Transgenic Fruit and Nut Tree Crops Review - Transgenic Horticultural Crops: Challenges and Opportunities - page 5

maintenance.of.the.embryogenic.callus,.and.transformation,.selection,.and.regenera-
tion.of.plants..The.large.number.of.cultivars.and.rootstocks.increases.the.complexity.
due.to.genotype.differences.observed.at.all.tissue.culture.steps.
In.walnut,.somatic.embryogenesis.techniques.were.developed.for. Juglans regia .
but. have. been. applied. to. other. Juglans . species.. In. this. system,. immature. walnuts.
are. cultured. on. a. basal. medium. and. secondary. embryos. that. originate. from. cells.
in. the. epidermal. layer. of. the. embryo. display. high. susceptibility. to. infection. with.
Agrobacterium . 16 . A. major. challenge. today. in. walnut. tissue. culture. is. to. obtain.
embryogenesis. from. maternal. tissue.. This. is. important. because. embryos. from.
zygotic.tissue.do.not.allow.the.exact.genotype.to.be.predicted..Repetitively,.embryo-
genic.cultures.have.been.obtained.from.immature.anther.tissue,.but.only.from.the.
“Chandler”. cultivar.. Recently,.a. modiied. protocol. allowed. a. somatic. embryo. line.
to. be. generated. from. immature. anthers. of. a. “Paradox”. hybrid. that. may. provide. a.
source.of.elite.rootstock.tissue.for.genetic.transformation. 17
In. grapevine,. anthers. collected. during. irst. pollen. mitosis. are. the. most. widely.
used.explant.for.culture.initiation,.with.the.embryogenic.callus.arising.from.diploid.
tissue. 18 .Embryogenic.callus.maintenance.has.been.accomplished.in.liquid,.semisolid.
or. solid. media. supplemented. with. a. variety. of. growth. regulator. combinations. 19-23 .
The.variety.of.protocols.developed.for.maintenance.of.embryogenic.cultures.is.due.
to.the.fact.that.the.production.of.suitable.embryogenic.cultures.for.transformation.
with. Agrobacterium .has.represented.a.greater.challenge.than.the.initiation.step,.with.
higher.transformation.eficiencies.being.obtained.with.embryogenic.lines.composed.
of.ine.cells,.arrested.in.a.very.early.pre-embryogenic.state. 7
m ethodS of t ranSformatIon
Agrobacterium -Mediated Transformation
The. vast. majority. of. transformation. protocols. use. A. tumefaciens . (biovar. 1). as. a.
vector.. Agrobacterium . strains. that. work. eficiently. for. walnut,. citrus,. and. apple.
transformation.include.disarmed.derivatives.of. the.tumorigenic. strains. A281.(e.g.,.
EHA101. and. EHA105). and. C58. (e.g.,. C58C1). 3,12,16 . The. disarmed. strains. possess.
the.Ti.plasmid-based. vir .genes.required.for.plant.transformation,.but.lack.the.native.
T-DNA.(which.is.associated.with.pathogenesis)..These.strains.are.transformed.with.
broad. host. range. binary. plasmids. that. contain. the. desired. T-DNA. region,. carry-
ing. the. gene. of. interest. and.selectable.marker. genes,.mainly. conferring.resistance.
to. antibiotics,. such. as. kanamycin. and. hygromycin. 24 . However,. given. the. public.
concern. with. the. introduction. of. antibiotic. resistance. genes. into. food,. methods. to.
eliminate.these.selectable.marker.genes.from.the.transformed.plants.and.strategies.
that. avoid. selection. with. antibiotics. are. being. developed.. In. citrus,. workable. trans-
formation.eficiencies.have.been.achieved.using.a.multi-auto-transformation.(MAT).
vector.combined.with.an.inducible.recombinase/recombination.sites.(R/RS)-speciic.
recombination.system.and.the.phosphomannose.isomerase/mannose.conditional.posi-
tive.selection.system. 25 .In.addition,.a.cotransformation.system.using.a.mixture.of.two.
Agrobacterium . strains. (one. harboring. the. gene. of. interest. and. the. other. containing.
both.positive.and.negative.selectable.markers.genes).has.been.used.to.obtain.transgenic.
grapevines.free.of.selectable.marker.genes. 26