Normal ovule fertilization and seed development
Morphological characteristics of the carpel and embryo sac:
The carpel of P. ludlowii consists of three parts: stigma, style, and ovary (Fig. 2a). The plant has a wet stigma which is curled in various forms, mostly about 360°. The stigma is formed by combining two parts of similar size and shape, and a narrow band 0.1–0.4 mm in width is formed in the middle (Fig. 2b). The surface of the stigma is densely covered with papillary cells (Fig. 2c). The style is joined to the stigma, and is approximately 1–3 mm long, with a hollow style canal in the center (Fig. 2e). The style canal is the growth channel for the pollen tube of P. ludlowii. The nuclei of inner canal cells of the style are large and can be clearly observed. Morphologically, these cells are regular and darker than other cells. Similar to the inner tube cells of Lilium regale (Hu et al. 1982) and Camellia oleifera (Gao et al. 2019), the inner tube cells of the style canal of P. ludlowii are glandular (Fig. 2f). Attached to the style is the ovary, in which there is a linear arrangement of two rows of inverted ovules (Fig. 2d), with a double integument, pellicle, and thick nucellus (Fig. 2g). At blooming, the embryo sac is mature and forms a typical seven-cell and eight-nucleus structure [Fig. 2h–k; Fig. 2h, two synergids; Fig. 2i, an egg cell; Fig. 2j, three antipodal cells; Fig. 2k, central cells (two polar nuclei)]. Subsequently, the two polar nuclei fuse to form a secondary nucleus (Fig. 2l), but occasionally do not fuse to form a secondary nucleus. Only one embryo sac was observed in each ovule in all experimental materials.
Pollen tube growth in the style and ovary
The flowers of P. ludlowii open during the day and close at night, a feature that facilitates pollination by wind and insects. We observed that 1–12 h after flowering, pollen grains fell on the stigma and germinated, the pollen tube penetrated deep into the stigma, where the pollen remained (Fig. 3a). After entering the stigma, the pollen tubes grew along the vascular bundles of the stigma and converged into the style (Fig. 3b). The tube then grew to the base through the stylistic tract and the layer of mucous secreted by the inner epidermal gland cells (Fig. 3c). After 36–48 h of flowering, the pollen tube reached the base of the style (Fig. 3d) and continued to grow toward the ovary. After penetrating the ovary, the pollen tube continued to grow along the placenta’s epidermal cells (Fig. 3e). After 48–60 h of flowering, when the pollen tube approached the ovule, it was bent nearly 90° to approach and penetrate the micropyle and pass through the nucellus into the embryo sac (Fig. 3f). A few aborted pollen grains, as well as some abnormal pollen tubes and self-pollinating pollen tubes could be seen on the stigma after pollination. However, due to the large amount of pollen, sufficient pollen tubes entered the ovary, enabling us to observe each ovule in the ovary with the pollen tube entering.
After passing through the apical nucellus from the degenerated synergid entering the embryo sac, the pollen tube released two sperm cells, one fusing with the egg nucleus and the other with the secondary nucleus (polar nucleus) of the central cell. The sperm nucleus gradually approached the egg nucleus, fused with it, and eventually formed the large nucleoli of the zygote completing the fertilization of the egg cell (Fig. 3g–i). This process was observed at 60–144 h after flowering. After zygote formation, a period of dormancy was required before the division stage. The fusion of the sperm nucleus and secondary nucleus was similar to that of the fusion of the sperm nucleus and egg nucleus (Fig. 3j, k). The difference was that the fertilization rate of the central cell was significantly higher than that of the egg cell, as determined by observing several successive sections. After fertilization of the central cell, the primary endosperm nucleus was formed; this nucleus then divided to produce the free endosperm nucleus (Fig. 3l). This process was observed from 60–108 h after flowering. The timing of double fertilization of the ovules in each ovary was not synchronous. During 108–144 h after flowering, some ovules in the double fertilization stage were also observed, but the number was small; the process was completed by day 7 after flowering.
