Growth and pigment analysis of tissue cultures derived from a genetic strain of soybean Glycine max (L.) Merrill

Growth studies and pigment analysis were made on tissue cultures derived from a genetic strain of soybean Glycine max (L.) Merrill. Optimal concentrations of growth hormones (NAA, 1 mg/1 and kinetin, 1 mg/1), ascorbic acid (75 mg/1), ([delta]-aminolevulinic acid (4 mg/1) and sucrose (30 gm/1) as supplements to a modification of Miller's salts (127) were determined on the basis of the following parameters: fresh and dry weight and chlorophyll formation. Other factors such as coconut milk, yeast extract, casein hydrolysate, a synthetic amino acid mixture and various carbohydrate sources were investigated in response to these parameters. It was evident that this callus strain required an exogenous supply of cytokinin, and glucose favored chlorophyll formation whereas in most cases fructose and sucrose supported both growth and chlorophyll formation. Also the data indicated that the physiological requirements for chlorophyll formation was inversely correlated with the requirements for rapid cell growth. Under the chemical environmental conditions employed, the callus variants demonstrated a degree of cytodifferentiation and genetic heterogeneity as observed by phase contrast microscopy. The pigmentation of these cultured phenotypes which were maintained under chlorophyllous and dark-grown conditions varied In response to the physical as well as chemical environmental conditions which were employed. Under continuous illumination the heterozygous variant in most cases demonstrated a greater potential for chlorophyll formation than the normal green and yellow phenotype. However, the dark-grown phenotypes demonstrated the characteristic ratio for chlorophyll synthesis as found in the parent material. The lethal mutant was more susceptible to photo-bleaching than the wild-type and light green phenotypes. This was evident by the increase in chlorophyll content under low light intensity. The greening ability of these variants demonstrated a two-phase accumulation period: one occurred at approximately day 3 while the second phase at day 12 corresponded with the deceleration phase of growth and the greening phase. The decline in chlorophyll b formation before the second accumulation phase was discussed in regards to plastid biogenesis of dark-grown callus cultures. The implication of these findings are discussed in respect to callus growth and chloroplast biochemistry of these variants and other cultured genera.