草莓花药培养作为脱毒途径的可行性研究
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摘要
草莓(Fragaria ananassa．Duch)是蔷薇科草莓属多年生草本植物，在园艺学上属于小浆果。草莓果实鲜艳亮丽，风味酸甜适中，芳香浓郁，营养丰富，是世界上七大水果之一。我国草莓生产近年来发展很快，栽培面积也不断扩大。草莓属无性繁殖作物，在生产过程中易感染病毒，据统计，草莓因病毒感染的种性退化一般减产30％-80％。用化学药物对病毒病进行防治效果甚微，故生产上多采用无病毒种苗来控制病毒病的蔓延。草莓花药培养是获得无病毒植株的重要途径之一，八倍体再生植株的细胞学起源是困扰草莓花药培养在脱毒苗生产中利用价值的一个重要因素，目前尚未有关于再生植株细胞起源的直接实验证据。本课题研究通过建立草莓花药培养再生体系，以及对再生植株倍性进行鉴定，并通过石蜡切片观察愈伤组织的细胞学起源，为探讨花药培养作为脱毒手段的可行性提供细胞学理论依据。其主要结果如下：
1．以花粉发育处于单核靠边期的草莓花蕾为外植体，研究了低温预处理对愈伤组织诱导的影响，结果表明，低温预处理3d既能显著提高愈伤组织的诱导率，又能保证所形成的愈伤组织的良好状态。
2．培养基的蔗糖浓度和激素配比对草莓花药培养具有显著影响。实验表明，无论是诱导愈伤组织形成还是分化不定芽，适宜的蔗糖浓度均为3％。愈伤组织诱导中，BA与KT的配合使用可以提高诱导率；而分化培养中，BA与ZT的配合使用有利于芽分化。本研究的优化培养基组合为：诱导培养基为MS+BA0.5mg／L+KT0.1mg／L+NAA2.0mg／L；分化培养基为MS+BA0.5mg／L+ZT2.0mg／L+NAA0.2mg／L。
3．实验采用20℃和25℃两种温度诱导了花药愈伤组织形成，结果表明，20℃诱导的愈伤组织结构紧密，颜色绿，活力强，易分化。
4．武汉地区草莓在上年10月至下年3月形成3次花序，第一花序较第二、三花序更适合花药培养。
5．对43个再生植株株系进行根尖染色体计数和DNA含量测定，结果表明43个株系中，有39个株系为八倍体(2n=8x=56)，占90.7％；4个株系为十六倍体
(Zn=16x=112)，占9.30，0，未发现单倍体。
通过石蜡切片观察愈伤组织的细胞起源表明，草幕花药培养初期，花粉细胞可
启动分裂，经几次分裂后便解体，未形成愈伤组织。愈伤组织均起源于药隔细
胞，因此，本实验中草墓花药再生植株均起源于体细胞。
花药苗较对照的生长势明显优越，栽培花药苗有提高产量和改善品质的作用。
对正常八倍体花药植株(Zn二8x=56)的田间观察表明，再生植株没有表型性状
的变异。
Strawberry (Fragaria ananassa. Duch) is a kind of renascent species classified to Rosaceae, Fragaria. With attractive colorful fruit, special flavor and rich nutriment, strawberry has being developed rapidly in our country and production area is getting larger and larger. Traditionally, strawberries are vegetative propagated from runners that are often infected by a number of aphid-born viruses in the perennial cultivation period. The loss of yield in strawberry due to virus-disease is estimated to be 30-80 percent per year. As we know there is little effect on controlling virus-disease by chemicals so using virus-free stocks is an efficient way to inhibit the epeidemics of the disease. Strawberry anther culture is an important method to obtain virus-free stocks, however, a problem encountered is that the plantlets from anther culture are not haploid but diploid. In previous studies the cytological origin of ploidy unreduced regenerate plantlets from anther culture is putative either from somatic cells, unre
duced microspores or be the result of spontaneous chromosome doubling. The cytological origin of plant regeneration is therefore a main factor of influencing anther culture to adopt in obtaining virus-free stocks commercially. The present research established a techniqal system of strawberry anther culture to look insight the ploidy variation and the cytological origin of the regenerated plants with purpose of providing both theoretic and technical bases for appraising virus-free stocks obtained by anther culture. The main results obtained are as follows:
1. The callus inducting rate was increased significantly and the quality of the callus was the best when the anthers at the uninucleate stage were pretreated in 4℃ for 3 days.
