INTERNATIONAL JOURNAL OF AGRICULTURE & BIOLOGY ISSN Print: 1560–8530; ISSN Online: 1814–9596 12–468/MFA/2012/14–6–870–878 http://www.fspublishers.org Full Length Article To cite this paper: Uzma, M.R. Khan, A. Muhammad, I. Hussain, S.H. Shah, T. Kumar, S. Inam, M. Zubair, H.U. Rehman, A. Sher, N. Rehman, S. Ahmed and G.M. Ali, 2012. Rapid in vitro multiplication of sugarcane elite genotypes and detection of sugarcane mosaic virus through two steps RT-PCR. Int. J. Agric. Biol., 14: 870‒878 Rapid in vitro Multiplication of Sugarcane Elite Genotypes and Detection of Sugarcane Mosaic Virus through Two Steps RT- PCR UZMA, MUHAMMAD RAMZAN KHAN 1 †, AISH MUHAMMAD†, IQBAL HUSSAIN†, SABIR HUSSAIN SHAH, TANWEER KUMAR, SAFEENA INAM, MUHAMMAD ZUBAIR‡, HAFEEZ-UR-REHMAN¶, ALTAF SHER†, NAZIA REHMAN†, SAJEELA AHMED† AND GHULAM MUHAMMAD ALI 1 † PARC Institute of Advanced Studies in Agriculture (PIASA), National Agricultural Research Centre (NARC), Islamabad, Pakistan †National Institute for Genomics and Advanced Biotechnology (NIGAB), National Agricultural Research Centre (NARC), Park Road, Islamabad, Pakistan ‡Sugarcane Program, Crop Sciences Institute, National Agricultural Research Centre (NARC), Park Road, Islamabad, Pakistan ¶Fruit Crops, Horticultural Research Institute, National Agricultural Research Centre (NARC), Park Road, Islamabad, Pakistan 1 Corresponding author’s e-mails: rkhan_75@yahoo.com; drgmali@yahoo.ca ABSTRACT A voluminous body of research has reported the establishment of efficient protocols for sugarcane multiplication through tissue culture. However, a reliable and reproducible in vitro plant production system remains obscured. Furthermore, validation of virus free nature of in vitro plants using molecular techniques is the most challenging one. Considering the need for high yielding cultivars due to land and constraints, this study was devised for mass multiplication of high yielding elite cultivars of sugarcane viz. HSF-240, YT-55 and YT-53. Use of 100% Clorox for surface sterilization of apical and lateral buds, and of cefotaxime (500 mg L -1 ) for controlling bacterial contaminants revealed complete sterilization of field grown explants. Culture initiation was dependent upon plant growth regulators (PGRs), genotype and type of explants. The highest shoot initiation frequency of 96% was obtained with combination of four plant growth regulators (0.1 mg L -1 BAP), (0.1 mg L -1 NAA), (0.1 mg L -1 Kn) and (0.1 mg L -1 GA 3 ). Maximum shoot number (17.4) was exhibited by HSF-240 on MS media when the concentrations of BAP, Kn and GA 3 were increased to 1 mg L -1 in combination with NAA (0.25 mg L -1 ) indicating preference for higher concentrations of PGRs. Half-strength MS media with 6% sucrose resulted in increased root length (9.2 cm) and root number (20.5) Hardening efficiency of 98.6% was achievable in sandy clay loam soil. Two steps reverse transcription PCR (RT-PCR) was successfully employed for detection of sugarcane mosaic virus (SCMV) in in vitro plants. These results have implications for understanding optimum conditions for in vitro mass production of sugarcane plants, molecular detection of SCMV in in vitro raised plants, and stable genetic transformation studies. © 2012 Friends Science Publishers Key Words: Sugarcane; Tissue culture; Plant growth regulators; Micropropagation; Sugarcane mosaic virus; RT-PCR Abbreviations: BAP: 6-Benzylaminopurine; NAA: Naphthalene acetic acid; Kn: Kinetin; GA 3 : Gibberellic acid; SCMV: Sugarcane Mosaic Virus; RT-PCR: Reverse Transcription Polymerase Chain Reaction INTRODUCTION Sugarcane (Saccharum officinarum L.) belongs to Poaceae family, and has been widely cultivated on tropical and subtropical regions globally. It is a high valued cash crop and exclusive source of 75% world sugar production (Lakshmanan et al., 2006). This crop provides many by- products for bio-factory such as alcohol, butanol, acetic acid, animal feed and paper besides, sugar and energy (Garcia et al., 2007). Sugarcane is genetically a complex crop that possesses highly variable chromosome number (octaploid; x = 10; 8x = 80). Being highly cross pollinated in nature, this crop requires specific, hot and humid climate for flowering (Gill et al., 2006). The area under cultivation and yield of this crop is becoming stagnant over the years. Hence it is desirable to sustain the yield without expansion in area. For this purpose, introduction of high yielding disease and virus free varieties with genetically improved traits in short period of time is the best strategy. Micropropagation bears high potential for rapid clonal