1、附件 2作者姓名：王君论文题目：青杨派树种多倍体诱导技术研究作者简介：王君，男，1982 年 8 月出生，2004 年 9 月师从于北京林业大学康向阳教授，于 2009 年 6 月获博士学位。中 文 摘 要我国青杨派树种资源丰富，普遍具有速生、易繁殖、耐干旱、抗寒冷等特点，但遗传改良程度相对较低。引入多倍体育种技术对青杨派树种进行遗传改良，为我国“三北” 地区提供生长迅速、材性优良、适应性强的林木良种，对当地生态环境的改善和经济水平的发展具有重要意义。本研究以哲引 3 号杨（Populus pseudo-simonii P. nigra Zheyin3#） 、小青杨（P. pseudo-sim
2、onii） 、小叶杨（ P. simonii） 、 通辽杨 （P. simonii P. nigra Tongliao）等青杨派树种及杂种雌、雄无性系为材料，围绕青杨派树种天然 2n 花粉发生及其细胞学机制、雌雄配子与合子染色体加倍，以及多倍体检测等多倍体育种技术进行了研究，取得了一定的进展。1. 首次发现我国青杨派树种存在天然 2n 花粉，其中 5 个小青杨无性系天然 2n 花粉发生频率介于 0.331.55%，9 个小叶杨无性系天然 2n 花粉发生频率为 0.483.10%，而通辽杨无性系则可产生 2.29%的 2n 花粉。进一步利用醋酸洋红压片和微管骨架免疫荧光分析揭示，小孢子母细胞减数
3、分裂末期 II 细胞成膜体缺失导致胞质分裂异常是 2n 花粉发生的主要细胞学机制。2. 明确了施加理化处理诱导小青杨、 通辽杨 等青杨派树种及杂种无性系花粉染色体加倍的最佳始处理时期和处理强度。其中，施加秋水仙碱进行处理，以小孢子母细胞减数分裂细线末期粗线期为最佳始处理时期，0.5%秋水仙碱溶液连续注射处理 5 次，每次间隔 4 h，可取得较好效果，小青杨 2n 花粉比率最高可达 60.69%， 通辽杨2n 花粉比率也达到 30%；施加高温处理以减数分裂终变期中期 I 为最佳始处理时期，处理强度不宜过大，以 38高温持续处理 24 h 较为合适，小青杨 2n 花粉比率可达 59%以上，而通辽杨
4、2n 花粉比率最高可达 50.28%。3. 提出以雌花芽发育形态为参照即时判别哲引 3 号杨大孢子发生进程的方法，明确了施加理化处理诱导大孢子染色体加倍的最佳始处理时期，首次通过大孢子染色体加倍途径获得青杨派杂种三倍体 159 株。其中，施加秋水仙碱溶液处理诱导大孢子染色体加倍共获得13 株三倍体，当雌花芽芽鳞微张、花序未露出，即大孢子母细胞减数分裂处于粗线期至双线期时，施加 0.3%0.5%的秋水仙碱溶液浸泡处理 24 h 效果较好；施加高温处理雌花芽诱导大孢子染色体加倍共获得三倍体 146 株，当雌花芽芽鳞张开、花序微露，即大孢子母细胞减数分裂处于双线期中期 I 时，施加 4144高温连续
5、处理 35 h 的诱导效果较好。4. 以哲引 3 号杨雌蕊柱头最佳授粉时期作为起始参照点估测胚囊发育进程，明确了施加理化处理诱导胚囊染色体加倍的最佳始处理时期，首次通过胚囊染色体加倍途径获得青杨派杂种三倍体 68 株。其中，当哲引 3 号杨雌花序长约 5.62cm，花序上所有苞片均外翻甚至脱落，柱头露出，所有柱头经过氧化物酶试纸溶液测试均呈蓝色阳性反应时， 哲引 3 号杨雌花序达到最佳授粉时期；施加秋水仙碱处理诱导胚囊染色体加倍共获得三倍体 23 株，以授粉后 5466 h 实施处理效果最好；施加高温处理诱导胚囊染色体加倍获得三倍体 45 株，以授粉后 6672 h 施加 41高温持续处理 4
6、6 h 或 44高温处理 2 h 的效果较好；细胞学分析表明，四核胚囊期是理化处理诱导哲引 3 号杨胚囊染色体加倍选育三倍体的最佳始处理时期，即通过抑制胚囊发育过程第三次有丝分裂实现 2n 雌配子的诱导，但也并不能完全排除第一次和第二次有丝分裂过程受抑制而形成 2n 雌配子的可能性。5. 首次提出以子房内絮状纤维发育状态即时判别合子发育进程的方法，在此基础上施加理化处理诱导 哲引 3 号杨北京杨 （P. beijingensis）合子染色体加倍，获得青杨派杂种同源四倍体 8 株。细胞学分析表明，授粉后 78 d，子房内絮状纤维包裹胚珠基部和中部，但尚未完全覆盖胚珠时，多数合子进入第一次有丝分裂
7、；在授粉后 7 d，以 0.5%秋水仙碱溶液处理 哲引 3 号杨 北京杨 果序 24 h，诱导获得四倍体 3 株；在授粉后 78 d，施加 3844高温持续处理哲引 3 号杨北京杨 果序 4 h，获得四倍体 5 株。6. 通过对大量青杨派杂种多倍体植株气孔密度、气孔大小以及幼叶间期细胞核染色中心数目的统计分析，提出气孔密度结合气孔大小鉴定法、幼叶细胞间期核染色中心计数鉴定法等青杨派多倍体快速检测方法，完善了杨树多倍体检测技术体系。其一，可综合利用气孔密度和气孔大小两个参数初步判别青杨派杂种三倍体，即将由上往下完全展开的第 15 叶叶背表皮在 40物镜下气孔数低于 40，且气孔面积大于 603.
