Ectopic expression of SsCYC in Nicotiana benthamiana and optimizing regeneration system of Sinningia speciosa
Date Issued
2014
Date
2014
Author(s)
Kuo, Wen-Hsi
Abstract
The native varieties of Sinningia speciosa (Gesneriaceae) bear zygomorphic flowers, but in horticultural varieties, large size showy actinomorphic flowers are selected due to human’s preference. CYCLOIDEA has been demonstrated to have a major genetic control in zygomorphy in Antirrhinum. In actinomorphic varieties, we found a small fragment deletion in its CYCLOIDEA homologue (∆SsCYC), which might indicate that the reversal to actinomorphy is a SsCYC loss of function mutant. I introduced CYC homologues from both zygomorphic cultivar (SsCYC) and actinomorphic cultivar (ΔSsCYC) into Nicotiana benthamiana, a closely related species to Sinningia speciosa, to verify whether ΔSsCYC has any effect on floral phenotype. I found that ectopic expression of SsCYC causes shorter longitudinal length of flowers, smaller floral opening diameters, induction of axillary shoots, curled leaves and dwarfism, agreed with CYC’s putative effects on cell proliferation or expansion. However, no visible phenotypic change could be observed in ΔSsCYC overexpression lines. I also optimized the genetic transformation system in Sinningia speciosa, focusing on tissue regeneration and Agrobacterium infection conditions. The MS medium supplied with 0.1 ppm naphthalene-acetic acid (NAA) and 1 ppm 6-benzylaminopurine (BA) was the best for shoot regeneration in both leaf and petiole explants. Eighty six percent and 56% regeneration rates were obtained from 6 mm diameter leaf explants and 5 mm petiole explants, respectively. Moreover, the orientation of petiole explants must be up-side down or horizontal to induce the shoot regeneration, which might relate to the endogenous basipetal distribution of auxin inside the petiole vascular tissue. It was found that the regenerative shoots of explants initiated from a single vascular parenchyma cell deep inside the regenerated tissue. This is different from other reported cases in Gesneriaceae species, in which their regenerative shoots usually originated from an epidermis cell or a glandular trichome basal cell. To explore whether Agrobacterium infection can enter regenerative tissue, transient transformation using GUS reporters was applied. In contrast to mature leaf or petiole explants, I found that young seedlings, especially those in cotyledonary stage or with the first pair of primary leaves have much higher success of transformation. This opens an opportunity that young seedlings are potential material for transformation. By paraffin sections, the positive signals of transformation were seen in epidermis, mesophyll and glandular trichome head cells but not inside the regenerative shoots. Alternatively, the induced callus tissue might be a better transformation material. This study provides a preliminary study on the functions of SsCYC genes and guidelines for further optimization of transformation system of Sinningia speciosa.
Subjects
Sinningia speciosa
symmetry
tissue culture
genetic transformation
Agrobacterium
Type
thesis
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