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Lec 8 - Genetic Modification of Crop Plants.docx

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Department
Environmental Science
Course Code
EESB16H3
Professor
Zachariah Campbell

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Genetic Modification of Crop
Plants
- DNA construct: artificially constructed segment of nucleic acid that is going to be
transplanted into a target tissue or cell
oDNA must integrate into genome of plant. This happens but we don’t
know why
oTransgenes are often linked to a strong constituitive promoter such as
Cauliflower Mosaic Virus 35S (CaMV35S). This promoter allows for
genes to be turned on all the time
oTransgenes can be linked to tissue specific promoters as well
These transgenes will only be turned on in some tissues (i.e.
anthers, seeds)
oTransgenes can be linked to condition specific promoters
These transgenes will only be turned on in presence of light, heat,
fluctuations of these parameters
- Dicot: a flowering plant with embryo that bears two cotyledons (seed leaves);
typically have broad, stalked leaves with netlike veins (daisies, oaks, etc.)
oMonocot: a flowering plant with embryo that bears one cotyledon (seed
leaf); typically have elongated stalkless leaves with parallel veins (grasses,
lilies, palms, etc.)
Engineering Dicots
- Totipoteny: the ability of a cell to give rise to unlike cells and so to develop a new
organism or part
- The Crown Gall Bacterium (agrobacterium) is causal agent of crown gall disease
(formation of tumours)
oSymptoms caused by insertion of small segment of DNA from a plasmid
into plant cell
These bacterium are pathogenic, not symbiotic (in the sense that
they don’t fix nitrogen like rhizozobes)
Serious pathogen of walnuts, grape vines, stone fruits, nut trees,
sugar beets, horse radish, rhubarb
oUsually, its contains a Ti plasmid (Ti=tumour inducing)
Ti plasmid lost when bacteria grown above 28oC
oPlasmid is integrated semi-randomly into genome of host cell. Region of
plasmid called T-DNA (phyto-oncogenes) is taken up by plant cell and
incorporated into one of its chromosomes.
The tumor morphology genes in Ti-plasmid are expressed causing
formation of gall
Ti-DNA carries genes for biosynthetic enzymes the produce
unusual AA like octopine or nopaline, as well as plant hormones
like auxin and cytokinins, and for opines
By altering hormone balance in plant cell, the division of cells
cannot be controlled by plant and tumour forms
- Flavr Savr Tomato, 1994  Calgene (Campbells Soup) inserted an antisense gene
into tomato strain using Agrobacterium tumefaciens
oAntisense gene had sequence complementary to that of naturally occurring
polygalacturonase gene (PG-ripening enzyme that degrades pectin)
Pectin is present in ripe fruits and extracted for use as setting agent
in jams and jellies. It is gelatinous polysaccharide
oAnti-PG mRNA and PG mRNA bind and prevent translation
Since antisense gene is complementary to PG gene, its RNA is
complementary to PG mRNA. It forms doublestranded RNA
structure, that cannot be translated into functional protein
Less PG, means more pectin, meaning fruits are ripe for longer
oTomato can redden and ripen longer, and still be picked in good condition
for long distance transportation
Was supposed to help with soup production but marketed as fresh
fruit, and sales flopped
There was controversy over presence of kanamycin gene (marker
gene) in the strain
- Virus resistant Yellow Crookneck Squash, 1994 Upjohn and Asgrow Seed
engineered squash strain to express viral coat protein genes, via Agrobacterium
oSpecific genetic modification caused yellow crookneck squash to be
resistant to infection by two plant virus. By inserting coat protein encoding
sequences from those two RNA plant virus, they introduced viral
sequences to plant and it developed its response to fight off virus
oImparted resistant to Watermelon Mosaic Virus, Zucchini Yellow Mosaic
Virus
- Laurate Canola, 1995  Canola (rapeseed) engineered with thioesterase (enzyme
that hydrolyzes thiol esters into acids and alcohols or phenols)
oGene derived from tree species allows for production of laurate from
precursors
Laurate: fatty acid used in shampoos, etc.
oAllowed canola to replace tropical palm and coconut oils
- Bromoxynil Resistant Cotton, 1995  Calgene, Rhone Poulenc Ag, Stoneville
Pedigree Seed
oCotton engineered with bacterial gene that encoded nitrolase enzyme
Nitrolase degrades herbicide bromoxynil, so introduction of this
gene allows farmers to use bromoxynil on this crop
oSeeds for bromoxynil resistant cotton are 41% more expensive than
conventional variety
- “Round Up Ready” Soybeans, 1996  produced by Monsanto as their patent for
glyphosate (herbicide =Round Up) was coming to an end
oUsually glyphosate kills plants by interfering with their ability to make
essential AA.
Roundup Ready Soybeans express enzyme that make those AA
Got those genes in there using Agrobacterium
oSoybeans engineered with bacterial genes for degradation of glyphosate
oSo farmer must buy seed AND Round Up from Monsanto
oSupposed to decrease overall herbicide use
- Bt protected Potato, Corn, Soybean  Monsanto, Ciba Seeds
oToxin genes from Bt engineered into wide variety of crops to provide built
in insect resistance
oBt gene of choice depends on pest of the particular crop
- Golden Rice, 2000  rice engineered to produce beta-carotene, the precursor for
Vitamin A
oInvolved 4 different enzymes
oThere isn’t enough Vitamin A produced to justify the hype because it is not
enough to cure VAD
- “Liberty Link” crops are resistant to glufosinate, through genetic engineering
oGlufosinate inhibits glutamine synthase enzyme in plants and animals,
making them tolerable to herbicides and glufosinate
oThe only other GMO herbicide tolerant gene other than RoundUpReady
oIt was a neurotoxin, teratogen, mobile and persistent, and leaves residues
on food
oDegrades to MPPA which even more persistent and still a neurotoxin
- Engineering of inducible male sterility in parent plant allows for easier
hybridization between male sterile plant and other parent
oEnhances ease of production of F1 hybrids
- To become resistant to drought, crop would need hundreds of genes altered, not
just one
oCurrently no verified existence of genetically modified crop for drought
resistance
oDrought resistance varieties still produced via classical crop breeding and
selection from diversity of strains
Genetic Use Restriction Technologies (GURT)
- GURT, or terminator technology, first developed and patented by USDA and
Delta and Pineland. The latter merged with Monsanto
oAKA suicide seeds
oGURT: proposed methods for restricting the use of genetically modified
plants by causing second generations to be sterile
- Two kinds of GURT:
oV-GURT – variety based – plants are engineered to kill their own seed
embryos, so that all offspring are sterile (unless an in house trigger
prevents this)

