辣椒全基因组测序材料Capsicum annuum cv.CM334再生体系的建立
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摘要
选用辣椒全基因组测序材料Capsicum annuum cv. CM334、高抗CMV辣椒材料C. frutescens cv.PBC688和高感CMV辣椒材料C. annuum cv. G29,以其幼苗的子叶、下胚轴作为外植体,研究了不同激素组合对辣椒组织培养再生的影响。结果显示:最佳的芽分化培养基为MS+5.0 mg/L 6-BA+1.0 mg/L IAA+5000.0mg/L DJ+10.0 mg/L AgNO_3;促进再生芽伸长的最优培养基为MS+0.5 mg/L 6-BA+1.0 mg/L IAA+2.0 mg/L GA_3+5000.0 mg/L DJ+10. 0 mg/L AgNO_3; 3个基因型子叶的平均芽分化率(5.9%)高于下胚轴的(2.4%);CM334的再生能力最强,以子叶和下胚轴作为外植体时,其芽分化率分别为10.74%和4.85%,芽伸长率分别为54.54%和53.33%,分别获得可移栽的生根再生植株30株和8株; PBC688和G29的再生能力较差,只获得少量的再生植株。
The pepper whole-genome sequencing material Capsicum annuum cv. CM334,the CMV-highly-resistant pepper material C. frutescens cv. PBC688,and the CMV-highly-susceptible pepper material C. annuum cv. G29 were used as experimental materials,the cotyledons and hypocotyls of their seedlings were used as explants,and the influences of different hormone combinations on the regeneration of these three pepper genotypes through tissue culture were studied. The results indicated that:the optimum culture medium for the bud differentiation of pepper materials was MS+5.0 mg/L 6-BA+1.0 mg/L IAA+5000.0 mg/L DJ+10.0 mg/L AgNO_3; the best culture medium for promoting the elongation of regenerative buds was MS+0.5 mg/L 6-BA+1.0 mg/L IAA+2.0 mg/L GA_3+5000.0 mg/L DJ+10.0 mg/L AgNO_3; the average bud differentiation rate( 5.9%) from the cotyledons of three pepper genotypes was higher than that( 2.4%) from their hypocotyls; CM334 had the strongest regenerative ability,when its cotyledons and hypocotyls were used as explants,the bud differentiation rate was 10.74% and 4.85%,respectively,the bud elongation rate was 54.54% and 53.33% separately,and 30 and 8 regenerative plantlets with roots were obtained,respectively; while PBC688 and G29 had worse regenerative ability,and only a little regenerative plantlets were obtained from them.
The pepper whole-genome sequencing material Capsicum annuum cv. CM334,the CMV-highly-resistant pepper material C. frutescens cv. PBC688,and the CMV-highly-susceptible pepper material C. annuum cv. G29 were used as experimental materials,the cotyledons and hypocotyls of their seedlings were used as explants,and the influences of different hormone combinations on the regeneration of these three pepper genotypes through tissue culture were studied. The results indicated that:the optimum culture medium for the bud differentiation of pepper materials was MS+5.0 mg/L 6-BA+1.0 mg/L IAA+5000.0 mg/L DJ+10.0 mg/L AgNO_3; the best culture medium for promoting the elongation of regenerative buds was MS+0.5 mg/L 6-BA+1.0 mg/L IAA+2.0 mg/L GA_3+5000.0 mg/L DJ+10.0 mg/L AgNO_3; the average bud differentiation rate( 5.9%) from the cotyledons of three pepper genotypes was higher than that( 2.4%) from their hypocotyls; CM334 had the strongest regenerative ability,when its cotyledons and hypocotyls were used as explants,the bud differentiation rate was 10.74% and 4.85%,respectively,the bud elongation rate was 54.54% and 53.33% separately,and 30 and 8 regenerative plantlets with roots were obtained,respectively; while PBC688 and G29 had worse regenerative ability,and only a little regenerative plantlets were obtained from them.
引文
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[2]曹亚从,张正海,王立浩,等.辣椒不同外植体处理方法对组织培养再生的影响[J].中国蔬菜,2012(8):32-39.
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[6]Manoharan M,Vidya C S S,Sita G L. Agrobacterium-mediated genetic transformation in hot chilli(Capsicum annuum L. var. pusa jwala)[J]. Plant Science,1998,131(1):77-83.
[7] Zhu Y X,Ouyang W J,Zhang Y F,et al. Transgenic sweet pepper plants from Agrobacterium mediated transformation[J]. Plant Cell Reports,1996,16(1-2):71-75.
