Nanoparticle-Mediated Delivery towards Advancing Plant Genetic Engineering

2018·2.8 MB·English

by  Francis J. Cunningham

Opinion
Nanoparticle-Mediated Delivery towards
Advancing Plant Genetic Engineering
Francis J. Cunningham,1,6 Natalie S. Goh,1,6 Gozde S. Demirer,1 Juliana L. Matos,2,3 and
Markita P. Landry1,3,4,5,*,@
Genetic engineering of plants has enhanced crop productivity in the face of Highlights
climatechangeandagrowingglobalpopulationbyconferringdesirablegenetic Plantbiotechnologyiskeytoensuring
traitsto agricultural crops. Efficient genetic transformation inplants remains a food and energy security; however,
biomolecule delivery and progeny
challenge due to the cell wall, a barrier to [342_TD$DIFF]exogenous biomolecule delivery.
regenerationcontinuetobekeychal-
Conventional delivery methods are inefficient, damaging to tissue, or are only lengesinplantgeneticengineering.
effective in a limited number of [343_TD$DIFF]plant species. Nanoparticles are promising
Conventional biomolecule delivery
materials for biomolecule delivery, owing to their ability to traverse plant cell
methodsinplantshavecriticaldraw-
wallswithoutexternalforce andhighlytunablephysicochemicalpropertiesfor backs,suchaslowefficiency,narrow
species range, limited cargo types,
diversecargoconjugationandbroadhostrangeapplicability.Withtheadventof
andtissuedamage.
engineerednucleasebiotechnologies,wediscussthepotentialofnanoparticles
asanoptimalplatformtodeliverbiomoleculestoplants[344_TD$DIFF]forgeneticengineering. Advancesinnanotechnologyhavecre-
atedopportunitiestoovercomelimita-
tions in conventional methods:
Current Biomolecule Delivery Methods for Genetic Engineering in Plants
nanoparticles are promising for spe-
Foodsecurityhasbeenthreatenedwithdecreasingcropyieldsandincreasing[346_TD$DIFF]foodconsumption cies-independent passive delivery of
inthewakeofpopulationgrowth,climatechange,increasingshortageofarableland,andcrop DNA,RNA,andproteins.
usageasrawmaterials[1,2].Classicalplantbreedingtoobtainplantswithpreferredgenotypes
The advent of nuclease-based gen-
requirescrossingandselectionofmultipleplantgenerations,whichdisallowsintroductionoftraits
omeediting(e.g.,CRISPR-Cas9)has
thatdonotcurrentlyexistinthespecies.Atechniquethatenablesspecifichorizontalgenetransfer usheredinaneweraofprecisegenetic
standstogreatlybenefittheagriculturalindustrybyconferringdesirabletraitstoplants,suchas engineering that, among other
impacts,hasenabledthedevelopment
increasedyield,abioticstresstolerance,anddiseaseandpestresistance[3].
ofgeneticallyengineeredcropswith-
outharshregulatoryrestrictions.
Geneticengineeringhasrecentlyseenmajoradvancesinanimalsystems,thoughprogresshas
lagged in plants[347_TD$DIFF]. When compared to the numerous and diverse gene and protein delivery Thepotentialofnanoparticlestoover-
methodsdevelopedforanimalsystems,significantlyfewermethodsexistforplants(Figure1, comelimitationsinconventionaldeliv-
erymakesthemexcellentcandidates
KeyFigure).Broadly,moderngenetictransformationofplantsentailstwomajorsteps:genetic fordeliveryofnuclease-basedgenome
cargodeliveryandregenerationofthetransformedplant,thenecessityanddifficultyofthelatter editingcargo,thusmakingnanoparti-
beinghighlydependentonwhatdeliverymethodisusedandwhetherstabletransformationis cledeliveryacriticaltechnologyforthe
advancement of plant genetic
desired. Regenerationprocedures involve threeparts: theinduction of competenttotipotent
engineering.
tissue, tissue culture to form calli [348_TD$DIFF](see Glossary), and selection and progeny segregation.
Regeneration protocols are dominated by complex hormone mixtures, which are heavily
speciesandtissuedependent,makingprotocoloptimizationthekeytoincreasingprocedure
efficacy.Thechallengeofgeneticcargodeliverytoplantsisattributedtothepresenceofthe
multilayeredandrigidplantcellwall,otherwiseabsentinanimalcells,whichposesanadditional 1DepartmentofChemicaland
physicalbarrierforintracellulardeliveryofbiomoleculesandisoneofthekeyreasonsforthe BiomolecularEngineering,University
ofCaliforniaBerkeley,Berkeley,CA
slowerimplementation andemployment ofgeneticengineeringtools inplants[4].
