玉米活体诱导单倍体受精情况和发育过程研究
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摘要
随着Stock6衍生系活体诱导玉米单倍体技术在玉米育种中的逐渐规模化利用,推动高频率单倍体诱导系的选育及染色体加倍技术的研究取得了长足进展,而诱导系杂交诱导单倍体的形成机制至今仍处于推测或假说状态。本研究以超甜玉米自交系Hua24为母本,以自主选育的高频率诱导系HZI6为父本进行杂交,通过整体染色透明技术、细胞学观察、SSR标记检测研究授粉之后F1籽粒早期、中途和成熟时卵细胞和中央细胞的受精情况及胚和胚乳发育特征,以期阐明单倍体的形成机理,其结果将为玉米高频诱导系的选育和该技术在育种中的规模化利用提供指导,并为其它植物开展单倍体相关研究提供借鉴,亦可深化对双受精过程的理解。本实验主要得到以下结果：
1.通过对诱导系HZI6、Hua24及普通玉米自交系相互杂交及自交果穗的籽粒发育情况调查发现：Hua24自交或Hua24×普通玉米果穗籽粒发育正常,HZI6×Hua24果穗籽粒发育正常,而HZI6自交、Hua24×HZI6,果穗部分籽粒发育不正常,说明籽粒发育的不正常是由诱导系的花粉引起的。
2. Hua24×HZI6授粉之后25-65小时的胚珠整体染色透明观察发现,平均54.19%的胚珠胚和胚乳都分裂,平均45.80%的胚珠胚或胚乳单方面发育,其中胚和胚乳都分裂的胚珠应该包括单倍体和二倍体胚的胚珠,根据12、15天调查的单倍体诱导率平均是4.77%,因此能正常分裂的二倍体胚和三倍体胚乳的胚珠基本占50%,另外一半只能单方面发育或形成单倍体籽粒。Hua24×HZI6授粉之后25小时和40小时时,胚和胚乳都分裂的胚珠中9%左右的助细胞中有一个精子,胚乳分裂、卵细胞未分裂的胚珠中,70%左右胚珠助细胞中有精子,基本上所有卵细胞核内都透明,可能这种助细胞中滞留的精子与单倍体的形成和胚或胚乳的单方面发育相关。
3. Hua24×HZI6授粉之后25-65h的胚珠整体染色透明观察发现,胚乳分裂、卵细胞未分裂的胚珠所占百分比平均为40.51%；授粉之后第8d的无胚籽粒为44.46%；授粉之后第12、15d时败育籽粒占42.46%,成熟时干瘪籽粒占44.31%,四者数据极为相似,说明授粉之后卵细胞未分裂的胚珠,在第8天时表现无胚,第12、15天时表现败育,成熟时表现干瘪。
4.Hua24×HZI6幼胚和根尖细胞学检测染色体是10或10与20共存的45个胚中,SSR分子标记检测都是母本Hua24的带型,细胞学检测时发现有核内有丝分裂现象,说明单倍体都是母本来源,20条染色体的细胞是由单倍体细胞发生核内有丝分裂自然加倍形成。
5. Hua24×HZI6发育籽粒幼胚压片或SSR标记检测发现大约有0.5-1%左右的胚中每个细胞染色体是一致的19或21条,并且19条染色体是少了父本的第8条染色体,因此推测诱导系花粉中有很少一部分非整倍体精子也能存活,并且能完成受精作用。第6d的胚乳压片观察发现,大约1.85%的籽粒中胚乳细胞染色体全是40条并且无胚,推测可能是两个精子都与中央细胞受精形成四倍体胚乳,而卵细胞没有分裂。
As the large-scale application of technology for inducing maize haploids in vivo by Stock6 derivative in maize breeding, the selection of inducers with high frequency and chromosome doubling technology have made great progress. However, the mechanism of inducing haploid is still in speculation or hypothesis state. In this study, the super-sweet maize inbred, Hua24 as the seed parent crossed with the high frequency inducer line, HZI6 which was independently cultivated by our research group as pollen parent. The fertilization condition of central cells and egg cells and development process of embryoes and endosperms at 25-65h,8d,12d,15d and at maturity were studied after Hua24 x HZI6 pollination. The goal was to reveal the mechanism of inducing haploid. The result would direct the selection of high frequency inducer and the application of technology for inducing maize haploids in vivo, and provide the example for the study of the other plants haploid. In addition, it would facilitate understanding the double fertilization more comprehensively. The results were as follow:
1. The line HZI6, Hua24 and normal Corn inbred lines were selected to hybrid each other or self-fertilization. The setting rate was examined. The high setting rate of Hua24 self-fertilized or Hua24×normal corn showed that the egg cells and sperms of Hua24 were normal. The high setting rate of HZI6 x Hua24 showed that the egg cells of HZI6 were normal. However, the setting rate of self-fertilization HZI6 and Hua24×HZI6 was very low and some kernels were abnormal, which showed that the kernels of abnormal development were caused by the pollens of inducer.
