秋水仙素对大蒜茎尖试管苗四倍体的诱导

doi:10.3864/j.issn.0578-1752.2014.15.012

摘要/Abstract

摘要： 【目的】大蒜为无性繁殖作物，难以采用常规育种方法选育新品种，而化学诱变作为种质创制的重要方法之一，在一些作物上获得了较好的效果。探讨秋水仙素对大蒜茎尖组培试管苗多倍体的诱变效应，旨在为创新大蒜种质提供理论依据和技术方法。【方法】选用山东‘苍山蒲棵’、四川‘正月早’和‘新疆紫皮’3个生态区域的大蒜品种，鳞茎经自然晾干通过休眠后，取鳞芽茎尖为外植体接种到不同处理的培养基上，采用二次饱和-D最优设计方法，研究秋水仙素不同浓度及处理时间对大蒜茎尖试管苗成苗率及染色体加倍率的影响，并建立相关回归模型；通过对回归模型进行解析，确定诱变大蒜四倍体的优化方案；采用流式细胞仪检测幼叶和根尖压片染色体计数相结合的方法进行染色体倍性鉴定。【结果】利用流式细胞仪检测的四倍体大蒜植株叶片DNA相对含量是二倍体对照大蒜‘苍山蒲棵’的2倍；通过染色体计数法鉴定二倍体植株根尖染色体为2n=2x=16，而四倍体植株染色体为2n=4x=32。在秋水仙素处理大蒜茎尖诱变四倍体过程中，随秋水仙素处理浓度的升高及时间的延长，3个大蒜品种试管苗成苗率均逐渐降低，且处理浓度的影响大于处理时间;品种间成苗率存在显著差异，以‘正月早’较高，‘新疆紫皮’次之，‘苍山蒲棵’较低，其处理间平均成苗率分别为48.69%、46.73%和41.83%。秋水仙素对大蒜四倍体的诱变效应以处理浓度大于处理时间，不同大蒜品种对秋水仙素的反应存在显著差异，虽然‘苍山蒲棵’、‘正月早’与‘新疆紫皮’的最高四倍体诱导率分别达15.69%、15.69%和23.53%，但大蒜四倍体平均诱导率则以‘正月早’最高，‘苍山蒲棵’次之，‘新疆紫皮’最低，分别为9.15%、7.84%和6.53%。经计算机模拟寻优，求得的3个大蒜品种试管苗成苗率均超过50%的处理方案为秋水仙素浓度为0.268%—0.694%、处理时间为61.1—113.8 h；3个大蒜品种四倍体诱导率均超过5%的秋水仙素处理浓度和时间分别为0.531%—0.580%和79.8 —112.2 h。【结论】不同大蒜品种对秋水仙素处理浓度和处理时间的反应不同，秋水仙素对大蒜四倍体的诱变效应亦存在显著差异，但均以处理浓度的影响大于处理时间，且处理浓度与处理时间存在互作效应。3个大蒜品种成苗率超过50%且四倍体率超过5%的秋水仙素处理浓度为0.531%—0.580%、处理时间为79.8—112.2 h。