[8]王玉文,杨美珠,潘乃穟,等.甜椒的离体再生及基因转化[J].植物学报,1991,33(10):780-786.
[9]EbidaA A I A,Hu C Y. In vitro morphogenetic responses and plant regeneration from pepper(Capsicum annuum L.cv. early California wonder)seedling explants[J]. Plant Cell Reports,1993,13(2):107-110.
[10]邓明华,周群初,刘志敏,等.辣椒子叶离体培养植株再生研究[J].长江蔬菜,2003(6):36-39.
[11]Sanatombi K,Sharma G J. In vitro plant regeneration in six cultivars of Capsicum spp. using different explants[J]. Biologia Plantarum,2008,52(1):141-145.
[12]Binzel M L,Sankhla N,Joshi S,et al. In vitro regeneration in chile pepper(Capsicum annuum L.)from‘halfseed explants’[J]. Plant Growth Regulation,1996,20(3):287-293.
[13]Ahmad N,Siddique I,Anis M. Improved plant regeneration in Capsicum annuum L. from nodal segments[J].Biologia Plantarum,2006,50(4):701-704.
[14] Dabauza M,Pena L. High efficiency organogenesis in sweet pepper(Capsicum annuum L.)tissues from different seeding explants[J]. Plant Growth Regulation,2001,33(3):221-229.
[15]Gammoudi N,Pedro T S,Ferchichi A,et al. Improvement of regeneration in pepper:a recalcitrant species[J]. In Vitro Cellular&Developmental Biology:Plant,2018,54(2):145-153.
[16]Anikumar M,Nair A S. Multiple shoot induction in Capsicum annuum L. cv. early California wonder[J]. Plant Cell Biotechnology and Molecular Biology,2004,5(3):95-100.
[17]Orlinska M,Nowaczyk P. In vitro plant regeneration of 4Capsicum spp. genotypes using different explant types[J]. Turkish Journal of Biology,2015,39(1):60-68.
[18]Arous S,Boussaid M,Marrakchi M. Plant regeneration from zygotic embryo hypocotyls of Tunisian chili(Capsicum annuum L.)[J]. Journal of Applied Horticulture,2001,3(1):17-22.
[19]Joshi A,Kothari S L. High copper levels in the medium improves shoot bud differentiation and elongation from the cultured cotyledons of Capsicum annuum L.[J]. Plant Cell,Tissue and Organ Culture,2007,88(2):127-133.
[20]Kim S,Park M,Yeoms I,et al. Genome sequence of the hot pepper provides insights into the evolution of pungency in Capsicum species[J]. Nature Genetics,2014,46(3):270-278.
[21] Guo G,Wang S,Liu J,et al. Rapid identification of QTLs underlying resistance to cucumber mosaic virus in pepper(Capsicum frutescens)[J]. Theoretical and Applied Genetics,2016,130(1):1-12.
[22]Cheng S,He S,Wang H,et al. Establishment of regeneration system and in vitro culture from pepper cotyledon and hypocotyl explants[J]. Southern Horticulture,2009,20(3):3-6.
[23]Long F,Zhang J. Plant regeneration from cotyledon and hypocotyl explants of pepper in vitro and high efficient system establishment of plant regeneration[J]. Journal of Gansu Agricultural University,2005,40(1):31-37.
[24]Szase A,Nervo G,Fari M. Screening for in vitro shootforming capacity of seedling explants in bell pepper(Capsicum annuum L.)genotypes and efficient plant regeneration using thidiazuron[J]. Plant Cell Reports,1995,14(10):666-669.
[25]黎定军,张宝玺,赵开军,等.辣椒子叶高效植株再生体系的建立[J].园艺学报,2002,29(1):25-29.
[26]成善汉,贺申魁,王红梅,等.辣椒子叶和下胚轴的离体培养及高效再生体系的建立[J].南方园艺,2009,20(3):3-6.
[27]余小林,李乃坚,黄自然,等.辣椒子叶离体培养和植株再生体系的建立[J].园艺学报,2000,27(1):42-46.
[28]孙月芳,陆瑞菊.杂种甜椒的离体快速繁殖[J].植物生理通讯,2001,37(3):215-216.
[29]何晓明,王鸣,王喆之.辣椒叶片离体培养与植株再生[J].西北农业大学学报,1996,24(1):93-96.
