Full script (Synopsis)

of
Seminar

Course No.: GP-591

Seminar Title: RECENT ADVANCES IN
GENETICALLY MODIFIED CORN RESEARCH



Submitted to: Submitted by:
Dr. J. G. Talati
Seminar co-ordinator,
Dept. of Agril. Biochemistry,
B.A.C.A., AAU, Anand.


Soni Nishit Vasantbhai
M.Sc. (Agri.) 3
rd
semester
Genetics & Plant breeding
Reg. No. : 04-1354-2010


Major Guide:
Dr. S M. Khanorkar
Associate Research Scientist
Main Maize Research Station
AAU, Godhra.


OUTLINE

INTRODUCTION
GENETICALLY MODIFIED PLANTS
AIMS OF BIOTECHNOLOGICAL RESEARCH AND DEVELOPMENT IN
MAIZE
DEVELOPING TRANSGENIC PLANTS
CASE STUDIES
RESISTANCE TO BIOTIC STRESS
RESISTANCE TO ABIOTIC STRESS
IMPROVEMENT IN NUTRITIONAL QUALITIES
PHARMACEUTICAL CORN
ADVANTAGES AND DISADVANTAGES
CONCLUSION
FUTURE PROSPECTS

















INTRODUCTION:
Maize is believed to be originated in Southern Mexico or Northern Guatemala.
(Weatherwax, 1955).
Maize was domesticated from its wild progenitor teosinte (Balsas teosinte).
Archeobotanical studies indicate that a single domestication event occurred
approximately 9,000 years ago in Central Mexico.


TAXONOMICAL STATUS
Taxonomy : Family Poaceae
Genus Zea
Species Zea mays ( L )
Ploidy level (Chromosome Number) : Randolph (1928) and Mc Clintock (1929)
Teosinte (Weedy) Maize (Cultivated)
* Zea mexicana (2n = 20) * Zea mays (2n = 20)
* Zea perennis (2n = 40)
* Zea luxurians (2n = 20)
* Zea diploperennis (2n = 20)

Teosinte x Maize (Cross compatible)

Why maize is Important?
Highest productivity (10.32 t/ha, USA) and production among cereals globally
Highest potential of carbohydrate production per unit/day
3
rd
most important cereal crop in India
Maize area has slowly expanded over past few years from 6.6 million ha in 2002 to
8.26 million ha in 2011
Uses as human food, animal and poultry feed and raw material industrial products
Staple food for tribal
Maize is the promising option for diversifying agriculture due to its resilience to
changing climate
Queen of cereals
(Anonymous, 2011a).
Area and Production of Maize
Area (million ha) Production (million tons) Productivity (kg/ha)
World 161 827 5136
India 8.26 21.23 2570
Gujarat 0.497 0.533 1072
(Anonymous, 2011b).
FACTORS AFFECTING PLANT PRODUCTION GLOBALLY
Crop losses due to weeds, pests and diseases in a globe

Out of the losses due to various pests, weeds account for nearly one-third
Projected increase in global demand from 1993-2020
o Cereal : 41%
o Roots & tubers : 40%

Disease
22%
Pest
29%
Weed
37%
Other
12%

GENETICALLY MODIFIED PLANTS

A transgenic plant contains a gene or
genes artificially inserted (Transgene)
instead of the plant acquiring them
through pollination.
Transgene may come from another
unrelated plant or a completely different
species.
Plants containing transgenes are often
called genetically modified or GM crops.

Table 2: Cultivation worldwide of maize and GM maize in million ha
Area Area GM Proportion GM
Soyabean 90 69 77%
Maize 161 42 26%
Cotton 33 16 49%

Increase in Global cultivation areas of GM Maize in million ha


Why GM crops?
Growing human population
Loss of farmable land
Remediation of soil
Enrich nutrient content

AIMS OF BIOTECHNOLOGICAL RESEARCH IN MAIZE
Biotic stresses
Herbicide tolerance
Insect resistance
Disease resistance
Abiotic stresses
Drought tolerance
Salt tolerance and Heavy metal tolerance
Freezing tolerance
Plant development
Male sterility
Altered flowering time
Quality traits
Oil content and thus increased energy yields
Enhanced proportion of the amino acids
Raising the content of vitamins
Phytase corn as animal feed
Renewable products, energy crops
Production of energy from starch by ethanol
Source of raw materials for industrial products like plastic industries
Production of pharmaceutically active substances
Molecular pharming





