丽格海棠快繁体系的建立和遗传转化初探
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摘要
本试验以丽格海棠(Begonia×Elatior)叶片、叶柄及茎段为外植体，通过诱导不定芽的形成，继而进行增殖培养，最后诱导生根、炼苗移栽，建立丽格海棠的快繁体系。以MS为基本培养基，研究了外植体类型，生长素、细胞分裂素配比对不定芽分化的影响，探索丽格海棠增殖培养适宜的激素配比；还对蔗糖与白砂糖的效果进行对比；最后，对丽格海棠试管苗生根的最佳培养基进行筛选。确定了丽格海棠组织培养的一系列技术参数组合，有效地提高了繁殖效率。在快繁体系的基础上进行了丽格海棠遗传转化方面的探索，以根癌农杆菌(Agrobacterium tumefaciens)介导丽格海棠叶片。研究结果表明：
①丽格海棠外植体在6-BA0.2—2mg/L和NAA0.05—1mg/L均有不同程度的分化。说明在一定范围里，丽格海棠对激素的要求不是很严格，不同配比均可分化。以MS+BA0.2mg/L+NAA0.1mg/L适宜的分化培养基；
②丽格海棠离体培养适宜的外植体类型为中度成熟叶片，其死亡率低、分化出芽快且分化率高，是适宜的外植体提供者。叶柄和茎段与叶片相比仅有少量芽的分化，在材料较少的情况下，利用叶柄、茎段作为外植体也是合适的；
③继代培养的时间不能超过60d，超过60d后继代苗有部分褐化死亡，30-40d继代既可有大量的有效苗又有较高的增殖率；
④优级白砂糖作为碳源对丽格海棠增殖生长以及生根的效果与分析纯蔗糖相比没有明显差异，从而可以利用白砂糖代替蔗糖来大大降低组培成本；
⑤丽格海棠试管苗生根较容易，不加任何激素的1/2MS培养基即可诱导其生根，但是所生的根比较细弱，NAA对其生根效果显著，丽格海棠试管苗生根适宜的培养基配方为：1/2MS+NAA0.05mg/L；
⑥在不添加蔗糖的生根培养基中丽格海棠组培苗所生根系与添加蔗糖的生根培养基中丽格海棠组培苗所生根系无明显差别，但是所长植株纤细，叶片薄弱颜色淡；
⑦丽格海棠对kan极其敏感，在筛选培养基与生根培养基中应选取较低的浓度；丽格海棠对Cef不敏感，因此在构建遗传转化体系时可以省去脱菌这一步，直接将共培养后的叶片直接接种到MS+500mg/L Cef+40mg/L Kan培养基中进行筛选培养；
⑧丽格海棠适宜的侵染时间为10min左右；
⑨经过3d预培养的外植体与未经预培养而直接侵染的外植体在分化时间、再生芽的频率没有明显差异，所以，为了缩短转化时间，丽格海棠转化可省去预培养；
⑩以丽格海棠的叶片为材料来进行遗传转化，进行GUS染色。未发现蓝色着色点，未将目的基因转入受体内，这和Sanae Kishimoto报道的试验结果一致。
This experimentation uses rieger begonia's (begonia×elatior) leaves, leafstalks and stems as explanted issue, inducing the formation of adventitious buds, culture and proliferate, and in the end induce the rhizogenesis, transplant reborn young plant, constitute the fast propagation system of rieger begonia. MS as the basic culture medium, this article analyses the affection of different explanted issue type, and different ratio of auxins and cytokines on the adventitious buds's differentiation, to explore the condign hormone ratio of rieger begonia multiplication culturing, and compare the different effect of white granulated sugar and sucrose. In the end, selects the optimal culture medium for rieger begonia's tube rhizogenesis, Ascertaines a series of technique parameter combination, effectively improves the breeding efficiency.Explore the genetic transformation of rieger begonia on the basis of fast propagation system, use agrobacterium tumefacien-mediated transformation of rieger begonia's leaf discs. The result shows that:
1) The rieger begonia's explanted issues undergo different degree of differentiation in 6-BA0.2-2mg/L and NAA0.5-1mg/L, which illuminates that within certain range the hormone for rieger begonia's differentiation is not strict, several compound portions can make it happen, MS+BA0.2mg/L+NAA0.1mg/L as the condign differentiation culture medium;
2) The condign explanted issue type for rieger begonia tissue culture is middle-typed mature lamina, which has low death rate, fast shooting and high differentiation ratio, being the good explanted issue provider. Compared with leaves, leafstalks and stems only has little differentiation of buds. When material is not abundance, leafstalks and stems as explanted issue also are appropriate.
