In Vitro Preservation of Transgenic Tomato

19 Pages·2017·5.8 MB·English

International Journal o f
Molecular Sciences
Article
In Vitro Preservation of Transgenic Tomato
(Solanum lycopersicum L.) Plants Overexpressing the
Stress-Related SlAREB1 Transcription Factor
AyedM.Al-Abdallat1,*,RidaA.Shibli1,MuhanadW.Akash1,ManarRabbaa1
andTamaraAl-Qudah2
1 DepartmentofHorticultureandCropScience,FacultyofAgriculture,theUniversityofJordan,
11942Amman,Jordan;[email protected](R.A.S.);[email protected](M.W.A.);[email protected](M.R.)
2 HamdiMangoCenterforScientificResearch,theUniversityofJordan,11942Amman,Jordan;
[email protected]
* Correspondence:[email protected];Tel.:[phone](ext.22331)
Received:18May2017;Accepted:1July2017;Published:21July2017
Abstract: Invitro preservation of transgenic tomato lines overexpressing the stress-responsive
transcriptionfactorSlAREB1wasstudiedbyusingslowgrowthandcryopreservationtechniques.
Slowgrowthpreservationwasperformedbyusingdifferentconcentrationsofsucrose(0,100,200,
300mm)andabscisicacid(0,4,8,12µm)inMurashigeandSkoog(MS)media,whilecryopreservation
wasconductedbyusingencapsulationdehydration,V-cryoplatesandseeds. Significantdifferences
wereobservedbetweentestedlinesgrownonMSmediasupplementedwith200mmsucrosewhere
transgeniclinesoverexpressingSlAREB1showedimprovedgrowthwhencomparedwithnegative
control. Theadditionofabscisicacid(ABA)tothepreservationmediaaffectednegativelytransgenic
linesgrowthanddevelopmentwhencomparedwithABA-freemedia. Inencapsulationdehydration,
non-cryopreservedtransgeniclinesoverexpressingSlAREB1pretreatedin0.8Msucrosefor1day
andsubjectedtodifferentdehydrationperiodsshowedsignificantlyhighersurvivalpercentages
whencomparedwithnegativecontrol. ForV-cryoplatestechnique,cryopreservedtransgeniclines
overexpressingSlAREB1treatedin0.3Msucrosefor3dayswithorwithoutcoldacclimatization
showedsignificantlyhighersurvivalpercentageswhencomparedwiththenegativecontrol. Seed
cryopreservationwasperformedsuccessfullywithaclearreductioningerminationpercentagein
transgenic lines overexpressing high levels of SlAREB1. In conclusion, transgenic tomato lines
overexpressingSlAREB1werefoundtoimprovetoleranceagainstdifferentabioticstressesassociated
withdifferentinvitropreservationprotocols.
Keywords: abscisic acid; encapsulation-dehydration; invitro preservation; Solanum lycopersicum;
transcriptionfactor;V-cryoplates
1. Introduction
Tomato(SolanumlycopersicumL.)isawarmseasonplant,whichisconsideredsensitivetocoldand
underseverefrostconditionstheentireplantcouldbedestroyed. Suchsensitivitytocoldandother
relatedabioticstressescanbeovercomebyseveralphysiologicalandbiochemicalprocessessuchasthe
accumulationofabscisicacid(ABA),osmolytesandlateembryogenesisabundantproteinsandgrowth
retardation [1]. Abscisic acid is known to regulate several physiological processes including seed
germination,stomataclosureandtolerancetodifferentabioticstresses[2]. ABAhasbeenreportedto
enhancetolerancetodifferentabioticstressesincludinglowtemperature,droughtandsalinitystresses
inseveralplantspecies.
Understanding the genetic basis of stress tolerance mechanisms in plants may offer the
opportunityto developnew varietieswith improvedadaption todifferentstresses includingcold
Int.J.Mol.Sci.2017,18,1477;doi:10.3390/ijms18071477 www.mdpi.com/journal/ijms
Int.J.Mol.Sci.2017,18,1477 2of19
anddrought. Inthisperspective, animportantstepincontrollingplanttoleranceresponsesisthe
transcriptionalactivationorrepressionofstress-responsivegenes[3]. Transcriptionalregulationof
stresstolerancegenesislargelymediatedbythespecificrecognitionofcis-actingpromoterelementsby
trans-actingsequencespecificDNA-bindingproteinsknownastranscriptionfactors. Inaddition,such
responsesaremediatedmainlythroughABA-dependentandABA-independentsignalsthatregulate
the expression of stress responsive genes involved in conferring stress tolerance phenotypes [3].
The basic strategy of generating tolerant plants is based on the activation of either one or both
pathwaysthroughtheintroductionofkeyregulatorygenesthataredirectlyinvolvedinactivating
molecularmechanismscontrollingabioticstresstolerance[4]. TheABAresponsiveelementbinding
(AREB) factors (also known as the ABF for ABA binding factors) are members of the bZIP family
of transcription factors that specifically bind to ARE cis-elements found in the promoter region of
manyABA-responsivegenes[5]. TheoverexpressionoftheAREBfactorsindifferentplantspecies
improvesfreezinganddehydrationtolerance[4]. Intomato,theoverexpressionofSlAREB1wasfound
toincreasedroughtandsaltstresstolerance[6]aswellastheinductionofchangesinorganicacid
accumulationandtheexpressionofgeneencodingenzymesinvolvedintheirsynthesis[7].
The loss of valuable plant genetic resources has prompted international actions to preserve
such valuable exotic plant material [8]. Invitro, preservation of plant germplasm can be
divided into medium-term preservation (slow growth preservation) and long-term preservation
(cryopreservation) [9]. In medium-term preservation, the plant material is subjected to growth
retardationconditionsthatslowdownplantgrowthanddevelopmentinvitro.Thisincludesgrowth
underlowtemperatures,reducedlightconditionsandmodificationofgaseousenvironments,and
the use of growth retardants such as ABA and osmoticums [10]. Medium-term preservation
was used successfully for many plant species where regeneration potential could be restored [11].
