Fig. 1. Calli induced from a young panicle. Fig. 2. Freeze-thawed callus. Fig. 3. Seedlings developing from freeze-thawed callus.
Biology Department, Wuhan University, Wuchang, Hubei, 430072 China
Cryo-preservation of cell and tissue cultures of plants is useful not only for maintaining morphogenetic potentiality and genetic stability over a long time, but also provides an ideal way to preserve germplasm resources of rare plants (Sala et al. 1979; Cella et al. 1982). China has rich natural resources of wild rice, especially of Oryza meyeriana Baill. This species has a strong resistance to bacterial blight. We have been engaged in cryo- or freeze-preservation of O. meyeriana calli which were induced from young panicles, and have obtained a large number of plantlets regenerating from freeze-thawed calli.
Young panicles at differentiation stages of secondary branchlets and spikelet primordia to stamen and pistil were cultured in the following media. Callus induction: N`6`-based medium containing 2,4-D 2 mg/L and sucrose 4.5% with 0.6% agar, adjusted at pH 5.8. After 30 days of culture for callus induction, the calli (Fig. 1) were precultured for 12 days and frozen according to the following procedure: O degC-cooling 1 degC/min-10 degC, 15min-cooling 1 degC/min-40 degC,60 min-liquid nitrogen. As cryo-protectant, solution of 10% dimethyl sufloxide and 0.5 M sorbitol was added to the calli before freezing.
Fig. 1. Calli induced from a young panicle.
Fig. 2. Freeze-thawed callus.
Fig. 3. Seedlings developing from freeze-thawed callus.
Regeneration of plantlets: MS (Murashige and Skoog)-based medium containing kinetin 2 mg/L and naphtyl-acetic acid (NAA) 0.5 mg/L plus sucrose 3.0%. After 8 days in liquid nitrogen, cryo-preserved calli were taken out quickly and were thrown into 40 degC water-bath for fast thawing. Through TTC-test (2,3,5-triphenyl-tetrazolium chloride, for cell viability), the survivorship of freeze-thawed cells was evaluated. It reached 80% of cells kept without freezing.
All the media were autoclaved at 120 degC for 20 minutes and were cooled before use.
The frozen thawed calli were incubated at 22 degC in dark to resume growth. A large number of proliferous calli were obtained after one or two subcultures. White and compact globular granules began to appear on the surface, showing the formation of embryonic callus (Fig. 2).
After transferring to regeneration medium, the embryonic callus produced plantlets with shoot and root (Fig. 3). We tested four regeneration media with different hormone contents. The above-mentioned one containing kinetin 2 mg/ L and NAA 0.5 mg/L was most suitable for differentiation of many plantlets. We have obtained 365 plants from 89 calli in total.
References
Cella, R., R. Colombo, M. G. Gaili, E. Nielsen, F. Rollo and F. Sala, 1982. Freeze-preservation of rice cells: A physiological study of freeze-thawed cells. Physiol. Plant. 55: 279-284.
Sala, F., R. Cella and F. Rollo, 1979. Freeze-preservation of rice cells grown in suspension culture. Physiol. Plant. 45: 170-176.