3) The time for subculture culturing can not exceed 60d, or the young plant would brown and die. Between 30-40s there are a great amount of effective young plants and the multiplication ratio is high.
4) The difference between advanced white granulated sugar and pure sucrose in rieger begonia's multiplication growth and rhizogenesis effect is not remarkable, so the white granulated sugar can replace pure sucrose for reducing the culturing cost greatly.
5) Rieger begonia's reborn young plant can take root easily, without any hormone the 1/2MS culture medium can induce the rooting, but the roots are very thin and weak. NAA can has great effect on the rooting , the condign culture medium formula for rieger begonia's reborn young plant rooting is 1/2MS+NAA0.05mg/L
6) With or without pure sucrose in the rooting culture medium ,cultured rieger begonia roots are not quite different, but in the latten the plants are thinner and the laminas are flimsier and the color is weaker.
7) Rieger begonia is quite sensitive to kan, the concentration of with should be relatively low in selective culture medium and rooting culture medium; rieger begonia is not sensitive to cef, so wash bacterium can be ignored in constructing the genetic transformation system, the cultured laminas can be directly inoculate in the MS+cef600mg/L+kan40mg/L culture medium to undergo the selective culturing;
8) The condign time of begonia elatior infection is about 10min.
9) The explanted issues undergoing 3d beforehand-culture or not before infection are not quite different in differentiation time, and re-rooting frequency, so in order to shorten the transformation time, rieger begonia transformation can ignore the beforehand-culture.
10) Using rieger begonia's leaves as material to undergo genetic transformation, no blue pigmentation stains appear under GUS pigmentation, and the targeted genes are not transformed into received body, which is consistent with the experiential results of Sanae Kishmoto.
①丽格海棠外植体在6-BA0.2—2mg/L和NAA0.05—1mg/L均有不同程度的分化。说明在一定范围里，丽格海棠对激素的要求不是很严格，不同配比均可分化。以MS+BA0.2mg/L+NAA0.1mg/L适宜的分化培养基；
②丽格海棠离体培养适宜的外植体类型为中度成熟叶片，其死亡率低、分化出芽快且分化率高，是适宜的外植体提供者。叶柄和茎段与叶片相比仅有少量芽的分化，在材料较少的情况下，利用叶柄、茎段作为外植体也是合适的；
③继代培养的时间不能超过60d，超过60d后继代苗有部分褐化死亡，30-40d继代既可有大量的有效苗又有较高的增殖率；
④优级白砂糖作为碳源对丽格海棠增殖生长以及生根的效果与分析纯蔗糖相比没有明显差异，从而可以利用白砂糖代替蔗糖来大大降低组培成本；
⑤丽格海棠试管苗生根较容易，不加任何激素的1/2MS培养基即可诱导其生根，但是所生的根比较细弱，NAA对其生根效果显著，丽格海棠试管苗生根适宜的培养基配方为：1/2MS+NAA0.05mg/L；
⑥在不添加蔗糖的生根培养基中丽格海棠组培苗所生根系与添加蔗糖的生根培养基中丽格海棠组培苗所生根系无明显差别，但是所长植株纤细，叶片薄弱颜色淡；
⑦丽格海棠对kan极其敏感，在筛选培养基与生根培养基中应选取较低的浓度；丽格海棠对Cef不敏感，因此在构建遗传转化体系时可以省去脱菌这一步，直接将共培养后的叶片直接接种到MS+500mg/L Cef+40mg/L Kan培养基中进行筛选培养；
⑧丽格海棠适宜的侵染时间为10min左右；
⑨经过3d预培养的外植体与未经预培养而直接侵染的外植体在分化时间、再生芽的频率没有明显差异，所以，为了缩短转化时间，丽格海棠转化可省去预培养；
⑩以丽格海棠的叶片为材料来进行遗传转化，进行GUS染色。未发现蓝色着色点，未将目的基因转入受体内，这和Sanae Kishimoto报道的试验结果一致。
This experimentation uses rieger begonia's (begonia×elatior) leaves, leafstalks and stems as explanted issue, inducing the formation of adventitious buds, culture and proliferate, and in the end induce the rhizogenesis, transplant reborn young plant, constitute the fast propagation system of rieger begonia. MS as the basic culture medium, this article analyses the affection of different explanted issue type, and different ratio of auxins and cytokines on the adventitious buds's differentiation, to explore the condign hormone ratio of rieger begonia multiplication culturing, and compare the different effect of white granulated sugar and sucrose. In the end, selects the optimal culture medium for rieger begonia's tube rhizogenesis, Ascertaines a series of technique parameter combination, effectively improves the breeding efficiency.Explore the genetic transformation of rieger begonia on the basis of fast propagation system, use agrobacterium tumefacien-mediated transformation of rieger begonia's leaf discs. The result shows that:
1) The rieger begonia's explanted issues undergo different degree of differentiation in 6-BA0.2-2mg/L and NAA0.5-1mg/L, which illuminates that within certain range the hormone for rieger begonia's differentiation is not strict, several compound portions can make it happen, MS+BA0.2mg/L+NAA0.1mg/L as the condign differentiation culture medium;
2) The condign explanted issue type for rieger begonia tissue culture is middle-typed mature lamina, which has low death rate, fast shooting and high differentiation ratio, being the good explanted issue provider. Compared with leaves, leafstalks and stems only has little differentiation of buds. When material is not abundance, leafstalks and stems as explanted issue also are appropriate.
