辣椒自然游离小孢子胚状体诱导研究
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摘要
为建立辣椒自然游离小孢子培养技术体系,以32个辣椒品种为试材,研究基因型(CL-1～CL-32)、基本培养基(MS、Nitsch and Nitsch、NLN、B5)、激素配比(ZT 0.5 mg·L~(-1)+IAA 0.8 mg·L~(-1)、ZT 0.5 mg·L~(-1)+IAA 1.0 mg·L~(-1)、ZT 1.0 mg·L~(-1)+IAA 1.0 mg·L~(-1)、ZT 1.5 mg·L~(-1)+IAA 1.5 mg·L~(-1))、活性炭添加量(0.1,0.5,0.8,1.0 g·L~(-1))对辣椒小孢子胚状体诱导的影响。结果表明,基因型是辣椒小孢子胚状体诱导的关键因素,供试的32个基因型中有18个诱导成功,诱导成功率56.25%,其中F1代诱导成功率达到66.67%,而自交系诱导成功率为0,各基因型中以CL-14诱导率最高,达到29.2%;不同基因型最适培养基不同,5个基因型(CL-4,CL-8,CL-10,CL-12,CL-14)中CL-4、CL-12、CL-14均以Nitsch and Nitsch为基本培养基添加适量激素诱导胚状体最佳;不同基因型的适宜激素浓度配比不同,3个基因型(CL-4、CL-8、CL-14)中CL-8、CL-14以添加0.5 mg·L~(-1) ZT和1.0 mg·L~(-1)IAA效果最好;适量添加活性炭可提高小孢子胚诱导率,CL-4、CL-8、CL-14分别以添加0.8,0.5,0.5 g·L~(-1)诱导效果最好。
To establish the shed-microspore culture technique system in pepper, the experiment was conducted with 32 pepper varieties as materials, the effects of genotypes(CL-1～CL-32), culture medium(MS, Nitsch and Nitsch, NLN, B5), hormones(ZT 0.5 mg·L~(-1)+IAA0.8 mg·L~(-1), ZT 0.5 mg·L~(-1)+IAA 1.0 mg·L~(-1), ZT 1.0 mg·L~(-1)+IAA 1.0 mg·L~(-1), ZT 1.5 mg·L~(-1)+IAA 1.5 mg·L~(-1)), active charcoal(0.1, 0.5,0.8, 1.0 g·L~(-1)) on microspore embryos induction were studied. The results showed that genotype was the key factor for microspore embryoid induction in pepper, 18 of 32 genotypes were embryonic, and the induction success rate was 56.25%; the induction success rate of F1 generation was 66.67%, while that of inbred lines were 0; the induction rate of CL-14 was the highest(29.2%). The optimum medium for different genotypes was different, Nitsch and Nitsch was best for CL-4, CL-12, CL-14 in 5 genotypes(CL-4, CL-8, CL-10, CL-12, CL-14). In 3 genotypes(CL-4, CL-8, CL-14), adding 0.5 mg L-1 ZT and 1.0 mg L-1 IAA was suitable for CL-8,CL-14 to produce embryos successfully. Adding activated carbon could improve the induction rate of microspore embryos, in which CL-4, CL-8 and CL-14 had the best induction effect by adding 0.8, 0.5, 0.5 g·L~(-1), respectively.
To establish the shed-microspore culture technique system in pepper, the experiment was conducted with 32 pepper varieties as materials, the effects of genotypes(CL-1～CL-32), culture medium(MS, Nitsch and Nitsch, NLN, B5), hormones(ZT 0.5 mg·L~(-1)+IAA0.8 mg·L~(-1), ZT 0.5 mg·L~(-1)+IAA 1.0 mg·L~(-1), ZT 1.0 mg·L~(-1)+IAA 1.0 mg·L~(-1), ZT 1.5 mg·L~(-1)+IAA 1.5 mg·L~(-1)), active charcoal(0.1, 0.5,0.8, 1.0 g·L~(-1)) on microspore embryos induction were studied. The results showed that genotype was the key factor for microspore embryoid induction in pepper, 18 of 32 genotypes were embryonic, and the induction success rate was 56.25%; the induction success rate of F1 generation was 66.67%, while that of inbred lines were 0; the induction rate of CL-14 was the highest(29.2%). The optimum medium for different genotypes was different, Nitsch and Nitsch was best for CL-4, CL-12, CL-14 in 5 genotypes(CL-4, CL-8, CL-10, CL-12, CL-14). In 3 genotypes(CL-4, CL-8, CL-14), adding 0.5 mg L-1 ZT and 1.0 mg L-1 IAA was suitable for CL-8,CL-14 to produce embryos successfully. Adding activated carbon could improve the induction rate of microspore embryos, in which CL-4, CL-8 and CL-14 had the best induction effect by adding 0.8, 0.5, 0.5 g·L~(-1), respectively.