Cryopreservationreferstotheconservationofplantgermplasmforindefiniteperiodsbykeepingitin
ultra-lowtemperatureconditionssuchasliquidnitrogen(−196◦C)[12]. Undersuchconditions,the
biochemicalprocesses,metabolicactivities,andcelldivisionofthecryopreservedbiologicalmaterial
will be arrested [13]. The cryopreserved material can retain its biological activities as soon as it
is allowed to re-grow under suitable conditions. Cryopreservation in liquid nitrogen is the only
currentlyavailablemethodthatensuressafe,cost-effectiveandlong-termconservationofvegetatively
propagatedplantsandforplantswithrecalcitrantseeds[10,14]. Cryopreservationisperformedby
usingdifferentmethodssuchasencapsulationdehydration,vitrification,encapsulation-vitrification
andcryoplates[15]. Forinstance,thecryopreservationofSolanumphurejashoottipswasachieved
successfullybyusingtheencapsulationdehydrationapproach[16].In2014and2015,Costeetal.[17,18]
succeededinthecryopreservationoffivetomatocultivarsbyusingencapsulationdehydrationand
dropletvitrification. Theyfoundthatthepreculturingofshoottipsinsucroseplaysanimportantrole
inincreasingtheefficiencyofcryopreservationbyenhancingtheresistancetodesiccationandliquid
nitrogenexposure. Recently,anewcryopreservationtechniquewasdevelopedbyusingaluminium
cryoplates that could be either based on vitrification (V-cryoplates) or dehydration-(D-cryoplates)
andbothwerefoundtogivehighsurvivalandregrowthpercentagesaftercryopreservationwhen
comparedtoothermethods[19–21]. Theutilizationofseedsasaplantmaterialincryopreservation
wasusedsuccessfullyindifferentplantspeciesincludingtomato[22,23].
Previously, the cryopreservation of transgenic plants was performed successfully with no
majoralternationsincryopreservedmaterialphenotypeorgenotypeaftertheirregeneration[24–26].
For instance, transgenic chrysanthemum plant with improved trehalose accumulation had higher
regeneration percentages when compared with wild type [26]. In this study, invitro preservation
of transgenic lines overexpressing the stress-related SlAREB1 transcription factors was studied by
using slow-growth and cryopreservation approaches. For slow-growth preservation, the effect of
differentlevelsofsucroseandABAonmicroshootdevelopmentafter12weeksofculturewasstudied.
Cryopreservationoftransgeniclineswasstudiedbyusingencapsulation-dehydration,V-cryoplates
andseeds. Tothebestofourknowledge,nopreviousreportsdescribingtheeffectofstress-related
transcriptionfactorsoninvitropreservationofthetomatoplantexist.
Int.J.Mol.Sci.2017,18,1477 3of19
2. Results
2.1. OverexpressionofSlAREB1inTomato
TotestwhetherSlAREB1canimproveinvitropreservationoftomatoplants,anoverexpression
constructwithSlAREB1CDS(GenBankaccessionnumber: AY530758)underthecontrolof35SCaMV
promoterwasintroducedintotomatocv. Moneymaker. Only5independenttransgeniclineswere
produced and were confirmed to be positive based on hygromycin selection. Three of them were
found to carry a single insertion event as revealed by real time PCR analysis (data not shown).
The transgenic lines grew normally when compared with tomato plants transformed with empty
plasmidandthisisconsistentwithOrellanaetal.(2010)[6],whoshowedthatthemajorityoftransgenic
lines overexpressing SlAREB1 had normal morphological phenotypes. Gene expression analysis
using quantitative RT-PCR analysis in transgenic tomato plants overexpressing SlAREB1 showed
highexpressionlevelsofthetargetedgenewhencomparedwithnegativecontrolplants(FigureS1).
However,acleardifferenceinSlAREB1expressionlevelsbetweenthetransgeniclineswasobvious
withSlAREB1#3lineshowingthehighestvalue. Thisparticularlineshowedadelayedgermination
phenotype when compared with other tested lines. The T3 generations of two homozygous lines
overexpressingdifferentlevelsofSlAREB1(SlAREB1#2andSlAREB1#3)andasingletransgenicline
harboringemptyT-DNAwereselectedandusedfurtherforinvitropreservationexperiments.
2.2. SlowGrowthPreservation
To test the effect of sucrose concentration on slow growth preservation of transgenic tomato
lines, microshoots were incubated for 12 weeks on MS media supplemented with different levels
of sucrose (0, 100, 200, 300 mm). Microshoots placed on media lacking sucrose showed a growth
arrest phenotype where no significant changes in plant height, roots number and leaves number
mean values were observed (Table 1). At 100 mm sucrose level, all tested plants showed normal
growthanddevelopmentwithrespecttoplantheight,leavesnumberandrootsnumber,although
nosignificantdifferenceswereobservedbetweentransgeniclinesandnegativecontrol(Figure1a;
Table1). Increasingsucroseconcentrationinthestoragemediato200mmsignificantlyreducedgrowth
innegativecontrolmicroshootswhencomparedwithtransgeniclinesoverexpressingSlAREB1after6
and12weeksofstorage(Figure1b;Table1). Theeffectof200mmsucroselevelonrootingwasalso
obviouswhereafullinhibitionofrootformationwasobservedinnegativecontrolmicroshootswhen
comparedwithSlAREB1transgeniclines. At300mmsucroselevel,microshootsoftransgenicline
SlAREB1#3significantlyproducedthehighestmeanvaluesforplantheight,rootnumberandleaf
numberindicatingitsabilitytogrowunderhighosmoticstressconditions(Table1). Atthesamelevel,
themicroshootsoftransgeniclineSlAREB1#2showedgrowthretardationphenotype. Forregrowth
potential, the SlAREB1#3 microshoots were able to give the highest percentage after 12 weeks of
culturingat200and300mmsucroselevelswhencomparedwithnegativecontrol(Table2).
