Highly efficient mesophyll protoplast isolation and PEG-mediated transient gene expression for rapid and large-scale gene characterization in cassava (Manihot esculenta Crantz)

Abstract

Background: Cassava (Manihot esculenta Crantz) is a major crop extensively cultivated in the tropics as both an important source of calories and a promising source for biofuel production. Although stable gene expression have been used for transgenic breeding and gene function study, a quick, easy and large-scale transformation platform has been in urgent need for gene functional characterization, especially after the cassava full genome was sequenced.

Methods: Fully expanded leaves from in vitro plantlets of Manihot esculenta were used to optimize the concentrations of cellulase R-10 and macerozyme R-10 for obtaining protoplasts with the highest yield and viability. Then, the optimum conditions (PEG4000 concentration and transfection time) were determined for cassava protoplast transient gene expression. In addition, the reliability of the established protocol was confirmed for subcellular protein localization.

Results: In this work we optimized the main influencing factors and developed an efficient mesophyll protoplast isolation and PEG-mediated transient gene expression in cassava. The suitable enzyme digestion system was established with the combination of 1.6% cellulase R-10 and 0.8% macerozyme R-10 for 16 h of digestion in the dark at 25 °C, resulting in the high yield (4.4 × 10⁷ protoplasts/g FW) and vitality (92.6%) of mesophyll protoplasts. The maximum transfection efficiency (70.8%) was obtained with the incubation of the protoplasts/vector DNA mixture with 25% PEG4000 for 10 min. We validated the applicability of the system for studying the subcellular localization of MeSTP7 (an H⁺/monosaccharide cotransporter) with our transient expression protocol and a heterologous Arabidopsis transient gene expression system.

Conclusion: We optimized the main influencing factors and developed an efficient mesophyll protoplast isolation and transient gene expression in cassava, which will facilitate large-scale characterization of genes and pathways in cassava.

Keywords: Cassava; Manihot esculenta; Protoplast isolation; Subcellular localization; Transient expression system.

Fig. 1

Schematic representation of the pA7-MeSTP7-GFP construct driven by the constitutive CaMV 35S promoter and terminated by nopaline synthase terminator (NOS)

Fig. 2

The viability assays of cassava mesophyll protoplasts using the optimized protocol, i.e. preparation of mesophyll protoplasts in an enzyme solution (1.6% cellulase + 0.8% macerozyme) incubated at 25 °C (45 rpm) for 16 h in darkness. a Bright field image of protoplasts; (b) Protoplasts stained with FDA; (c) Merged image of protoplasts (Scale bar = 100 μm).

Fig. 3

High-efficiency transformation of cassava mesophyll protoplasts with pA7-GFP plasmid. a Bright field image of protoplasts; (b) Image of GFP; (c) Image of GFP merged with chlorophyll autofluorescence (Scale bar = 100 μm)

Fig. 4

Effects of PEG4000 concentration (a) and transfection time (b) on cassava protoplast transformation efficiency. Values are expressed as mean ± SEM, the error bars represent at least five independent replicates. The different letters indicate significant differences (P ≤ 0.05) according to the Duncan test

Fig. 5

Subcellular localization of MeSTP7 in cassava mesophyll protoplasts. Transient expression of GFP, showing that the GFP is distributed throughout the nucleus and cytoplasm (a-d). The laser-scanning confocal microscopy images are the bright field image (a), fluorescence image (b), merged image (c) and autofluorescence image (d), respectively. The transient expression of GFP-fused MeSTP7 protein, showing that the MeSTP7-GFP fusion protein is likely localized to plasma membrane (e-h). The laser-scanning confocal microscopy images are the bright field image (e), fluorescence image (f), merged image (g) and chloroplast fluorescence (H), respectively. The bars = 5 μm.)

Fig. 6

Subcellular localization analysis of MeSTP7 in Arabidopsis mesophyll protoplasts. Transient expression of GFP, showing that the GFP is distributed throughout the nucleus and cytoplasm (a-d). The laser-scanning confocal microscopy images are the bright field image (a), fluorescence image (b), merged image (c) and chloroplast fluorescence image (d), respectively. The transient expression of GFP-fused MeSTP7 protein, showing that the MeSTP7-GFP fusion protein is likely localized to cytoplasm membrane (e-h). The laser-scanning confocal microscopy images are the bright field image (e), fluorescence image (f), merged image (g) and chloroplast fluorescence image (h), respectively. The bars = 10 μm