PhCHS5 and PhF3'5'H Genes Over-Expression in Petunia (Petunia hybrida) and Phalaenopsis (Phalaenopsis aphrodite) Regulate Flower Color and Branch Number

Abstract

Flower breeders are continually refining their methods for producing high-quality flowers. Phalaenopsis species are considered the most important commercially grown orchids. Advances in genetic engineering technology have provided researchers with new tools that can be used along with traditional breeding methods to enhance floral traits and quality. However, the application of molecular techniques for the breeding of new Phalaenopsis species has been relatively rare. In this study, we constructed recombinant plasmids carrying flower color-related genes, Phalaenopsis Chalcone synthase (PhCHS5) and/or Flavonoid 3',5'-hydroxylase (PhF3'5'H). These genes were transformed into both Petunia and Phalaenopsis plants using a gene gun or an Agrobacterium tumefaciens-based method. Compared with WT, 35S::PhCHS5 and 35S::PhF3'5'H both had deeper color and higher anthocyanin content in Petunia plants. Additionally, a phenotypic comparison with wild-type controls indicated the PhCHS5 or PhF3'5'H-transgenic Phalaenopsis produced more branches, petals, and labial petals. Moreover, PhCHS5 or PhF3'5'H-transgenic Phalaenopsis both showed deepened lip color, compared with the control. However, the intensity of the coloration of the Phalaenopsis lips decreased when protocorms were co-transformed with both PhCHS5 and PhF3'5'H. The results of this study confirm that PhCHS5 and PhF3'5'H affect flower color in Phalaenopsis and may be relevant for the breeding of new orchid varieties with desirable flowering traits.

Keywords: Flavonoid 3′,5′-hydroxylase; Petunia; Phalaenopsis; anthocyanin; chalcone synthase.

Figure 1

Construction of recombinant plasmids carrying the PhCHS5 and PhF3′5′H sequences for the transformation of Petunia. (a) Confirmation of the construction of the pCAMBIA2301-sense-CHS5 plasmid by PCR. Regarding the plasmid with PhCHS5 in the forward direction, lane 1 presents the fragment amplified with the Cav35s-primerF and CHS-XR’ primers. Lane 2 presents the approximately 750-bp non-specific band amplified by PCR with the Cav35s-primerF and CHS-XF primers. (b) Confirmation of the transformation of A. tumefaciens cells with PhF3′5′H by PCR. Lanes 1 to 8 represent transformed A. tumefaciens colonies that were analyzed by PCR. (c) Petunia leaf explants during the transformation procedure. (d) Mature transgenic plant. (e) Transgenic Petunia tissue culture. (f) Rooting of transgenic Petunia plants. (g) Preliminary identification of PhCHS5-transformed Petunia plants by PCR. Each lane represents a different Petunia strain. The red arrow indicates the PhCHS5 stripe; WT for wild type; +stand for vector with NPTII gene (h) Preliminary identification of PhF3′5′H-transformed Petunia plants by PCR. Each lane represents a different Petunia strain. The black arrow indicates the PhF3′5′H stripe; WT for wild type; Ev stand for vector with only NPTII gene without PhF3′5′H.

Figure 2

Phenotypic analysis and anthocyanin content determination of Petunia mutants PhCHS5 and PhF3′5′H. (a) Anthocyanin content of Petunia hybrida transformed by PhCHS5. (b) The phenotype of Petunia transformed by PhF3′5′H. (c) Anthocyanin content of Petunia hybrida transformed by PhF3′5′H. WT: Wild Type. The data were expressed as mean ± standard error and repeated three times for each sample. * p < 0.05.

Figure 3

Induction of the Phalaenopsis protocorm. (a) Swollen axillary bud. (b) Seedling generated directly from an axillary bud. (c) Protocorm development. (d) Protocorm enlargement. (e) Seedling cluster. (f–i) Protocorms from YD4 (f,g), YD2 (h) and YD1 (i), respectively.

Figure 4

Screening of Phalaenopsis protocorms carrying PhCHS5 and PhF3′5′H after transformations. (a) PhCHS5-transgenic Phalaenopsis protocorms. The image on the right is a close-focus image of the image on the left (b) Confirmation of the transformation of Phalaenopsis protocorms with PhCHS5 by PCR for Kan gene.M stands for marker, 1–10 stands for transformation seedlings. (c) PhF3′5′H-transgenic Phalaenopsis protocorms. Figure 1 is a close-focus image, and Figure 2, Figure 3 and Figure 4 are three representative repeating telefocal images (d) Molecular confirmation of the presence of PhF3′5′H in the transformed samples by PCR for Kan gene. M stands for marker, 1–5 stands for transformation seedlings.

Figure 5

Phenotypes of the transgenic Phalaenopsis samples. (a,b) Branch number of the transgenic plants. (c) Flower phenotypes of the transgenic plants. WT: Wild Type. The data were expressed as mean ± standard error and repeated three times for each sample. **** p < 0.0001.