TotesttheeffectofABAconcentrationonslowgrowthpreservationoftransgenictomatolines,
microshootswereincubatedfor12weeksonMSmediasupplementedwithdifferentlevelsofABA
(0,4,8and12µm). SlAREB1#3microshootshadsignificantlylowerrootsandleavesnumberat4µm
ABAlevelafter6and12weeksofstoragewhencomparedwithABA-freemedia(Table3).Furthermore,
onlySlAREB1#3microshootscultivatedonMSmediasupplementedwith4µmABAlevelfor12weeks
showed a significantly lower plant height when compared with negative control and SlAREB1#2
microshoots. Adecreaseinmicroshootsheightwasrecordedafter6weeksofculturingwhenABA
wasaddedtothemediacomparedtothecontrol(ABA-freemedia),whichwasmorepronouncedafter
12weeksonlyathighABAlevels(8and12µm)(Table3). Forrootsandleavesnumbers,significant
reductionsinalltestedlineswereobservedathighABAlevels(8and12µm)after6and12weeks
ofstoragewhencomparedwithABA-freemedia. Forregrowthpotential,theadditionofABAtothe
preservationmediadidn’taffecttheregrowthpercentagesofalltestedlineswhencomparedwith
ABA-freemedia(Table2).
Int.J.Mol.Sci.2017,18,1477 4of19
Table1. Effectsofsucroseconcentrationonplantheight, rootsnumberandleavesnumberofinvitroconservedmicroshootsofnegativecontrol, SlAREB1#2,
SlAREB1#3transgenictomatoplantsafter6and12weeksofstorage.
Sucrose PlantHeight(cm) RootsNumber LeavesNumber
Concentration(mm)
Control SlAREB1#2 SlAREB1#3 Control SlAREB1#2 SlAREB1#3 Control SlAREB1#2 SlAREB1#3
6weeks
0 1.00a*±0.00** 1.00a±0.00 1.21a±0.10 0.00a±0.00 0.00a±0.00 0.00a±0.00 1.00a±0.00 1.00a±0.00 1.14a±0.67
100 5.32a±0.59 5.85a±0.42 5.99a±0.66 7.14a±1.12 7.66a±1.55 7.89a±2.49 7.86a±1.28 7.29a±1.67 6.43a±1.37
200 1.71b±0.24 6.13a±1.18 6.47a±0.50 0.00b±0.00 10.71a±2.31 9.57a±1.86 3.57b±1.03 6.29a±1.23 5.71a±1.23
300 1.14b±0.13 1.97b±0.36 3.84a±0.92 0.00b±0.00 0.57b±0.56 3.43a±0.83 2.00b±0.61 1.57b±0.83 6.00a±1.22
12weeks
0 1.00a±0.00 1.00a±0.00 1.34a±0.11 0.00a±0.00 0.00a±0.00 0.00a±0.00 1.00a±0.00 1.00a±0.00 1.43a±0.77
100 8.47a±1.10 8.55a±0.68 9.10a±1.10 10.14a±2.39 9.23a±1.29 10.86a±1.32 12.29a±2.71 11.46a±2.77 10.43a±1.58
200 2.54b±0.21 7.27a±1.29 8.77a±0.72 0.14b±0.04 13.57a±3.33 13.43a±3.03 5.57b±1.59 10.86a±2.79 9.43a±1.24
300 1.24b±0.12 2.22b±0.44 5.27a±0.76 0.00b±0.00 0.86b±0.73 7.43a±2.10 2.57b±0.56 2.43b±0.56 8.57a±2.19
*BasedonTukey’sHSDtest,differentletterindicatessignificantdifferenceamongtransgeniclines(p<0.05)foreachcombinationofsucroseconcentrationandnumberofweeks.**Values
arethemean±SD.
Table2.Regrowthpercentagesofinvitroconservedmicroshootsofnegativecontrol,SlAREB1#2,SlAREB1#3transgenictomatoplantsafter12weeksfromculturing
onmediawithdifferentlevelsofsucroseandabscisicacid(ABA).
Transgenic SucroseConcentration(mm) ABAConcentration(µm)
Lines 0.00 100 200 300 0 4 8 12
Control 0.00a*±0.00** 78.6a±10.97 38.5b±13.00 21.4b±10.97 71.4a±12.07 71.4a±12.07 81.8a±10.31 83.0a±9.96
SlAREB1#2 0.00a±0.00 81.8a±10.31 71.4a±12.07 38.5b±13.00 76.9a±11.26 78.6a±10.97 84.6a±9.64 83.0a±9.96
SlAREB1#3 0.00a±0.00 85.7a±9.35 78.6a±10.97 92.3a±7.12 85.7a±9.35 69.2a±12.97 78.6a±10.97 78.56a±10.97
*BasedonTukey’sHSDtest,differentletterindicatessignificantdifferenceamongtransgeniclines(p<0.05)foreachconcentration.**Valuesarethemean±SD.
Int.J.Mol.Sci.2017,18,1477 5of19
Table3.EffectsofABAconcentrationonplantheight,rootsnumberandleavesnumberofinvitroconservedmicroshootsofnegativecontrol,SlAREB1#2,SlAREB1#3
transgenictomatoplantsafter6and12weeksofstorage.
ABAConcentration PlantHeight(cm) RootsNumber LeavesNumber
(µm)
Control SlAREB1#2 SlAREB1#3 Control SlAREB1#2 SlAREB1#3 Control SlAREB1#2 SlAREB1#3
6weeks
0 5.04a*±0.55** 5.14a±1.31 5.01a±0.70 5.86a±1.42 6.57a±2.01 5.57a±1.03 6.14a±1.42 5.71a±1.58 5.29a±1.00
4 2.91a±0.66 2.46a±0.37 2.11a±0.31 5.43a±1.81 4.57a±1.20 1.86b±1.55 5.14a±1.42 5.29a±1.80 2.86b±1.13
8 1.41a±0.11 1.43a±0.15 1.53a±0.25 0.00a±0.00 0.14a±0.40 0.00a±0.00 2.71a±0.52 3.14a±0.95 3.00a±0.86
12 1.46a±0.20 1.51a±0.13 1.43a±0.18 0.00a±0.00 0.29a±0.80 0.00a±0.00 2.57a±0.83 3.29a±0.80 3.14a±0.40
12weeks
0 7.19a±1.00 7.33a±1.75 6.93a±0.56 9.86a±2.69 8.71a,b±1.18 7.57b±2.10 13.00a±3.56 11.71a±2.91 11.00a±1.83
4 6.90a±1.19 7.14a±1.61 2.26b±0.32 8.43a±2.79 6.86a±0.95 1.86b±1.55 9.71a±2.42 8.29a±1.58 3.86b±1.42
8 1.53a±0.15 1.94a±0.29 1.60a±0.15 0.14a±0.40 0.57a±0.83 0.14a±0.40 5.43a±0.83 5.14a±0.95 4.43a±0.83
12 1.73a±0.27 1.73a±0.26 1.61a±0.20 0.00a±0.00 0.29a±0.52 0.00a±0.00 5.29a±0.80 4.14a±0.73 3.71a±1.00
*BasedonTukey’sHSDtest,differentletterindicatessignificantdifferenceamongtransgeniclines(p<0.05)foreachcombinationofABAconcentrationandnumberofweeks.**Values
arethemean±SD.
