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低温诱导毛白杨花粉染色体加倍选育三倍体

来源：花匠小妙招时间：2025-12-01 18:35

摘要:

目的

建立低温诱导毛白杨花粉染色体加倍选育三倍体的技术体系，为林木三倍体育种提供理论指导。

方法

以山西朔州的毛白杨为研究对象，利用醋酸洋红染色法，在掌握雄花芽减数分裂规律的基础上，施加4 ℃低温处理毛白杨雄花芽，诱导产生2n花粉；利用免疫荧光技术研究低温胁迫处理条件下花粉母细胞微管骨架动态变化，探索低温诱导毛白杨花粉染色体加倍的内在机制；利用获得的低温诱导型2n花粉与毛白杨雌株进行授粉杂交，通过流式细胞仪和体细胞染色体计数法检测倍性，筛选获得三倍体。

结果

（1）毛白杨花粉母细胞在温室水培条件下完成减数分裂需4 ~ 5 d，其花粉母细胞减数分裂进程与雄花芽外部形态和花药颜色变化有着明显的对应关系。（2）减数分裂时期和持续处理时间对毛白杨2n花粉诱导率具有显著影响；处理时期与持续处理时间的交互作用对毛白杨2n花粉诱导率无显著影响。（3）利用4 ℃低温对发育至减数分裂Ⅱ中期的花粉母细胞进行2 d的持续处理是低温诱导毛白杨花粉染色体加倍，获得2n花粉的最佳处理组合，最高可获得9.51%的2n花粉。（4）利用获得的低温诱导型2n花粉与毛白杨雌株进行授粉杂交，筛选获得1株三倍体。（5）4 ℃低温处理可干扰花粉母细胞减数分裂Ⅱ末期辐射状微管的形成，导致四分体时期相邻子核之间的微管部分缺失。

结论

本研究首次利用低温诱导获得毛白杨2n花粉，并通过低温诱导型2n花粉杂交成功筛选获得三倍体1株，验证了利用低温诱导杨树花粉染色体加倍选育三倍体的可行性。

Abstract:

Objective

This study aims to establish a technical system for triploid production of Populus tomentosa by inducing pollen chromosome doubling with low temperature, which provides theoretical guidance for triploid breeding of forest tree species.

Method

Taking P. tomentosa in Shuozhou, Shanxi Province of northern China as the research object, based on the observation of male meiosis of pollen mother cells (PMCs), aceto-carmine staining method was used, and 4°C low temperature treatment was applied to male flower buds of P. tomentosa to induce the production of 2n pollen. Immunofluorescence technology was utilized to study dynamic changes of microtubule in PMCs under low temperature stress conditions, exploring intrinsic mechanism of low temperature-induced chromosome doubling in P. tomentosa pollen. Using the obtained low-temperature induced 2n pollen for pollination hybridization with female Populus tomentosa plants, the ploidy was detected by flow cytometry and somatic chromosome counting to screen for triploidy.

Result

(1) The microsporocyte of P. tomentosa completed meiosis in about 4 to 5 d under greenhouse hydroponic conditions.The meiotic process of PMCs showed a clear correspondence with morphology of male flower buds and dynamic change of anther color. (2) Meiotic stage and duration had significant effects on frequency of cold-induced 2n pollen. However, meiotic stage × duration interaction had no significant effect on frequency of cold-induced 2n pollen. (3) The optimum treatment combination to induce pollen chromosome doubling was successfully developed, and the PMCs were treated by 4 ℃ for two days when the meiotic stage was at metaphase Ⅱ. The highest average frequency of cold-induced 2n pollen was 9.51%. (4) One triploid was detected by crossing cold-induced 2n pollen with haploid female gametes of P. tomentosa clone SX-1. (5) After treating via low temperature, radial microtubule arrays (RMAs) were interfered in some treated PMCs at telophase Ⅱ, resulting in partial lack of RMAs between the four formed daughter nuclei at tetrad stage.

Conclusion

It is the first time for 2n pollen induction by low temperature in P. tomentosa and one triploid is successfully obtained, which shows that inducing pollen chromosome doubling to breed triploid by low temperature in Populus is feasible in a certain.

图 1 不同水培时间毛白杨雄花芽不同部位形态变化

Figure 1. Morphological changes in different parts of male flower buds of Populus tomentosa at different hydroponic times

图 2 毛白杨花粉母细胞减数分裂进程

Figure 2. Meiosis process of pollen mother cells in P. tomentosa

黑色箭头指向为2n花粉。

图 3 毛白杨低温诱导型2n花粉（a）与对照组中天然2n花粉（b）

Figure 3. Low temperature-induced 2n pollen (a) and natural 2n pollen (b) of control group in P. tomentosa

