Variability among plants and their progeny regenerated from protoplasts of Su/su heterozygotes of Nicotiana tabacum

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Abstract
Mesophyll protoplasts of Nicotiana tabacum, heterozygous for the sulfur locus (Su/su), were isolated and more than 2,200 calli were cultured. More than 8,000 regenerated shoots were analyzed for leaf colour. Cell culture regimes included media for normal and stressed growth conditions with both short and long culture periods. An additional treatment included N-methyl-N′-nitro-N-nitrosoguanidine mutagenesis. An analysis of the regenerated shoots showed that an extended culture period led to an enhanced frequency of variant colony types, i.e. colonies producing both parental (Su/su) and non-parental (Su/Su or su/su) plants. NNG at 10 mg/l also enhanced the frequency of variant colony types. In some treatments there was also an increase in non-morphogenic colonies but this was independent of genetic changes at the sulfur locus. The frequency of dark green spots and twin spots, presumed to result from somatic crossing-over, was higher in the leaf cells of regenerated plants after both prolonged cell culture and chemical mutagenesis. Genetic analysis of the progeny of selfed regenerants revealed additional tissue culture induced variability with respect to segregation ratios of the different sulfur phenotypes. About two thirds of the lines tested segregated in accordance with a 1∶2∶1 Mendelian ratio. The remainder deviated from the expected segregation pattern and some lines also showed heterogeneity between progeny families derived from different seed capsules of the same plant. These results demonstrate that genetic changes affecting a specific locus and segregation patterns in progeny of regenerated plants are induced during cell culture.