2. Sucrose concentration and the hormone ratio of the medium had significant effects on strawberry anther culture. Results showed that 3% sucrose not only promoted callus induction but also benefited to adventitious shoots differation. In callus induction, BA and KT combination could increase the inducting rate of callus and BA combined with ZT was favor for the shoot differation. In the present research,
MS+BA0.5mg/L+KT0.1mg/L+NAA2.0mg/L was proved to be more optimal in callus induction and MS+BA2.0mg/L+ZT2.0mg/L+NAA2.0mg/L was promising to adventitiou shoots differation respectively.
3. Inoculating temperature of 20℃ was piror to 25℃ to obtain more vigorous callus as well as to improve the subsequent shoot regeneration.
4. From October to the next March in Wuhan, strawberry has 3 anthotaxy normally. The results indicated that the first anthotaxy is more suitable for anther culture than of the second or the third in callus induction.
5. Among the 43 anther derived lines investigated, 39 were octoploid (2n=8x=56), 4 hexadecaploid (2n=16x=112), and no haploid (2n=4x=28) was observed.
6. The uninucleate pollen could start devision in the early inoculate time, but only after several divisions, the microspore disintegrated, and thus, there were no callus originated from pollens. All plantlets from anther culture in the present research were proved to originate from somatic cells.
7. Field trial showed that plantlets obtained from the anther culture had excellent growth vigor and potential productivity with improvement in yield and quality but without morphological variation compared to the wild types.
1．以花粉发育处于单核靠边期的草莓花蕾为外植体，研究了低温预处理对愈伤组织诱导的影响，结果表明，低温预处理3d既能显著提高愈伤组织的诱导率，又能保证所形成的愈伤组织的良好状态。
2．培养基的蔗糖浓度和激素配比对草莓花药培养具有显著影响。实验表明，无论是诱导愈伤组织形成还是分化不定芽，适宜的蔗糖浓度均为3％。愈伤组织诱导中，BA与KT的配合使用可以提高诱导率；而分化培养中，BA与ZT的配合使用有利于芽分化。本研究的优化培养基组合为：诱导培养基为MS+BA0.5mg／L+KT0.1mg／L+NAA2.0mg／L；分化培养基为MS+BA0.5mg／L+ZT2.0mg／L+NAA0.2mg／L。
3．实验采用20℃和25℃两种温度诱导了花药愈伤组织形成，结果表明，20℃诱导的愈伤组织结构紧密，颜色绿，活力强，易分化。
4．武汉地区草莓在上年10月至下年3月形成3次花序，第一花序较第二、三花序更适合花药培养。
5．对43个再生植株株系进行根尖染色体计数和DNA含量测定，结果表明43个株系中，有39个株系为八倍体(2n=8x=56)，占90.7％；4个株系为十六倍体
(Zn=16x=112)，占9.30，0，未发现单倍体。
通过石蜡切片观察愈伤组织的细胞起源表明，草幕花药培养初期，花粉细胞可
启动分裂，经几次分裂后便解体，未形成愈伤组织。愈伤组织均起源于药隔细
胞，因此，本实验中草墓花药再生植株均起源于体细胞。
花药苗较对照的生长势明显优越，栽培花药苗有提高产量和改善品质的作用。
对正常八倍体花药植株(Zn二8x=56)的田间观察表明，再生植株没有表型性状
的变异。
Strawberry (Fragaria ananassa. Duch) is a kind of renascent species classified to Rosaceae, Fragaria. With attractive colorful fruit, special flavor and rich nutriment, strawberry has being developed rapidly in our country and production area is getting larger and larger. Traditionally, strawberries are vegetative propagated from runners that are often infected by a number of aphid-born viruses in the perennial cultivation period. The loss of yield in strawberry due to virus-disease is estimated to be 30-80 percent per year. As we know there is little effect on controlling virus-disease by chemicals so using virus-free stocks is an efficient way to inhibit the epeidemics of the disease. Strawberry anther culture is an important method to obtain virus-free stocks, however, a problem encountered is that the plantlets from anther culture are not haploid but diploid. In previous studies the cytological origin of ploidy unreduced regenerate plantlets from anther culture is putative either from somatic cells, unre
duced microspores or be the result of spontaneous chromosome doubling. The cytological origin of plant regeneration is therefore a main factor of influencing anther culture to adopt in obtaining virus-free stocks commercially. The present research established a techniqal system of strawberry anther culture to look insight the ploidy variation and the cytological origin of the regenerated plants with purpose of providing both theoretic and technical bases for appraising virus-free stocks obtained by anther culture. The main results obtained are as follows:
1. The callus inducting rate was increased significantly and the quality of the callus was the best when the anthers at the uninucleate stage were pretreated in 4℃ for 3 days.
