羽衣甘蓝小孢子培养及红叶性状的SRAP标记
摘要
羽衣甘蓝是一种重要的观赏植物和蔬菜作物，但由于其为绿体春化植物，花芽分化所需时间长，两年才能完成一个有性生活周期，利用常规育种手段培育一个自交系需要6~8年。采用双单倍体育种方法替代自交系选育过程，可以有效地缩短育种年限，提高育种效率，小孢子培养便是一种高效的培育双单倍体的细胞育种技术，因此，优化羽衣甘蓝小孢子培养技术体系，有重要的实践意义。叶色是羽衣甘蓝最重要的观赏性状，开发与叶色紧密连锁的分子标记，是探索叶色形成机理和开展分子标记辅助育种研究的重要前提。
本项研究以16个羽衣甘蓝杂交种为试材进行小孢子培养，分析小孢子胚状体的发生与发育、以及小孢子植株再生的影响因素，筛选准确高效的小孢子植株倍性鉴定方法，探索人工诱导染色体加倍方法，鉴定培养所得DH系的自交亲和性及用其配制杂交种的园艺学性状；利用小孢子培养创制的DH系配制杂交组合，构建F2分离群体，筛选与红叶性状连锁的SRAP标记。获得了以下结果：
1．优化了羽衣甘蓝小孢子胚诱导体系。研究证明了基因型是影响羽衣甘蓝游离小孢子培养中胚状体形成的关键因素。在供试的16个基因型材料中15个基因型获得了小孢子胚状体，但出胚品种的胚诱导率相差很大，最高的‘皱叶红心’和最低的‘白鸠’两者胚诱导率相差达1.94胚·蕾~(-1)。花蕾长度在3.01~3.50mm范围内时，小孢子所处单核中晚期的比例最高，达67.31%。因此，在羽衣甘蓝小孢子培养时，选蕾长度应集中在3.01~3.50mm范围内，有助于胚状体诱导成功，此时花瓣与花药比在2/3~4/5。花蕾经4℃低温预处理可以提高胚产量，以处理24h效果最好，时间过长则会起反作用；小孢子培养前先在33℃热激处理24h有利于小孢子启动胚状体发育途径。培养过程中添加和更换培养基均能显著提高胚产量，加液培养更有利于提高胚产量。不同材料对激素反应不尽相同。‘波浪叶红心’在不含激素的培养基中胚诱导率最高；‘红欧’以在添加0.10mg·L~(-1)6-BA的培养基中培养效果最好；‘白鸥’则在6-BA0.10mg·L~(-1)+NAA0.10mg·L~(-1)浓度组合的培养基中反应最好。振荡培养有利于胚状体的发生和发育，既能提高小孢子胚诱导率，又能促进子叶形胚发生，提高子叶形胚的比例，减少畸形胚发生率。
2．建立了羽衣甘蓝小孢子胚高频再生植株体系。B5固体培养基最适合羽衣甘蓝小孢子胚的再生。小孢子胚形成后，转至B5培养基，萌发很快，胚芽迅速分化，有利于胚发育成再生植株。子叶形胚植株再生频率显著高于其它类型的胚。鱼雷形胚可以发育成子叶形胚，然后再发育成苗；也可以先形成愈伤组织然后再分化成植株。心形胚、球形胚和畸形胚只有很小一部分能发育形成植株。最适宜的转胚时间是小孢子培养后的第25天。尽量减少胚状体在NLN液体培养基中滞留的时间，可以获得高的植株再生频率。小孢子胚转至固体培养基后，先在4℃低温培养一定时间，可以显著提高植株再生频率。‘白欧’和‘皱叶白心’的小孢子胚在4℃处理5d时效果最好；而‘皱叶红心’的胚在4℃处理2d后再生频率最高，但是低温处理时间不宜过长。接种到添加活性炭培养基上的胚状体萌发较快，再生植株生长健壮，成活率高。但是，添加活性炭并不能显著提高植株再生频率。添加适当浓度的AgNO_3能促进小孢子胚发育形成植株。‘红欧’在添加3.0mg·L~(-1)AgNO_3处理中获得最高植株再生频率，‘皱叶红心’在添加5.0mg·L~(-1)AgNO_3的处理中植株再生频率最高。
3．研究确定了羽衣甘蓝小孢子植株倍性鉴定及加倍方法。小孢子再生植株鉴定方法可以应用染色体计数的直接鉴定方法与形态学特征、气孔特征、花粉粒大小与活力、自交结实情况和流式细胞仪间接鉴定方法相结合。小孢子再生植株群体中，单倍体、双单倍体和四倍体同时存在，但不同基因型间再生植株的自然加倍率差别很大，对于自然加倍率低的基因型有必要进行人工加倍处理。在不同人工染色体加倍方法中，秋水仙素处理小孢子方法效果最好，在50mg·L~(-1)处理36h时加倍效率为52.9%，显著高于对照；秋水仙素处理切根小植株处理效果最差，小植株死亡率很高；在试管苗浸根试验中以1000mg·L~(-1)的秋水仙素处理植株根部4h的加倍效果最好，为44.8%，但这种方法容易造成根部损伤，移栽田间后生长较弱。
4．鉴定筛选出一批优异的羽衣甘蓝DH系。通过游离小孢子培养体系的优化，获得10个品种羽衣甘蓝来自不同胚状体的小孢子植株239株，经园艺学性状鉴定筛选出优异DH系18个。经自交亲和指数测定，所有DH系均符合自交不亲和系标准。利用DH系试配杂交组合，筛选出综合性状优良的杂交组合7个。
5．筛选获得了与红叶性状紧密连锁的SRAP标记。通过遗传分析证明了羽衣甘蓝红叶与白叶性状为一对基因控制的质量性状，红叶为显性。采用SRAP+BSA法，筛选获得了3个与红叶基因Re紧密连锁的SRAP标记，它们位于Re基因的两侧，遗传距离分别为2.2cM、6.4cM和3.7cM。
Kale is an important ornamental plant and vegetable. The flower bud differentiation ofkale takes a long time and it takes two years to accomplish a cycle of sexual life as it is avernalization plant. Six to eight years needed to cultivate an inbred line using conventionalmeans of breeding. Breeding method of double haploid utilized can effectively shorten thebreeding period and raise the efficiency of breeding instead of breeding process for inbredlines. Microspore culture is a technique of cell breeding to cultivate double haploid efficiently.Thus, the optimization of system for culture technique in kale has practical significance.Color of leaves as the most important ornamental trait in kale, and exploiting the molecularmarker closely linked to color of leaves forms an important premise of exploring themechanism of color conformation and carrying out research of breeding assisted bymolecular marker.
