HORTSCIENCE 56(1):85–93. 2021. https://doi.org/10.21273/HORTSCI15479-20 crosporogenesis and formation of the male gametophyte in P. orientalis.Thefemale cone and ovule development were detected Bud Initiation, Microsporogenesis, by Zhang et al. (2000). There were no reports on the reproductive biology of P. orientalis in Megasporogenesis, and Cone the past 20 years, and parts of the development processes were lacking, such as the detailed Development in Platycladus orientalis date, internal and external morphological characteristics of cone initiation, sporogenesis Ting Liao, Guobin Liu, Liqin Guo, Ye Wang, Yanwu Yao, and Jun Cao and seed formation process, and so on. Owens Beijing Academy of Forestry and Pomology Sciences, No. 12 A Rui Wang and Marje (1977), Owens and Molder (1974, Fen, Fragrance Hills Haidian District, Beijing 100093, P. R. China; and 1980), and Owens and Pharis (1967) reported the sexual reproduction process of Thuja Beijing Academy of Agricultural and Forestry Sciences, No. 11 Shuguang plicata and Chamaecyparis nootkatensis in Huayuan Middle Road Haidian District, Beijing, 100097, P. R. China detail; in addition, gibberellin-induced cone Additional index words. bud initiation, microsporogenesis, megasporogenesis, cone develop- formation was studied. There is a lack of comprehensive research on the bud initiation, ment, Platycladus orientalis cone development, and sexual reproduction of Abstract. As a native tree species with a strong adaptability, Platycladus orientalis is a P. orientalis, and even less research at the gene species of choice for afforestation and landscaping in northern China. However, it level. develops mostly male cones and few female cones. In addition, its reproductive charac- The cone development and reproductive teristics are not yet clear, which limits further breeding work. To systematically clarify biological characteristics in pine and Chinese the reproductive biology characteristic and fertilization mechanism of P. orientalis, the fir were studied comprehensively and in de- present study comprehensively investigated the process of micro and macro-sporogenesis tail in gymnosperms in recent years. Mor- in male and female cones from bud initiation to fertilization, and seed development. The phologic and anatomical observations in the specific time in each developmental stage, including bud initiation, microsporogenesis, process of ovulate strobilus generation and megasporogenesis, and cone and seed development, was determined, and the abortive development were researched in Pinus tabu- phenomenon during development was discovered in both male and female cones. In liformis (Zhang et al., 2017). Embryonal addition, this research showed that the microspore mother cells were dormant in winter development revealed the annual cycle of at meiosis stage, and the male gametophyte started to develop when dormancy ended. ovulate cone development in Pinus sibirica The tapetum developed normally and belonged to the secretory type. The optimal in the western Sayan Mountains (Tretyakova treatment time for male and female cones transformation by artificial induction was et al., 2004). An embryological study from late June to mid-July. This finding provided a theoretical basis for hybridization, revealed the systematic significance of the breeding, improvement of seed yield and quality, and artificial induction of male and primitive gymnosperm Ginkgo biloba (Wang female cone transformation in P. orientalis. et al., 2011a). Morphological and structural changes during female and male cone devel- opment in Cunninghamia lanceolate were Plants in the Cupressaceae family, which northern China. Its wood has moderate hard- reported in detail by Jiang et al. (2017) and are widely distributed in the southern and ness and strong corrosion resistance, and its Zhu et al. (2018). Furthermore, recent inves- northern hemispheres, are the only gymno- seeds, roots, leaves, and bark can be used as tigations of gene level in P. tabuliformis had sperms distributed worldwide (Zheng and Fu, traditional Chinese medicine. As a monoe- been researched deeply. High-throughput 1978). P. orientalis, a monotypic genus be- cious tree species, P. orientalis produces a gene expression profile chip in male and longing to Cupressaceae, is one of the most very large amount of pollen, which easily female cones were detected, and the specif- commonly used trees worldwide. Moreover, causes allergic symptoms in spring; however, ically expressed genes and expression pattern as one of the city trees in Beijing, it is an a low number of female cones limits the were analyzed in different cone development important local tree species for afforestation, breeding process. Therefore, it is necessary stages (Niu et al., 2013). Analysis of