引文
[1]王玉英,孙敬三,王敬驹,等.小黑麦和辣椒花粉植株的诱导[J].中国科学,1973(16):104-107.
[2]GEORGEL,NARAYANASWAMYS. Haploid Capsicum through experimental androgenesis[J].Protoplasma, 1973, 78:467-470.
[3]KELL K, SVEN B A. Effects of donor plant temperature,photoperiod, and age on anther culture response of Capsicum annuum L.[J].Euphytica, 1993, 67:105-109.
[4]MITYKO J, FARI M. Problems and results of doubled haploid plant production in pepper(Capsicum annuum L.)via anther and microspore culture[J]. Acta horticulturae, 1997, 447:281-288.
[5]ERCAN N, SENSOY F A, SENSOY S. Influence of growing season and donor plant age on anther culture response of some pepper cultivars(Capsicum annuum L.)[J].Sci hortic, 2006, 110:16-20.
[6]王立浩,张宝玺,郭家珍,等.辣椒花药培养中若干影响因素的研究[J].园艺学报,2004,31(2):199-204.
[7]李春玲,蒋钟仁.甜椒花培新品种‘海花三号’的育成[J].园艺学报,1990,17(1):39-44, 82.
[8]张树根,沈火林,蒋钟仁,等.辣椒花药培养单倍体育种技术研究进展[J].辣椒杂志,2006(3):1-5, 8.
[9]PAUK J, LANTOS C, SOMOGYI G, et al. Tradition, quality and biotechnology in Hungarian spice pepper(Capsicum annuum L.)breeding[J]. Acta agron hung, 2010, 58(3):259-266.
[10]GEMES J A, KRISTOF Z, VAGI P, et al. In vitro anther and isolated microspore culture as tool in sweet and spice pepper breeding[J]. Acta horticulturae, 2009, 829:61-64.
[11]BUYUKALACA S, COMLEKCIOGLU N, ABAK K, et al.Effects of silver nitrate and donor plant growing conditions on production of pepper(Capsicum annuum L.)haploid embryos via anther[J].Eur j hortic sci, 2004, 69:206-209.
[12]LANTON C, JUHASZ A G, VAGI P, et al. Androgenesis induction in microspore culture of sweet pepper(Capsium annuum L.)[J].Plant biotechnol rep, 2012, 6:123-132.
[13]成研,巫东堂,马蓉丽,等.不同游离方式辣椒小孢子的胚胎发生[J].山西农业科学,2012,40(7):705-708.
[14]SUPENA E D J, SUHARSONO S, JACOBSEN E, et al.Successful development of a shed-microspore culture protocol for doubled haploid production in Indonesian hot pepper(Capsicum annuum L.)[J].Plant cell rep, 2006b, 25:1-10.
[15]SUPENA E D J, CUSTERS J B M. Refinement of shed-microspore culture protocol to increase embryos production in hot pepper(Capsicum annuum L.)[J].Sci hortic, 2011, 130:769-774.
[16]ESIN A, TOLGA Y, NEDIM M, et al. Comparison of different androgenesis protocols for doubled haploid plant production on ornamental pepper(Capsicum annuum L.)[J].Turk journal of biology, 2016, 40:944-954.
[17]黄亚杰,李素文,肖瑜,等.辣椒花药培养的初步研究[J].河南农业科学,2014,43(7):112-115, 125.
[18]曹鸣庆,李岩,刘凡.基因型和供体植株生长环境对大白菜游离小孢子胚胎发生的影响[J].华北农学报,1993,8(4):1-6.
[19]戴希刚,施雪萍,包满珠.基因型与培养条件对羽衣甘蓝小孢子胚胎发生的影响[J].植物生理学报,2012,48(11):1113-1119.
[20]张玉苗.不同茄子材料小孢子脱分化及影响因子研究[D].北京:中国农业科学院,2011.