94720,USA
2DepartmentofPlantandMicrobial
Amongstconventionalplantbiomoleculedeliveryapproaches,Agrobacterium-mediatedand Biology,UniversityofCalifornia,
Berkeley,CA94720,USA
biolistic particle delivery are the two most established and preferred tools for plant genetic
3InnovativeGenomicsInstitute(IGI),
transformations(Box1).Currentbiomoleculedeliverymethodstoplantsexperiencechallenges Berkeley,CA94720,USA
882 TrendsinBiotechnology,September2018,Vol.36,No.9 https://doi.org/10.1016/j.tibtech.2018.03.009
©2018ElsevierLtd.Allrightsreserved.
thathindertheirscopeofuse(Table1).Methodssuchaselectroporation[349_TD$DIFF],biolistics,Agro- 4CaliforniaInstituteforQuantitative
bacterium-mediated delivery, or cationic delivery typically target immature plant tissue ([350_TD$DIFF]calli, Biosciences(QB3),Universityof
CaliforniaBerkeley,Berkeley,CA
meristems, or embryos). These methods require the regeneration of genetically modified 94720,USA
progeny plants, which can be time-consuming and challenging, [351_TD$DIFF]whereby efficient protocols 5Chan-ZuckerbergBiohub,San
Francisco,CA94158,USA
have only been developed for a narrow range of plant species. Biolistic particle delivery
6Theseauthorscontributedequallyto
circumvents the cell wall via mechanical force, but often damages portions of target tissue thiswork
in the process and [352_TD$DIFF]yields low levels of gene expression [353_TD$DIFF]that is often sparse and sporadic. @Twitter:@Landry_lab
Agrobacterium-mediated delivery is subject to orthogonal challenges, the largest being that
Agrobacteriumdisplaysnarrowhostandtissuespecificity,evenbetweenspecificcultivarsof *Correspondence:
[email protected](M.P.Landry).
thesamespecies[5].Agrobacteriumgenerallyexperienceslowertransformationefficiencyfor
bothdeliveryandregenerationinmonocotyledonousplants(monocots)overdicotyledon-
ous plants (dicots). Additionally, Agrobacterium yields random DNA integration, which can
cause disruption of important genes, or insertion into sections of the genome with poor or
unstable expression [6]. Random DNA integration, however, can be prevented by utilizing
magnifectionwithnonintegratingviruses[7],orbyusingaplasmiddeficientintransferDNA(T-
DNA)insertion [8].
Insum,plantgeneticengineeringhaslaggedbehindprogressinanimalsystems;conventional
methodsofbiomoleculedeliverytoplantsremainchallengedbyintracellulartransportthrough
cellwalls,andinturnlimitplantgenetictransformationefficacy.Todate,plantbiotechnology
lacks a method that allows passive delivery of diverse biomolecules into a broad range of
plant phenotypes and species without the aid of external force and without causing tissue
damage.Wepositnanotechnologyasakeydriverinthecreationofatransformationaltoolto
address deliverychallenges andenhancetheutility ofplant geneticengineering.
Nanoparticle-Mediated Biomolecule Delivery in Animal Systems
Nanoparticles asMolecularTransportersinLivingSystems
Nanotechnology has advanced a variety of fields, including manufacturing, energy, and
medicine. Of particular interest isthe useof nanoparticles(NPs) (Box 2) asmolecular trans-
portersincells,anareathathaslargelyfocusedonmoleculardeliveryinanimalsystems.NPs
allow manipulation on a subcellular level, giving rise to a previously unattainable degree of
controloverexogenousinteractionswithbiologicalsystems.Therefore,theimpactofNPsas
drug andgene deliveryvehicles inanimalshasbeennothing shortof revolutionary.
ThesmallsizeofNPsandtheirhighlytunablechemicalandphysicalpropertieshaveenabled
NP engineering for NPs to bypass biological barriers and even localize NPs in subcellular
domainsofCHOandHeLacells,amongothers[10–13].NPsserveasnonviral,biocompatible,
andnoncytotoxicvectorsthatcantransportarangeofbiomolecules[smallmolecules,DNA,
siRNA,miRNA,proteins,andribonucleoproteins(RNPs)][14–19]tobiologicalcells.Tothisend,
variousfeaturesofNPs,includingsize,shape,functionalization,tensilestrength,aspectratio,
andcharge,havebeentunedforefficientintracellularbiomoleculedeliverytoanimalsystems.
Furthermore, ‘smart’ NPs have been developed to achieve responsive release of cargo for
increased control of site-specificity [20]. Various NPs have been manufactured and are
responsive to a range of stimuli, including temperature [21], pH [22], redox [23], and the
presence of enzymes[24].
Outlookand ImplicationsforNanocarriersinPlant Science
IncontrasttotheproliferatestudiesdemonstratingNP-mediateddeliveryinanimals,analogous
researchinplantsisrelativelysparse(Figure1),owingtothetransportchallengeimposedbytheplant
cellwall,whichrendersbiomoleculedeliverymorechallengingthanformostmammaliansystems.
TrendsinBiotechnology,September2018,Vol.36,No.9 883
Nevertheless, knowledge gained from biomolecule delivery to animals provides a blueprint for Glossary
translationtoplantsystems,andcouldaccelerateadvancementsinNP-mediatedplantbiomolecule
Callus:amassofundifferentiated
delivery. NP-mediated delivery may overcome the three foremost limitations of current delivery cellsthatcanbeusedtoregenerate
techniquesinplantsystemsbycontrollingNPsizetotraversethecellwall,tuningchargeandsurface plants.
Cultivars:shortforcultivated
propertiestocarrydiversecargo,andgreaterbreadthinutilityacrossplantspecies.
varieties,agroupofplantswith
desiredcharacteristicsthathave
NP-mediateddeliveryinanimalshassuccessfullycarriedmanytypesofcargoindiscriminately, beenselectedfromanaturally
[354_TD$DIFF]wherebycertainmethodsforplants,suchasAgrobacterium,[355_TD$DIFF]canonlydeliverDNA.Forinstance, occurringspeciesandarepassed
WangandcolleaguesreportNP-mediatedRNPdelivery[356_TD$DIFF]tomammaliancellsvialipidencapsulation throughpropagation.