her- border trees, and landscaping unique to and meaningful to conduct a comprehensive maphrodite in P. tabuliformis provided the and detailed study on the reproductive biol- basis and new ideas for molecular biology ogy characteristics and ontogeny to provide research on the cone development of conif- Received for publication 29 Sept. 2020. Accepted further information for production and culti- erous species (Niu et al., 2016). for publication 6 Nov. 2020. var breeding of P. orientalis. To understand the reproductive biology Published online 9 December 2020. At present, there have been some studies and ontogeny more systematically, and to This study was funded by The Youth Research about the embryo development, palynol- explore the mechanisms of pollination and Foundation of Beijing Academy of Agricultural ogy, and leaf structure of Cupressaceae fertilization, the present research comprehen- and Forestry Sciences of China (No. QNJJ201923) plants, but the micrographs were unclear sively and systematically reported the cone and The Science and Technology Innovation Abil- due to the relatively long time frame and initiation, formation, and development pro- ity Construction Projects of Beijing Academy of the limitations of microscopy over which cesses and the phenomenon of megaspore Agricultural and Forestry Sciences of China (No. KJCX20200207, KJCX20200114). the studies were conducted. Most of these and microspore abortion in P. orientalis by We are grateful to senior engineer Jin Bai for studies focused on morphological research using a paraffin section method. This study identification of experimental materials in this and plant classification (Gadek and Quinn, provides a theoretical basis and precondition research. 1985, 1988; Kumrann, 1994; Sugihara, for improving seed yield and quality, achiev- 1992). In P. orientalis, research had mainly ing gender transformation, hybridization, and T.L. and J.C. conceived and designed the experi- focused on breeding, cultivation tech- breeding in P. orientalis. ments; T.L. and G.L. performed the experiments; niques, physiological and ecological char- L.G. and Y.W. collected the samples of different acteristics, diseases, and insect pests. There Materials and Methods stages; Y.Y. took photos in field; T.L. analyzed the were a few reports on the reproductive data and photos; and T.L. wrote the manuscript. J.C. is the corresponding author. E-mail: caojun@ development of P. orientalis; however, The new cultivar of P. orientalis ‘Dieye’, baafs.net.cn. they were not comprehensive. Cone initia- used in the present research, was from a This is an open access article distributed under the tion, development, and phenology of P. coniferous plant resource nursery at the Bei- CC BY-NC-ND license (https://creativecommons. orientalis werereportedbyDongetal. jing Academy of Forestry and Pomology org/licenses/by-nc-nd/4.0/). (1992). Cao et al. (1997) studied the mi- Sciences, Beijing, China. The tree was HORTSCIENCE VOL. 56(1) JANUARY 2021 85 15 m high and 20 years old, and was guishable from the vegetative buds in ap- male cones were round, with a light brown situated on alkaline soil (pH 7.1–8.2) and pearance and morphology (Fig. 1D and L). apex (Fig. 2L). From mid-November to subjected to a typical subhumid continental Subsequently, the subepidermal cells at the mid-December, the MMCs underwent mei- monsoon climate. The mean annual temper- base of the distal axial plane divide continu- osis. The nucleoli were particularly prom- ature was 11 to 13 °C, and the mean annual ously to produce the microsporangium pri- inent with low condensed chromatin. No rainfall was 626 mm. Rainfall occurred mordium (Fig. 1E), at which point the cell wall formed between the two nuclei, primarily from June to August, with 80% of initiation of the male cones was complete. whereas the tapetum cells were still flat, and the annual precipitation recorded during this When the microsporangium primordium first some had two nucleoli (Fig. 2G and H). In period. There were 180 to 200 frost-free appeared, the male cones were still indistin- mid-January of the next year, tetrahedral days in the year. guishable from the vegetative buds in ap- tetrad shapes were observed by meiosis of Male and female cones at different devel- pearance and morphology (Fig. 1F and M). the MMCs, and the tapetum cells near the opmental stages were collected from bud With the growth of the microsporangium, in sporogenous tissue started to disintegrate, initiation to fertilization, and female cones mid to late July, the male cones expanded after which mononuclear pollen grains up to seed maturity, which was from June gradually and developed from a flat shape to a formed (Fig. 2I). The male cone morphology 2018 to the following Aug. 2019. Depending round