番茄小孢子发育时期鉴定和游离方法的研究

为寻找番茄小孢子单核期的鉴定方法以及将小孢子从花药中游离出来而不影响其活性的最佳方法，采用醋酸洋红染色法、酒精-盐酸-醋酸洋红染色法、DAPI 染色法，分别染小孢子单核期细胞核，pH6.8-8.0的中性红染小孢子单核期的液泡；采用挤压法、挤压离心法、自然散出法，分别从花药中游离出小孢子。实验结果醋酸洋红染细胞核着色不好，酒精-盐酸-醋酸洋红染细胞核着色清晰，DAPI染色细胞核清晰，但成本高且受时间限制，中性红染色液泡效果好，但受细胞活性影响；挤压法游离小孢子提前脱壳率较高，挤压离心法游离小孢子提前脱壳率最高，自然散出法游离小孢子脱壳率最小。所以，采用酒精-烟酸-醋酸洋红法鉴定番茄小孢子发育时期效果最好，采用自然散出法游离番茄小孢子可减少小孢子脱壳率，提高小孢子活性。

Research on the method for developmental stage identification and free for the microspore of tomato

The identification method for the uninucleate stage of tomato microspore, and the best method to free microspore from anther are studied in this paper. Carmine acetate dyeing, Alcohol-Hydrochloride-Carmine acetate dyeing, DAPI staining were used to stain the cell nuclear of microspore in uninucleate stage. Neutral Red with pH value 6.8-8.0 was used to stain the vacuole. Squeezing method, squeezing centrifuge and Natural spill out were used to free the microspore from anther. Results show that Carmine acetate dyeing is not effective in coloring compared to Alcohol Hydrochloride-Carmine acetate dyeing. Clear coloring can also be obtained by DAPI staining and Neutral Red staining. However, these two methods are limited by high cost, testing time or the activity of microspore. Lowest in advance hulling rate can be reached by Natural spill out , compared to squeezing and squeezing centrifuge method. Therefore, Alcohol-Hydrochloride-Carmine acetate dyeing is the best identification method for the DEVELOPMENTAL STAGES of tomato microspore. And Natural spill out can be used to free the microspore, which can reduce the hulling rate and improve the activity.