黄瓜属种间遗传资源的创制及其细胞学和分子遗传学定性研究
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摘要
本项研究在黄瓜属种间杂交取得突破性进展的基础上，以黄瓜属种间双二倍体Cucumis hytivus Chen & Kirkbride、及其双亲(栽培黄瓜“北京截头”和野生种C.hystrix Chakr.)等为核心材料，成功地培育了不同基因型的种间杂种F_1(“二早子”/C.hystrix)、黄瓜属异源三倍体以及两个“黄瓜—C.hystrix”单体异附加系；建立了种间杂种F_1和异源三倍体的无性繁殖体系；并运用细胞遗传学和分子标记等分析手段完成了对这些新合成的特异种间材料的定性研究。
1．黄瓜属种间杂种F_1离体繁殖以及定性研究
运用不同的授粉方式、以4个生态型黄瓜栽培种“A309”、“二早子”、“白丝条”、“北京截头”作亲本，与野生种C.hystrix进行正、反杂交，结合胚胎拯救技术，最终获得“二早子”/野生种C.hystrix的种间杂种F_1。通过体细胞染色体记数(2n=2x=19)和POD同工酶分析，并结合田间形态学观察，确定了“二早子”/野生种C.hystrix杂种的真实性。
在对正反交杂种F_1的形态和育性差异进行比较研究的同时，研究了杂种F_1的花粉母细胞减数分裂过程。结果表明：当以黄瓜栽培种为母本时，杂种植株呈现雌雄高度不育，染色体加倍后杂种的育性仍然非常低；而当以C.hystrix为母本时，杂种植株虽然仍高度不育，但受外来花粉刺激后能结大量的果实，加倍后雌雄育性均得到恢复。减数分裂研究发现，正反杂种减数分裂行为无明显差异，终变期和中期Ⅰ，主要以17条单价体(Ⅰ)和1个二价体(Ⅱ)构成；整个花粉母细胞的减数分裂行为异常，经常可见后染色体滞后和中期纺锤丝定向紊乱。
利用胚胎拯救获得的杂种F_1无菌苗的茎尖或腋芽建立了杂种F_1的快速繁殖体系。离体繁殖的结果表明，杂种F_1不定芽增殖具有杂种优势，极易增殖。在只添加BA 0.5～1.0mg·L~(-1)的培养基上，其增殖系数达7.6以上。高于这个范围，增殖系数随着浓度的增加而下降。而在无激素的MS基本培养基上不分化出丛生芽。
2．异源三倍体的培育及离体繁殖研究
通过胚胎拯救和体细胞突变加倍获得的完全可育的黄瓜属双二倍体种(Cucumis hytivus Chen and Kirkbride，2n=4x=38)是转移野生种有用性状的桥梁。对C.hytivus与栽培黄瓜的回交后代进行鉴定和定性研究是黄瓜属作物种间基因渐渗的主
摘要
要内容。将双二倍体(母本)和栽培黄瓜“北京截头”(e sarivus ev.Beijin自ietou.，
2n二2x=14)(父本)的杂种胚进行培养，获得无菌苗。将无菌苗的顶芽和腋芽用
于离体快繁，结果表明，Ms+2.2 mg·L一，6一BA和Ms+3.0 mg·L一，KT+0.2
mg·t-IN从为最佳增殖培养基，繁殖系数分别为6.53和“3;丛生芽在Ms+0.2
mg·L-16一BA的培养基上伸长，大约10d后取整齐一致的芽用于生根，在1/2 Ms+
0.2 mg·L’IBA上生根率达92.80，0.
驯化成活的异源三倍体组培苗形态整齐但不同于父母本的植株.其体细胞染色体
数为2n=3x=26·异源三倍体单性结实能加匕“双亲”更强;果刺黑色，与.c.
匆tl’vus相同;叶深绿色，不同于双二倍体特有的淡黄绿色而与“北京截头”一致;
花粉粒的醋酸洋红可染率不到10%.
