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Dongmei Wei, Chao Gao and Deyi Yuan

The mechanism by which calcium regulates anther development remains unclear. This study investigated the relationship between calcium distribution and anther development in oil tea (Camellia oleifera Abel.) by using the potassium antimonite technique. Before the onset of microsporogenesis, abundant minute calcium precipitates appeared on the plasma membranes of microspore mother cells. Meanwhile, numerous precipitates accumulated in the tapetal cells. After meiosis, calcium precipitates appeared in young microspores. During microspore development, calcium precipitates mainly appeared in the small vacuoles of the cytoplasm. At the late microspore stage, a large vacuole formed, and the number of precipitates in the microspore decreased. The number of precipitates in the tapetal cells decreased as microsporogenesis proceeded. Then, calcium precipitates in the bicellular pollen cytoplasm again increased in number. During bicellular pollen development, the number of calcium precipitates decreased. As the pollen grains matured, only a few calcium precipitates were evident in the pollen cytoplasm. The results of this study, which show the spatial and temporal features of calcium distribution during the anther development of C. oleifera, suggest that calcium distribution is related to anther development.

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Chao Gao, Rui Yang and Deyi Yuan

Camellia oleifera is an important woody edible oil plant in southern China. In this study, the developmental differences in ovules at different positions in the ovary of C. oleifera were observed. The developmental type and characteristics of aborted ovules, ratios of normal and aborted ovules, and their developmental differences after flowering were examined. Ovules near the stylar end and in the middle exhibit normal development and are able to form embryo sacs; lower ovules near the pedicel end are usually aborted. The proportion of abortion of four examined cultivars ranges from 10.2% to 33.3%. Aborted ovules can be divided into four categories: 1) nascent egg apparatus lacking distinguishable cells; 2) completely absent egg apparatus structure consisting of flocculent tissue; 3) lack of tissue, comprising only integument cells; and 4) the inner integument not constituting a micropyle channel, with incomplete egg apparatus development and generating abnormal ovules. At 120 days after pollination (DAP), significant distinguishable size differences were found between fertile and aborted ovules; aborted ovules ceased growth at 180 DAP. On fruit maturation, aborted seeds were still attached to the placenta.

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Huan Xiong, Feng Zou, Sujuan Guo, Deyi Yuan and Genhua Niu

Chinese chestnut (Castanea mollissima), which is native to China, has been cultivated as a nontimber forest tree species for 4000 years. This species has been found to display self-sterility, which results in a significantly lower seed set following self-pollination (SP) compared with that following cross-pollination (CP). Self-sterility can be induced by prezygotic or postzygotic late-acting self-incompatibility (LSI) or early-acting inbreeding depression (EID). To elucidate the causes of self-sterility in chestnut, we investigated pollen–pistil interactions, fertilization, and early ovule development following SP and CP by using a paraffin section technique and fluorescence microscopy. The fruit set percentage and seed characteristics also were evaluated among different pollination treatments. The results show that there were no significant differences in pollen tube behavior following SP vs. CP, regardless of the stigmatic or stylar level. Double fertilization was significantly greater following CP (18.09%) than SP (2.58%). The significantly lower percentages of ovule penetration and double fertilization in the selfed vs. crossed ovules support a prezygotic LSI mechanism in C. mollissima. The fruit set resulting from chase-pollination (CHP; 53.85% to 63.64%) was greater than that resulting from SP (12.12% to 14.00%). In addition, the distribution of aborted seed sizes after SP showed a widely clumped pattern. Abortion occurred at different stages during seed development rather than at a uniform stage, which supported the idea that EID was operating in C. mollissima. Levels of self-sterility in the chinese chestnut trees ranged from 88.2% to 90.5%. Thus, partial prezygotic LSI and EID contributed to self-sterility in the C. mollissima ‘Yanshanzaofeng’, with prezygotic LSI rejecting part of the self-pollen in the ovary and EID aborting part of the self-fertilized seeds.

