Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto, Japan.
PLoS One. 2011;6(8):e23541. doi: 10.1371/journal.pone.0023541. Epub 2011 Aug 11.
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.
METHODOLOGY/PRINCIPAL FINDINGS: We 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/SIGNIFICANCE: The 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.
体细胞变异的起源以前没有被质疑过，即外植体中的“预先存在的突变”和组织培养过程中产生的“新诱导的突变”没有被区分开来。这主要是因为以前没有可靠的分子方法来估计或量化变异。
方法/主要发现：我们采用花瓣斑驳的圣帕特里夏‘Thamires’（圣帕特里夏属）作为模式植物。基于黄酮类化合物 3'，5'-羟化酶（F3'5'H）启动子区域的转座子切除前后序列的差异，我们使用定量实时 PCR 估计突变（转座子切除）细胞的百分比。在高度或低度斑驳花植株的叶片薄片作为外植体中，突变细胞的百分比分别为 4.6%和 2.4%，尽管它们再生体中的蓝色突变体的发生率超过 40%。在组织培养过程中，培养外植体中的预先存在的突变细胞百分比明显低于总再生体中的突变植物百分比。
结论/意义：使用定量实时 PCR 可以估计突变细胞的百分比。突变的起源成功地区分；证实体细胞变异主要是由组织培养过程中产生的新的基因突变引起的。
