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Fig. 3 | Plant Methods

Fig. 3

From: Creating a novel petal regeneration system for function identification of colour gene of grape hyacinth

Fig. 3

Overexpression of the GUS gene in transgenic petals of grape hyacinth. The GUS gene was ligated into the vector pFGC5941 under the control of the CaMV 35S promoter. The Basta gene was used as a selection marker gene (a). PCR amplification of a 1216 bp DNA fragment of the GUS gene from the transgenic and non-transgenic lines (b). Total RNA was extracted from young petals prior to full expansion. Then, the mRNA accumulation of the GUS gene was assessed by semi-quantitative and quantitative real-time PCR. Actin was used as a reference gene (c, d). GUS protein activity in the transgenic and non-transgenic petals (e). Each bar represents means ± standard deviation from three dependent replicates. The symbols ‘**’ and ‘***’ above bars indicate statistically significant differences at P ≤ 0.01 and P ≤ 0.001, respectively, by the student’ s t-test. Control: non-transgenic petals, GUS1-3: transgenic petals overexpressing the GUS gene. Production of transgenic grape hyacinth petals via Agrobacterium mediated transformation and flower organogenesis system (f). After pre-culture and co-cultivation with A. tumefaciens carrying CaMV35S::GUS, the flower explants turned green (I). The majority of them showed transient GUS expression (VI). Then, the BIArcell clusters developed around the flower stalk on the selection medium (II). Histochemical staining indicated that these cell clusters showed GUS activity (VII). BIAr cell cluster gave rise to violet-blue petals (III, IV, V). The non-transgenic and BIAr petals, prior to full expansion, were excised and used for later analysis (IV). The GUS histochemical assay showed that blue staining was detected in resistant petals (IX, X), whereas no blue spots were observed in the non-co-cultivated controls (VIII). Scale bar in d: 100 μm

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