CHLOROPHYLL AND CAROTENOID ACCUMULATION IN THREE CHLOROPHYLLOUS CALLUS PHENOTYPES OF GLYCINE MAX

Effects of exogenous carbohydrates and various medium supplements on chlorophyll and carotenoid accumulation in three chlorophyllous callus phenotypes of Glycine max (L.) Merrill were studied. Glucose (filtered), at 3%, supported the highest level of chlorophyll and carotenoids in the NG and Y phenotypes, while only moderate levels of chlorophyll accumulated in the LG phenotype. Sucrose (filtered and autoclaved), at 3%, supported phenotypical levels of chlorophyll and carotenoids for all phenotypes. Ascorbic acid, at 75 mg/l, stimulated chlorophyll-carotenoid accumulation for all phenotypes, while 8-aminolevulinic acid was slightly toxic for pigment biosynthesis in the NG and LG phenotypes. In contrast, 8-aminolevulinic acid supported chlorophyll-carotenoid accumulation in the Y phenotype. A negative correlation (rxy) was evident between chlorophyll formation and callus growth for all phenotypes. Light intensity of 3,000 lux suppressed chlorophyll accumulation in the Y callus phenotype while an increase in pigment formation occurred in the NG and LG callus phenotypes. In comparison, light intensity of 700 lux supported chlorophyll accumulation in the Y callus phenotype. Carotenoid accumulation appeared to be coupled with chlorophyll formation in all callus phenotypes except for the Y callus phenotype when grown under the higher light intensity. All phenotypes accumulated chlorophylls a and b, aand ,8-carotene, lutein plus zeaxanthin, violaxanthin and neoxanthin as indicated by their respective absorption maxima. Under cultural conditions, the genetic mutation (yll) in these soybean callus phenotypes appeared to regulate carotenoid formation which in turn influenced chlorophyll stability. PIGMENT biosynthesis has been studied in several nuclear and chloroplast mutants to determine more fully the compartmental controls of chloroplast structure and function. Chlorophyll deficient nuclear mutants (xantha) of barley, when grown under etiolated conditions, have been shown to possess a blockage in the chlorophyll pathway, while other barley mutants (tigrina) have been found to accumulate higher levels of protochlorophyllide than the normal plant (Wettstein et al., 1974). These etiolated mutants contained different characteristic levels of carotenes, while many accumulated a molar equivalent of total carotenoids as compared to the wild type (Nielsen and Gough, 1974). Because these seedlings were shown to be photosensitive to a prolonged period of high light intensity, it was suggested that the corresponding genes influenced chlorophyll formation and possibly carotene biosynthesis in developing plastids (Nielsen, 1974). 1 Received for publication 24 March 1978; revision accepted 31 July 1978. The authors thank Drs. Jerry M. Modisette, James H. Ray, and James E. Mann for their valuable assistance during this study and Prof. Paul G. Mahlberg for his critical review of this report. 2 This work, in part, was submitted as partial fulfillment of a Ph.D. Degree to the Department of Biology, University of Houston, Houston, Texas. I Present address: Department of Biology, Indiana University, Bloomington, Indiana 47401. Some mutants severely deficient in colored carotenoids have been shown to be photobleached when placed under high light intensities. Troxler et al. (1969) described an albescent maize mutant that possessed a normal protochlorophyllide synthesizing mechanism; however, only phytoene and phytofluene accumulated under etiolated conditions. When subjected to low light conditions, these mutant seedlings contained similar levels of chlorophyll and carotenoids as found in the wild type. However, the lack of colored carotenoids in this mutant resulted in photobleaching of seedlings grown under high light intensity. Weber and Weis (1959) described a gene (yll) that conditioned the accumulation of chlorophyll in soybean and segregates in a Mendelian ratio of 1 (normal green):2 (light green): 1 (yellow, lethal). Both the light green and yellow phenotypes contained reduced levels of carotenoids while the lethal phenotype accumulated only trace amounts of :3-carotene (Keck et al., 1970). Recently this homozygous recessive mutant has been shown to be photosensitive to high light intensities (Noble, 1975). In the present study we investigated the metabolic blockage of the yll trait in these soybean phenotypes when grown as callus. Effects of carbohydrates, ascorbic acid, 8-aminolevulinic acid and light intensity on growth and on the formation of chlorophyll and carotenoids in these cultured phenotypes were investigated.