Anthocyanin accumulation and phenylalanine ammonia lyase (PAL) activity in callus cultures

In continuation of our earlier studies (MGNL, 1984) with anthocyanin pigment synthesis in tissue cultures of homozygous Pr and pr genotypes, the present study was undertaken to see the effect of different hormones on anthocyanin synthesis both in callus cultures and in endosperm grown in vitro, and phenylalanine ammonia Iyase (PAL) activity in callus and suspension cultures.

Fresh 10 day old immature endosperms were collected from field grown plants and inoculated onto medium. Callus cultures of seedling root and node were also established from one week old germinated kernels. All the cultures were maintained on MS medium containing different concentrations (0.5-4 mg/l) and combinations of various hormones such as IAA, NAA, 2,4-D and BAP and kinetin. Anthocyanins from the tissues were extracted in methanolic HCl (0.1%), and the content is expressed in OD units at A530. Results are presented in the Table. In the case of endosperms 1 mg/l IAA + 1 mg/l KN containing MS media enhanced the pigment production about five times (0.661) over the control (0.126), whereas callus cultures from seedling root accumulated more pigment (0.074) on 2 mg/l 2,4-D MS medium. Of the different auxins and cytokinins tested, for fresh endosperms low levels of IAA (0.5 mg/l) and 1.0 mg/l KN resulted in accumulation of more pigment (i.e., 1.585 and 2.584, respectively) than in callus cultures of the same. Earlier studies in Daucus carota (Alfermann and Rienhard, Experentia, 1971) have indicated that the auxin may participate in the induction of necessary enzymes, and in turn in the synthesis of anthocyanins; however, the involvement of phytochrome was not ruled out.

PAL activity was studied in callus and suspension cultures, and also at the root forming stage. During callus formation, activity was greater (4.04 units) than during cell suspension (0.07) and the root forming stage (0.4) of calli. There was no appreciable synthesis of anthocyanins in the callus stage where the PAL activity was high. During pigment synthesis, calli cultured on MS + 2 mg/l 2,4-D exhibited 0.47 units of enzyme activity. Further studies on PAL activity in different mutants of anthocyanin may reveal the role of PAL in biosynthesis of anthocyanin.


P. Suprasanna, K.V. Rao and G.M. Reddy

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