8、10 m2 的植株判定为三倍体植株；其二，可利用植株幼叶细胞间期核染色中心数目初步判别青杨派杂种多倍体，在实际应用中，可将染色中心数目最大值大于 40 且小于 60 的植株初步判定为三倍体，而将染色中心数目最大值大于 60 则判定为四倍体。7. 对部分多倍体植株 2 年生实生苗地径和株高测量分析表明，无论是大孢子染色体加倍还是胚囊染色体加倍，获得的哲引 3 号杨北京杨 杂种三倍体与相同杂交组合二倍体平均值相比均具有一定的生长优势，其中生长最佳的三倍体植株地径和株高分别超过二倍体平均值 152%和 59%；而四倍体植株则与相同杂交组合二倍体植株在生长方面则表现相当。同时， 哲引 3 号杨 北京杨
9、 杂种三倍体体也并非株株皆优，仍需要对其进行无性系测定，选择生长表现优良的无性系。关键词：青杨派，多倍体诱导，秋水仙碱，高温，倍性检测Techniques of Polyploid Induction in Populus spp. (Section Tacamahaca)Wang JunABSTRACTIn China, there are many species of Populus (section Tacamahaca), most of which have good properties in growth, propagation, drought tolerance and
10、cold resistance. However, the extent of genetic improvement in the section Tacamahaca is low. Polyploid breeding is one of the most important approaches for improvement of Populus. Introduction of techniques on polyploid breeding to improvement of section Tacamahaca is significant for ecological imp
11、rovement and economic development of the Three-North Regions of China (northeastern, northwestern and northern China). In order to provide more forest tree varieties with fast growth, good pulping characteristics and great adaptability for those regions, some male and female clones of section Tacama
12、haca, including P. pseudo-simonii, P. simonii, P. simonii P. nigra Tongliao and P. pseudo-simonii P. nigra Zheyin3#, were studied on occurrence of spontaneous 2n pollen and its cytological mechanism, chromosome doubling of gametes and zygote, and fast-determination of ploidy level. Major results as
13、follows,1. Occurrence of spontaneous 2n pollen in section Tacamahaca was first reported in China. The frequency of 2n pollen ranged from 0.33% to 1.55% in 5 male clones of P. pseudo-simonii, from 0.48% to 3.10% in 9 male clones of P. simonii, and was 2.29% in P. simonii P. nigra Tongliao. Analysis o
14、f microtubular cytoskeletons during microsporogenesis revealed that abnormal cytokinesis resulted from lack of phragmoplasts in telophase II was the major cytological mechanism for 2n pollen formation.2. The optimal treating periods and treating intensities for pollen chromosome doubling with colchi
15、cine and high temperature both in P. pseudo-simonii and P. simonii P. nigra Tongliao were confirmed respectively. When colchicine solution was used to induce 2n pollen, the period from late leptotene to pachytene during microsporogenesis was the optimal treating period, and the rate of 2n pollen ind
16、uction was 60.69% in P. pseudo-simonii and up to 30% in P. simonii P. nigra Tongliao by injecting male flower buds with 0.5% colchicine solution for 5 times with 4 h intervals; When high temperature was used, the period from diakinesis to metaphase I was the most efficient period, and the rate of 2n
17、 pollen induction was up to 59% in P. pseudo-simonii and 50.28% in P. simonii P. nigra Tongliao by treating with 38 for 24 h.3. A method for instant determination of megaspore mother cell meiotic process was supplied by cytological analysis combined with morphological characteristics of female flowe
18、r buds. The optimal treating period and treating intensity for megaspore chromosome doubling using colchicine and high temperature in P. pseudo-simonii P. nigra Zheyin3# were confirmed respectively, and 159 triploid hybrids were obtained. Thirteen triploids were produced by megaspore chromosome doub
19、ling with colchicine solution. When the bract scales of the flower bud dehisced a little but the catkin did not come out, the megaspore mother cells were at the period from pachytene to diplotene, which was the optimal treating period for megaspore chromosome doubling with colchicine, and the optima
20、l mode was to immerse buds with 0.3% to 0.5% colchicine solution for 24 h. One hundred and forty six triploids were produced by megaspore chromosome doubling with high temperature. When the bract scales dehisced and the catkin came out a little, the megaspore mother cells were at the period from dip