2. Based on the ovule whole stain-clearing 25-65h after Hua24 x HZI6 pollination, ovules of an average 54.19% that embryoes and endosperms both divided, ovules of an average 45.80% that embryoes or endosperms alone developed, ovules of embryoes and endosperms both divided should include haploids and diploids, which the rate of haploid induction was 4.77% according to the data of 12th and 15th day after pollination. So the ovules of normal diploid embryo and triploid endosperm were about 50%, and the other half ovules developed only embryo or endosperm or developed into haploids. About 9% of ovules had one sperm in synergid in embryo and endosperm ovules of division, in ovules of the endosperms division and egg cells no division, the 70% of ovules had sperms in synergid cells, and the egg cell is clearing, is similar as the unpollination.
3. Based on the ovule whole stain-clearing 25-65h after Hua24 x HZI6 pollination, the ovule of 37.74%-43.57%, an average of 40.51% of the egg cells that didn't divide while the endosperm divided normally.44.68% of seeds developed without embryos at 8th day after pollination, seeds of 42.46% were abortive at 12th day and 15th day after pollination, seeds of 44.31% were wizened at the seed mature stage. These values were very similar. So speculated that the ovule which the egg cells had no division after pollination, no embryo at 8 th day, abortion at 12th and 15th day, wizened at mature.
4. Hua24 x HZI6 embryoes and root tips of 10 or 10 and 20 coexistence, SSR markers dections suggested that all the bands were materal Hua24, and endomitosis phenomenon at cytology detection. These suggested that the haploids were maternal origin. The cells of 20 chromosomes were doubled naturally during development by endomitosis.
5. Young embryoes of about 0.5-1.0% by cytological or SSR markers detection were aneuploidy, of which chromosomes of every cell were 19 or 21 in aneuploidy embryoes, Presumably, the sperms of inducer HZI6 were 11 or 9 chromosomes. That is to say, and the embryoes of 19 chromosomes lacks the the eighth chromosome, so Speculated that fraction of aneuploidy sperms in inducer HZI6 could also live, even accomplished fertilization. The endosperms of about 1.85% had 40 chromosomes and the embryoes had no development by cytological detection the 6th day after Hua24 x HZI6 pollination It was speculated that two sperms fertilized the central cells and egg cell had no fertilization.
1.通过对诱导系HZI6、Hua24及普通玉米自交系相互杂交及自交果穗的籽粒发育情况调查发现：Hua24自交或Hua24×普通玉米果穗籽粒发育正常,HZI6×Hua24果穗籽粒发育正常,而HZI6自交、Hua24×HZI6,果穗部分籽粒发育不正常,说明籽粒发育的不正常是由诱导系的花粉引起的。
2. Hua24×HZI6授粉之后25-65小时的胚珠整体染色透明观察发现,平均54.19%的胚珠胚和胚乳都分裂,平均45.80%的胚珠胚或胚乳单方面发育,其中胚和胚乳都分裂的胚珠应该包括单倍体和二倍体胚的胚珠,根据12、15天调查的单倍体诱导率平均是4.77%,因此能正常分裂的二倍体胚和三倍体胚乳的胚珠基本占50%,另外一半只能单方面发育或形成单倍体籽粒。Hua24×HZI6授粉之后25小时和40小时时,胚和胚乳都分裂的胚珠中9%左右的助细胞中有一个精子,胚乳分裂、卵细胞未分裂的胚珠中,70%左右胚珠助细胞中有精子,基本上所有卵细胞核内都透明,可能这种助细胞中滞留的精子与单倍体的形成和胚或胚乳的单方面发育相关。
3. Hua24×HZI6授粉之后25-65h的胚珠整体染色透明观察发现,胚乳分裂、卵细胞未分裂的胚珠所占百分比平均为40.51%；授粉之后第8d的无胚籽粒为44.46%；授粉之后第12、15d时败育籽粒占42.46%,成熟时干瘪籽粒占44.31%,四者数据极为相似,说明授粉之后卵细胞未分裂的胚珠,在第8天时表现无胚,第12、15天时表现败育,成熟时表现干瘪。
4.Hua24×HZI6幼胚和根尖细胞学检测染色体是10或10与20共存的45个胚中,SSR分子标记检测都是母本Hua24的带型,细胞学检测时发现有核内有丝分裂现象,说明单倍体都是母本来源,20条染色体的细胞是由单倍体细胞发生核内有丝分裂自然加倍形成。
5. Hua24×HZI6发育籽粒幼胚压片或SSR标记检测发现大约有0.5-1%左右的胚中每个细胞染色体是一致的19或21条,并且19条染色体是少了父本的第8条染色体,因此推测诱导系花粉中有很少一部分非整倍体精子也能存活,并且能完成受精作用。第6d的胚乳压片观察发现,大约1.85%的籽粒中胚乳细胞染色体全是40条并且无胚,推测可能是两个精子都与中央细胞受精形成四倍体胚乳,而卵细胞没有分裂。
