International Journal of Science and Research (IJSR) ISSN (Online): 2319-7064 Index Copernicus Value (2015): 78.96 | Impact Factor (2015): 6.391 Volume 6 Issue 5, May 2017 www.ijsr.net Licensed Under Creative Commons Attribution CC BY Extraction of High Quality DNA from Mucilaginous Plants with a New Improved Method, Suitable for Detection of Geminiviruses and Downstream Applications Buddhadeb Roy 1 , Ang Rinzing Sherpa 2 1 West Bengal State University, Berunanpukuria, Malikapur, Barasat, 24 North Parganas, Kolkata, 700126, West Bengal, India 2 West Bengal State University, Berunanpukuria, Malikapur, Barasat, 24 North Parganas, Kolkata, 700126, West Bengal, India Abstract: High quality genomic DNA extraction from higher plants is interfered by the presence of secondary metabolites, which reduces the yield and quality of the DNA and the downstream processes such as DNA amplification, restriction digestion and cloning. We describe an alternative protocol for genomic DNA extraction from fresh and dry mucilaginous plant leaves that is amenable to PCR- based genetic analysis. It is relatively simple quick, rapid, less time consuming and cost effective method for DNA isolation from leaves of mucilaginous plant using modified cetyl trimethylammonium bromide (CTAB) method for extraction of DNA from leaf materials. DNA isolated using this method showed consistency in yield and compatibility with PCR for detection of geminiviruses from different mucilaginous plant species. The method was compared for efficacy with other reported methods and it was found to be superior over the existing methods described for isolation of DNA from mucilaginous hosts. Thus the method described could be used on a wider scale for reliable and consistent detection of geminiviruses from mucilaginous hosts for characterization and variability study. Keywords: Geminivirus, Malachra sp., DNA extraction, mucilaginous plants 1. Introduction A large number of not only crop plant but also much wild plants are susceptible to infection by geminiviruses. The family Geminiviridae contains four genera viz Mastrevirus, Topocuvirus, Curtovirus and Begomovirus. Begomovirus is the largest genus of all viral taxonomy with respect to the number of species that it includes. In fact, currently 322 species have been recognized by the International Committee on Taxonomy of Viruses (ICTV) (http://www.ictvonline.org/virusTaxonomy.asp ). Geminiviruses contains one or two small circular single stranded DNA (approx.2.7-3.0 kbp). The worldwide expansion of agriculture has also resulted in the emergence and spread of numerous insect pests and diseases including many destructive viral diseases. Geminiviruses are insect- transmitted viruses that have emerged, over the past two decades and are one of the most economically important emerging and re-emerging viruses [1]. Isolation of good quality DNA is a prerequisite for the applications such as different molecular techniques when studying any organism or pathogen or diagnosis. Plants produce secondary metabolites that interfere not only with extraction of high quality genomic DNA but also with the subsequent reactions such as PCR and related genetic analyses [2][3]. Since the biochemical composition of plant tissues vary in different species, DNA isolation protocols need to be optimized for each species [4]. The family Malvaceae containing mucilaginous plant like Malachra capitata, Hibiscus rosa-sinensis, bhendi or okra (Abelmoschus esculentus) possess high amount of foliar mucilage. DNA isolation from mucilaginous plant is difficult due to the high content of polyphenols and polysaccharides which are found to be not only co- precipitate with the DNA but also involves in the degradation of DNA due to endonucleases that hampers restriction digestion of DNA, PCR amplification and cloning. There are many protocols for DNA preparations from various sources of tissue have been published over the last few decades. We have tried five different protocols for the isolation of DNA from mucilaginous plant M. capitata [5][6][7][8] but we found that most of the protocols are not very suitable for the isolation of DNA from M. capitata. With this background we have developed a simple, rapid, less time consuming and cost effective method for DNA isolation from leaves of mucilaginous plants such as Bhendi (Abelmoschus esculentus), Jatropha gossipiifolia, Croton bonplandianus, Jute (Corchorus sp.), Sida sp and M. capitata. The aim of our study was to develop a rapid and cost efficient method for extraction of genomic DNA from leaves of mucilaginous plant, gives pure viral DNA without ultra- purification. 2. Materials and Methods Plant samples for DNA isolation: Initially, for standardization of the method, geminivirus infected M. capitata leaves with yellow mosaic typical of geminivirus symptoms were chosen as the model plant sample. After standardization, the method was validated with different mucilaginous plants samples with typical symptoms of geminivirus infection. All plant materials used in this study were collected from different parts of the North 24 Parganas, West Bengal, India. Plant materials used for this purpose were Bhendi (Abelmoschus esculentus), Jatropha gossipiifolia, Croton bonplandianus, Jute (Corchorus sp.), Sida sp and M. capitata infected with virus and showing typical geminivirus symptoms. Paper ID: ART20173077 121