不结球白菜小孢子培养及其胚胎发生机制
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摘要
不结球白菜(Brassica campestris ssp. chinensis Makino)是十字花科芸薹属中最重要的蔬菜作物之一。原产于中国,不结球白菜营养丰富,在中国、韩国、日本等东亚国家普遍栽培,近年来欧美等国家也广泛引种,逐渐成为世界性的重要蔬菜。不结球白菜具有明显的杂种优势,了解小孢子胚胎发生的机理、提高小孢子胚胎发生频率及可重复性,从而建立稳定的培养体系,在杂种优势利用上具有重要的意义。具体的研究结果如下：
1.小孢子胚胎诱导技术研究
以抽薹较早的4个不结球白菜品种为试材,对可能影响其小孢子胚胎发生的主要因素进行了研究,筛选出了稳定的培养程序并利用该程序对18个品种进行培养以验证其可靠性。结果表明：从植株始花开始不断采摘幼嫩小花序进行培养,可以使植株上的花序长时间保持初花状态,延长适宜取材的时期,并有利于减少或克服部分品种由于裂蕾等导致的污染；2d或3d32.8℃预处理以及NLN培养基中添加适宜浓度的活性炭能够提高小孢子的胚胎发生频率；使用前将B5培养基以及NLN培养基的pH值调整至5.8可大大提高小孢子胚胎发生频率；B5培养基和/或NLN培养基添加0.05mg·L-16-BA有利于提高不结球白菜小孢子胚胎发生频率；利用该培养程序可使基因型反应频率达到88.9%,并使多数品种的胚胎发生频率达到或超过10胚/皿。
2.小孢子胚胎萌发及炼苗移栽
利用试验中获得的小孢子胚胎对部分影响胚胎萌发成苗的因素以及炼苗移栽技术进行了研究,结果表明：诱导期间的热处理时间不同会影响小孢子成苗,热处理2d和3d后诱导产生的胚胎移栽成苗率较热处理1d的高；培养25d的胚胎在固体培养基上萌发率最高；胚胎根部插入培养基较平放培养基表面胚胎成苗率更高,低温预处理不能增加胚胎成苗率；含1.2%琼脂的B5培养基中添加0.4g·L-1活性炭和0.05mg·L-16-BA有利于小孢子胚胎直接成苗,‘青梗’小孢子胚胎成苗率可达93.51%；直接成苗的小孢子植株经过生根后更有利于移栽,移栽过程中注意保湿可使成活率达到74%以上。移栽后的多数小孢子植株能够现蕾开花。
3.小孢子再生植株倍性鉴定
利用流式细胞仪对移栽成活的123株再生株进行倍性鉴定,对其中的113株进行了叶绿体计数分析,将两种方法鉴定的结果进行比较分析并进一步用形态法和减数分裂观察法进行验证,结果表明：不结球白菜小孢子培养再生植株中存在单倍体、二倍体和四倍体等不同倍性的植株,总自然加倍率为55.3%,但不同品种加倍率有一定的差异,‘华京’自然加倍率最低,为57.5%,‘华冠’为73.1%,‘夏绿2号’为80.0%,‘青梗’最高,达94.7%；叶绿体数目鉴定法与流式细胞仪鉴定的结果一致性很高,均达到92.8%以上。对结果一致的103株进一步分析则表明：无论是单一品种分别分析还是各品种综合分析,结果均表明不同倍性植株叶绿体平均数之间差异极显著；根据各不同倍性植株叶绿体平均数分布推断叶绿体平均数平均值(四舍五入)在不同倍性植株的分界为：单倍体≤9；10≤二倍体<16；四倍体等多倍体则≥16。叶绿体数分界法鉴定结果与田间形态鉴定倍性结果吻合,较流式细胞仪法更简便,该方法既可以在苗期利用莲座叶也可以在抽薹期利用薹生叶进行植株倍性鉴定。不同倍性植株在形态上存在很大的区别,主要表现为植株倍性越高植株生长势越强,花瓣先端也随倍性增加而变宽。单倍体减数分裂中期I出现10个单价体,中期Ⅱ观察到单价体5/5分离,花粉呈不规则的扁盘状；二倍体减数分裂正常,终变期、中期I可见到10个二价体,中期II出现两组各10条染色体,花粉三裂状；而四倍体中期II、后期Ⅱ及末期Ⅱ中染色体数目明显多于10条,中期II可观察到两组染色体中一组染色体数目为20个,其花粉常出现四裂或多裂。
4.小孢子形成、发育及胚胎发生细胞学研究
采用H33258荧光染色法研究了不结球白菜花粉母细胞减数分裂及其雄配子体发育过程,并用FDA荧光染色法对小孢子游离后的发育过程进行了追踪观察。结果表明；不结球白菜花粉母细胞减数分裂的胞质分裂方式为同时型,终变期二价体多为棒状和环状。终变期、中期I和中期Ⅱ是染色体数目鉴定的最佳时期。1.5-2.49mm长的花蕾中花粉处于单核早至中期,2.5-3.0mm长的花蕾中花粉处于单核靠边期至2核早期。FDA染色反应显示单核靠边期小孢子活力最强,适宜进行培养。多数游离小孢子培养经历的发育过程与合子胚相似,并且胚胎发育迅速,6-9d产生大量球胚,13d开始出现子叶形胚胎。其发育途径既有白菜中普遍报道的B途径,也有第1次不对称分裂后营养细胞发育的A途径；第1次不对称分裂后,观察到营养细胞和生殖细胞共同发育的现象即E途径,未发现生殖细胞单独发育。不同品种胚胎发育的途径存在一定的差异。
Non-heading Chinese cabbage (Brassica campestris ssp. chinensis Makino) is one of the most important vegetable crops in the Brassica genus. Non-heading Chinese cabbage, known as Pak Choi, originated in China, is among the most popular vegetables crops in eastern Asia like China, Korea and Japan and is now common in Europe and America. Like the other Brassicas, Non-heading Chinese cabbage is a good source of nutrients. Non-heading Chinese cabbage obtained the character of heterosis, so it's very important to study on the embryogenesis mechanism and optimize the isolated microspore culture technology for the crop breeding. The results are as follows:
