钝叶酢浆草花粉活力测定与贮藏温度研究

Pollen vitality and storage temperature study of Oxalis obtusa

关键词:  钝叶酢浆草，花粉活力，离体萌发，亚历山大染色法，贮藏温度

Key words:  Oxalis obtusa, pollen viability, in vitro germination, Alexander staining method, storage temperature

摘要:

为提高钝叶酢浆草杂交育种的效率，该研究以6个钝叶酢浆草品种花粉为实验材料，采用离体萌发法，通过正交试验筛选适宜花粉萌发的培养条件；采用TTC染色法、醋酸洋红染色法、I2-KI染色法和亚历山大染色法测定花粉活力，与离体萌发法测定结果进行对比，评估各染色方法的可靠性；在常温（28 ℃）、4 ℃、-20 ℃、-80 ℃温度分别贮藏花粉0、1、2、5、10、15、20、25、30、40、50、60 d，检测不同温度对花粉活力的影响，确定最佳的花粉贮藏温度。结果表明：（1）钝叶酢浆草花粉离体萌发的最适培养条件为20 g·L-1琼脂+100 g·L-1蔗糖+200 mg·L-1硼酸+200 mg·L-1氯化钙，温度25 ℃，时间4 h。（2）离体萌发法能够有效测定钝叶酢浆草花粉活力，各染色方法与之对比发现，TTC染色法不能使花粉染色，I2-KI染色法和醋酸洋红染色法测定结果显著高于离体萌发法，亚历山大染色法测定结果与离体萌发法无显著差异，可以快速、准确地测定花粉活力。（3）在不同温度下钝叶酢浆草花粉活力下降的速度为常温＞4 ℃＞-20 ℃＞-80 ℃，最佳贮藏温度为-80 ℃。该研究结果可为钝叶酢浆草人工授粉、杂交育种及花粉收集贮藏提供理论依据，有效解决花期不遇、异地授粉等问题。

Abstract:

To improve the hybrid-breeding efficiency in Oxalis obtusa, this study identified the culture conditions for optimal germination of O. obtusa pollen in vitro, established an efficient pollen vitality staining method, and investigated optimal pollen storage temperature for six cultivars. An orthogonal experiment was used to screen the culture conditions suitable for pollen germination. Different pollen viability staining method including TTC staining, Aceto-carmine staining, I2-KI staining, and Alexander staining, were carried out and results were compared with those of the in vitro germination assay to evaluate their reliability. To determine the effect of temperatures on pollen vitality and an optimal pollen storage temperature, the pollens stored at different temperatues (28 ℃, 4 ℃, -20 ℃, and -80 ℃) for different period (0, 1, 2, 5, 10, 15, 20, 25, 30, 40, 50, 60 d) were assayed. The results suggest: (1) The optimal culture condition for the in vitro germination of O. obtusa pollens is 20 g·L-1 agar+100 g·L-1 sucrose+200 mg·L-1 H3BO3+200 mg·L-1 CaCl2, at 25 ℃ for 4 h. (2) The in vitro germination method significantly affect the pollen vitality of O. obtusa. It is found that the pollen vitality ratio determined by I2-KI staining or Aceto-carmine staining is significantly higher than that determined by in vitro germination method. The TTC staining procedure does not work on O. obtusa. The Alexander staining revealed a pollen vitality ratio that has no significant difference from that by the in vitro germination method. (3) The decline rates of the O. obtusa pollen viability at different temperature are ordered as: room temperature > 4 ℃ > -20 ℃ > -80 ℃. To summarize, this study systematically studied the optimal culture conditions for the in vitro germination of O. obtusa pollens and determined that the Alexander method is the most efficient and accurate staining method for pollen viability, and the optimal storage temperature is -80 ℃. This research established important theoretical basis to improve artificial pollination, hybrid breeding, pollen collection and pollen storage of the O. obtusa, which can potentially solve the problem of missing flowering period for cross pollination.