Int.J.Mol.Sci.2017,18,1477 6of19
Int. J. Mol. Sci. 2017, 18, 1477 6 of 19

FFiigguurree 11. .SSloloww ggrroowwthth pprreesseerrvvaatitoionn oof ftwtwoo trtraannssggeennicic lilnineess oovveerreexxpprreessssiinngg SSlAlARREEBB11 ((SSllAARREEBB11##22 aanndd
SSlAlARREEBB11##33)) aanndd nneeggaattiivvee ccoonnttrrooll aafftteerr 1122 wweeeekkss ooff ccuultltuurriinngg. .(a(a) )GGrroowwthth oonn MMSS mmeeddiaia ssuupppplelemmeenntteedd
wwiitthh 110000 mmmm ssuuccrroossee wwiitthhoouutt AABBAA; ;((bb)) GGrroowwtthh oonn MMSS mmeeddiiaa ssuupppplleemmeenntteedd wwitithh 220000 mmmm ssuuccrroossee
withoutABA;(c)GrowthonMSmediasupplementedwith100mmsucroseand4µmABA;(d)Growth
without ABA; (c) Growth on MS media supplemented with 100 mm sucrose and 4 μm ABA; (d)
onMSmediasupplementedwith100mmsucroseand8µmABA.
Growth on MS media supplemented with 100 mm sucrose and 8 μm ABA.
22.3.3. .CCrryyoopprreesseerrvvaattioionn
TThhee eeffffeecctt ooff ccrryyoopprreesseerrvvaattioionn oonn ttrraannssggeennicic totommaatoto lilnineess wwaass sstutuddieiedd bbyy uussiningg eennccaappssuulalatitoionn--
ddeehhyyddrraatitoionn, ,VV-c-rcyryoopplaltaetse sanadn dseesedesd. Fs.orF eonrcaepnscualpastuiolnat-idoenh-ydderhaytdiorna,t imonic,romsihcoroostsh oofo ttrsaonfsgterannics glienneisc
wlienrees swuberjeecsteudb jteoc tdeidffetorednitf tfreeraetnmtetrnetast mincelnutdsiinngc lcuodminbgincaotimonbsin oaft itownos soufctrwosoe sluevcreolss e(0l.e4v aenlsd( 00..84 Man)d,
i0n.c8uMba)t,ioinnc puebraitoidon (1p aenrido d3 (d1aayns)d a3ndd adyesh)yadnrdatdioenh ypderriaotdio n(1,p 3e,r i6o hd) (b1e,f3o,r6e hsu)bbjeefcotirnegs uthbejemct tino gliqthueimd
ntiotrloiqguenid onri trwoigtehnouotr (wroitohmou tte(mropoemrattuemrep).e rIantiutiraell)y., Inthiteia lmlyo,itshtuerme ociostnuterentc oonf tetnretaotefdtr ebaetaeddsb ewaadss
dweatesrmdeitneerdm iannedd afonudnfdo uton ddteocrdeeacsree awseithw iitnhcirnecarseinagsi ntghet hpeeprieordio dofo fddeheyhdyrdartaiotino n(F(Figiguurer eSS22).) .FFoorr
eennccaappssuullaatteedd sshhoooott ttiippss wwiitthhoouutt lilqiquuiidd nnititrrooggeenn,, aa ccoommpplelettee ssuurrvvivivaall ppeerrcceennttaaggee ((110000%%)) wwaass oobbsseerrvveedd
wwiitthh nneeggaattiivvee cocnontrtorlo,lS, lASlRAERBE1B#21,#a2n, danSldA RSElABR1#E3Bt1r#e3a tetrdeiante0d.4 iMn s0u.4cr oMse fsourcoronseed afoyra nodned edhayyd raantedd
dfoehry0dorra3tehd afonrd 0i nor0 3.4 hM ansdu cirno 0se.4f Mor sthurcereosdea fyosr atnhdrede edhayydsr aantedd dfeohry0dhra(tFeidg uforer 20) h. O(Fnigtuhreeo 2th).e Ornh athned ,
oetnhcearp hsualnadte, denshcoapotsutilpasteodf nsehgoaottiv teipcso notfr onletrgeaattievde icno0n.4trMol sturecarotesde fionr 30.d4a Mys asnudcrdoeshe yfdorra t3e ddafoyrs 3aanndd
d6ehhypdroradtuedce fdorlo 3w aenrds u6 rhv ipvraoldpuecrecden ltoawgeesr wsuhrevnivcaol mpepracreendtawgietsh wSlhAeRnE cBo1m#p2aarnedd SwlAithR ESBlA1#R3EsBh1o#o2t atnipds .
