陆地棉隐性核不育系花器发育的形态解剖特征及生理生化特性研究
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摘要
杂种优势是植物界普遍存在的一种现象,植物雄性不育系在农作物杂交育种上有着广泛的应用前景,是杂交育种最重要的方法之一。实践证明棉花具有明显的杂种优势。由于棉花上还没有理想的雄性不育三系,隐性核不育系是目前应用于棉花杂种优势最重要的途径之一,为了揭示棉花细胞核雄性不育的细胞分子学机制,为棉花雄性不育系的选育提供理论依据,本研究采用石蜡切片技术和一系列生化方法,从形态学,细胞学和生理生化水平,对三个棉花隐性核雄性不育系(抗A_3、阆A和1355A)花粉败育机理进行了较系统的研究。研究结果如下:
1.三个不育系中不育株的花器均略小于可育株,不育株雄蕊的花丝极短,花药小而干瘪且紧缩在花柱下部。3个不育系的不育株和可育株相比,花器官在花瓣大小、花丝长度和柱头超出花药长度的差异极显著;花药个数和不育株中花粉数量差异分别为,1355A极显著,抗A3显著,阆A不显著;子房大小的差异分别为,抗A3显著,阆A不显著,1355A不显著;胚珠个数和大小的差异分别为,抗A3不显著,阆A显著,1355A显著。三个不育系中不育株的花器官形态比较发现,抗A3退化较好且制种产量潜力大,阆A退化不够好但具早熟性,1355A退化很好又具形态抗虫性但配合力较低。我们应根据这三个不育系各自的特点,有针对性地对其加以选育,使其在生产上发挥自身优势,以获得最佳利用效果。
2.对三个不育系的不育株和可育株小孢子发生的细胞学观察结果表明,抗A3不育株小孢子发生败育的时期主要是减数分裂Ⅰ时期;造孢细胞和PMC退化导致雄性不育的主要细胞形态学特征是:PMC在形成过程中相互粘连,减数分裂时染色体不配对、PMC出现出芽式增殖、绒毡层细胞过早退化或膨大畸形、胼胝质沉积、出现细胞质穿壁现象,单核期细胞质解体出现高度液泡化。阆A不育株小孢子发生败育的时期主要是单核靠边期;造孢细胞和PMC退化导致雄性不育的主要细胞形态学特征是:初生造孢细胞解体不能形成正常的PMC,减数分裂时期染色体分离杂乱、细胞质分裂异常,单核期细胞核和细胞质解体、外壁不能正常发育长出刺,在双核期营养细胞和生殖细胞先后解体。1355A不育株小孢子发生败育的时期主要是单核期;造孢细胞和PMC退化导致雄性不育的主要细胞形态学特征是:在单核期细胞核和细胞质解体、外壁发育异常不能长出刺且外壁增厚明显,双核期营养细胞和生殖细胞先后解体。
3.对三个不育系的不育株和可育株的生理生化变化研究发现:三类不育株花药中可溶性糖含量偏高,缺乏淀粉积累,表明可溶性糖的运转或淀粉的合成受阻,细胞处于饥饿状态而导致退化解体;不育株的可溶性蛋白质含量远低于可育株,表明其蛋白质合成受阻,致使细胞原生质体组成异常,不能进行正常的生命代谢,最终导致细胞退化解体;不育株的POD同工酶在花药发育过程中酶带条数少于可育株,且POD活性明显偏高,这表明了不育株与可育株之间体内代谢强度的差异,也说明酶的变化与雄性不育现象有着直接的关系。三类不育株花药的MDA含量和电导率变化从PMC减数分裂时期到成熟花粉时期都呈上升趋势,且上升趋势相同,MDA含量和花药提取液的电导率从PMC减数分裂期到双核期变化较快,以后趋于平缓,由此可以推断三类不育株花粉败育的关键时期可能均为PMC减数分裂时期到双核期。对三类不育株这一系列生理生化变化的比较发现,各不育株的不育基因控制其体内的生理生化变化从而导致败育的侧重点不同,ms_(14)基因对抑制细胞内可溶性糖的运转或淀粉的合成,POD同工酶的合成及其活性,MDA的合成均有明显的作用,ms_2基因对抑制细胞内可溶性蛋白质的合成有明显的作用。
Heterosis is a common phenomenon in plant kingdom, male-sterile line has wide application in crops' cross-breeding, and it's also one of the most important methods of cross-breeding. Practice testified that cotton possessed obvious heterosis. Since there isn't an ideal triple male-sterile line in cotton at present, Recessive Genic Male-sterile Line turns into the major way of utilizing cotton's heterosis. This research was in order to announcing physiology, cytology and biochemistry mechanism of Recessive GMS in cotton, and providing theory foundation for induction and seed selection. This research adopted paraffin sections and a series of biochemistry methods, performed systematic study on mechanism of pollen abortion in three kinds of cotton Recessive GMS line (Kang A_3, Lang A and 1355A) from morphology, cytology and biochemistry. The results were as follows:
1. In all of the three kinds of GMS line, sterilities compared with fertilities, flowers were smaller, filaments were shorter, anthers were smaller and dryer, and sterility anthers tightened on the lower part of stigma. The sterilities had a very significant difference in size of petal, length of filament and length of stigma overstep anther. The differences of anther quantity and pollen amount in sterilities were respectively that 1355A had an very significant difference, Kang A_3 had a significant difference, and Lang A had no significant difference. The differences of ovary size were respectively that Kang A3 had a significant difference, Lang A and 1355A had no significant difference. The differences of ovule size and quantity were respectively that Kang A_3 had no significant difference, Lang A and 1355A had significant difference. Compared with the three kinds of GMS line, the results showed that, the degeneration of Kang A_3 was fairly good, and yielding potentiality of seed was large. The degeneration of Lang A was not good, but Lang A had early-maturing character. The degeneration of 1355A was very good, and 1355A had a particular leaf shape which possessed insect-resistance, but combining ability of 1355A was fairly low. We should select them according to their own character, brought their selves preponderance into play to obtained the best utilize effect in production.
