AgNO_3、GA_3、S-3307对水稻花药培养的组合效应及染色体倍性鉴定新方法
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摘要
花药培养技术的应用为水稻育种提供了一条快速有效的途径。但目前花培育种在花药培养方法上仍存在着工作量大、工作效率低的矛盾，技术上仍存在花药培养力低、绿苗加倍率低以及后代鉴定方法贫乏等问题，限制了花培技术在育种中的应用。本文针对以上问题，结合实验室条件，做了一些研究，结果如下：
1．比较了AgNO_3、S-3307和GA_3三种物质对水稻花药培养的单一和组合作用效应。在愈伤组织诱导和分化培养基中分别添加AgNO_3、S-3307和GA_3，分别促进了愈伤组织诱导率和绿苗分化率的提高；其中AgNO_3对水稻花培愈伤诱导率的作用出现反弹现象，即先抑制诱导率，而后为促进作用。而三种物质两两组合中，都可以得到较佳组合，与对照相比，显著提高了愈伤组织诱导率和绿苗分化率；S-3307分别与其它两种物质组合时，两种物质之间表现为互相抑制作用；AgNO_3和GA_3两两组合则为互相促进作用。
2．对气孔大小和气孔密度、保卫细胞叶绿体数目以及一系列植株形态进行观测统计分析，探索快速鉴定水稻花培植株染色体倍性鉴定简便有效的方法。对水稻花培植株单倍体和二倍体植株的气孔性状观测发现，单倍体和二倍体植株的气孔长度和叶绿体数在1％水平差异显著。对水稻再生植株不同叶位的叶长和叶宽的观测发现，倒2叶长可以区分水稻花培单倍体和二倍体植株；颖花性状、花药大小、穗长等数据的统计分析表明，颖长、颖宽、花药长、穗长也可作为水稻倍性鉴定的指标。为了提高叶片气孔性状鉴定植株倍性水平的效率，在对观测植物叶片气孔的各种方法进行比较研究的基础上，探索出用于水稻叶片气孔性状观测的“印迹液撕取法”。该方法步骤简单易掌握，可通过显微镜摄影直接在照片上观测气孔大小、气孔密度和叶绿体数等性状。大大提高了水稻植株染色体倍性水平早期鉴定的可行性。
Since the first haploid plants were regenerated from rice anthers in 1968, anther culture has been widely integrated in breeding programmes of this cereal crop, thus permitting rapid production of homozygous DH lines from F1 hybrids. However, two problems were encountered with the development of rice anther culture. The first was low efficiency of anther culturability of rice, especially of indica. The second was cumbersome ploidy determination methods and chromosome duplication of regenerated plantlets obtained from rice anther culture. Some researches had been done and the primary results were summarized as following:
1. The single and combinated effects of AgNO3, S-3307 and GA3 on anther culture of indica rice varieties were studied. After AgNO3 S-3307 and GA3 were supplemented to the callus induction medium or regeneration medium separately, the ratio of callus induction (CI) and frequency of green plantlet regeneration (GPR) increased significantly. Interestingly, a rebounding phenomenon of CI was found when AgNO3 was supplemented to the callus induction medium. The combination of two of the three supplements could promote CI and GPR. compared with CK. When S-3307 combinated with one of the other two, inhibitory actions were observed. The combination of AgNO3 and GA3 could promote CI and GPR. Thus, the supplement on rice anther culture might be optimal or not, dependent on the different combinated effects of materials supplemented to the medium.
2. Stomatal (stomatal length, stomatal width, stomatal frequency, chloroplast number of guard cell) and morphological characters (plant height, length and width of penultimate leaves, length and width of antepenultimate leaves, glume length, glume width, anther length, anther width, panicle length) were evaluated in order to explore rapid indirect methods to identify haploids from diploids in anther-derived japonica rice plants. The results revealed that seven traits from the fourteen characters could be used to determine the ploidy levels of rice plants successfully. Of stomatal characters, stomatal length and chloroplast number could identify haploid plants from diploids. Of a series of morphological characters, five traits were available in determination of ploidy levels of rice plants. The results and methods performed in this study indicated that the efficiency of determining ploidy levels of anther-derived rice
plants could be increased rapidly and the early determination of anther-derived rice plants could benefit anther culture before chromosome-doubling treatment.