[21]李春玲,佟曦然,朱至清,等.辣(甜)椒游离小孢子培养中的雄核发育和胚胎发生[J].园艺学报,2008,35(11):1613-1620.
[22]韩阳,叶雪凌,冯辉.大白菜小孢子培养影响因素研究[J].中国蔬菜,2006(7):16-18.
[2]GEORGEL,NARAYANASWAMYS. Haploid Capsicum through experimental androgenesis[J].Protoplasma, 1973, 78:467-470.
[3]KELL K, SVEN B A. Effects of donor plant temperature,photoperiod, and age on anther culture response of Capsicum annuum L.[J].Euphytica, 1993, 67:105-109.
[4]MITYKO J, FARI M. Problems and results of doubled haploid plant production in pepper(Capsicum annuum L.)via anther and microspore culture[J]. Acta horticulturae, 1997, 447:281-288.
[5]ERCAN N, SENSOY F A, SENSOY S. Influence of growing season and donor plant age on anther culture response of some pepper cultivars(Capsicum annuum L.)[J].Sci hortic, 2006, 110:16-20.
[6]王立浩,张宝玺,郭家珍,等.辣椒花药培养中若干影响因素的研究[J].园艺学报,2004,31(2):199-204.
[7]李春玲,蒋钟仁.甜椒花培新品种‘海花三号’的育成[J].园艺学报,1990,17(1):39-44, 82.
[8]张树根,沈火林,蒋钟仁,等.辣椒花药培养单倍体育种技术研究进展[J].辣椒杂志,2006(3):1-5, 8.
[9]PAUK J, LANTOS C, SOMOGYI G, et al. Tradition, quality and biotechnology in Hungarian spice pepper(Capsicum annuum L.)breeding[J]. Acta agron hung, 2010, 58(3):259-266.
[10]GEMES J A, KRISTOF Z, VAGI P, et al. In vitro anther and isolated microspore culture as tool in sweet and spice pepper breeding[J]. Acta horticulturae, 2009, 829:61-64.
[11]BUYUKALACA S, COMLEKCIOGLU N, ABAK K, et al.Effects of silver nitrate and donor plant growing conditions on production of pepper(Capsicum annuum L.)haploid embryos via anther[J].Eur j hortic sci, 2004, 69:206-209.
[12]LANTON C, JUHASZ A G, VAGI P, et al. Androgenesis induction in microspore culture of sweet pepper(Capsium annuum L.)[J].Plant biotechnol rep, 2012, 6:123-132.
[13]成研,巫东堂,马蓉丽,等.不同游离方式辣椒小孢子的胚胎发生[J].山西农业科学,2012,40(7):705-708.
[14]SUPENA E D J, SUHARSONO S, JACOBSEN E, et al.Successful development of a shed-microspore culture protocol for doubled haploid production in Indonesian hot pepper(Capsicum annuum L.)[J].Plant cell rep, 2006b, 25:1-10.
[15]SUPENA E D J, CUSTERS J B M. Refinement of shed-microspore culture protocol to increase embryos production in hot pepper(Capsicum annuum L.)[J].Sci hortic, 2011, 130:769-774.
[16]ESIN A, TOLGA Y, NEDIM M, et al. Comparison of different androgenesis protocols for doubled haploid plant production on ornamental pepper(Capsicum annuum L.)[J].Turk journal of biology, 2016, 40:944-954.
[17]黄亚杰,李素文,肖瑜,等.辣椒花药培养的初步研究[J].河南农业科学,2014,43(7):112-115, 125.
[18]曹鸣庆,李岩,刘凡.基因型和供体植株生长环境对大白菜游离小孢子胚胎发生的影响[J].华北农学报,1993,8(4):1-6.
[19]戴希刚,施雪萍,包满珠.基因型与培养条件对羽衣甘蓝小孢子胚胎发生的影响[J].植物生理学报,2012,48(11):1113-1119.
[20]张玉苗.不同茄子材料小孢子脱分化及影响因子研究[D].北京:中国农业科学院,2011.
[21]李春玲,佟曦然,朱至清,等.辣(甜)椒游离小孢子培养中的雄核发育和胚胎发生[J].园艺学报,2008,35(11):1613-1620.
[22]韩阳,叶雪凌,冯辉.大白菜小孢子培养影响因素研究[J].中国蔬菜,2006(7):16-18.