Abstract:

Objective: Optimization of an in vitro germination protocol to effectively determine the viability of Paeonia ludlowii pollen, the effects of different storage conditions and incubation temperature on pollen longevity were compared to determine the appropriate temperature for pollen storage in the short, mid and long term, and the physiological mechanism of the programmed death of P. ludlowii pollen at different storage temperatures was analyzed, so as to provide a basis for cross breeding and germplasm resource conservation. Method: The fresh pollen of P. ludlowii from Tibet was used to examine pollen morphology by scanning electronic microscope (SEM). Pollen germination was characterized by in vitro culture to investigate the effects of different storage temperatures and times on pollen viability and activity of superoxide dismutase(SOD), peroxidase(POD), and catalase(CAT), as well as the contents of ascorbic acid(AsA) and malondialdehyde(MDA). Result: The rate of pollen plumpness of P. ludlowii is high under the natural conditions, and the deformity rate is only 5.6%, and the sexual reproductivity is relatively high. The factors influencing the pollen germination were as follows: sucrose > boric acid > CaCl₂ > GA₃. The longevity of pollen stored at room temperature was only 24 days, the longevity of pollen stored at 4 ℃ was about 80 days, and the longevity of pollen stored at -20 ℃ was 120-184 days. The pollen longevity for storage at -80 ℃ was over one year, and the suitable temperature for long-term preservation of pollen was at -196 ℃. The germination rate of pollen decreased rapidly before and after the drastic changes of pollen protective enzyme, MDA content and AsA content. SOD, CAT, POD activities and AsA content at -196 ℃ remained stable and the ability of eliminating active oxidation was strong. Correlation analysis showed that SOD activity was the most important physiological factor that affected the pollen longevity during storage, and membrane peroxidation was the main physiological factor that caused pollen death. The three protective enzymes and AsA under different storage temperatures had different effects: POD served as a sensitive protective enzyme at room temperature, SOD served as a sensitive protective enzyme at - 20 ℃ and - 80 ℃, whereas both SOD and CAT served as sensitive protective enzymes at 4 ℃. The order of the effects of three protective enzyme activities and AsA content on pollen germination rate was as follows: SOD > CAT > AsA > POD. Conclusion: The rate of pollen plumpness was correlated with the germination rate. The highest germination rate in Paeonia ludlowii was about 92.10% of the pollen treated with 120 g·L^-1 sucrose+45 mg·L^-1 boric acid + 55 mg·L^-1 GA₃ + 30 mg·L^-1 CaCl₂. The room temperature was suitable for the short-term storage of P. ludlowii pollen within 24 days, 4 ℃, -20 ℃ were suitable for the mid-term storage of pollen in 80-120 days. -80 ℃ was suitable for transannual storage of pollen, and -196 ℃ was suitable for long-term storage of pollen. The generation and removal of reactive oxygen species within pollen cells were at an equilibrium when the pollen was stored at -196 ℃, and the stability of cell membrane system was the physiological responses to keeping high pollen germination rate. The cell membrane peroxidation and damage caused by excessive accumulation of reactive oxygen species and free radicals were the main reasons for the decrease of pollen germination rate after storage at room temperature, 4 ℃, -20 ℃ and -80 ℃.

Key words: Paeonia ludlowii, pollen germination, pollen longevity, pollen storage, protective enzymes, MDA, AsA