• Journal of Plant Biology
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• v.33 no.4
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• pp.253-257
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• 1990

Callus-inducing ability of Alnus hirsuta was examined by culturing various tissues (leaf, hypocotyl, cotyledon and seed) on NT (Nagata & Takebe) medium, supplemented with 2.5$\mu$M 2,4-D. Leaf-originated callus was cultured on media varying in auxin (IBA and NAA) and cytokinin (BAP) concentrations to examine the effects of auxin and cytokinin on callus growth. Maximum growth was obtained at 10 $\mu$M IBA+10$\mu$M BAP and 10$\mu$M NAA without cytokinin. Cell suspensions established from cotyledon-originated callus yielded viable protoplasts after incubation for 16-18 hours in an enzyme mixture (1% (w/v) Onozuka R-10 0.5% (w/v) Macerozyme, CPW salts and 13% (w/v) mannitol, pH 5.8). Protoplasts were cultured on NT medium, supplemented with glucose, hormones and coconut milk. After 6 weeks of culture, protoplasts sustained cell divisions to form microcallus, which showed various colors from red to white.

• Journal of Plant Biology
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• v.37 no.2
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• pp.237-244
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• 1994

Callus tissues derived from protoplast of Alnus hirsuta were shown to produce red pigments under the continuous light. The R4 cell line with high yield of the pigments was selected. One of the red pigments was identified as anthocyanin; glucose and galactose as sugar moiety and cyandin as aglycone, by spectroscopic analysis, cellulose TLC and GLC. Both callus growth and anthocyanin accumulation were remarkably stimulated by 2,4-D. The highest anthocyanin yield was observed when $1\;\mu\textrm{M}$ 2,4-D in combination with $0.1\;\mu\textrm{M}$ BAP was supplemented to the culture medium. In case of inorganic salts, anthocyanin yield was enhenced by reducing concentration of phosphate from 5.0 mM to 2.5 mM and by increasing the ratio of ${NO_3}^{-}$ type to 80% for total nitrogen source.source.