Dicotyledonousplants:oneofthe
[25].Additionally,plastidengineeringisnotachievablewithAgrobacterium,whichonlytargetsthe twomajorgroupsoffloweringplants.
plantnucleargenomeandcannottargetthechloroplastormitochondrialgenomes.Conversely, Theeponymoustermoriginatesfrom
targetingmoietiescanbeattachedtoNPstoobtainsubcellularlocalizationandmodificationofthe thepresenceoftwoembryonic
leavesupongermination.
desired genome. Hoshino and coworkers demonstrate the delivery of quantum dots to the
Additionally,dicotscanbe
nucleusandmitochondriaofVero[357_TD$DIFF]kidneycellsusingrespectivelocalizingsignalpeptides[26]. distinguishedfrommonocotsbya
Active targeting and controlled release is not achievable with conventional plant biomolecule numberofcharacteristicsthat
includeleafveins,vascularbundles,
deliverymethods,buthasbeendemonstratedinanimalsystemswithNP-baseddelivery.Davis
rootdevelopment,floralbundles,and
andcolleaguesdesignedapolymericNPwithahumantransferrinprotein-targetingligandand
pollen.Seemonocotyledonous
polyethylene-glycol(PEG)ontheNPexteriortodeliversiRNAtohumanmelanomatumorcells, plants.
specifically [15]. [358_TD$DIFF]Additionally, Lai and coworkers accomplished stimuli-responsive controlled Electroporation:aphysical
transfectionmethodwherean
releaseofdrug moleculesand neurotransmittersencapsulated within mesoporoussilica NPs
electricfieldisappliedtocreate
(MSNs)to[359_TD$DIFF]neuroglialcells [27].Drawing inspirationfromprogress inNP-mediateddeliveryfor
temporaryporesincellmembranes
animalsystems,NP-mediatedcontrolleddeliveryandreleaseofbiomoleculeswithoutspecies fortheuptakeofgeneticcargointoa
limitationsinplantsisaforthcominggoal. cell.
Explant:anysegmentofaplantthat
isremovedtoinitiateaculture.
NP-Mediated Biomolecule Delivery to Plants
Inplanta:atransformationparadigm
NP–Plant Interactions involvingthegenetictransformation
To date, most literature on NP–plant systems focuses on plant-based metallic nanomaterial ofanysegmentofaplantwithout
theneedfortissuecultureand
synthesis[28],agrochemicaldelivery[29],andNPuptake,showingbothvaluableanddeleterious
regeneration.
effectsonplantgrowth[30,31].Dicotandmonocotplantsexhibitvariabledegreesofdirectuptake Magnifection:deliveringvirus
ofmanyNPtypes,includingMSNs[32],carbonnanotubes(CNTs)[33],quantumdots[34],and vectorsusingAgrobacteriumT-DNA
metal/metal oxide NPs [35–37]. Once uptaken, certain types of NPs exhibit phytotoxicity via transfer.
Meristems:regionsoftissue
vascularblockage,oxidativestress,orDNAstructuraldamage[30].Conversely,NPshavebeen
containingundifferentiatedcells.
shown to improve root [360_TD$DIFF]and leaf growth, and chloroplast production [31]. Tradeoffs between Monocotyledonousplants:oneof
phytotoxicityandgrowthenhancementasafunctionofspecies,growthconditions,NPproper- thetwomajorgroupsofflowering
plantsthathaveoneembryonicleaf
ties,anddosagearenotwellunderstoodandcallformorestudieswithafocusonNPphysicaland
upongermination.Monocotsinclude
chemicalproperties.ClosingtheknowledgegapinplantphysiologicalresponsetoNPuptakeis
cropsthatmakeupthemajorityofa
important and should be pursued in parallel with the enhancement of plant science using balanceddiet,suchasrice,wheat,
engineered nanomaterials, as the ‘nanorevolution’ in targeted delivery to animals suggests andbarley.Seedicotyledonous
plants.
tremendouspotentialforanalogousprogressinplants.
Passivedelivery:transportofcargo
acrosscellwallandmembranetoan
Heuristics forNanocarrierDesign intracellularlocationwithouttheuse
Whileacompletestructure–functionlandscapeofphysicalandchemicalNPpropertiesthatdrive ofmechanicalforce.
Protoplasts:plantcellswiththeir
cargoloadingandcellularinternalizationremainselusive,aheuristicapproachtonanocarrierdesign
cellwallsremoved,typicallythrough
is a useful starting point. NP uptake and transport throughout plant tissue is limited by pore eithermechanicalorenzymatic
diameters,settingsizeexclusionlimits(SELs)forvarioustissuesandorgansthatarediscussed means.
extensivelyintheliterature[30,38–43].Thecellwalliscommonlythoughttoexcludeparticles>5– Recalcitrant:aspeciesofplantthat
isdifficulttogeneticallytransform
20nm,althoughrecentlyNPsupto50nmindiameterhavebeenreportedascellwall-permeable
andregenerateintomatureplants.
through unclear mechanisms [38,41]. For genetic engineering applications, where cytosolic or Oftenusedinthecontextof
nuclearlocalizationisnecessarytoaffectgenefunction,theplasmaandnuclearmembranes[361_TD$DIFF]pose Agrobacterium-mediated
additionalbarrierstodelivery.Inpractice,thecellwall(SEL<50nm)playsadominantroleinNPsize transformation.
