RECENT ADVANCES IN GENETICALLY MODIFIED CORN RESEARCH

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. TalatiSeminar co-ordinator,Dept. of Agril. Biochemistry,B.A.C.A., AAU, Anand.

Soni Nishit Vasantbhai M.Sc. (Agri.) 3rd semesterGenetics & Plant breedingReg. No. : 04-1354-2010

Major Guide:Dr. S M. Khanorkar

Associate Research ScientistMain 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 – ZeaSpecies – 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

3rd 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

teosinte

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…

Disease22%

Pest 29%

Weed37%

Other12%

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%

Oerke et al.,2004Germany

GENETICALLY MODIFIED PLANTS

How the GM Plants Produced?

• 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

Source: ISAAA Brief No 41-2009

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

Source : http://www.gmo-compass.org/eng/database/plants/52.maize.html

Case studiesResistance to Biotic Stress

Insect Resistance

Virus Resistance

Herbicide Tolerance

Resistance to Abiotic Stress

Drought Tolerance

Salt Tolerance

Freezing Tolerance

Improving nutritional qualities

Pharmaceutical corn

BIOTIC STRESSES

Tissue culture required to generate transgenic plants.

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.

Case 1: Transgenic maize for South western corn borer (Diatraea grandiosella) resistance

cry1B-cry1Ab gene in Maize by micro projectile bombardment

Expression analysis bySouthern Blot Analysis

L 1: Plasmid DNAL 2-10: T0 transgenic plants L 11: CML216 : non-transformed, Pstl : molecular-weight marker

Transgenic Maize

pAc35SAct cry1B-1Ab

Genes expressed protein that active against south western corn borer

Bohorova et al., 2001Mexico

Fig 1 : cry1B-1Ab gene expressed in transgenic maize resistance to South western Corn Borer(SWCB)

TG CONTROLCONTROL TG

Result : The co-expression of the cry1B-1Ab gene has been obtained in

17 transgenic plants that confers resistance to south western corn borer.

Transgenic

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.Case 2 : Resistance to Western corn

root borer (Diabrotica virgifera) through RNA interferenceV-ATPase cassettes containing WCR gene in Maize by Agrobacterium-mediated

transformation

Transgenic Maize

WCR genes caused mortality when targeted for suppression using dsRNAs in the WCR feeding assay.

Northern analysisPCR

Map of the expression cassette

Detection of Transformed Plants

Baum et al., 2007USA

Gene expression analyzed by

Quantitative RT- PCR

Target genes: gene encoding V-type ATPase A, demonstrated rapid knockdown of endogenous mRNA within 24 h of ingestion and triggered a specific RNAi response with low concentrations of dsRNA.

Fig 2: Northern blot analysis of transgenic events expressing the WCR V-ATPase-A dsRNA transcript.

Non transgenic Transgenic

Fig 3: Comparison of transgenic and non-transgenic plant for average root damage

Graph 1: Mean root damage ratings for eight transgenic events, the parental inbred line (negative control) and the corn rootworm-protected Cry3Bb event MON863

Result : -> There was no RNA from the WCR V-ATPase-A cassette in events 1 and 2, which had the most root damage.

-> Transgenic corn plants engineered to express WCR dsRNAs show a significant reduction in WCR feeding damage, suggesting that the RNAi can be

exploited to control insect pests.

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.

Case 3: Maize streak virus-resistant transgenic maize

rep1-219Rb- gene from mutated MSV replication in Maize by Particle bombardment

Transgenic Maize

Southern analysisPCR

Transcript verified by Gene expression analyzed by RT- PCR

MSV rep plasmid prep1-219Rb- consists of rep1-219Rb- gene between the maize ubiquitin (Ubi) promoter & A. tumefaciens nopaline synthase (Nos) terminator in pAHC17

vector pAHC17

Shepherd et al., 2007South Africa

Graph 2: (A) Chlorotic areas on leaves and (B) Percentage alive of transgenics (♦) and non transgenics (■)

Fig 4: Comparison of transgenics and control under non-symptomic and symptomic conditions at 20 dpi.

T NT

NT T

non-symptomaticsymptomatic

(A)

(B)

dpi= days to post inoculation

Shepherd et al., (2007) introduced rep1-219Rb- 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.

