摘要
本研究以慈姑(Sagittaria trifolia Linn.var.sinensis Makino)茎尖为外植体,进行了组织培养快速繁殖技术的研究,筛选出诱导茎尖分化的最适启动培养基,并探讨了继代增殖培养基的最佳激素配比;同时,以试管苗为基础,对试管慈姑诱导形成的主要影响因素进行了初步研究。试验主要研究结果如下:
1.外植体的消毒处理试验结果表明,慈姑茎尖消毒的最佳方法是:以72%农用硫酸链霉素处理20min,再用70%乙醇表面消毒30s,最后用0.1%升汞处理5min。可使成活率达到53.7%,而污染率降至43.9%。
2.建立了有效的慈姑组培扩繁体系。以MS为基本培养基,通过调节6-BA和NAA的不同浓度配比,得出适宜慈姑茎尖分化的启动培养基为MS+6-BA1.0mg/L+NAA0.3mg/L,分化率为88.1%;继代增殖的最适培养基为MS+6-BA3.0mg/L+NAA0.1mg/L,最佳的继代周期为6周,增殖系数达8.87。
3.通过正交设计研究蔗糖浓度、激素配比、温度和光照时间4因子对株高、根状茎、试管慈姑等的影响,结果表明,4因子中仅蔗糖浓度对株高有显著影响,随着蔗糖浓度的增加试管苗高度逐渐降低;4因子中仅蔗糖浓度和温度2因子对单株试管苗抽生根状茎的条数有显著影响,以蔗糖浓度30g/L或60g/L和温度22℃时效果较好,可得到数量较多的根状茎;4因子对结慈姑株率和单株结慈姑数都有显著影响,以组合蔗糖浓度=60g/L,激素配比6-BA:NAA=0:0,温度=22℃,光照时间=0h,结慈姑株率和单株结慈姑数均为最高值,为诱导试管慈姑的最佳组合。
4.组培苗移栽试验结果显示:组培苗所结慈姑较种球繁殖苗所结慈姑在平均单个球茎鲜重上增加57.63%。
The shoot tips of Sagittaria trifolia Linn. var. sinensis Makino were used as explant in the study to establish an effective mass propagation technique of tissue culture. We gained the favorable induction medium for shoot tips, and studied the best plant growth regulator proportion for regeneration medium. Meanwhile, we preliminarily studied the major influence factors to microtubers of Sagittaria trifolia Linn. var. sinensis. The main results were as the following:1. The best surface sterilization method for inoculation shoot tips was immersed in 72% streptomycin sulphate for 20 minutes, then dipped in 70% ethanol for 30s, and finally agitated in 0.1% HgCl_2 for 2 minutes. The effect was excellent and the survival rate could up to 53.7% and the pollution rate reduced to 43.9%.2. Effective rapid propagation system in vitro of Chinese Arrowhead was set up. Through regulating the concentration of 6-BA and NAA in MS medium, We found out that the favorable induction medium for shoot tips was MS + 6-BA 1.0 mg/L + NAA 0.3mg/L, and the differentiation rate was 88.1%; The optimum medium for regeneration was MS + 6-BA 3.0 mg/L + NAA 0.1mg/L, and the best multiplicative period was 6 weeks, the propagation coefficient could up to 8.87.3. Through orthogonal experiment, carried on research on sucrose concentration. 6-BA:NAA, temperature and illumination time 4 factors to the influence of height of plantlet, microrhizome and microtuber. The results showed that only sucrose concentration could influence to height of plantlet significantly, and with the rising of sucrose concentration, height of plantlet presented the downward trend; Both sucrose concentration and temperature 2 factors could influence to number of microrhizome per plantlet significantly, when we used 30g/L or 60g/L sucrose concentration and 22℃temperature, we got much more microrhizome; All of 4 factors could influence to ratio of microtuber and number of microtuber per plantlet significantly, and the combination of sucrose concentration=60g/L, 6-BA:NAA=0:0, temperature=22℃, illumination time =0h was the best combination to induce tubers in vitro of Sagittaria trifolia Linn. var. sinensis Makino, then we could gain the highest value of both ratio of microtuber and number of microtuber per plantlet.
4. Transplantation experiment of planlets showed that the fresh weight on per tuber formed by plantlets increased 57.63% compared with by seedlings.