SElAncRaEpBsu1l#a3t esdhoshoto otitptsip. Esnocfanpesgualtaivteedc oshnotrootl tliipnse otrfe nateegdatiniv0e. 8coMntsruocl rloinsee ftorera1tedda yinw 0i.t8h M0h sudcerhoysde rfaotrio 1n
dsahyo wweitdh a0 shli gdhethryeddruatcitoionn shinowsuerdv iav aslligphert creendtuagcteisonw ihne nsucrovmivpala rpeedrcwenitthagSelsA wREheBn1 ctroamnpsgaernedic wlinitehs
SblAutRaEcBl1e atrrarnesdguecntiioc nlinweass boubts ae rcvleeadr irnedshuocotitotnip wsadse ohbysderravteedd ifno rsh3oaontd ti6psh d(Fehigyudrreaste2da nfodr 33) .anOdn 6t hhe
(oFtihgeurrehsa n2d a,nndo n3-c).r yOonp rtehsee rovtehdeer nhcaanpdsu, lnaotend-csrhyoooptrteispesrvofedSl AenRcEaBp1s#u3lattreadn ssgheonoict ltiinpes torefa StelAdRinE0B.18#M3
tsruancrsogseenfioc rl3indea ytsresahtoewd eidn a0s.i8g nMifi csaunctrionscer efaosre i3n sduaryvsi vsahlopwerecden taa gseigsnwifhiceanncto minpcraeraedsew iinth snuergvaivtiavle
pceorncternotlaagneds wSlhAeRnE cBo1m#2pashreodo twtiipths .negative control and SlAREB1#2 shoot tips.
IInn ggeenneerraal,l ,tthhee hhiigghheesstt rreeggrroowwtthh ppeerrcceennttaaggeess iinn tthhee nnoonn--ccrryyoopprreesseerrvveedd ttrraannssggeennicic lliinneess wweerree
oobbsseerrvveedd inin sshhoooott ttiippss ttrreeaatteedd iinn 00.4.4 MM ssuuccrroossee ffoorr oonnee ddaayy aanndd ddeehhyyddrraatteedd ffoorr 00 hh ((FFiigguurree 44)).. TThhee
ddeehhyyddrraatitoionn oof fshsohooto ttiptisp fsorf o6r h6 rehsurletseudl tiend siignnisfiigcannifit craendtucretidounc itnio rnegirnowretghr powertchenptaergceesn otfa aglels teosfteadll
ltinesetse datl i0n.e4s Mat 0s.u4cMrosseu clerovseel lienvceulbiantceudb aetiethdeeri tfhoer r1f oorr1 3o rda3ydsa wyshwenh ecnomcopmapreadre wdiwthit h0 0h.h .HHoowweveveer,r ,
ttrraannssggeenniicc SSllAARREEBB11##22 aanndd SSllAARREEBB11##33 sshhoooottt tipipsst rtereaateteddi nin0 .04.4M Ms uscurcorsoesfeo fro3r d3a dyasyasn adndde dhyehdyradtreadtefodr
f3orh 3s hho wsheodwsiegdn isfiicganniftilcyahnitglyh ehrirgehgerro wretghrpoewrcthen ptaegrecsenwtahgeensc owmhpenar ecdomwpitahrende gwatiitvhe ncoengatrtoivle(F cigounrtero4l) .
(IFrirgeuspree c4t)iv. eIrorefsipnecuctbivaeti oonf tiinmcuebaantdiodne thimyder aatniodn dpeehryioddr,atthioent rpeeartimoden, tthine 0tr.8eaMtmseunctr oisne 0r.e8s uMlt esducinrotshee
raebsuseltnecde ionf trhege raobwsethncine oefn creagprsouwlatthed in-d eenhcyadprsauteladtende-gdaethivyedcroantetrdo nlsehgoaotitvtei pcson(Ftrigoul rsehso4ota tnidps5 ()F.iOgnurtehse
4o athnedr 5h).a Ondn, tthrea nostgheenr ihcaSnldA, RtrEaBn1sg#2enainc dSlSAlARERBE1B#12# a3nsdh oSolAtRtipEsB1tr#e3a stehdooint t0ip.8s Mtresautecdro isne 0f.o8r M1 dsuacyroansed
fdoer h1y ddarya taenddf odre0hyhdwraetreeda fbolre 0to hr wegerroew ab(Flei gtou rreesg4roawnd (F5i)g.uMreesa n4 wanhdil e5,).S MlAeRaEnBw1h#i3les,h SoloAtRtiEpBs1t#re3a sthedooitn
tips treated in 0.8 M sucrose for 3 days and dehydrated for 0 and 3 h showed a significant ability to
regrow when compared with other tested lines. On the contrary to non-cryopreserved treatments,
Int.J.Mol.Sci.2017,18,1477 7of19
0.8Msucrosefor3daysanddehydratedfor0and3hshowedasignificantabilitytoregrowwhen
Int. J. Mol. Sci. 2017, 18, 1477 7 of 19
comparedwithothertestedlines. Onthecontrarytonon-cryopreservedtreatments,neithersurvival
norregrowthwereobservedinalltransgeniclinessubjectedtocryopreservationinliquidnitrogen
neither survival nor regrowth were observed in all transgenic lines subjected to cryopreservation in
(Figures3and5).
liquid nitrogen (Figures 3 and 5).
Int. J. Mol. Sci. 2017, 18, 1477 7 of 19
neither survival nor regrowth were observed in all transgenic lines subjected to cryopreservation in
liquid nitrogen (Figures 3 and 5).

