MODIFIED EXTRACTION METHOD FOR DETECTION OF COCONUT CADANG-CADANG VIROID (CCCVd) VARIANTS

Head of Corporate Implementation and Consultancy Unit, Malaysian Palm Oil Board. 6, Persiaran Institusi, Bandar Baru Bangi, 43000 Kajang, Selangor, Malaysia. Tel: 03-8769 4574 Fax: 03-8926 1337 E-mail: [email protected] Website: www.mpob.gov.my he oil palm is an important perennial oil crop for a number of countries in the tropics. A recent finding reported the presence of Coconut cadang-cadang viroid (CCCVd) variant in oil palm. The viroid as reported by Vadamalai (2005) implicated the movement of oil palm planting materials in Malaysia although there was no evidence of the viroid causing devastating disease loss compared to the loss recorded in the Philippines. In reality, the infected oil palm is only found isolated within a planting block (Sundram et al., 2017). The viroid sequence in oil palm was also found with a few base changes when compared to the viroid sequence in coconut of the Philippines. The viroid present in the form of free RNA strands replicating with protein of the host, was difficult to be detected and the available techniques were unable to give reliable and consistent detection. Therefore this technology improved the existing Natrium Chloride EDTA Tris-HCL Mercaptoethanol (NETME) extraction developed originally for coconut, to detect CCCVd variants consistently in oil palm. Extraction of high quality RNA is necessary in generating cDNA libraries, characterisation and detection by Reverse Transcriptase – Polymerase Chain Reaction (RTPCR) or investigating gene expression profile (Gehrig et al., 2000). However, high phenolic and/ or polysaccharides compounds level give rise to poor RNA quality or no absence of RNA. Based on these submissions, it is therefore paramount that extraction methods produces high concentration of RNA, thus the absolute need of optimisation. NETME is a suitable choice for simpler extraction with small amount of leaves sample (2-5 g) used (Vadamalai, 2005; Joseph, 2012). NETME extraction method undergoes purification step through Polyacrylamide gel electrophoresis (PAGE) that is time consuming and laborious (Hanold and Randles, 1991; Vadamalai, 2005). It requires additional two to three days procedure and is proven to be inconsistent in detection when repeated. Modification and optimisation of the existing NETME extraction method gave rise to shortened extraction time and provide a higher quality RNA for better detection of CCCVd variants in oil palm with the complete elimination of PAGE purification process.