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| Study on Molecular Detection and Elimination of Three Lily(Lilium longiflorum) Viruses |
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Abstract Viral diseases cause adverse effects on the yield and quality of lilies grown in China. However, we still can't detect and eliminate these viruses effectively. The viral detection methods used these days are either time-consuming and expensive or sometime provide false-negative results occasionally. On the other hand, difficulties in operation mechanisms and low virus-free rate are the major constraints restricting the development of virus elimination techniques. Addressing these limitations, we carried out this study to establish effective ways to detect and eliminate three major viruses in lilies namely, Cucumber mosaic virus(CMV), Lily symptomless virus(LSV) and Lily mottle virus(LMoV). In vitro plantlets of Lilium longiflorum cv. Raizan 1 were used as subject material. Specific primer pairs were designed according to the gene sequence of the three viruses and 18S rRNA, and used for multiplex RT-PCR. Several reaction components (dNTPs, Mg2+ and Taq DNA polymerase), annealing temperature and cycle numbers were optimized to conduct multiplex RT-PCR effectively. Among couple of used reaction systems, the most effective and precise reaction system was as follow: dNTPs(2.5 mmol/L) 1.5 μL, Mg2+(25 mmol/L) 1.5 μL, Buffer 1.875 μL, cDNA 1 μL, Taq DNA polymerase 0.2 μL and each primer 0.5 μL, adding H2O to a final volume of 25 μL. The optimal reaction procedure wasas follow: 94℃ denaturation temperature for 5 min;94℃ for 30 s,52.4℃ annealing temperature for 30 s,extension phase with 72℃ for 30 s,30 PCR cycles;72℃ for 10 min,and kept at 4℃ to terminate the reaction. Thus, we established an efficient method to detect the three viruses simultaneously. The 18S rRNA was used as an internal control to avoid false-negative during the procedure. Heat treatment (38℃ for 14 h, 32℃ for 10 h, all in dark) combining with bulblet culture could eliminate more than 90% of the CMV in lilies, but remains ineffective in eliminating LMoV or LSV. After heat treatment, shoot tips were cut with a different size range of ≤1 mm, 1~2 mm and ≥2 mm respectively and culture in light or dark conditions. The results illustrated that culturing shoot tips of ≤1 mm size under light condition was the most effective way to eliminate all the three viral strains. High virus -free rate of 93.3% was observed in this method. Additionally, short culture cycles, easy handling and enhanced virus-elimination rate makes it an ideal method for producing virus-free lilies.
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Received: 26 July 2012
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