Protoplast Isolation, Fusion, Culture and Transformation in the Woody Plant <i>Jasminum</i> spp.
Mohamed A. A. Ahmed,
Miao Miao,
Emmanouil D. Pratsinakis,
Hongliang Zhang,
Wei Wang,
Yuan Yuan,
Meiling Lyu,
Junaid Iftikhar,
Ahmed F. Yousef,
Panagiotis Madesis,
Binghua Wu
Affiliations
Mohamed A. A. Ahmed
College of Horticulture and the Fujian Provincial Key Laboratory of Plant Functional Biology, Fujian Agriculture and Forestry University, Fuzhou 350002, China
Miao Miao
College of Horticulture and the Fujian Provincial Key Laboratory of Plant Functional Biology, Fujian Agriculture and Forestry University, Fuzhou 350002, China
Emmanouil D. Pratsinakis
Laboratory of Agronomy, School of Agriculture, Faculty of Agriculture, Forestry and Natural Environment, Aristotle University of Thessaloniki, GR-54124 Thessaloniki, Greece
Hongliang Zhang
College of Horticulture and the Fujian Provincial Key Laboratory of Plant Functional Biology, Fujian Agriculture and Forestry University, Fuzhou 350002, China
Wei Wang
College of Horticulture and the Fujian Provincial Key Laboratory of Plant Functional Biology, Fujian Agriculture and Forestry University, Fuzhou 350002, China
Yuan Yuan
College of Horticulture and the Fujian Provincial Key Laboratory of Plant Functional Biology, Fujian Agriculture and Forestry University, Fuzhou 350002, China
Meiling Lyu
College of Horticulture and the Fujian Provincial Key Laboratory of Plant Functional Biology, Fujian Agriculture and Forestry University, Fuzhou 350002, China
Junaid Iftikhar
College of Horticulture and the Fujian Provincial Key Laboratory of Plant Functional Biology, Fujian Agriculture and Forestry University, Fuzhou 350002, China
Ahmed F. Yousef
Department of Horticulture, College of Agriculture, University of Al-Azhar (Branch Assiut), Assiut 71524, Egypt
Panagiotis Madesis
Laboratory of Molecular Biology of Plants, School of Agricultural Sciences, University of Thessaly, GR-38446 Thessaly, Greece
Binghua Wu
College of Horticulture and the Fujian Provincial Key Laboratory of Plant Functional Biology, Fujian Agriculture and Forestry University, Fuzhou 350002, China
Plant protoplasts are significant for plant cell culture, somatic cell fusion, genetics, and breeding studies. In addition, in vitro plant regeneration has great importance for developmental biology, manifesting potential applications in agriculture and biotechnology. In this regard, we present a well-established protocol regarding protoplast isolation, cell culture and protoplast fusion of Jasminum spp. In particular, different tissues of Jasminum samab L. and Jasminum mesnyi were employed for protoplast isolation, and stem explants provided a high callus induction rate in a short period of time. The best source for protoplast isolation was calli tissues. The optimized isolation protocol consisted of digesting callus in an enzyme solution containing 0.4 M mannitol, 0.2 M MES, 1 M CaCl2, 0.2 M KCL and 1 M NaH2PO4, 1.5% Cellulases onozuka R-10, 0.4% Macerozyme R-10 and 0.8% Pectinase for 4 h at 26 °C in the dark, providing a yield of 23.8 × 106 Protoplast/gFW with 88% viability. Protoplasts were cultured both in liquid and agarose medium under optimum conditions, leading to microcalli formation after eight weeks. A 5% protoplast-fusion rate can be achieved when cultured in 40% (w/v) PEG-MW6000 supplemented with 0.1 M CaCl2, 0.1 M sorbitol and 1 M Tris for 20 min. Furthermore, we developed an efficient PEG-mediated transformation protocol for jasmine protoplasts. The best results regarding protoplast transformation were obtained when the protoplast concentration was 4 × 105 cells/mL and the exogenous plasmid DNA added had a concentration of 10 µg DNA/100 µL protoplast solution, followed by the application of 40% PEG-4000 for 10 min.
