Mustafa Abubakar S, Ssenku Jamilu E, Ssemanda Paul, Ntambi Saidi, Dinesh-Kumar Savithramma P, Tugume Arthur K
Department of Plant Sciences, Microbiology and Biotechnology, College of Natural Sciences, Makerere University, Kampala P.O. Box 7062, Uganda.
Department of Plant Biology, College of Biological Sciences, University of California, Davis, CA 95616, USA.
Methods Protoc. 2022 Jun 26;5(4):54. doi: 10.3390/mps5040054.
The existing methods of callose quantification include epifluorescence microscopy and fluorescence spectrophotometry of aniline blue-stained callose particles, immuno-fluorescence microscopy and indirect assessment of both callose synthase and β-(1,3)-glucanase enzyme activities. Some of these methods are laborious, time consuming, not callose-specific, biased and require high technical skills. Here, we describe a method of callose quantification based on Sandwich Enzyme-Linked Immunosorbent Assay (S-ELISA). Tissue culture-derived banana plantlets were inoculated with pv. () bacteria as a biotic stress factor inducing callose production. Banana leaf, pseudostem and corm tissue samples were collected at 14 days post-inoculation (dpi) for callose quantification. Callose levels were significantly different in banana tissues of -inoculated and control groups except in the pseudostems of both banana genotypes. The method described here could be applied for the quantification of callose in different plant species with satisfactory level of specificity to callose, and reproducibility. Additionally, the use of 96-well plate makes this method suitable for high throughput callose quantification studies with minimal sampling and analysis biases. We provide step-by-step detailed descriptions of the method.
现有的胼胝质定量方法包括对苯胺蓝染色的胼胝质颗粒进行落射荧光显微镜观察和荧光分光光度法、免疫荧光显微镜观察以及对胼胝质合成酶和β-(1,3)-葡聚糖酶活性的间接评估。其中一些方法费力、耗时、不具有胼胝质特异性、存在偏差且需要高技术技能。在此，我们描述一种基于夹心酶联免疫吸附测定（S-ELISA）的胼胝质定量方法。将组织培养获得的香蕉组培苗接种pv. ()细菌作为诱导胼胝质产生的生物胁迫因子。在接种后14天（dpi）采集香蕉叶片、假茎和球茎组织样本用于胼胝质定量。除了两种香蕉基因型的假茎外，接种组和对照组香蕉组织中的胼胝质水平存在显著差异。这里描述的方法可用于不同植物物种中胼胝质的定量，对胼胝质具有令人满意的特异性水平和可重复性。此外，使用96孔板使该方法适用于高通量胼胝质定量研究，且采样和分析偏差最小。我们提供了该方法的详细分步说明。