During the late development of fertile ovules in P. ludlowii, the primary endosperm nucleus was the first to change. After the fertilization of the secondary nucleus (polar nucleus), the primary endosperm nucleus split to form several free endosperm nuclei inside the embryo sac (Fig. 4a). Subsequently, the free endosperm nuclei were divided repeatedly, and the number of free nuclei increased continuously, during which no cell wall was formed (Fig. 4b–d). Until 45 days after flowering, the free nuclear endosperm began to cellularize, at which point the internal ovule was liquid or semi-liquid (Fig. 4e). The cellularization was completed at approximately 55 days after flowering (Fig. 4f). Thereafter, the volume of the endosperm increased rapidly; the endosperm reached its final shape and size approximately 75 days after flowering and the interior of the ovule gradually changed from liquid or semi-liquid to a solid state.
The endozygote of the fertile ovules began to divide after the end of dormancy (Fig. 4g). The zygote divided first to form the binuclear proembryo and then divided repeatedly and synchronously. The number of free nuclei increased continuously, and the proembryo grew gradually. During this period, no cell wall was formed (Fig. 4h, i). The free nuclear stage of the proembryo lasted from zygotic dormancy to 30 days after flowering. The free nuclei then began to cellularize, generally beginning at the chalazal end and advancing toward the micropylar end (Fig. 4j). At 55 days after flowering, embryo development reached the globular embryo stages (Fig. 4k, l). Thereafter, the embryonic somatic cells divided and differentiated rapidly, and the embryonic development successively went through the heart-shaped embryo (Fig. 4m), torpedo-shaped embryo stage (Fig. 4n), and cotyledon embryo stage (Fig. 4o), until the final seeds were mature.
Abnormalities during fertilization and seed development
During the growth and development of P. ludlowii, some pistils were abnormal in different ways, for example, ovule bursting, caused by the rupture of the carpel (Fig. 5a), stigma exposure before anthesis (Fig. 5c), and carpel deformity (Fig. 5b, d–f).
These abnormal pistils were present before flowering and could not be fertilized normally. Among nearly 500 flowering branches and about 1,500 open flowers (some buds failed to open normally), approximately 30 had abnormal pistils, accounting for 0.02%.
During the study, repeated experiments and many dissections showed that the appearance of carpels in the ovaries of some test materials was the same as that of other carpels, but there were sterile ovules inside. These ovules were present on the day of flowering, when the embryo sac matured, rather than being abnormal during post-pollination development. These ovules were markedly different from other ovules in morphology; that is, they were smaller in size or deformed (Fig. 5g–i). Some had no embryo sac, and there were no clear spaces between the outer and inner integument (Fig. 5j, m). Moreover, some of the ovules had embryo sac structures, and the space was small, without any obvious organizational structure (Fig. 5k, l, n, o). In the materials observed, not all carpels had sterile ovules, and according to the statistics, the carpels with sterile ovules accounted for 10.59–15.73% of all the test materials.
In addition, through several sections and repeated experiments, we found some abnormal embryo sacs in the experimental materials during fertilization. The main characteristics of these abnormal embryo sacs were as follows. (1) At 84 h after anthesis, the pollen tube had reached the ovary, and most ovules had begun fertilization, but the two synergistic cells in the individual embryo sac remained intact without degeneration. The embryo sac, which could not enter the physiological state of fertilization in time, could not be fertilized normally (Cheng 1996). (2) At 8–12 days after flowering, free nuclear proembryo and endosperm were formed in the fertilized ovules, and the number of free nuclei increased significantly, but the secondary nuclei in some of the embryo sacs remained unfertilized, and no endosperm free nuclei were formed. These ovules could not develop into seeds normally without double fertilization. The ovules containing abnormal embryo sacs accounted for approximately 1.2% of all experimental materials, and only a few carpels had abnormal embryo sacs.
