Back
 AS  Vol.7 No.12 , December 2016
Optimization Conditions of Wheat Mesophyll Protoplast Isolation
Abstract:
The research object of this study is “ML7113” wheat leaf, which is used to isolate protoplast with enzyme hydrolysis method. Three main effectors—the concentration of mannitol, enzymolysis time and centrifugal force, affect the production and vitality of protoplast. While the production and vitality of wheat protoplasts were detected by the hemacytometer and the FDA staining respectively. Results showed that, with the increasing concentrations of mannitol during 0.2 M - 0.4 M, protoplast yield increases and when the concentration is 0.4 M, the protoplast vitality can be up to 95%; with the extension of enzymolysis time in 2 h to 8 h, protoplast yield reaches a maximum in 6 h, but its vitality achieves the maximum in 4 h; considering a combination of these two factors impacting on protoplast, we obtain the best time to digest for 4 h; meanwhile, with the increasing of the centrifugal force from 500 rpm - 2000 rpm, its comprehensive effect of protoplast vitality and yield is the highest when the centrifugal force is 1000 rpm for 2 min (replicated three times). So 0.4 M mannitol, 4 h enzymolysis time and 1000 rpm for 2 min centrifugal force are the best separation condition.
Cite this paper: Jia, X. , Zhang, X. , Qu, J. and Han, R. (2016) Optimization Conditions of Wheat Mesophyll Protoplast Isolation. Agricultural Sciences, 7, 850-858. doi: 10.4236/as.2016.712077.
References

[1]   Shewry, P.R. (2009) Wheat. Journal of Experimental Botany, 60, 1537-1553.
https://doi.org/10.1093/jxb/erp058

[2]   Yu, Y., Guo, G., Lv, D., et al. (2013) Transcriptome Analysis during Seed Germination of Elite Chinese Bread Wheat Cultivar Jimai 20. BMC Plant Biology, 14, 1-19.

[3]   Mark, T. and Peter, L. (2010) Breeding Technologies to Increase Crop Production in a Changing World. Science, 327, 818-822.
https://doi.org/10.1126/science.1183700

[4]   Cocking, E.C. (1960) A Method for the Isolation of Plant Protoplasts and Vacuoles. Nature, 187, 962-963.
https://doi.org/10.1038/187962a0

[5]   Nagata, T. and Takebe, I. (1970) Cell Wall Regeneration and Cell Division in Isolated Tobacco Mesophyll Protoplasts. Planta, 92, 301-308.
https://doi.org/10.1007/BF00385097

[6]   Liu, J.H. and Deng, X.X. (1999) Plant Protoplast Asymmetric Fusion and Its Application in Breeding. Life Sciences, A01, 88-91.

[7]   Davey, M.R., Anthony, P., Power, J.B., et al. (2005) Plant Protoplasts: Status and Biotechnological Perspectives. Biotechnology Advances, 23, 131-171.
https://doi.org/10.1016/j.biotechadv.2004.09.008

[8]   Kielkowska, A. and Adamus, A. (2012) An Alginate-Layer Technique for Culture of Brassica oleracea L. Protoplasts. In Vitro Cellular & Developmental Biology—Plant, 48, 265-273.
https://doi.org/10.1007/s11627-012-9431-6

[9]   Karamian, R. and Ranjbar, M. (2010) Somatic Embryogenesis and Plantlet Regeneration from Protoplast Culture of Muscari neglectum Guss. African Journal of Biotechnology, 10, 4602-4607.

[10]   Sinha, A. and Caligari, P.D.S. (2005) Enhanced Protoplast Division by Encapsulation in Droplets: An Advance towards Somatic Hybridisation in Recalcitrant White Lupin. American Psychologist, 146, 441-448.
https://doi.org/10.1111/j.1744-7348.2005.040097.x

[11]   Korlach, J. and Zoglauer, K. (1995) Developmental Patterns during Direct Somatic Embryogenesis in Protoplast Cultures of European Larch (Larix decidua Mill.). Plant Cell Reports, 15, 242-247.
https://doi.org/10.1007/BF00193728

[12]   Aoyagi, H. (2011) Application of Plant Protoplasts for the Production of Useful Metabolites. Biochemical Engineering Journal, 56, 1-8.
https://doi.org/10.1016/j.bej.2010.05.004

[13]   Guan, Q.Z., Guo, Y.H., Wei, Y.X., Meng, F. and Zhang, Z. (2010) Regeneration of Somatic Hybrids of Ginger via Chemical Protoplast Fusion. Plant Cell Tissue & Organ Culture, 102, 279-284.
https://doi.org/10.1007/s11240-010-9730-8

[14]   Huang, H., Wang, Z., Cheng, J., et al. (2013) An Efficient Cucumber (Cucumis sativus, L.) Protoplast Isolation and Transient Expression System. Scientia Horticulturae, 150, 206-212.
https://doi.org/10.1016/j.scienta.2012.11.011

[15]   Chen, H. and Han, R. (2016) Characterization of Actin Filament Dynamics during Mitosis in Wheat Protoplasts under UV-B Radiation. Scientific Reports, 6, Article Number: 20115.
https://doi.org/10.1038/srep20115

[16]   Peng, X.Q., Tang, R. and Xie, X.M. (2015) Advances on the Isolation of Protoplast in Gramineous Plant. Chinese Agricultural Science Bulletin, 31, 252-257.

[17]   Cao, J., Yao, D., Lin, F. and Jiang, M. (2014) PEG-Mediated Transient Gent Expression and Silencing System in Maize Mesophyll Protoplasts: A Valuable Tool for Signal Transduction Study in Maize. Acta Physiologiae Plantarum, 36, 1271-1281.
https://doi.org/10.1007/s11738-014-1508-x

[18]   Yang, J.W., Fu, J.X., Li, J., et al. (2014) A Novel Co-Immunoprecipitation Protocol Based on Protoplast Transient Gene Expression for Studying Protein-Protein Interactions in Rice. Plant Molecular Biology Reporter, 32, 153-161.
https://doi.org/10.1007/s11105-013-0633-9

[19]   Ma, F. and Li, J. (1999) Protoplast Culture and Plant Regeneration of Chinese Plum. Acta Universitatis Agriculturalis Boreali-Occidentalis, 27, 61-65.

[20]   Firoozabady, E. (1986) Rapid Plant Regeneration from Nicotiana, Mesophyll Protoplasts. Plant Science, 46, 127-131.
https://doi.org/10.1016/0168-9452(86)90119-6

[21]   He, S., Lang, Z. and Li, Z. (2013) Optimized Condition for Protoplast Isolation from Maize, Wheat and Rice Leaves. Chinese Journal of Biotechnology, 29, 224-234.

 
 
Top