Application of pollen isolation in apomixis germplasm identification of date plum (Diospyros lotus) by sealing stigma with white latex

Abstract:【Objective】Diospyros lotus is an excellent persimmon rootstock widely used in North China. Some individual date plum plants do have apomixis characteristics which has great theoretical and practical significance for breeding. A research team of diospyros plants is working on the screening and utilization of apomixis germplasm and trying to improve work efficiency by exploring other possible pollen isolation methods to replace the traditional sulfuric acid paper bagging method. This study aimed to make a thorough inquiry into the effectiveness of white latex in pollen isolation and provide technical support for apomixis germplasm identification and in-depth analysis of apomixis reproductive characteristics of date plum.【Methods】Flowers at large bud stage were selected from mature female plants and the corolla was removed carefully to expose the stigma of female flowers on a calm and sunny day. Three pollen isolation methods including sulfuric acid paper bag, nylon net bag and white latex stigma seal were used for pollen isolation, and normal artificial pollination was used as the control. The morphology of stigmas and quantity of pollens on them were observed under the fluorescence microscope, compared with the control: artificial pollination flowers. In addition, the apomictic ability of 22 individual date plum germplasms were identified by sealing stigma with white latex to test and verify the feasibility of large-scale or mass application.【Results】The white latex did not affect the ovary development  of date plum and succeeded in blocking the contact between stigma and pollinated pollen. By using this method called white latex pollen isolation, apomixis evaluation of 22 date plum plants was completed in one year just by one person. Under conventional artificial pollination, male xy-1 pollen could germinate normally on the stigma of female plant xx-10, and 2 h after pollination, many pollen tubes had germinated on the stigma; 3 h after pollination, the pollen tube reached the middle of the style; at 5 h, the pollen tube extended to the bottom of the style, and at 8 h, the pollen tube could be seen at the base of the ovary. After sealing the stigma with white latex, artificial pollination was performed at 1, 3, 5, 8, 12, 24, 48 and 72 h, respectively, and no pollen grains were observed on the tested stigma samples under a fluorescence microscope, indicating that white latex spot coating on stigmata was indeed completely effective in isolation of pollinated pollen. The results also showed that xx-8, xx-10, xx-20, xx-24, xx-29, xx-31 and xx-33 could not set fruit normally after pollen isolation. It was preliminarily determined that these individual plants did not have apomictic reproductive ability. Two individual plants of xx-13 and xx-15, after bagging, set fruit and did not produce seed, indicating that they did not have apomictic reproductive ability, but may have parthenogenetic bearing ability. The individual fruit of xx-18 date plum had seeds, but none of the seeds became seedlings in that year. Further evidence is needed to identify its apomixis reproductive ability. The remaining 12 date plums could produce viable seeds after pollen isolation, indicating that they had apomixis reproductive characteristics. Among them, xx- 7 and xx- 37 were highly apomixis reproductive plants, and more than 40 seeds could be obtained from 100 female flowers isolated.【Conclusion】In this study, the exact effect of white latex on pollen isolation of date plum was verified by fluorescence microscopic observation, and an efficient pollen isolation technology suitable for date plum was successfully established. The white latex is available and efficient in pollen isolation and apomixis germplasm identification of date plum and it can improve the identification efficiency of apomixis germplasm and provide technical support for in-depth analysis of apomictic characteristics of date plum. White latex has the characteristics of good film forming and fast curing speed, forming a protective film after air drying, and can completely and effectively isolate foreign pollens. Compared with the sulfuric acid paper bagging isolation method, there is no need for multiple bagging and bagging, saving a lot of labor and time. Compared with polyvinyl alcohol, white latex is a mature commodity, which is more common in the market and convenient to purchase with low cost. We can try to apply this method to other fruit crops to carry out relevant research.