2. Sucrose concentration and the hormone ratio of the medium had significant effects on strawberry anther culture. Results showed that 3% sucrose not only promoted callus induction but also benefited to adventitious shoots differation. In callus induction, BA and KT combination could increase the inducting rate of callus and BA combined with ZT was favor for the shoot differation. In the present research,
MS+BA0.5mg/L+KT0.1mg/L+NAA2.0mg/L was proved to be more optimal in callus induction and MS+BA2.0mg/L+ZT2.0mg/L+NAA2.0mg/L was promising to adventitiou shoots differation respectively.
3. Inoculating temperature of 20℃ was piror to 25℃ to obtain more vigorous callus as well as to improve the subsequent shoot regeneration.
4. From October to the next March in Wuhan, strawberry has 3 anthotaxy normally. The results indicated that the first anthotaxy is more suitable for anther culture than of the second or the third in callus induction.
5. Among the 43 anther derived lines investigated, 39 were octoploid (2n=8x=56), 4 hexadecaploid (2n=16x=112), and no haploid (2n=4x=28) was observed.
6. The uninucleate pollen could start devision in the early inoculate time, but only after several divisions, the microspore disintegrated, and thus, there were no callus originated from pollens. All plantlets from anther culture in the present research were proved to originate from somatic cells.
7. Field trial showed that plantlets obtained from the anther culture had excellent growth vigor and potential productivity with improvement in yield and quality but without morphological variation compared to the wild types.
引文
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68. Mullin R H, Smith S H, Frazier N W, Schlegel D E, Mccall S R. Meristem culture frees strawberries of mild yellow edge, pallidosis, and mottle diseases. Phytopathology, 1974, 64: 1425-1429
69. Nehra N S, Stashnoff C. Direct shoot regeneration from strawberry leaf disks. J Amer Soc Hort Sci, 1989, 114(6): 1014-1018
70. Niemirowicz -Szczytt K, Zakrzewska Z, Malepszy S and Kubicklbm. Characters of plants obtained from Fragaria × ananassa in anther culture. Acta Horticulturae 1983, 131, 231-237
71. Quail A M, Spiegel S, Jelkmann W. Development of monoclonal antibodies specific
for strawberry mild yellow edgfe potexvirus. Acta Horticulturae, 1994, 235:872-876
72. Quarta R, Nati D and Paoloni F M. Strawberry anther culture. Acta horticulturae 1991, 300:335-339
73. Rosati R, Evereux V and Laneri U. Anther culture of strawberry. Hortiscience 1975, 10:119-200
74. Sato T, Nishio T, Hirea M. Plant regeneration from isolated microspore culture of Chinese cabbage (Brassica Campestres aap. Pekinensis). Plant Cell Rep., 1989, 8: 486-488
75. Siebel J J, Pauls K. Acomparison of anther and microspore culture as a breeding tool in Brassica naprs. Theoretical and Applied Genetics, 1989, 78(4): 473-479
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77. Sorvari S. The effect of starch gelatinized nutrient medija in barley anther cultures. Annales Agriculturae Fenniae 1986, 25:127-133
78. Spiegel S, Cohen J and Converse R H. Detection of strawberry mild yellow-edge virus by serological specific electron microscopy. Acta Hortic. 1993186:65
79. Stache-smith R and Mellor F C Eradication of potato viruses X and S by thermotherapy and axillary bud culture. Phytopathology, 1968, 58:199-203
80. Svensson and Johansyson L B. Anther culture of Fragaria × ananassa: Environmental factors and medium components affection microspore division and callus production. Journal of Horticulturae science, 1994, 69(3): 417-426
81. Takahata Y. High frequency embryogenesis and plant regeneration in isolated microspore culture of Brassica oleracea L. Plant Science, 1991, 74:235-242
