Establishment of a Protoplasts-Based Transient Expression System in Banana (Musa spp.)

Abstract

The breeding of triploid banana cultivars with improved traits, such as yield and disease resistance, remains a major challenge for breeders. One reason is that the molecular study and functional gene analysis in bananas fall behind due to the difficulties of its genetic manipulation. The plant protoplast-based transient transformation has been documented and widely used as a versatile and convenient system for functional gene analysis in many plant species. However, an efficient high-quality protoplast isolation and transformation system is still lacking for bananas. Here, we established an efficient protoplast isolation and transformation method for bananas by selecting proper source materials, optimizing conditions for enzymatic hydrolysis and PEG-mediated transfection. We found the best source materials for banana protoplasts’ isolation are young suckers, which give a yield of protoplasts ranging from 2.5 × 106 to 10.1 × 107 g−1 fresh weight after 5 to 6 h of enzymolysis. The yield is sufficient for most assays that have been established in protoplasts-based systems, such as protein subcellular localization and protein interaction assays. Moreover, using the established transient gene expression system in banana protoplasts, we validated the subcellular localization of Arabidopsis VESICLE SORTING RECEPTOR 1 (VSR1) and the protein self-interaction of Arabidopsis CNGC20 on the cell membrane. The results indicated this system works well and could be routinely used for the functional characterization of banana genes.

Advances in Quercus ilex L. breeding: the CRISPR/Cas9 technology via ribonucleoproteins

“…The results in holm oak showed that the best transfection efficiency (62%) was achieved with 10 µg of plasmid and with GFP expression recorded 24h from the transfection process. By contrast, in Liriodendron ( Huo et al., 2017 ) and banana ( Zhao et al., 2022 ) the highest values were attained with 20 μg. Finally, our transfection values were higher than those previously obtained with protoplasts of European chestnut tree ( Pavese et al., 2022 ) and Cymbidium Orchids ( Ren et al., 2020 ).…”

“…DNA transfection can be performed by PEG, electroporation, particle bombardment, and DNA microinjection, however, the use of PEG-mediated transient editing showed higher transfection efficiency and it is cost-effective and simple in terms of releasing RNPs into the protoplasts ( Shen et al., 2014 ; Subburaj et al., 2022 ). PEG has been applied to transfect protoplasts of many different plant species including woody species such as poplar ( Tan et al., 2013 ), Liriodendron ( Huo et al., 2017 ), European chestnut ( Pavese et al., 2022 ) and banana ( Zhao et al., 2022 ). The protoplast to plasmid DNA ratio is an essential factor influencing transfection efficiency ( Burris et al., 2016 ), and generally the amount of plasmid DNA to be used is between 10 to 20 μg ( Huo et al., 2017 ).…”

Function of ALA Content in Porphyrin Metabolism Regulation of Ananas comosus var. bracteatus

“…Porphyrin metabolism provides the vital pigments for chlorophyll and heme [ 34 ]. Its biosynthesis begins with the formation of 5-aminolevulinic acid (ALA) as the first step [ 35 ]. The study examined the porphyrin metabolism characters in the PT and WT of Ananas comosus var.…”

Using A Protoplast Transformation System to Enable Functional Studies in Mangifera indica L.

“…Protoplast transformation systems can be applied in studies on cell signaling pathways in response to hormones [7], immediate transcriptome responses, and regulatory networks [8]. Protoplast-based biochemical and molecular studies have been applied to carnations [9], cotton [10], citruses [11], eggplants [7], oil palms [12], pineapples [13], Mangifera indica L. [14], bananas [15], etc. This is because the versatility and ability of protoplast transient expression systems can be developed and applied to many non-model plants that have transformation platforms that are difficult to use [16].…”

“…In this case, the cellulase R-10 degrades natural cellulose and dissolves plant cell walls [23], the macerozyme R-10 contains pectinase and hemicellulose that degrade the plant tissues to single cells [24], and the mannitol provides a suitable osmotic pressure and thereby ensures the integrity of the protoplasts [25]. Moreover, the digestion time affects the protoplast yield, and prolonging the digestion time may or may not increase the protoplast yield because an extended digestion time may result in protoplast breakage [15]. The isolated protoplast potency and viability rate are dependent on the digestion solution's pH value.…”