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Description
Genetic Modification of Crop PlantsDNAconstructartificiallyconstructedsegmentofnucleicacidthatisgoingtobetransplantedintoatargettissueorcelloDNAmustintegrateintogenomeofplantThishappensbutwedontknowwhyoTransgenesareoftenlinkedtoastrongconstituitivepromotersuchasCauliflowerMosaicVirus35SCaMV35SThispromoterallowsforgenestobeturnedonallthetimeoTransgenescanbelinkedtotissuespecificpromotersaswellThesetransgeneswillonlybeturnedoninsometissuesieanthersseedsoTransgenescanbelinkedtoconditionspecificpromotersThesetransgeneswillonlybeturnedoninpresenceoflightheatfluctuationsoftheseparametersDicotafloweringplantwithembryothatbearstwocotyledonsseedleavestypicallyhavebroadstalkedleaveswithnetlikeveinsdaisiesoaksetcoMonocotafloweringplantwithembryothatbearsonecotyledonseedleaftypicallyhaveelongatedstalklessleaveswithparallelveinsgrassesliliespalmsetcEngineering Dicots TotipotenytheabilityofacelltogiverisetounlikecellsandsotodevelopaneworganismorpartTheCrownGallBacteriumagrobacteriumiscausalagentofcrowngalldiseaseformationoftumoursoSymptomscausedbyinsertionofsmallsegmentofDNAfromaplasmidintoplantcellThesebacteriumarepathogenicnotsymbioticinthesensethattheydontfixnitrogenlikerhizozobesSeriouspathogenofwalnutsgrapevinesstonefruitsnuttreessugarbeetshorseradishrhubarboUsuallyitscontainsaTiplasmidTitumourinducingoTiplasmidlostwhenbacteriagrownabove28CoPlasmidisintegratedsemirandomlyintogenomeofhostcellRegionofplasmidcalledTDNAphytooncogenesistakenupbyplantcellandincorporatedintooneofitschromosomesThetumormorphologygenesinTiplasmidareexpressedcausingformationofgall
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