Abstract
BackgroundCassava (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.MethodsFully 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.ResultsIn 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.ConclusionWe 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.
Highlights
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
Protoplast yield increased with increasing concentrations of cellulase R-10 and macerozyme R-10, but protoplast viability decreased with increasing enzyme concentrations
For obtaining the highest number viable protoplasts for the subsequent transfection experiments, the optimum combination of enzymes for cassava protoplast isolation was determined as 1.6% cellulase + 0.8% macerozyme
Summary
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. Cassava (Manihot esculenta Crantz) is a perennial and woody shrub of the Euphorbiaceae cultivated in tropical and subtropical regions for its starchy storage roots [21, 22]. These roots have been used as the sources for dietary carbohydrate, starch processing and potential biofuel production. The cassava sequencing data set has been publicly released [24] and the determination of gene function has been a major goal of cassava molecular biology in genomic and post-genomic era, it will necessitate efficient high-throughput transient gene expression for gene function analysis
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