Influence of bacterial density during preculture on Agrobact

ORIGINAL PAPER

In?uence of bacterial density during preculture

on Agrobacterium -mediated transformation of tomato

Nan Gao ?Weishou Shen ?Yu Cao ?Yanhua Su ?Weiming Shi

Received:7December 2008/Accepted:8July 2009/Published online:25July 2009óSpringer Science+Business Media B.V.2009

Abstract An improved bacterial preculture protocol for Agrobacterium -mediated genetic transformation was developed for an economic tomato cultivar (Solanum lyco-persicum L.cv.Zhongshu No.4).Frequencies of transient gene expression and stable transformation were in?uenced by the density of Agrobacterium preculture and not the density of Agrobacterium used for infection.The improved protocol presented in this study depends on the use of an overnight-grown Agrobacterium preculture density of OD 600nm =1.0,diluted 1/10th with Luria-Bertani (LB)liquid medium,and grown for an additional 4h.Cultures are collected and resuspended in a liquid cocultivation medium-I,adjusted to OD 600nm =aadc476f011ca300a6c390f3ing this modi?ed Agro-bacterium preparation,transient b -glucuronidase expression was higher than 90%,and transformation ef?ciency reached 44.7%.This improved transformation is simple,repeatable,does not require a feeder layer,and most notably,the trans-formation frequency is stable and highly ef?cient.Keywords Density of bacterial cells ánptII á

Solanum lycopersicum L.áTransient b -glucuronidase (GUS)expression

Introduction

Tomato (Solanum lycopersicum L.)is an economically important crop.However,abiotic stresses,including salinity,heat,drought,and nutrient de?ciencies (such as phosphate and nitrate)often constrain fruit productivity (Raghothama 2000;Abel et al.2002;Zhu 2002;Bhatnagar-Mathur et al.2008).Developing transgenic plants is an effective approach for improving abiotic stress tolerance (Park et al.2005;Bhatnagar-Mathur et al.2008).Estab-lishment of an ef?cient transformation system is essential.The ?rst successful transformation of tomato was reported by Horsch et al.(1985)using Agrobacterium -mediated transformation.To date,various factors affecting the ef?-ciency of Agrobacterium -mediated transformation have been investigated.These include cocultivation temperature (Dillen et al.1997),type (Frary and Earle 1996;Ellul et al.2003;Park et al.2003;Wu et al.2006)and developmental status of explants (Chyi and Phillips 1987;Hamza and Chupeau 1993;Tabaeizadeh et al.1999;Dan et al.2006;Sun et al.2006),use of feeder-layer cells (van Roekel et al.1993;Qiu et al.2007),addition of phenolic compounds

(Lipp Joa

?o and Brown 1993;Cortina and Culia ′n ˇez-Macia `2004;Sun et al.2006;Wu et al.2006),vector constructs (van Roekel et al.1993;Qiu et al.2007),Agrobacterium strains (van Roekel et al.1993;Wroblewski et al.2005),and concentration (Ellul et al.2003;Dan et al.2006;Qiu et al.2007;Wu et al.2006)and composition of the medium (Hamza and Chupeau 1993;Frary and Earle 1996;Ling et al.1998;Vidya et al.2000;Krasnyanski et al.2001;Pozueta-Romero et al.2001;Park et al.2003;Cortina and

Culia

′n ˇez-Macia `2004;Wu et al.2006).Current transfor-mation ef?ciencies ranges from 6to 40%(Hamza and Chupeau 1993;Frary and Earle 1996;Ling et al.1998;Vidya et al.2000;Krasnyanski et al.2001;Park et al.2003;

N.Gao áY.Cao áY.Su áW.Shi (&)

State Key Laboratory of Soil and Sustainable Agriculture,Institute of Soil Science,Chinese Academy of Sciences,210008Nanjing,China e-mail:wmshi@aadc476f011ca300a6c390f3

N.Gao áY.Cao

Graduate School of the Chinese Academy of Sciences,100039Beijing,China

W.Shen

College of Chemistry and Environmental Science,Nanjing Normal University,210097Nanjing,China

123

Plant Cell Tiss Organ Cult (2009)98:321–330DOI 10.1007/s11240-009-9566-2

Sun et al.2006).In spite of these successes,most protocols rely on the use of either cumbersome feeder layers from petunia,tobacco,or tomato,complex media formulations, or time-consuming successive subcultures.Hence,opti-mizing the frequency of transformation of tomato is highly desirable.

