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Gemmae Formation and Characteristics of the Endangered Plant Cystopteris chinensis
Xiaohong CHEN, Zejuan GUO, Xia ZHAO, Wenrui MENG
Bulletin of Botanical Research    2024, 44 (5): 655-661.   DOI: 10.7525/j.issn.1673-5102.2024.05.002
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Viviparous reproduction is a beneficial supplement to spore reproduction of pteridophytes, which enriches the reproduction and regeneration system and affects the population genetic diversity and environmental adaptability. In this study, Cystopteris chinensis, a national key protected wild plant, was selected as materials, and morphological and anatomical methods were used, the morphological and anatomical characteristics of the gemmae with the unknown process behind the pinna were examined, and the reproductive structure and development process were revealed, and the viviparous origin and ecological adaptation mechanism of the gemmae were traced. The results showed that: (1)The gemmae and sporangium groups appeared in the middle of the last veins on the abaxial leaf pinna, but the gemmae were located in the apical pinnate lobes. The gemmae formation included three stages: initiation, expansion and maturation, the gemmae gradually grew from yellow-green globule to dark green spindle-shaped, and matured when the base turned black to form a separate layer. (2)The gemmae originated from the parenchyma cells in the extension area of the vascular bundle sheath, which continuously divided and grew to form the gemmae primordium, and gradually broke through the lower epidermis of the leaf to form spherical gemmae. With the gemmae expansion, the middle and lower cells differentiated into storage cells and vascular bundles, and the apex differentiated into growth points to form buds. (3)Few gemmae could directly sprout on the leaves to form new autotrophs. After falling to the soil for 1 week, the mature gemmae began to germinate, which grew cylindrical roots at the base, and the fist bud germinated at the top. The residual gemmae still germinated into new leaves in the second year, which were different from those produced by stem buds.

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Megasporogensis, Microsporogenesis and Development of Male and Female Gametophytes of Endangered Berchemiella wilsonii
Kangxin WANG, Shiqin WANG, Yimin WANG, Zhijie CHENG, Faju CHEN, Yubing WANG
Bulletin of Botanical Research    2024, 44 (5): 662-669.   DOI: 10.7525/j.issn.1673-5102.2024.05.003
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To explore whether there were any endangered links in sporogenesis and development of male and female gametophyte of endangered Berchemiella wilsonii, the reproductive biology of B. wilsonii was observed using paraffin section technique. The results showed that the flower of B. wilsonii had five stamens, four anthers, and the mature anther wall was composed of one inner layer, two middle layers and one tapetal layer. The tapetum developed normally with regularly arranged cells closely connected with the middle layer cells containing multiple nuclei in each cell. The cytoplasmic division of microspore mother cells was synchronous during meiosis, and mainly formed tetrahedral tetrads, the mature pollen grains were bicellular, round or oval in shape, which most of them had three germinating tubes. There were a few abnormal tapetum development and a few abortive pollen during anther development. B. wilsonii had two locule in ovary, only one chamber developed. The ovule was anatropous double integuments and crassinucellate. The chalazal end megaspore developed into functional megaspores,which underwent three times consecutive mitosis and further developed into polygonum embryo sac with seven cells and eight nucleus. Ovary ovule development was basically synchronized. The process of the mega-microsporogenesis and development of female and male gametophyte of B. wilsonii were normal, which was not the reason for the low seed setting rate.

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Anatomical Structure and Cell Wall Components Changes in Chestnut ‘Yanshan Hongli’ Female Flowers at Developmental Stages
Qingjiyan WANG, Jin LI, Tian LI, Yu XING, Ling QIN, Kefeng FANG
Bulletin of Botanical Research    2024, 44 (5): 670-680.   DOI: 10.7525/j.issn.1673-5102.2024.05.004
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To investigate the anatomical characteristics of female flowers and the changes of cell wall components of Castanea mollissima during the growth and development, ‘Yanshan Hongli’ was used as materials, and the internal morphological changes were observed by paraffin sections and the changes of cell wall components were observed by fluorescence staining. According to its internal microstructure changes, the collected samples were determined at the four most important development stages: flower primordium differentiation stage, stigma primordium differentiation stage, stigma elongation stage and flowering stage. The results showed that the fluorescence intensity of cellulose in the cell wall of female flowers gradually increased during the development. Especially during the process of stigma elongation, the fluorescence intensity of cellulose in stigma and ovary was significantly enhanced. In addition, the fluorescence intensity of esterified pectin was always higher than that of acidic pectin during the four development stages and the fluorescence intensity of pectin in stigma and ovary was stronger than that of other parts. These results revealed the changes of cell wall components during the growth and development of chestnut female flowers, and provided important clues for further understanding the mechanism of chestnut growth and development.