2. The results of cytological observation to microspore development in the three sterilities and fertilities indicated that: PMC meiosis stage was chief abortion period of Kang A_3. The main cytological character were that PMC adhesioned mutually when took its shape; chromosome didn't take correct match, PMC propagated in bud pattern, tapetum degenerated too early or grow expand and deformity, callose precipitated, arose cytomixis in PMC meiosis stage; cytoplasm disintegrated and highly vasculized in uniucleus stage. Late uniucleus stage was chief abortion period of Lang A. The main cytological character were that, original sporogneous cells disintegrated and couldn't formed normal PMC; chromosomes separated in a jumble and cytoplasm fissioned abnormal in meiosis stage; nucleus and cytoplasm disintegrated, the exine spine couldn't be produced in uniucleus stage; nutrition cells and reproduction cells disintegrated successively in binucleus stage. Uniucleus stage was chief abortion period of 1355A.The main cytological character were that, nucleus and cytoplasm disintegrated, the thickness of cell wall increased obviously, the exine spine couldn't be produced in uniucleus stage; nutrition cells and reproduction cells disintegrated successively in binucleus stage.
3. After biochemistry studied on the three sterilities and fertilities, we discovered that: Sterilities had higher contents of soluble sugar and less starch accumulation. This phenomenon indicated that transferred of soluble sugar or composed of starch was prevented, cells were in starved condition then leaded to cells degeneration and disintegrate. Sterilities had lower contents of soluble protein, and we could know that composed of protein was prevented, which caused component of protoplasm abnormal and couldn't perform normal metabolism, leaded to cells degeneration and disintegrate at last. During anther development, there were less bands of peroxidase isozymes and higher POD activity in sterilities. This indicated the difference of metabolize intensity between sterilities and fertilities, and also proved that abnormal component of peroxidase isozymes may have direct associate with abortion. Contents of MDA and electrical conductivity investigation in sterilities appeared a ascend tendency from PMC meiosis period to mature pollen period and had a similar ascend tendency, which was contents of MDA and electrical conductivity had a faster change from PMC meiosis period to binucleus stage, then tended to stable. We could infer that the key period of pollen abortion may be from PMC meiosis period to binucleus stage. Compared with these biochemical changes in the three sterilities, we could find that the emphasis point of different sterile gene which controlled various biochemical changes were different, ms_(14) gene had significant restrain affect on transferring of soluble sugar or composing of starch, synthesizing of peroxidase isozymes, POD activity and synthesizing of MDA. ms_2 gene had significant restrain affect on synthesizing of soluble protein.