[30]曹冬孙,贾士荣.青椒子叶培养及植株再生[J].园艺学报,1993,20(2):171-175.
[31]Li D,Zhao K,Xie B,et al. Establishment of a highly efficient transformation system for pepper(Capsicum annuum L.)[J]. Plant Cell Reports,2003,21(8):785-788.
[32]曾华,胡宗利,陈国平,等.辣椒离体再生及遗传转化研究进展[J].生命科学研究,2011,15(6):542-549.
[33]Bairwa V K,Kachhwaha S,Kothari S L. Phloroglucinol mediated shoot bud elongation in Capsicum annuum L.[J]. National Academy Science Letters,2012,35(4):331-335.
[34]缪武,刘志敏.辣椒离体培养研究进展[J].辣椒杂志,2005,3(2):1-4.
[35]罗素兰,王鹏程,张转,等.辣椒离体高效再生体系及其卡那霉素筛选体系的建立[J].海南大学学报:自然科学版,2003,21(1):51-57.
[36]董兆龙,陈沁,刘文轩,等.辣椒子叶和下胚轴离体培养再生[J].上海大学学报:自然科学版,2003,9(2):148-152.
[37]Elwan M W M,Elhamahmy M A M. Improved productivity and quality associated with salicylic acid application in greenhouse pepper[J]. Scientia Horticulturae,2009,122(4):521-526.
[38]叶志彪,李汉霞,张健,等.辣椒转基因植株再生[J].植物学报,1993,35(增刊):88-93.
[39]Kim S,Kim S R,Aa C S,et al. Constitutive expression of rice MADS box gene using seed explants in hot pepper(Capsicum annuum L.)[J]. Molecules and Cells,2001,12(2):221-226.
[2]曹亚从,张正海,王立浩,等.辣椒不同外植体处理方法对组织培养再生的影响[J].中国蔬菜,2012(8):32-39.
[3]龙凤,张金文.辣椒子叶和下胚轴的离体培养及高效再生体系的建立[J].甘肃农业大学学报,2005,40(1):31-37.
[4]唐亮,陈沁,邓志瑞,等.辣椒茎尖离体培养及植株再生[J].上海大学学报:自然科学版,2004,10(5):497
[5]Ezura H,Nishimiya S,Kasumi M. Efficient regeneration of plants independent of exogeneous growth regulators in bell pepper(Capsicum annuum L.)[J]. Plant Cell Reports,1993,12(12):676-680.
[6]Manoharan M,Vidya C S S,Sita G L. Agrobacterium-mediated genetic transformation in hot chilli(Capsicum annuum L. var. pusa jwala)[J]. Plant Science,1998,131(1):77-83.
[7] Zhu Y X,Ouyang W J,Zhang Y F,et al. Transgenic sweet pepper plants from Agrobacterium mediated transformation[J]. Plant Cell Reports,1996,16(1-2):71-75.
[8]王玉文,杨美珠,潘乃穟,等.甜椒的离体再生及基因转化[J].植物学报,1991,33(10):780-786.
[9]EbidaA A I A,Hu C Y. In vitro morphogenetic responses and plant regeneration from pepper(Capsicum annuum L.cv. early California wonder)seedling explants[J]. Plant Cell Reports,1993,13(2):107-110.
[10]邓明华,周群初,刘志敏,等.辣椒子叶离体培养植株再生研究[J].长江蔬菜,2003(6):36-39.
[11]Sanatombi K,Sharma G J. In vitro plant regeneration in six cultivars of Capsicum spp. using different explants[J]. Biologia Plantarum,2008,52(1):141-145.
[12]Binzel M L,Sankhla N,Joshi S,et al. In vitro regeneration in chile pepper(Capsicum annuum L.)from‘halfseed explants’[J]. Plant Growth Regulation,1996,20(3):287-293.
[13]Ahmad N,Siddique I,Anis M. Improved plant regeneration in Capsicum annuum L. from nodal segments[J].Biologia Plantarum,2006,50(4):701-704.
[14] Dabauza M,Pena L. High efficiency organogenesis in sweet pepper(Capsicum annuum L.)tissues from different seeding explants[J]. Plant Growth Regulation,2001,33(3):221-229.
[15]Gammoudi N,Pedro T S,Ferchichi A,et al. Improvement of regeneration in pepper:a recalcitrant species[J]. In Vitro Cellular&Developmental Biology:Plant,2018,54(2):145-153.