图 4 减数分裂Ⅱ中期处理组花粉与对照组花粉直径分布

Figure 4. Pollen diameter distribution of metaphase Ⅱ treatment group and control group

a. 减数分裂前期；b. 终变期；c. 减Ⅰ中期；d. 减Ⅰ后期；e. 减Ⅰ末期；f. 减Ⅱ前期；g. 减Ⅱ中期；h. 减Ⅱ后期；i. 减Ⅱ末期；j. 低温处理后的减Ⅱ中期；k. 低温处理后的减Ⅱ后期；l ~ o. 低温处理后的异常减Ⅱ末期。

图 5 毛白杨花粉母细胞减数分裂过程中微管骨架的动态变化

Figure 5. Dynamic changes of microtubule skeleton during meiosis of PMCs in P. tomentosa

图 6 低温诱导毛白杨花粉染色体加倍子代的倍性分析

Figure 6. Ploidy analysis of offsprings derived from pollen chromosome doubling induced by low temperature

表 1 毛白杨减数分裂进程与雄花芽外部形态及其花药颜色变化的对应关系

Table 1 Corresponding relationship between meiosis stage process and male flower bud morphological and anther color change of Populus tomentosa

水培时间/h
花芽（序）变化
花芽颜色
小花颜色
减数分裂时期
顶部
基部
75花芽略微膨大，芽鳞尚未开裂
嫩绿色
嫩绿色
嫩绿色
细线期
93芽鳞开裂，花序微露
黄绿色
微红色
微红色
双线期
101花序明显露出
微红色
浅红色浅红色减Ⅰ后期
104花芽进一步膨大，花序露出
浅红色
鲜红色
鲜红色
减Ⅱ后期
105花序露出芽鳞约1/3
鲜红色
深红色
深红色
四分体时期

表 2 不同处理组合的2n花粉诱导率

Table 2 Induction rates of 2n pollen derived from different treatments

减数分裂时期处理天数/d 2n花粉诱导率/% 处理编号 1 1.36 ± 1.18 M1 粗线期 2 3.65 ± 1.58 M2 3 3.17 ± 1.59 M3 1 4.67 ± 0.67 M4 终变期 2 8.69 ± 5.96 M5 3 4.45 ± 2.78 M6 1 2.60 ± 1.30 M7 减Ⅰ中期 2 5.39 ± 2.45 M8 3 4.13 ± 2.07 M9 1 5.51 ± 3.91 M10 减Ⅰ后期 2 5.57 ± 2.69 M11 3 3.44 ± 2.94 M12 1 2.90 ± 1.22 M13 减Ⅰ末期 2 4.62 ± 2.86 M14 3 3.30 ± 1.04 M15 1 2.88 ± 0.71 M16 减Ⅱ前期 2 4.17 ± 3.61 M17 3 2.78 ± 0.96 M18 1 5.11 ± 3.68 M19 减Ⅱ中期 2 9.51 ± 1.33 M20 3 5.50 ± 1.80 M21 1 3.33 ± 0.76 M22 减Ⅱ后期 2 5.12 ± 3.33 M23 3 2.83 ± 0.58 M24 1 2.38 ± 1.86 M25 减Ⅱ末期 2 6.07 ± 1.52 M26 3 4.50 ± 2.18 M27 对照 0 0.33 ± 0.24 CK

表 3 不同处理组合毛白杨2n花粉诱导率的方差分析

Table 3 Variance analysis on induction rates of pollen in P. tomentosa under different treatment combinations

变异来源
df F P 减数分裂时期
8 2.510 0.021* 处理天数
2 8.023 0.001** 减数分裂时期 × 处理天数
16 0.454 0.958 注：** 代表影响极显著（P < 0.01），* 代表影响显著（P < 0.05）。

表 4 不同减数分裂时期和不同处理天数毛白杨2n花粉诱导率的多重比较

Table 4 Multiple comparisons in induction rates of 2n pollen under different meiotic stages and varied treating days in P. tomentosa

减数分裂
时期平均2n花粉
诱导率/% 处理天
数/d 平均2n花粉
诱导率/% 粗线期 2.73 ± 1.64b 1 3.42 ± 2.17b 终变期 5.93 ± 3.90ab 2 5.87 ± 3.21a 减Ⅰ中期 4.04 ± 2.05b 3 3.79 ± 1.83b 减Ⅰ后期 4.84 ± 2.98ab 减Ⅰ末期 3.61 ± 1.82b 减Ⅱ前期 3.28 ± 2.02b 减Ⅱ中期 6.71 ± 3.01a 减Ⅱ后期 3.76 ± 2.02b 减Ⅱ末期 4.32 ± 2.28b 注：同列不同小写字母代表差异显著（P < 0.05）。

表 5 低温诱导型毛白杨2n花粉与毛白杨雌配子（SX-1）杂交创制三倍体

Table 5 Crossing 2n pollen induced by low temperature in P. tomentosa with female gametes in P. tomentosa (SX-1) to produce triploids

序号2n花粉诱导率/%
杂交组合
种子数
成苗数
三倍体数
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