1．比较了AgNO_3、S-3307和GA_3三种物质对水稻花药培养的单一和组合作用效应。在愈伤组织诱导和分化培养基中分别添加AgNO_3、S-3307和GA_3，分别促进了愈伤组织诱导率和绿苗分化率的提高；其中AgNO_3对水稻花培愈伤诱导率的作用出现反弹现象，即先抑制诱导率，而后为促进作用。而三种物质两两组合中，都可以得到较佳组合，与对照相比，显著提高了愈伤组织诱导率和绿苗分化率；S-3307分别与其它两种物质组合时，两种物质之间表现为互相抑制作用；AgNO_3和GA_3两两组合则为互相促进作用。
2．对气孔大小和气孔密度、保卫细胞叶绿体数目以及一系列植株形态进行观测统计分析，探索快速鉴定水稻花培植株染色体倍性鉴定简便有效的方法。对水稻花培植株单倍体和二倍体植株的气孔性状观测发现，单倍体和二倍体植株的气孔长度和叶绿体数在1％水平差异显著。对水稻再生植株不同叶位的叶长和叶宽的观测发现，倒2叶长可以区分水稻花培单倍体和二倍体植株；颖花性状、花药大小、穗长等数据的统计分析表明，颖长、颖宽、花药长、穗长也可作为水稻倍性鉴定的指标。为了提高叶片气孔性状鉴定植株倍性水平的效率，在对观测植物叶片气孔的各种方法进行比较研究的基础上，探索出用于水稻叶片气孔性状观测的“印迹液撕取法”。该方法步骤简单易掌握，可通过显微镜摄影直接在照片上观测气孔大小、气孔密度和叶绿体数等性状。大大提高了水稻植株染色体倍性水平早期鉴定的可行性。
Since the first haploid plants were regenerated from rice anthers in 1968, anther culture has been widely integrated in breeding programmes of this cereal crop, thus permitting rapid production of homozygous DH lines from F1 hybrids. However, two problems were encountered with the development of rice anther culture. The first was low efficiency of anther culturability of rice, especially of indica. The second was cumbersome ploidy determination methods and chromosome duplication of regenerated plantlets obtained from rice anther culture. Some researches had been done and the primary results were summarized as following:
1. The single and combinated effects of AgNO3, S-3307 and GA3 on anther culture of indica rice varieties were studied. After AgNO3 S-3307 and GA3 were supplemented to the callus induction medium or regeneration medium separately, the ratio of callus induction (CI) and frequency of green plantlet regeneration (GPR) increased significantly. Interestingly, a rebounding phenomenon of CI was found when AgNO3 was supplemented to the callus induction medium. The combination of two of the three supplements could promote CI and GPR. compared with CK. When S-3307 combinated with one of the other two, inhibitory actions were observed. The combination of AgNO3 and GA3 could promote CI and GPR. Thus, the supplement on rice anther culture might be optimal or not, dependent on the different combinated effects of materials supplemented to the medium.
2. Stomatal (stomatal length, stomatal width, stomatal frequency, chloroplast number of guard cell) and morphological characters (plant height, length and width of penultimate leaves, length and width of antepenultimate leaves, glume length, glume width, anther length, anther width, panicle length) were evaluated in order to explore rapid indirect methods to identify haploids from diploids in anther-derived japonica rice plants. The results revealed that seven traits from the fourteen characters could be used to determine the ploidy levels of rice plants successfully. Of stomatal characters, stomatal length and chloroplast number could identify haploid plants from diploids. Of a series of morphological characters, five traits were available in determination of ploidy levels of rice plants. The results and methods performed in this study indicated that the efficiency of determining ploidy levels of anther-derived rice
plants could be increased rapidly and the early determination of anther-derived rice plants could benefit anther culture before chromosome-doubling treatment.
引文
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陈英．籼稻花药培养基的筛选，见：植物细胞工程与育种．北京工业大学山版社，1990：25-30
陈兆贵，韦鹏霄．光(温)敏核不育系水稻花药培养及遗传育种的研究进展．广西农业生物科学，1994，18(1)：34-37
冯英，薛庆中．直接产生抗除草剂转基因水稻纯系的新方法．农业生物技术学报，2001，9(4)：330-333
葛台明，余毓君．小麦花药培养的基因型和培养基效应研究．华中农业大学学报，1996，15(5)：400-413
何平，沈利爽，陆朝福，陈英，朱立煌．水稻花药培养力的遗传分析及基因定位．遗传学报，1998，25(4)：337-344
何涛，罗科，韩思怀，郭学兴．水稻花约培养中培养力相关因素的研究．西南农业学报，2002，15(4)： 15-18
胡忠，梁汉兴，黄仕周，佃静波．水稻花约培养方法的改进，见：花约培养学术讨论会论文集．科学出版社，1977：93-98
季彪俊，江树业，陈启锋，李维明，祁建民，毛大梅．活性炭在水稻花药培养中的作用．福建农业大学学报，1998，27(1)：16-19
李浚明主编．组织培养教程．北京农大出版社，1995：119-147
李文泽，胡含．在花药培养中预处理的作用机理，遗传，1995，17(增刊)：13-18
李欣，于恒秀，杨成根，程祝宽，顾铭洪．生根粉与植物激素在粳籼杂交花药培养中的应用研究．江苏农业研究，2001，22(2)：1-6
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