Transgenic WM3 × 7E plants (♦)Non-transgenic WM3 × 7E (□) and WM3 plants (■)

Graph 3: Symptomatic infection rates

Graph 4: Percentage of plants

Grew to maturity (First column) Yielded seed (Second column) Symptomatic plants that grew to maturity (Third column) Yielded seed (Forth column)

Case 4: Maize Dwarf Mosaic Virus (MDMV) resistant by RNA interference

P1 protein (protease) gene of MDMV in Maize by Agrobacterium-mediated transformation

Transcript verified by

Transgenic Maize

Zhang et al., 2010China

The expression construct was under the control of the ubiquitin promoter and nos terminator, generating the expression vector pASP150.


pASP150

PCR analysis of transformed plantsLane 1 : non-transformed control 18-599Lane 2 : positive control of expression vector pASP150Lane 3-20 : transformed plants

putative transgenic T2 lines positive in PCR Lane 1 : non-transformed control 18-599lanes 2–14 : transformed plant lineslane 15 : plasmid control of hpRNA expression

vector pASP150

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.

Table 3: MDMV resistance of T2 transformed plant lines

Fig 5: Performance of MDMV symptom in T2 plants and controls in field

T2 plant line H2

Non-transformed

control (18-599)

Susceptible control

(Mo17)

Resistant control

(H9-21)

Case 5: Development of Glyphosate-Tolerant Corn Event NK 603

Dual CP4 EPSPS transgene cassettes in Maize by Particle bombardment

Transgenic Maize

Heck et al., 2005USA


5-Enol-pyruvylshikimate-3-phosphate synthase from Agrobacterium sp. CP4 (CP4 EPSPS) confers tolerance to the nonselective herbicide glyphosate

Southern analysis

PV-ZMGT32

Western blot analysis

Transgene expression analyzed by

Fig 6: Southern analysis NK603 transgenic

Corn genomic DNA was digested with the restriction enzymes,

L 1) Non transformed; L 2) Transgenic B73 L 3) Transgenic NK603 B73 BC5S3 L 4) Transgenic B73 BC5S3. rice Act1 promoter probe; CP4 EPSPS probe; e35S promoter probe; NOS termination sequence probe.

Fig 7: Western blot analysis of CP4 EPSPS protein expressed over five generations of NK603

Corn inbred B73 was used as a recurrent parent in all generational material except for hybrid

L 1) molecular weight markersL 2,3,4) 5 , 2.5 , 1 ng of E. coli-expressed CP4 EPSPS; L 5) buffer blank; L 6) NK603 B73 BC0F1 L 7) LH82 x NK603 B73 BC1F1; L 8) NK603 B73 BC1F1; L 9) NK603 B73 BC5F1; L 10) LH82 x NK603 B73 BC2F3;L 11) B73 nontransgenic ;L 12) LH82 x B73 nontransgenic

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

Table: 4. Segregation data and analysis of progeny of line

NK603

1 Data expressed as number of positive and negative plants based on glyphosate sprays, except for the BC2F3 and BC4F3 generations where data are number of homozygous positive ear-rows, number of homozygous negative ear rows and number of segregating ear-rows based on glyphosate sprays.ns not significant at p= 0.05 (chi square = 3.84, 1 df).

# not significant at p= 0.05 (chi square = 6.99, 2 df).** significant at p = 0.01 (chi square = 6.63, 1 di).

Case 6: Transgenic maize for Drought tolerance by pollen tube

pathwayTPS (Trehalose synthase) gene from Saccharomyces cerevisiae H. , in Maize inbred line Zheng58 by Pollen tube pathway transformation

Expression analysis

Transgenic Maize


Gene has special function of preserving the vitality of organisms & protecting the cell membrane and protein structure effectively, so as to make organisms remain intracellularly moist in extreme cases

Dong et al., 2011China

PCR analysis of transformed plantsLane P : PlasmidLane N : Negative controlLane 1-10 : transformed plants

Analysis of T3 transgenic plants. Lane P : Plasmid Lane CK : Negative controlLane 1-3: Transgenic plants

Fig 8: Comparisons of control and transgenic maize plants in Greenhouse

Non-transgenic Line Transgenic Line

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 µg / 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.

