The Cytological Study On Late-acting Self-incompatibility In Camellia Oleifera

Camellia oleifera Abel.is a woody plant that produces edible oil;it is endemic to Southern China.It plays an important role in maintaining the supply of grain and oil in China,as well as in promoting ecological sustainability.However,during the cultivation of Camellia oleifera,the low fruit-to-flower ratio leads to unstable and low yield.This has become a major issue that hinders the healthy and efficient development of camellia industry in China.Previous preliminary studies have shown that camellia is self-incompatible,which might be one of the main factors contributing to the low fruit-to-flower ratio.Further,self-incompatibility(SI)in Camellia oleifera was remarkably different from that of conventional gametophytic self-incompatibility(GSI).In Camellia oleifera,SI response occurred in the ovary instead of the style and exhibited the characteristics of late-acting self-incompatibility(LSI).This study aimed to identify the type and characteristics of LSI in Camellia oleifera by conducting fluorescence microscopy and artificial pollination and by comprehensively evaluating the numerical data.The microstructures of the pistils at the cellular level were compared between self-pollination and cross-fertilization by performing paraffin sectioning with optical microscopy and whole stain-clearing technique with confocal laser microscopy.Moreover,the ultrastructure of the pistils at the subcellular level was compared between self-pollination and cross-fertilization by using scanning electron microscopy and ultrathin sectioning along with transmission electron microscopy.By analyzing the characteristics and cellular mechanism of LSI in Camellia oteifera,this study aimed to provide a scientific framework for improving the yield of Camellia oleifera by regulating its fruit-to-flower ratio.The main findings of this study are as follows.1.Identification of LSI in Camellia oleifera.The results of fluorescence microscopy,artificial pollination,and comprehensive evaluation of the numerical data indicated that the pollen tubes reached the base of the styles 40 h after self-pollination and cross-pollination.However,the pollen tubes from self-pollinated flowers showed slower growth,whereas those from cross-pollinated flowers showed normal growth.Between 40 and 48 h after pollination,the pollen tubes from cross-pollinated flowers continued to grow from the base of the style to the ovules and entered the embryo sac.However,the growth of the pollen tubes from self-pollinated flowers was slow or inhibited near the ovary at the base of the style;they also showed a range of incompatibility responses,such as being abnormally bright and having swollen,split,curled,layered,and wavy tips,when they stopped.growing in the ovary.Field experiments showed that the fruit-dropping peak was between 30 and 50 days after self-pollination,and the amount of fruits dropped accounted for two-third of the pollinated flowers,but fruit-dropping was at a lower rate in cross-pollinated plants.The fruit-to-flower ratio and seed-producing ratio of ripe fruits were significantly higher after cross-pollination than after self-pollination.After 120 days of pollination,the early-stage fruits after the rare success of self-pollination only had one to two developing ovules,and the remaining ovules showed browning and were shriveled.However,most of the ovules showed normal development in the early-stage fruits of cross-pollinated plants.Further,180 days after pollination,the immature embryos in cross-pollinated plants showed more advanced development than those of the self-pollinated plants.The pollen-ovule ratio value indicated that cross-pollination was obligatory in the reproductive system of camellia;the out-crossing index value showed that camellia belonged to the cross-pollination category of plants that need pollination agents;compatibility index suggested that camellia had self-incompatibility for pollination.The results from these three indices indicated that camellia had LSI and showed prezygotic isolation.2.The microscopic characteristics of LSI in Camellia oleifera.Paraffin sectioning with optical microscopy and whole stain-clearing technique with confocal laser microscopy showed that the embryo sacs were already well developed when the flowers were blossoming.The double fertilization observed in crossed-pollinated plants and an extremely small number of self-pollinated plants did not show noticeable differences,but the timings of these events were different.The sperm cell and polar nuclei(secondary nucleus)merged and formed primary endosperm nuclei 54 to 120 h after cross-pollination;however,this process was observed 84 to 144 h after self-pollination.Moreover,66 to 144 h after cross-pollination,sperm cells and eggs cells merged to form fertilized eggs,but this process was observed 102 to 150 h after self-pollination.After self-pollination,the majority of the ovules in the ovary died or degenerated during their development,because the pollen tubes failed to enter the embryonic sac and double fertilization did not occur.For the unfertilized flowers in which the stamens were removed,the cells in the entire embryonic sac deteriorated,and their ovules shrank and deformed.These observations were similar to those observed in unfertilized ovules and embryonic sacs in self-pollinated plants.The infertile ovules found in the ovaries were already present when the embryonic sac matured,instead of developing after pollination.These types of ovules were commonly located in the middle and lower sections of the ovary.These infertile ovules continued to grow in size when the fertilized ovary underwent development.However,their volume stopped increasing approximately four months after pollination,and they gradually shrank and browned during the later stage of development and became one of the sources of aborted seeds.3.The ultrastuctural characteristics of LSI in Camellia oleifera.The results of ultrathin sectioning with transmission electron microscopy and scanning electron microscopy showed that,after self-pollination,when the pollen tube began to reach the base of the styles,some parts of the pollen tubes started to swell and showed uneven tube walls.After the pollen tubes entered the ovaries and stopped growing,they showed swelling,splitting,curling,layered,and wavy patterns.These phenomena were the result of the thickening of some parts of the pollen tube walls;they showed 'spotted','sheet-like' and 'bands-like' thickening and 'armor-like'cracking.The pollen tubes in self-pollinated flowers were thicker than those in cross-pollinated flowers.Furthermore,in cross-pollinated flowers,the pollen tubes had higher level of vacuolization and cell organelles showed degeneration;however,in self-pollinated flowers,the pollen tubes were blurred,and their organelles became dissociated and unidentifiable.When the fertilized eggs were formed,a few starch granules were observed around the nuclei;thirty days after pollination,the starch granules started to enlarge and their number increased,and they surrounded the nuclei closely.The cellular membrane of the chalaza as well as the cell wall started to thicken;fifty days after pollination,the cell wall thickening increased further.Thirty days after self-pollination,a clear reduction of extracellular secretion and the amount and types of cell organelles in the micropyle opening of the unfertilized ovules was noted.Moreover,the secretion and spherical amyloplast disappeared in the micropyle opening passages.In the unfertilized flowers,a very small amount of extracellular secretion was noted.The secretion found in the gap of the passage in the micropyle opening,as well as the amyloplasts,essentially disappeared.The residual synergids in the embryonic sac deformed and degenerated;their cytoplasm dispersed,large vacuoles degenerated,and membrane structures dissociated.Moreover,no complete organelles were found in the egg cells,and only some traces of degeneration were observed;the nucleoplasm of the polar nuclei(secondary nuclei)gradually degenerated.