1.三个不育系中不育株的花器均略小于可育株,不育株雄蕊的花丝极短,花药小而干瘪且紧缩在花柱下部。3个不育系的不育株和可育株相比,花器官在花瓣大小、花丝长度和柱头超出花药长度的差异极显著;花药个数和不育株中花粉数量差异分别为,1355A极显著,抗A3显著,阆A不显著;子房大小的差异分别为,抗A3显著,阆A不显著,1355A不显著;胚珠个数和大小的差异分别为,抗A3不显著,阆A显著,1355A显著。三个不育系中不育株的花器官形态比较发现,抗A3退化较好且制种产量潜力大,阆A退化不够好但具早熟性,1355A退化很好又具形态抗虫性但配合力较低。我们应根据这三个不育系各自的特点,有针对性地对其加以选育,使其在生产上发挥自身优势,以获得最佳利用效果。
2.对三个不育系的不育株和可育株小孢子发生的细胞学观察结果表明,抗A3不育株小孢子发生败育的时期主要是减数分裂Ⅰ时期;造孢细胞和PMC退化导致雄性不育的主要细胞形态学特征是:PMC在形成过程中相互粘连,减数分裂时染色体不配对、PMC出现出芽式增殖、绒毡层细胞过早退化或膨大畸形、胼胝质沉积、出现细胞质穿壁现象,单核期细胞质解体出现高度液泡化。阆A不育株小孢子发生败育的时期主要是单核靠边期;造孢细胞和PMC退化导致雄性不育的主要细胞形态学特征是:初生造孢细胞解体不能形成正常的PMC,减数分裂时期染色体分离杂乱、细胞质分裂异常,单核期细胞核和细胞质解体、外壁不能正常发育长出刺,在双核期营养细胞和生殖细胞先后解体。1355A不育株小孢子发生败育的时期主要是单核期;造孢细胞和PMC退化导致雄性不育的主要细胞形态学特征是:在单核期细胞核和细胞质解体、外壁发育异常不能长出刺且外壁增厚明显,双核期营养细胞和生殖细胞先后解体。
3.对三个不育系的不育株和可育株的生理生化变化研究发现:三类不育株花药中可溶性糖含量偏高,缺乏淀粉积累,表明可溶性糖的运转或淀粉的合成受阻,细胞处于饥饿状态而导致退化解体;不育株的可溶性蛋白质含量远低于可育株,表明其蛋白质合成受阻,致使细胞原生质体组成异常,不能进行正常的生命代谢,最终导致细胞退化解体;不育株的POD同工酶在花药发育过程中酶带条数少于可育株,且POD活性明显偏高,这表明了不育株与可育株之间体内代谢强度的差异,也说明酶的变化与雄性不育现象有着直接的关系。三类不育株花药的MDA含量和电导率变化从PMC减数分裂时期到成熟花粉时期都呈上升趋势,且上升趋势相同,MDA含量和花药提取液的电导率从PMC减数分裂期到双核期变化较快,以后趋于平缓,由此可以推断三类不育株花粉败育的关键时期可能均为PMC减数分裂时期到双核期。对三类不育株这一系列生理生化变化的比较发现,各不育株的不育基因控制其体内的生理生化变化从而导致败育的侧重点不同,ms_(14)基因对抑制细胞内可溶性糖的运转或淀粉的合成,POD同工酶的合成及其活性,MDA的合成均有明显的作用,ms_2基因对抑制细胞内可溶性蛋白质的合成有明显的作用。
Heterosis is a common phenomenon in plant kingdom, male-sterile line has wide application in crops' cross-breeding, and it's also one of the most important methods of cross-breeding. Practice testified that cotton possessed obvious heterosis. Since there isn't an ideal triple male-sterile line in cotton at present, Recessive Genic Male-sterile Line turns into the major way of utilizing cotton's heterosis. This research was in order to announcing physiology, cytology and biochemistry mechanism of Recessive GMS in cotton, and providing theory foundation for induction and seed selection. This research adopted paraffin sections and a series of biochemistry methods, performed systematic study on mechanism of pollen abortion in three kinds of cotton Recessive GMS line (Kang A_3, Lang A and 1355A) from morphology, cytology and biochemistry. The results were as follows:
1. In all of the three kinds of GMS line, sterilities compared with fertilities, flowers were smaller, filaments were shorter, anthers were smaller and dryer, and sterility anthers tightened on the lower part of stigma. The sterilities had a very significant difference in size of petal, length of filament and length of stigma overstep anther. The differences of anther quantity and pollen amount in sterilities were respectively that 1355A had an very significant difference, Kang A_3 had a significant difference, and Lang A had no significant difference. The differences of ovary size were respectively that Kang A3 had a significant difference, Lang A and 1355A had no significant difference. The differences of ovule size and quantity were respectively that Kang A_3 had no significant difference, Lang A and 1355A had significant difference. Compared with the three kinds of GMS line, the results showed that, the degeneration of Kang A_3 was fairly good, and yielding potentiality of seed was large. The degeneration of Lang A was not good, but Lang A had early-maturing character. The degeneration of 1355A was very good, and 1355A had a particular leaf shape which possessed insect-resistance, but combining ability of 1355A was fairly low. We should select them according to their own character, brought their selves preponderance into play to obtained the best utilize effect in production.