关键词: 大蒜 , 试管苗 , 秋水仙素 , 四倍体

Abstract: 【Objective】Garlic is a vegetative crop. It is difficult to use conventional method to breed new varieties. Chemical mutagenesis plays an important role in germplasm innovation, and the effect is admirable in some crops. This paper mainly discussed the mutagenic effect of colchicine on in vitro shoot-tips of garlic with the aim at finding a theoretical and methodical basis for obtaining new garlic germplasm. 【Method】 Garlic cultivars of Cangshan Puke, Zhengyuezao, and Xinjiang purple were collected from different regions. After dormancy broken, the selected shoot-tips from bulbs of garlic were cultured in different media and proceeded to plant regeneration. The rate of plantlet regeneration and tetraploid in vitro shoot-tips were studied in three garlic varieties induced by colchicine exposure. Correlative model of different colchicine concentrations and time duration was established by applying D-optimal design, so as to determine the optimum scheme of the tetraploid induction of garlic. The plant ploidy was identified by Flow Cytometry Analysis and chromosome counts of the root-tips.【Result】The result showed that the DNA content of tetraploid garlic leaf was twice that of diploid garlic Cangshan Puke by flow cytometry analysis. The diploid chromosome number was 2n=2x=16 and the tetraploid chromosome number was 2n=4x=32 by chromosome counting. In colchicine treatment process, the rate of plantlet regeneration was significantly decreased when treated with colchicine at higher concentration and after longer duration. And the concentration of colchicine had a stronger effect on plantlet regeneration than time duration. The difference among the three garlic varieties was significant. As an outstanding example, the plantlet of Cangshan Puke survived significantly fewer than Zhengyuezao and Xinjiang purple. And the plantlet rates were 41.83%, 48.69% and 46.73%, respectively. The concentration of colchicine had a more significant effect on tetraploid induction than the treatment duration. The difference of tetraploid rate among the three varieties was also significant. The tetraploid rates were 15.69%, 15.69% and 23.53% for Cangshan Puke, Zhengyuezao, and Xinjiang purple, respectively. Among the cultivars, the tetraploid rate of Zhengyuezao was the highest (9.15%), Cangshan Puke took the second place (7.84%), and Xinjiang purple was in third place (6.53%). By applying simulation technology, the results showed that over 50% of plantlets had been regenerated and survived when all the varieties were treated by colchicine at a concentration of 0.268%-0.694% after 61.1-113.8 h. While the tetraploid rate was more than 5% when treated at a concentration of 0.531%-0.580% after 79.8-112.2 h. 【Conclusion】 Garlic varieties showed different reactions when treated at various colchicine concentrations and time durations. The difference of mutagenic effect among these was significant. It showed that immersion concentration affected more apparently than the immersion time did. Mutual effect between the concentration and treatment time of colchicine was found. By applying intersection method analysis, the result showed that when treated with colchicine at concentration of 0.531%-0.580% and after 79.8 h-112.2 h duration, more than 50% of plantlets were regenerated and survived, while the tetraploid rate was more than 5%.

Key words: garlic , in vitro plantlet , colchicine , tetraploid

孔素萍1, 2, 曹齐卫2, 孙敬强2, 刘波2, 徐坤1. 秋水仙素对大蒜茎尖试管苗四倍体的诱导[J]. 中国农业科学, 2014, 47(15): 3025-3033.

KONG Su-Ping-1, 2 , CAO Qi-Wei-2, SUN Jing-Qiang-2, LIU Bo-2, XU Kun-1. The Tetraploid Induction of Shoot-Tip Plantlets with Colchicine in Garlic[J]. Scientia Agricultura Sinica, 2014, 47(15): 3025-3033.

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参考文献

[1]Pooler M R，Simon P W. True seed production in garlic. Sex Plant Reprod, 1994, 7: 282-286.

[2]Etoh T. Variation of chromosome pairings in various clones of garlic, Allium sativum L. Mem Fac Agr Kagoshima University，1979, 15: 63-72.

[3]Etoh T. Studies on the sterility in garlic(Allium sativum L.). Mem Fac Agr Kagoshima University, 1985, 21: 77-132.

[4]Etoh T, Noma Y, Nishitarumizu Y, Wakamoto T. Seed productivity and germinability of various garlic (Allium sativum L.) clones collected in Soviet central Asia. Mem Fac Agr Kagoshima University, 1988, (24): 129-139.

[5]Adaniya S, Shirai D. In vitro induction of tetraploid ginger (Zingber offcinale Roscoe) and its pollen fertility and germinability. Scientia Horticulture, 2001, 88: 277-287.

[6]马国斌, 王鸣. 西瓜和甜瓜茎尖离体诱导四倍体. 中国西瓜甜瓜, 2002(4): 4-5.

Ma G B, Wang M. The induction of tetraploid of Watermelon and muskmelon with shoot tips in vitro. China Watermelon and Muskmelon, 2002(4): 4-5. (in Chinese)

[7]周朴华, 何立珍, 刘选明. 组织培养中用秋水仙素诱发黄花菜同源四倍体的研究. 中国农业科学, 1995, 28(4): 49-55.

Zhou P H, He L Z, Liu X M. A Study on the induction of auto tetraploid of daylily with colchicine in vitro. Scientia Agricultura Sinica, 1995, 28(4): 49-55. (in Chinese)

[8]安飞飞, 凡杰, 李庚虎, 简纯平, 李开绵. 华南8号木薯及其四倍体诱导株系叶片蛋白质组分及叶绿素荧光差异分析. 中国农业科学, 2013, 46(19): 3978-3987.

An F F, Fan J, Li G H, Jian C P, Li K M. Comparison of leave proteome and chlorophyll fluorescence of Cassava cv. SC8 and its tetraploid mutants. Scientia Agricultura Sinica, 2013, 46(19): 3978-3987. (in Chinese)

[9]李豆豆, 刘芝龙, 黄明忠, 陈积天, 唐燕琼, 杨光穗. 鹤顶兰四倍体植株的诱导与鉴定. 园艺学报, 2013, 40(10): 2033-2038.

