Figure 1.
Saintpaulia ‘Thamires’ and the genetic background of a flower color change.
(A) An original cultivar of ‘Thamires’. Bar = 3 cm. (B) Magnification of a variegated petal. Bar = 1,000 µm. (C) Schematic representation of the F3′5′H sequence in variegated ‘Thamires’ plants (not to scale). When the transposon is present (GenBank: AB596833), F3′5′H expression is disturbed, and cells become pink. On the other hand, when the transposon is excised (AB596834), F3′5′H expression resumes, and cells become blue. Arrows represent primers. In real-time PCR, only the post-excised sequence (157 bp) could be detected by shortening the elongation time.
Table 1.
Flower phenotypes of the leaf laminae culture-derived regenerants of Saintpaulia ‘Thamires’.
Figure 2.
Comparison of amplification efficiencies and examination of the comparative Ct method within petal samples.
(A) Standard curves obtained by a dilution series of petal DNA from solid blue mutants. Closed circles indicate the results for a post-transposon excision sequence (“target”), and open circles indicate the results for CHS-A (“reference”). (B) Difference between target Ct and reference Ct within petal samples. Slopes (0.055) smaller than 0.1 were valid (ABI User Bulletin #2). Identical procedures were used to validate the method in other plant parts (data not shown).
Figure 3.
Comparison of estimates and measured values of mutated cell percentages.
Data from 9 plants with different petal variegations are shown. For real-time PCR results, the average value for solid blue mutants was considered to be 100%, and values for other phenotypes are shown in comparison with those for solid blue mutants. Black circles represent the results for petals, and green circles represent the results for leaf laminae. Data for petals assessed using two different methods showed strong correlation (coefficient of correlation; 0.95), and these results validated the approach. In all cases, mutation rates in the leaf laminae were lower than those in the petals. Mutation rates in the leaf laminae of V1, V2, V3 and V4 were 0.34, 0.82, 0.46 and 0.94 respectively. Leaf laminae of V5 and V9 were used for in vitro shoot regeneration.
Figure 4.
Mutated cell percentages in different layers of the petiole in the two variegated types.
Mutated cell percentages were calculated by real-time PCR analysis. The average value for solid blue mutants (whole petiole was examined) was considered to be 100%, and values for other phenotypes are shown in comparison with those for solid blue mutants. V6, V7 and V8 plants were used for highly variegated plants and V1, V2, V3 and V4 plants were used for low variegated plants. Data presented as mean ± standard deviation. No statistical differences were not observed between epidermal and inner layers in both variegated types.
Figure 5.
Mutated cell percentages in different parts of the two variegated types.
Variegated areas of petals were determined by image analysis in highly and low variegated plants. Real-time PCR analysis was used to determine mutated cell percentages. The average value for solid blue mutants (whole petiole was examined) was considered to be 100%, and values for other phenotypes are shown in comparison with those to solid blue mutants are shown. V6, V7 and V8 plants were used for highly variegated plants, and V1, V2, V3 and V4 plants were used for low variegated plants. Data presented as mean ± standard deviation. Different letters indicate statistical significance (P<0.05) as determined by ANOVA followed by Ryan's test.
Figure 6.
Illustration of 2 conflicting models of the distribution of mutated cells.
This figure represents the longitudinal section of a leaf lamina, and the intensity of dots represents the frequency of mutation, or transposon excision. In model A, the epidermis contains a large amount of mutated cells, and the inner layers contain few mutations. In model B, the mutation rate is not high, but mutated cells are homogenously distributed. We observed that model B was more suitable for explaining the results shown in Figure 4.
Figure 7.
Tissue culture results (left) and origins of solid blue mutations (right).
Percentages were calculated based on the results of medium and highly variegated plants. Solid blue was the most frequent form of somaclonal variations of ‘Thamires.’ Most of these variations were derived from newly induced mutations arising from the tissue culture process. The numbers in this figures are calculated by the result of Table 1.aTotal number of somaclonal variations should be include solid blue mutants, other phenotypes, and altered-variegation mutants. bOthers include periclinal chimeras and solid pink mutants.