小麦/中间偃麦草体细胞杂交早期及愈伤组织杂种染色质消减/渐渗研究
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摘要
植物体细胞杂交技术能够转移不同亲缘关系物种间所具有的优良性状,利用该技术向作物转移有价值的基因,已经进行了30多年的尝试,获得了一些用于作物改良的新种质和新品系(种),跨越了有性杂交中的不亲和性和花期不遇等障碍,在作物育种中显示出强大的优势。小麦是重要的粮食作物,对小麦体细胞杂交机制进行深入的理论探讨具有重要意义。本实验室建立了利用不对称体细胞杂交介导的不同亲缘关系禾谷类植物的异(外)源染色质直接渐渗到小麦基因组中,从而创制小麦渐渗系的技术体系。已开展的不对称体细胞杂交工作大多关注于对外源目的性状转移的研究,尚无利用该技术创制外源基因渐渗系并对渐渗的影响因素如异源染色质消减、渐渗发生的时期及影响因素等进行系统深入的研究。本论文选取与小麦亲缘关系较近的异属植物中间偃麦草(Elytrigiaintermedium(Host)Nevski)进行了扬麦158(+)中间偃麦草的对称与不对称(UV辐照)体细胞杂交,获得了体细胞杂种愈伤组织;利用细胞学和基因组原位杂交技术(GISH,Genomic in situ hybridization),对体细胞杂交后1、2、3周的融合产物及获得的杂种愈伤组织进行了染色质消减、渐渗的初步分析。结果表明,对称/不对称体细胞杂种中,供体(中间偃麦草)染色质主要以微核的形式在细胞间期发生消减;染色质还存在在有丝分裂后期形成微核、多核体中形成微核、滞后染色体(桥)及自融合中发生核外排等现象;另UV 1min组融合1周的材料中发现,扬麦158自融合来源的细胞有丝分裂末期形成的两个子细胞的过程中,由于细胞分裂周期不同步,首先完成有丝分裂的细胞将还在处于分裂期的细胞中的少数几条染色体(滞后染色体)随机带入该细胞中,该结果间接支持了前人推测的两细胞周期不同步是导致体细胞杂种中染色质消减的原因之一。在UV辐照1min组合中,培养2周的融合体中观察到一个细胞中存在2-4个微核而无主核的细胞,推测是由于UV辐照使细胞中遗传物质发生变异,导致有丝分裂中形成的微核数量增加,在分裂后期被包裹细胞膜内,在随后的细胞分裂中微核逐渐丢失。这也是有丝分裂过程中染色质发生消减的一个间接证据。综合分析对称/不对称体细胞杂交产物出现的各种变化,初步确定异源染色质的消减和渐渗主要发生在原生质体融合7天内或更早,消减和渐渗过程可延续至融合培养2-3周或更长时间。
对再生愈伤组织进行了形态学、酯酶/过氧化物酶、RAPD(Randomly amplifiedpolymorphism DNA,随机扩增多态性DNA)和SSR(Simple Sequence Repeat简单序列重复)标记分析,发现,在得到的178个再生克隆中有17个是体细胞杂种,杂种表型偏向扬麦158,并有少量中间偃麦草染色质渐渗入小麦基因组中;该结果与GISH分析结果相符合。
Plant somatic hybridization not only allows bypassing normal crossfertilization but also increases our ability to investigate the mechanism of chromatin elimination and introgression between relatively remote species. This technique has successfully allows for the improvement of a crop species by the transfer of agronomical relevant traits. For wheat, works carried out in our laboratory had generated many intra-, interspecies and generic somatic hybrid plants. The system of wheat introgression lines has established by transferring exogenous chromatin from other cereals by somatic hybridization in our laboratory. In this work, preliminary report describes a somatic hybrid route to introgression involved in wheat and Elytrigia intermedium (Host) Nevski, which will be more efficient than sexual crossing.