3.单体异附加系的培育及其细胞遗传学和分子标记研究
以异源三倍体离体茎尖为材料，采用不同浓度梯度(0 .0%，0.2%，0.4%，0.8%)
的秋水仙素处理。在驯化成活的252棵植株中有4棵变异植株，编号分别为02-
517、02一87、02一13和02一43.02一517植株叶形为戟形，02一87为深陷的掌状形，两棵
植株的果刺均为白色，不同于异源三倍体的五角心状形和黑刺，果实比异源三倍体
的长，更接近普通黄瓜.经细胞学鉴定发现，02一517和02一87为单体异附加系，而
02一13和02一43为混倍体。
单体异附加系02一517和02一87在终变期和中期I染色体构型主要(94%以上)是
711+1I，三价体(m)的频率为4一5%;后期I不均等分裂，一极为8条染色体，而
另一极为7条。在后期n，18.2~2 5.2%花粉母细胞中的外源染色体滞后.
POD同工酶和RAPD标记分析表明，02一517和02一87中的外源染色体来源于野
生种C hystrix.引物E一19，N一20，A一9，A一11，Allg，AH13，AJ18和^J20在02一517和
02一87能扩增出具有与野生种共有条带，而在栽培黄瓜“北京截头”中则不能扩增出
该条带.其中引物E一19扩增出的野生种c hystrix染色组条带为300 bp的片段.引
物A巧，N一12，N一20和AJ 13能在02一517和02一87中扩增出各自的特征带.
4.不同倍性种间杂种的离体繁殖研究
以杂种F，、异源三倍体和双二倍体为试材，探讨了不同倍性的黄瓜属种间杂种
离体繁殖率的差异.试验结果表明:种间杂种F;和异源三倍体具有杂种优势，两种
材料不管是在离体条件下还是在田间，长势都要超过栽培黄瓜和双二倍体C
勺tivlls，杂种Fl和异源三倍体在单一细胞分裂素BA的培养基上繁殖系数能达到最
大值，浓度分别为0.5一1 .0 mg·L一，和1.5一2.2mg·LI，增殖系数分别为8.6和
摘要
6.5。双二倍体生长缓慢，以培养基Ms+TDz 0.05mg·L一，和Ms十2.2 mg·L一，BA
效果最好，增殖系数分别为4.6和3.9。不同倍性的种间杂种材料在1/2 MS+0.2
mg·L一，IBA上生根率达80%以?
Based on the achieving interspecific hybridization progress aiming at gene introgression from C. hystrix Chakr to C. sativus, the amphidiploid C. hytivus Chen & Kirkbride and its parental C. sativus L. cv. Beijingjietou and wild species C. hystrix et al. were used as core material for further investigation. Novel genotype interspecific hybrids FI, allotriploids and two monosomic alien addition lines have been sucessfully produced in the present study. At the same time, micropropagation system of FI and allotriploid has been established. The development new germplasm were characterized by cytogenetic and molecular - marker analyses.
1. Micropropagation and characterization of Cucumis sativus X Cucumis hystrix hybrids
Four cucumber cultivars (A309, Erzaozi, Baisitiao and Beijingjietou) were crossed with C. hystrix with different pollination methods. Young embryos were rescued in vitro, and interspecific hybrids were only obtained from the cross between Erzaozi and C. hystrix. The metaphase chromosomes of FI hybrids were counted to be 19. The hybrid was also identified by morphological comparison and electrophoresis of isozyme.
The difference in fertility and morphology between the reciprocal plants was found, and the meiosis behaviors in FI were investigated. When cucumber was used as the female parent, both tetraploid and diploid hybrid plants were highly sterile and did not set any fruits. However, When C. hystrix was used as female parent, there were not female flowers in the diploid (2n = 2x = 19) hybrids and the male male flower would be wilt before flowering. In pollen mother cells (PMCs) of the diploid hybrids, the major chromosome configuration was 17 univalents and 1 bivalent at diakinesis and metaphse I (M I). The abnormal meiotic behaviors such as chromosome lagging and spindle misorientation were frequently observed.
Apical and axillary buds of hybrids FI, which was obtained through embryo rescuing, were used as explants to establish the micropropagation system. The result indicatied that hybrids F1(dihaploid) proliferated quickly. The proliferation ratio was more than 7.6 on the media with supplemented 0.5 -1.0 mg·L-1 BA only. The multiplication ratio would
reduced if the concentration was higher than over 1.0 mg·L-1. The shoots would not multiplictate on free plant growth regulator media.