3) The time for subculture culturing can not exceed 60d, or the young plant would brown and die. Between 30-40s there are a great amount of effective young plants and the multiplication ratio is high.
4) The difference between advanced white granulated sugar and pure sucrose in rieger begonia's multiplication growth and rhizogenesis effect is not remarkable, so the white granulated sugar can replace pure sucrose for reducing the culturing cost greatly.
5) Rieger begonia's reborn young plant can take root easily, without any hormone the 1/2MS culture medium can induce the rooting, but the roots are very thin and weak. NAA can has great effect on the rooting , the condign culture medium formula for rieger begonia's reborn young plant rooting is 1/2MS+NAA0.05mg/L
6) With or without pure sucrose in the rooting culture medium ,cultured rieger begonia roots are not quite different, but in the latten the plants are thinner and the laminas are flimsier and the color is weaker.
7) Rieger begonia is quite sensitive to kan, the concentration of with should be relatively low in selective culture medium and rooting culture medium; rieger begonia is not sensitive to cef, so wash bacterium can be ignored in constructing the genetic transformation system, the cultured laminas can be directly inoculate in the MS+cef600mg/L+kan40mg/L culture medium to undergo the selective culturing;
8) The condign time of begonia elatior infection is about 10min.
9) The explanted issues undergoing 3d beforehand-culture or not before infection are not quite different in differentiation time, and re-rooting frequency, so in order to shorten the transformation time, rieger begonia transformation can ignore the beforehand-culture.
10) Using rieger begonia's leaves as material to undergo genetic transformation, no blue pigmentation stains appear under GUS pigmentation, and the targeted genes are not transformed into received body, which is consistent with the experiential results of Sanae Kishmoto.
引文
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76．潘会掌，张启翔．花卉种质资源与遗传育种研究进展．北京林业大学学报，2000，22(1)：81—86
77．周国辉，李华平．转基因植物及其应用．热带作物学报，2000，21(3)：71-76
78. Ovadis M. Zuker A. Tzfira T. et al. Generation of transgenic carnation plants with novel characteristics by combining microprojectile bombardment with Agrobacterium tumefaciens transformation, plant biotechnology and in vitro biology in the 21 stcentury. Proceedings of the Ⅸth lntenational Congress of the International Association of Plant Tissue Culture and Biotechnology, Jerusalem, Israel. 1998
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85．马忠华，罗如新，夏怡丰，等．邻单胞菌邻苯二酚1，2-双加氧酶基因(tfd C)的克隆及其在大肠杆菌中表达．微生物学报．2000，40(6)：579-585
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63. Nishihara M , ho M, Tanaka I, et al. Expression of the beta-glucuronidase gene in pollen of lily(Lilium longiflorum), tobacco (Nicotiana tabacum), Nicotiana rustica, and peony(Paeonia lactiflora)by particle bombardment. Plant Physiology. 1993, 102 (2): 357—361
64. Yang J, Lee H J, Shin D H, et al. Genetic transformation of Cymbidium orchid by particle bomhardment. Plant Cell Reports. 1999. 18(12): 978-984
65. Wilmink A. van de Ven B C E, Dons J J M, et al. Expression of the GUS-gene in the monocot tulip after introduction by panicle bombardment and Agrobacterium. Plant Cell Reports. 1992. 11(2): 76-80
66. Tanaka T, Nishihara M , Seki M , et al. Successful cxpression in pollen of various plant species of in vitro synthesized mRNA introduced by panicle bombardment. Plant Molecular Biology, 1995. 28(2): 337—341