Li D D, Liu Z L, Huang M Z, Chen J T, Tang Y Q, Yang G S. Induction and determination of tetraploid plants of Phaius tankervilleae. Acta Horticulturae Sinica, 2013, 40(10): 2033-2038. (in Chinese)

[10]杨寅桂, 庄勇, 陈龙正, 娄群峰, 陈劲枫. 蔬菜多倍体育种及其应用. 江西农业大学学报, 2006, 28(4): 534-538.

Yang Y G, Zhuang Y, Chen L Z, Lou Q F, Chen J F. Vegetable polyploid and polyploidy breeding. Acta Agriculturae Universitatis Jiangxiensis, 2006, 28(40): 534-538. (in Chinese)

[11]程金水．园林植物遗传育种学．北京: 中国林业出版社, 2000

Cheng J S. Genetic and Breeding of Ornamental Plants. Beijing: China Forestry Publishing House, 2002.(in Chinese)

[12]周香君, 程智慧. 蒜瓣茎尖注射秋水仙素的诱变效果和诱变方法的建立. 西北农业学报, 2008, 17(5): 267-271.

Zhou X J, Cheng Z H. Establishment of Induction gystem of garlic clove-tip by injection with colchicine and effect of mutation. Acta Agriculturae Boreali-occidentalis Sinica, 2008, 17(5): 267-271.(in Chinese)

[13]谢晓玲, 邓自发. 秋水仙素对大蒜生长的影响及多倍体诱导效应分析. 安徽农业科学, 2009, 37(9): 4191-4194.

Xie X L, Deng Z F. Analysis on the influences of colchicine on the growth of Allium sativum and its polyploid induction effect. Journal of Anhui Agriculturae Sinica, 2009, 37(9): 4191- 4194. (in Chinese)

[14]王晓桐, 张金凤. 大蒜多倍体诱导的探究性实验. 青海农林科技, 2010(2): 4-7, 55

Wang X T, Zhang J F. Preliminary study on induction of polyploidy in Allium sativum. Qinghai Forestry Science and Technology, 2010(2): 4-7, 55. (in Chinese)

[15]佘建明, 吴鹤鸣, 陆维忠, 周邢扬, 丁犁平, 璞冶民. 秋水仙素对大蒜离体培养细胞染色体倍性的影响. 核农学通报, 1991, 12(4): 162-164.

She J M, Wu H M, Lu W Z, Zhou X Y, Ding L P, Pu Y M. The effect on chromosome ploidy of in vitro cell of garlic with colchicine. Nuclear Agricultural Science Bulletin, 1991, 12(4): 162-164. (in Chinese)

[16]张素芝, 李纪蓉. 秋水仙素诱导大蒜四倍体的研究. 核农学报, 2006, 20(4): 303-308.

Zhang S Z, Li J R. Study on colchicine induced tetraploid in garlic (Allium sativum L.). Journal of Nuclear Agricultural Sciences, 2006, 20(4): 303-308. (in Chinese)

[17]付金娥, 覃斯华, 李天艳,黄金艳, 李文信, 何毅, 洪日新. 秋水仙素诱变薄皮甜瓜同源四倍体研究. 中国瓜菜, 2008(2): 11-15.

Fu J E, Qin S H, Li S H, Li T Y, Huang J Y, Li W X, He Y, Hong R X. Autotetraploid induction of oriental melon with colchicine. Chinese Cucurbits and Vegetables, 2008(2): 11-15. (in Chinese)

[18]蒋洪恩, 刘孟军. 秋水仙碱诱导枣多倍体的研究. 园艺学报, 2004, 31(5): 647-650.

Jiang H E, Liu M J. Studies on polyploid induction of chinese jujube with colchicine. Acta Horticulturae Sinica, 2004, 31(5): 647-650. (in Chinese)

[19]于锡宏, 张艳梅, 蒋欣梅, 齐月. 秋水仙素诱导榨菜四倍体的效应. 东北农业大学学报, 2013, 44(10): 88-92.

Yu X H, Zhang Y M, Jiang X M, Qi Y. Study on effects of colchicines on tetraploid inducement of tuber mustard. Journal of Northeast Agricultural University, 2013, 44(10): 88-92. (in Chinese)

[20]张文霞, 戴灼华. 洋葱根尖有丝分裂标本制备与观察实验. 遗传学实验指导. 北京: 高等教育出版社, 2007.

