| Vol. 6 |
Arun P. ARYAN and Thomas W. OKITA
Institute of Biological Chemistry, Washington State University, Pullman, Washington 99164- 6340, U.S.A.
Transformation of rice by direct DNA uptake of a selectable marker by protoplast has been achieved (Zhang and Wu 1988), although it remains to be a reproducible and routine procedure. One of the major problems in this protoplast transformation system is the loss of regenerability of cells after a short period. In order to establish a good long lasting totipotent cell line, a number of explant tissues such as young embryos, mature embryos, young inflorescence, root tips, and anthers of different rice lines were screened. Our initial results showed that among these various explants, the callus derived from anthers at the mononucleate stage was most embryogenic. A comparison of three different Japonica lines (i.e., T-309, Nipponbare and 77-170) on optimized anther culture medium (basic MS salts + 5.0 mg/l thiamine HCl + 2 mg/l glycine + 2 mg/l 2, 4-5 + 1 mg/l NAA and 90 g/l sucrose, pH 5.6) showed that after a cold treatment (six days at 5°C) the anthers incubated at 25°C produced compact calli in 3-4 weeks. Among these three lines, the percentage of callus-forming anthers were Nipponbare (ca. 5%), T-309 (ca. 20%) and 77-170 (ca. 90%). All of these calli were highly totipotent with line 70-170, giving the best results. Callus derived from ten anthers of this line were placed on regeneration medium which gave 124 plants (122 normal green and 2 albinos) of which 58 were transferred to the green house. All of the plants were fertile and set normal seeds.
The anther-derived callus of this line has been tested regularly for its regenerability and this trait has been well maintained for the last six months. Thus, line 77-170 appears to be a good source of embryogenic calli with minimum problems of genetic abberations which results in the albino phenotype. This callus is currently being used for initiating suspension cultures for protoplasting and DNA transformation studies or by direct DNA introduction by the particle gun.
Reference
Zhang, W. and R. Wu, 1988. Efficient regeneration of transgenic plants from rice protoplasts and correctly regulated expression of the foreign gene in plants. Theor. Appl. Genet. 76: 835-840.
| Vol. 6 |