82. Thomas J E and Dietxgen R G. Purification, charcterization and serological detection of virus. Journal of phytopathhoogy, 1990, 128:203-208
83. Thompson J R, Leooone G, Lindneer J L, Jelkmann W and Schoone C D. Characterization and complete nucleotidee sequence of strawberry mottle virus: a tentative member of a new family. Theor Appl Genet, 1994, 88:973-980
84. Toyoda H, Horikoshi K. Selectionn for Fusariu mediate disease resistance from regenerates derived from leaf callus of strawberry Plant CelReports, 1991,10:167-170
85. Velchev V and Milanov E. Regeneration of callus cultures from anthers and pistils in strawerry Gradinarska Ⅰ Lozarska Naula, 1984, 21:29-35
86. Vergne P, Riccardi F, Beckert M C, Dumas C. Identification of 32-KD anther marker protein for and rogenic response in maize. Zea, mays L Thoer. Appl. Genet, 1993, 86: 843-850
87. Vine S J. Improved culture of apical tissues for production of virus-free strawberries. Horticulture Science, 1968, 43:293-297
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67. Morel G M. Producing virus-free cymbidiums Am. Orchid Soc. Bull. 1960, 29:495-497
68. Mullin R H, Smith S H, Frazier N W, Schlegel D E, Mccall S R. Meristem culture frees strawberries of mild yellow edge, pallidosis, and mottle diseases. Phytopathology, 1974, 64: 1425-1429
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72. Quarta R, Nati D and Paoloni F M. Strawberry anther culture. Acta horticulturae 1991, 300:335-339
73. Rosati R, Evereux V and Laneri U. Anther culture of strawberry. Hortiscience 1975, 10:119-200
74. Sato T, Nishio T, Hirea M. Plant regeneration from isolated microspore culture of Chinese cabbage (Brassica Campestres aap. Pekinensis). Plant Cell Rep., 1989, 8: 486-488
75. Siebel J J, Pauls K. Acomparison of anther and microspore culture as a breeding tool in Brassica naprs. Theoretical and Applied Genetics, 1989, 78(4): 473-479
76. Simon I, Racz E and Zatyko J M. Prelinubary notes on somaclonal variation of strawberry Fruit. Science Reports, Poland, 19871, 4:151-154
77. Sorvari S. The effect of starch gelatinized nutrient medija in barley anther cultures. Annales Agriculturae Fenniae 1986, 25:127-133
78. Spiegel S, Cohen J and Converse R H. Detection of strawberry mild yellow-edge virus by serological specific electron microscopy. Acta Hortic. 1993186:65
79. Stache-smith R and Mellor F C Eradication of potato viruses X and S by thermotherapy and axillary bud culture. Phytopathology, 1968, 58:199-203
80. Svensson and Johansyson L B. Anther culture of Fragaria × ananassa: Environmental factors and medium components affection microspore division and callus production. Journal of Horticulturae science, 1994, 69(3): 417-426
81. Takahata Y. High frequency embryogenesis and plant regeneration in isolated microspore culture of Brassica oleracea L. Plant Science, 1991, 74:235-242
82. Thomas J E and Dietxgen R G. Purification, charcterization and serological detection of virus. Journal of phytopathhoogy, 1990, 128:203-208
83. Thompson J R, Leooone G, Lindneer J L, Jelkmann W and Schoone C D. Characterization and complete nucleotidee sequence of strawberry mottle virus: a tentative member of a new family. Theor Appl Genet, 1994, 88:973-980
84. Toyoda H, Horikoshi K. Selectionn for Fusariu mediate disease resistance from regenerates derived from leaf callus of strawberry Plant CelReports, 1991,10:167-170
85. Velchev V and Milanov E. Regeneration of callus cultures from anthers and pistils in strawerry Gradinarska Ⅰ Lozarska Naula, 1984, 21:29-35
86. Vergne P, Riccardi F, Beckert M C, Dumas C. Identification of 32-KD anther marker protein for and rogenic response in maize. Zea, mays L Thoer. Appl. Genet, 1993, 86: 843-850
87. Vine S J. Improved culture of apical tissues for production of virus-free strawberries. Horticulture Science, 1968, 43:293-297