Exploring the Diversity of Ovule Development in the Novel Rice Mutant ShuangLi Using Confocal Laser Scanning Microscopy

Low energy N⁺ ion beam implantation has been used to create the novel rice mutant "<i>shuangli</i>", which produces partially fertile spikelets containing double grains. Abnormal ovule development is a major cause of partial fertility and grain diversity in rice mutants. To elucidate the developmental mechanism of ovule diversity in <i>shuangli</i>, ovules undergoing development were stained using eosin Y and H33342 and observed using confocal laser scanning microscopy. Different developmental abnormalities were observed in the ovary, embryo sac, and ovule. Abnormal development was observed in 35.18% of the ovary structures, primarily manifesting as "tumor" like cell clusters, "false ovaries", stamen degeneration, and double ovaries. In the embryo sac, abnormal development occurred in about 17.35% of the megaspore cells, including the formation of three nuclei, two daughter cells of asynchronously divided dyads, multiple megaspore tetrads, and "narrow and elongated" cavities. At the female gametogenesis stage, the abnormal development rate was 27.53%, mainly involving the degeneration of the central polar nucleus, egg apparatus, antipodal cell mass, or female germ unit. In <i>shuangli</i>, abnormal development occurred in 28.06% of the ovule structures, including lateral tissue, nucellar tissue, double ovules and double embryo sacs. Of the observed lateral tissues, 8.27% did not differentiate into sexual reproductive tissue, which affected the fertilization of the embryo sac, leading to atrophy and degeneration. A new abnormal tissue similar to the inner integument was found on both sides of the nucellar tissue, and the two specialized nucellar tissues appeared to have "staggered" growth within a single ovary. Of the examined ovules, 10.79% exhibited different types of double ovules, including heart-shaped, "anatropous", "conjoined" structures. However, the double ovules typically developed synchronously, explaining the production of different sizes of the two grains in <i>shuangli</i>. In addition, "double" embryo sacs from two "twinborn" nucelli were found in one ovule, and the frequency of "double" embryo sacs was 3.60%. Therefore, ovule development diversity may result in fertilization or gradual degeneration after fertilization, explaining the lower fertility of <i>shuangli</i> at the embryological level.