Embryological Studies On Bupleurum Chinense DC.

Bupleurum chinense DC. which belongs to Bupleurum L. in Umbelliferae is a traditional chinese herb for its dry root. It has many functions for medical purposes. It is mainly distributed in province of HeBei,ShaanXi,GanSu and so on. There have had many reports about Bupleurum L. on biology, phytochemistry, pharmacology and cultivation in recent years. The seeds of Bupleurum chinenses are difficult to germination, and the rate of seedlings grown from them are very low in the course of cultivation. While, there are few reports on the biological characters of the seeds, especially, there are no reports about embryology of Bupleurum chinense. The aim of present study was to systematically study the megasporogenesis, microsporogenesis and development of male and female gametophytes, the development of embryo and endosperm in Bupleurum chinense DC. with method of paraffin section. And we also studied its floral biology and the afterripening of seeds in order to provide scientific basis and new materials on cultivation and biological charaters of Bupleurum chinense. The results are as follows:1. Megasporogenesis and development of female gametophytesThe ovary of Bupleurum chinense DC. is anatropous, single integument and thin nucellus. Megasporogenesis is started from an archesporial cell under the nucellus epidermis. The archesporial cell is different from other nucellus cells with large volume and dense cytoplasm and a big nuclear. The archesportial cell dose not divide and develops into megaspore mother cell directly. The megaspore mother cell undergoes meiosis to form a linear tetrad. Normally, the chalazal megaspore functions and forms the female gametophyte, but sometimes the second chalazal megaspore also functions. During 8-nuclei embryo sac stage, some nucellus cells on the base and two sides keep their own cytoplasms and shapes longer and then become into podium. The inner integument cells grow into endothelium. There are two synergids and one egg at the micropylar end of embryo sac. The egg has a large vacuole at the chalazal end and a nuclear at the micropylar end. The synergids are kept during two polar nucleus are fusing together. The filiform apparatus of synergids is apparent. Two polar nucleuses are located at the center part of the embryo sac and they have different orientations, sometimes they are at transverse direction, sometimes at longitudinal direction, and sometimes at oblique direction. There are three Antipodal cells at the early stage of megasporogenesis. They become pyramid and intrude into nucellus with the development of embryo sac.2. Microsporogenesis and development of male gametophytesThere are four microsporangiums in Bupleurum chinense DC. The structure of anther primodium is very simple, its outmost layer is epidermis and the inner layers are a group of active meristem cells. Then the antherogenous occurres because the outside cells at four comers divide quickly. A group of archesporial cells are formed interior to epidermis at each comer. The archesporial cells divide periclinally to form primary sporogenous cells and primary parietal cells. Primary parietal cells divide periclinally into four layer cells of the anther wall, which continue to growing and become into epidermis, endothecium(1 layer), middle layer(1 layer, disappeared earlier) and tapetum(1 layer). During the meiotic prophaseâ… of microspore mother cells, the nucleus of tapetum divides into two. The cells of tapetum are glandular and secrete many fat-sphere bodies into pollen sac. The primary sporogenous cells develop into microsporocyte by mitosis. The microsporocyte undergoes simultaneous meiotic division and produces tetrahedral tetrad. The tetrad is tetrahedral arrangement and surrounded by callose. Then the tetrad is separated by callose decomposition and each of them is a microspore which develops into the mature pollen who is 3-cell type. At this time, the anther wall just is consisted of epidermis and endothecium. With the spliting of the wall between neighbor anther sacs, the mature pollens are spilled out.3. The development of embryo and endospermThe division of zygote is latter than that of endosperm nucleus. There have been a few of endosperm nucleus at the stage of the zygote dividing. The apical cell and basal cell are formed by the transverse mitosis of zygote. Then they formed five-celled proembryo. The two cells from basal cell are not divided again, and the second cell from apical cell is divided into two cells by the transverse mitosis, which are showing different stages of mitosis. The proembryo then becomes 7-celled after the mitosis of the two cells. The suspensor is not well developed and dissolved early. Then the multi-celled proembryo is formed. The development of embryo belongs to the solanad type. The development of the endosperm is of nuclear type. The two polar cells assemble at the center of embryo sac and then fuse into the center cell. After the fertilization, the primary endosperm nuclei keep away from the micropylar end and start the first mitosis. The division of primary endosperm nucleus is prior to that of zygote. Most of endosperm nuclei at early stage are circular or ellipsoid or irregular. They form a layer and surrounded the embryo sac. The early mitosis of endosperm nucleus is synchronous, and there are a lot of endosperm nuclei in the center of embryo sac at the later stage of mitosis of endosperm. Above all, it is observed that endosperm nuclei in the micropylar end have larger volume and more nucleolus than the others. And they are surrounded by dense cytoplasm. The embryo sac enlarges quickly along with the increase of endosperm nuclei. Endosperm cells are formed from outside of embryo sac to the center. Finally, the embryo sac is full of endosperm cells. Then the volume of endosperm cells becomes larger and cytoplasm becomes denser, showing the characters of mature cells. It is showed that the ergastic material of endosperm cells is mainly protein and some fat by the method of histochemistry. At the stage of proembryo, the wall of ovary includes many layers: exterior epidermis cells, many layers of parenchyma cells, 1-2 layers of rectangular parenchyma cells. Single integument also includes many layer cells and'degenerates gradually from the center of embryo sac to the outside along with the development of embryo. When the fruits mature, the wall of ovary develops into peel, the exterior epidermis cells develop to exterior peel, many layers of parenchyma cells develop into middle peel in which secretory canals and vascular bundles are distributed. And 1-2 layer rectangular parenchyma cells develop into inner peel, whose wall becomes lignified.4. The statistics of the embryo development stage after seeds were collectedAccording to the statistics of the embryo development stage after seeds were collected, it is found that in all the seeds observed, embryo of 20% of the seeds belong to globular proembryo stage, 70% is heart-shaped embryo, 10% is torpedo embryo stage. The results showed that the developmental stare of the embryo in the collected seeds of Bupleurum chinense is different and the seeds have morphological post-maturation. This is the main reason why the seeds of Bupleurum chinense are difficult to germinate and the ratio of germination is very low.5. Floral biology of Bupleurum chinense DC.The floral biology of Bupleurum chinense DC. was studied. The results of elementary observation and statistics showed: the annual plant can blossom and bear the same year, the quantity of blossom between annual and biennial is similar. But the time of bud bursting is different, the biennial is earlier than the annual and the blooming dates of both are 3 months. The inflorescence of Bupleurum chinense DC. is compound umbel, which is terminal and axillary. The terminal inflorescences usually blossom early. When the terminal inflorescences are at blooming date, the axillary inflorescences start to blossom. There are many kinds of flower visitors and they are large, which is effective for pollination. The fruits located on different branches are at different developmental stages. The terminal inflorescences on the stem and branches of the first and second level develop earlier and their seeds are better than that of the terminal inflorescences on the branches of the third and forth level.