| Microspore development during in vitro anther
culture of Wheat T. Shimada Research Institute of Agricultural Resources, Ishikawa Agricultural College, Nonoichi-machi, Ishikawa 921, Japan Haploid plants of wheat have been obtained by anther culture since 1973 (Ouyang et al 1973, Picard and Debuyser 1973). We have cultured the anthers of some Japanese wheat cultivars and found that the frequencies of anthers producing pollen embryoids varied with genotypes (Shimada and Otani 1988) and the yield of green plantlets from pollen embryoids were very low (Shimada and Otani 1989). In this experiment, the development of microspores during in vitro anther culture was observed. Materials and Methods Spikes of Triticum aestivum cv. Norin 61 were collected from greenhouse-grown plants and sterilized in 70% ethanol for 1 - 2 minutes. Anthers containing microspores in the late-uninucleate stage were inoculated on Potato-2 (Chung et al 1978) medium and cultured at 28C in the dark. To observe the division occurring in vitro in the microspores, anthers were taken randomly from the cultures after inoculation. The anthers were placed on a glass slide in a few drops of acetocarmine and tapped softly through the coverglass. Results and Discussion Almost all microspores cultured at the stage of late-uninucleate started nuclear division within the first few days (Fig. 1a, 2). The first mitosis produced either identical nuclei by equal division of the microspore nucleus or a vegetative like and a generative like nuclei which were recognized by the different intensities of acetocarmine staining (Fig. 1b). Fig. 2 shows the development of microspores which indicates the relationship between culture period and the number of nuclei. After five to seven days of culture the microspores with four to five nuclei whose cell membrane was obscure were observed (Fig. 1c). Although the surviving microspores decreased drastically after a week, some of them with multinuclei developed into multicellular microspores (Fig. 1d). At 14 days, microspores with over 20 cells were observed, where the cell division occurred inside the exine of microspores (Fig. 1e). Some multicellular microspores broke the exine and grew into proembryoids after three weeks in culture (Fig. 1f) About 30% of the anthers had more than 30 viable multicellular microspores (Fig. 1e). However, the frequencies of anthers with embryoids in Norin 61 was 10.7% and average number of embryoids in one anther was around two (Shimada and Otani 1988). Only a few percents of multicellular microspores could develop into embryoids. There are two steps in the development of microspores which are from the multinuclear to the multicellular stage, and from the multicellular stage to proembryoids. At both steps the frequencies of surviving microspores decreased drastically. More than 100 multicellular microspores which would be potential embryoids were observed in some anthers. The induction frequency of pollen plants could be increased to satisfy the requirements for breeding by further improvement of the satisfactory conditions in anther culture. References Chuang CC, Ouyang JW, Chia A, Chou SM and Ching CK (1978) A set of potato media for wheat anther culture. In: Proc Symp Plant Tissue Culture, Science Press, Peking pp. 51-56 Ouyang JW, Hu H, Chung CC and Tseng CC (1973) Induction of pollen plants from anthers of Triticum aestivum L. cultured in vitro, Sci Sinica 16: 79-95 Picard E and Debuyser J (1973) Obtention de plntules haploides de Triticum aestivum L. a patir de cultures d'antheres in vitro. CR Acad Ser D 281 127-130 Pan JL, Gao GH and Dan H (1983) Initial types of pollen cells and their development in anther culture. In "Cell and tissue culture techniques for cereal improvement" Proc. Workshop cosponcered by the Institute of Genetics, Academia Sinica and the International Rice Research Institute, Science Press, Beijing pp. 117-129 Shimada T and Otani M (1988) Efficiency of potato medium on induction of pollen embryoids in anther culture of Japanese wheat cultivars. Japan J Breed 38: 212-222 Shimada T and Otani M (1989) Varietal differences in green plant regeneration ability of wheat pollen embryoids. Japan J Breed 39: 187-194 |