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Chao Gao, Deyi Yuan, Ya Yang, Bifang Wang, Dongming Liu and Feng Zou

Camellia oleifera is an important plant species that produces edible oils. Understanding the double fertilization of this plant is critical for studies concerning crossbreeding, self-incompatibility, and the biological mechanisms underlying hybridization. We aimed to characterize pollen tube growth and double fertilization in C. oleifera. The female and male parent cultivars (Huashuo and Xianglin XLC15, respectively) were used for artificial pollination. Growth of the pollen tube in the style, ovary, and ovule from pollination to fertilization and the cytological characteristics of female and male gamete fusion during double fertilization were observed using fluorescence and scanning electron microscopy (SEM). Numerous pollen grains germinated 2 to 4 hours after pollination. The pollen tubes entered the interspaces between the papillar cells, grew along the stylar canal, and aggregated at the one-third site of the style. They grew in the gradually narrowing stylar canal, entering the locule. The tubes turned 90° and entered the embryo sac through the micropyle; subsequently, they entered a degenerated synergid, where the spermatids were released. One sperm nucleus fused with the polar nucleus, forming the primary endosperm nucleus, whereas the other sperm fused with the egg, forming the zygote. The polar nucleus was fertilized earlier than the egg. Double fertilization of C. oleifera is characterized as pre-mitotic gametogony. The current results lay a theoretical foundation for studies concerning the crossbreeding and embryology of C. oleifera and provide fundamental data concerning the reproductive biology of the genus Camellia.

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Huan Xiong, Ping Chen, Zhoujun Zhu, Ya Chen, Feng Zou and Deyi Yuan

Camellia oleifera is an important woody tree species in China that produces edible oil. Although sterile male C. oleifera plants play an important role in hybrid breeding, the possible cytological characteristics of pollen abortion remain unknown. To characterize the pollen abortion process, a genic petaloid-type sterile male C. oleifera ‘X1’ plant was investigated using a cytological method. The results showed that in male-fertile plants, the anthers were full and butterfly shaped, the pollen viability was as high as 97.5%, and the development of the tapetum and anther vascular bundles was normal. However, in male-sterile C. oleifera ‘X1’, petaloidy in the anther was observed, and the pollen vitality was as low as 4.5%. Pollen abortion in sterile C. oleifera ‘X1’ anthers occurred from the microspore stage to the mature pollen period. Further cytological analyses revealed an abnormally enlarged tapetum and retarded tapetum degeneration, suggesting that insufficient nutrients were provided for microspore development. Moreover, the anther vascular bundles displayed hyperplasia, and the pollen sac area became increasingly smaller, causing most anthers to be sterile and to have few pollen grains. Taken together, the results indicate that petaloid-type male sterility in C. oleifera may be attributed to abnormal development of the tapetum and anther vascular bundles. The findings clarify the pollen abortion period and the cytological characteristics of petaloid-type cytoplasmic male sterility in C. oleifera, and lay a solid foundation for the male sterile line in C. oleifera hybrid breeding.

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Huan Xiong, He Sun, Feng Zou, Xiaoming Fan, Genhua Niu and Deyi Yuan

Castanea henryi is an important woody grain tree species native to China. The objective of the current study was to find the suitable plant growth regulators (PGRs) and the optimal concentrations for direct organogenesis by using axillary shoots and cotyledonary nodes. Seeds were collected from the field, sterilized, and germinated in vitro. Axillary shoots and cotyledonary nodes of 3-week-old seedlings were used as explants. To find the suitable PGR for adventitious shoot induction, 0.5 mg·L–1 6-benzylaminopurine (6-BA), 0.1 mg·L–1 indole-3-acetic acid (IAA), 0.1 mg·L–1 2,4-dichlorophenoxyacetic acid (2,4-D), or 0.1 mg·L–1 1-naphthaleneacetic acid (NAA) was supplemented to Murashige and Skoog (MS) medium containing 0.65% agar and 3% sucrose. A high induction percentage of adventitious shoots (85.67%) was obtained from cotyledonary nodes supplemented with 0.1 mg·L–1 2,4-D. The type of explant influenced shoot proliferation rates and quality. Apical explants produced more and longer shoots than nodal segments. For shoot multiplication, 1 mg·L–1 6-BA + 0.05 mg·L–1 indole-3-butyric acid (IBA) supplemented with MS medium produced 12.33 and 6.25 shoots per explant, respectively, from apical and nodal explants. For shoot elongation and strengthening, 2 mg·L–1 6-BA + 0.05 mg·L–1 IBA supplemented with MS medium was the best combination, producing shoots with a mean length of 3.50 cm, a diameter of 0.46 cm, and about eight leaves per shoot. The greatest rooting of 76.70% and 11.33 roots per shoot was achieved when cultured in MS medium supplemented with 3.5% perlite + 1.5 mg·L–1 IBA. For acclimatization of the rooted plantlets in the greenhouse, a survival rate of 80% was achieved. This protocol—from multiplication to acclimation—is helpful to realize mass propagation of high-quality trees of chinquapin for increasing production and nut quality.