IMPACT OF IMPROVED DNA EXTRACTION METHOD FROM CITRUS LEAVES MIDRIB AND PCR FOR THE DETECTION OF CITRUS GREENING (CANDIDATUS LIBERIBACTER)

Abstract

Citrus greening/ yellow shoot disease/ HLB, caused by Candidatus liberibacter asiaticus, showing mosaic/ mottling pattern on leaves, stunting of plants, de-shape, pre-mature fruit drop and yellowing of reticulate venation as characteristic symptoms, is the most concerned disease prevailing in citrus groves of Punjab, Pakistan. For the detection of pathogen and downstream studies, a high-quality DNA is required. In citrus, due to the variety of species, different age groups in plants, thick waxy cuticle of leaves, high production of phenolics, polysaccharides and other compounds, it is very difficult to extract good quality DNA from leaves and especially from main midrib where the fastidious bacterium is residing. CTAB and SDS are two devised methods for the extraction of total genomic DNA from citrus leaves while the current research suggests some modified protocols for the detection of DNA from infected/ healthy samples. 100mg leaf midrib sample was crushed in liquid nitrogen, homogenized in 500µl CTAB, incubated at 60°C in water bath for 45 minutes, centrifuged at 12000rpm for 5 minutes, supernatant was transferred to new tube and 5µl RNase-A was added, incubated at 37°C for 20 minutes. Equal volume of chloroform/ isoamyl alcohol (24:1) was added, vortexed for 5 minutes, centrifuged for 2 minutes at 12000rpm for phase separation and upper phase was transferred to new tube where 500µl of chilling isopropanol was added, kept at -20°C for 15 minutes, centrifuged at 12000rpm, pallet was washed twice with 70% ethanol. CTAB method was modified by increasing the incubation time i.e., 45 minutes for cell lysis in water bath for determination the variation in quality of DNA and the amount of beta mercaptethanol was also doubled from the normal one. Modified protocols have been proved excellent for the extortion of total genomic DNA from citrus. In SDS, addition of TE buffer, RNAase, Isoamylalcohol: Chloroform (24:1) along with 20% SDS and 2M Sodium Acetate gave high quality DNA from citrus leaves. Hence, DNA was extracted by four different ways but the modified CTAB and SDS methods gave improved quality DNA as confirmed by its quantification through nanophotometer. The DNA was quantified by nanophotometer and presence was observed on gel documentation apparatus. Hence, DNA was found suitable for PCR and RFLP analysis and long-term storage on -80 ⁰C.