Figure 2. Survival percentages of encapsulated-dehydrated shoot tips of non-cryopreserved s hoots
Figure2. Survivalpercentagesofencapsulated-dehydratedshoottipsofnon-cryopreservedshoots
tips (−LN) of negative control, SlAREB1#2 and SlAREB1#3 transgenic tomato plants as affected by air
tips(F−igLuNre) 2o. fSnuervgiavtailv peecrocennttraogle,sS loAf RenEcBap1s#u2laatnedd-dSelAhyRdEraBte1d# 3shtroaont stigpesn oicf ntoonm-cartyoopprleasnetrsveads ashffoeocttse dby
adierhdyetdihprysa dt(i−roLanNti od) noufrd nauetigroaanttii voaenf tceaorfnt eptrrroepl,t rrSeeltAarteRmaEteBmn1et# 2nw tainwtdhi tS0hl.A40 R.a4EnaBdn1 #d03. 08t.r 8aMnMs sguseuncriccor otsoesme cacotonon cpcelenanntrttrasa taitsoi onanf ffefocortre odon nbeey ooarirr tthhrreeee
ddaayyssd ((evvhaayllduureeasst iaoanrree d ttuhhreea tmimonee aaannft e±±r pSSrDDet))r.. eBaBtaamsseeedndt oownni thTT uu0kk.4ee ayyn’’ssd HH0.SS8 DDM ttseeussctt,,r oddsieiff ffceeorrneecnnettn tllreeatttttieeorrn i infnoddr iioccnaatetee ossr s stiihggrnneieifif iccaanntt
differdeanycse ( vaamluoensg a trrea tnhseg meneiacn l i±n eSsD ()p. B<a 0s.e0d5 )o fno rT euakcehy’ sc oHmSbDi nteastti,o dni foffe rseunctr loestete cr oinndceicnattreas tsioignn,i nfiucamntb er of
differenceamongtransgeniclines(p<0.05)foreachcombinationofsucroseconcentration,numberof
daysd, aifnfedr eanicre d aemhoyndgr atrtaionnsg denuirca ltinioens .( p < 0.05) for each combination of sucrose concentration, number of
days,andairdehydrationduration.
days, and air dehydration duration.

FigurFeig3u.rSeu 3r. vSiuvravlivoafl towf otwtora tnrasngsegneincicl ilnineesso ovveerreexxpprreessssiinngg SSlAlARREBE1B (1Sl(ASlRAERBE1#B21 a#n2da SnldARSElAB1R#E3B) a1n#d3 )and
nFiegguarnteiev g3ea. tcSivouenr vtcroiovnlatr(lo -ovl fe( -tvwceoo nc ottrrnaotnrl)oslgs) hesnohioocot ltti intpiepsss aoafvftteeerrre eexnnpccraaeppssssuiunlalgati toSinolA ndRedEheyBhd1yr a(dStriloaAntiR otErneBat1trm#e2ae ntamtn oedfn S1tl AodfaRy1E (Bd11 aD#y3) )( 1anDd)
innecguabitnaivctiueo bncaotiinnotn0r o.i8nl M0(-.8v seMu cc srouoncstreroosaeln) a dsnhd0o h0o htd edtihephysyd darfratatetiiroo nnen wwciiattphh s((u++LlLaNtNi)o )onro wrdweithhityohudotr ualittqiluoiiqndu tnirdietranotigmternoe ng(−et LnoNf( −)1. LdNa)y. (1 D)
incubation in 0.8 M sucrose and 0 h dehydration with (+LN) or without liquid nitrogen (−LN).
Int.J.Mol.Sci.2017,18,1477 8of19
Int. J. Mol. Sci. 2017, 18, 1477 8 of 19
Int. J. Mol. Sci. 2017, 18, 1477 8 of 19

Figure 4. Regrowth percentages of encapsulated-dehydrated shoot tips of non-cryopreserved shoots
Figure4.Regrowthpercentagesofencapsulated-dehydratedshoottipsofnon-cryopreservedshoots
Figure 4. Regrowth percentages of encapsulated-dehydrated shoot tips of non-cryopreserved shoots
tips (−LN) of negative control, SlAREB1#2 and SlAREB1#3 transgenic tomato plants as affected by air
tips(−LN)ofnegativecontrol,SlAREB1#2andSlAREB1#3transgenictomatoplantsasaffectedby
tips (−LN) of negative control, SlAREB1#2 and SlAREB1#3 transgenic tomato plants as affected by air
dehydration duration after pretreatment with 0.4 and 0.8 M sucrose concentration for one or three
airdehydrationdurationafterpretreatmentwith0.4and0.8Msucroseconcentrationforoneorthree
dehydration duration after pretreatment with 0.4 and 0.8 M sucrose concentration for one or three
ddaayyss ((vvaalluueess aarree tthhee mmeeaann ±± SSDD)).. BBaasseedd oonn TTuukkeeyy(cid:48)′ss HHSSDD tteesstt,, ddiiffffeerreenntt lleetttteerr iinnddiiccaatteess ssiiggnniifificcaanntt
days (values are the mean ± SD). Based on Tukey′s HSD test, different letter indicates significant
difference among transgenic lines (p < 0.05) for each combination of sucrose concentration, number of
differenceamongtransgeniclines(p<0.05)foreachcombinationofsucroseconcentration,numberof
difference among transgenic lines (p < 0.05) for each combination of sucrose concentration, number of
days, and air dehydration duration.
days,andairdehydrationduration.
days, and air dehydration duration.

Figure 5. Regrowth of two transgenic lines overexpressing SlAREB1 (SlAREB1#2 and SlAREB1#3) and
FFiigguurree 55. .RRegergorwowtht hofo tfwtow toratnrasgnesngiecn liicnelisn oevseorvexeprerxepssriensgs iSnlgARSlEABR1E (BSl1A(RSElABR1#E2B a1n#d2 SalnAdRSElBA1R#E3)B a1n#d3)
negative control (-ve control) shoot tips after encapsulation dehydration treatment of 1 day (1 D)
naengdatnivegea ctiovnetrcooln (t-rvoel (c-ovnetcroonl)t rsohlo)osht otioptst iapfstearf teenrceanpcsauplsautiloanti odnehdyehdyradtriaotnio tnretaretmatemnet notfo 1f 1ddaya y(1(1 DD))
incubation in 0.8 M sucrose and 0 h dehydration with (+LN) or without liquid nitrogen (−LN).
incubationin0.8Msucroseand0hdehydrationwith(+LN)orwithoutliquidnitrogen(−LN).
incubation in 0.8 M sucrose and 0 h dehydration with (+LN) or without liquid nitrogen (−LN).
