The templates used in the in vitro transcription reactions were synthesized through long-distance PCR (LD-PCR) in 50 μL PCR cocktails containing 1X HF Buffer of Phusion DNA Polymerase (Finnzymes, Espoo, Finland) with 1.5 mM MgCl 2 (Finnzymes, Espoo, Finland), 0.2 mM dNTP mix, 0.5 μM forward primer, CGT7dG (5 -CCGAGCTCGTAATAC-GACTCACTATAGGTTTTA-3 ), 0.5 μM reverse primer, CGMMV 3 -UTR (5 -TGGGCCCCTACCCGGGGAAAA-GGGGGGAT-3 ), 10-20 ng of DNA template, and 1 U of Phusion DNA Polymerase (Finnzymes, Espoo, Finland). The reaction was set up in 0.2 mL tubes, and the thermal cycling was conducted with initial denaturation at 98 • C for 60 seconds, followed by 30 cycles of 98 • C denaturation for 10 seconds, annealing at 63 • C for 20 seconds and elongation at 72 • C for 1 minute and 50 seconds, and finally an extension step at 72 • C for 5 minutes. The amplified product was purified through phenol-chloroform extraction followed by ethanol precipitation. The pellet was dissolved in an appropriate volume of RNase-free distilled water to 1 μg/μL and stored at −20 • C till further use. The in vitro transcription was carried out using the (Ambion, Calif, USA) High Yield Capped T7 RNA Transcription Kit according to the manufacturer protocol. Aliquots of the in vitrosynthesized transcripts were denatured and electrophoresed alongside RNA markers showing its integrity and the expected transcript size of approximately 6.5 kb. Since no DNase I treatment was done, traces of DNA template of the transcription reactions were detected. 2.5. Inoculation with RNA Transcripts. One transcription reaction was used to inoculate 2 plantlets by gently rubbing the reaction mixture over carborundum-dusted first leaf and cotyledons of 10-day-old plantlets. Mock inoculation was done by gently rubbing distilled water onto carborundumdusted first leaves. The excess inoculum was rinsed off using distilled water from the leaf surfaces 60 minutes after inoculation.