884 TrendsinBiotechnology,September2018,Vol.36,No.9
[362_TD$DIFF]internalizationlimitations,asthecellmembraneSELismuchlarger(>500nm)[38].NPchargeand Transgene:agenetakenfroman
shape greatly influence cell membrane translocation and thus these properties are central to organismandtransferredintothe
genomeofanother.Consequently,
nanocarrieroptimization[44].Plantcellularuptakecanoccurthroughenergy-dependent(endocy-
transgeneintegrationresultsin
tosis) and energy-independent (direct penetration) pathways that are not well understood. It is
transgenicplants.
commonlyreportedthatinternalizationisfasterandmoreefficientforcationicNPsversusanionic
NPs, due to [363_TD$DIFF]cationic NP binding with the negatively charged cell membrane [44]. This charge
preferencehasbeendemonstratedinprotoplastsandwalledplantcells[45,46].
Endosomalescapeiscriticalforsubcellulardelivery,asvesicle-entrappedNPscanbetraffickedfor
degradationorexocytosis[364_TD$DIFF],andremaininaccessiblefordownstreamprocessingiftrappedinthe
endosome.SubcellularlocalizationofNPsinplantsisnotwellunderstoodbutwilldependonthe
uptakepathway,asendocyticproteinsandvesiclecargoplayaroleinendosomefate[47],whereby
directcellpenetrationbypasses[365_TD$DIFF]endosomalvesicleformationentirely.Seragandcolleaguesreport
CNTinternalization[366_TD$DIFF]inprotoplaststhroughbothdirectpenetrationandendocytosis[367_TD$DIFF],supportingprior
demonstrationsinmammaliancellsthathighaspectratioNPsundergovesicle-freeinternalization
[48,49].However,[368_TD$DIFF]forSeragandcolleagues,directpenetrationwasonlyobservedforcellwall-
impermeablemultiwalledCNTsinprotoplasts[369_TD$DIFF][48,49],motivatingfurtherstudiesforplantcellwall
internalizationbyhighaspectratioNPs.Wongandcolleagueshavedemonstratedpassiveinternali-
zationofsingle-walledCNTsinextractedchloroplasts[129]throughamechanismdependentonNP
sizeandzetapotential[130].Cationic,pH-bufferingpolymersarewell-knownendosomedisruption
agents[50]thatcanfunctionasligandstoimproveendosomalescape.Changandcolleaguesreport
energy-independentinternalizationtowalledrootcellsbyorganicallyfunctionalizedsphericalMSNs
[51].Notably,endocytosedsingle-walledCNTsinplantsaretraffickedtovacuolesbutlocalizeinthe
cytosolwhenloadedwithDNA[33,48].
MostNPs[370_TD$DIFF]areamenabletosurfaceadsorption(physisorption)ofbiomoleculesasasimpleconju-
gationstrategy.However,physisorptionmaybeunstabledependingonthespecificNPandcargo,
andthuselectrostaticinteractionsarepreferablefornoncovalentcargoloading[52].Cationicsurface
chemistrynotonlyenhancesendocyticuptakeandescape[371_TD$DIFF],butisalsoamenabletoelectrostatic
loadingofgeneticcargo[372_TD$DIFF]viaattractionwithnegativelychargedDNA[373_TD$DIFF]andRNA.CovalentNPsurface
functionalizationistypicallyachievedbyoneofmanyof‘click’chemistries[53].Notably,covalent
attachmentofthiolatedDNAandproteinstogoldNPshasshownrecentsuccess[54]butthefield
remainsopentonewstrategiesforcovalentbioconjugation,especiallyforapplicationsinplants.As
analternativetosurfacefunctionalization,porousNPssuchasMSNscanbeinternallyloadedwith
macromoleculesorsmallchemicalsalike,forcontrolledintracellularrelease[55].
NPswithsomeorallofthepropertiesmentionedabovehavedemonstratedsuccessfulbiomolecule
deliveryinplantsand[374_TD$DIFF]aregoodstartingpointsforchoosingtheappropriateNP,ligand,andcargofora
givenapplication.However,itshouldbenotedthatnanocarrierdesignisacomplex,multivariable
optimizationprocess,suchthatsuccesswilllikelyrequiretweakingoftheseheuristicsfordifferent
systemsuntilacompleteNPstructure–functionrelationshipisestablishedforplantsystems.
