Weekly Publication of

Cotton
Association Edited & Published by Amar Singh

of India 2013 No. 29 15th October, 2013 Published every Tuesday

Cotton Exchange Building, 2nd Floor, Cotton Green, Mumbai - 400 033
Phone: 30063400 Fax: 2370 0337 Email: [email protected]
www.caionline.in

Testing Seed Quality of Bt Cotton

(Dr. K.R. Kranthi, Director of Central Institute cotton hybrids that express Cry1Ac, Cry2Ab, Cry1C
for Cotton Research (CICR), Nagpur has completed and fusion gene (Cry1Ac) have been approved by
the GEAC for commercial cultivation in India.
his Ph.D in Entomology from IARI, New Delhi. He The GEAC approved the following GM Bt cotton
has more than 20 years of experience in the field of events:
cotton research. 1. Monsanto: MON531 (Cry1Ac) event Bollgard;
The views expressed in this column are his own 2. Monsanto: Mon15985 (Cry1Ac+Cry2Ab2) event
in Bollgard-II;
and not that of Cotton Association of India) 3. JK seeds, India: JK Event-1 (Cry1Ac);
Bt Cotton 4. Chinese Academy of Agricultural Sciences,
China: GFM Cry1A (Cry1Ac), introduced by
Bt cotton is genetically modified (GM) cotton Nath seeds India;
variety/hybrid that has crystal (Cry) protein toxin 5. NRCPB, New Delhi and UAS Dharwad, India:
producing genes derived from a soil bacteria called BNLA601 (Cry1Ac) event;
Bacillus thuringiensis (Bt). The bacterial 6. Metahelix, India: Event 9124
species was first discovered by Prof (Cry1C).
Ishiwata in 1901 in Japan. Many crystal The GEAC has thus far approved
proteins produced by the bacteria are the cultivation of 1128 Bt cotton
toxic as stomach poison to many species hybrids for commercial cultivation
of insects. When insects feed on the in India. However, about 25 to 30
toxins, they cause holes in the membrane hybrids developed by 6 to 7 major
that lines the insect stomach. companies occupy more than 70.0% of
The genetically modified (GM) the area. It is estimated that about 85%
technology was developed first by of the current area is under Bollgard-
Monsanto and released commercially II (Cry1Ac+Cry2Ab) and rest under
in USA, Mexico and Australia in 1996. Bollgard (cry1Ac) being produced and
Subsequently, the technology was marketed by 44 Indian seed companies.
introduced into China (1997), South
Africa (1998), Argentina (1998), India (2002), Bt testing kits developed by CICR
Colombia (2002), Brazil (2005), Costa-Rica (2008),
Burkina Faso (2009) and recently in Pakistan and Bt detection kits were developed and
Mynamar in 2010. Thus 13 countries cultivated Bt commercialized by Central Institute for Cotton
cotton in 161 lakh hectares in 2012, which accounts Research (CICR). Patents for the Bt detection kit
for 48% of the global cotton area. Bt Cotton was were granted in the following countries vide patent
introduced into India in the year 2002 and became numbers (Inventor: Dr K. R. Kranthi: Patents: South
extremely popular to the extent that about 95% Africa, 2007: Patent No. 2004110268. /ZA200410268;
of India’s cotton area is under Bt cotton hybrids China, 2008: Patent No. ZL 03817641.6CN1672049;
with almost all of the area under Monsanto’s Bt Mexico, 2008: Patent No. MXPA04011769; Uzbekistan,
technology. Incidentally, cotton is the only GM crop 2008: Patent No. WO03102208 and South Korea, 2008:
approved for commercial cultivation in India. Bt- Patent No. KR20050026396).
2 15th October, 2013 C o t t o n S t a t i s t i cs & N e w s

