Institute of Genetics, Academia Sinica, Beijing 100012, China
The genetic manipulation of rice via cell culture has been a limited approach because of the inherent difficulty of initiating and maintaining actively growing suspension cultures capable of protoplasting and subsequent regeneration into plants. Although such culture conditions have been developed for Japonica genotypes of rice (Masuda et al. 1989 and references cited therein), there have been only two reports (Kyozuka et al. 1988; Kee et al. 1989) describing the successful regeneration of Indica type protoplasts. Both studies required the use of a nurse culture for maintenance of protoplast's viability and growth. In this report, we describe the culture conditions necessary for the regeneration of Indica protoplasts without the use of a nurse culture.
Calli were induced from the young infloresence tissue of Indica rice DGZ on N6 media containing 2 mg/l of 2, 4-D, 3% sucrose and 1% agar. After 5-6 weeks of culture, the calli obtained was yellowish in color and exhibited high totipotency when plated on regeneration media. The calli, 1.0-1.5 g, was then infused with 30 ml of AA liquid media containing 5 mg/l of thiamine-HCl and 0.5 mg/l of pyridoxine-HCl, the elevated vitamin levels enhancing cell growth. After 2 months of culture, the cell suspension culture consisted of small clumps, averaging about 100 small spherical shaped cells with dense cytoplasm and thin cell walls. Cell lines enriched in these traits routinely yielded more than 11X 107 protoplasts/g FW possessing uniform cytoplasmic density and size. The protoplasts were cultured at cell densities between 1 to 2.5 X 105 protoplasts/ml in KM8p media solidified with 0.8% agarose (Table 1). The protoplasts embedded in agarose beds were incubated in 6 cm plates containing about 1.0 - 1.5 ml of KM8p media at 27 deg.C in darkness.
Table 1. Protoplast culture medium KM8p,
supplements to basal medium
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Supplement Concentration
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Casein hydrolysate 250 mg/l
Coconut water 20 ml/l
Calf serum 8 Ml/l
Agarose 0. 8%
Glucose 0.35 M
2, 4-D 1.0 mg/l
NAA 0.5 mg/l
Zeatin 0.5 mg/l
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(cf. Kao 1977)
Plating efficiencies were determined 8 days after protoplasting,
although in most instances the first cell division occurred at 3-5 days. We
have observed that the absence of ammonium ions was critical for the survival
and growth of these protoplasts. In the presence of normal levels of ammonium
ions, the plating efficiency was less than 1% (Table 2). Reduction of this
cation from the media enhanced the frequency of protoplast division and the
highest level was observed in the absence of ammonium ion. Reduction in
ferric ion to 1/2 the level specified in the MS medium also enhanced
protoplast growth and division. After two weeks of incubation, the osmotic
pressure of the incubation media was reduced by the addition of 1 ml of KM8p
media containing 0.25 M glucose. The reduction in osmolarity as well as
the addition of calf serum significantly increased cell growth and colony
formation. The use of agarose was also critical for protoplast survival. When
the protoplasts are cultured in liquid media alone, many of the protoplasts
aggregated together and the plating efficiency fell to about 1-3%.
Table 2. Effect of ammonium ions on the plating
efficiency of protoplasts
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NH/4+ (Mg/1) Dividing Frequency (%)
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200 0.5+/-0.2
100 2+/-1.4
67 6+/-1.5
0 38+/-2.5
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After 35 days of incubation, the colonies reached a size of about 1 mm
in diameter. The colonies were then transferred onto N6 medium plates
containing 2 mg/l of 2, 4-D, 3% sucrose and 300 mg/l of glutamine. The
calli could be maintained on this media for several months without any
significant loss of totipotency. After 3 weeks of incubation, the calli
were then transferred onto LMB differentiation medium which contained the
macro-salts of N6, micro-nutrients of MS, vitamins of B5, 2 mg/l of kinetin,
0.5 mg/l of BAP, and 3% sucrose. Green spots were observed at 7-10 days
with shoots appearing after 2 weeks. Whole green plants were obtained after
6-7 weeks and grown to maturity, yielding normal seed set.References
Lee, L., R. Z. Schroll, H. D. Grimes and T. K. Hodges, 1989. Plant regeneration for indica rice (Oryza sativa L.) 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: Genotype differences in culture response. Theor. Appl. 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.