Abstract
BackgroundThe origin of somaclonal variation has not been questioned previously, i.e., “pre-existing mutations” in explants and “newly induced mutations” arising from the tissue culture process have not been distinguished. This is primarily because there has been no reliable molecular method for estimating or quantifying variation.Methodology/Principal FindingsWe adopted a petal-variegated cultivar of Saintpaulia ‘Thamires’ (Saintpaulia sp.) as the model plant. Based on the difference between the pre- and post-transposon excision sequence of the promoter region of flavonoid 3′, 5′-hydoroxylase (F3′5′H), we estimated mutated (transposon-excised) cell percentages using a quantitative real-time PCR. Mutated cell percentages in leaf laminae used as explants was 4.6 and 2.4% in highly or low variegation flower plants, respectively, although the occurrences of blue color mutants in their regenerants were more than 40%. Preexisting mutated cell percentages in cultured explants were considerably lower than the mutated plant percentage among total regenerants via tissue culture.Conclusions/SignificanceThe estimation of mutated cell percentages became possible using the quantitative real-time PCR. The origins of mutations were successfully distinguished; it was confirmed that somaclonal variations are mainly caused by newly generated mutations arising from tissue culture process.
Highlights
Tissue culture is a well known and efficient method of plant propagation, but the resulting regenerants often has a number of mutations termed ‘‘somaclonal variations’’ [1]
A primer set for the Chalcone synthaseA (CHS-A) gene was adopted as reference
In the tissue cultures mentioned above, approximately 40% progenies were solid blue mutants, and there was no significant difference between the progenies from the two parents (Table 1)
Summary
Tissue culture is a well known and efficient method of plant propagation, but the resulting regenerants often has a number of mutations termed ‘‘somaclonal variations’’ [1]. In the strict meaning of the term, somaclonal variations are thought to be derived from ‘‘newly induced mutations’’ arising from the tissue culture process as well as from ‘‘pre-existing mutations’’ in explants. Generation of somaclonal variations is attributed to genetic and epigenetic modifications in DNA. Tissue culture is reported to activate silent TEs, resulting in somaclonal variations [3]. The origin of somaclonal variation has not been questioned previously, i.e., ‘‘pre-existing mutations’’ in explants and ‘‘newly induced mutations’’ arising from the tissue culture process have not been distinguished. This is primarily because there has been no reliable molecular method for estimating or quantifying variation
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