Preparation of Protoplast and Establishment of Transient Expression System in Gerbera hybrida

doi:10.11983/CBB16155

Abstract

Abstract: Gerbera hybrida has been used as model plant to study the evolution and development of composite inflorescences. To establish an efficient protoplast isolation and transient transformation system in G. hybrida, leaves of cultivar ‘Shenzhen No. 5’ in vitro seedlings were used as materials and the effects of different concentrations of cellulase R10 and macerozyme were tested to screen a suitable preparation method. The best enzyme solution concentration consisted of cellulase R10 2.0% (w/v), macerozyme 0.3% (w/v), and 0.4 mol·L^-1 mannitol and more than 2.2×10⁷∙g^-1FW protoplasts with high activity were obtained after 4 h treatment under 25°C condition. Subcellular localization and protein-protein interaction assays were performed using these protoplasts and revealed 80% transformation rate. Collectively, this study established an efficient protoplast isolation and transient assay system that can provide an important platform to facilitate gene function studies in G. hybrida.

Aihua Song, Wenbin Zhang, Shulan Sun, Lingfei Li, Xiaojing Wang. Preparation of Protoplast and Establishment of Transient Expression System in Gerbera hybrida[J]. Chinese Bulletin of Botany, 2017, 52(4): 511-519.

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URL: https://www.chinbullbotany.com/EN/10.11983/CBB16155

https://www.chinbullbotany.com/EN/Y2017/V52/I4/511

Figures/Tables 4

Figure 1 Isolation efficiency of protoplast in Gerbera hybrid^(A) Leaves isolated from two-month-old in vitro seedlings (Bar=1 cm); (B) Protoplast yields under different cellulase R10 concentrations with 0.3% macerozyme; (C) Protoplast yields under different macerozyme concentrations with 2.0% cellulase R10; (D) Morphological characteristics of protoplasts, isolated from 2.0% cellulase R10 +0.3% macerozyme, observed by optical microscope (Bar=200 μm)

Figure 2 Transformation efficiency of protoplast in Gerbera hybrid^Transformation efficiency of protoplast observed by laser confocal scanning microscope. From left to right are GFP fluorescence, chloroplast autofluorescence, and a merge of above. Arrows indicate the protoplasts with unsuccessful transformation. Bar=100 μm

Figure 3 Subcellular localization of GFP and YFP in protoplasts from Gerbera hybrid^(A) Subcellular localization of GFP by laser confocal scanning microscope, from left to right are GFP fluorescence, chloroplast autofluorescence, bright, and a merge of above; (B) Subcellular localization of YFP by laser confocal scanning microscope, from left to right are YFP fluorescence, chloroplast autofluorescence, bright, and a merge of above. Bar=10 μm

Figure 4 Analysis of interaction of IBH1 and HBI1 by bimolecular fluorescence complementation (BiFC)^IBH1-nYFP and HBI1-cYFP represent IBH1 and HBI1 connected to the N-terminal fragment and the C-terminal fragment of YFP, respectively. IBH1-nYFP+cYFP and HBI1-cYFP+nYFP were used as negative control. Bar=10 μm

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