DEVELOPING TRANSGENIC PLANTS




Case studies
Resistance to Biotic Stress
Insect Resistance
Virus Resistance
Herbicide Tolerance
Resistance to Abiotic Stress
Drought Tolerance
Salt Tolerance
Freezing Tolerance
Improving nutritional qualities
Pharmaceutical corn
Transfer of Bt genes: Even though the toxin
does not kill the insect immediately, treated
plant parts will not be damaged because the
insect stops feeding within hours.
RNA interference technology: RNA
interference (RNAi) is a process within living
cells that moderates the activity of their genes
RNAi is highly selective upon degrading an
mRNA target if the exogenously added dsRNA
shares sequences of perfect homology with the
target
Agrobacterium mediated method:
Transformation with Agrobacterium
tumefaciens is the preferred method for
delivery of transgene into a wide range of plant
species including maize.
Particle bombardment method: Both
immature zygotic embryos and immature
embryo derived callus can be transformed by
using this method.
Pollen tube pathway: DNA can be transferred
by cutting the stigma following pollination and
applying the DNA solution on the severed
style.
BIOTIC STRESSES


Bohorova et al., (2001) introduced fusion cry1B-cry1Ab gene in maize by micro projectile
bombardment. Southern blotting results indicated that gene was integrated into maize
genome. They reported co-expression of the cry1B-1Ab gene which was obtained in 17
transgenic lines and confers resistance to south western corn borer.



Baum et al., (2007) introduced WCR gene containing V-ATPase cassettes in maize by
agrobacterium mediated transformation. Detection of transformed plants was done by PCR
and southern blot analysis while transgene expression analysis was done by RT-PCR. They
demonstrated that ingestion of double-stranded (ds)RNAs supplied in an artificial diet
triggers RNA interference in western corn root borer, resulting in larval stunting and
mortality. Transgenic corn plants engineered to express WCR dsRNAs show a significant
reduction in WCR feeding damage.




Shepherd et al., (2007) introduced rep
1-219Rb-
gene from mutated MSV replication in maize
by particle bombardment. Transcript verified by PCR and southern blot analysis while
transgene expression done by RT-PCR. Transgenic plants displayed a significant delay in
symptom development, a decrease in symptom severity and higher survival rates than non-
transgenic plants after MSV infection.


Zhang et al., (2010) transferred P1 protein (protease) gene of MDMV by agrobacterium-
mediated transformation. They showed that integration of the hpRNA expression construct
was certified for nine transgenic lines by southern blotting. Transgenic lines h2, 13, and h1
increased significantly compared to non-transformed control line 18-599 and not
significantly different from the highly resistant control line H9-21 for disease incidence.




Heck et al., (2005) transferred dual CP4 EPSPS transgene cassettes in maize by particle
bombardment. They showed that NK603 event exhibited high glyphosate tolerance with a
minimum of target sequence disruption which remained stable over more than eight
generations. It showed lack of chlorosis and malformation injury after two sequential
applications of 1.68 kg acid equivalents (a. e.) ha
-1
glyphosate.
ABIOTIC STRESSES



Dong et al., (2011) introduced TPS gene into maize inbred line Zheng58 by pollen-tube
pathway. PCR, Southern blots confirms integration of trasgene. They reported that
transgenic plants contain higher proline content more than 25% except for one, reaching
196.20 g / g, which was about 2.5 times higher than that of the non transformed control
plants and also chlorophyll content of some transgenic plants was far higher, reaching
2.330 mg/g, twice higher than control plants suggesting drought resistance capability of
some transgenic plants is better than that of non-transgenic plants.




Wu et al., (2008) introduced BADH gene construct into maize by pollen-tube pathway
which was composed of only the BADH gene, expression regulatory sequence and T-DNA
border sequence at both sides. Southern blotting results indicated that the BADH gene was
integrated into maize genome. Transgenic lines of progeny were tolerance under salt stress
higher and that contains higher glycine betaine and chlorophyll content than wild type in
salt stress.


Chen et al., (2007) introduced OsNHX1 gene from rice & bar genes in maize by particle
bombardment. PCR, Southern and Northern blots confirms integration. The maize plants
over-expressing OsNHX1 accumulated more biomass when grown in the presence of
200mM NaCl in greenhouse conditions. Higher Na+ and K+ content was observed in
transgenic leaves than in wild type leaves when treated with 100~200mM NaCl, while the
osmotic potential and the proline content in transgenic leaves was lower than in wild-type
maize. A field trial revealed that the transgenic maize plants produced higher grain yields
than the wild-type plants.