67. Hsia ChiN, Korban S S, Hsia C N. Microprojectile mediated genetic transformation of Rhododendron hybrids. American Rhododendron Society Journal, 1998, 52(4):187—191
68. Zuker A, Chang P L, Ahroni A. et al. Transformation of carnation by microprojectile bombardment. Scientia Horticulturae. 1995, 64(3): 177-185
69. Yepes L M , Mittak V, Pang ShenkZhi. et al. Biolistic transformation of chrysanthemum with the nucleocapsid gene of tomato spotted wilt virus. Plant Cell Reports. 1995. 14(11): 694-698
70. Marchant R, Davey M R. Lucas J A, et al. Expression of a chitinase transgene in rose(Rosa hybrida L.)reduces development of blackspot disease(Diplocarpon rosae Wolf) Molecular Breeding, 1998. 4(3): 187-194
71. de Villiers S M, Kamo K, Thomson J A, et al. Biolistic transformation of chinchefinchee (Ornithogalum) and regeneration of transgenic plants. Physiologia Plantarum, 2000, 109(4); 450-455
72. de Lin H S. Jeu M J. Jacobsen E, et al. Development of a plant regeneration system applicable for gene transformation in the ornamental Alstroemeria. Acta Horticulturae, 2000(508): 61-67
73．杜娟，王罡，王萍，等．玉米遗传转化系统的研究进展．遗传．2001，23(1)：69～72
74. Inokuma C, Sugiura K, Imaizumi N, et al. Transgenic Japanese lawngrass (Zoysia japonica Steud. ) plants regenerated from protoplasts. Plant Cell Reports, 1998, 17(5):334—338
75. Steinhart W L, Mistretta A. Genetic engineering in orchids: expression of a beta-glucuronidase reporter gene following plasmid transformation of floral protoplasts. Plant Physiol, 1992, 99(Supp): 47
76．潘会掌，张启翔．花卉种质资源与遗传育种研究进展．北京林业大学学报，2000，22(1)：81—86
77．周国辉，李华平．转基因植物及其应用．热带作物学报，2000，21(3)：71-76
78. Ovadis M. Zuker A. Tzfira T. et al. Generation of transgenic carnation plants with novel characteristics by combining microprojectile bombardment with Agrobacterium tumefaciens transformation, plant biotechnology and in vitro biology in the 21 stcentury. Proceedings of the Ⅸth lntenational Congress of the International Association of Plant Tissue Culture and Biotechnology, Jerusalem, Israel. 1998
79. Hoshino Y, Turkan I. Mii M. Transgenic bialaphos—rcsistant snapdragon (Antirrhinum majus L. ) produced by Agrobacterium rhizogenes transformation. Scientia Horticulturae, 1998, 76(1—2): 37-57
80. Dolgov S V, Mityshkina T U, Rukavtsova E B, et al. Production of transgenic plants of Chrysanthemum morifolium Ramat with the gene of Bac.thuringieusis delta—endotoxin, ornamental plant improvement, Acta Horticulturae, 1995(420); 46-47
81. Winefield C, Lewis D, Arathoon S, et al. Alteration of Petunia plant form through the introduction of the rolC gene from Agrobacterium rhizogenes. Molecular Breeding, 2000, 5(6): 543—551
82. Giovannini A, Zottini M , Morreale G.et al. Ornamental traits modification by rol genes in Osteospernmum ecklonis transformed with Agrobacterium tumefaciens. In vitro Cellular and Developmental Biology Plant, 1999. 35(1): 70—75
83．汪开治．利用转基因植物降解土壤中的残留炸药．生物技术通报．1999(51：48-49
84．马忠华，张云芳，徐传祥，等．早熟禾的组织培养和基因枪介导的基因转化体系的初步建立．复旦学报(自然科学版)．1999．38(5)：540—544
85．马忠华，罗如新，夏怡丰，等．邻单胞菌邻苯二酚1，2-双加氧酶基因(tfd C)的克隆及其在大肠杆菌中表达．微生物学报．2000，40(6)：579-585
86．李春秀，石大兴，王米力．丽格海棠组织培养外植体再生体系建立关键技术的研究．四川农业大学学报．2002．20(4)：330—333．
87．韩超，方正．外植体和激素对丽格海棠组培不定芽分化的影响．河北农业大学学报．2005．28(3)：38-41
88. Kishimoto , Ryutaro Aida , Michio Shibata Sanae. Agrobacterium tumefaciens-mediated transformation of Elatior Begonia (Begoniaxhiemalis Fotsch). Plant science. 2002. (162): 697-703