As the large-scale application of technology for inducing maize haploids in vivo by Stock6 derivative in maize breeding, the selection of inducers with high frequency and chromosome doubling technology have made great progress. However, the mechanism of inducing haploid is still in speculation or hypothesis state. In this study, the super-sweet maize inbred, Hua24 as the seed parent crossed with the high frequency inducer line, HZI6 which was independently cultivated by our research group as pollen parent. The fertilization condition of central cells and egg cells and development process of embryoes and endosperms at 25-65h,8d,12d,15d and at maturity were studied after Hua24 x HZI6 pollination. The goal was to reveal the mechanism of inducing haploid. The result would direct the selection of high frequency inducer and the application of technology for inducing maize haploids in vivo, and provide the example for the study of the other plants haploid. In addition, it would facilitate understanding the double fertilization more comprehensively. The results were as follow:
1. The line HZI6, Hua24 and normal Corn inbred lines were selected to hybrid each other or self-fertilization. The setting rate was examined. The high setting rate of Hua24 self-fertilized or Hua24×normal corn showed that the egg cells and sperms of Hua24 were normal. The high setting rate of HZI6 x Hua24 showed that the egg cells of HZI6 were normal. However, the setting rate of self-fertilization HZI6 and Hua24×HZI6 was very low and some kernels were abnormal, which showed that the kernels of abnormal development were caused by the pollens of inducer.
2. Based on the ovule whole stain-clearing 25-65h after Hua24 x HZI6 pollination, ovules of an average 54.19% that embryoes and endosperms both divided, ovules of an average 45.80% that embryoes or endosperms alone developed, ovules of embryoes and endosperms both divided should include haploids and diploids, which the rate of haploid induction was 4.77% according to the data of 12th and 15th day after pollination. So the ovules of normal diploid embryo and triploid endosperm were about 50%, and the other half ovules developed only embryo or endosperm or developed into haploids. About 9% of ovules had one sperm in synergid in embryo and endosperm ovules of division, in ovules of the endosperms division and egg cells no division, the 70% of ovules had sperms in synergid cells, and the egg cell is clearing, is similar as the unpollination.
3. Based on the ovule whole stain-clearing 25-65h after Hua24 x HZI6 pollination, the ovule of 37.74%-43.57%, an average of 40.51% of the egg cells that didn't divide while the endosperm divided normally.44.68% of seeds developed without embryos at 8th day after pollination, seeds of 42.46% were abortive at 12th day and 15th day after pollination, seeds of 44.31% were wizened at the seed mature stage. These values were very similar. So speculated that the ovule which the egg cells had no division after pollination, no embryo at 8 th day, abortion at 12th and 15th day, wizened at mature.
4. Hua24 x HZI6 embryoes and root tips of 10 or 10 and 20 coexistence, SSR markers dections suggested that all the bands were materal Hua24, and endomitosis phenomenon at cytology detection. These suggested that the haploids were maternal origin. The cells of 20 chromosomes were doubled naturally during development by endomitosis.
5. Young embryoes of about 0.5-1.0% by cytological or SSR markers detection were aneuploidy, of which chromosomes of every cell were 19 or 21 in aneuploidy embryoes, Presumably, the sperms of inducer HZI6 were 11 or 9 chromosomes. That is to say, and the embryoes of 19 chromosomes lacks the the eighth chromosome, so Speculated that fraction of aneuploidy sperms in inducer HZI6 could also live, even accomplished fertilization. The endosperms of about 1.85% had 40 chromosomes and the embryoes had no development by cytological detection the 6th day after Hua24 x HZI6 pollination It was speculated that two sperms fertilized the central cells and egg cell had no fertilization.
引文
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