2. The results of cytological observation to microspore development in the three sterilities and fertilities indicated that: PMC meiosis stage was chief abortion period of Kang A_3. The main cytological character were that PMC adhesioned mutually when took its shape; chromosome didn't take correct match, PMC propagated in bud pattern, tapetum degenerated too early or grow expand and deformity, callose precipitated, arose cytomixis in PMC meiosis stage; cytoplasm disintegrated and highly vasculized in uniucleus stage. Late uniucleus stage was chief abortion period of Lang A. The main cytological character were that, original sporogneous cells disintegrated and couldn't formed normal PMC; chromosomes separated in a jumble and cytoplasm fissioned abnormal in meiosis stage; nucleus and cytoplasm disintegrated, the exine spine couldn't be produced in uniucleus stage; nutrition cells and reproduction cells disintegrated successively in binucleus stage. Uniucleus stage was chief abortion period of 1355A.The main cytological character were that, nucleus and cytoplasm disintegrated, the thickness of cell wall increased obviously, the exine spine couldn't be produced in uniucleus stage; nutrition cells and reproduction cells disintegrated successively in binucleus stage.
3. After biochemistry studied on the three sterilities and fertilities, we discovered that: Sterilities had higher contents of soluble sugar and less starch accumulation. This phenomenon indicated that transferred of soluble sugar or composed of starch was prevented, cells were in starved condition then leaded to cells degeneration and disintegrate. Sterilities had lower contents of soluble protein, and we could know that composed of protein was prevented, which caused component of protoplasm abnormal and couldn't perform normal metabolism, leaded to cells degeneration and disintegrate at last. During anther development, there were less bands of peroxidase isozymes and higher POD activity in sterilities. This indicated the difference of metabolize intensity between sterilities and fertilities, and also proved that abnormal component of peroxidase isozymes may have direct associate with abortion. Contents of MDA and electrical conductivity investigation in sterilities appeared a ascend tendency from PMC meiosis period to mature pollen period and had a similar ascend tendency, which was contents of MDA and electrical conductivity had a faster change from PMC meiosis period to binucleus stage, then tended to stable. We could infer that the key period of pollen abortion may be from PMC meiosis period to binucleus stage. Compared with these biochemical changes in the three sterilities, we could find that the emphasis point of different sterile gene which controlled various biochemical changes were different, ms_(14) gene had significant restrain affect on transferring of soluble sugar or composing of starch, synthesizing of peroxidase isozymes, POD activity and synthesizing of MDA. ms_2 gene had significant restrain affect on synthesizing of soluble protein.
引文
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