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

Abstract

Mangoes (Mangifera indica L.) are an important kind of perennial fruit tree, but their biochemical testing method and transformation technology were insufficient and had not been rigorously explored. The protoplast technology is an excellent method for creating a rapid and effective tool for transient expression and transformation assays, particularly in plants that lack an Agrobacterium-mediated plant transformation system. This study optimized the conditions of the protoplast isolation and transformation system, which can provide a lot of help in the gene expression regulation study of mango. The most beneficial protoplast isolation conditions were 150 mg/mL of cellulase R-10 and 180 mg/mL of macerozyme R-10 in the digestion solution at pH 5.6 and 12 h of digestion time. The 0.16 M and 0.08 M mannitol in wash solution (WI) and suspension for counting (MMG), respectively, were optimal for the protoplast isolation yield. The isolated leaf protoplasts (~5.4 × 105 cells/10 mL) were transfected for 30 min mediated by 40% calcium-chloride-based polyethylene glycol (PEG)-4000-CaCl2, from which 84.38% of the protoplasts were transformed. About 0.08 M and 0.12 M of mannitol concentration in MMG and transfection solutions, respectively, were optimal for protoplast viability. Under the florescence signal, GFP was seen in the transformed protoplasts. This showed that the target gene was successfully induced into the protoplast and that it can be transcribed and translated. Experimental results in this paper show that our high-efficiency protoplast isolation and PEG-mediated transformation protocols can provide excellent new methods for creating a rapid and effective tool for the molecular mechanism study of mangoes.

Optimization of preparation and transformation of protoplasts from Populus simonii × P. nigra leaves and subcellular localization of the major latex protein 328 (MLP328)

“…During the process of sugarcane protoplast preparation, Wu et al [ 51 ] showed a trend of first increasing and then decreasing protoplast yield and viability with increasing enzymolysis time. Adjei et al [ 52 ] found that the optimal enzymolysis time for mango protoplasts was 12 h, while Wang et al [ 53 ] used 2 h for separation of passion fruit protoplasts. In this experiment, the enzymatic hydrolysis of plant tissue was observed every half hour to ensure that the protoplasts were not damaged or insufficiently digested due to enzymolysis time.…”

Improved Protoplast Production Protocol for Fungal Transformations Mediated by CRISPR/Cas9 in Botrytis cinerea Non-Sporulating Isolates

“…Moreover, the inclusion of suitable osmotic stabilizers in the isolation buffer is crucial for maintaining protoplast integrity and viability [55]. Osmotic stabilizers such as sorbitol or sucrose help counteract the osmotic shock experienced by protoplasts upon cell wall removal, thereby preventing cellular rupture and improving survival rates [56].…”