【作者】 杨红花；

【导师】 陈学森；

【作者基本信息】 山东农业大学 ， 果树学， 2004， 博士

【摘要】 核果类果树的李、杏属间具有丰富的种质资源,对李、杏属间优良品种(早熟 、优质)进行属间的远缘杂交,可以创造新的种质资源,丰富生物的遗传多样性,并为进一步育种及研究提供种质。针对果树远缘杂交过程中存在的杂交不亲和性及杂种不育性等问题,在前人研究的基础上,研究了不同处理对核果类果树远缘杂交亲和性的效应,并探讨了李、杏属间杂交过程中发生的生理生化变化以及花粉与柱头的互作,建立了李、杏属间远缘杂种的胚培养技术体系,建立了杂种鉴定检测方法,并分析了杂种后代与其父母本之间的遗传变异及规律,最终将一批远缘杂交新种质定植于大田,同时对自然界存在的杏梅种质资源进行了 RAPD 分析。主要研究结果如下: (1)不同处理对远缘杂交亲和性的效应结果表明,铃铛花期授粉坐果率显著高于初花期授粉的坐果率;同一杂交组合,正反交坐果率存在差异或差异显著;母本对远缘杂交坐果率影响较大,差异显著,父本对远缘杂交亲和性影响较小,选择自交亲和性强及自然坐果率高的品种(系)及种为杂交母本, 容易克服远缘杂交不亲和性。同时选择花粉活力或萌芽率高的品种(系)作父本,容易提高杂交坐果率;适宜场强的静电场、He-Ne激光处理及 Coγ 射线与 He-Ne 激光联合处理花粉均能显著提高花粉离体萌芽率,用上 60述处理的花粉进行的远缘杂交坐果率也明显高于对照;60Coγ 射线处理则降低了花粉离体萌芽率,远缘杂交的坐果率也低于对照;赤霉素 GA3、β-萘乙酸及 NaCl 溶液处理母本柱头或子房不能提高核果类果树的杂交亲和性。 (2)对李、杏属间杂交过程中的生理生化变化研究,结果表明,授粉后远缘杂交组合的 SOD 活性均随着授粉时间的延长,先上升后下降,呈现波动性变化,不同组合之间存在差异,而对照的 SOD 活性的高峰出现时期均早于处理的酶活性高峰时期;远缘杂交授粉组合的 POD 活性也是波动性变化,但其 POD 活性高于对照,并且引起的POD 保护酶活性高峰期也比对照提前;李×杏杂交组合授粉后可溶性蛋白含量的变化一直处于缓慢的上升趋势,而反交组合在一个较大幅度的下降过程后才开始缓慢上升;远缘杂交组合的 MDA 含量均随着授粉时间的延长,呈增加的趋势,而对照的 MDA 含量基本处于一个相对稳定的水平。这些生理生化的变化可能与杂交不亲和性的存在以及花粉与柱头的识别障碍有关。 (3)李、杏属间杂交花粉与柱头的互作表明,李杏杂交存在一定程度的花粉和柱头不亲和及“拒绝”反应,并且在柱头的表现要强于花柱内部。表现在远缘杂交授粉后 6 小时,花粉粒未萌发;授粉后 12 小时,仅有少量萌发,未进入花柱;授粉后 48 小时,花粉管少量开始萌发伸长,并出现末端膨大;授粉 72 小时后,少量花粉管能够正常生长进入花柱中部,有些花粉管末端出现膨大或在花柱中缠绕生长,或盘绕、扭曲,生长受抑制,未能进入子房到达胚珠。对照无论是自交授粉以及自然授粉组合,花粉粒在柱头上的萌发率均相当高,表明远缘杂交授粉后柱头对花粉的抑制作用比自交及品种间杂交要强,其作用机理更为复杂。 1<WP=10>李、杏属间远缘杂交及种质创新的研究 (4)对李、杏远缘杂交杂种胚败育的时期进行了调查,建立了李、杏远缘杂种胚培养(抢救)技术体系,并对部分杂种进行了鉴定。结果表明,李、杏杂种胚的败育时期从第 3 周开始;不同杂交组合获得的杂种胚发育之间存在着差异,并存在败育现象;1周后子房黄化乃至败育脱落的主要原因在于柱头上附着的异源花粉粒没有萌发,不能完成受精,胚胎观察结果亦发现败育胚胎的子房内胚珠和胚囊结构发生异常变化,呈不规则的三角状或存在多胚珠现象,部分胚囊内含有部分萎缩的胚珠或为空腔;不同胚龄李、杏杂种胚的萌发及生长不同,PF 值>0.5 的杂种胚可以萌发及生长,PF 值<0.5 的幼胚不能正常萌发;去种皮处理下,杂种胚的成苗数高达 80%,而未去种皮的处理,杂种胚未见萌发;在远缘杂种胚抢救技术体系中,杂种胚最佳萌发及分化培养基为 MS+6-BA 2mg·L-1+IAA 0.3 mg·L-1,胚萌发及分化率达 80%;多丛芽诱导及增殖培养基为 MS+6-BA1.5 mg·L-1+IAA 0.3 mg·L-1,同时发现李×杏杂交获得杂种胚多丛芽诱导及增殖效率明显高于杏×李,而静电场处理获得的李、杏远缘杂种胚在萌发、分化速度及多丛芽诱导频率上也明显高于对照;杂种胚培苗随着继代时间的延长叶形发生变化;生根培养基为1/2MS+IAA 0.8 mg·L-1,成功地将一批杂种胚培苗移栽到大田;经叶片形态及 Sallele-specific PCR、SSR 分别结合 RAPD 技术对杂种进行苗期亲缘关系鉴定,结果表明,杂种与其父母本之间存在差异,所获杂种为真杂种。同时证明任何两种方法的结合都可以有效的鉴定杂种。 (5)对参试杏梅种质资源的 RAPD 分析表明,杏梅与其近缘种之间亲缘关系较近,多态性相对较少;聚类分析结果显示大部分的杏梅与中国李的遗传距离较近,与形态学上的描述基本一致,因此杏梅种质资源在自然界中产生时,其母本可能为李;同时 S 等位基因的扩增结果显示杏梅与李的扩增条数一致,都为一条,因此推断杏梅与李的进化趋同性可能较杏更近。更多还原