CICR developed simple ‘dip-stick’ kits and

‘ELISA’ (Enzyme Linked Immuno-Sorbent Assay) to
enable farmers, seed testing officers, researchers, seed
companies and regulators detect Bt-seed quality and
Bt-toxin expression in plants under field conditions.
ELISA test kits have been developed by CICR for
detection and quantitative testing of Cry1Ac, Cry2Ab
and Cry1C proteins, in the test material such as seeds,
leaves or other plant parts. CICR developed PCR
tests to detect specific gene and events for five major
events based on the data available in public domain.
Event specific data was unavailable for Metahelix
Event 9124 (Cry1C). CICR developed ‘dip-stick-strip
test kits’ for instantaneous 10-min detection test at
‘on-site’ conditions for leaves or seeds containing
Cry1Ac, Cry2Ab and Cry1C proteins. Additionally
another test called “GUS-reporter test” has been
developed as a 30-min easy reagent based test to detect
the reporter protein/gene associated with Cry2Ab.
The dip-stick kits can be used to regulate quality
while the ELISA kits are used to quantify the levels
of Bt-toxin expression so as to identify the time when
Bt-crop shows poor expression of Bt-toxin, when
farmers need to take up appropriate control measures.
The Bt-detection kits have been commercialised sold either in the name of the GEAC approved brands
and became extremely popular with farmers and seed or most of them as unapproved brands. The role of
testing agencies, as evidenced by the fact that more CICR and ICAR in regulating Bt-seed quality in India
than 40,000 kits have been used by stake holders. The has been widely acknowledged.
Bt-detection kits enabled regulation, streamlining
and ensuring Bt-cotton seed quality for farmers in the Seed testing methods of the approved events
country. All seed testing laboratories in India have
Three kinds of tests are used commonly in India
been using the kits and more than 6000 seed lots have
to detect the purity of GM seeds.
been tested using the kits. Legal cases have been filed
1. Antibody based tests:
in courts of several cotton growing states of north,
a. ELISA (Enzyme Linked Immuno-
central and south India and are under review. In the
Sorbent Assay) and
absence of the testing kits, illegal Bt-seed would have
b. Dip-stick-strip immuno-sorbent test
been rampant and proliferated without any control.
2. PCR (polymerase Chain Reaction) based tests:
It has been widely acknowledged that the kits acted
a. Regular PCR
as deterrents for spurious seed traders. It is estimated
b. Real-Time qPCR (quantitative PCR)
that the cotton yield losses due to illegal seed trade
3. Biochemical tests
and Bt-spurious-seed trade would have reached
a. GUS (Glucuronidase Assay) to detect
about Rs 250 crores worth each year, if the kits
the GM reporter enzyme
were not available. The kits assisted the technology
developers of Bt-cotton to introduce the technology
and establish it in the market to an extent of 90% Gazette notifications for GM seed testing
coverage, that resulted in cotton yields doubling to There are ten main gazette notifications that
31.5 million bales (170 kg lint per bale) in just 5 years relate to seed testing of GM crops.
from a meager 16.5 million bales in 2001. The sub- On the 12th November, 2003, S.O. 1300(E).-In
standard seed samples have now decreased to 5.23% exercise of the powers conferred by Sub-section
in 2007-08 as compared to 69% in 2003-04 apparently (1) of Section 4 of the Seeds Act, 1966 (54 of 1966),
due to the constant vigil and continuous testing. the Central Government declared the laboratory of
One significant advantage of the simple ‘dip-stick Central Institute of Cotton Research (CICR), Indian
test developed by CICR, has been that it empowered Council of Agricultural Research (ICAR), Nagpur as
farmers, extension workers and seed testing agencies the Central Seed Laboratory to carry out the functions
with a rapid test that can be conducted ‘on-the-spot’ of ascertaining the presence or absence of cry1Ac
directly in shops or in fields. Therefore it served as gene in Cotton seeds under the said Act with effect
a strong deterrent to the manufacturers and traders from the date of publication for the whole of lndia. 2.
who would have otherwise continued to produce In pursuance of clause (c) of rule 5 of the Seeds Rules,
substandard and spurious seeds which were being 1968, the Central Government also entrusted the
C o t t o n a ssoc i a t i o n o f i n d i a 15th October, 2013 3