2. An allotriploid derived from a amphidiploid X diploid mating in Cucumis : production, micropropagation and verification
A fully fertile interspecific hybrid (Cucumis hytivus Chen and Kirkbride, 2n = 4x = 38) between Cucumis hystrix Chakr. (2n = 2x = 24) and C. sativus L. (2n = 2x - 14) was previously produced by means of FI (2n =19) embryo rescue and subsequent chromosome doubling. This amphidiploid, a new synthetic species, may serve as a genetic bridge in Cucumis, and thus is a source for broadening the genetic base of C. sativus. The identification and characterization of fertile progeny possessing lower ploidy levels would facilitate bridge among Cucumis species. Putative allotriploids (2n = 26) were recovered from C. hytivus X C. sativus matings by means of embryo culture. Experiments were designed to confirm their genetic constitution, describe their morphology, and establish an efficient protocol for their micropropagation. Apical and axillary buds of these putative allotriploid plants were used as explants to establish a micropropagation system for subsequent verification and characterization of ploidy. Of the array o
f micropropagation media tested, the most effective for the induction of adventitious buds (desginated Stage II) was a Murashige and Skoog (MS) growth media containing 3.0 mg· L-1 BA + 0.2 mg · L-1 NAA or containing 2.2 mg · L-1 BA only. The mean number of adventitious buds per explant in the two media was 6.8 and 6.5, respectively. Shoots resulting from adventitious buds produced roots (Stage III) in relative abundance (39 of 42, 92.8%) on half-strength MS medium containing 0.2 mg · L-1 IBA. The survivorship of rooted plantlets after acclimatization as assessed
1．黄瓜属种间杂种F_1离体繁殖以及定性研究
运用不同的授粉方式、以4个生态型黄瓜栽培种“A309”、“二早子”、“白丝条”、“北京截头”作亲本，与野生种C.hystrix进行正、反杂交，结合胚胎拯救技术，最终获得“二早子”/野生种C.hystrix的种间杂种F_1。通过体细胞染色体记数(2n=2x=19)和POD同工酶分析，并结合田间形态学观察，确定了“二早子”/野生种C.hystrix杂种的真实性。
在对正反交杂种F_1的形态和育性差异进行比较研究的同时，研究了杂种F_1的花粉母细胞减数分裂过程。结果表明：当以黄瓜栽培种为母本时，杂种植株呈现雌雄高度不育，染色体加倍后杂种的育性仍然非常低；而当以C.hystrix为母本时，杂种植株虽然仍高度不育，但受外来花粉刺激后能结大量的果实，加倍后雌雄育性均得到恢复。减数分裂研究发现，正反杂种减数分裂行为无明显差异，终变期和中期Ⅰ，主要以17条单价体(Ⅰ)和1个二价体(Ⅱ)构成；整个花粉母细胞的减数分裂行为异常，经常可见后染色体滞后和中期纺锤丝定向紊乱。
利用胚胎拯救获得的杂种F_1无菌苗的茎尖或腋芽建立了杂种F_1的快速繁殖体系。离体繁殖的结果表明，杂种F_1不定芽增殖具有杂种优势，极易增殖。在只添加BA 0.5～1.0mg·L~(-1)的培养基上，其增殖系数达7.6以上。高于这个范围，增殖系数随着浓度的增加而下降。而在无激素的MS基本培养基上不分化出丛生芽。
2．异源三倍体的培育及离体繁殖研究
通过胚胎拯救和体细胞突变加倍获得的完全可育的黄瓜属双二倍体种(Cucumis hytivus Chen and Kirkbride，2n=4x=38)是转移野生种有用性状的桥梁。对C.hytivus与栽培黄瓜的回交后代进行鉴定和定性研究是黄瓜属作物种间基因渐渗的主
摘要
要内容。将双二倍体(母本)和栽培黄瓜“北京截头”(e sarivus ev.Beijin自ietou.，
2n二2x=14)(父本)的杂种胚进行培养，获得无菌苗。将无菌苗的顶芽和腋芽用
于离体快繁，结果表明，Ms+2.2 mg·L一，6一BA和Ms+3.0 mg·L一，KT+0.2
mg·t-IN从为最佳增殖培养基，繁殖系数分别为6.53和“3;丛生芽在Ms+0.2
mg·L-16一BA的培养基上伸长，大约10d后取整齐一致的芽用于生根，在1/2 Ms+
0.2 mg·L’IBA上生根率达92.80，0.
