芽形成效果最佳的培养基作为处理组，同时设置一组对照。通过SDS-聚丙烯酰胺凝胶电泳进行蛋白组分的分析。30d的对照组中出现了两个特异性蛋白，分子量为36KD左右和29KD左右。75d的对照组中出现了一个分子量
约12KD的特异性蛋白。90d对照组中的特异性蛋白分子量约41KD;处理组中三
个特异性蛋白分子量分别为54KD左右、13KD左右和12KD左右。另外，105d
的处理组中也出现了三个特异性蛋白，分子量分别约为82KD、42KD和28KD。
Wild Dendrobium nobile Iindl take several years to flower. In vitro flowering by plantlets produced through tissue culture can decrease the juvenile phases from a vegetative to a reproductive stage. Studing molecular mechanisms of D. nobile Lindl floral induction, floral meristem formation, and floral organ development are important not only in theory but also in application.
The objective of this research is to study the effects of PP333, ABA, TDZ, BA, nitrogen concentrations and sucrose concentrations on flowering in vitro of D. nobile Lindl. The results of our research show that BA had an indistinctive effect on in vitro flowering of D. nobile lindl. The highest frequency of floral bud induction was about 34.3% if induced by TDZ only. 1/2MS medium with less nitrogen sources (1/10N) and more phosphor concentration (5P) and supplemented with 40g/L sucrose could make the frequency of floral buds induction increased greatly reaching 76.3% under the induction of TDZ. Effects of PP333 or ABA pretreatment on in vitro flowering of D. nobile plantlets induced by TDZ were not obvious. The number of floral buds was increased if pretreated by "PP333+ABA". The optimal way for inducing floral buds is to preculture the plantets on 1/2MS medium supplemented with 0.5mg/L ABA and 0.5mg/L PP333 for 35 days, then transferred them onto 1/2MS medium supplemented with 1.0mg/L PP333 and 0.1mg/L TDZ.
The frequency of floral buds induction increased sharply reaching 62.2% during a period of 150 days. All the mediums were added to 30g/L sucrose and 9g/Lagar. The pH was adjusted to 5.6-5.8 with IN NaOH. In addition, the frequency of floral bud induction was also affected by root excision. Most of the plantlets able to form floral buds were not the original ones but those grew up from new stems. On the top of them, a single flower or a inflorescence including 2~3 small flowers could be observed. MS medium supplemented with 0.5mg/L NAA and different concentrations of PP333 could make D. nobile plantlets thrived. Floral buds were also formed during the process of thriving treatment. If thriving the plantlets before precultured, the frequency and the sum of normal flowers were sharply raised at
the same conditions of pretreatment and induction.
After having screened the optimal medium for inducing flower buds initiation on D. nobile plantlets, we observed the process of early stage of floral bud differentiation. The whole process was divided into 8 stages. After 20 days of culture, the floral bud was still undifferentiated and it would differentiate in 30 days. Floral anlage was formed after 40 days of culture. After that, sepal anlage, petal anlage and habenaria reniformis appeared gradually after 50-80 days of culture.
The components of the proteins were analyzed by SDS-PAGE. When plantlets were cultured in comparison mediums, two special proteins about 36KD and 29KD appeared in 30 days, and a special protein about 12KD appeared in 75 days. In addition, three special proteins about 54KD, 13KD and 12KD appeared when plantlets were cultured in the optimal medium for 90 days, and another three special proteins about 82KD, 42KD and 28KD could be observed after cultured in the same medium for 105 days.
