【作者】 李艳华；

【导师】 康向阳；

【作者基本信息】 北京林业大学 ， 林木遗传育种， 2007， 博士

【摘要】 以毛新杨、银腺杨、银毛杨等为材料,在掌握有关树种大孢子发生和雌配子体发育等细胞学规律的基础上,就白杨雌配子染色体加倍技术进行了研究,并取得了一定的进展。(1)首次提出利用雌、雄花芽发育进程的时序性相关性,以相同生境以及培养条件下的小孢子发育进程为参照,从而相对准确地实现白杨大孢子母细胞减数分裂进程即时判别的技术方法。研究表明,不同白杨的大孢子母细胞减数分裂进程存在差异性,而同一树种的大、小孢子母细胞减数分裂进程存在时序性相关。当白杨小孢子母细胞减数分裂至四分体时,大孢子母细胞开始进入减数分裂细线期;而当小孢子发育至单核靠边期上时,大孢子母细胞减数分裂达到粗线期。(2)通过小孢子发育进程参照系的设立,实现了大孢子染色体加倍诱导白杨三倍体技术的实用化。当相同生境以及培养条件下的小孢子母细胞减数分裂达到四分体后,适时采用秋水碱溶液对雌花芽进行浸泡处理,可保证白杨大孢子染色体加倍处理能集中于一个较短的时间段内进行,从而大大减轻了工作量,同时提高三倍体诱导效率和效果。在银腺杨大孢子染色体加倍中,共获得了12株银腺杨×毛白杨杂种三倍体,三倍体得率最高达16.7%。(3)证明了授粉后施加秋水仙碱处理诱导白杨三倍体的细胞学机理是胚囊染色体加倍。在银腺杨等白杨派树种中,对授粉24-36 h的雌花序进行秋水仙碱溶液处理可获得三倍体植株。通过大量的常规切片观察证明,尽管银腺杨、毛新杨、银毛杨等白杨授粉后胚囊发育进程表现为不同步性,但大多在授粉后48h内完成由大孢子到8核胚囊的3次有丝分裂,表明秋水仙碱溶液抑制胚囊发育中的3次有丝分裂导致雌配子染色体加倍,是授粉后施加秋水仙碱处理获得三倍体的细胞学机理。(4)提出以最佳授粉时期为实施诱导胚囊染色体加倍处理的起始参照点,为适时进行胚囊染色体加倍提供了技术保障。在温室水培条件下,毛新杨、银腺杨、银毛杨等白杨树种雌蕊柱头的可授期一般在1-3d;进入最佳授粉时期时的柱头开裂角度约为180。左右,此时柱头发亮且表面有大量分泌物存在,在适宜授粉时期内,联苯胺-过氧化氢染色后柱头变蓝,并有大量气泡产生。在育种实践中,可根据柱头形态以及联苯胺-过氧化氢染色后柱头颜色变化和气泡产生情况等,即时判别有关白杨派树种的最佳授粉时期。(5)在揭示授粉后施加秋水仙碱处理诱导白杨三倍体的细胞学机理的同时,还获得了3株来源于胚囊染色体加倍的白杨杂种三倍体,并获得了一大批有待检测的胚囊染色体加倍处理后的杂种苗木。更多还原

【Abstract】 Chromosome doubling of female gamete in white poplars were studied in Populns tomentosa × P. balleana, P. alla × P. glandulosa and P. alba × P. tomentosa, based on megasporogenesis and megagametogenesis of cytogenetics.Now the research has made progress.（1） The meiosis stages of megaspore could be discriminated timely through observing the meiosis of microspore under the same environmental conditions, according to the association of female and male buds during development. Results indicated : The meiosis stages of megaspore of white poplars are different, while there was a association between MMCs and PMCs meiosis in the same hybrid. Megaspore mother cell just enters leptotene as The meiosis of microspore mother cell develop into tetrads . The meiosis of megaspore mother cell develop into pachytene while that of microspores develop into a single nuclear adjacent to cell wall.（2）It is practical that triploid white poplars induced by the chromosome doubling of megaspore based on the development of microspore. Under the same environmental conditions when the meiosis of microspore mother cell develop into tetrads the female buds were dipped duly with colchicine solution, which guaranteed that chromosome doubling of megaspore in white poplar could be accomplished in a short time. So much workload was cut down, and induction efficiency of triploid white poplars was also improved. 12 triploid poplars were identified among the hybrid progenies by chromosome doubling of megaspore. A high doubling efficiency of triploids （16.7%） was obtained in （P. alba × P. glandulosa） × P. tomentosa.（3） It was testified that chromosome doubling of embryo sac was the cytology mechanism of triploid induction by colchicine solution after pollination. Triploid could be obtained by the female catkins were treated with colchicine solution 24-36h after pollination in white poplar （P. alla×P. glandulosa）. Asynchronism in development of embryo sac among Populus tomentosa×P. balleana, P.alla×P. glandulosa, P. alba×P. tomentosa was found by many paraffin sections observed. 48h after pollination, most them developed into eight- nuclear embryo sac after undergoing 3 times mitosis. All facts indicated that chromosome doubling of female gametes resulted from restriction of mitosis of embryo sac by colchicine solution.（4）The paper provided that the optimal receptivity of stigma was by way of the initial point for chromosome doubling of embryo sac, so which assured that a technique for chromosome doubling in embryo sac. The stigma receptive stage of white poplars（Populus tomentosa×P. balleana, P. alla×P. glandulosa, P. alba×P. tomentosa） lasted about 1 to 3 days when floral branches were cultured in water in greenhouse. The stigma of the optimal receptive stage of the three poplars were shining with a great deal secretion and opened to the flat stage -about 180°.The receptivity of the stigma was indicated by the apperance of a blue colour and air bubble with benzidine-H₂O₂ solution. In breeding, the optimal pollination stage of white poplars could be discriminated timely based on the morphological characteristics of the stigma and the changes of colour and air bubble of stigma after stained with benzidine-H₂O₂ solution.（5） The cytology mechanism of triploid induction by colchicine solution treatment after pollination was revealed. As the same time, 3 triploid white poplars were identified by chromosome doubling of embryo sac. Now many hybrid seedlings were going to be identified by chromosome counts.更多还原