1. Study on the isolated microspore culture technology of non-heading Chinese cabbage
Factors affected on microspore culture were studied on four early bolting cultivars of non-heading Chinese cabbage. Established a stable and reproducible embryogenesis system of microspore culture technology, then applied this program to18materials. The results showed that kept picking small inflorescence from the early flowering season can remained on the fresh inflorescence for a long time, and help reduce or overcome contaminate which came from the crack bud. There were some factors that could improve the induction rate of microspore-derived embryos significantly, such as pre-culture under32.8℃for2-3days; with1%of active charcoal in the NLN culture medium; the value of pH were5.8and add6-BA with0.05mg·L-1both for NLN culture medium and B5.The response frequency of genotype was88.9%, and the embryogenesis frequency of almost cultivars can reached or exceeded ten embryos per dish with the optimized program.
2. Plant regeneration and transplanting in non-heading Chinese cabbage
The microspore-derived embryos we had obtained were used as materials for research the effect on the development of the embryoids and plantlet formation. Results showed that: heat treatment time would affect the survival rate of plantlet, the rate of heat treatment for2days and3days was higher than survival rate for1day in'qinggeng'; After cultured for25days, the percentage of embryos germination was highest in the solid medium; Seedling rate of embryonic roots into the medium was higher than keeping flat on the medium, and cold treatment didn't help improve the survival number; Improved B5added with1.2%agar,0.4g-L"1AC and0.05m g-L-16-BA could promote the development of the embryoids and plantlet formation. The survival rate of the 'qinggeng' plantlets was93.51%after being transplanted into soil. The average survival rate was more than74%, when the plantlets were induced roots and transferred under wet environment. Most of the plantlets after transplanted could grow to flower.