引文
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61. Arndt F, Rusch R, Stilfried H V. A New Cotton Defoliant. Plant Phsiology, 1976, 57:99
62. Mok M C, D W S Mok, D J Armstrong. Cytokinin activity of N-phenyl-N-1,2,3-thiadiazol-5-ylurea (thidiazuron). Phytochemistry, 1982, 21:1509～1511
63．苑兆和，杨世忠，王家喜。TDZ在组织培养中的生物学效应．果树科学，1995，12(增刊)：138～140
64．徐晓峰，黄学林．TDZ：一种有效的植物生长调节剂．植物学通报，2003，20(2)：227～237
65．王关林，方宏筠，那杰．高活性细胞激动素TDZ在植物组织培养中的应用．植物学通报，1997，14(3)：47～53
66．杨正业．棉花脱叶剂TDZ简介．植物生理学通讯，1989，6：63-64
67. Wang G Y. Xu Z H. Chua N H. In vitro flowering of Cybidium ensifoloium. 4th Asia Pacific Orchid Conference Programme and Abstracts. Registrant' s Handbook. 1992,84
68．曹尚银，汤一卒，张俊昌．GA_3和PP_(333)对苹果花芽形态建成及其内源激素比例变化的影响．果树学报，2001，18(6)：313～316
69. Krekule J, Kohli P K. The condition of the apical meristem of seedings responsive to a promotive effect of abscisic acid on flowering in the short day plant Chemopodium rubrum. Zeitschrift fiir Pflanzenphysiologie, 1981, 103:45～51
70. Purse J G Phloem exudates of Perilla crispa and its effect on flowering of P. crispa shoot explants. J Exp Bot, 1984, 35:227～238
71．芦建国，陈昕．PP_(333)对盆栽矮牵牛的矮化和促进开花效应．浙江林业科技，1996，23(3)：17～20
72. Clarke A E, Dennis E, Mol J. Forefronts of flowering. Plant Cell,1992, 4:867-872
73．黄先群，黄燕芬，唐丽，颜怀珍，沈永珍．BA和PP_(333)对油菜试管苗生长的影响。贵州农业科学，1999，27(1)：10～13
74．张宪省，郑成超，Neill S D O．与受粉有关的因子调节乙烯生物合成基因在兰花花器官中的表达．植物学报，1999，41(5)：468～471
75. Murthy B N S, Murch S J, Saxena Praveen K. Thidiazuron: a potent regulator of in vitro plant morphogenesis. In Vitro Cell. Dev. Biol.—Plant, 1998, 34:267～275
76．徐华松．TDZ对莴苣(Lactuca sativa L)器官发生及乙烯形成的影响．广西植物，1999，19(1)：92～94
77．陈肖英．叶庆生，刘伟．TDZ研究进展(综述)．亚热带植物科学，2003，32(3)：59～63
78. Grochowska M J, Karaszewska A, Jankowska B. Dormant pruning influence on auxin, gibberellin and cytokinin levels in apple trees. J Amer Soe Hort Sci, 1984, 109:312-1317
79. Ammar S, Benhadis A, Tripathi B K. Floral induction in date palm seeding (Phoenea dactylifera
var. Deglet Nour) cultured in vitro. Can J Bot, 1990, 65:137-142
80. McDaniel C N. Developmental physiology of floral initiation in Nicotiana tabacum L. J Exp Bot, 1996, 297:465～475
81．李明军．PP_(333)对玉米试管苗生长的调控．植物生理学通讯，1997，33(4)：269-271
82．赵成章，郑康乐，戚秀芳．多效唑对水稻未成熟胚愈伤组织诱导、分化和壮苗培养的影响。植物学报，1990，32(5)：407
83．陈龙清，张雨琴，袁芳亭．PP3_(333)及矮壮素对地被菊试管苗生根的影响(简报)．植物生理学通讯，2000，36(5)：425～427
84．王正询，段敏妍．多效唑对香蕉试管苗生长的影响．植物生理学通讯，1994，30(5)：346～348
85．李正巧，朱鹿鸣．PP_(333)、GA_3、BA对马铃薯试管苗生长调节作用的研究．作物学报，1994，20(1)：59～65
86．罗琼，朱力煌．水稻花发育的分子生物学研究进展．遗传，2002，24(1)：87～93