Degeneration of the embryo and endosperm
There were some ovules in the carpel, which could complete double fertilization and showed zygote and primary endosperm nuclear division in the early stages but could not develop further in the late stages. At 9 days after flowering, some aborted ovules similar in size to normal ovules showed abnormalities in the embryo sac and degeneretion of the free nuclei of the endosperm (Fig. 6a, b). On day 10 after flowering, the free nuclei of the endosperm continued to degenerate (Fig. 6c). Some abortive ovules of the same size as normal ovules showed shrinkage and deformity in their outer tepals after dewatering and embedding (Fig. 6d). When no free nuclear embryo was observed in the embryo sac, the endosperm developed abnormally, and the free endosperm nuclei were rare and irregular. The cytoplasm of the endosperm gradually disintegrated and was in a state of severe degeneration (Fig. 6e, f). In the material at 11 days after flowering, apart from the phenomenon of endosperm abortion, the free endosperms in some of the embryo sacs aggregated after nuclear division but did not separate (Fig. 6g). At 12 days after flowering, the endosperm of the fertilized ovules degenerated. In some embryo sacs, only the free nuclear proembryo developed to the stage of dinucleus proembryo, but the free endosperm nuclei were rare, the cytoplasm was significantly reduced, and the embryo sac was in a severely degenerated state; consequently, the embryo sac became narrow (Fig. 6h). Embryo sacs were distinct from the fertile ovules at the same period (Fig. 4h). On the same day, with the development of the ovaries and the enlargement of the ovules, the ovules’ volume in the ovaries began to show differences (Fig. 6i). A few aborted ovules developed normally until approximately 20 days after flowering, and the volume of the ovules was slightly larger than that of other early aborted ovules and slightly smaller than that of fertile ovules (Fig. 6j, indicated with red arrows). The free nuclei of the embryo and endosperm in these aborted ovules were not as developed as the other fertile ovules and tended to degenerate (Fig. 6k). At this time, less than one-third of the fertile ovules were observed in the ovary, and abortive ovules were observed in all carpels. The results indicated that the abortion of embryo and endosperm occurred mainly in the early stage, 9–12 days after flowering.
Late development of abortive ovules
After 12 days, the embryo sacs of some aborted ovules gradually contracted toward the nucellus, the cavity of the embryo sac became narrow and long, degenerated, and necrotic, and filled with a brown material (Fig. 7a, b). At 30 days after flowering, the embryo, nucellar, and integument gradually degenerated (Fig. 7c). Some embryo sacs were filled with large cells, with only a narrow gap in the middle, with darker staining (Fig. 7d–g). With the growth of the ovule, the free nuclear embryo and endosperm of some ovules that began to abort at approximately 20 days after flowering degenerated completely, and the outer envelope had also gradually degenerated, finally forming a hollow shell with no embryo or endosperm in the embryo sac. The outer cover gradually degenerated, finally forming a hollow shell without an embryo and endosperm in the embryo sac (Fig. 7h–j). Although the embryo and endosperm were not formed in the aborted ovules, the external morphology of the aborted seeds significantly differed (Fig. 7k, indicated with the red arrow). In addition, when harvesting seeds, we also found a few seeds with external morphology and volume similar to those of the fertile seeds, but they were deformed after applying pressure. After peeling, the seeds were hollow inside, without embryo or endosperm structure (Fig. 7l, indicated with the green arrow). This could be attributed to embryo and endosperm atrophy during seed maturity.
Position and rate of aborted ovules in P. ludlowii
The total number of seeds in a single carpel ranged from 6–23, and the number of normal seeds ranged from 0–8 (2–4 more). More than two-thirds of the ovules were aborted (Table 1, Fig. 8). According to the statistics of the rate of abortion seeds (Table 1), the seed abortion rate in the ovaries in 2019 and 2020 was 69.14% and 74.01%, respectively, and the abortion rate in wild populations was also high (66.39%), indicating that seed abortion occurred independent of the growing environment.
The statistical results of the position of aborted seeds in P. ludlowii (Table 1) showed that, in 2019, the abortion rate in the lower part of the ovary was the highest (77.08%) and the abortion rates in the middle and upper parts of the ovary were 66.98% and 66.25%, respectively. Moreover, the differences among the three parts were significant. In 2020, the abortion rate was the highest in the middle part of the ovary (83.33%), followed by the lower part (78.59%) and the upper part (70.86%). These results showed that there was no specific position for seed abortion in the ovary of P. ludlowii, and that seed abortion was random.Moreover, in 2021, the abortion rate in the lower part of the ovary was the highest (78.95%) and the abortion rates in the upper and middle parts were 74.90% and 71.65%, respectively.