引文
1.白淑霞,安忠民,冯学赞,王静.马铃薯试管薯诱导因子研究.中国马铃薯,2001,15(5):271-273
2.白淑霞,安忠民,王静,冯学赞.不同培养方式对马铃薯试管苗生长与试管薯诱导的影响.中国生态农业学报,2002,10(2):40-41
3.曹碚生,蔡汉,李良俊,丁存明.温度和光照等因素对荸荠试管球茎形成的影响.扬州大学学报(自然科学版),2000,3(2):45-48
4.曹侃,赵有为,翁训珠.茨菇生物学特性及其丰产栽培技术的初步研究.园艺学报,1962,Z1:305-312
5.陈果香.夏茭白套栽慈姑的研究.长江蔬菜,1990(1).36-38
6.陈家宽.中国慈姑属的系统与进化植物学研究.武汉:武汉大学出版社,1989,1-63
7.催翠,何凤发,王季春,周清元,黄远新,唐道斌.光照时间和碳源对试管薯形成的影响.西南农业大学学报,2001,23(6):547-548
8.崔瑾,李式军.水杨酸对芋试管球茎发育的影响.南京农业大学学报,2003,26(1):97-99
9.党玉丽,刘忠玲,宁爱民,宛新生,周占芳.不同苗龄及碳源对马铃薯试管薯诱导的影响.河南农业大学学报,2004,38(3):292-295
10.杜光伟,易清明,陈家宽.运用AP-PCR对中国慈姑属内亲缘关系的研究植物.分类学报,1998,36(3):216-221
11.丰锋.提高试管苗移栽成活率的技术研究.中国南方果树,2001,30(4):63-64
12.郭得平,应振土,Shah GA.植物激素与马铃薯块茎形成.植物生理学通讯,1991,27(2):130-133
13.韩德俊,陈耀锋,李春莲,任慧利.水杨酸对马铃薯试管微薯形成的影响研究.曲北植物学报,1999,19(3):428-433
14.胡云海,蒋先明.植物激素对微型薯形成的影响.马铃薯杂志,1992,6(1):14-22
15.黄新芳,孔庆东,柯卫东,刘义满,周国林.慈姑种质资源保存应注意的问题.种子科技,1997(3),40
16.黄新芳,孔庆东,柯卫东.华夏慈姑种质资源及其研究.长江蔬菜,1998a(11):1-3
17.黄新芳,孔庆东,柯卫东.华夏慈姑种质资源及其研究.长江蔬菜,1998b(12):1-3
18.黄忠兴,王海.慈姑栽培技术要点.云南农业,1999(6):8-9
19.金波主编.中国多年生蔬菜.中国农业出版社,1997,200-201
20.柯卫东,黄新芳,李双梅,叶元英,刘玉平,孔庆东.水生蔬菜种质资源研究概况.长江蔬菜(2001年增刊),水生蔬菜学术及产业化研讨会论文集,武汉,2001a,15-24
21.柯卫东,彭静,刘玉平,黄新芳.试管藕诱导技术研究.武汉植物学研究,2001b,19(2):173-175
22.柯卫东,刘义满,吴祝平主编.绿色食品水生蔬菜标准化生产技术.中国农业出版社,2003,188-199
23.孔庆东主编.中国水生蔬菜品种资源.中国农业出版社,2004,145
24.李浚明.植物组织培养教程(第2版).中国农业大学出版社,2002,267
25.李曙轩主编.蔬菜栽培学各论(南方本).农业出版社,1985,401
26.连勇.马铃薯试管薯诱导与应用.马铃薯杂志,1995,9(4):237-240
27.连勇,邹颖,杨宏福,金黎平,卞春松.马铃薯试管薯发育机理的研究——外源诱导剂对试管薯形成的影响.马铃薯杂志,1996a,10(3):130-133
28.连勇,邹颖,杨宏福,金黎平,卞春松.马铃薯试管薯发育机理的研究——温度对试管薯形成的影响.马铃薯杂志,1996b,10(3):133-137
29.连勇,刘蕾,屈冬玉,金黎平,纪颖彪,卞春松,杨琳.GA_3、IAA和C/N对马铃薯试管根状茎及试管薯形成的影响.马铃薯杂志,1999,13(1):3-6
30.连勇,邹颖,东惠茹,金黎平,林桓.马铃薯试管薯形成过程中儿种内源激素的变化.园。。艺学报,2002,29(6):537-541
31.梁艳,陈典,黄晓梅,陈青奇.大蒜试管微鳞苇形成的激素筛选研究.东北农业大学学报,2005,36(5):589-592
32.缪建国,潘宜春.里下河地区晚水慈姑高产技术.长江蔬菜,1999(8):11-12
33.刘高琼,李式军,张学平.大蒜试管鳞茎微繁技术研究.南京农业大学学报,1996,19(3):31-36
34.刘高琼,李式军,张学平.温光条件对离体大蒜鳞茎形成和内源激素变化的效应.园艺。学报,1997,24(2):165-169
35.柳俊,谢从华,黄大恩.马铃薯试管块茎形成机制的研究——暗处理与光照时间对试管块茎形成的影响.马铃薯杂志,1994,8(3):138-141
36.柳俊,谢从华,黄大恩,廖勇,吴承金.马铃薯试管块茎形成机制的研究——BA对试管块茎形成与膨大的影响.马铃薯杂志,1995,9(1):7-11
37.柳俊.马铃薯试管块茎的形成机理及块茎形成调控.[博士学位论文].武汉:华中农业大学图书馆,2001
38.刘仁祥.活性炭和无机盐对马铃薯试管薯的诱导效应(简报).植物生理学通讯,2001,37(4):295-298
39.刘玉平,柯卫东.慈姑的组织培养.植物生理学通讯,2002,38(3):244
40.刘玉平,柯卫尔,黄新芳,彭静.试管芋诱导的研究.园艺学报,2003,30(1):43-46
41.龙庆海,赵兵荣.早稻茬慈姑的配套栽培技术.江苏农业科学,1987(12),25-26
42.罗列,王永龙,徐爱琴,芮根华,徐霞,陈浩.慈姑叶根茎同生关系及其配套栽培技术的研究.安徽农业科学,2004,32(5):950-952
43.蒙美莲,刘梦芸,门福义,张宏志.赤霉素和脱落酸对马铃薯块茎形成的影响.马铃薯杂志,1994,8(3):134-137