Table 5: Proline and Chlorophyll content in T3 plants

Case 7: Transgenic maize for Salt tolerance by pollen tube pathway

BADH gene (betaine aldehyde dehydrogenase) of Suaeda liaotungensis kitag in Maize by Pollen tube pathway transformation

Transgenic Maize

Wu et al., 2008China

Transcript verified bySouthern analysis

RT-PCR

Transgene expression analyzed by

PCR of transformed samples

The construct of transformation element and PCR primer landing sites

BADH catalyzes the conversion of betaine aldehyde to glycinebetaine

L 1–3: DNA from transformed samplesL 4: DNA from the wild typeL 5: ddH2OL 6: pCAMBIA1301-BADH plasmids DNA

(A) HindIII(B) EcoRI

Fig 9: Southern blot analysis of transgenic plants and the wild type L: 1 transgenic plants, L : 2 wild type, L 3: M DNA Molecular Marker

Fig 10: RT-PCR analysis of transgenic samples and the wild type plants

L 1–3: DNA from transformed samplesL 4: DNA from the wild typeL 5: ddH2OL 6: pCAMBIA1301-BADH plasmids DNA

Fig 11: Improved tolerance of transgenic seedlings of maize to salt stress

Wild-type seedling

without salt stress

Fig 12: The growth of maize roots after salt stress

Wild-type seedlings with salt stress

Transgenic seedlings with salt stress

Transgenic Nontransgenic

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.

Table 6: Stress tolerance assay for transgenic maize progeny seedlings grown in the medium with 250 mM NaCl

Table 7: Glycine betaine, relative electrical conductivity and chorophyll content of maize BADH transgenic progeny plants under 250 mM NaCl 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

Case 8: Transgenic maize for Salt tolerance OsNHX1 gene & bar genes in Maize by Particle bombardment

Expression analysis

Transgenic Maize

Chen et al., 2007China

Northern analysisPCR

The Oryza sativa Na+/H+ antiporter gene OsNHX1 gene was under the control of the cauliflower mosaic virus (CaMV) 35S promoter, and the terminator region contained the polyadenylation signal of the nopaline synthase gene (Nos).

Southern analysis

L 1-4: Genomic DNA from transgenic plants Q 31-5, Q31-33, Z3-1, Z3-7L 5: Genomic DNA from non transformed plant

analysis of T1 generation transgenic plantsL 1-3: T1 transgenic plants Z3-1, Z3-7, Q 31-5L 4: non transformed plant

Control T1 plant

Control T1 plant

Fig 13: Non-transformed and Z3-1 transgenic plant (T1) treated with 200 mM Nacl

10 days 16 days

Table 8: Effects of 200mM NaCl on biomass of T1 plants & wild type plant

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.

Case 9: Freezing tolerance in transgenic maizetobacco NPK1 gene in Maize by Agrobacterium-mediated

transformation

Transgenic Maize

pSHX004

Quantitative RT-PCR

PCR

Shou et al., 2004San Diego

transgenic seedlings screened by expression analysis

Table 9: Comparison of transgenic plants and non transgenic segregates

NA, not available *Events were selected for freezing test

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 2°C lower freezing temperature compared with their non-transgenic lines, which would dramatically minimize yield loss due to frost damage.PLANTS ENHANCED WITH NUTRIENTS

Fig 14: (A) A4–9-transgenic and non transgenic seedling under graduated freezing . (B) A4–15 transgenic and non transgenic seedlings after 4-h constant freezing

treatment (-5°C).

Result : Two NPK1-transgenic maize events were able to withstand up to 2°C lower freezing temperature compared with their non-transgenic

plants. The 2°C improvement in the freezing tolerance would dramatically minimize yield loss due to frost damage that often occurs in spring and fall seasons, thereby stabilizing the productivity.

Case 10: Transgenic multivitamin corn through bio fortification of

endospermFour Genes and selectable marker bar in Maize by Particle bombardment.


Transgenic Maize

Naqvi et al., 2009Spain

Four genes encoding enzymes in the metabolic pathways for the vitamins

β-carotene, ascorbate and folate

Northern analysis

PCR

pUC18-based expression cassettes

L1: transgenic L 2: wild-type (WT) M37W corn.

Genes to be used for transformation

β-carotene levels: Corn phytoene synthase (psy1) and Pantoea ananatis carotene desaturase gene (crtl)Ascorbate levels : Rice dehydroascorbate reductase (dhar) Folate levels : E. coli folE gene encoding GTP cyclohydrolase (GCH1)

Orange-yellow phenotype of the transgenic endosperm. Normal phenotype of the WT M37W endosperm. (C) Comparison of WT and transgenic cobs,

showing significant increases in the levels of key carotenoids in the transgenic cobs.

Fig 15: Accumulation of carotenoids in the endosperm of transgenic corn line L-1.

Naqvi et al., (2009) introduced 5 constructs; selectable marker bar & 4 genes (psy1, crtl for -carotene, β 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 ofβ ascorbate and double the normal amount of folic acid.

