Pollen chromosome doubling induced by low temperature to produce triploid in Populus tomentosa

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 the P. tomentosa in Shuozhou, Shanxi 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 the male flower buds of P. tomentosa to induce the production of 2n pollen. Immunofluorescence technology was utilized to study the dynamic changes of the microtubule in PMCs under low temperature stress conditions, exploring the intrinsic mechanism of low temperature-induced chromosome doubling in P. tomentosa pollen. Triploid produced by pollinating the obtained low temperature-induced 2n pollen with female P. tomentosa, and were screened by flow cytometry and somatic chromosome counting.

Result (1) The microsporocyte of P. tomentosa completes meiosis in about 4 to 5 days under greenhouse hydroponic conditions.The meiotic process of PMCs shows a clear correspondence with the morphology of male flower buds and the dynamic change of anther color. (2) Meiotic stage and duration had significant effects on the frequency of cold-induced 2n pollen. However, meiotic stage × duration interaction had no significant effect on the frequency of cold-induced 2n pollen. (3) The optimum treatment combination to induce pollen chromosome doubling, the PMCs were treated by 4 ℃ for two days when the meiotic stage was at metaphase Ⅱ, was successfully developed. 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 treated 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 was the first time for 2n pollen induction by low temperature in P. tomentosa and one triploid was successfully obtained, which showed that inducing pollen chromosome doubling to breed triploid by low temperature in Populus was feasible in a certain.