3. Studies on the determination of ploidy level of microspore-derived plants
The relationship between the ploidy level of microspore-derived plants and chloroplast number in stomatal guard cells was studied in non-heading Chinese cabbage. We had determined123plants by the FCM (Flow Cytometry) and113plants by chloroplast counting, then morphology identifying and chromosome counting through meiosis were used to test accuracy. The results showed there were haploid, diploid, triploid, tetraploid and chimera for the plantlets. The average rate of spontaneously doubled haploid was55.3%. Different cultivars had different rate, as follow,57.5%of huajing,73.1%of huaguan,80.0%of xialv No.2and the highest rate was qinggeng (94%). The accuracy of the two methods for identification of different ploidy plants was92.8%. The chloroplast average number in stomatal guard cells varied significantly among the different ploidy stoma in the same variety. A correlation has been established between ploidy and chloroplast number in the stomatal guard cells. In every single stoma of microspore-derived plants, the chloroplast number for a haploid should not be more than9, that for diploids should be10to16, and polyploids should be more than16. The result of this method was the same with the morphology identifying method, and was simpler than by the FCM. Furthermore, the accuracy of this method was reliable and did not vary with the plants growth conditions (seedling or bolting stage). The multiploids plants grew faster and the petal was larger.10univalents were observed in the haploid cells at metaphase I stage, and they symmetrically divide at metaphase Ⅱ stage. The shape of pollen from haploid was irregular; the germinated groove was deeper than diploids and there was little fluorescence area (no DNA); For the normal form of diploids,10bivalents could be observed at diakinesis and metaphase Ⅰ stage; But more than10chromosomes could be observed in the tetraploid cells at metaphase Ⅱ stage and so on. For the normal form of tetraploid,20bivalents could be observed.
4. Cytological studies on pollen mother cell meiosis, embryogenesis and development
Chromosomal meiotic behaviors and male gametophytes development of non-heading Chinese cabbage were studied using chromosome preparation and fluorescence staining technique. The research showed that:the cytokinesis and chromosomes meiosis of pollen mother cells of non-heading Chinese cabbage was simultaneous type; diakinesis bivalents was mostly rod and ring. A few lagging bivalents appeared in anaphase Ⅰ and the spindle arranged in vertical, parallel and an acute angle in metaphase Ⅱ. The microspores in tetrad stage showed the arrangement of decussate type, isobilateral type and tetrahedral type. Diakinesis, metaphase Ⅰ and metahase Ⅱ was the best period for identification of chromosome numbers. The length of buds which were from1.5to2.49mm was usually at the stage of uniuclaete microsore in center. Uniucleate microsore at periphery and early2-celled pollen existed in buds long from2.5to3.0mm.2.0～3.0mm long flower buds were adapt for anther or microspore culture. Most of microspore embryos development was same to the zygotic embryos, and the development process was very fast. The embryos grow to globular stages after6-9days of culture, and to cotyledonary stages after13days. There were two kinds of male gametophyte development, one was asymmetrical division (names A pathway), other was B pathway. Both A and B pathway were observed in our study. After the first asymmetrical division, we found that both the vegetative and germ cells could be inducted to embryos, or only the vegetative cells could be inducted, but not the germ ones.
