Optimal Parameters for the Isolation of Carrot Mesophyll Protoplasts In Vitro
https://doi.org/10.18619/2072-9146-2025-3-5-9
Abstract
The isolation of protoplasts from diverse plant species is a widely employed technique. The purpose of this work is to develop an efficient system for isolating and purifying mesophyll protoplasts from Daucus carota. Main factors influencing the qualitative and quantitative dimensions of protoplast isolation procedures attempted to be optimized, using the well-established protoplast fusion technique as the foundation for the comprehensive analysis, including sorbitol concentration during the preplasmolysis stage and the duration of the enzymolysis process, those key variables affect the yield and survivability of the protoplasts. This research employed "Vil-1" carrot leaves as the primary source material to isolate protoplasts through enzymolysis. The data revealed that higher concentrations of sorbitol led to increased protoplast yield, with the optimal concentration being 0.5 M, which resulted in up to 95% protoplast vitality. Furthermore, prolonging the enzymolysis duration to 6 hours maximized both protoplast yield and vitality. The optimal conditions for isolating protoplasts were determined to be 0.5 M sorbitol pre-treatment for one hour, combined with a mixture of 1% cellulase, 0.1% pectinase, and a 6-hour incubation period.
About the Authors
N. Aljaramany
Russian State Agrarian University – Moscow Timiryazev Agricultural Academy
Russian Federation
Russian Federation
Naseem Aljaramany – PhD student
Timiryazevskaya Str., 49, Moscow, 127434
S. G. Monakhos
Russian State Agrarian University – Moscow Timiryazev Agricultural Academy
Russian Federation
Russian Federation
Sokrat G. Monakhos – Head of the Department of Botany, Breeding and Seed Production
Timiryazevskaya Str., 49, Moscow, 127434
References
1. Que F., Hou X.L., Wang G.L., Xu Z.S., Tan G.F., Li T., Wang Y.H., Khadr A., Xiong A.S. Advances in research on the carrot, an important root vegetable in the Apiaceae family. Horticulture Research. 2019;(6):1-15. https://doi.org/10.1038/s41438-019-0150-6
2. Iorizzo M., Senalik D.A., Ellison S.L., Grzebelus D., Cavagnaro P.F., Allender C., Simon P.W. Genetic structure and domestication of carrot (Daucus carota subsp. sativus) (Apiaceae). American Journal of Botany. 2013;(5):930–938. https://doi.org/10.3732/ajb.1300055
3. Cocking E.C. A Method for the Isolation of Plant Protoplasts and Vacuoles. Nature. 1960;(87):962-963. https://doi.org/10.1038/187962a0
4. Nagata T., Takebe I. Cell Wall Regeneration and Cell Division in Isolated Tobacco Mesophyll Protoplasts. Planta. 1970;(4):301-308. https://doi.org/10.1007/BF00385097
5. Liu J.H., Deng X.X. Plant Protoplast Asymmetric Fusion and Its Application in Breeding. Life Sciences. 1999;(14):88-91. https://doi.org/10.4236/ojapps.2024.143048
6. Davey M.R., Anthony P., Power J.B. Plant Protoplasts: Status and Biotech nological Perspectives. Biotechnology Advances. 2005;(23):131-171. https://doi.org/10.1016/j.biotechadv.2004.09.008
7. Tee C.S., Lee P.S., Kiong A.L.P, Mahmood M. Optimisation of protoplast isolation protocols using in vitro leaves of Dendrobium crumenatum (pigeon orchid). Agricultural research. 2010;(5):2685-2693. https://doi.org/10.5897/AJAR.9000516.
8. Murashige T., Skoog F. A revised medium for rapid growth and bioassays with tobacco tissue culture. Plant Physiology. 1962;(2):473- 497. https://doi.org/10.1111/j.1399-3054.1962.tb08052.x
9. Baranski R., Klocke E., Ryschka U. Monitoring the expression of green fluorescent protein in carrot. Acta Physiologiae Plantarum. 2007;(29):239–246. https://doi.org/10.1007/s11738-007-0030-9
10. Ingeborg L., Stefan S., Brigitte S., George K. Plasmolysis: Loss of Turgor and Beyond. Plants. 2014;(3):583–593. https://doi.org/10.3390/plants3040583
11. Huang H., Wang Z., Cheng J. An Efficient Cucumber (Cucumis sativus L.) Protoplast Isolation and Transient Expression System. Scientia Horticulturae. 2013;(150):206-212. https://doi.org/10.1016/j.scienta.2012.11.011
12. Jia X.Y., Zhang X.H., Qu J.M., Han R. Optimization Conditions of Wheat Mesophyll Protoplast Isolation. Agricultural Sciences. 2016(7):850-858. https://doi.org/10.4236/as.2016.712077
13. Aljaramany N., Vishnyakova A.V., Monakhos S.G. Somatic hybridization in agricultural crops improvement: An environmentally amiable era in biotechnology. Caspian Journal of Environmental Sciences (CJES). 2024;(22):1233-1241. https://doi.org/10.22124/cjes.2024.8236
14. Huo A., Chen Z., Wang P., Yang L., Wang G., Wang D., Liao S., Cheng T., Chen J., Shi J. Establishment of transient gene expression systems in protoplasts from Liriodendron hybrid mesophyll cells. PLOS ONE. 2017;(12):1-14. https://doi.org/10.1371/journal.pone.0172475
15. Firoozabady E. Rapid Plant Regeneration from Nicotiana, Mesophyll Protoplasts. Plant Science. 1986;(46):127-131. https://doi.org/10.1016/0168-9452(86)90119-6
16. Peng X.Q., Tang R., Xie X.M. Advances on the Isolation of Protoplast in Gramineous Plant. Chinese Agricultural Science Bulletin. 2015;(31):252-257. https://doi.org/10.11924/j.issn.1000-6850.20141921
Review
For citations:
Aljaramany N., Monakhos S.G. Optimal Parameters for the Isolation of Carrot Mesophyll Protoplasts In Vitro. Vegetable crops of Russia. 2025;(3):5-9. https://doi.org/10.18619/2072-9146-2025-3-5-9
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