59. Plant regeneration from protoplasts of Indica and Japonica rices

Xiang-hui Li1,2 , Arun ARYAN1, Cheng-kin XIANG1 and Thomas OKITA1

1) Institute of Biological Chemistry, Washington State University, Pullman, Washington 99164-6340, U.S.A.

2) Institute of Genetics, Academia Sinica, Beijing 100012, China

With the recent success of protoplast culture (Lee et al. 1989; Kyozuka et al. 1988; Masuda et al. 1989 and references cited therein) and gene transfer (Zhang et al. 1988; Toriyama et al. 1988) techniques in rice by several laboratories, we directed our efforts to improve the frequency of protoplast regeneration from both subspecies indica and japonica of Oryza sativa L., limiting step in this process, using standard protoplast culture methods. In this paper, we report the regeneration of plants from protoplasts of Japonica type rice cv. J-02428 and Indica type rice cv. DGZ under different media conditions.

Calli were initiated from young inflorescence tissue of Indica type cv. DGZ and japonica type cv. J02408 rice on solid N6 medium containing 2 mg/l 2, 4-D, 3% sucrose and 0.8% agar. After subculture for 5-6 weeks, 1.5 g of calli possessing a slight yellowish color and exhibiting high regenerative capacity were suspended into AA2 liquid medium. The suspension cell line was subcultured on a gyrotory shaker at 100 rpm under dim light, with the medium replaced at 5- to 7-day intervals. The embryogenic cell suspension lines were then maintained on AA2 or grown on a modified N6 media containing the amino acids of AA2, unmodified N6, GM, D2, or R2. After subculturing over a 9-10 month period, the cell lines grown in AA2 and modified N6 media yielded cytoplasmically-densed protoplasts at a density of 4- 10X 106 per ml. The amino acids specified by AA medium were absolutely necessary for the compact growth of highly cytoplasmic- dense cells and produced the highest yield and percentage of viable protoplasts as compared to the other media (GM, N6, R2) tested without the amino acids supplements. The addition of casein hydrolysate (0.025%) and coconut water (55), although not absolutely necessary, resulted in faster growth of the liquid suspension cultures and, hence, was routinely added to the AA2 or modified N6 media.

The protoplasts of J02428 and DGZ released by an enzyme mixture solution were cultured in either KM (Kao, 1977), D2, N6 or R2 media with or without agarose using normal protoplast culture techniques. The plating density of the protoplasts was 1-6 X 105/ml. The osmoticum was varied somewhat by the presence of glucose in a range between 0.4-0.52 M.

Table 1. Plating efficiencies from protoplasts of rice
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Variety      Culture media       Frequency of cell division
                                  (%, at 10-11 days)
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J02428 Japonica  KM               25.0
J02428    "      KM(agarose)      34.0
J02428    "      D2               32.0
J02428    "      R2(agarose)      32.0
DGZ     Indica   KM               23.0
DGZ       "      KM(agarose)      37.0
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The highest plating efficiency (33.0% for J02428, 37.0% for DGZ) of protoplast growth was observed on modified KM medium solidified with 0.2 % agarose. A somewhat lower plating efficiency was observed for both genotypes on KM medium without agarose. This observation differed significantly from an earlier study by Sun et al. (this issue) in which the growth of DGZ protoplasts was strikingly dependent on the presence of agarose in KM. D2 and R2 media were also suitable for the growth of J02428 which yielded plating efficiencies similar to KM medium with agarose (Table 1).

After incubation for three weeks, fresh KM medium containing 18 g/l glucose and 20 g/l sucrose was added to reduce the osmoticum which enhanced the growth rate of the cells. After 3-4 weeks, the calli (about 1 mm in size) were grown in liquid N6 medium containing 1 mg/l of 2, 4-D for 3-4 weeks of growth, followed by a similar growth period on solid N6G medium containing 0.6 mg/l of BAP and 2 mg/l of 2, 4-D. Incubation of the calli, first in liquid N6 media followed by N6G medium enhanced the growth of the calli while maintaining its embryogenic character. Furthermore, the amount of embryogenic calli capable of regeneration obtained after these treatments was substantially increased as no plants were obtained from calli which pre-incubated under these culture conditions. The protocalli were then plated on hormone-free N6SS medium. Within 3-4 weeks after transfer, green spots appeared on many of the calli. In the case of J02428, the frequency of differentiation was 37.2-56.9% (amount of green spots with shoots/number calli X 100) while for Indica type rice it was somewhat lower at 34.8%. In a number of instances many of the small green shoots did not develop into whole plants. The frequency of plant regenerations, a month after transferring onto N6SS, were 21.8-27.8% for J02428 and 15.0-21.7% for DGZ. Calli derived from protoplasts incubated in modified KM media with agarose yielded substantially higher frequencies of differentiation and regeneration than cells incubated in the absence of agarose. Approximately 30 protoplast-derived plants from J02428 have been transferred to the greenhouse. All of the plants had normal morphology, and have flowered and set seed. Likewise, more than 15 protoplast-derived plants from the /indica genotype, DGZ, have been grown to maturity.

Table 2. Differentiation and plant regeneration from protoplasts 
         of rice
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Varieties         Media         %Frequency of  % Frequency of
                              differentiation plant regeneration
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J02428(Japonica)  KM(Agarose)-N6SS  56.9              27.8
J02428(Japonica)  KM  -N6SS         37.2              21.8
DGZ (Indica)      KM(Agarose)-N6SS  34.8              21.7
DGZ (Indica)      KM  -N6SS      not determined       15.0
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The method described here is direct and relatively simple as it does not require the use of feeder cells for protoplast growth and viability.

References

Lee, L., R. E. Schioll, H. D. Gtimes and T. K. Hodges, 1989. Plant regeneration from indica rice protoplasts. Planta 178: 325-333.

Kao, K. N., 1977. Chromosomal behavior in somatic hybrids of soybean-Nicotiana glauca. Mol. Gen. Genet. 150: 225-230.

Kyozuka, J., Z. Otoo and K. Shimamoto, 1988. Plant regeneration from protoplasts of indica rice: genotypic differences in culture response. Theor. Appi. Genet. 76: 887-890.

Masuda, K., A. Kudo-Shiratori and M. Inoue, 1989. Callus formation and plant regeneration from rice protoplasts purified by density gradient centrifugation. Plant Sci. 62: 237-246.

Toriyama, K., Y. Arimoto, H. Uchimiya and K. Hinata, 1988. Transgenic rice plants after direct gene transfer into protoplasts. Bio/Technology 6: 1072-1074.

Zhang, H. M., H. Yang, E. L. Rech, T. J. Golds, A. S. Davis, B. J. Mulligan, E. C. Cocking and M. R. Davey, 1988. Transgenic rice plants produced by electroporation-mediated plasmid uptake into protoplasts. Plant Cell Reports 7: 379-384.