An efficient plating system for rapid isolation of mutants from plant cell suspensions

Abstract

A plating system for cell suspensions of soybean, SB-1, (Glycine max L. cv. 'Mandarin') and Datura innoxia D.I. (Mill) was developed using feeder cells. The characteristics of the system are: a) the efficiency of plating (EOP) is high (0.5-0.6), b) over a range of 10-300 plated clumps the EOP is constant, c) the growth rate of plated cells resembles that of suspension cultures (generation time 24 hr.). Clumps with few or with many cells have similar plating efficiencies.Employing the plating system, a mutant resistant to 8 azaguanine (8AG) was isolated from SB-1 in 7 days and purified and tested within an additional 3 weeks. Feeder plates were used to selectively re-isolate 8 AG resistant and maltose utilizing mutants from a 1000-fold excess of wild type cells.The plating technique also can be utilized to isolate auxotrophic mutants since free amino acids are not produced by the feeder suspension. Other applications of this plating technique are discussed.