Central Institute of Cotton Research, Indian Council

of Agricultural Research, Nagpur to act as a referral
laboratory for Bacillus thuringiensis Cotton seeds
(Bt. Cotton seeds).
A sample of 25 gm seeds would be drawn from
450 gm seed packet and ten seeds will be tested for
the Cry toxin. The minimum limits of quantification
were stipulated at 420 ng/gm seed or 420 ng per sq
cm of a leaf disc for the sample to be qualified as
positive for Bt.
On 5th November 2005 -S.O. 1567(E)-ln exercise
of the powers conferred by Section6 of the Seeds
Act, 1966 (Act 54 of 1966), the Central Government,
after consultation with the Central Seed Committee
specified the purity in terms of quantum of gene
express of Bacillus thuringienisis (Bt.) Protein (Toxin) 2. PCR test: 30 seeds to be tested from working
as 90 per cent in Bacillus thuringiensis cotton seed lot sample size of 25 g drawn from a single packet of 450
for labelling of Bacillus thuringienisis Cotton Seed. g. A minimum number of 27 seeds tested positive for
On 21st September 2006, six gazette notifications primers specific to the gene (cry1Ac, cry2Ab, cry1C
(G.S.R.584 (E) to 589(E) dated September 21, 2006) and fusion cry1Ac gene (cry1Ab+cry1Ac)) may be
were issued to empower all the seed Inspectors/ taken as the acceptable value for 90% gene purity. If
analysts and laboratories notified under Seed Act, only 25-26 seeds are positive, 30 freshly drawn seeds
also under EPA, 1986. from the same working sample may be re-tested again
On 8th May 2008, S.O.1107(E)- In exercise of the on PCR. The total number of positive seeds from the
powers conferred by Sub-section (1) of section 4 of the two tests should be equivalent to or more than 54 out
Seeds act, 1966 (54 of 1966), the Central Government of the total 60 seeds tested from the working sample.
made the following amendment in the notification of 3. Event specific PCR (only for referral
the Government of India, DAC number S,O, 1300(E) purposes): 10 seeds to be tested from working sample
dated 12th November 2003 and published in the size of 25 g drawn from a single packet of 450 g. A
Gazette of India, Extraordinary, Part II, Section 3, minimum number of 9 seeds tested positive for the
Sub-section (ii) namely: In the said notification, in event may be taken as the acceptable value for 90%
paragraph 1, in line No 7, for the words and figures event purity.
‘cry1Ac’, the following words shall be substituted, 4. Gus-Reporter test: 90 seeds to be tested from
namely –all types of Bacillus thuringiensis. working sample size of 25 g drawn from a single
packet of 450 g. A minimum number of 81 seeds tested
Methods to test Bt cotton positive for the test protein GUS (ß-Glucuronidase)
A working sample of 25 g should be taken in a which is the reporter gene for Bollgard-II may be taken
random manner from the seed packet. as the acceptable value for 90% gene purity of Cry2Ab.
1. ELISA test / dip-stick-strip test: 90 seeds to
be tested from working sample size of 25 g drawn Suggestions:
from a single packet of 450 g. A minimum number of The current gazette notification does not include
81 seeds tested positive for the test protein, Cry1Ac, essential requirement of 90% purity for each of the
Cry2Ab, Cry1C and fusion-gene protein Cry1Ab- Cry toxins in Bollgard-II. It only mentions that the
Cry1Ac, may be taken as the acceptable value for hybrid seeds should contain 90% of Bt toxins, which,
90% gene purity. in effect are present even in F-2 seeds. This should be
rectified. The state seed testing laboratories must be
provided with Bt testing fw acilities. Seed analysts
should be trained in Bt testing methods. Only Andhra
Pradesh has been able to set up such facilities. Gazette
notifications must be issued for DNA based PCR
testing to include all GM events approved in India.
Unless and until seed testing is done rigorously, there
is every possibility that the crop suffers in farmers’
feilds. Therefore, the seed testing laboratories in all
the cotton growing states must develop Bt testing
facilities and test for the presence of Bt in random
samples to ensure that the best quality seed reaches
farmers.