ORIGINAL ARTICLES   

Minerva Biotecnologica 2014 September;26(3):115-26

Copyright © 2014 EDIZIONI MINERVA MEDICA

language: English

Protein extraction for callus and node cultures of Vanilla planifolia Andrews

Tan B. C. ¹, Chin C. F. ¹, Liddell S. ², Alderson P. ³

¹ School of Biosciences, Faculty of Science, The University of Nottingham Malaysia Campus, Jalan Broga, Semenyih, Selangor Darul Ehsan, Malaysia; ² Division of Animal Sciences, School of Biosciences, Faculty of Science, Sutton Bonington Campus, University of Nottingham, Leicestershire, UK; ³ Division of Plant Sciences, School of Biosciences, Faculty of Science, Sutton Bonington Campus, University of Nottingham, Leicestershire, UK

AIM: Vanilla planifolia is an economically important orchid that has been cultivated for its flavouring pods. The low percentage of callus formation in regeneration protocol has prompted an urgency to investigate the problems associated with callus initiation at the biochemical and molecular level.
METHODS: To establish a routine procedure for the application of proteomic analysis to Vanilla explants, four protein extraction methods, namely TCA-acetone, phenol, combination of TCA-acetone with phenol (TCA-acetone-phenol) and direct lysis using urea buffer, were qualitatively and quantitatively evaluated.
RESULTS: The TCA-acetone-phenol protocol yielded the highest protein contents of 2.7 and 2.8 mg/g of fresh weight from callus and nodal extracts respectively. Whereas 2-DE analysis revealed that the highest number of protein spots with a clearer 2-D gel profile was detected on TCA-acetone gel for callus (251 spots) and node (256 spots). Inclusion of PVPP in TCA-acetone solution during extraction procedure, treatment of the protein extracts with nuclease mix and solubilisation of the extracts in rehydration buffer containing DeStreak provided well resolved 2-D protein patterns for both types of sample. A total of 9 proteins were successfully identified using mass spectrometry analysis.
CONCLUSION: This protocol is applicable and provides enhanced 2-DE based proteomic analysis of Vanilla callus and node tissues and is expected to be applicable to other recalcitrant plant tissues as well.