Sodium azide is a potent seed mutagen (A. Kleinhofs et al., Mutation Research 55:165, 1978) that has been shown to be mutagenic in maize (R.W. Briggs, personal communication; K.A. Hibberd and C.E. Green, MNL 52:73, 1978). Azide has potential in tissue culture mutagenesis for inducing biochemical mutants because it is a point mutagen and is not known to be highly mutagenic in mammals. Azide mutagenicity in friable, embryogenic (type II) cultures was evaluated using selection for lysine plus threonine resistance described by Hibberd and Green (PNAS 79:559, 1982) as a model selectable marker. Two friable, embryogenic callus lines, designated B and S, were initiated from advanced generations of A188 x B73 crosses. Callus was incubated in citrate buffered azide solution, pH 4.45, for 1 h on a gyratory shaker. After washing, 0.5 g fr. wt. of callus was plated on a 7 cm Whatman #1 filter paper overlying non-selective medium. After 3 d recovery, the filter paper with cells was transferred to selective medium containing 3 mM lysine and threonine (LT). After 10 wk culture on LT medium, surviving colonies were scored as resistant variants (Tables 1 and 2). The LD50 azide for B and S callus lines was 1.1 and 2.5 mM, respectively. Even after adjusting the number of variants selected for azide toxicity, the recovery of LT resistant variants decreased with increasing azide. This may be because the plating procedure preferentially identified pre-existing, spontaneous variants which existed as relatively large cell aggregates at the time of plating. Any azide-induced mutants, existing as 1-8 cell colonies when selection was applied, may not have been selected due to decreased selection efficiency of the small cell colonies. The decrease in selected variants may have reflected the toxic effects of azide reducing the pre-existing population of LT resistant variants in our cultures and reducing the viable cell plating density. We are conducting further experiments to evaluate this explanation.
A more likely hypothesis is that azide was not highly mutagenic in the maize tissue cultures. Azide is known to be metabolically activated in barley embryos into the presumptive mutagenic metabolite, o-azidoalanine. To determine if maize callus synthesized the azide mutagenic metabolite, extracts of azide treated callus were compared with embryo extracts from azide treated B73 x A188 kernels for mutagenic metabolite synthesis using the Ames Mutagenicity Test. Free azide is volatilized in this assay so only levels of mutagenic metabolite were determined. Kernels were pre-soaked 8.5 h and then treated with 1 mM azide. Embryos were dissected and extracted for mutagenic metabolite. A 1 mM azide kernel treatment induced light green, albino, brown-spotted or yellow-gold stripes in 56% of the plants arising from the treated kernels indicating that somatic mutations were induced by azide treatment of embryo tissues. Callus was treated with 1 mM azide as before and sampled over time. Mutagenic metabolite levels 4 h after azide treatment were similar in callus and embryos (Table 3). Mutagenic metabolite levels declined in the callus to slightly above control levels of 20 h after treatment, which was before callus growth had resumed. Embryo metabolite levels remained high up to 40 h after azide treatment, by which time germination had resumed as indicated by the appearance and elongation of the radicle. Friable, embryogenic maize callus appeared to possess an active detoxification pathway, which is hypothesized to degrade the azide mutagenic metabolite before respiratory-arrested cells resumed growth and were mutagenized. The lack of evidence for azide mutagenesis at the Ltr loci in callus and the potent mutagenicity in kernels correlates with the decrease of mutagenic metabolite in callus tissue and higher levels persisting in embryos of treated seed, suggesting that azide mutagenesis is tissue specific in maize.
Using the filter paper selection procedure, 180 LT variant callus lines have been selected. Plants have been regenerated from 20 of the most promising lines and each line will be genetically characterized to determine if the trait can be recovered in the progeny of regenerated plants. Since selections were carried out in two different callus lines, we can be confident that mutants selected will represent at least two different genetic events. Multiple selections within the same line may not represent different mutations. Thus, to maximize selection of different alleles and genes conferring LT resistance in the absence of a mutagenic treatment, we suggest selecting among different callus lines to insure isolation of different spontaneous mutations.
Stanton B. Dotson and David A. Somers
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