Cytological Characterization of Nucleus Development in Microspore Embryogenesis and Improvement in Healthy Embryo Production using Polyethylene Glycol in Brassica napus

摘要

This study characterized microspore embryogenesis at each stage of cell development in oilseed Brassica napus (cvs. Zheshuang 758 and Zheshuang 72). Isolated microspores of B. napus were cultured with various concentrations of polyethylene glycol (PEG 6000) as an osmoticum and sucrose (0.1%) as carbohydrate source in liquid NLN culture medium. The most embryos were produced from NLN culture medium with 6.25% PEG 6000. Approximately 9% of the microspores swelled after one day heat treatment at 30°C in darkness. After 2 days culture, some swollen microspores began to divide into 2-cell and 3-cell proembryos with “T” shaped and gradually into multi-cellular proembryos, which further developed into globular, hearted, and torpedo embryos. Two kinds of division were observed under electron microscope: symmetrical division and asymmetrical division. It showed that 3 weeks culture was suitable for microspore embryogenesis. The optimal concentration of 6-benzyl-aminopurineand naphthalene acetic acid for shoot regeneration was 2.5 mg·L-1 and 0.4 mg·L-1, respectively. The highest doubling frequency was observed in Zheshuang 758 (78%) and Zheshuang 72 (73%), when 200 mg·L-1 colchicine was employed in culture medium. The ploidy level of microspore-derived plants could be checked by flowcytometer or staining the lower epidermis of leaves with fluorescein diacetate by observing fluorescence of guard cell chloroplasts with a microscope under ultraviolet light. From these findings, it can be concluded that the swelling of microspore cell and symmetric division are key factors to start microspore embryogenesis and healthy embryos could be produced using PEG 6000 in microspore culture.