Shou et al., (2004) transferred tobacco NPK1 gene in maize by agrobacterium-mediated
transformation. They demonstrated that constitutive expression of the Nicotiana PK1 gene
enhances freezing tolerance in transgenic maize plants. Two NPK1-transgenic maize events
were able to withstand up to 2C lower freezing temperature compared with their non-
transgenic lines, which would dramatically minimize yield loss due to frost damage.
PLANTS ENHANCED WITH NUTRIENTS



Naqvi et al., (2009) introduced 5 constructs; selectable marker bar & 4 genes (psy1, crtl for
-carotene, dhar for ascorbate and GCH1 for folate) in maize by particle bombardment.
Transcript was verified by PCR and northern blot analysis. They reported that transgenic
kernel contained 169-fold the normal amount of -carotene, 6-fold the normal amount of
ascorbate and double the normal amount of folic acid.




Huang et al., (2008) introduced inverted repeat sequence of LKR/SDH cDNA & CordapA on
expression cassette in maize for expression of a lysine. Suppression of LKR/SDH in the
endosperm tissue increases free lysine to 1324 ppm (~30 folds increase). The combination
of CordapA expression and LKR/SDH suppression in a single transgene produces over
4000 ppm free lysine (~100 folds increase), the highest ever reported in corn kernels.





Bicar et al., (2007) introduced -lactalbumin expression construct in maize by particle
bombardment. They reported that total protein content in endosperm from transgenic
kernels was not significantly different from non-transgenic kernels, whereas the lysine
content of the lines examined was 2947% greater in transgenic kernel endosperm. The
content of some other amino acids was changed to a lesser extent.










PHARMACEUTICAL CORN
One day, we may get vaccinated just by eating a few kernels of GM corn and that would be
good news for people with needle phobia.


Streatfield et al., (2002) introduced Lt-B gene encoding the barley -amylase secretion of
an E. coli strain of human origin in maize by agrobacterium-mediated transformation.
Transcript verified by PCR and southern blot analysis confirming that transgene was
successfully inserted. They reported that Lt-B was highly expressed in transgenic corn seed
at up to 1.8% of the total soluble protein (TSP) in T1 plant and this was further increased
approximately 9.2% TSP in a single seed of T3 plant five-fold through plant breeding.
Phytase corn
Phosphorus , essential element for growth and development of all animals.
However, phosphorus in corn is enclosed in an indigestible form (phytate) which is
not readily absorbed by animals. As a result, farmers add the enzyme phytase as an
additive in animal feed to release phosphorus from phytate. It is estimated that the
addition of phytase can increase 60% phosphorus absorption.
Currently, phytase is produced by microorganisms and purchased separately with
corn. The insertion of a phytase gene into corn allows the plant to produce
kernels containing high levels of the phytase enzyme. This, on one hand, can help
improve the nutritional value of livestock feed also better digestion of phytate
can reduce phosphorous pollution caused by animal waste. Since the animal can
absorb more phosphorous directly from their feed, the need for phosphate
supplements and cost of feed can also be reduced.
Recently, the phytase corn has passed a safety evaluation in China and has been
granted with a safety certification for commercial production. It is expected that
this GM phytase corn will soon be available on market as feed for livestock animals.

RECENT ADVANCES IN TRANSGENIC MAIZE RESEARCH




SIGNIFICANCE OF BACKCROSS BREEDING IN TRANSGENIC TECHNOLOGY
It enables breeders to transfer a desired trait such as transgene from one
donor parent into favoured genetic background of another recurrent parent.
If trait is dominant gene, process requires four rounds of backcrossing within
seven seasons and if recessive gene is transferred it requires more seasons.
Number of donor parent genes, that are removed and recurrent parent genes
recovered in the genetic makeup of the plant can be calculated using the
number of backcross generation utilized.
ADVANTAGES
To check environmental pollution by reduce use of pesticide
Biological defence against diseases, stresses, pests, weeds, herbicides, and viruses
Increased crop yield and thus food security
Increase in food carbohydrate content
Use of Land and Recovery for forestry and recreation
Improvement in nutritional quality and health benefits as nutritional security
Development of Speciality corns e.g. PHARMA CORN and PHYTASE CORN
Manufacture of edible vaccines, drugs and source of raw materials for industries
including bio fuel and bio-plastic industries
Encourages the development of scientific research and job creation
DISADVANTAGES
Gene flow / Transgene Escape: Maize being cross pollinated crop transfer its
pollen to other normal maize plant causing transgene escape.
Insect Resistance: Insect have ability to adopt resistance to environmental
pressure of insect resistant varieties.
Human health problems: Toxicity, allergencity and human health problems
Increased weediness: Possibility of becoming super weeds from herbicide
resistant varieties.
Loss of Biodiversity/reduction of cultivars: mono-culturing leads to biological
desert.
Non-target effects: Some transgenic plants may cause effect on non target parasites
and predators.


