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 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.