1.小孢子胚胎诱导技术研究
以抽薹较早的4个不结球白菜品种为试材,对可能影响其小孢子胚胎发生的主要因素进行了研究,筛选出了稳定的培养程序并利用该程序对18个品种进行培养以验证其可靠性。结果表明：从植株始花开始不断采摘幼嫩小花序进行培养,可以使植株上的花序长时间保持初花状态,延长适宜取材的时期,并有利于减少或克服部分品种由于裂蕾等导致的污染；2d或3d32.8℃预处理以及NLN培养基中添加适宜浓度的活性炭能够提高小孢子的胚胎发生频率；使用前将B5培养基以及NLN培养基的pH值调整至5.8可大大提高小孢子胚胎发生频率；B5培养基和/或NLN培养基添加0.05mg·L-16-BA有利于提高不结球白菜小孢子胚胎发生频率；利用该培养程序可使基因型反应频率达到88.9%,并使多数品种的胚胎发生频率达到或超过10胚/皿。
2.小孢子胚胎萌发及炼苗移栽
利用试验中获得的小孢子胚胎对部分影响胚胎萌发成苗的因素以及炼苗移栽技术进行了研究,结果表明：诱导期间的热处理时间不同会影响小孢子成苗,热处理2d和3d后诱导产生的胚胎移栽成苗率较热处理1d的高；培养25d的胚胎在固体培养基上萌发率最高；胚胎根部插入培养基较平放培养基表面胚胎成苗率更高,低温预处理不能增加胚胎成苗率；含1.2%琼脂的B5培养基中添加0.4g·L-1活性炭和0.05mg·L-16-BA有利于小孢子胚胎直接成苗,‘青梗’小孢子胚胎成苗率可达93.51%；直接成苗的小孢子植株经过生根后更有利于移栽,移栽过程中注意保湿可使成活率达到74%以上。移栽后的多数小孢子植株能够现蕾开花。
3.小孢子再生植株倍性鉴定
利用流式细胞仪对移栽成活的123株再生株进行倍性鉴定,对其中的113株进行了叶绿体计数分析,将两种方法鉴定的结果进行比较分析并进一步用形态法和减数分裂观察法进行验证,结果表明：不结球白菜小孢子培养再生植株中存在单倍体、二倍体和四倍体等不同倍性的植株,总自然加倍率为55.3%,但不同品种加倍率有一定的差异,‘华京’自然加倍率最低,为57.5%,‘华冠’为73.1%,‘夏绿2号’为80.0%,‘青梗’最高,达94.7%；叶绿体数目鉴定法与流式细胞仪鉴定的结果一致性很高,均达到92.8%以上。对结果一致的103株进一步分析则表明：无论是单一品种分别分析还是各品种综合分析,结果均表明不同倍性植株叶绿体平均数之间差异极显著；根据各不同倍性植株叶绿体平均数分布推断叶绿体平均数平均值(四舍五入)在不同倍性植株的分界为：单倍体≤9；10≤二倍体<16；四倍体等多倍体则≥16。叶绿体数分界法鉴定结果与田间形态鉴定倍性结果吻合,较流式细胞仪法更简便,该方法既可以在苗期利用莲座叶也可以在抽薹期利用薹生叶进行植株倍性鉴定。不同倍性植株在形态上存在很大的区别,主要表现为植株倍性越高植株生长势越强,花瓣先端也随倍性增加而变宽。单倍体减数分裂中期I出现10个单价体,中期Ⅱ观察到单价体5/5分离,花粉呈不规则的扁盘状；二倍体减数分裂正常,终变期、中期I可见到10个二价体,中期II出现两组各10条染色体,花粉三裂状；而四倍体中期II、后期Ⅱ及末期Ⅱ中染色体数目明显多于10条,中期II可观察到两组染色体中一组染色体数目为20个,其花粉常出现四裂或多裂。
4.小孢子形成、发育及胚胎发生细胞学研究
采用H33258荧光染色法研究了不结球白菜花粉母细胞减数分裂及其雄配子体发育过程,并用FDA荧光染色法对小孢子游离后的发育过程进行了追踪观察。结果表明；不结球白菜花粉母细胞减数分裂的胞质分裂方式为同时型,终变期二价体多为棒状和环状。终变期、中期I和中期Ⅱ是染色体数目鉴定的最佳时期。1.5-2.49mm长的花蕾中花粉处于单核早至中期,2.5-3.0mm长的花蕾中花粉处于单核靠边期至2核早期。FDA染色反应显示单核靠边期小孢子活力最强,适宜进行培养。多数游离小孢子培养经历的发育过程与合子胚相似,并且胚胎发育迅速,6-9d产生大量球胚,13d开始出现子叶形胚胎。其发育途径既有白菜中普遍报道的B途径,也有第1次不对称分裂后营养细胞发育的A途径；第1次不对称分裂后,观察到营养细胞和生殖细胞共同发育的现象即E途径,未发现生殖细胞单独发育。不同品种胚胎发育的途径存在一定的差异。
Non-heading Chinese cabbage (Brassica campestris ssp. chinensis Makino) is one of the most important vegetable crops in the Brassica genus. Non-heading Chinese cabbage, known as Pak Choi, originated in China, is among the most popular vegetables crops in eastern Asia like China, Korea and Japan and is now common in Europe and America. Like the other Brassicas, Non-heading Chinese cabbage is a good source of nutrients. Non-heading Chinese cabbage obtained the character of heterosis, so it's very important to study on the embryogenesis mechanism and optimize the isolated microspore culture technology for the crop breeding. The results are as follows:
1. Study on the isolated microspore culture technology of non-heading Chinese cabbage
Factors affected on microspore culture were studied on four early bolting cultivars of non-heading Chinese cabbage. Established a stable and reproducible embryogenesis system of microspore culture technology, then applied this program to18materials. The results showed that kept picking small inflorescence from the early flowering season can remained on the fresh inflorescence for a long time, and help reduce or overcome contaminate which came from the crack bud. There were some factors that could improve the induction rate of microspore-derived embryos significantly, such as pre-culture under32.8℃for2-3days; with1%of active charcoal in the NLN culture medium; the value of pH were5.8and add6-BA with0.05mg·L-1both for NLN culture medium and B5.The response frequency of genotype was88.9%, and the embryogenesis frequency of almost cultivars can reached or exceeded ten embryos per dish with the optimized program.