驯化成活的异源三倍体组培苗形态整齐但不同于父母本的植株.其体细胞染色体
数为2n=3x=26·异源三倍体单性结实能加匕“双亲”更强;果刺黑色，与.c.
匆tl’vus相同;叶深绿色，不同于双二倍体特有的淡黄绿色而与“北京截头”一致;
花粉粒的醋酸洋红可染率不到10%.
3.单体异附加系的培育及其细胞遗传学和分子标记研究
以异源三倍体离体茎尖为材料，采用不同浓度梯度(0 .0%，0.2%，0.4%，0.8%)
的秋水仙素处理。在驯化成活的252棵植株中有4棵变异植株，编号分别为02-
517、02一87、02一13和02一43.02一517植株叶形为戟形，02一87为深陷的掌状形，两棵
植株的果刺均为白色，不同于异源三倍体的五角心状形和黑刺，果实比异源三倍体
的长，更接近普通黄瓜.经细胞学鉴定发现，02一517和02一87为单体异附加系，而
02一13和02一43为混倍体。
单体异附加系02一517和02一87在终变期和中期I染色体构型主要(94%以上)是
711+1I，三价体(m)的频率为4一5%;后期I不均等分裂，一极为8条染色体，而
另一极为7条。在后期n，18.2~2 5.2%花粉母细胞中的外源染色体滞后.
POD同工酶和RAPD标记分析表明，02一517和02一87中的外源染色体来源于野
生种C hystrix.引物E一19，N一20，A一9，A一11，Allg，AH13，AJ18和^J20在02一517和
02一87能扩增出具有与野生种共有条带，而在栽培黄瓜“北京截头”中则不能扩增出
该条带.其中引物E一19扩增出的野生种c hystrix染色组条带为300 bp的片段.引
物A巧，N一12，N一20和AJ 13能在02一517和02一87中扩增出各自的特征带.
4.不同倍性种间杂种的离体繁殖研究
以杂种F，、异源三倍体和双二倍体为试材，探讨了不同倍性的黄瓜属种间杂种
离体繁殖率的差异.试验结果表明:种间杂种F;和异源三倍体具有杂种优势，两种
材料不管是在离体条件下还是在田间，长势都要超过栽培黄瓜和双二倍体C
勺tivlls，杂种Fl和异源三倍体在单一细胞分裂素BA的培养基上繁殖系数能达到最
大值，浓度分别为0.5一1 .0 mg·L一，和1.5一2.2mg·LI，增殖系数分别为8.6和
摘要
6.5。双二倍体生长缓慢，以培养基Ms+TDz 0.05mg·L一，和Ms十2.2 mg·L一，BA
效果最好，增殖系数分别为4.6和3.9。不同倍性的种间杂种材料在1/2 MS+0.2
mg·L一，IBA上生根率达80%以?
Based on the achieving interspecific hybridization progress aiming at gene introgression from C. hystrix Chakr to C. sativus, the amphidiploid C. hytivus Chen & Kirkbride and its parental C. sativus L. cv. Beijingjietou and wild species C. hystrix et al. were used as core material for further investigation. Novel genotype interspecific hybrids FI, allotriploids and two monosomic alien addition lines have been sucessfully produced in the present study. At the same time, micropropagation system of FI and allotriploid has been established. The development new germplasm were characterized by cytogenetic and molecular - marker analyses.