Nanomaterials forPlantGenetic Engineering
NPsarevaluablematerialsforintracellularbiomoleculedelivery,owingtotheirabilitytocross
biologicalmembranes,protectandreleasediversecargoes,and[375_TD$DIFF]achievemultifacetedtargeting
viachemicalandphysicaltunability.SuchpropertieshaveenabledNPstorevolutionizetargeted
deliveryandcontrolledreleaseinmammaliansystems.However,nanocarrierdeliveryinplants
remainslargelyunderexploredduetothecellwall,whichistypicallyovercomebychemicalor
mechanical aid (Figure 1). Passive biomolecule delivery to plants is promising for minimally
invasive, species-independent[376_TD$DIFF], in vivo genetic engineering of plants, especially for transient
TrendsinBiotechnology,September2018,Vol.36,No.9 885
KeyFigure
Nanoparticle (NP)-Mediated Genetic Cargo Delivery to Animals and Plants
(A) NPs classes commonly employed in gene(cid:415)c cargo delivery
Bio-inspired NPs Metallic / Magne(cid:415)c NPs
• Calcium phosphate Gene(cid:415)c cargo has been delivered in: • Gold
• Chitosan • Silver
Both animal and plant systems
• Liposomes • Iron oxide
Gene(cid:415)c cargo delivereda–d Animal systems only Gene(cid:415)c cargo deliveredt–x
Plant systems only
Neither system
DNA RNA Protein RNP DNA RNA Protein RNP
Carbon-based NPs Silicon-based NPs Polymeric NPs
• Single-walled carbon • Silica spheres • Polyethylene-glycol (PEG)
nanotubes • Mesoporous silica NPs •Poly(lac(cid:415)c-co-glycolic acid)
• Mul(cid:415)walled carbon nanotubes (MSNs) (PLGA)
• Fullerenes • Silicon carbide • Polyethylenimine (PEI)
Gene(cid:415)c cargo deliverede–g Gene(cid:415)c cargo deliveredh–k Gene(cid:415)c cargo deliveredi–s
DNA RNA Protein RNP DNA RNA Protein RNP DNA RNA Protein RNP
(B) Modes of NP-mediated cargo delivery
Electropora(cid:415)on /
Biolis(cid:415)c Magnetofec(cid:415)on Microinjec(cid:415)on
sonopora(cid:415)on / optopora(cid:415)on
Ca(cid:415)onic transfec(cid:415)on Incuba(cid:415)on Infiltra(cid:415)on
Figure1.(A)NPscommonlyusedforbiomoleculedeliveryinbothanimalandplantsystemscoverfivemajorcategories:bio-inspired,carbon-based,silicon-based,
polymeric,andmetallic/magnetic.Weprovideavisualcomparisonofdeliveryofvariousgeneticcargo[DNA,RNA,proteins(site-specificrecombinasesornucleases),
andribonucleoprotein(RNP)]witheachofthefiveNPtypesacrossanimalandplantsystems.ItisevidentthatNP-mediateddeliveryhasbeenutilizedwithagreater
varietyofgeneticcargoinanimalsthaninplants.(b)NP-mediatedcargodeliveryisconductedvia[321_TD$DIFF]severalmeans.Physicalmethodsinclude[322_TD$DIFF]creatingtransientporesin
thecellmembranewithelectricfields,soundwaves,orlight,magnetofection,[323_TD$DIFF]microinjection,andbiolisticparticledelivery[324_TD$DIFF].Nonphysicalmethodsinclude[325_TD$DIFF]theuseof
cationiccarriers,incubation,andinfiltration.a[64],b[86],c[87],d[88],e[89],f[68],g[90],h[91],i[45],j[92],k[58],l[93],m[94],n[95],o[96],p[97],q[98],r[99],s[81],t[100],u[63],
v[101],w[102],x[54].
886 TrendsinBiotechnology,September2018,Vol.36,No.9
Box1.CommonGeneDeliveryMethodsinPlants
Agrobacterium-MediatedTransformation
Agrobacteriumtumefaciensisasoilbacteriumthatinfectsawiderangeofdicots,causingcrowngalldisease.The
formationofagallonthehostplantisachievedviathestabletransfer,integration,andexpressionofbacterialDNAin
infectedplants.EngineeringoftheAgrobacteriumplasmidbysubstitutionofthegall-inducingvirulencegeneswith
genesofinterestconferstheabilityofAgrobacteriumtotransformthehostplant.Forthisreason,Agrobacteriumhas
beenharnessedasatoolforplantgenetictransformationsincetheearly1980s[107].
GenetictransformationoccursthroughaprocessinvolvingT-DNAexport,targeting,andinsertionintotheplantnuclear
genome.TheexportofT-DNAfromthebacteriumtotheplantcellisfacilitatedbytheactivityofvirulencegenespresent
in the tumor inducing-plasmid of Agrobacterium, but are not themselves transferred. These virulence genes are
expressedinthepresenceofphenolicinducers,suchasacetosyringone[334_TD$DIFF],producedbywoundedplantcells.Agro-
bacteriumattachestoplantcells,wherebordersequencesoneithersideoftheT-strand(asingle-strandedcopyofthe
T-DNAsequence)arecleaved.TheT-strandisthencarriedbyatransporterwithanuclearlocalizationsequenceand
integratedintotheplantnucleargenome.Integrationoccursatrandompositionsinthegenomevianonhomologous
recombination,arepairpathwayfordouble-strandedbreaksinDNA.
GeneGun-MediatedTransformation
Aformofbiolisticparticledelivery(alsocalledparticlebombardment),thegenegun,isaphysicalmethodthatis
commonly[335_TD$DIFF]utilizedforplantgenetictransformations.Developedin1982bySanford[336_TD$DIFF]andcolleagues[108],theprocess
involvesgoldortungstenmicroparticles(ormicrocarriers)coatedwithgeneticcargothatareacceleratedbypressurized
helium(He)gasintoplantcells,rupturingcellwallsandmembranes.Thegenegunconsistsofthreemainparts:arupture
disk,macrocarrier(holdingmicrocarrierparticles),andstoppingscreen.Therupturediskisamembranedesignedto
burstatacriticalpressureofHegas.WhenHegasisacceleratedtothedesiredpressure,therupturediskbursts,
creatingashockwavethatpropelsthemacrocarriertowardstheplantcells.Themacrocarrier’smomentumisstopped
bythestoppingscreen,whichallowsgeneticcargo-loadedmicrocarrierstopassandentertheplantcells.
