Cytological Studies On The Somatic Embryogenesis Of Gossypium Hirsutum L.

Because of the high transformation efficiency and genetic stability of foreign gene, Agrobacterium-mediated transformation method is considered as the most common technique which has been applied to cotton genetic transformation. However, it requires an efficient plant regeneration procedure. The high genotype-dependence of embryogenic potential, the difficulty embryogenic callus induction, and the high ratio of abnormal somatic embryos restrict the application of biotechnology on cotton development and fundamental research. In view of this, we studied histological structure and ultrastructure of different type callus during the course of Agrobacterium-mediated cotton genetic transformation systematically via such techniques as scanning electron microscope (SEM), transmission electron microscope (TEM) and so on. In this study, enhanced foreign green fluorescent protein (EGFP) was served as the reporter gene to identify the transformant.1) Expression of EGFP was observed in callus, embryogenic callus, non-embryogenic callus and somatic embryo. As EGFP is a stable, cell-autonomous fluorescent protein, positive transformant could be identified by fluorescence microscope non-destructively easily.2) On surface of embryogenic callus, series of nodule-like structures developed, which was formed by small, tightly packed and hemispherical cells. Results of the histological observation revealed that early embryogenic callus displayed similar cellular morphology and embryogenic callus could develop embryonic nodule-like structure through further sub-culturation. Organelles of embryogenic callus cells were located near the nucleus and contained dense cytoplasm. Plastid was degraded into proplastid-like structures with some starch grains. Rough surfaced endoplasmic reticulum (RER) and a series of small vacuoles could also be observed.In contrast, non-embryogenic callus was characterized by high variant of cell morphology and loose cell organization on the surface. The nucleus was located in a narrow strip of cytoplasm between cell wall and the large vacuole which is located in the center. Chloroplasts of hard callus developed well organized membrane system and chloroplasts in cell of overgrown callus were degraded into proplastids which exhibited some vacuole-like structures.3)In the cells of hypocotyls, chloroplasts had abnormal structure. In the cell of callus, plastids were degraded into amyloplast. In the cell of embryogenic callus, chloroplasts were degraded into plastids. When somatic embryo formed, endomembrane system of chloroplast emerged. Once somatic embryo developed into cotyledonary embryos, endomembrane system of chloroplast developed grana lamella and starch grains accumulated in chloroplast stroma. Abnormal development of chloroplast (plastid) was related with the occurrence of non-embryogenic callus closely and there were structural abnormalities of chloroplasts in cells of albino and vitrified somatic embryo.4)As while embryogenic callus were induced, fine fibrils of extracellular matrix(ECM) could be observed. The structural arrangement of the ECM changed during culture. During the formation of a series of multi-cellular embryonic clusters, the ECM was the most abundant and was arranged as network-like layer connected by coarse strands outside of callus. While nodule-like structures formed, ECM was disturbed gradually, and while embryo formed,the ECM network disappeared completely. The ECM was not observed on the surfaces of callus and non-embryogenic callus.