Int.J.Mol.Sci.2017,18,1477 9of19
Int. J. Mol. Sci. 2017, 18, 1477 9 of 19
To test the effect of cryopreservation using V-cryoplates technique on the transgenic tomato
To test the effect of cryopreservation using V-cryoplates technique on the transgenic tomato
lines,shoottipsweretreatedin0.3Msucrosefortwoincubationperiods(1or3days)withoutcold
lines, shoot tips were treated in 0.3 M sucrose for two incubation periods (1 or 3 days) without cold
acclimatizationandin0.3Msucroseforthreedayswithacoldacclimatizationpretreatmentfor3days.
acclimatization and in 0.3 M sucrose for three days with a cold acclimatization pretreatment for 3
Thetreatedshoottipsweresubjectedthentoeitherliquidnitrogen(cryopreservation)orkeptatroom
days. The treated shoot tips were subjected then to either liquid nitrogen (cryopreservation) or kept
temperature(withoutliquidnitrogen). Irrespectivetoincubationtimeorcoldacclimatization, the
at room temperature (without liquid nitrogen). Irrespective to incubation time or cold
V-cryoplatestechniqueproducedhighsurvivalpercentagesinalltestedtransgeniclinesthatwerenot
acclimatization, the V-cryoplates technique produced high survival percentages in all tested
subjectedtoliquidnitrogentreatment(Figures6and7). Inaddition,nomajordifferencesinregrowth
transgenic lines that were not subjected to liquid nitrogen treatment (Figures 6 and 7). In addition,
percentageswereobservedbetweenthetreatednon-cryopreservedtransgeniclines(Figures6and8).
no major differences in regrowth percentages were observed between the treated non-cryopreserved
Nevertheless,regrowthpercentagesofnon-cryopreservedshoottipswereconsiderablylowerthan
transgenic lines (Figures 6 and 8). Nevertheless, regrowth percentages of non-cryopreserved shoot
observed survival percentages (Figure 6). Cryopreserved shoot tips (with liquid nitrogen) of all
tips were considerably lower than observed survival percentages (Figure 6). Cryopreserved shoot
transgeniclinesshowedlowersurvivalpercentageswhencomparedwithnon-cryopreservedshoot
tips (with liquid nitrogen) of all transgenic lines showed lower survival percentages when compared
tips(Figures6and7). Ontheotherhand,cryopreservedtransgenicSlAREB1#2andSlAREB1#3shoot
with non-cryopreserved shoot tips (Figures 6 and 7). On the other hand, cryopreserved transgenic
tips treated in 0.3 M sucrose for 3 days with or without cold acclimatization showed significantly
SlAREB1#2 and SlAREB1#3 shoot tips treated in 0.3 M sucrose for 3 days with or without cold
highersurvivalpercentageswhencomparedwiththenegativecontrol. Noregrowthofcryopreserved
acclimatization showed significantly higher survival percentages when compared with the negative
shoottipswasobservedinalltransgeniclines(Figures6and8).
control. No regrowth of cryopreserved shoot tips was observed in all transgenic lines (Figures 6 and 8).

Figure 6. Survival and regrowth percentages of V-cryoplates treated non-cryopreserved and
Figure 6. Survival and regrowth percentages of V-cryoplates treated non-cryopreserved and
cryopreserved shoots tips of negative control, SlAREB1#2, SlAREB1#3 transgenic tomato plants as
cryopreservedshootstipsofnegativecontrol,SlAREB1#2,SlAREB1#3transgenictomatoplantsas
affected by pretreatment with 0.3 M sucrose concentration for one day or three days with or without
affectedbypretreatmentwith0.3Msucroseconcentrationforonedayorthreedayswithorwithout
ccoolldd aacccclliimmaattiizzaattiioonn ((vvaalluueess aarree tthhee mmeeaann ±± SSDD)).. BBaasseedd oonn TTuukkeeyy′(cid:48)ss HHSSDD tteesstt,, ddiiffffeerreenntt lleetttteerr iinnddiiccaatteess
significant difference among transgenic lines (p < 0.05) for each combination of cryopreservation and
significantdifferenceamongtransgeniclines(p<0.05)foreachcombinationofcryopreservationand
cold acclimatization treatment.
coldacclimatizationtreatment.
For seed cryopreservation experiments, 100 seeds of each transgenic tomato line were subjected
Forseedcryopreservationexperiments,100seedsofeachtransgenictomatolineweresubjected
to dissection by using silica gel until they reached an equilibrium state where the initial fresh weight
todissectionbyusingsilicageluntiltheyreachedanequilibriumstatewheretheinitialfreshweight
is equal to dry weight. Thereafter, the treated seeds were subjected then to either liquid nitrogen
is equal to dry weight. Thereafter, the treated seeds were subjected then to either liquid nitrogen
(cryopreservation) or kept at room temperature (without liquid nitrogen). Seed cryopreservation was
(cryopreservation) or kept at room temperature (without liquid nitrogen). Seed cryopreservation
performed successfully with the ability of all transgenic lines to germinate after liquid nitrogen
wasperformedsuccessfullywiththeabilityofalltransgeniclinestogerminateafterliquidnitrogen
treatment (Figure 9; Table 4). However, a significant difference exists between tested transgenic lines
treatment (Figure 9; Table 4). However, a significant difference exists between tested transgenic
where SlAREB1#3 seeds produced lower percentages when compared with negative control and
lines where SlAREB1#3 seeds produced lower percentages when compared with negative control
SlAREB1#2. This was also observed in non-cryopreserved seeds indicating that the reduced
and SlAREB1#2. This was also observed in non-cryopreserved seeds indicating that the reduced
germination in SlAREB1#3 is most likely related to the genotype itself. This was further confirmed by
germinationinSlAREB1#3ismostlikelyrelatedtothegenotypeitself. Thiswasfurtherconfirmedby
performing a germination test on tested transgenic lines seeds (without silica gel treatment)
incubated on filter paper with distilled water (data not shown).
Int.J.Mol.Sci.2017,18,1477 10of19
performingagerminationtestontestedtransgeniclinesseeds(withoutsilicageltreatment)incubated
onfilterpaperwithdistilledwater(datanotshown).