ACHIEVEMENTS
Total 65 events approved for GM maize across the world.
Companies involved
in GM corn
commercialization
Insect
resistance
Herbicide
resistant
Insect +
Herbicide
resistant
Drought
tolerance
High
Lysine
content
Monsanto MON89034,
MON863
NK603 MON89034 x
NK603
MON87460 -
Syngenta Seeds MIR604 ,
Bt 11
GA21 Bt11 x
MIR162 x
GA21
- 3272
Pioneer HiBred 1507, 59122 98140 59122 x
NK603
- -
Mycogen Seeds
(Dow Agro
Sciences)
- - 1507 x
59122 ,
TC6275
- -
Bayer Crop Science
(Aventis Crop
Science)
CBH-351 T25 - - -
Renessen LLC - - - - LY038
DeKalb Genetics
Corporation
DLL25
(B16)
DBT418


Utilizations of GM maize
Different GM maize lines (Events) approvals worldwide
For cultivation As food stuff/feed
Japan 30 35
Canada 35 26
Philippines 5 35
Corea 2 29
USA 27 29
Europe 2 17
Mexico - 29
Taiwan - 23
Australia - 16
China - 12
Brazil 11 11
Argentina 10 9
South Africa 4 9
Colombia 1 5
Russia - 4
El Salvador - 3
Egypt 1 1
Uruguay 2 2
Traits Herbicide tolerance, Insect resistance, virus resistance, high Lysine content,
amylase producing

FIELD TRIALS WITH GM MAIZE
Worldwide
Total 7200
Period 1996-2010
Countries Argentina, Australia, Canada, Japan, Columbia, China, South Africa,
Kenya, India, Cuba, Uganda, Tanzania, Zimbabwe

STATUS OF GM CORN RESEARCH IN INDIA
GEAC, in December 2010, gave permission for field trials of BRL I & BRL-II of GM corn
developed by Monsanto, Syngenta and Pioneer.


PERMITS ISSUED FOR FIELD TRIALS OF GM CORN IN 2011

Company
Name
Gene/Event Trial Hybrids
Monsanto
India Ltd.
Stacked cry2Ab2 and
cry1A.105 genes (Event
MON 89034) &
CP4EPSPS genes (Event
NK603)
BRL-II
trial
Two transgenic corn hybrids
namely Hishell and 900M Gold
containing
stacked events MON89034 x
NK603
Syngenta
Biosciences
Pvt. Ltd.
StackedBt11 x GA21
event (cry1Ab &
mepsps genes)
BRL-I
2nd
year
trial
Corn hybrids namely NK6607
containing GA 21 event
(mepsps gene) and one
transgenic corn hybrid namely
NK6240 containing stacked Bt11
x GA21 event(cry1Ab &
mepspsgenes)
Pioneer
Overseas
Corporation
cry1F, cp4epsps & PAT genes
[stacked events TC1507 x
NK603 (DAS-01507-1 x
MON-00603-6)]
BRL-I
2nd
year
trial
Hybrids namely 30V92HR and
30B11HR








CONCLUSION:
Biotechnology can be tool of plant breeding for providing food for increasing
population and reducing malnutrition problems in developing countries like Africa
and South Asian countries.
RNAi provides a unique mode of action for the control of insect pests that could
complement the current strategy of expressing B. thuringiensis (Bt) insecticidal
proteins.
The transgenic NK603 event has remained stable over more than eight generations
through glyphosate tolerance testing.
Vector free maize transformation system based on pollen tube pathway avoids
biosafety dispute and TPS transgenic plants contain higher proline and chlorophll
content in drought stress, while BADH transgenic plants contains higher glycine
betaine and chlorophyll content under salt stress.
The transgenic kernels contained 169-fold the normal amount of -carotene, 6-fold
the normal amount of ascorbate, and double the normal amount of folate.
GM corn can help in minimizing environmental pollution and also use for producing
vaccines and drugs.
Tissue culture is essential for regeneration of transformed plant.
Transgene instability may leads to alteration of nutritional values of corn.


FUTURE PROSPECTS:
Need to evaluate carefully GM corn for toxicity to non target organisms using
scientific methods on whole ecosystems, rather than on individual test species
before commercial release.
Bio safety measures should be critically studied before adoption of transgenic corn
technology.
Need to develop transgenic corn for important diseases like Downy mildew and
bacterial diseases.
Need for strengthening development of transgenic corn used for industrial
applications in bio fuels and bio-plastic industries.
Need for stacking or pyramiding multiple genes conferring different stresses into
the same plant.
A certain level of national research and regulatory capacity are pre-requisites, along
with effective IPR management and input supply systems, especially for transgenic
seeds.









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