Prior to fusion, callus-derived protoplasts of E.intermedium were irradiated with ultraviolet light (UV, 380μW/cm~2) for 0s, 30s and 1min respectively, and then were fused with suspension-derived protoplasts of common wheat by PEG (Neomycin phosphate transferase) method. Seventeen somatic hybrid cell lines from total 178 regenerated clones were verified by peroxidase/esterase isoenzymes, cytological observation, RAPD (Randomly amplified polymorphism DNA), SSR (Simple Sequence Repeat) and GISH (Genomic in situ hybridization) analysis. The phenotypes of them all were resembled to wheat, and a few chromatins of E. intermedium were observed to introgression into genomics of wheat.
Fashions of chromatin elimination of E.intermedium in early stage and regenerated calli of wheat somatic hybridization was analysised by cytological and GISH method. The results showed that donor chromatin were mainly eliminated in interphase through micronucleus formation in early stage somatic hybridization, other patterns of chromatin elimination of donor in early stage of wheat including micronucleus formation in anaphase, micronucleus formation in multi-nucleus and segregation of parents. The lagging chromosomes, chromosome bridges, nucleus efflux and abnormal nucleus were also expressed in some symmetric and asymmetric somatic hybrid clones. Interestingly, desynchronized cell cycle and a random chromosomes distribution in dividing cells were noticed in asymmetric somatic hybrid cell lines. Two-four micronucleus without main nucleus was found in 2 weeks-culture by UV treatment for 1min, which meant that this micronucleus might be lost gradually during the following cell division.
In conclusion, the main period of donor chromatin elimination and introgression took place during the early stage, in other word, were from 5 to 15 days after fusants were cultured.
对再生愈伤组织进行了形态学、酯酶/过氧化物酶、RAPD(Randomly amplifiedpolymorphism DNA,随机扩增多态性DNA)和SSR(Simple Sequence Repeat简单序列重复)标记分析,发现,在得到的178个再生克隆中有17个是体细胞杂种,杂种表型偏向扬麦158,并有少量中间偃麦草染色质渐渗入小麦基因组中;该结果与GISH分析结果相符合。
Plant somatic hybridization not only allows bypassing normal crossfertilization but also increases our ability to investigate the mechanism of chromatin elimination and introgression between relatively remote species. This technique has successfully allows for the improvement of a crop species by the transfer of agronomical relevant traits. For wheat, works carried out in our laboratory had generated many intra-, interspecies and generic somatic hybrid plants. The system of wheat introgression lines has established by transferring exogenous chromatin from other cereals by somatic hybridization in our laboratory. In this work, preliminary report describes a somatic hybrid route to introgression involved in wheat and Elytrigia intermedium (Host) Nevski, which will be more efficient than sexual crossing.
Prior to fusion, callus-derived protoplasts of E.intermedium were irradiated with ultraviolet light (UV, 380μW/cm~2) for 0s, 30s and 1min respectively, and then were fused with suspension-derived protoplasts of common wheat by PEG (Neomycin phosphate transferase) method. Seventeen somatic hybrid cell lines from total 178 regenerated clones were verified by peroxidase/esterase isoenzymes, cytological observation, RAPD (Randomly amplified polymorphism DNA), SSR (Simple Sequence Repeat) and GISH (Genomic in situ hybridization) analysis. The phenotypes of them all were resembled to wheat, and a few chromatins of E. intermedium were observed to introgression into genomics of wheat.
Fashions of chromatin elimination of E.intermedium in early stage and regenerated calli of wheat somatic hybridization was analysised by cytological and GISH method. The results showed that donor chromatin were mainly eliminated in interphase through micronucleus formation in early stage somatic hybridization, other patterns of chromatin elimination of donor in early stage of wheat including micronucleus formation in anaphase, micronucleus formation in multi-nucleus and segregation of parents. The lagging chromosomes, chromosome bridges, nucleus efflux and abnormal nucleus were also expressed in some symmetric and asymmetric somatic hybrid clones. Interestingly, desynchronized cell cycle and a random chromosomes distribution in dividing cells were noticed in asymmetric somatic hybrid cell lines. Two-four micronucleus without main nucleus was found in 2 weeks-culture by UV treatment for 1min, which meant that this micronucleus might be lost gradually during the following cell division.
In conclusion, the main period of donor chromatin elimination and introgression took place during the early stage, in other word, were from 5 to 15 days after fusants were cultured.
引文
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