The document summarizes a study comparing the biosafety of genetically modified (GM) canola. The objectives are to analyze root exudates of GM and non-GM crops, investigate gene flow, evaluate the effect of GM crops on soil microbes and fertility, and assess impacts on succeeding crops and associated weeds. Methods described include growing GM and non-GM canola in hydroponics and soil pots. The study will analyze root exudates, DNA, protein profiles, and effects on soil chemistry, microbes, and nutrients. It aims to determine if GM canola adversely affects weeds, soil health, or succeeding crops.

1. Comparative Biosafety Evaluation of
GM Canola
Presented by: Saira Karimi
M. Phil 2nd
Plant Sciences

2. INTRODUCTION
With the advent of molecular gene technology, biologists have been able to
move novel gene constructs that are coupled with novel promoters into crop
plant genomes, creating transgenic plants.
enables the plants to express novel compounds, including insecticidal substances
that kill certain organisms when they feed on the transgenic crop.
The toxins released by these genetically modified plants are controversial and their effect
on associated weeds and soil rhizosphere is not totally explored. Therefore, the present
investigation is aimed on determination of adverse effects of genetically modified canola
on soil beneficial microbes and fertility, succeeding crop species as well as behavior of
gene flow from genetically modified maize and canola into surrounding interspecific
weeds.

3. Also , the hypothesis of present investigation is that whether
gene flow occurs from genetically modified maize and canola
into natural plants and genetically modified canola adversely
affect the associated weed species, soil rhizosphere beneficial
microbes and soil nutrient status and their effects on
succeeding crop species.

4. Objectives
To analyzed the
root exudates of
To investigate
genetically
phenomenon of
modified and
gene flow
non modified
crops.
Effect of
To evaluate the
genetically
effect of
modified crop
genetically
on soil
modified crops
beneficial
on soil fertility
microbes

5. Their seeds are used to
produce edible oil that is fit
Canola is one of two cultivars for human consumption
of rapeseed or Brassica because it has lower levels of
campestris erucicacid than
(Brassica napus L. and B. traditional rapeseed oils and
campestris L.). to produce livestock feed
because it has reduced levels
of the toxic glucosinolates.
Fig 1: Canola blooms

6. Weed control and environmental
benefits
improved nutritional
value
Canola is now the third
most important winter
weed control options
grain crop grown in
Australia
GM canola with
effect on wheat tolerance to either
critically important for glyphosate or
the cropping belt. glufosinate-ammonium
herbicides
Canola field in Temora, new
south whales

7. Why canola?
• The development of transgenic crops has especially raised
some issues more especially the problem of environmental
safety.
• Things that aren’t immediately obvious in the environment
are the vast number of microorganisms that live in the soil.
• What will the impact be of GM crops on these?
Soil microorganisms play a vital role in natural cycles such as the
carbon cycle and the nitrogen cycle. They are a source of plant
nutrition, nutrient cycling, pesticide and pollutant
decomposition and the source of soil fertility.

8. GM canola
• Herbicide-resistant GM canola is grown on about 80% of the
acres in western Canada
• GM canola was first introduced in 1995.
• Growers reported an average 10% yield increase (3 bu/ac) for
their GM canola compared to conventional canola (2001
study).
• The factors that contributed to this increase included better
yielding varieties, earlier seeding and better weed control.

9. • More experiments are needed to systematically examine the
relationship among Bt toxin, soil microorganisms, and soil
biochemical properties (Liu, 2009).
• Transgene products have also been shown to be released
directly from the plant roots either from sloughed or
damaged root cells as well as through root exudation.
Transgenic Bt crops was found to release a Bacillus
thuringiensis insecticidal endo-toxin from its roots (Saxena
and Stotzky, 2000).

10. • Possibility of transgene flow from engineered crops to
their wild relatives with undesirable consequences was
independently recognized by several scientists (e.g.
Colwell et al., 1985; Ellstrand, 1988; Dale, 1992).
• When genetic crossing between a transgenic plant and a
non-modified strain occurs there is also some worry that
the novel genetic material will disturb the entire genome,
creating unknown and potentially dangerous side-effects
(Mann 2002). Consumers also worry about the
possibility for transgenic DNA to be toxic or easily
integrated into their own genomes.

11. Hydroponic Experiment
The root exudates will be analyzed according to method
described by Sun et al (2003).
A hydroponic experiment is designed using two varieties
of GM canola (B.napus), named Tornado and Telfor.
Plant are given regular aeration by an aeration pump.
Hoagland s solution is changed weekly.
I started this experiment on 1st April now it is in 4th week
Two non Gm varieties are also hydroponically grown as a
control named Dunkled and Pakola.

