Protoplast Isolation, Fusion, Culture and Transformation in the Woody Plant Jasminum spp
| Autor: | Emmanouil D. Pratsinakis, Binghua Wu, Miao Miao, Meiling Lyu, Junaid Iftikhar, Ahmed Fathy Yousef, Mohamed A A Ahmed, Panagiotis Madesis, Hongliang Zhang, Wei Wang, Yuan Yuan |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2021 |
| Předmět: |
0106 biological sciences
0301 basic medicine callus induction Agriculture (General) protoplast culture Plant Science 01 natural sciences S1-972 03 medical and health sciences chemistry.chemical_compound Jasminum mesnyi Pectinase biology Chemistry fungi food and beverages Protoplast biochemical phenomena metabolism and nutrition biology.organism_classification protoplast isolation PEG Somatic fusion Horticulture Transformation (genetics) 030104 developmental biology woody plants Callus Agarose Agronomy and Crop Science 010606 plant biology & botany Food Science Explant culture |
| Zdroj: | Agriculture, Vol 11, Iss 699, p 699 (2021) Agriculture Volume 11 Issue 8 |
| ISSN: | 2077-0472 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|