【Abstract】 Prunus and Armeniaca are abundant in germplasm resources in stone fruits. Newgermplam will be created if by using excellent cultivars (maturing early, good quality) indistant hybridization as parents between plum and apricot, thus can enrich the geneticdiversity, and also occupy germplasm for further breeding program and related studies. Aimedat the arising problems such as the cross-incompatibility and hybrid’s sterility during distanthybridization of fruit trees, effects of different treatments on cross-compatibility of distanthybridization in stone fruits were studied based on the former studies, then physiological andbiochemical changes during hybridization between plum and apricot were primarily discussed,and rescue and culture system of hybrids’ embryos were also conducted, further the geneticvariation and principle between the hybrids and their parents were analyzed and methods ofidentification of hybrids were established. Some hybrids were transplanted into the fieldsuccessfully. The plum-apricot germplasm resources in nature were analyzed by RAPDmarkers. The main results were as followings:(1) The effects of different treatments on cross-compatibility showed that the settingpercentage in balloon stage was obviously higher than that in early blooming stage; Thedifferences of fruit set between positive and reverse crosses were significant; Female parentaffected cross compatibility more remarkably than male parent in distant hybridization. Thecross incompatibility were easily overcome by using the cultivars with high level ofself-compatibility and high open-pollinated setting percentage as female parent, and thesetting percentage could be increased by using the cultivars(lines) with high pollen vigor andpollen germination as male parent;The appropriate electrostatic field, He-Ne laser andcompound treatment with 60Co γ-ray and He-Ne laser could improve the pollen germinationrate in vitro effectively, the fruit set percentage was higher than control; while 60 Co γ-raytreatment reduced the pollen germination rate in vitro, and the fruit set percentage obtained bydistant hybridization was lower than control. The results of the treatments on maternal ovaryor stigma indicated that plant growth regulators (GA and β-NAA) and NaCl solution can’timprove cross-compatibility among stone fruits.(2) The physiological and biochemical changes during intergeneric hybridizationbetween plum and apricot were discussed, and the results showed that SOD activities of crosscombinations were increased in the former period and then decreased in the latter withfluctuant change, and differences were also observed in different combinations and the peaksof SOD activity of control appeared earlier than those of the treated. The POD activities hadfluctuant changed either, while the POD activities of the hybrid cross combinations werehigher than control and the peaks of the treated appeared also earlier than that of control. The 3<WP=12>李、杏属间远缘杂交及种质创新的研究content of soluble protein in Plum × apricot combination increased slowly, while that of thereverse combination decreased in a great range and then increased slowly. The content ofMDA in distant hybridization combinations increased with the pollination period prolonged,while the control was still in a stable level during the pollination period. These changes werecorrelated with the cross incompatibility and recognition barriers between pollen and stigmaduring hybridization.(3) The interaction of distant pollen and stigma showed that the incompatibility andrejection responses had occurred between each other in a certain extent, and were stronger onstigma than inside of style. The pollen grain could not germinate after 6h of pollination, andlittle germinated but could not enter the style after 12h. Little pollen tube began to germinateand grow b更多还原