Protoplast culture of rice (Oryza sativa L.) using media solidified with agarose
J. A. Thompson, R. Abdullah* and E. C. Cocking Plant Genetic Manipulation Group, Department of Botany, University of Nottingham, University Park, Nottingham NG7 2RD, U.K. Received 17 February 1986; revised 7 July 1986; accepted 25 August 1986. ; Available online 28 January 2003.
Abstract
A procedure is described for the reproducible establishment of rice cell suspension cultures from callus of embryo origin. Protoplasts were readily isolated from cell suspension of four rice cultivars, including japonica and indica types, when maintained in an amino acid-based culture medium. Sustained protoplast division from two japonica genotypes has been obtained in agarose solidified culture medium. An increase in the agarose concentration from 0.6% to 1.2% (w/v) produced a marked improvement in protoplast survival, division and plating efficiency. Protoplast division and plating efficiency frequencies of up to 26% and 0.5%, respectively, were obtained at the higher agarose level. The protoplast-derived calli were similar in appearance to explant-derived morphogenic callus and produced distinct embryo-like structures.
Kinya Toriyama and Kokichi Hinata Laboratory of Plant Breeding, Faculty of Agriculture, Tohoku University, Tohoku 980, Japan Received 3 June 1985; revised 22 July 1985; accepted 31 July 1985. ; Available online 29 January 2003.
Abstract
A finely dispersed cell suspension was obtained from anther-derived rice callus cultured in AA medium, which contained an amino acid mixture as the sole nitrogen source. AA medium promoted the release of protoplasts effectively.
These protoplasts, when cultured initially in B5 medium for 8–10 days followed by transfer to AA medium, showed sustained cell division and gave rise to callus with a high frequency. The critical factor in the protoplast culture seemed to be the nitrogen source in the culture medium. Green-spot and root formation could be obtained from the protoplast-derived callus.
Plant Physiol, November 1999, Vol. 121, pp. 805-812
The original callus cultures were derived from rice (Oryza sativa L. cv Sasanishiki) embryos in 1996. The stock suspensioncultures were maintained by transferring approximately 1 g freshweight of cells to 125 mL of fresh R-2 medium containing 3% (w/v)Suc and 4.5 µM 2,4-D (Ohira et al., 1973) in a 500-mL flask at7-d intervals. The suspensioncultures were shaken on a rotaryshaker at 120 rpm at 25°C. When the cells were treated with variousreagents, 7-d-old cells were further cultured with nitrogen-freeR-2 medium for 2.5 d and used as the inoculum (Watanabe et al.,1996). To reduce variations among individual cultures, the nitrogen-starvedcells were first pooled in a batch with mixing and inoculatedinto 20 mL of individual nitrogen-free R-2 medium in a 100-mLflask. Afterward, the cells were treated with various reagentsfor the time required (as described below and in the figure legends).MES-NaOH buffer (50 mM, pH 5.8) was supplied to media to reducethe pH change during the subsequent treatments with various reagents.Suspension-cultured cells were harvested by vacuum filtrationthrough Miracloth (Calbiochem-Novabiochem, San Diego) and weighed.The collected cells were quick-frozen in liquid nitrogen and storedat 80°C.
YATAZAWA, M., K. FURTHASHI, N. KuJRIHARA, AND Y. OHNISm. 1967. Growth of callus tissue from rice-root in vitro. Plant Cell Physiol. 8: 363-373.
Plant Physiol. (1988) 88, 26-29
Cultured Rice Cells. The cell line Oc derived from roots of
Oryza sativa seedlings (2), kindly provided by Dr. Sh6no, was maintained in liquid MS medium2 (21) supplemented with 1 mg/L 2,4-D and 3% sucrose. Five mL of the suspension culture
were transferred into 50 mL of the fresh MS medium in a 200-
mL Erlenmeyer flask every week. The suspension cells were
cultured on a gyratory shaker (80 rpm) at 25°C in the dark.
21. MURASHIGE T, F SKOOG 1962 A revised medium for rapid growth and bioassays
with tobacco tissue cultures. Physiol Plant 15: 473-497
Plant Physiol. (1991) 95, 1294-1297
Rice (Oryza sativa) cell suspension cultures Calrose 76 and
the lysine-rich mutant, 4C, were maintained in Murashige
and Skoog's medium (16) supplemented with 1 mg/L 2,4-D
and 2% sucrose as a carbon source. The cells were grown on
a gyratory shaker (New Brunswick Scientific,2 Edison, NJ) at
107 ± 2 rpm under continuous light (15 Umolm-2.s-') at 27
± 1°C.
16. Murashige T, Skoog F. (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15: 473-497