| Cypripedium japonicum is a kind of perennial herb that belongs to Orchidaceae, Cypripedioideae, Cypripedium. It is mainly distributed in East Asia, such as China, Japan and Korea. It is well known for its fan-shaped leaves and rounded leaf type, big showy and exotic flowers. So they are of high cultivated and ornamental value. The rhizomes of C. japonicum can also be used as medicine. C. japonicum seeds aborts highly, with low natural germination rate. The update is very poor, and it mainly relies on division propagation to maintain individual survival. However, people unreasonably dig wild populations, resulting in population decline and habitat destruction. Division propagation is not able to maintain the stability of population any more. Currently, C. japonicum has been listed as an endangered species.Two wild populations of C. japonicum from West Tianmu Mountain National Nature Reserve in Zhejiang province were used as materials. This study aimed to explore the mechanism of C. japonicum seed abortion, by the method of observation on seed embryological development process and determination of physiological and biochemical changes. The findings could be drawn as follows:C. japonicum rhizome had a dormant bud which was yellow-green in the top, with coleoptile coating the lower part. The bud grew above the ground within the first year of October. Dormant buds developed fully and produced milky white flower buds. They would flower within the second year of April. While, dormant buds which could not develop well only produced fan-shaped leaves in the second year, and there were no floral organs. Due to space and nutrients limitations, selective abortion probably occurred in the groups.Ultrastructure of ovules showed that the archesporial cell was of large volume. Nucleus occupied most of the space, with evident nucleolus and dense cytoplasm and organelles. Megasporocyte located at the micropyle end, and nucleolus disappeared gradually. The vacuoles were rich in the cytoplasm. Mitochondria and plastids densely distributed. After the formation of embryo sac, vacuole occupied a large space, the nucleus was relatively small, with sparse cytoplasm and organelles. Mitochondria and plastids were visible. During the ovule developmental stage, protein and polysaccharide existed all the way. The stain of starch gradually deepened, without the presence of lipids. Ovule abortion rate was little in C. japonicum, which was not the direct cause to seed abortion.Pollen grain presented prolate, smooth surface with a small amount of glue materials and without any characteristics. Pollen wall divided into the inner wall with cellulose pectin composition and the outer wall of rod-shaped pillar structure, with covering layer outside. Germ cells were nearly round, of large and dense nucleus; vegetative cells were curved, with nuclei and cytoplasm dispersion. Pollen cells contained a large number of mitochondria, plastids, vesicles and other organelles, and were rich in starch, protein and polysaccharide. Pollen tubes grew along the direction of elongation of the ovary wall after germination. Fertilization basically completed at 50 DAP. Pollen development was normal and did not hinder the reproductive process.Mature seeds of C. japonicum were brown and spindle-shaped, with mesh of fine lines on the surface. Seed embryo was not fully differentiated, and remained in the globular stage. The vacuole accounted for 3/4 cell space, with sparse cytoplasm. Only lipid particles and a large number of starch grains distributed in the cytoplasm. The viability of seeds was as high as 76%, but in vitro germination was difficult. Seed embryo gradually entered into dormancy. Delayed differentiation led directly to the seed abortion.Soluble protein content in C. japonicum seeds was low and peaked as 774.03?g ? g-1 twenty days after pollination(DAP) and minimized as 373?g ? g-1 at 85 DAP. Starch content rose rapidly from the initial 2.65% to 21.82%, and maintained at about 20%. Soluble sugar content changed between 1.72% and 3.09%.The peak of GA3, ZR and IAA occurred at 20 DAP, 30 DAP and 40 DAP, respectively. ABA content reached a peak at the time of 115 DAP. ABA contents were significantly negatively correlated with three acceerative hormones(P<0.05). The ratio of(IAA+GA3+ZR) and ABA peaked at 20 DAP. Then this ratio decreased.SOD activity rose rapidly to a peak as 451.18 U /(g ? min) 20 DAP, and then decreased until 85 DAP. SOD activity and soluble protein trends ketp consistent. Both POD activity and CAT activity showed a single peak curve.In summary, embryo development was a critical period of abortion. At this time, low protein and soluble sugar levels, endogenous hormone imbalance and low enzyme activity may lead to seed abortion. |
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