Table 10 : Comparison of levels of carotenoid and other vitamins

Values are in microgram per DW

Huang et al., 2008USA

Case 11: Genetically Modified High Lysine Corn

Fig 16: A two-front approach to increase lysine content in corn kernels by a single transgene

DHDPS: dihydrodipicolinate synthaseLKR: lysine-ketoglutarate reductaseSDH: saccharopine dehydrogenase

Incorporated Inverted repeat sequence of LKR/SDH cDNA in expression cassette CordapA (from Corynebacterium glutamicum) in Maize

Transcript expression by

Transgenic Maize

The free lysine level in plant cells is thought to be regulated by lysine feedback inhibition of DHDPS and feed-forward activation of LKR/SDH.

Western blot analysis

Total protein extracted from individual kernels was run on Western blots & probed with either CordapA or LKR/SDH specific antibodies. The arrows indicate kernels showing the coordinated expression of CordapA and suppression of LKR/SDH.

Table 12: Lysine accumulation in mature corn kernels with modified lysine pathways

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.Case 12: Transgenic maize

endosperm containing a milk protein with improved amino

acid balanceα-lactalbumin expression construct in Maize by Particle bombardment.


Transgenic Maize

Bicar et al., 2007USA

α- lactalbumin was under control of maize gamma zein promoter and the nos terminator. The expression vector designated P64.

Western analysis

Southern analysisEvaluation of α-lactalbumin accumulation in generations by

analysis of F1 , F2 & F3 progenies from 2 events.L 1 : DNA from an F1

L 2 : DNA from an F2 L 3-4 : DNA from 2 F3 sibling progenies. Lane P : 5 pg of plasmid DNA representing 1 copy per diploid genome

L 1 : Human α-lactalbuminL 2 : P64-18 (negative) L 3 : P64-18 (positive) L 4.: P64-14 (negative)L 5 : P64-14 (positive) L 6 : Untransformed maize inbred B73

Bicar et al., (2007) introduced -lactalbumin expression construct in maize by particleα 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 29–47% greater in transgenic kernel endosperm. The content of some other amino acids was changed to a lesser extent.

* = difference between positive and negative

Table 13: Endosperm amino acid composition and total protein content of

α-lactalbumin protein positive and negative plants kernels in event 14

PHARMACEUTICAL CORNOne 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.

Case 13: Development of an edible vaccine in corn against enterotoxigenic strains of E. coliLt-B gene of an E. coli strain of human origin in Maize by Agrobacterium-

mediated transformation


Transgenic Maize

Streatfield et al., 2002Texas

The Lt-B transformation vector was designated as PGN7101.


Analysis of T0 plants L 1-9: transformed plantsL 10 : controlL 11 : Lt-B plant transformation vector PGN7101

Analysis of T0 plants L 1-9: transformed plantsL 10 : control

Streatfield et al., (2002) introduced Lt-B gene encoding the barley -amylase secretion ofα 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.

The highest expressing lines of Lt-B corn was backcrossed through two further generations, into elite inbred lines, Lancaster and Stiff Stalk lines.

RESULT :Breeding improved the agronomic quality of the transgenic plants and also further boosted accumulation of Lt-B in the seed by 1.9- to 2.6-fold per generation. In the highest expressing example of T3 seed carrying Lt-B, accumulation is elevated to 9.2% TSP in a single seed, an approximately five-fold improvement over expression in T1 seed.

Fig 17: Expression levels of Lt-B achieved in corn lines transformed with PGN7101 in T1, T2 and T3 generation

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

- - 1507 x

59122 ,

- -

Sciences) 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,

http://www.isaaa.org/gmapprovaldatabase/cropevents/default.asp?CropID=7&Crop=Maize



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 INDIAGEAC, 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)



Bihar (two locations)Tamil Nadu Karnataka Andhra Pradesh Bihar (two locations)Tamil NaduKarnatakaAndhra Pradesh Uttar PradeshRajasthanGujaratMadhya Pradesh

Source: THE HINDU News article, NEW DELHI, march 9, 2011





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.

Source: http://igmoris.nic.in/GENES_EVENTS2011.htm

“It is pretty clear that GM maize is absolutely necessary to increase production and productivity and to save marginal farmers from the high cost of agriculture”

-Dr. Sain Dass Former Project director,

DMR, New Delhi.

• The transgenic kernels contained 169-fold the normal amount of β-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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RECENT ADVANCES IN GENETICALLY MODIFIED CORN RESEARCH

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