Microspore culture was conducted using16F1hybrids of kale as materials in this study.We studied the initiation and development of microspore embryos and the effect factors forregeneration of microspore embryo. The comparison of identifications for ploidy fromdifferent plant was made. The artificial induction for chromosome doubling method wasexplored. Identification of self-compatibility from DH lines and the horticultural traits forhybrid combinations from DH lines was taken. Hybrid combinations prepared using DH linesto build F2segregating populations to filter out SRAP markers on red leaf genes. The mainresults are as follows:
1. Optimization for mbryogenesis system of microspore in kale was conducted. It wasrevealed that genotype played a key role in the conformation of embryoids from isolatedmicrospore culture of kale.15microspore embryos obtained in the16tested materials.While, the differences of embryo rate for varieties were great. The difference of embryo ratebetween the highest ‘Zhouyehongxin’ and the lowest ‘Baijiu’ was1.94embryos per bud.The highest proportion of medium and late uninucleate period for microspore was67.31%,when the range of bud length for kale was from3.01to3.50mm. Thus, the range of budlength should be3.01~3.50mm when microspore culture of kale was taken, and contributingfor embryo induction. The ratio between petals and anthers was2/3~4/5. The production ofembryo was enhanced by low temperature（4℃）pretreatment on bud and24hours of treatment was the best result. Counterproductive was occurred when processing time wastoo long. Microspore treated with heat shock of33℃for24h before cultured contributed forstarting the developmental pathway of embryo. Adding and replacing the medium forculture of isolated microspore significantly improved the production of embryos. Addingliquid medium culture contributed to increasing production of embryos. The response forvarious materials to hormone was different. The embryo rate for ‘Bolangyehongxin’ inmedium without hormone was the highest. In vitro culture of0.10mg·L~(-1)of6-BA mediumwhen ‘Hongou’ added was the best, and0.10mg·L~(-1)of6-BA+0.10mg·L~(-1)of NAA mediumwhen ‘Baijiu’ added was the best. Shaking culture contributed for the initiation anddevelopment of embryos. The embryo rate of microspore and initiation of cotyledonaryembryo were promoted, as well as the proportion of cotyledonary embryo. In addition, theincidence of deformed embryos was reduced by shaking culture.