Int. J. Mol. Sci. 2017, 18, 1477 10 of 19
Int. J. Mol. Sci. 2017, 18, 1477 10 of 19

FigurFeig7u.rSeu 7r. vSiuvravlivoafl towf otwtora tnrasngsegneincicli lnineesso ovveerreexxpprreessssiinngg SSlAlARREBE1B 1(S(lASlRAERBE1#B21 a#n2da SnldARSlEABR1#E3B) 1a n#d3) and
negatnievgeFaitcgiouvrnee tc r7oo. nSltu(r-rovvl ie(v-avcloe o ncf totrwnotolr) otrla)af natesfgtreerVn iV-cc -lrciynryoeosp pollvaaettereessx ttprrreeeaaststmimnegen nSt ltAoofR f3E 3Bd1ad y(aS yilnAciRnuEcbBua1tb#ioa2n tai noindn 0Si.ln3A M0R.E3 sBuM1c#r3os) usaenc rdaon sde and
coldacoclcdnlie magcacatlitivimze aacttoiinzoatnrtoifolo n(r- vf4oer dc 4oa ndytasroywls) iwatfhitteh(r+ (VL+-LNcNry))oo oprrla wwteiistt hhtrooeuuattt mlliiqeqnuuti diod fn 3nit dirtoargyoe ginne c(nu−Lb(−aNtiL)o. nN i)n. 0.3 M sucrose and
cold acclimatization for 4 days with (+LN) or without liquid nitrogen (−LN).

FigurFeig8Fu.irgReu e8rge. Rr8o.e wRgretoghwrootwhft tohwf o tofw ttwor aotrn tarsnagsnegsngeinecnicilc iln ilnienesess oo ovvveeerrreeexxxppprrreeessssssiininnggg SSlSlAAlRAREREBBE11B ( S(1SlAl(ASRlREAEBRB1#E12#B 2a1 na#dn2 dSal SAnlRdAERSBEl1AB#31R)#E a3Bn) da1n #d3 )and
negatnievgenaetcgiovaneti vtcreoo nclot(rn-otvlr oe(l- vc(o-ev nceto crnootnlr)torlao)fl )at efatrfetreV rV- Vc-rc-ycrryoyopoppllalaatteteesss tttrrreeeaaatttmmmeeennntt toofof 3f3 d3daadyy ai nyinccuinubcbautaitobinoa ntiin oi n0n .30i .n3M M0 s.u3 scuMrocsreos usaenc rdaon sde and
cold acclimatization for 4 days with (+LN) or without liquid nitrogen (−LN).
coldacoclcdli maccaltiimzaattiizoantifoonr f4ord 4a dyasyws iwthith(+ (L+LNN))o orr wwiitthhoouutt lliiqquuidid nnitirtorgoegne (n−L(−NL). N).
Table 4. Germination percentages of non-cryopreserved and cryopreserved seeds of negative control,
Table 4. Germination percentages of non-cryopreserved and cryopreserved seeds of negative control,
Table4.SGlAeRrEmBi1n#a2,t iSolAnRpEeBr1c#e3n ttraagnsegseonficn toomn-actroy polapnrtess. ervedandcryopreservedseedsofnegativecontrol,
SlAREB1#2, SlAREB1#3 transgenic tomato plants.
SlAREB1#2,SlAREB1#3transgenictomatoplants.
Non-Cryopreserved Cryopreserved
Transgenic Lines NonN-uCmryboepr roefs Deravyesd NuCmrbyeorp oref sDearvyes d
Transgenic Lines N5on-CryoNpurem7s beervr eofd Days10 14 5 7NumCbreyro opf rD1e0sa yesr ved 14
Transgenic Control 60 a5 * ± 7.07 ** 80 7a ± 7.07 84 a1 0± 5.47 100 a1 ±4 0.00 60 a ±5 7.07 78 a ± 77.07 86 a ± 51.04 7 100 a ± 01.040
Lines CoSnlAtrRoEl B1#2 60 a 5*0 ± a 7 ±.0 17N2 .*2u*4 m b8e07r a2o ±af ±7D .40.7a4 y7 s 8748 a a ± ± 5 5.4.477 19040 aa ± ± 5 0.4.070 606 0a a± ±1 27..2047 727 8a a± 4±. 47N7.0 7u m7b6 8ea6r ± ao 5 ±f.4 5D7. 4a7y s96 1a0 ±0 8 a.4 ±9 0.00
SlARSlEABR1E#B25 1#3 50 a2 5± b1 2±. 274.07 7 724 a4 b± 4± .54.74 7 107580 ab ±± 51.04.70 0 951444 ab ±± 85..4497 3600 ba ±±5 71.20.72 4 427 2b a± ±5. 4477.4 7 50 b7 6± a1 0±. 050.4 71 074 b9 6± a5. 4±7 8 .49 14
SlA*R BEBa1s#e3d on Tu2k5 eby ±’ s7 .0H7 SD t4e4s bt, ±d 5i.f4f7e ren50t ble ±t 1te0r.0 0i ndi5c4a tbe ±s 8s.4i9g nifi3c0a nb t± d7.i0f7f ere4n2c be ±a 5m.47o ng5 t0r ba n± s1g0.e0n0 ic l7in4 ebs ± 5 .47
Control 60a*±7.07** 80a±7.07 84a±5.47 100a±0.00 60a±7.07 78a±7.07 86a±5.47 100a±0.00
SlAREB1#2* B(aps5 e<0d a0 .o±0n51) 2Tf.o2ur4k eeayc’hs 7cH2oaSnDc±e n4te.t4rs7att, idonif.7f *8e*ra eV±natl5 ul.e4e7tst earr ei n9th4deai cm±ate5ea.s4n 7s ±ig SnD6if.0 icaa±nt1 2d.i2f4fere7n2cea ±am4.o47ng tr7a6nasg±e5n.i4c7 line9s6 a±8.49
SlAREB1#3(p < 02.505b)± fo7r. 0e7ach co4n4cbe±nt5r.a4t7ion. 5*0* bV±alu10e.0s0 are t5h4eb m±e8a.4n9 ± SD3.0 b±7.07 42b±5.47 50b±10.00 74b±5.47
*BasedonTukey’sHSDtest,differentletterindicatessignificantdifferenceamongtransgeniclines(p<0.05)for
eachconcentration.**Valuesarethemean±SD.