44.裴鉴,单人骅.华东水生维管束植物.北京:科学出版社,1952,18-19
45.施国新,徐祥生,陈维培.慈姑主茎和根状茎的形态解剖学研究.南京师大学报(自然科学版),1989a,12(1):73-81
46.施国新,徐祥生,陈维培.慈姑根状茎的起源和球茎的膨大研究.武汉植物学研究,1989b,7(3):205-209
47.孙祥钟.中国植物志.第8卷.北京:科学出版社,1992,128-136
48.王爱勤,周歧伟,何龙飞,许鸿源,杨美纯.百合试管结鳞茎的研究.广西农业大学学报,1998,17(1):71-75
49.王军.马铃薯快繁技术的某些进展.中国马铃薯种薯生产研讨会论文集,1992,9-15
50.汪俏梅,郭得平编著.植物激素与蔬菜的生长发育.中国农业出版社,2002,52-55
51.韦启光,韦兆统,喻忠刚,覃金桥,韦宗赛.莲藕套种慈姑栽培技术.当代蔬菜,2006,2:27
52.鄢铮,郭德章.植物激素对马铃薯试管薯形成的影响.中国马铃薯,2004,18(2):84-86
53.夏光华,方家齐.夏茭后茬慈姑高产栽培技术.江苏农业科学,1985(8),23-24
54.肖汝松.慈姑优质高产栽培技术.云南农业,1998(3):13
55.熊正琴,李式军,周燮,刘高琼,黄保健.茉莉酸甲酯和水杨酸促进大蒜试管鳞茎的形成.园艺学报,1999,26(6):408-409
56.颜素珠主编.中国水生高等植物图说.北京:科学出版社,1983,220-225
57.杨文玉.不同组织培养条件对马铃薯试管微型薯的影响.马铃薯杂志,1996,10(1):20-23
58.叶静渊.我国水生蔬菜的栽培起源与分布.长江蔬菜(2001年增刊),水生蔬菜学术及产业化研讨会论文集,武汉,2001,4-12
59.张志军,李会珍,姚宏亮,周伟军.多效唑对马铃薯试管苗生长和块茎形成的影响.浙江大学学报(农业与生命科学版),2004,30(3):318-322
60.赵有为主编.中国水生蔬菜.中国农业出版社,1997,66-69
61.庄志鸿,刘建.百合鳞片试管结鳞茎的研究.福建农业科技,2002,2:26
62. Abbot A J, Belcher A R. Potato tuber formation in vitro. Plant tissue culture and its agricultural application. 1986,113-122
63. Allemann J, Hammes P S. Effect of photoperiod on tuberization in the Livingstone potato (Plectranthus esculentus N.E.Br. Lamiaceae). Field Crops Research, 2006, 98:76-81
64. Dobranszki J, Mandi M. Induction of in vitro tuberization by short day period and dark treatment of potato shoots grown on hormone-free medium. Acta Biol Hung, 1993,44:411-420
65. Dodds J H, Silva-Rodriguez D, Tovar P. Micropropagation of potato (Solanum tuberosum L.). Baja YSP (ed) Biotechnology in agriculture and forestry, 1992,19:91-106
66. Ewing E E. Heat stress and the tuberization stimulus. Am Potato J, 1981,58:31-49
67. Garner N, Blake J. The induction and development of potato microtubers in vitro on media free of growth regulating substances. Annals of Botany, 1989, 63:663-674
68. Gopal J, Minocha J L, Dhaliwal H S. Microtuberization in potato (Solarium tuberosum L.). Plant Cell Reports, 1998,17:794-798
69. Hussey G, Stacey N J. Factors affecting the formation of in vitro tubers of potato (Solarium tuberosum L.). Ann Bot, 1984,53:565-578
70. Jimenez E, Perez N, Feria de M, Barbon R, Capote A, Chavez M, Qyuiala E, Perez J C. Improved production of potato microtubers using a temporary immersion system. Plant Cell Tissue and Organ Culture, 1999, 59(1): 19-23
71. Khuri S, Moorby J. Investigation into the role of sucrose in potato cv. Estima microtuber production in vitro . Ann Bot, 1995,75:295-303
