Protoplast isolation and transient transformation system for Ginkgo biloba L

Abstract

Ginkgo biloba L. has a unique evolutionary status. Owing to its high medicinal and ornamental value, ginkgo has also recently become a research hotspot. However, the large genome and long juvenile period, as well as the lack of an effective genetic transformation system, have hindered gaining a full understanding of the comprehensive functions of ginkgo genes. At present, heterologous expression of genes in model plants is the primary method used in ginkgo-related research; however, these distant plant model relatives limit reliable interpretation of the results for direct applications in ginkgo breeding. To overcome these limitations, in this study, an efficient isolation and transient expression system for ginkgo protoplasts was established. A large number of intact and homogeneous ginkgo mesophyll protoplasts were isolated using 2% cellulase and 0.25% pectinase in 0.4 M mannitol. The activity of these protoplasts remained above 90% even after 24 h. Furthermore, when the concentration of the polyethylene glycol 4000 solution was 30%-40% (w/v), the transformation efficiency of the protoplasts reached 40%. Finally, the reliability of the system was verified using subcellular localization, transient overexpression, and protein interaction experiments with ginkgo genes, thereby providing a technical platform for the identification and analysis of ginkgo gene functions. The proposed method partially compensates for the limitations associated with the lack of a genetic transformation system and provides technical support to expand research on elucidating the functions of ginkgo genes.

Keywords: Ginkgo biloba L.; PEG-mediated transformation; protein interaction; protoplasts; transient expression.

Figure 1

Isolation of protoplasts from different tissues of ginkgo seedlings. (A) Two-week-old ginkgo seedling; scale bar = 2 cm. (B) True leaves, tender stems, and young roots (from top to bottom) enzymatically hydrolyzed in enzymolysis solution. (C) Protoplast status in different tissues under bright-field microscopy; scale bars = 50 μm.

Figure 2

(A–E) Optimization of the ginkgo protoplast isolation system. (A) Effects of different plant materials on protoplast isolation efficiency. The enzymatic solution composition was 0.4 M mannitol, 2% (v/v) cellulose, and 0.5% (v/v) pectinase; the enzymatic hydrolysis conditions were constant temperature of dark enzymatic hydrolysis of 28°C for 3 h; the tissue mass in each group was approximately 0.5 g. (B) Effect of mannitol concentration on protoplast yield and activity. The enzymatic solution was consistent with that described in (A). Approximately 0.3 g leaves from 2-week-old seedlings were subjected to enzymatic hydrolysis for 3 h. The bar chart shows protoplast production and the broken line shows protoplast activity in 24 h. Y×A is the product of the number of isolated protoplasts and their average activity, which was calculated as the number of active protoplasts in 24 h. (C) Effects of cellulase and pectinase contents on protoplast yield and activity. The mannitol concentration was 0.4 M; 0.3 g leaves from 2-week-old seedlings were subjected to enzymatic hydrolysis for 3 h. The two dotted lines indicate the protoplast yield of 10.0 × 10⁵/gFW and activity of 80%, respectively. (D) Effect of enzymatic hydrolysis time on protoplast yield and activity. Enzymatic solution composition: 0.4 M mannitol, 2% (v/v) cellulose, and 0.25% (v/v) pectinase. Enzymatic hydrolysis conditions: allowed to stand or shaken at 1 ×g at 28°C for 3–6 (h) Different lowercase letters above bars in (A–D) indicate a significant difference (p < 0.05). (E) Fluorescein diacetate (FDA) staining was used to detect the activity of ginkgo protoplasts after 24 h; scale bar = 50 μm. The left image shows the bright field and the right image shows the FITC channel. Green light represents the active protoplasts. (F–H) Optimizing the conditions of the ginkgo protoplast transient transformation system. (F) Effects of different concentrations of PEG 4000 on transformation efficiency. Conversion solution composition: 10 μg plasmid, approximately 5 × 10⁴ ginkgo protoplasts, and 10%–50% PEG 4000 solution. Transformation conditions: allowed to stand at 25°C for 30 min. (G) Influence of transformation time on transformation efficiency. The transformation solution was 30% (w/v) PEG 4000 and the same components described above for (F). Transformation conditions: allowed to stand at 25°C for 10–40 min. Different lowercase letters above bars in (F–G) indicate a significant difference (p < 0.05). (H) 35S::GFP transformation of ginkgo protoplasts confirmed under a fluorescence microscope at 10× magnification; scale bar = 50 μm. The upper image shows the bright field and the lower image shows the merge of the FITC and Rhod channels. Red light represents autofluorescence and green light represents the protoplasts successfully transformed. (I) RT-qPCR results for the Chr3.406.1 and Ghr5.1249.1 genes that were transiently overexpressed in ginkgo protoplasts (TO); ***p < 0.001.

Figure 3

Subcellular localization for different vectors in ginkgo protoplasts (scale bars = 10 μm). The protoplasts transformed by three vectors are displayed in different channels. The FITC channel shows the location of the GFP fusion gene, the Rhod channel shows the autofluorescence of protoplasts, and the Bright channel shows the cell state. Merge1 is the combination of FITC and Rhod channels and Merge2 is the integration of FITC, Rhod, and Bright channels. DAPI is the nuclear marker.

Figure 4

Protein–protein interaction assays in ginkgo protoplasts. Constructed pairs of Chr3.406.1-YFP^C with Chr5.1249.1-YFP^N (predicted), Chr3.406.1-YFP^C with YFP^N (negative control), YFP^C with Chr5.1249.1-YFP^N (negative control), and YFP^N with YFP^C (negative control) were transiently transformed in ginkgo protoplasts. The BiFC fluorescence is indicated by the YFP signal (FITC channel, channel imaging shown in green). Individual and merged images of YFP and chlorophyll autofluorescence as well as bright-field images of protoplasts are shown. DAPI is the nuclear marker. Scale bars = 20 μm.