Agriculture Reference
In-Depth Information
Chloroplast. transformation. offers. several. advantages.
77,80-82
. First,. each. plant. cell.
contains.a.large.number.of.chloroplasts,.with.some.having.up.to.100.chloroplasts.per.
cell.
83
.Also,.each.chloroplast.contains.50-100.or.more.copies.of.its.own.genome
84
..As.
a.consequence,.if.a.trait.gene.is.incorporated.into.the.chloroplast.genome,.the.gene.can.
be.enriched.up.to.100,000.copies.per.each.cell..High.copy.number.of.transgenes.per.
cell.leads.to.high.expression.levels..For.example,.47%.leaf.total.proteins.were.the.
Bt
.
cry2Aa2
.protein.if.the.
Bt
.
cry2Aa2
.gene.was.introduced.into.the.choloroplast.genome.
of. tobacco.
85
. Second,. transgene. expression. via. chloroplast. transformation. is. more.
stable.because.gene.silencing.that.can.affect.expression.of.nuclear.transgenes.appears.
to.have.little.effect.on.transgenes.that.are.introduced.into.the.chloroplast.genome.
86
.
Third,. for. plant. species. in. which. chloroplasts. are. inherited. exclusively. through. the.
female.line,.chloroplast.transformation.can.provide.an.effective.means.of.preventing.
transgenes.from.transferring.to.non-transgenic.crops.or.wild.relatives.through.pollen.
In. spite. of. the. technical. challenges. in. transforming. chloroplasts,. stable. incor-
poration. of. trait. genes. into. chloroplast. genomes. has. been. reported. in.
Arabidopsis
thaliana
,.cotton,.poplar,.rice,.and.soybean.
87,88
.Also,.chloroplast.transformation.has.
been.described.in.a.number.of.horticultural.crops.such.as.cabbage,
89
.caulilower,
90
.
carrot,
91
.egg.plant,
92
.lettuce,
93,94
.bladderpod,
95
.petunia,
96
.tobacco,
97-100
.potato,
101
.and.
tomato.
102-104
.Genetic.transformation.of.chloroplasts.should.provide.an.effective.tool.
to.reduce.pollen-mediated.transgene.low.in.these.horticultural.species.whose.chlo-
roplasts.are.inherited.from.female.parents..On.the.other.hand,.problems.associated.
with. the. seed-mediated. transgene. escape. will. not. be. addressed. with. a. chloroplast.
transformation.strategy.
G
ene
d
eletor
t
echnoloGy
Gene.deletor.technology.refers.to.techniques.that.can.eliminate.all.transgenes.in.a.
particular. organ. or. in. the. entire. plant. when. transgene. functions. are. completed. or.
the.transgene.presence.is.of.concern.
105-108
.The.principle.of.the.gene.deletor.technol-
ogy.is.illustrated.in.Figure.14.1..All.transgenes,.such.as.trait.and.selection.marker.
genes,.are.placed.within.the.two.
loxP-FRT
.hybrid.sites..In.addition,.the.
FLP
.or.
Cre
.
recombinase.gene.under.the.control.of.a.conditionally.active.(e.g.,.organ-.or.tissue-
speciic,.or.developmental.stage.speciic).gene.promoter.sequence.is.inserted.into.the.
two.
loxP-FRT
.sites..If.a.pollen.and.seed-speciic.gene.promoter.such.as.
PAB5
.from.
Arabidopsis
109
. is. used. to. control. the. expression. of. the.
FLP
. gene,. the. FLP. protein.
will.be.produced.only.in.pollen.and.seed..We.have.shown.that.transgenic.plants.have.
transgenes.present.and.functional.in.all.organs.during.the.plant.life.cycle.except.in.
seed. and. pollen.. Because. of. pollen-. and. seed-speciic.
FLP
. expression,. all. trans-
genes.except.an.86-bp.nonprotein.encoding.
loxP-FRT
.fusion.sequence.are.excised.
from.pollen.and.seed..The.excised.DNA.sequence,.consisting.of.one.loxP-FRT.site,.
the.trait.gene,.the.selection.marker.gene,.and.the.
FLP
.gene.should.be.destroyed.by.
nonspeciic.nucleases.in.the.cell.
110,111
Site-speciic.recombination.systems.(e.g.,.bacterial.phage.
Cre/loxP
.and.
Saccha-
romyces cerevisiae FLP/FRT
). function. through. interactions. of. a. recombinase. with.
its.speciic.recognition.sites.
112-115
.Recombinase-mediated.excision.between.directly.
oriented.recognition.sites.results.in.removal.of.the.intervening.DNA.leaving.one.
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