Direct leaf-peeling method for areca protoplasts: a simple and efficient system for protoplast isolation and transformation in areca palm (Areca catechu)

Abstract

Background Areca palm (Areca catechu) is a woody perennial plant of both economical and medicinal importance grown in tropical and subtropical climates. Yet, the molecular biology study of areca palm is extremely impeded by its unavailability of a transformation method. An efficient protoplast isolation and transformation system could be highly desirable to overcome this barrier. Results Here, we described a simple and efficient method for protoplast isolation and transformation from the perennial plant areca palm. A high yield of protoplasts (2.5 × 107 protoplasts per gram of fresh leaf tissues) was obtained from the fresh light green leaflet from the newly-emerged leaf digested overnight in the enzyme solution [2% (w/v) cellulase R10, 0.5% (w/v) macerozyme R10, 0.7 M mannitol, 10 mM CaCl2, 20 mM KCl, 20 mM MES and 0.1% (w/v) BSA, pH 5.7] by the direct leaf-peeling method. The isolated areca protoplasts maintain viability of 86.6% and have been successfully transformed with a green fluorescent protein (GFP)-tagged plasmid (pGreen0029-GFP, 6.0 kb) via the polyethylene glycol (PEG)-mediated transformation. Moreover, the mannitol concentration (optimal: 0.7 M) was determined as a key factor affecting areca protoplast isolation. We also demonstrated that the optimal density of areca protoplast for efficient transformation was at 1.0–1.5 × 106 cells/ml. With the optimization of transformation parameters, we have achieved a relatively high transformation efficiency of nearly 50%. Conclusion We have established the first efficient protocol for the high-yield isolation and transformation of areca palm protoplasts. This method shall be applied in various biological studies of areca palm, such as gene function analysis, genome editing, protein trafficking and localization and protein–protein interaction. In addition, the protoplast system offers a great genetic transformation approach for the woody perennial plant-areca palm. Moreover, the established platform may be applied in protoplast isolation and transformation for other important species in the palm family, including oil palm and coconut.

Cotyledon Peeling Method for Passion Fruit Protoplasts: a versatile cell system for transient gene expression in passion fruit (Passiflora edulis)

“…In this study, we found that a passion fruit protoplast density of 1.0 × 10 6 cells/ml resulted in the best transformation efficiency (Fig. 2C, D), which is similar to maize and areca palm (Gentzel et al, 2020;Wang et al, 2023). Different from this, the protoplast density is adjusted to 2-5 × 10 5 cells/ml in the classic protoplast system of Arabidopsis and tobacco (Yoo et al, 2007;Wu et al, 2009).…”

“…Plant protoplasts are the plant single cells excluding the rigid cell wall, and serve as a versatile singlecell-based system for transient gene expression (Davey et al, 2005;Dai and Wang, 2022;Wang et al, 2023). Foreign plasmid DNA of interest can be easily delivered into protoplasts typically by polyethylene glycol (PEG)-mediated transformation, electroporation and microinjection (Yoo et al, 2007;Priyadarshani et al, 2018;Dai and Wang, 2022).…”

Optimization of protoplast isolation and transient expression systems for lettuce (Lactuca sativa L.) and other important dicotyledon vegetables

“…Many efficient protoplasts isolation protocols for model plant based on mature tissues have been developed, including Arabidopsis thaliana [2], rice [3], cassava [11], and orchid [1]. Protoplasts have also been successfully isolated from some non-model plants, e.g., Mucuna pruriens, coffee, Medicago sativa [12,13], Panicum virgatum [14], Areca catechu [15]and Cannabis sativa [16].…”

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

“…At present, protoplasts have been isolated from various plant tissues [ 10 ], with leaves being the most common [ 11 ]. The methods for separating leaf protoplasts have also been established in many plants, such as Arabidopsis [ 12 ], castor ( Ricinus communis L.) [ 13 ], Areca palm ( Areca catechu ) [ 14 ], Eggplant ( Solanum melongena L.) [ 15 ], Cymbidium orchid [ 16 ], and Perennial ryegrass ( Lolium perenne L.) [ 17 ].…”