2. System for high-frequency regeneration of microspore-derived plant in kale was built.B5solid medium was most suitable for regeneration of microspore embryo in kale. Whenmicrospore embryo formed, and then transferred onto B5solid medium, after that, quicklygerminates and rapid differentiation of germ was found. It contributed for embryo developinginto regeneration plant. The regeneration frequency of cotyledonary embryo was significantlyhigher than other types of embryos. Torpedo-shaped embryos can develop into cotyledon,then into seedling. It also develops into callus, and then differentiates into plant. Only a smallfraction of heart-shaped embryo, globular embryo and embryonic malformations can developinto plant. The most appropriate time of embryo transfer was the25thday after microsporeculture. Higher regeneration frequency of plant observed when embryos stranded in NLNliquid medium was minimized. Microspore embryo was transferred into solid medium, andthen cultivated under low temperature（4℃）pretreatment for a certain period couldsignificantly increase the regeneration frequency of plant. Microspore embryo of ‘Baiou’ and‘Zhouyebaixin’ pretreated for5d was best, and the regeneration frequency of‘Zhouyehongxin’ pretreated for2d was the highest. However, the period for pretreatment oflow temperature should not be too long. Germination of embryos inoculated into the medium,which added activated carbon was faster, and the regeneration plant grew stronger andreached higher survival percentage. Howeve, addition of activated carbon did notsignificantly increase the percentage of plant regeneration. Appropriate concentration of AgNO_3was added to promote microspore embryo to develop into plant. The regenerationfrequency of ‘Hongou’ when3.0mg·L~(-1)of AgNO_3added was the highest, and when‘Zhouyehongxin’ added with5.0mg·L~(-1)of AgNO_3was the highest.
3． Identification and doubling methods for microspore-derived ploidy in kale werestudied. The application of combination for directs identification of chromosome count andindirect identification of characteristics of morphological and stoma, size and vitality ofpollen grain, from outcrossing and flow cytometry to evaluate the microspore regenerationplant. Haploid, double haploid and tetraploid existed among plant groups of microsporeregeneration. However, natural doubling rate from different genotypes of regenerated plantmade a great difference. It is necessary to carry out artificial redouble for natural doublinglow genotypes. Small spores treated with colchicines among artificial chromosome doublingwere the best. Doubling efficiency was52.9%when treated with concentration of50mg· L~(-1)colchicine for36h and it was significantly higher than control. Cut root plants treated withcolchicines was the worst, and it is difficult for genetics and breeding research as a highmortality rate obtained. During baptist root test of plant, the redouble effect was44.8%and wasbest when the root immersed in1000mg·L~(-1)of colchicine for4h. While, this method was likely tocause root damage and there was a weak growth after transplanting field.
4．Identification for DH lines population of kale was made. Two hundred and thirty nineof microspore-derived plntas were from10species of embryos in kale by optimization forsystem of isolated microspore culture. Eightteen excellent DH lines were identificatedaccording to horticultural traits. All DH lines fitted with standard of self-incompatibilitybased on the investigation of self-compatibility index. Seven hybrized combinations withexcellent comprehensive properties selected using DH lines to obtain the combination.
