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龙源期刊网 http://www.qikan.com.cn 葫芦种子无菌苗组培体系建立
作者:楼均威 张香琴 宋慧
来源:《广西农学报》2017年第01期
摘要:【目的】建立葫芦种子无菌苗组培体系,为葫芦高频再生体系提供外植体。【方法】以葫芦种子为材料,研究了(A)完整种子、(B)切半种子、(C)萌发种子和(D)剥壳种仁四种种子处理方式和升汞灭菌时间对无菌苗获得的影响。【结果】葫芦种子种皮坚硬,直接作为外植体灭菌困难。接种葫芦完整种子和切半种子,均表现为污染率高(4.4%和51.1%)而萌发率低(0%和4.4%);接种萌发种子虽然萌发率最高(100%)但污染率也最高(66.7%);种子经过剥壳处理和0.1%升汞消毒5min,污染率低(0%)、萌发率高(84.4%)。【结论】剥壳种仁是获得葫芦无菌苗的最佳外植体,经0.1%升汞灭菌5min,接入MS培养基可高效获得无菌苗,而(A)完整种子、(B)切半种子和(C)萌发种子均不宜作为获取葫芦无菌苗的外植体。 关键词:葫芦;种仁;无菌苗
中图分类号:S642文献标识码:A文章编号:1003-4374(2017)01-0047-03 Abstract:【Objective】In order to establish a sterile seedling generation system of Lagenaria siceraria so that explants can be provided for the high frequency regeneration system of Lagenaria siceraria.【Method】This experiment,with Lagenaria siceraria seeds as its subjects,studies how the four forms of Lagenaria siceraria seeds [(A)intact seeds,(B)half seeds,(C)germinating seeds and (D)husk-free seeds]and the time span of sterilizing with mercuric chloridge effect on the acquisition of germ-free buds.【Result】In result,as the husk of Lagenaria siceraria seeds is hard,the explants produced by seeds with husk are hard to sterilize.The intact seeds and half seeds have high pollution rate (4.4% and 51.1%)and low germinating rate (0% and 4.4%);though the germinating seeds had the highest germinating rate (100%),they have the highest pollution rate (66.7%)too;the seeds without husk which are sterilized with 0.1% mercuric chloridge solution for 5 minutes are not polluted,and 84.4% of those seeds are germinated.【Conclusion】In conclusion,husk-free seeds are the ideal explants which can provide germ-free buds through being cultivated in MS culture media after being sterilized with 0.1% mercuric chloridge solution for 5 minutes,and (A)intact seeds,(B)half seeds and (C)germinating seeds are not the suitable explants for being cultivated as Lagenaria siceraria germ-free buds. Key words: Lagenaria siceraria;husk-free seeds;germ-free buds葫蘆(Lagenaria siceraria),隶属葫芦科葫芦属,别名瓠子、扁蒲、瓠瓜。它根系发达,高抗西瓜枯萎病,作为砧木已广泛应用于西瓜嫁接生产[1],有效提高西瓜的适应性。培育抗病耐逆、抗早衰的砧木品种一直是葫芦育种的重要目标,但经过长期定向选育,葫芦种群遗传背景日渐狭窄,很多
龙源期刊网 http://www.qikan.com.cn 优异抗性在日益恶化的生态条件下逐渐退化,大大降低葫芦抗性育种的亲本选择范围,减缓选育进程[2-3]。
转基因技术是种质创新和品种改良的便捷途径,建立无菌苗组培体系更是利用遗传转化导入优良基因的重要前提[4]。瓜类作物中,西瓜、黄瓜、甜瓜和南瓜具有较高经济价值,研究较深入,已经成功建立了离体再生体系[5-9],葫芦因为近年来能够作为砧木有效解决瓜类生产连作障碍问题,逐渐得到重视,但是建立葫芦无菌苗组培体系、高频再生体系等基础研究还很薄弱,制约了转基因技术在葫芦抗性育种中的应用。为此,本试验旨在参考其他瓜类作物相关研究的基础上,以葫芦种子为材料,建立无菌苗组培体系。 1材料和方法 1.1试验材料
葫芦种子由宁波市农业科学院蔬菜研究所繁育提供。 1.2试验方法
1.2.1外植体处理方式选择颗粒饱满、大小一致的葫芦种子,利用四种方式处理外植体(图1),包括(A)完整种子、(B)切半种子、(C)萌发种子和(D)剥壳种仁。 1.2.2外植体灭菌时间种子处理后,首先用清水洗净,在超净工作台上用75%酒精表面消毒30s,无菌水清洗一次,置于0.1%升汞中消毒,消毒时间分别设置5、15、10、20和30min(见表1),之后用无菌水清洗4次。每个处理以5颗种子/瓶的接种密度,接种于MS培养基上,重复三次。接种后在光照培养箱培养,条件为25℃和12h/d光周期。
1.2.3数据统计与分析分别在接种7天、20天和30天,统计污染种子数目、萌发种子数目和无菌苗数,依照下列公式计算污染率、萌发率。 污染率=(污染种子数/