12. Protocol for hydroponics
Hoagland's Solution (Plant Nutrient Solution)
Component Stock Solution mL Stock Solution/1L
2M KNO3 202g/L 2.5
2M Ca(NO3)2 x 4H2O 236g/0.5L 2.5
Iron (Sprint 138 iron chelate) 15g/L 1.5
2M MgSO4 x 7H2O 493g/L 1
1M NH4NO3 80g/L 1
Minors: 1
H3BO3 2.86g/L
MnCl2 x 4H2O 1.81g/L
ZnSO4 x 7H2O 0.22g/L
CuSO4 0.051g/L
H3MoO4 x H2O or 0.09g/L
Na2MoO4 x 2H2O 0.12g/L
1M KH2PO4 (pH to 6.0 136g/L 0.5
with 3M KOH)
1) Make up stock solutions and store in separate bottles with appropriate label.
2) Add each component to 800mL deionized water then fill to 1L.
3) After the solution is mixed, it is ready to water plants.

13. Hydroponics

14. Effect of GMO`s on soil chemistry
Pot experiments will be
conducted using GM canola. As I
included hydroponic also, under
control conditions as well as
three different soil types will be
used.
The soil will be
Homogenized before used.
The soil will be sampled
at the start of experiment
and also after completion

15. Chemical analysis
The soil will be analyzed for The soil nutrient status as
bulk density organic matter affected by GM canola will
content, pH, total N,P,K, be determined by the
ammonium and nitrate. method of Schwab (1977)

16. Effect on soil chemistry of genetically modified
(GM) vs. non-GM maize
A study was conducted to find the effects of genetically
modified (GM) maize (Zea mays L.) expressing the
Bacillus thuringiensis Berliner Cry1Fa2 protein (Bt) and
glyphosate herbicide tolerance on soil chemistry
(organic matter, N, P, K and pH), compared with non-GM
controls, were assessed in field and pot experiments
Results indicate that growing GM crops instead of
conventional crops may alter soil chemistry, but not
greatly, and that effects will vary with both the specific
genetic modification and the soil.

17. Soil Micro-organisms isolation and
characterization
The rhizosphere
contains majority of
Rhizosphere
micro biota in soil
microbes will be
and plant-microbe
under taken on the
interaction in the
basis of Colony
rhizosphere are
Counting by the
among the major
method of Miller
factors the regulate
(1972)
health and growth
of plants.

18. ISB News Report
PLANT RESEARCH NEWS
A STRATEGY FOR TRANSGENE CONTAINMENT
IN PLANTS BASED ON THE REPRESSION OF A
SEED-LETHAL COMPONENT
Johann P. Schernthaner

19. Gene flow
The protein profile
of modified vs. non
modified crop
specie will be
The DNA of GM determined by PCR
canola and non GM and SDS-PAGE.
variety named
Dunkled and Pakola
will be extracted
through CTAB
method.

20. Protocol for SDS-PAGE
Materials
To Pour Gels: 30% acrylamide, 10% SDS, 10% APS (make fresh each time)
TEMED, 1.5 M Tris, pH 8.8 (resolving gel),1.0 M Tris, pH 6.8 (stacking gel)
5x SDS Running Buffer (1 L).Tris 15 g.,Glycine 72 g
SDS 5 g, Coomassie Blue Stain, 10% (v/v) acetic acid
0.006% (w/v) Coomassie Blue dye
90% ddH2O, Isopropanol Fixing Solution
10% (v/v) acetic acid,25% (v/v) isopropanol, 65% ddH2O
SDS sample loading buffer (40 ml), ddH2O 16 ml, 0.5 M Tris, pH 6.8 5 ml
50% Glycerol 8 ml, 10% SDS 8 ml, 2βmercaptoethanol 2 ml (add immediately before use)
bromophenol blue, 10% (v/v) acetic acid

21. Protocol
1. Prepare polyacrylamide gel according to
standard protocol.
2. Load samples and run gel @ 25 mA (2
gels run @ 50 mA) in 1x SDS Running
Buffer.
3. At this point, the gel can either be
transferred to a membrane (see
Western protocol) or stained with
Coomassie (see below).
4. Place gel in a plastic container. Cover
with isopropanol fixing solution and
shake
at room temperature. For 0.75 mm-thick
gels, shake 10 to 15 min; for 1.5
mmthick gels, shake 30 to 60 min.
5. Pour off fixing solution. Cover with
Coomassie blue staining solution and
shakeat RT for 2 hr. Apparatus used for SDS-PAGE
6. Pour off staining solution. Wash gel
with 10% acetic acid to destain,
shaking at RT ON

22. Confirmation of Stable gene integration
For the purpose to confirm the
stable integration of insecticidal
gene (BT cry1A and cry2A)in
canola molecular analysis like
PCR and Southern Blot will be
perform.

23. Southern blotting