1. Micropropagation and characterization of Cucumis sativus X Cucumis hystrix hybrids
Four cucumber cultivars (A309, Erzaozi, Baisitiao and Beijingjietou) were crossed with C. hystrix with different pollination methods. Young embryos were rescued in vitro, and interspecific hybrids were only obtained from the cross between Erzaozi and C. hystrix. The metaphase chromosomes of FI hybrids were counted to be 19. The hybrid was also identified by morphological comparison and electrophoresis of isozyme.
The difference in fertility and morphology between the reciprocal plants was found, and the meiosis behaviors in FI were investigated. When cucumber was used as the female parent, both tetraploid and diploid hybrid plants were highly sterile and did not set any fruits. However, When C. hystrix was used as female parent, there were not female flowers in the diploid (2n = 2x = 19) hybrids and the male male flower would be wilt before flowering. In pollen mother cells (PMCs) of the diploid hybrids, the major chromosome configuration was 17 univalents and 1 bivalent at diakinesis and metaphse I (M I). The abnormal meiotic behaviors such as chromosome lagging and spindle misorientation were frequently observed.
Apical and axillary buds of hybrids FI, which was obtained through embryo rescuing, were used as explants to establish the micropropagation system. The result indicatied that hybrids F1(dihaploid) proliferated quickly. The proliferation ratio was more than 7.6 on the media with supplemented 0.5 -1.0 mg·L-1 BA only. The multiplication ratio would
reduced if the concentration was higher than over 1.0 mg·L-1. The shoots would not multiplictate on free plant growth regulator media.
2. An allotriploid derived from a amphidiploid X diploid mating in Cucumis : production, micropropagation and verification
A fully fertile interspecific hybrid (Cucumis hytivus Chen and Kirkbride, 2n = 4x = 38) between Cucumis hystrix Chakr. (2n = 2x = 24) and C. sativus L. (2n = 2x - 14) was previously produced by means of FI (2n =19) embryo rescue and subsequent chromosome doubling. This amphidiploid, a new synthetic species, may serve as a genetic bridge in Cucumis, and thus is a source for broadening the genetic base of C. sativus. The identification and characterization of fertile progeny possessing lower ploidy levels would facilitate bridge among Cucumis species. Putative allotriploids (2n = 26) were recovered from C. hytivus X C. sativus matings by means of embryo culture. Experiments were designed to confirm their genetic constitution, describe their morphology, and establish an efficient protocol for their micropropagation. Apical and axillary buds of these putative allotriploid plants were used as explants to establish a micropropagation system for subsequent verification and characterization of ploidy. Of the array o
f micropropagation media tested, the most effective for the induction of adventitious buds (desginated Stage II) was a Murashige and Skoog (MS) growth media containing 3.0 mg· L-1 BA + 0.2 mg · L-1 NAA or containing 2.2 mg · L-1 BA only. The mean number of adventitious buds per explant in the two media was 6.8 and 6.5, respectively. Shoots resulting from adventitious buds produced roots (Stage III) in relative abundance (39 of 42, 92.8%) on half-strength MS medium containing 0.2 mg · L-1 IBA. The survivorship of rooted plantlets after acclimatization as assessed
引文
1．陈劲枫，罗向东，余纪柱，等．异源三倍体黄瓜的离体繁殖和鉴定，植物生理学通讯．2003a，32(2)：109～112．
2．陈劲枫，罗向东，钱春桃，等．附加Cucumis hystix Chakr．染色体的两个黄瓜单体异附加系．园艺学报．2003b(在版)
3．陈劲枫，钱春桃．利用几种园艺作物卷须制片鉴定染色体数目的研究．园艺学报，2002，29(4)：378～380．
4．陈劲枫，庄飞云，娄群峰，等．Cucumis属植物种间正反交差异研究．园艺学报．2002b，29(5)：483～485．
5．钱春桃，陈劲枫，庄飞云，等．弱光条件下黄瓜属种间杂交新种的某些光合特性．植物生理学通讯．2002．38(4)：336～338．
6．陈劲枫，林茂松，钱春桃等．黄瓜属野生种及其与黄瓜种间杂交后代抗根结线虫初步研究．南京农业大学．2001，24(1)：21-24．
7．陈振光，王家福．柑橘合子胚早期离体培养获得植株．福建农业大学学报．1986，15(4)：271-276．
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