玉米体细胞抗盐突变体的筛选及耐盐性鉴定
摘要
玉米是一种主要农作物，同时是盐敏感作物，提高玉米的耐盐性，培育玉米耐盐品种具有重要的意义。本试验利用玉米幼胚作为外植体诱导愈伤组织，并以NaCl为筛选剂，采用多步选择法，先在含1.0％-1.5％-2.0％NaCl的筛选培养基上连续筛选三次，然后转入无盐培养基上培养三代，得到耐盐愈伤组织变异系。分化出的R_0代再生植株，经温室缓苗后移栽到大田，于开花期自交授粉得到R_1代种子。测定不同浓度盐胁迫下愈伤组织相对生长量、干物质累积、游离脯氨酸含量和Na~+、K~+含量，并测定1.0％NaCl胁迫下R_0代再生株叶片的可溶性糖和相对电导率以及不同浓度盐胁迫下R_1代种子的发芽率、R_1代幼苗的株高、根条数、主根长和游离脯氨酸含量等与耐盐性相关的指标，结果如下：
1)随着盐胁迫强度的增加，对照和耐盐变异系的相对生长量下降，但同样胁迫下变异系的相对生长量仍高于对照，变异系干物质的积累大于对照；随着NaCl浓度的升高，对照和变异体的脯氨酸含量均增加，且变异体脯氨酸含量的增幅大于对照；对照和耐盐愈伤组织变异体细胞内Na~+含量在0-1.0％的NaCl浓度范围内随NaCl浓度的升高急剧增加，当NaCl浓度继续增加时，对照细胞内Na~+含量明显降低，而耐盐愈伤组织变异体细胞内仍维持相当高的Na~+水平。
2)在相同的盐胁迫下耐盐变异体R_0植株叶片的相对电导率低于对照，可溶性糖含量和脯氨酸含量高于对照，愈伤组织水平的高脯氨酸特性在R_0代再生植株得以保持，表明变异系的R_0代再生植株是耐盐的。玉米种子发芽对盐极其敏感，随着盐浓度的增加，R_1代种子发芽率下降，但对照发芽率下降幅度大于耐盐变异体R_1种子，变异系R_1代种子在0.4--2.0％NaCl浓度培养基中的发芽率均高于对照，变异系与对照的苗高、根的条数和主根长随着盐浓度的增加都呈下降趋势，然而相同盐浓度下变异系上述三项指标均高于对照。R_1代
2000级硕＿．L研究生毕业论文
再生植株叶片脯氨酸含量较愈伤组织和巳；代再生植株明显降低，尽管R；代再
生植株游离脯氨酸绝对含量下降，但变异系仍比对照系高，说明变异系积累
脯氨酸的能力强于对照。由此看来我们得到的耐盐变异体的R；代种子确实是
耐盐的，R；代幼苗也具有较强的耐盐性，表明耐盐变异体的耐盐性是可以遗
传的，筛选是有效的。
Maize is important crop, and it is sensitive to salinity. In our country,amount of plough are salines. To improve the adaptive of maize to salinity, breeding new NaCl-tolerant varieties of Maize is urgent. An project on selecting salt-resistant Maize was put on in our lab. The calli were induced from the immature embroys of maize. After the calli were propagated,they were subgenerated in the N6 medium containing NaCl for three times to select NaCl-tolerant variants. The content of NaCl was increased from 1.0%-1.5%-2.0% one by one, then the NaCl-tolerant variants were transferred to NaCl-free medium for three times subgeneration. The relative growth rate of calli ,the content of proline , the ratio of dry material and waterish weight were analysised.The results showed that when increasing NaCl concentration ,the relative growth rate decreased, the proportion of dry weight to fresh weight, the content of pro rised. While suffering the same salt stress, the varietants accaculate more pro and grew more
quickly than the control. It proved the calli were NaCl-tolerant indeed. RO plants were regenerated from NaCl-tolerant callus variants and RI seeds were obtained. The relative electronic conductivity of leaves of the RO variant was lower than the control, but solution sugar content was higher than the control under 1.0%NaCl stress. The germinating rate of R1 generation seeds, the variants were higher than the control, especially the germinating rate of the variant was 20% under 1.6% NaCl pressure, but that of control was zero. In Comparing of the control under the same NaCl stress, the variants seedling had more roots, their seedling were higher. The pro content of the calli and RO plantlet were ten times higher than that of R1 plantlet. However the pro content of the Variants were always higher than that of control. All of the indexes showed that the calli variants and their regenerated plants are
salt-tolerant, and the character of salt-tolerant was inheritable.