5．SRAP marker linked to trait of red leaves closely was obtained by filtering. The lawwas found through genetic analysis indicated that a couple of dominant gene on quality traitcontrol the red and white leaf trait of kale. Marker analysis was carried out with BSA strategyand3SRAP markers linking to Re gene were developed with an F2population derived fromcrossing D05×D10. These markes, Me8Em4、Me8Em17and Me9Em11, were mapped to bothsides of Re with genetic distances of2.2cM,3.7cM and6.4cM, respectively.
本项研究以16个羽衣甘蓝杂交种为试材进行小孢子培养，分析小孢子胚状体的发生与发育、以及小孢子植株再生的影响因素，筛选准确高效的小孢子植株倍性鉴定方法，探索人工诱导染色体加倍方法，鉴定培养所得DH系的自交亲和性及用其配制杂交种的园艺学性状；利用小孢子培养创制的DH系配制杂交组合，构建F2分离群体，筛选与红叶性状连锁的SRAP标记。获得了以下结果：
1．优化了羽衣甘蓝小孢子胚诱导体系。研究证明了基因型是影响羽衣甘蓝游离小孢子培养中胚状体形成的关键因素。在供试的16个基因型材料中15个基因型获得了小孢子胚状体，但出胚品种的胚诱导率相差很大，最高的‘皱叶红心’和最低的‘白鸠’两者胚诱导率相差达1.94胚·蕾~(-1)。花蕾长度在3.01~3.50mm范围内时，小孢子所处单核中晚期的比例最高，达67.31%。因此，在羽衣甘蓝小孢子培养时，选蕾长度应集中在3.01~3.50mm范围内，有助于胚状体诱导成功，此时花瓣与花药比在2/3~4/5。花蕾经4℃低温预处理可以提高胚产量，以处理24h效果最好，时间过长则会起反作用；小孢子培养前先在33℃热激处理24h有利于小孢子启动胚状体发育途径。培养过程中添加和更换培养基均能显著提高胚产量，加液培养更有利于提高胚产量。不同材料对激素反应不尽相同。‘波浪叶红心’在不含激素的培养基中胚诱导率最高；‘红欧’以在添加0.10mg·L~(-1)6-BA的培养基中培养效果最好；‘白鸥’则在6-BA0.10mg·L~(-1)+NAA0.10mg·L~(-1)浓度组合的培养基中反应最好。振荡培养有利于胚状体的发生和发育，既能提高小孢子胚诱导率，又能促进子叶形胚发生，提高子叶形胚的比例，减少畸形胚发生率。
2．建立了羽衣甘蓝小孢子胚高频再生植株体系。B5固体培养基最适合羽衣甘蓝小孢子胚的再生。小孢子胚形成后，转至B5培养基，萌发很快，胚芽迅速分化，有利于胚发育成再生植株。子叶形胚植株再生频率显著高于其它类型的胚。鱼雷形胚可以发育成子叶形胚，然后再发育成苗；也可以先形成愈伤组织然后再分化成植株。心形胚、球形胚和畸形胚只有很小一部分能发育形成植株。最适宜的转胚时间是小孢子培养后的第25天。尽量减少胚状体在NLN液体培养基中滞留的时间，可以获得高的植株再生频率。小孢子胚转至固体培养基后，先在4℃低温培养一定时间，可以显著提高植株再生频率。‘白欧’和‘皱叶白心’的小孢子胚在4℃处理5d时效果最好；而‘皱叶红心’的胚在4℃处理2d后再生频率最高，但是低温处理时间不宜过长。接种到添加活性炭培养基上的胚状体萌发较快，再生植株生长健壮，成活率高。但是，添加活性炭并不能显著提高植株再生频率。添加适当浓度的AgNO_3能促进小孢子胚发育形成植株。‘红欧’在添加3.0mg·L~(-1)AgNO_3处理中获得最高植株再生频率，‘皱叶红心’在添加5.0mg·L~(-1)AgNO_3的处理中植株再生频率最高。
3．研究确定了羽衣甘蓝小孢子植株倍性鉴定及加倍方法。小孢子再生植株鉴定方法可以应用染色体计数的直接鉴定方法与形态学特征、气孔特征、花粉粒大小与活力、自交结实情况和流式细胞仪间接鉴定方法相结合。小孢子再生植株群体中，单倍体、双单倍体和四倍体同时存在，但不同基因型间再生植株的自然加倍率差别很大，对于自然加倍率低的基因型有必要进行人工加倍处理。在不同人工染色体加倍方法中，秋水仙素处理小孢子方法效果最好，在50mg·L~(-1)处理36h时加倍效率为52.9%，显著高于对照；秋水仙素处理切根小植株处理效果最差，小植株死亡率很高；在试管苗浸根试验中以1000mg·L~(-1)的秋水仙素处理植株根部4h的加倍效果最好，为44.8%，但这种方法容易造成根部损伤，移栽田间后生长较弱。
4．鉴定筛选出一批优异的羽衣甘蓝DH系。通过游离小孢子培养体系的优化，获得10个品种羽衣甘蓝来自不同胚状体的小孢子植株239株，经园艺学性状鉴定筛选出优异DH系18个。经自交亲和指数测定，所有DH系均符合自交不亲和系标准。利用DH系试配杂交组合，筛选出综合性状优良的杂交组合7个。
5．筛选获得了与红叶性状紧密连锁的SRAP标记。通过遗传分析证明了羽衣甘蓝红叶与白叶性状为一对基因控制的质量性状，红叶为显性。采用SRAP+BSA法，筛选获得了3个与红叶基因Re紧密连锁的SRAP标记，它们位于Re基因的两侧，遗传距离分别为2.2cM、6.4cM和3.7cM。
Kale is an important ornamental plant and vegetable. The flower bud differentiation ofkale takes a long time and it takes two years to accomplish a cycle of sexual life as it is avernalization plant. Six to eight years needed to cultivate an inbred line using conventionalmeans of breeding. Breeding method of double haploid utilized can effectively shorten thebreeding period and raise the efficiency of breeding instead of breeding process for inbredlines. Microspore culture is a technique of cell breeding to cultivate double haploid efficiently.Thus, the optimization of system for culture technique in kale has practical significance.Color of leaves as the most important ornamental trait in kale, and exploiting the molecularmarker closely linked to color of leaves forms an important premise of exploring themechanism of color conformation and carrying out research of breeding assisted bymolecular marker.
