大白菜花药和游离小孢子培养技术体系的研究
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摘要
生物技术的不断发展和完善，为大白菜育种提供了新的思路和手段，花药培养和游离小孢子培养技术的应用大大加快了大白菜的育种进程。本课题以13份春、夏大白菜为材料，从2001年春到2002年冬进行花药和游离小孢子培养，同时对花培材料的快速繁殖、倍性鉴定、染色体加倍及大白菜真叶的再生进行了相关的研究探讨。结果如下：
1．花蕾外观特征与小孢子发育时期的关系：通过细胞学观察表明，当花蕾长为2.0-3.0mm，花瓣与花药长度之比为1／2～4／5时，50-70％的小孢子发育处于单核晚期，最适合进行花培。
2．花药培养：不同的基因型材料具有不同的胚状体的诱导能力，在接种的13份材料中，126最容易诱导得到胚状体，诱导效率达13.71％；不同的培养基的培养效果也有差异，本研究中以蔗糖浓度为10％、有机成份加倍的Keller培养基的诱导效率最高，可达到17.58％，MS和Nitch培养基的诱导效率不高；花药培养胚状体的出现主要在接种后的15～30天之间。
3．游离小孢子培养：采用NLN-13液体培养基进行游离小孢子培养。由培养结果可见基因型不同，胚状体的诱导率有极大的差异，最易形成胚状体的材料是123，诱导效率达16.51％；在本实验中，3-5℃低温预处理48小时对胚状体的诱导无显著的影响；而细胞分裂素6-BA0.2mg／L和生长素NAA0.05mg／L的应用则可以明显提高胚状体的诱导效果。
4．胚状体成苗、试管苗快繁和移栽：胚状体转移到琼脂1.0％、蔗糖2.0％、6-BA0.2mg／L的B_5培养基上成苗；花药培养得到的胚状体成苗率为72.73％，而游离小孢子培养的只有54.61％，花药培养的成苗率显著优于游离培养的。花粉植株的快速繁殖以B_5+6-BA1.0mg／L+NAA0.25mg／L的繁殖系数最高，可达500％；大白菜试管苗玻璃化现象严重，用透气膜代替一般的封口膜可以大大降低玻璃化，提高胚状体成苗率；移栽成活率达到90％以上。
5．花粉植株的鉴定：应用形态学和解剖学特征可以快速初步鉴定花粉植株；染色体鉴定单倍体植株n=10，正常二倍体2n=20；流式细胞仪可以快速而准确地测定单倍体材料DNA含量为二倍体的一半，鉴定结论极为可靠；结合上述几种鉴定方法，可以在短期内对大量的花培材料进行快速而准确的鉴定。
6.染色体加倍:大白菜的花粉植株自然加倍率为40-70%，基因型不同，加倍效
率也有差异。利用秋水仙素结合组织培养诱导试管苗加倍，以秋水仙素浓度为
loom叭、处理时间10天为宜。
7大白菜真叶的再生的初步探讨:真叶的再生以BS+6.BA2.omg几+N AAO.smg/I‘
论AI.om叭+ABAO.25 mg几+AgNo3 5.om叭的处理效果最好，再生效率可达600/0
左右。
The Chinese cabbage ( Brassica campestris ssp. pekinensi ) breeding took on a new look along with the development of biotechnology-. Homozygous lines established by anther and isolated microspore culture could provide new solutions for improvement of Chinese cabbage breeding. Thirteen cultivars of spring and summer Chinese cabbage were used in the study of anther and isolated microspore culture. Meanwhile, ploidy identification, propagation, chromosome doubling of regenerated plants by colchicine and regeneration of leaf were also studied.
The main results are as follows:
1. The cytological observations of pollen in different developmental stages of Chinese cabbage were undertook. The results showed that most of the pollen were in the late uninucleate stage when the ratio of petal to anther length was between 1/2-4/5 in Chinese cabbage. The stage of late uninucleate was optimal for anther and isolated microspore culture.
2.Anther culture: The effects of genotype and cultural medium on embryoid induction were investigated in spring and summer Chinese cabbage. The results showed it was optimal to induce the embryoid in Keller medium with double organic ingredients and 10% sucrose. The capacity of embryoid production varied significantly in different genotypes, and genotype 126 was an ideal material for embryoid induction.
3.Isolated microspore culture: Embryoids were obtained from four cultivars, and the embryoid induction frequency varied significantly among different cultivars. Low-temperature pretreation on donor plant (3-5 "C for 48 hours) in NLN liquid medium did not yield more embryoids.The embryoid induction frequency reached the highest when isolated microspores were cultured in NLN-13 supplemented with 6-BA 0.2mg/L and NAA0.05mg/L.
4. Regeneration from embryoids. Propagation and transplantation: The frequency of plants regenerated from embryoids was 72.73% for anther culture, while 54.61% for isolated microspore culture. Propagation of regenerated plants: a large number of plants
could be obtained by induction of shoot in the 85 medium supplemented with 6-BA1.0mg/L and NAA0.25mg/L. More than 90% of the plantlets survived during transplantation after acclimation in plastic pot for more than a week.
5 .Ploidy identification: The ploidy character of the plantlets were primarily identified by observing the phenotypic appearance as well as the stoma density on the leaf surface. The ploidy feature were further determined by chromosome microscopical observation and Flow Cytometer technology. Chromosome identification showed that the number of haploid plantlets chromosome is n=10,while 2n=20 for diploid. The DNA count of haploid plantlets was equal to half of diploid .
6.Chromosome doubling: The ratios of natural chromosome doubling of varied from 40% to 70% for different genotypes. It was optimal for chromosome doubling inducement by culturing the plantlets in 85 medium with colchicine 100mg/L for 10 days .
