Selecting and optimizing a reliable DNA extraction method for isolating viral DNA in okra (&lt;em&gt;Abelmoschus esculentus&lt;/em&gt;)

T C Jeyaseelan,M W Shaw,D M De Costa,E C Jeyaseelan

doi:10.4038/vingnanam.v14i1.4140

Abstract

Detection and identification of viruses in okra plant is important to manage viral diseases. Good quality of DNA is essential for the PCR based detection and subsequent genomic sequencing. However, the DNA extraction has been greatly affected by mucilaginous substances present in okra plant parts. Objective of this study is to find out an extraction method that can eliminate mucilaginous materials and yield good quality DNA. Five different protocols were tested with okra leaf samples having okra yellow vein mosaic disease (OYVMD) symptoms. Quantity and purity of extracted DNA was tested using Nanodrop spectrophotometer. Precise quantity comparison was done by measuring total plant DNA in relation to the copy number of ACT2 gene using quantitative polymerase chain reaction (qPCR). The extracted DNA samples were used as template for detection of Bhendi yellow vein mosaic virus and Bhendi yellow vein mosaic betasatellite using specific primers. A modified protocol (modified method 1), introduced in this study, produced better yield and quality of DNA compared to other tested protocols. The method yielded 32 μg DNA for 100 mg leaf powder and the ratios A260/A280 and A260/A230 were 1.8 and 2.1, respectively. The qPCR quantification further confirmed higher quantity of DNA yield in this modified protocol. End point PCR with virus specific primers yielded very bright bands for Bhendi yellow vein mosaic virus and Bhendi yellow vein mosaic betasatellite. In conclusion, the modified method 1 can be used to extract good quality and quantity of DNA from okra.

Highlights

Detection and identification of plant viruses is the basis to manage plant diseases and to predict the crop loss by the infection
Five different DNA extraction protocols were tested to find out the protocol which can yield good quality and quantities of DNA extracted from okra leaves affected by okra yellow vein mosaic disease (OYVMD)
The protocol 4, one of the methods that we introduced in this study, yielded higher concentration of DNA with good quality (Table 3); the average concentration of extracted total DNA was 85.8 ng/μl and the ratios A260/A280 and A260/A230 were 1.8 and 2.1 respectively

Summary

Introduction

Detection and identification of plant viruses is the basis to manage plant diseases and to predict the crop loss by the infection. Better quality and enough quantity of virus genomic DNA or RNA are pre-requisite since the genomic components of the viruses are mixed with cellular constituents of infected plant tissues (Swanson et al, 1992). DNA extraction from some plant tissues remains difficult due to the contamination of secondary metabolites (Cavallari et al, 2014). Plants such as Okra (Abelmoschus esculentus), Mesta (Hibiscus cannabinus), Jute (Corchorus sp.) and Sida (Sida sp.) possess large amounts of viscous mucilage, which often have secondary metabolites, such as polysaccharides, phenolic compounds, tannins, and alkaloids (Höfer et al, 1997; Jose and Usha 2003; Ghosh et al, 2008; Roy et al, 2009). The concentration and viscosity of mucilage varies from one plant species to other or among different cultivars of one species is due to the chemical complexity and variation in the physiological properties (Muralikrishna et al, 1989; Kawamura et al, 2000; Axel Diederichsen et al, 2006)

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Conclusion