Selection of cell lines with high DAPI stainability from rice (Oryza sativa L.) suspension culture for protoplast isolation and regeneration

Abstract

Suspension culture plays an important role in high yield of protoplast isolation. From our routine maintenance of rice suspension lines, we found growth rates of most suspension lines were not quite stable even after subculture for 3-4 months. A DNA specific binding fluorochrome-DAPI(4',6-diamidino-2-phenylindole) was used to elucidate the nuclear status of cell lines during culture. Suspension lines derived from anther calli were used for this study. The results show that cell lines tend to have differential DAPI stainability during culture. The DAPI stainability is classified into high, medium and low groups based on percentage of suspension cells exhibiting nuclear fluorescence immediately after staining. Variations in regeneration ability and protoplast yield exist among three groups. High DAPI stainability cell lines tend to have higher regeneration ability and higher protoplast yields than the others. The average protoplast yields of high DAPI stainability lines are 2.5 x 10(7) protoplasts/g.fresh wt. cells and 7.8 x 10(6) protoplasts/g.fresh wt. cells for cultivars Hsin-Chu 56 (HC56) and Taipei 309 (TP309) respectively. A maximum yield up to 2-3 x 10(8) protoplasts/g.fresh wt. cells was also noted occassionally. Plating efficiency apparently increased when selection for high DAPI stainability and medium size of nucleus were applied. The non-stained cells were believed to be nonvital or aged cells in a contrast study on FDA test.