UnlikeAgrobacterium-mediatedtransformation,biolisticdeliverycanresultintransformationofthenuclear,plastidal,or
mitochondrialgenomesduetothenonspecificlocalizationofgeneticcargo.Consequently,moreDNAneedstobe
deliveredwithbiolisticdeliverythan[337_TD$DIFF]Agrobacterium-mediateddeliverywhentargetingthenucleargenome.
expression in somatic tissue (Table 2). The potential of NP-based plant delivery methods is
underscoredbythelimitationsofinvitroplantstudiesingeneral,whereinregenerationcapacity
varieswidelyacrossspecies,genotype,andevenwithinasingleplantdependingondevelop-
mentalageofsourcetissue[56].Currently,stabletransformationrequiresprogenyregeneration
fromembryogeniccalliregardlessofthedeliverymethod(Table2).Thus,paralleloptimizationof
deliveryandregenerationisnecessarytoimproveefficiencyandexpandstabletransformation
capabilitiestoallplantspecies.
In2007,TorneyandcolleagueswerethefirsttodemonstrateNP[377_TD$DIFF]co-deliveryofDNAandchemicals
toNicotianatabacumplantsviabiolisticdeliveryof100–200-nmgold-cappedMSNs[45].Inthis
study,achemicalexpressioninducerwasloadedintoMSNpores((cid:1)3nm)thatweresubsequently
covalentlycappedwithgoldNPs.ThecappedMSNswerethencoatedwithGFPplasmidsand
deliveredbygeneguntoN.tabacumcotyledons,whereinGFPexpressionwastriggeredupon
uncappingandreleaseoftheexpressioninducer[45].Thisseminalpaperdemonstratedproofof
conceptthatstrategiescommonforNPdeliveryofDNAtomammaliansystemscanbeadaptedto
plants.Notably,goldMSNswerealsousedforbiolisticco-deliveryofDNAandproteins,namely
GFPandCre-recombinase,demonstratingtheabilityofMSNstodeliverproteinsforgeneediting
[58].Manydeliverystrategiesstillrequireagenegun,electromagneticfield,orprotoplastPEG-
transfection[58–63]asNPstructure–functionparametershavenotyetbeenfullyoptimizedto
[378_TD$DIFF]passivelybypassthecellwall(Table3).However,for[379_TD$DIFF]systemswheremechanicalorchemicalaidis
necessaryfor[380_TD$DIFF]NPinternalization,thesmallsizeandhighsurfaceareaofnanocarriersstilloffers
TrendsinBiotechnology,September2018,Vol.36,No.9 887
8
8 Table1.ScopeofUseSummaryforPlantBiomoleculeDeliveryMethods
8
T Deliverymethod Adverseeffectsofdelivery Targetspecies/tissue Cargotypeandsizea Limitations
re
nd Physical
s
inB Biolisticparticle- Damagetotargettissue&cargo,low Dependsontissuetypeb/ DNA,siRNA,miRNA, Targetingleavesrequiresdetachmentfrom
iote (genegun) penetrationdepth,random calli,embryos,leaves ribonucleoproteins plant,whichlimitstimetoobservedelivery
c
hn mediateddelivery integration (RNPs),largecargo effects;targetingembryosrequireslaborious
o
lo size regenerationprotocols,theeffectivenessof
g
y, whichishighlyspecies/cultivar-dependent
S
e
pte Electroporation Damagetotargettissue,nonspecific Unlimited/protoplastsc[328_TD$DIFF], Nucleicacids(DNA, Limitedcargo-carryingcapacity
mb transportofmaterialthroughpores meristems,pollengrains siRNA,miRNA)
e
r mayleadtoimpropercellfunction
2
0
1
8, Chemical
V
o
l. Polymer-mediated Highchargedensitiesinduce Speciesamenableto Nucleicacids(DNA, Regenerationishighlyinefficientformost
3
6, delivery cytotoxicity protoplastregeneration/ siRNA,miRNA) speciesintransientstudiesandrequires
N
o. protoplastsc tissueculture
9
Biological
Agrobacterium- Canleadtoapoptosisandnecrosis, Narrowrangeofplant LimitedtoDNA,large Leaf-targeteddeliveryistransientandgene
mediateddelivery randomintegration species,especially cargosize editsarenottransmittedtoprogeny,butallow
restrictedfrommonocotsd[329_TD$DIFF]/ diversebiologicalstudies;requirestissue
matureplants,immature culture(exceptArabidopsis)togenerate
tissue,protoplasts progeny;exhibitshighhost-specificity
Viraldelivery Virusintegration(canbemitigatedby Hostplantspecies Nucleicacids(DNA, Highlylimitedcargo-carryingcapacity
usingnonintegratingviruses) restrictions/matureplants, siRNA,miRNA),very
meristems limitedcargosize
aWhilemostbiomoleculedeliverymethodstoplantscandeliveravarietyofgeneeditingreagents,DNAplasmidsarearguablythemostcommoncargoofinterest;DNAloadingcapacitiesareausefulmetric
fortheupperlimitforcargosizeseachmethodcansustain.
bWhilebiolisticparticle-mediateddeliverycantheoreticallybeutilizedinunlimitedtargetspecies,theabilitytotargetspeciesdependsonthetargettissue(byextension,cellwallstructuralstrength)and
capabilityofavailableequipment.
cTheuseofprotoplastsastargettissuenecessitatesregenerationprotocolsandprogenysegregationthataretime-consumingandarechallengedbythelimitedplantspeciesamenabletoprotoplast
regeneration.
dProgresshasbeenmadeonincreasingtransformationefficiencyinrecalcitrantmonocots[9].