Microspore culture was conducted using16F1hybrids of kale as materials in this study.We studied the initiation and development of microspore embryos and the effect factors forregeneration of microspore embryo. The comparison of identifications for ploidy fromdifferent plant was made. The artificial induction for chromosome doubling method wasexplored. Identification of self-compatibility from DH lines and the horticultural traits forhybrid combinations from DH lines was taken. Hybrid combinations prepared using DH linesto build F2segregating populations to filter out SRAP markers on red leaf genes. The mainresults are as follows:
1. Optimization for mbryogenesis system of microspore in kale was conducted. It wasrevealed that genotype played a key role in the conformation of embryoids from isolatedmicrospore culture of kale.15microspore embryos obtained in the16tested materials.While, the differences of embryo rate for varieties were great. The difference of embryo ratebetween the highest ‘Zhouyehongxin’ and the lowest ‘Baijiu’ was1.94embryos per bud.The highest proportion of medium and late uninucleate period for microspore was67.31%,when the range of bud length for kale was from3.01to3.50mm. Thus, the range of budlength should be3.01~3.50mm when microspore culture of kale was taken, and contributingfor embryo induction. The ratio between petals and anthers was2/3~4/5. The production ofembryo was enhanced by low temperature（4℃）pretreatment on bud and24hours of treatment was the best result. Counterproductive was occurred when processing time wastoo long. Microspore treated with heat shock of33℃for24h before cultured contributed forstarting the developmental pathway of embryo. Adding and replacing the medium forculture of isolated microspore significantly improved the production of embryos. Addingliquid medium culture contributed to increasing production of embryos. The response forvarious materials to hormone was different. The embryo rate for ‘Bolangyehongxin’ inmedium without hormone was the highest. In vitro culture of0.10mg·L~(-1)of6-BA mediumwhen ‘Hongou’ added was the best, and0.10mg·L~(-1)of6-BA+0.10mg·L~(-1)of NAA mediumwhen ‘Baijiu’ added was the best. Shaking culture contributed for the initiation anddevelopment of embryos. The embryo rate of microspore and initiation of cotyledonaryembryo were promoted, as well as the proportion of cotyledonary embryo. In addition, theincidence of deformed embryos was reduced by shaking culture.