Zhang W X, Dai Z H. The Experiment of Onion Root Tip Mitosis Specimen Preparation and Observation. Genetics Experiment Guidance. Beijing: Higher Education Press, 2007. (in Chinese)

[21]唐启义. DPS数据处理系统: 实验设计、统计分析及数据挖掘. 北京: 科学出版社, 2010.

Tang Q Y. Data Processing System: Experimental Design, Statistical Analysis and Data Mining. Beijing: Science Press, 2010. (in Chinese)

[22]郭启高, 张钟灵, 周虹, 梁国鲁, 宋明. 秋水仙碱诱导生姜多倍体的研究. 西南农业大学学报，2000, 22(5): 400-402.

Guo Q G, Zhang Z L, Zhou H, Liang G L, Song M. In vitro induction of polyploidy from colchicine-treated Zingiber officinale Roscse. Journal of Southwest Agricultural University, 2000, 22(5): 400-402. (in Chinese)

[23]Sarathum S, Hegele M, Tantiviwat S, Nanakorn M. Effect of concentration and duration of colchicine treatment on polyploidy induction in Dendrobium scabrilingue L. Europe Journal of Horticultural Sciences, 2010,75(3): 123-127.

[24]Saikat G, Nirmal M, Somnath B, Prakash K D. Induction and identification of tetraploids using in vitro colchicine treatment of Gerbera jamesonii Bolus cv. Sciella. Plant Cell Tissue Organ Culture, 2011,106: 485-493.

[25]李晓艳, 张志东, 李亚东, 吴林, 刘海广. 秋水仙素诱导离体培养越橘多倍体研究. 东北农业大学学报, 2010, 41(1): 38-42.

Li X Y, Zhang Z D, Li Y D, Wu L, Liu H G. Study on polyploid induction of blueberry in vitro with colchicine treatment. Journal of Northeast Agricultural University, 2010, 41(1): 38-42. (in Chinese)

[26]董飞, 陈运起, 刘世琦, 高莉敏, 王传增, 陈伟. 秋水仙素诱导大葱多倍体的研究. 园艺学报, 2011, 38(12): 2381-2386.

Dong F, Chen Y Q, Liu S Q, Gao L M, Wang C Z, Chen W. Colchicines induced polyploid plants and identification in welsh onion. Acta Horticulturae Sinica, 2011, 38(12): 2381-2386. (in Chinese)

[27]郑思乡, 章海龙, 董志渊, 雷小云, 胡秀, 周丽娟.东方百合多倍体诱导及种球繁育的研究. 西南农业大学学报: 自然科学版, 2004, 26(3): 260-263.

Zheng S X, Zhang H L, Dong Z Y, Lei X Y, Hu X, Zhou L J. Polyploidy induction and in vitro bulb propagation of lilium oriental hybrids. Journal of Southwest Agricultural University: Natural Science, 2004, 26(3): 260-263. (in Chinese)

[28]Milene M P, Carlos R C, Wellington R C. A practical and reliable procedure for in vitro induction of tetraploid tomato. Scientia Horticulturae, 2009, 122: 501-505.

[29]王晓红, 谭晓风. 用秋水仙碱诱导非洲菊多倍体的研究. 中南林学院学报, 2005, 25(4): 57-61.

Wang X H, Tan X F. Study of gerbera jamesonii inducing polyploid plants with colchicine. Journal of Central South Forestry University, 2005, 25(4): 57-61. (in Chinese)

[30]刘庆忠, 赵红军, 刘鹏, Mong R, Freddi H. 秋水仙素处理离体叶片获得皇家嘎拉苹果四倍体植株. 果树学报, 2001, 18(1): 7-10.

Liu Q Z, Zhao H J, Liu P, Mong R, Freddi H. Regeneration of tetraploid plants of royal gala apple variety from in vitro leave treated with colchicine. Journal of Fruit Science, 2001, 18(1): 7-10.(in Chinese)

[31]童俊, 叶要妹, 冯彪, 袁玮. 秋水仙素诱导三种紫薇多倍体的研究. 园艺学报, 2009, 36(1): 127-132.

Tong J, Ye Y M, Feng B, Yuan W. Colchicines induced polyploid plants and their identification in three species of Lagerstroemia indica. Acta Horticulturae Sinica, 2009, 36(1): 127-132. (in Chinese)