2. Plant regeneration and transplanting in non-heading Chinese cabbage
The microspore-derived embryos we had obtained were used as materials for research the effect on the development of the embryoids and plantlet formation. Results showed that: heat treatment time would affect the survival rate of plantlet, the rate of heat treatment for2days and3days was higher than survival rate for1day in'qinggeng'; After cultured for25days, the percentage of embryos germination was highest in the solid medium; Seedling rate of embryonic roots into the medium was higher than keeping flat on the medium, and cold treatment didn't help improve the survival number; Improved B5added with1.2%agar,0.4g-L"1AC and0.05m g-L-16-BA could promote the development of the embryoids and plantlet formation. The survival rate of the 'qinggeng' plantlets was93.51%after being transplanted into soil. The average survival rate was more than74%, when the plantlets were induced roots and transferred under wet environment. Most of the plantlets after transplanted could grow to flower.
3. Studies on the determination of ploidy level of microspore-derived plants
The relationship between the ploidy level of microspore-derived plants and chloroplast number in stomatal guard cells was studied in non-heading Chinese cabbage. We had determined123plants by the FCM (Flow Cytometry) and113plants by chloroplast counting, then morphology identifying and chromosome counting through meiosis were used to test accuracy. The results showed there were haploid, diploid, triploid, tetraploid and chimera for the plantlets. The average rate of spontaneously doubled haploid was55.3%. Different cultivars had different rate, as follow,57.5%of huajing,73.1%of huaguan,80.0%of xialv No.2and the highest rate was qinggeng (94%). The accuracy of the two methods for identification of different ploidy plants was92.8%. The chloroplast average number in stomatal guard cells varied significantly among the different ploidy stoma in the same variety. A correlation has been established between ploidy and chloroplast number in the stomatal guard cells. In every single stoma of microspore-derived plants, the chloroplast number for a haploid should not be more than9, that for diploids should be10to16, and polyploids should be more than16. The result of this method was the same with the morphology identifying method, and was simpler than by the FCM. Furthermore, the accuracy of this method was reliable and did not vary with the plants growth conditions (seedling or bolting stage). The multiploids plants grew faster and the petal was larger.10univalents were observed in the haploid cells at metaphase I stage, and they symmetrically divide at metaphase Ⅱ stage. The shape of pollen from haploid was irregular; the germinated groove was deeper than diploids and there was little fluorescence area (no DNA); For the normal form of diploids,10bivalents could be observed at diakinesis and metaphase Ⅰ stage; But more than10chromosomes could be observed in the tetraploid cells at metaphase Ⅱ stage and so on. For the normal form of tetraploid,20bivalents could be observed.
4. Cytological studies on pollen mother cell meiosis, embryogenesis and development
Chromosomal meiotic behaviors and male gametophytes development of non-heading Chinese cabbage were studied using chromosome preparation and fluorescence staining technique. The research showed that:the cytokinesis and chromosomes meiosis of pollen mother cells of non-heading Chinese cabbage was simultaneous type; diakinesis bivalents was mostly rod and ring. A few lagging bivalents appeared in anaphase Ⅰ and the spindle arranged in vertical, parallel and an acute angle in metaphase Ⅱ. The microspores in tetrad stage showed the arrangement of decussate type, isobilateral type and tetrahedral type. Diakinesis, metaphase Ⅰ and metahase Ⅱ was the best period for identification of chromosome numbers. The length of buds which were from1.5to2.49mm was usually at the stage of uniuclaete microsore in center. Uniucleate microsore at periphery and early2-celled pollen existed in buds long from2.5to3.0mm.2.0～3.0mm long flower buds were adapt for anther or microspore culture. Most of microspore embryos development was same to the zygotic embryos, and the development process was very fast. The embryos grow to globular stages after6-9days of culture, and to cotyledonary stages after13days. There were two kinds of male gametophyte development, one was asymmetrical division (names A pathway), other was B pathway. Both A and B pathway were observed in our study. After the first asymmetrical division, we found that both the vegetative and germ cells could be inducted to embryos, or only the vegetative cells could be inducted, but not the germ ones.
引文
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