7.Regeneration of leaf: The experiment showed it was optimal for leaf regeneration when the treatmeat was in the 85 medium supplemented with 6-BA 2.0mg/L, NAA 0.5mg/L, GA1.0mg/L, ABA 0.25mg/L and AgNO3 5.0mg/L.
1．花蕾外观特征与小孢子发育时期的关系：通过细胞学观察表明，当花蕾长为2.0-3.0mm，花瓣与花药长度之比为1／2～4／5时，50-70％的小孢子发育处于单核晚期，最适合进行花培。
2．花药培养：不同的基因型材料具有不同的胚状体的诱导能力，在接种的13份材料中，126最容易诱导得到胚状体，诱导效率达13.71％；不同的培养基的培养效果也有差异，本研究中以蔗糖浓度为10％、有机成份加倍的Keller培养基的诱导效率最高，可达到17.58％，MS和Nitch培养基的诱导效率不高；花药培养胚状体的出现主要在接种后的15～30天之间。
3．游离小孢子培养：采用NLN-13液体培养基进行游离小孢子培养。由培养结果可见基因型不同，胚状体的诱导率有极大的差异，最易形成胚状体的材料是123，诱导效率达16.51％；在本实验中，3-5℃低温预处理48小时对胚状体的诱导无显著的影响；而细胞分裂素6-BA0.2mg／L和生长素NAA0.05mg／L的应用则可以明显提高胚状体的诱导效果。
4．胚状体成苗、试管苗快繁和移栽：胚状体转移到琼脂1.0％、蔗糖2.0％、6-BA0.2mg／L的B_5培养基上成苗；花药培养得到的胚状体成苗率为72.73％，而游离小孢子培养的只有54.61％，花药培养的成苗率显著优于游离培养的。花粉植株的快速繁殖以B_5+6-BA1.0mg／L+NAA0.25mg／L的繁殖系数最高，可达500％；大白菜试管苗玻璃化现象严重，用透气膜代替一般的封口膜可以大大降低玻璃化，提高胚状体成苗率；移栽成活率达到90％以上。
5．花粉植株的鉴定：应用形态学和解剖学特征可以快速初步鉴定花粉植株；染色体鉴定单倍体植株n=10，正常二倍体2n=20；流式细胞仪可以快速而准确地测定单倍体材料DNA含量为二倍体的一半，鉴定结论极为可靠；结合上述几种鉴定方法，可以在短期内对大量的花培材料进行快速而准确的鉴定。
6.染色体加倍:大白菜的花粉植株自然加倍率为40-70%，基因型不同，加倍效
率也有差异。利用秋水仙素结合组织培养诱导试管苗加倍，以秋水仙素浓度为
loom叭、处理时间10天为宜。
7大白菜真叶的再生的初步探讨:真叶的再生以BS+6.BA2.omg几+N AAO.smg/I‘
论AI.om叭+ABAO.25 mg几+AgNo3 5.om叭的处理效果最好，再生效率可达600/0
左右。
The Chinese cabbage ( Brassica campestris ssp. pekinensi ) breeding took on a new look along with the development of biotechnology-. Homozygous lines established by anther and isolated microspore culture could provide new solutions for improvement of Chinese cabbage breeding. Thirteen cultivars of spring and summer Chinese cabbage were used in the study of anther and isolated microspore culture. Meanwhile, ploidy identification, propagation, chromosome doubling of regenerated plants by colchicine and regeneration of leaf were also studied.
The main results are as follows:
1. The cytological observations of pollen in different developmental stages of Chinese cabbage were undertook. The results showed that most of the pollen were in the late uninucleate stage when the ratio of petal to anther length was between 1/2-4/5 in Chinese cabbage. The stage of late uninucleate was optimal for anther and isolated microspore culture.
2.Anther culture: The effects of genotype and cultural medium on embryoid induction were investigated in spring and summer Chinese cabbage. The results showed it was optimal to induce the embryoid in Keller medium with double organic ingredients and 10% sucrose. The capacity of embryoid production varied significantly in different genotypes, and genotype 126 was an ideal material for embryoid induction.
3.Isolated microspore culture: Embryoids were obtained from four cultivars, and the embryoid induction frequency varied significantly among different cultivars. Low-temperature pretreation on donor plant (3-5 "C for 48 hours) in NLN liquid medium did not yield more embryoids.The embryoid induction frequency reached the highest when isolated microspores were cultured in NLN-13 supplemented with 6-BA 0.2mg/L and NAA0.05mg/L.
4. Regeneration from embryoids. Propagation and transplantation: The frequency of plants regenerated from embryoids was 72.73% for anther culture, while 54.61% for isolated microspore culture. Propagation of regenerated plants: a large number of plants
could be obtained by induction of shoot in the 85 medium supplemented with 6-BA1.0mg/L and NAA0.25mg/L. More than 90% of the plantlets survived during transplantation after acclimation in plastic pot for more than a week.
5 .Ploidy identification: The ploidy character of the plantlets were primarily identified by observing the phenotypic appearance as well as the stoma density on the leaf surface. The ploidy feature were further determined by chromosome microscopical observation and Flow Cytometer technology. Chromosome identification showed that the number of haploid plantlets chromosome is n=10,while 2n=20 for diploid. The DNA count of haploid plantlets was equal to half of diploid .
6.Chromosome doubling: The ratios of natural chromosome doubling of varied from 40% to 70% for different genotypes. It was optimal for chromosome doubling inducement by culturing the plantlets in 85 medium with colchicine 100mg/L for 10 days .
7.Regeneration of leaf: The experiment showed it was optimal for leaf regeneration when the treatmeat was in the 85 medium supplemented with 6-BA 2.0mg/L, NAA 0.5mg/L, GA1.0mg/L, ABA 0.25mg/L and AgNO3 5.0mg/L.
引文
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