Transgenic Fruit and Nut Tree Crops Review - Transgenic Horticultural Crops: Challenges and Opportunities - page 4

genotypes..Most.of.the.basic.protocols.were.developed.several.years.ago.and.subse-
quent.efforts.have.been.oriented.toward.optimization.or.adaptation.of.the.technique.
to.new.genotypes..Unfortunately,.the.time.required.to.produce.transgenic.fruit.trees.
is.too.long,.especially.for.fruit.character.analysis,.and.there.is.a.need.for.faster.sta-
ble.transformation.systems.. Transient.expression.systems.are.less.time-consuming.
methods.that.are.receiving.more.attention.as.a.tool.for.gene.functional.analysis..The.
production.of.marker-free.transgenic.plants.is.another.growing.area.of.interest.due.
mainly.to.consumer.concerns.
Genetic Transformation via Organogenesis
Although.a.common.factor.in.many.transformation.protocols.is.the.use.of.embryo-
genic. cultures. as. starting. material. for. transformation,. some. species. have. been.
successfully. transformed. through. the. use. of. organogenesis.. This. system. offers.
an.alternative.approach.for.genotypes.that.are.recalcitrant.to.plant.regeneration.
by.somatic.embryogenesis,.although.the.probability.of.obtaining.chimeric.plants.
is.higher.
Current. protocols. for. the. production. of. transgenic. citrus. involve. the. transfor-
mation. of. cells.in. seedling. stem.pieces.and. regeneration. of.shoots. from.organo-
genic.cultures..The.irst.reliable.protocol.reported.the.transformation.of.the.citrus.
relative. Poncirus trifoliata . by. cocultivation. of. etiolated. epicotyl. segments. with.
Agrobacterium tumefaciens . 8 .A.similar.transformation.procedure.with.modiica-
tions.has.been.used.to.produce.transgenic.plants.of.other.citrus.genotypes. 9 .In.general,.
rooting. of. transgenic. shoots. in. citrus,. except. for. P. trifoliata . and. grapefruit,. is.
rather.ineficient.but.can.be.alleviated.by.the.use.of.shoot.tip.grafting. 9,10 .Mature.
tissues.have.also.been.used.as.explant.material.in.order.to.maintain.genotype.iden-
tity.and.overcome.the.long.juvenile.growth.phase..Buds.collected.from.trees.are.
grafted.onto.seedlings.of.a.vigorous.rootstock.grown.under.glasshouse.conditions;.
new.shoots.elongated.from.them.are.then.used.as.starting.material. 9
Transformation.via.organogenesis.has.also.been.applied.in.several.other.fruit.tree.
species..For.example,.apple.transformation.was.irst.reported.by.James.et.al.,.who.
used. Agrobacterium -mediated.transformation.of.leaf.disks.from.the.apple.cultivar.
'Greensleeves'. 11,12 .Although.somatic.embryogenesis.is.the.most.prevalent.regenera-
tion.method.used.in.grape.genetic.transformation,.transgenic.plants.of.table.grape.
cultivars. 'Silcora'. and. 'Thompson. Seedless'. have. also. been. produced. via. organo-
genesis.. The. method. is. based. on. the. formation. of.meristematic. bulk.tissue. with. a.
high. regenerative. capacity,. using. adventitious. shoots. as. a. starting. material.. Shoot.
regeneration.is.obtained.30.days.after.meristematic.bulk.slices.are.inoculated.with.
Agrobacterium . 13 . Alternatively,. shoot. tips. subjected. to. wounding. and. then. cocul-
tivated. with. Agrobacterium . have. been. shown. to. rebuild. complete. meristems. and.
produce.non-chimeric.transgenic.'Thompson.Seedless'.plants. 14
Genetic Transformation via Somatic Embryogenesis
Embryogenic. cultures. are. the. preferred. starting. materials. for. transformation. with.
either. Agrobacterium .inoculation.or.microprojectile.bombardment.because.they.are.
the.most.responsive.tissues,.with.competence.for.in.vitro.regeneration.and.genetic.
transformation. 15 . The. entire. process. involves. induction. of. somatic. embryogenesis,.