Absence of genetically modified (GM) maize (Zea mays L.) in seed

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
4. Event-specific real-time PCR (esrt-PCR)
CG
CG
T 25
Mahendra
Nagar Dhangadhi
ract1intron
P-ract1
NK 603
INDIA
3’
5’
CTP2
CP4 EPSPS
Zmhsp
70
T-Nos P-E35S
3’
5’
P-35S
pat
pUC18 Vector
T-35S
100 bp
5’
CG
MON 810
CTP2 CP4 EPSPS
hsp70intron
V
106 bp
CG
3’
P-35S
T-Nos
CG
CG
cry1A(b)
Kaski
Nepal
Bhairahawa
Zein
Gorkha
72 bp
Ktm valley
Hetaunda
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.)
Chitwan
INDIA
Total area: 147,181 sq km
Total population: 28,875,140
72bp
INDIA
Biratnagar Jhapa
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.
Amplification plots
Amplification plots
10
10
Threshold level
1
NK 603
1
T 25
ΔCt
2. Samples handling process in Laboratory
0.1
0.1
0.01
0.01
ΔCt
Nepalganj
84 bp
0.001
0.0001
0.001
NTC/samples
0.0001
NTC/samples
0.00001
0.00001
(A)
0.000001
2
3
2
4
0.000001
6
8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40
2
10
10
1
MON 810
0.001
0.0001
10
NTC/samples
0.00001
(C)
2
4
(D)
6
8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40
0.000001
2
4
6
8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40
Cycles
6
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
5. Conclusions
3. Event- and construct specific mPCR
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
bp
bp
700
600
500
400
300
570
458
415
293
260
224
150
200
100
90
72
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.
Cycles
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
8
7
NTC
0.001
0.0001
0.00001
0.000001
8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40
0.01
ΔCt
ΔCt
4
6
0.1
0.01
9
4
Amplification of PC and suspected 5 samples
with zein primers and probe
1
0.1
1
(B)
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.
For further information
Men-Chi Chang (email: menchi@ntu.edu.tw) and
Hari K. Shrestha (presenter, email: hariks_nepal@yahoo.com)

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