2. System for high-frequency regeneration of microspore-derived plant in kale was built.B5solid medium was most suitable for regeneration of microspore embryo in kale. Whenmicrospore embryo formed, and then transferred onto B5solid medium, after that, quicklygerminates and rapid differentiation of germ was found. It contributed for embryo developinginto regeneration plant. The regeneration frequency of cotyledonary embryo was significantlyhigher than other types of embryos. Torpedo-shaped embryos can develop into cotyledon,then into seedling. It also develops into callus, and then differentiates into plant. Only a smallfraction of heart-shaped embryo, globular embryo and embryonic malformations can developinto plant. The most appropriate time of embryo transfer was the25thday after microsporeculture. Higher regeneration frequency of plant observed when embryos stranded in NLNliquid medium was minimized. Microspore embryo was transferred into solid medium, andthen cultivated under low temperature（4℃）pretreatment for a certain period couldsignificantly increase the regeneration frequency of plant. Microspore embryo of ‘Baiou’ and‘Zhouyebaixin’ pretreated for5d was best, and the regeneration frequency of‘Zhouyehongxin’ pretreated for2d was the highest. However, the period for pretreatment oflow temperature should not be too long. Germination of embryos inoculated into the medium,which added activated carbon was faster, and the regeneration plant grew stronger andreached higher survival percentage. Howeve, addition of activated carbon did notsignificantly increase the percentage of plant regeneration. Appropriate concentration of AgNO_3was added to promote microspore embryo to develop into plant. The regenerationfrequency of ‘Hongou’ when3.0mg·L~(-1)of AgNO_3added was the highest, and when‘Zhouyehongxin’ added with5.0mg·L~(-1)of AgNO_3was the highest.
3． Identification and doubling methods for microspore-derived ploidy in kale werestudied. The application of combination for directs identification of chromosome count andindirect identification of characteristics of morphological and stoma, size and vitality ofpollen grain, from outcrossing and flow cytometry to evaluate the microspore regenerationplant. Haploid, double haploid and tetraploid existed among plant groups of microsporeregeneration. However, natural doubling rate from different genotypes of regenerated plantmade a great difference. It is necessary to carry out artificial redouble for natural doublinglow genotypes. Small spores treated with colchicines among artificial chromosome doublingwere the best. Doubling efficiency was52.9%when treated with concentration of50mg· L~(-1)colchicine for36h and it was significantly higher than control. Cut root plants treated withcolchicines was the worst, and it is difficult for genetics and breeding research as a highmortality rate obtained. During baptist root test of plant, the redouble effect was44.8%and wasbest when the root immersed in1000mg·L~(-1)of colchicine for4h. While, this method was likely tocause root damage and there was a weak growth after transplanting field.
4．Identification for DH lines population of kale was made. Two hundred and thirty nineof microspore-derived plntas were from10species of embryos in kale by optimization forsystem of isolated microspore culture. Eightteen excellent DH lines were identificatedaccording to horticultural traits. All DH lines fitted with standard of self-incompatibilitybased on the investigation of self-compatibility index. Seven hybrized combinations withexcellent comprehensive properties selected using DH lines to obtain the combination.
5．SRAP marker linked to trait of red leaves closely was obtained by filtering. The lawwas found through genetic analysis indicated that a couple of dominant gene on quality traitcontrol the red and white leaf trait of kale. Marker analysis was carried out with BSA strategyand3SRAP markers linking to Re gene were developed with an F2population derived fromcrossing D05×D10. These markes, Me8Em4、Me8Em17and Me9Em11, were mapped to bothsides of Re with genetic distances of2.2cM,3.7cM and6.4cM, respectively.
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