72. Koda Y, Okazawa Y. Influences on environmental, hormonal and nutritional factors on potato tuberization in vitro. Crop Sci ,1983,52:582-591
73. Koda Y K, Omer E A, Yoshihara T, Shibata H, Sakamura S, Okazawa Y. Isolation of a specific potato tuber-inducing substance from potato leaves. Plant Cell Physiol, 1988, 29(6): 1047-1051
74. Krauss A. Tuberization and abscisic acid content in Solanum tuberosum as affected by nitrogen nutrition. Potato Res, 1978, 21:183-193
75. Kumar D, Wareing P F. Studies on tuberization in Solanum andigena. II. Growth hormones and tuberization. New Phytol, 1974, 73:833-840
76. Lovell P H, Booth A. Effect of gibberellin acid on growth, tuber formation and carbohydrate distribution in Solanum tuberosum L. New Physiol, 1967, 66: 525-537
77. Muhammad A A, Trevor A V. Induction of microtubers in vitro from stem segments of Solanum tuberosum L., S. commersonii Dun. And S. acaule Bitt. Scientia Horticulturae, 1997,70:231-235
78. Ranalli P, Bassi F, Ruaro G, Del Re P, Dicandilo M, Mandolino G. Microtuber and minituber production and field performance compared with normal tubers . Potato Res, 1994,37:383-391
79. Sharma N, Kaur N, Gupta A K. Effects of gibberellic acid and chlorocholine chloride on tuberisation and growth of potato (Solanum tuberosum L.). J of the Science of Food and Agriculture, 1998,78(4) :466-470
80. Silva J A B, Otoni W C, Martinez C A, Dias L M, Silva M A P. Microtuberization of Andean potato species (Solanum spp.) as affected by salinity. Scientia Horticulturae, 2001,89:91-101
81. Simoko 1. Effects of kinetin, paclobutrazol and their interactions on the microtuberization of potato stem segments cultured in vitro in the light. Plant Growth Regulation, 1993,12:23-27
82. Takahashi K, Fujino K, Kikuta Y, Koda Y. Expansion of potato cells in response to jasmonic acid. Plant Sci, 1994, 100:3-8
83. Vreugdenhil D, Struik P C . An integrated view of the hormonal regulalion of tuber formation in potato (Solanum tuberosum L.). Physiol Plant, 1989, 75:525-531
84. Yu W C, Joyce P J, Cameron D C, McCown BH. Sucrose utilization during potato microtuber growth in bioreactors. Plant Cell Reports, 2000, 19:407-413
85. Zobayed S M A, Armstrong J, Armstrong W. Micropropagation of potato: Evaluation of closed, diffusive and forced ventilation on growth and tuberization. Annals of Botany, 2001,87: 53-59