21、lotene to metaphase I, which was the optimal treating period for treating with high temperature, and treating with 41 to 44 for 3 h to 5 h was the most efficient.4. Process of embryo sac development in P. pseudo-simonii P. nigra Zheyin3# was estimated by timing from its optimal pollination period. T
22、he optimal treating period for embryo sac chromosome doubling with colchicine and high temperature in P. pseudo-simonii P. nigra Zheyin3# were confirmed respectively, and 68 triploid hybrids were obtained. When female catkins of P. pseudo-simonii P. nigra Zheyin3# had become approximately 5.62 cm lo
23、ng and all stigmas were exposed and turned to blue by testing with Peroxtesmo Ko solution, the catkin reached to the optimal pollination period. Twenty three triploids were produced by embryo sac chromosome doubling with colchicine solution; the optimal treating period was 5466 h after pollination.
24、Forty five triploids were produced by embryo sac chromosome doubling with high temperature; the optimal treating period was 6672 h after pollination, and treating with 4144 for 24 h was efficient. The cytological analysis showed that four-nucleate embryo sac stage was the optimal treating stage for
25、embryo sac chromosome doubling with colchicine and high temperature. The mechanism was to induce 2n egg through inhibiting the third mitotic division during embryo sac development. However, the possibility of 2n egg formation by mitotic inhibition at the first or second division cannot be excluded.5
26、. A method to instantly judge the process of zygote development by seed-hair development in ovary was first proposed in (P. pseudo-simonii P. nigra Zheyin3#) P. beijingensis, and 8 tetraploids were obtained by zygote chromosome doubling with colchicine and high temperature. Cytological analysis reve
27、aled that majority of zygotes started the first mitotic division at 78 d after pollination, when the seed-hair enclosed the base and middle of ovules but did not cover the whole ovules; Three tetraploid plants were produced by soaking the catkins 7 d after pollination with 0.5% colcincine for 24 h;
28、Five tetraploids were produced by treating the catkins 78 d after pollination with 3844 for 4 h respectively. 6. Based on the statistical analysis in the large population of induced polyploids in section Tacamahaca, methods for fast determination of ploidy level in section Tacamahaca were establishe
29、d. One was the method of combination between stomata density and stomatal size to preliminarily screen triploid plants, i.e. if a plant has less than 40 stomatas under 40 objective in stomata density and more than 603.10 m2 in stomatal area in back of the 15th completely expanded leaf from upper to
30、lower, it could be determined as triploid. The other was detection of polyploids by counting the number of chromocenters in interphase nuclei of young leaves. In practice, when the maximum of chromocenter number in a plant is more than 40 and less than 60, it will likely be determined as triploid, a
31、nd when the maximum is more than 60, the plant will likely be determined as tetraploid.7. Based on the measurement both basal diameter and stem height of 2-year-old seedlings of some induced allo-polyploids of section Tacamahaca, it was showed that the growth of the triploids respectively derived fr
32、om both megaspore chromosome doubling and embryo sac chromosome doubling was better than that of the diploids in average; that the most excellent triploid exceeded the mean of the diploids by 152% and 59% in basal diameter and stem height respectively; and that the growth of the tetraploids was just as much as the diploids. However, not all of the triploids were good in growth, so it is necessary to make the clone test and select plus clones in future.Key words: Populus spp. (section Tacamahaca), polyploid induction, colchicine, high temperature, ploidy level determination
