1. Absence of Genetically Modified (GM) Maize (Zea mays L.) in Seed Samples from Nepal Hari Kumar Shrestha, Men-Chi Chang and Kae-Kang Hwu* Department of Agronomy, National Taiwan University * Corresponding author. E-mail: khwu@ntu.edu.tw, Tel: +886-2-33664761, Fax: +886-2-23620879 To determined the current status of GM maize in Nepal, we surveyed and collected forty six maize samples in Nepal. Then, an event and construct-specific multiplex polymerase chain reaction (mPCR) method (Shrestha et. al., 2008) was used for monitoring eight GM maize lines. The event-specific real-time PCR was also used to verify the suspected samples from mPCR analysis. Our result showed that none of the samples was contaminated with GM maize. Therefore, currently, no risk concern of GM maize in Nepal has to be taken care. This was the first report of GM maize testing in Nepal and would create awareness about food safety and provide a basic guideline for future understanding, formulating and implementing GMO quality control in Nepal. 1. Seed materials collection in Nepal In total, 46 samples were collected from different areas of Nepal and tested for the presence of GM maize, including seed traders ( ) 24 samples; National Maize Research Programme ( ) 12 samples, and farmers’ saved seed ( ) 10 samples. 2. Samples handling process in Laboratory Note: 1. Composite sample; 2. Mixing and dividing; 3. Counting of 100 seeds and weighing; 4. Weighing of 2000 seeds; 5. Grinding of 2000 seeds; 6. Mixing and sampling; 7. Samples packets after grinding; 8. Fine grinding, DNA extraction, and measurement, 9. Multiplex PCR, 10. Real-time PCR 4. Event-specific real-time PCR (esrt-PCR) For singlet real-time PCR analysis (ABI 7500), specific primer pairs for detection of each GM maize NK 603 (A), T 25 (B), MON 810 (C) and Zein (internal control) (D) were used. The reference sample was mixture of the above GM maize DNA at final conc. of 0.67%. Five previously suspected samples (4, 6, 10, 32 and 45) from mPCR were further analyzed. Only the reference samples showed the detectable level of amplification. Whereas; no signal was detected from non-template control (NTC) and other suspected maize samples. 5. Conclusions None of GM maize samples was found in Nepal seed samples. For safety consumption and environmental concern, however, it is still necessary to continuously analyzed other maize varieties from seed markets and produced in other locations of Nepal. This is the first GM testing of Nepal and will be a basic starting report to support further formulation and implementation of GM maize quality control and regulation in Nepal (Shrestha and Wulff, 2007). 6. Acknowledgements Nabin CTD Shrestha (NPQP), M.N. Shrestha (SQCC) and their staff, MoAC, and Laxmi, Padam K, Som N., Jeevan K. and Kamala D. Shrestha; Nepal. Government of Taiwan; DoA, NTU; Hsin Yi Chang, and labmates; Taiwan. Organizers of RAFA 2009, European Union. 7. References Shrestha HK, Hwu K-K, Chang M-C. J. Agric. Food Chem. 56: 8962-8968. Shrestha CB, Wulff E. 2007. UDV J.NR. 104.M.46, Copenhagen, Denmark. 3. Event- and construct specific mPCR The sensitivity of mPCR method was confirmed to be as low as 0.25% (Shrestha et. al., 2008). This method was also used for rapid and specific screening of 8 GM maize lines: Event 176 (570 bp), Bt 11 (458 bp), TC 1507 (415 bp), NK 603 (293 bp), T 25 (260 bp), MON 863 (224 bp), MON 810 (150 bp), GA 21 (90 bp) and Zein (72 bp) in all samples. Multiplex-PCR result showed that no specific bands present in most samples, except number 4, 6, 10, 13, 23, 32 and 42. Sample numbers 4 and 32 were suspected to be contaminated with NK 603; 6 with T 25; 45 with MON 810, and 10, 13, 23 with unknown, which were absent in purified DNA samples (data not shown). Therefore, it was concluded that most of the samples were non-GM maize. Event-specific primers and probes [NK 603 (106 bp): NK 603 08F/R/P; T 25 (100 bp): T 25 08F/R/P; MON 810 (84 bp): MON 810F/R/P, and Zein (72 bp): Zein04Se/07R/08P] were designed using primer express software 3.0; where Zein primers were adapted from Shrestha et. al., 2008 and others were newly designed. (The CG denotes DNA flanking sequence of corn genome and F/R/P represents Forward/Reverse/Probe.) For further information Men-Chi Chang (email: menchi@ntu.edu.tw) and Hari K. Shrestha (presenter, email: hariks_nepal@yahoo.com) Total area: 147,181 sq km Total population: 28,875,140 MON 810 Amplification of PC and suspected 5 samples with zein primers and probe NTC NTC/samples (C)(D) Cycles 10 1 0.1 0.01 0.001 0.0001 0.00001 0.000001 ΔC t 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 10 1 0.1 0.01 0.001 0.0001 0.00001 0.000001 ΔC t 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 T 25 NTC/samples (A)(B) Threshold level NK 603 NTC/samples Amplification plots 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 10 1 0.1 0.01 0.001 0.0001 0.00001 0.000001 ΔC t 10 1 0.1 0.01 0.001 0.0001 0.00001 0.000001 ΔC t Nepal Chitwan Mahendra Nagar Dhangadhi Nepalganj Bhairahawa Gorkha Hetaunda Ktm valley Biratnagar Jhapa INDIA Kaski NK 603 T 25 MON 810 Zein M, S, 1, 2, 3, 4, S, 5, 6, 7, 8, S, 9, 10,11,12, S, 13,14, 15,16, S, 17,18,19,20, S, 21,22,23,24, S,M, S, 25, 26, 27, 28, S, 29, 30, 31, 32, S, 33, 34, 35, 36, S, 37, 38, 39, 40, S, 41, 42,43, 44, S, 45, 46, S, NG, Q, S, M 570 458 415 293 260 224 150 90 72 100 200 300 400 500 600 700 bp 3’ 106 bp P-ract1 ract1- intron CTP2CP4 EPSPSP-E35ST-Nos Zmhsp 70 CTP2CP4 EPSPST-NosV 5’ pUC18 Vector P-35ST-35S 5’ 3’ pat 100 bp CG 72bp CG P-35Scry1A(b)hsp70intron 84 bp 5’ 3’ CG 72 bp 1 2 3 4 5 6 7 8 9 10