【作者】 陈洪伟；

【导师】 康向阳；

【作者基本信息】 北京林业大学 ， 林木遗传育种， 2008， 博士

【摘要】 为综合利用白杨派树种生长迅速、材质优良和胡杨派树种抗盐碱的特性,本研究以白杨派树种毛新杨(P. tomentosa×P. bolleana)、银毛杨(P. alba×P. tomentosa)、银腺杨(P. alba×P. glandulosa)等为主要研究对象,选择耐盐木本植物胡杨(P. euphratica Oliv.)为外源DNA供体,就白杨花粉管通道法导入胡杨外源DNA技术进行系统研究,为通过花粉管通道法选育耐盐白杨杂种无性系打下一定的理论与技术基础。主要研究结果如下:(1)针对胡杨远距离取样问题,开展了胡杨叶片非低温取样保存方法研究,证明硅胶干燥和SDS DNA提取液均可以将胡杨叶片保存20d以上;考虑在花粉管通道导入中胡杨DNA的用量较大,宜选用冬季采取枝条水培出的鲜叶或硅胶干燥保存10d内的叶片;此时,采用DNA提取率相对较高的SDS方法,并添加2%β-巯基乙醇抑制DNA褐化,可以保证获得大量高质量胡杨DNA。(2)通过对不同发育状态的雌花序定期授粉观察表明,杨树雌蕊柱头可授性及最佳可授期与柱头形态存在一定关联性。当雌蕊柱头发育至最佳可授期时,毛新杨雌花序柱头外露明显,柱头4裂且夹角约为180°;银腺杨雌花序苞片张开,柱头外露且发亮,柱头4裂片开裂角度180°;银毛杨花序苞片张开,柱头外露,柱头4裂成60°～90°。在最佳授粉期授粉并以此为参照点进行处理,保证导入时机准确。参考作物育种经验,以毛新杨、银腺杨和银毛杨等为受体进行胡杨外源DNA花粉管通道导入处理时,毛新杨、银腺杨的有效处理时期在授粉后36～72h之间;银毛杨的有效处理时期在授粉后36～60h之间。而从施加花粉管通道导入外源DNA处理的实际结果看,授粉后30h开始处理即可获得转化株。(3)以毛新杨、银毛杨、银腺杨雌株为母本,毛白杨和新疆杨雄株为父本,就白杨花粉管通道导入胡杨DNA以及与秋水仙碱诱导胚囊染色体加倍联合处理等技术条件进行了研究。各处理组合共收获14721粒种子,大田存苗883株;施加花粉管通道导入胡杨外源DNA处理导致花序的平均结实率降低,其原因可能是由于授粉雌花序切柱头时造成的机械损伤影响,或者外源DNA处理液中残存的微量有机试剂的毒害作用;从获得的杂种后代表型变异看,大多倾向于父本或母本,只发现2株倾向于胡杨披针形叶的特异植株;在采用花粉管通道导入与染色体加倍联合处理获得的杂种后代中未检出三倍体或四倍体。(4)对白杨花粉管通道导入胡杨DNA的部分杂种后代进行AFLP分子标记检测,在授粉后24～54h开始处理、持续处理2～18h的杂种后代中,经AFLP分析检出了70株分子水平存在变异的株系,包括具A型新谱带(仅在处理后代中存在而在父本、母本、对照和供体胡杨中不存在的条带)的62株,具B型新谱带(仅在处理后代和供体胡杨中存在而在父本、母本、对照中不存在的条带)的8株。2株叶形特异植株在目前的分子检测中未发现变异。进一步对几组具有AFLP特异带的杂种后代和胡杨供体进行DNA同源序列比对分析,发现AFLP检测技术存在“假阳性”现象,表明花粉管通道导入外源基因组DNA研究只根据分子标记谱带特异性鉴别转化体并不准确。经杂种后代和胡杨供体进行DNA同源序列比对分析,以及对处理后代、供体、父母本和对照SCAR标记分析,以毛新杨为母本的杂种无性系中检测出1株外源胡杨DNA部分片段整合的杂种后代(MM10-2),推测其整合机理为同源重组。更多还原

【Abstract】 For integrating properties of fast growth and good timber characters in Section Populus and salt-alkali-resistance in Section Turanga of the genus Populus, techniques of pollen-tube pathway were systematically studied to introduce the exogenous DNA from P. euphratica to white poplars, including P. tomentosa×P. bolleana、P. alba×P. tomentosa and P. alba×P. glandulosa. These studies made some theoretical and technical bases for breeding of salt-resistant white poplars by pollen-tube pathway. Major results as follows:(1) Studies on the noncryogenic preservation of leaves tissue for DNA extraction of P. euphratica have been carried out, to settle the problems of preserving plant materials far away. The results indicated that, DNA from leaves stored in salica gel and SDS DNA extraction buffer for 20 days was good integrity and high purity . However, fresh leaves from branches cultured in water in winter or leaves preserved in salic gel within 10 days should be used and DNA extraction is employed by SDS method, to meet the needs in introduction of exogenous DNA via pollen-tube pathway. Furthermore, 2%β-Mercaptoethanol should be added to restrain brown of DNA.(2) Pollination tests of female catkins at different development stages show that, the receptivity and the optimal pollination stage of the stigmase are releated to morphological characteristics of the stigmas. At the optimal pollination stage, stigmas of P. tomentosa×P. bolleana catkins are evident、bright, slivers of stigmas flat about 180°; P. alba×P. glandulosa bracts expand, stigmas are evident、bright, and slivers of stigmas flat 180°; P. alba×P. tomentosa bracts expand too, stigmas are evident、bright, and slivers of stigmas splay about 60°～90°. Pollination at the the optimal stage could help accurate the starting introduction time. According to breeding of crops, when introduction of exogenous DNA into white poplars via pollen-tube pathway is carried out, the effective treatment time of P. tomentosa×P. bolleana and P. alba×P. glandulosa is both 36～72h after pollination, and P. alba×P. tomentosa is 36～60h after pollination.(3) Transformation of exogenous DNA of P. euphratica via pollen-tube pathway and combination of the introduction and chromosome doubling of embryo sac were studied at different time after pollination and treated for different hours, with P. tomentosa×P. bolleana、P. alba×P. bolleana and P. alba×P. glandulosa as female parents, P. tomentosa and P. bolleana as male parents. A total of 14,721 seeds were harvested and 883 offspring were conserved in field. Seed sets decreased after transformation of exogenous DNA via pollen-tube pathway, which may be resulted from mechanical damage when stigmas cut or poison of retained organic reagents in DNA liquor. Analysis of phenotype characteristics showed that most of offspring were similar to parents and only 2 ones presented variation of leaves similar to lanceolate leaves of P. euphratica. In the progeny, no polyploid was detected.(4) 70 offspring from treatments started from 24 to 54 hours after pollination and treated for 2 to 18 hours were detected as putative transformed plants at molecular level by amplified fragment length polymophysm (AFLP). Among them, there were 62 individuls with new bands of type A (presenting only in treated progenies but not parents、CK and dornor) and another 8 ones possessing new bands of type B (presenting only in treated progenies and dornor but not parents、CK). However, no molecular variation was found in those 2 offspring with phenotypical variation. The sequencing results and alignment results of couples specific bands showed that the results of AFLP might be“false positive”, so it was not reliable to detect transformed plants with exogenous genomic DNA via pollen-tube pathway only relying on specific bands detected in primary molecular marker. The result showed that individual MM10-2 was hybrid progeny which was integrated by homologous recombination of exogenous P. euphratica partial DNA.更多还原