Box2.Nanoparticles
Nanoparticles(1–100nminatleastonedimension)canbeengineeredwithvariedcompositions,morphologies,sizes,
andcharges,enablingtunablephysicalandchemicalproperties.Rangingfromzerotothreedimensional,NPsarenovel
toolsthathaveawiderangeofapplications,includingbutnotlimitedtoenergystorage,sensingdevices,andbiomedical
applications[109,110].
Inadditiontotheirhighdegreeoftunability,NPspossessseveraladvantagesthatvalidatetheirrecentwidespreaduse,
withparticularemphasisinthebiomedicalindustry.MostNPscanbepreparedwithconsistentpropertiesforlowbatch-
to-batchvariability,andcanbedesignedtotargetbiologicalsystems,tissues,cells,orsubcellularstructureswithhigh
specificity[52].Moreover,NP-mediatedgeneanddrugdeliverycanovercomecommonissuesfacedwithviralvectors;
NPsare[338_TD$DIFF]oftenlessimmunogenicandoncogenicandcancarrydiverseand[339_TD$DIFF]largercargo,althoughtheincreasedNP
sizes[340_TD$DIFF]whenbiomoleculesaresurface-loadedraisethechallengeofbypassingbiologicalbarriers[111].Furthermore,the
effectsofNPusehaveyettobethoroughlystudied,thoughexistingresearchpointsto[341_TD$DIFF]nanoparticlechemistry,size,and
doseastunableparameterstocontrolcytotoxicity[112,113].
NPsaretypicallyclassifiedbasedonmorphologyandchemicalproperties.Themostcommoncategoriesinclude
polymeric[114],lipid[115],magnetic[116],metallic[117],andcarbon-basedNPs[118].NPscanbesynthesizedwith
eitheratop-downorbottom-upapproachusingtechniquessuchaslithography[119],deposition[120],andself-
assembly[121].
InNP-baseddelivery,avarietyofstrategiesareemployedtoloadNPswiththedesiredcargo.Physicaltechniquessuch
asencapsulationorentrapmentarecommonlyusedindrugdeliverytoensuretheprogressivereleaseofdrugs.
ChemicaltechniqueswheretheNPsurfaceismodifiedforcargograftingareindevelopment,includingnoncovalent
conjugation(electrostaticinteraction[122],p-pstacking[123])andcovalentconjugation[23].
superiorperformance overconventionalmethods.For instance,Torneyand colleagues’ [381_TD$DIFF]MSN
study achieved transgene expression with 1000(cid:3)less DNA than [382_TD$DIFF]the tens to hundreds of
microgramsofDNAtypicallyrequiredforconventionalPEG-transfectioninprotoplasts[45].
AfewrecentexamplesshowpromiseforNP-mediatedpassivedeliverytoplantsinvitro[64–66]
andinvivo[51,67]in,forexample,N.tabacumprotoplasts[66]andArabidopsisthalianaroots
[51,67],respectively(Table3).Demirerandcolleagueshaverecentlyachievedpassivedelivery
of DNA plasmids and [383_TD$DIFF]protected siRNA using functionalized CNT [384_TD$DIFF]NPs for transient GFP
expressioninErucasativa(arugula)leavesandtransientsilencingofconstitutivelyexpressed
GFP in transgenic Nicotiana benthamiana leaves [68]. This study also demonstrates CNT-
mediated transient GFP expression in Triticum aestivum (wheat), indicating the potential for
passiveNPdeliveryinbothmodelandcropspecieswithhighefficiencyandlowtoxicity.While
many more studies are needed to optimize NP properties and functionalization, these early
resultsarepromisingforfurtherexplorationofNPsasaplantbiomoleculedeliveryplatformthat
addressestheshortcomingsofconventionalmethods.Furthermore,withtheadventofnucle-
ase-basedgeneeditingtechnologies(Box3),itisofgreatinteresttooptimizethedeliveryof
these revolutionary [385_TD$DIFF]genome engineering tools by exploring NP-based delivery strategies for
[386_TD$DIFF]diversebiomolecularcargoes.
Genome Editing has Enabled a New Era of Plant Science
Engineered Nucleasesfor PlantGenome Editing
Engineered nuclease systems, namely ZFNs, TALENs, and CRISPR-Cas, have emerged as
[387_TD$DIFF]breakthrough genome editing [388_TD$DIFF]tools owing to their high genetic engineering specificity and
efficiency(Box3),wherebyCRISPR-Cashas [389_TD$DIFF]demonstrated increasedsimplicity,affordability,
andmultiplexingcapabilitiesoverTALENsandZFNsinplants[69,70].Since2012,CRISPR-Cas
hasshownsuccessforgenomeeditinginbothmodelandcropspecies,includingA.thaliana,N.
