Yong-Jiong JIA, Toshinori ABE and Yuzo FUTSUHARA
Faculty of Agriculture, Nagoya University, Chikusa-ku, Nagoya, 464 Japan
Pollen culture is useful for producing haploid plants (Chen et al. 1980). It also offers an efficient experimental system for mutation induction and genetic manipulaion. We attempted to obtain haploid plants by isolated pollen culture.
Three Japonica varieties, Sasanishiki, Nipponbare and Reimei, were used. Panicles were sampled at the mid-uninucleate microspore stage, and the anthers were kept at 10°C for 10 days. They were then cultured on the N6 medium supplemented with NAA (2-4 mg/1), kinetin (0.3 mg/1), casein hydrolysate (1 g/1), and 5% sucrose at pH 5.7, in a 6.0 X 1.5 cm petri dish. Pollen grains were released from the anthers by pressing them gently, and the remaining somatic tissues were removed using a nylon mesh with a pore size of 40μm. The pollen suspension thus obtained was centrifuged at 80 G. The pellets were washed with culture medium, precipitated again by centrifuging and finally resuspended in a medium supplemented with NAA or 2,4-D (2 mg/1), kinetin (0.3 mg/1), casein hydrolysate (1 g/1), and sucrose (3 or 5%). Pollen density was adjusted at 5 X 104 per ml. The pollen suspension (2-3 ml) was poured directy into a petri dish or on a 1 mm depth of agarose N6 culture medium. These were incubated under a low light intensity (ca. 500 lux).
Pollen grains precultured for 10 days in the anther were divisible into two types, one with rich cytoplasm and the other poor (Fig. 1). The former type grew larger and performed nuclear division (Fig. 2). In three weeks after the nuclear division, a cell mass consisting of 8-16 cells appeared out of the exine (Fig. 3). One month after this, the cell cluster developed to form a cell colony which was visible (Fig. 4). Many calli with diameters ranging from 0.5 cm to 2 cm were formed after two months (Fig. 5).
These calli were transferred to 100 ml flasks containing an N6 regeneration medium supplemented with kinetin (1 mg/1), casein hydrolysate (1 g/1) and 3% sucrose. Several plantlets (Sasanishiki 7, Reimei 3, and Nipponbare 1) were obtained from the calli in petri dishes (Fig. 6). The reimi calli subcultured for about one year on the AA medium (Muller and Grafe 1978) showed competence for regeneration on the medium containing kinetin (1 mg/1). The regeneration process would be due to somatic embryogenesis. Chromosome numbers in the root tips of green regenerated plants were 12, indicating that they were haploids (Fig. 7). This method of pollen culture is effective in obtaining haploid plants.
Fig. 1. Isolated pollen grains released after preculture of the anther for 7 days. One of the two contains rich cytoplasm, and the other is poor.
Fig. 2. An enlarged pollen grain containing two or more nuclei within the exine, cultured for 10 days.
Fig. 3. An androgenic mass of 8-16 cells extruding out of the broken exine, after 10 days in culture.
Fig. 4. An androgenic colony after one month in culture.
Fig. 5. Calli formed in a petri dish after two months in culture.
Fig. 6. A plantlet emerging from the pollen callus, two months after transfer to the regeneration medium.
Fig. 7. Giemsa-stained chromosomes in the root tip of a regenerated plantlet.
Chen, Y., R. Wang, W. Tian, O. Zuo, S. Zeng, D. Lu and S. Zheng, 1980. Studies on pollen culture in vitro and induction of plantlets in Oryza sativa subsp. keng. Acta Genetica Sinica 7: 46-54. (Chinese/English)
Muller, A. J.and R. Grafe, 1978. Isolation and characterization of cell lines of Nicotiana tabacum lacking nitrate reductase. Mol. Gen. Genet. 161: 67-76.