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Adaptability of Floral Characteristics to a Fly Pollinator in Bulbophyllum andersonii (Orchidaceae)
Jinrui HE, Xiong ZHANG, Yinling LUO, Yan LUO, Qingqing LI
Bulletin of Botanical Research    2024, 44 (5): 681-691.   DOI: 10.7525/j.issn.1673-5102.2024.05.005
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To reveal the reproductive characteristics and pollination mechanisms of Bulbophyllum andersonii, the pollination biology including flowering phenology, breeding system, pollinator, flower morphological characteristics, and flower volatile components were investigated in a wild population in Xishuangbanna, Yunnan in 2023. The results were as follows: the blooming period of B. andersonii was in mid-July, and the individual flowering period was five days. The breeding system was outcrossing and dependent on pollinators, and the natural seed setting rate was 26.06%. Bagging experiments indicated self-incompatibility, no apomixis and no spontaneous autogamy. Floral features such as the hinge structure of the labellum, pollination channels, and broad lateral sepals played important roles in pollination. Gampsocera sp.(Chloropidae) was the only effective pollinator and exhibited lip-licking behavior during the flower visit. Using Scanning Electron Microscopy, a large number of special particles were observed on the lips of B. andersonii, which might be the food rewards for the flower visitors. Flower volatile odors primarily contained monoterpenoids and aromatics, among which 1,2-Dimethoxybenzene and N, N-diethyl-m-toluamide might be related to the flower visiting behavior of insects. The results indicated that the flower morphology and flower scent of B. andersonii were adaptable to pollination by Gampsocera sp., which seemed to be apt to a specialized pollination system.

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Somatic Embryo Induction and Histocytological Observation of Isodon rubescens
Wange WU, Baoyu JI, Lixin PEI, Conglong LIAN, Chengming DONG, Suiqing CHEN
Bulletin of Botanical Research    2024, 44 (5): 692-701.   DOI: 10.7525/j.issn.1673-5102.2024.05.006
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Establishing the somatic embryogenesis system of Isodon rubescens to give full play to its value in traditional Chinese medicine, and promote the stability and utilization of its resources. The leaves of I. rubescens were used as explants to dedifferentiate into embryonic callus and further proliferate to induce somatic embryo. The histological and cytological observation of embryonic callus and somatic embryo at each stage was made by paraffin section, and the effect of hormones on plant regeneration was tested. The results showed that in B5+1.0 mg·L-1 2,4-D medium, leaves could dedifferentiate into light yellow and granular embryonic callus. In MS+1.0 mg·L-1 2,4-D+0.2 mg·L-1 6-BA medium, the embryogenic callus had the best proliferation effect, and the maximum net proliferation was 2.81 g. The optimal medium for somatic embryo induction was MS+1.5 mg·L-1 TDZ+0.2 mg·L-1 NAA, and the highest induction rate was 91.33%. In MS+0.5 mg·L-1 6-BA+0.5 mg·L-1 IBA medium, embryogenic callus containing somatic embryos could differentiate into small plants. In histological observation, the embryonic callus was yellowish or yellowish white granules with large and obvious nuclei. Two kinds of somatic embryogenesis were observed in the inner and outer origin of the callus, and the later developed into spherical, heart-shaped, torpedo-shaped, cotyledon-shaped embryo or cluster embryo structures containing starch granules. This study successfully established the indirect somatic embryogenesis system of I. rubescens, which laid a foundation for optimizing the “quality” and “quantity” of embryogenic callus, and also provided the theoretical basis for the research of molecular breeding and somatic embryo technology.

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Meristematic Nodule Induction and Adventitious Shoot Differentiation in Distant Hybrid Tree Peony
Yu XIE, Wanting LIU, Jun CUI, Fangyun CHENG, Yuan ZHONG
Bulletin of Botanical Research    2024, 44 (5): 702-710.   DOI: 10.7525/j.issn.1673-5102.2024.05.007
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To explore a simple and suitable method for inducing meristematic nodules in multiple cultivars, the petioles of inter-subsectional and inter-sectional distant hybrid tree peony were used as explants, and the conditions for the differentiation of meristematic nodules into adventitious shoot in some cultivars were also identified. The results showed that meristematic nodules were induced by one step in 10 cultivars with 52.08%- 83.33% induction rates, the thin layers of petioles of 11 cultivars were continuously cultured on SH medium containing 0.5 mg·L-1 NAA+1.0 mg·L-1 CPPU+0.5(or 0) mg·L-1 TDZ for 60-100 d in the dark, and meristematic nodules induced by one step were generated indirectly from the callus. Adventitious shoot differentiation rate of three cultivars was 6.67%-33.33% when meristem nodules were added to the modified WPM medium containing 0.5(or 1) mg·L-1 CPPU+0.5 mg·L-1 TDZ under light condition and sub-cultured every 15 days. Lower concentrations of CPPU and TDZ together promoted adventitious shoot differentiation, but high concentrations of CPPU alone were not conductive to adventitious shoot differentiation. The general medium for one-step induction of meristematic nodules was screened, and adventitious shoot differentiation was achieved in some cultivars, which provided an important reference for the establishment of plant regeneration system for distant hybrid tree peonies.

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