Technical systemic optimization of pollen chromosome doubling induced by low temperature in Populus tomentosa

Objective This paper aims to determine the appropriate temperature range for low-temperature-induced chromosome doubling in pollen of Populus tomentosa to enhance the induction rate of 2n pollen, thereby improving polyploid breeding technology system of forest through inducing pollen chromosome doubling of P. tomentosa by low temperature.

Method Based on an investigation of differential sensitivity at different developmental stages of male flower buds to low temperature in P. tomentosa, aceto-carmine staining method was used, the experimental conditions for inducing 2n pollen by low temperature were systematically optimized by taking male floral branches of P. tomentosa collected from Shuozhou, Shanxi Province of northern China. Furthermore, triploids were generated by pollinating the obtained low-temperature-induced 2n pollen with female P. tomentosa and Populus alba × P. glandulosa, and were determined by flow cytometry and somatic chromosome counting.

Result (1) Compared with pollen mother cell stage, pollen mother cells (PMCs) of P. tomentosa at meiotic stage exhibited higher sensitivity for low temperature. The optimal temperature for inducing 2n pollen ranged from −13 to −7 ℃. (2) The dominant meiotic stage, temperature, and duration significantly affected the induction rate of low temperature-induced 2n pollen. The optimal treatment combination for inducing pollen chromosome doubling in Populus tomentosa by low temperature was determined to be 5 h of continuous treatment of pollen mother cells at −13 ℃ during metaphase II. The highest induction rate of 2n pollen was (31.11 ± 2.93)%. (3) Twenty-six triploids were produced by crossing low temperature-induced 2n pollen with haploid female gametes of P. tomentosa and Populus alba × P. glandulosa.

Conclusion In this study, to increase induction rate of 2n pollen, the technical system for inducing pollen chromosome doubling by low temperature in P. tomentosa has been optimized and 26 triploids were produced by crossing low temperature-induced 2n pollen. Therefore, an efficient and stable method to produce triploids for woody plants is established. These results lay a solid foundation for the development of theoretical frameworks and technical applications in triploid breeding of forest trees.