1)随着盐胁迫强度的增加，对照和耐盐变异系的相对生长量下降，但同样胁迫下变异系的相对生长量仍高于对照，变异系干物质的积累大于对照；随着NaCl浓度的升高，对照和变异体的脯氨酸含量均增加，且变异体脯氨酸含量的增幅大于对照；对照和耐盐愈伤组织变异体细胞内Na~+含量在0-1.0％的NaCl浓度范围内随NaCl浓度的升高急剧增加，当NaCl浓度继续增加时，对照细胞内Na~+含量明显降低，而耐盐愈伤组织变异体细胞内仍维持相当高的Na~+水平。
2)在相同的盐胁迫下耐盐变异体R_0植株叶片的相对电导率低于对照，可溶性糖含量和脯氨酸含量高于对照，愈伤组织水平的高脯氨酸特性在R_0代再生植株得以保持，表明变异系的R_0代再生植株是耐盐的。玉米种子发芽对盐极其敏感，随着盐浓度的增加，R_1代种子发芽率下降，但对照发芽率下降幅度大于耐盐变异体R_1种子，变异系R_1代种子在0.4--2.0％NaCl浓度培养基中的发芽率均高于对照，变异系与对照的苗高、根的条数和主根长随着盐浓度的增加都呈下降趋势，然而相同盐浓度下变异系上述三项指标均高于对照。R_1代
2000级硕＿．L研究生毕业论文
再生植株叶片脯氨酸含量较愈伤组织和巳；代再生植株明显降低，尽管R；代再
生植株游离脯氨酸绝对含量下降，但变异系仍比对照系高，说明变异系积累
脯氨酸的能力强于对照。由此看来我们得到的耐盐变异体的R；代种子确实是
耐盐的，R；代幼苗也具有较强的耐盐性，表明耐盐变异体的耐盐性是可以遗
传的，筛选是有效的。
Maize is important crop, and it is sensitive to salinity. In our country,amount of plough are salines. To improve the adaptive of maize to salinity, breeding new NaCl-tolerant varieties of Maize is urgent. An project on selecting salt-resistant Maize was put on in our lab. The calli were induced from the immature embroys of maize. After the calli were propagated,they were subgenerated in the N6 medium containing NaCl for three times to select NaCl-tolerant variants. The content of NaCl was increased from 1.0%-1.5%-2.0% one by one, then the NaCl-tolerant variants were transferred to NaCl-free medium for three times subgeneration. The relative growth rate of calli ,the content of proline , the ratio of dry material and waterish weight were analysised.The results showed that when increasing NaCl concentration ,the relative growth rate decreased, the proportion of dry weight to fresh weight, the content of pro rised. While suffering the same salt stress, the varietants accaculate more pro and grew more
quickly than the control. It proved the calli were NaCl-tolerant indeed. RO plants were regenerated from NaCl-tolerant callus variants and RI seeds were obtained. The relative electronic conductivity of leaves of the RO variant was lower than the control, but solution sugar content was higher than the control under 1.0%NaCl stress. The germinating rate of R1 generation seeds, the variants were higher than the control, especially the germinating rate of the variant was 20% under 1.6% NaCl pressure, but that of control was zero. In Comparing of the control under the same NaCl stress, the variants seedling had more roots, their seedling were higher. The pro content of the calli and RO plantlet were ten times higher than that of R1 plantlet. However the pro content of the Variants were always higher than that of control. All of the indexes showed that the calli variants and their regenerated plants are
salt-tolerant, and the character of salt-tolerant was inheritable.
引文
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2．颜昌敬著．植物组织培养手册．1990，上海：上海科学技术出版社
3. Steward F C, M O Mapes, K Mears. Growth and Organized development of cultured cells.Ⅲ．Interpretations of the growth from free cell to carrot plant. Amer. J. Bot, 1988; 45: 709-712
4．生物技术．1991，农业出版社
5．植物生物技术原理与方法．1990，湖南科学技术出版社
6．《细胞工程》俞大新著
7．李海燕．细胞和组织培养技术在植物育种中的应用．天津农林科技，1999，151(5)：45～47
8．高效转基因体系还要尽量避免体细胞无性系变异的发生．东北农业大学学报，31(2)：191～198
9．河南科技．1997年第11期，5～7
10．朱庆麟，陈新民．玉米胚乳愈伤组织诱导及器官发生研究初报．植物生理学通讯．1985(4)：30～32
11．谷明光．诱导玉米花粉植株的初步研究．遗传学报，1975，2(2)：138～142
12．郭仲琛，陆文梁．玉米花粉愈伤组织产生胚状体的细胞形态学研究．植物学报，1984，26(1)：19～23
13. Coumans M.P et al, 1989, Plant Cell Report, 7:618～621
14. Pesitelli S.M, et al,1989, Plant Cell Report, 7:673～676
15. Pesictelli S.M, et al, 1990, Plant Cell Report, 8:628～631
16. Gaillard A,et al, 1991, Plant Cell Report
17. Afele J.C, et al, 1992 Plant Cell Tissue and Organ culture. 28:87～90
18．母秋华，杜娟等．高培养力玉米种质资源及生物技术后代保存利用的研究．玉米科学，1996，4(3)
19．李学红，苏玲，张举红．玉米芽尖培养和丛生芽发生．1997，32(4)：461～465
20. From M.E. Taylor L .P, Walbor V. stable transformation of maize after gene transfer by electroporation. Nature. 1986,319:791～793
21. From M.E, Morrish F, Armstrong C, et al, Bio-Tech. 1990,8:833～839
22. Rhodes C.A, Pierce D. A, Mettler I J, et al, Science, 1988,240:204～207
23. C.E. Green and R.L. Phillips, Plant Regeneration from Tissue of Maize, Crop Science, 1975,15,417～418
24．谢友菊，陈鹏．玉米体细胞胚状体诱导及其植株再生的研究．1986，13(2)：113～119
25．母秋华．提高玉米花粉植株诱导频率的研究．遗传，1981，(4)：25～28
26．母秋华．玉米复合生物技术育种的研究．玉米科学，1994(4)
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