[16]Anikumar M,Nair A S. Multiple shoot induction in Capsicum annuum L. cv. early California wonder[J]. Plant Cell Biotechnology and Molecular Biology,2004,5(3):95-100.
[17]Orlinska M,Nowaczyk P. In vitro plant regeneration of 4Capsicum spp. genotypes using different explant types[J]. Turkish Journal of Biology,2015,39(1):60-68.
[18]Arous S,Boussaid M,Marrakchi M. Plant regeneration from zygotic embryo hypocotyls of Tunisian chili(Capsicum annuum L.)[J]. Journal of Applied Horticulture,2001,3(1):17-22.
[19]Joshi A,Kothari S L. High copper levels in the medium improves shoot bud differentiation and elongation from the cultured cotyledons of Capsicum annuum L.[J]. Plant Cell,Tissue and Organ Culture,2007,88(2):127-133.
[20]Kim S,Park M,Yeoms I,et al. Genome sequence of the hot pepper provides insights into the evolution of pungency in Capsicum species[J]. Nature Genetics,2014,46(3):270-278.
[21] Guo G,Wang S,Liu J,et al. Rapid identification of QTLs underlying resistance to cucumber mosaic virus in pepper(Capsicum frutescens)[J]. Theoretical and Applied Genetics,2016,130(1):1-12.
[22]Cheng S,He S,Wang H,et al. Establishment of regeneration system and in vitro culture from pepper cotyledon and hypocotyl explants[J]. Southern Horticulture,2009,20(3):3-6.
[23]Long F,Zhang J. Plant regeneration from cotyledon and hypocotyl explants of pepper in vitro and high efficient system establishment of plant regeneration[J]. Journal of Gansu Agricultural University,2005,40(1):31-37.
[24]Szase A,Nervo G,Fari M. Screening for in vitro shootforming capacity of seedling explants in bell pepper(Capsicum annuum L.)genotypes and efficient plant regeneration using thidiazuron[J]. Plant Cell Reports,1995,14(10):666-669.
[25]黎定军,张宝玺,赵开军,等.辣椒子叶高效植株再生体系的建立[J].园艺学报,2002,29(1):25-29.
[26]成善汉,贺申魁,王红梅,等.辣椒子叶和下胚轴的离体培养及高效再生体系的建立[J].南方园艺,2009,20(3):3-6.
[27]余小林,李乃坚,黄自然,等.辣椒子叶离体培养和植株再生体系的建立[J].园艺学报,2000,27(1):42-46.
[28]孙月芳,陆瑞菊.杂种甜椒的离体快速繁殖[J].植物生理通讯,2001,37(3):215-216.
[29]何晓明,王鸣,王喆之.辣椒叶片离体培养与植株再生[J].西北农业大学学报,1996,24(1):93-96.
[30]曹冬孙,贾士荣.青椒子叶培养及植株再生[J].园艺学报,1993,20(2):171-175.
[31]Li D,Zhao K,Xie B,et al. Establishment of a highly efficient transformation system for pepper(Capsicum annuum L.)[J]. Plant Cell Reports,2003,21(8):785-788.
[32]曾华,胡宗利,陈国平,等.辣椒离体再生及遗传转化研究进展[J].生命科学研究,2011,15(6):542-549.
[33]Bairwa V K,Kachhwaha S,Kothari S L. Phloroglucinol mediated shoot bud elongation in Capsicum annuum L.[J]. National Academy Science Letters,2012,35(4):331-335.
[34]缪武,刘志敏.辣椒离体培养研究进展[J].辣椒杂志,2005,3(2):1-4.
[35]罗素兰,王鹏程,张转,等.辣椒离体高效再生体系及其卡那霉素筛选体系的建立[J].海南大学学报:自然科学版,2003,21(1):51-57.
[36]董兆龙,陈沁,刘文轩,等.辣椒子叶和下胚轴离体培养再生[J].上海大学学报:自然科学版,2003,9(2):148-152.
[37]Elwan M W M,Elhamahmy M A M. Improved productivity and quality associated with salicylic acid application in greenhouse pepper[J]. Scientia Horticulturae,2009,122(4):521-526.
[38]叶志彪,李汉霞,张健,等.辣椒转基因植株再生[J].植物学报,1993,35(增刊):88-93.
[39]Kim S,Kim S R,Aa C S,et al. Constitutive expression of rice MADS box gene using seed explants in hot pepper(Capsicum annuum L.)[J]. Molecules and Cells,2001,12(2):221-226.