The feasibility of somatic cell genetics implies the expression of genetic markers at the cellular level. In our laboratory we have begun to analyze the in vitro expression of genes involved in anthocyanin biosynthesis (Gavazzi and Racchi, Maydica 26:175-184, 1981). Here I report preliminary observations on the expression of a homozygous A C R pr stock in tissue culture.
Callus was induced from ten-day-old endosperms and maintained under continuous light (3000 lux) at 26 C. The medium used was R. M. (Linsmaier and Skoog, 1965) supplemented with thiamine (0.1 mg/l), sucrose (30 g/l) and 2,4-D (2 mg/l).
Explants turned brownish during the first month of culture and maintained the color through successive passages. After six subcultures, small pieces of callus were transferred to a liquid medium (MS supplemented with yeast extract (2 g/l) and 2,4-D (1 mg/l)) on a rotary shaker (120 rpm) and grown under continuous light (12000 lux) at 25 C. Pigment concentration of cultures on liquid and solid media was then compared to that of the aleurone layer.
The determinations (expressed as A 530 nm/mg fresh weight) accomplished on extracts in methanolic 1% HCl are the following:
Growth on solid rather than liquid medium appears the more suited to pigment development.
Thin layer chromatography of aglycones obtained from callus using formic and Forestal as developing solvents showed, in addition to pelargonidin and cyanidin, two yellow pigments not present in aglycones obtained from aleurone. Characterization of these compounds is in progress.
Milvia Luisa Racchi
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