benthamiana,N.tabacum(tobacco),Oryzasativa(rice),T.aestivum(wheat),Zeamays(corn),
Solanumlycopersicum(tomato),andSorghumbicolor,amongothers[71,72].Notably,CRISPR-
Casmutationsassmallas1bphavebeenconservedthroughthreeplantgenerations[73,74],
TrendsinBiotechnology,September2018,Vol.36,No.9 889
8
90 Table2.ChallengesinPlantGeneticEngineeringandProposedAdvantagesofNPDelivery
T Desiredoutcome Nonheritablea Heritable
ren (somatic/transientexpression) (germline/stabletransformation)
d
s
in Targetedtissue Leaves Roots Protoplasts Zygoticembryo Somaticembryogeniccalli
B
iote [331_TD$DIFF]Tissue-specificbiologicaland (cid:4) Cellwall (cid:4) Cellwall (cid:4) Cellwalldegradation (cid:4) Cellwall (cid:4) Cellwall
c
hn experimentalchallenges (cid:4) Inefficientcellularuptake (cid:4) Inefficientcellularuptake protocol (cid:4) Inefficient[332_TD$DIFF]cellularuptake (cid:4) Inefficient[332_TD$DIFF]cellularuptake
o
log (cid:4) Epidermalbarrier (cid:4) Inefficientcellularuptake (cid:4) Embryocollection/calli (cid:4) Totipotency/calliinduction
y,S induction (cid:4) Calliregeneration
ep (cid:4) Calliregeneration
te
m
b ProposedadvantagesofNP (cid:4) NP-cellwallpermeability (cid:4) NP-cellwallpermeability (cid:4) TunableNPproperties (cid:4) NP-cellwallpermeability (cid:4) NP-cellwallpermeability
e
r2 delivery (cid:4) NP-stomatapermeability (cid:4) CationicNProot andligandsforsubcellular (cid:4) TunableNPproperties (cid:4) TunableNPproperties
0
18 [57] accumulation[46] targeting andligandsforsubcellular andligandsforsubcellular
,
Vo (cid:4) AnionicNPsroot-to-shoot (cid:4) Passiveuptakewithout targeting targeting
l.
36 vasculartranslocation[46] genegunorprotoplasts
,N (cid:4) Passiveuptakeordirect (cid:4) TunableNPproperties
o
.9 mesophyllinjection andligandsforsubcellular
withoutgenegunor targeting
protoplasts
(cid:4) TunableNPproperties
andligandsforsubcellular
targeting
aWhile these somatic tissues (leaves, roots, protoplasts) are most commonly targeted for transient expression experiments, heritable outcomes may be derived [333_TD$DIFF]through somatic embryogenesis
(dedifferentiationofsomatictissue).
Table3.SelectSummaryofNP-MediatedGeneticEngineeringinPlants
NPtype Cargo Plantspecies;cell/tissuetype Deliverymethod Comments Year Refs
Withexternalaid GoldcappedMSNs GFPplasmid;chemical N.tabacumcotyledons;Z. Biolistic Co-deliveryandcontrolledreleaseof 2007 [45]
expressioninducer maysembryos DNAandchemicals
Poly-L-lysinecoated GFPplasmid DioscoreazingiberensisC.H. Sonoporation 5%transientexpressionefficiency; 2008 [60]
starchNPs Wrightcallisuspension someintegrationoccurs
Gold-platedMSNs GFPandmCherryplasmids; Alliumcepaepidermistissue Biolistic DNAandproteinco-delivery 2012 [59]
GFPprotein
MagneticgoldNPs b-glucuronidase(GUS) Brassicanapusprotoplasts Magneticfield TransientGUSexpression 2013 [61]
plasmid andwalledcellsuspension
Gold-platedMSNs AmCyan1andDsRed2 Z.maysembryos Biolistic DNAandproteinco-delivery;both 2014 [58]
plasmids;Creprotein transientandstableexpression
Dimethylaminoethyl Yellowfluorescentprotein N.tabacumandCeratodon PEGtransfection Bothtransientandstableexpression 2017 [62]
methacrylate(DMAEM) (YFP)andGFPplasmids purpureusprotoplasts
polymerNPs
MagneticFe3O4NPs Selectablemarkergene Gossypiumhirsutumpollen Magneticfield (cid:1)1%efficiencyforgeneratingstable 2017 [63]
plasmids transgenicseeds
Invitrowithoutexternalaid Polyamidoamine GFPplasmid AgrostisstoloniferaL.calli Passive 48.5%cellsshowedtransient 2008 [65]
(PAMAM)dendrimerNPs expression
CalciumphosphateNPs GUSplasmid Brassicajunceahypocotyl Passive 80.7%stabletransformation 2012 [64]
(CaPNPs) explants efficiency
Organically YFPplasmid N.tabacumprotoplastsand Passive Bothtransientandstableexpression 2015 [66]
functionalizedCNTs leafexplants
Invivowithoutexternalaid Organically mCherryplasmid A.thalianaroots Passive 46.5%transientexpressionefficiency 2013 [51]
functionalizedMSNs
PAMAMdendrimerNPs Double-strandedDNAfor A.thalianaroots Passive Developmentalgenesilencingledto 2014 [67]
T RNAinterference systemicphenotypes
re
nd Polymerfunctionalized GFPplasmid;siRNAfor E.sativa,N.benthamiana, Passive 95%transientsilencingefficiency; [68]
s
in CNTs transgenicGFPsilencing andT.aestivumleaves transientexpressioninmatureleaves
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