Agrobacterium tumefaciens has been widely used to transform numerous plant species,including tomato.The ability of Agrobacterium to transform plants is under highly regulated genetic control,especially for the vir gene (Hansen et al.1994;Gelvin2003;Yuan et al.2007).Over the years,optimal conditions for Agrobacterium vir gene induction have been extensively investigated(Joubert et al. 1995;Hwang and Gelvin2004;Yuan et al.2007).In addition,bacterial concentrations used for in?ltration have also been optimized(Pozueta-Romero et al.2001;Wu et al. 2006;Qiu et al.2007).For tomato transformation,Agro-bacterium concentrations usually range between0.01and 1.0at OD600nm(Lipp Joa?o and Brown1993;Frary and Earle1996;Ling et al.1998;Krasnyanski et al.2001;Po-zueta-Romero et al.2001;Park et al.2003;Dan et al.2006; Wu et al.2006;Qiu et al.2007),and overnight-grown Agrobacterium cultures are usually diluted prior to cocul-tivation(Krasnyanski et al.2001;Dan et al.2006;Wu et al. 2006).McCormick et al.(1986)incubated explants in a 1:20dilution of Agrobacterium C58C1(Rif r)culture;while, Lipp Joa?o and Brown(1993)incubated explants in a1:10 dilution,grown for about4h with Agrobacterium C58C1 (Rif r)culture.On the other hand,Frary and Earle(1996) diluted an overnight-grown Agrobacterium strain LB4404 culture one to twofold to reach OD600nm=0.4to obtain an optimal infection concentration;whereas,Raj et al.(2005)?oated precultured cotyledons in a1:15dilution of Agro-bacterium strain LB4404.Sun et al.(2006)used a1:20–40 dilution of Agrobacterium C58C1(Rif r)culture;while,Qiu et al.(2007)diluted an overnight-grown culture of Agro-bacterium strain EHA101to OD600nm=0.2.In our pilot experiment on tomato transformation,we found that when using cotyledons as explants,transformation frequencies varied greatly,even with the same Agrobacterium strain at the same inoculation concentration and length of inocula-tion followed by the same cocultivation conditions.These results indicated that there might be other factors that in?uenced transformation frequency of tomato.

This study reports on the effects of density of Agro-bacterium preculture of tomato cotyledons under different compositions of cocultivation media on frequency of transformation of tomato.

Materials and methods

Plant material

Seeds of tomato(Solanum lycopersicum L.)cultivar Zhongshu No.4were surface-sterilized for30s with75% ethanol,15min in a2%NaClO,followed by eight washes with sterilized-distilled water.Sterile seeds were grown on a germination medium(Table1unless otherwise stated). Cultures were kept at22–24°C under a16h photoperiod with a light intensity of72l mol m-2s-1.

Cotyledons were excised from6to8-day-old seedlings, and cotyledonary explants,*25mm2,were incubated, placed abaxially,on a preculture medium as described in Table1.Preculture was conducted for a period of2days at 22–24°C under a16h photoperiod and a light intensity of 72l mol m-2s-1.

Agrobacterium strain and cloning vectors Agrobacterium strain LB4404carrying the pBI121binary vector(Jefferson et al.1987)was used.The binary vector contains the selectable marker gene neomycin phospho-transferase II(nptII),driven by the nopaline synthase(nos) promoter,and a b-glucuronidase(GUS)gene under the control of the cauli?ower mosaic virus35S promoter (CaMV35S).Besides pBI121,three pBI121-derived vectors, designated B,C,and D(Fig.1),were also constructed to.

Table1Media used in tomato tissue culture and Agrobacterium transformation,unless otherwise stated.All media were prepared in MS basal medium including vitamins(Murashige and Skoog1962)

Culture medium Additional components

Germination medium30g l-1sucrose,8g l-1agar,pH5.8

Preculture medium2mg l-1ZT,30g l-1sucrose,8g l-1agar,pH5.8

Co-cultivation medium-I30g l-1sucrose,pH5.8

Co-cultivation medium-II100l M AS,2mg l-1ZT,0.1mg l-1IAA,30g l-1sucrose,8g l-1agar,pH5.2

Selection medium50mg l-1kanamycin,200mg l-1timentin,1mg l-1ZT,0.1mg l-1IAA,30g l-1sucrose,8g l-1agar,pH5.8 Rooting medium-I50mg l-1kanamycin,200mg l-1timentin,0.2mg l-1IAA,30g l-1sucrose,8g l-1agar,pH5.8

Rooting medium-II50mg l-1kanamycin,30g l-1sucrose,8g l-1agar,pH5.8

ZT zeatin,IAA indole-3-acetic acid,AS acetosyringone

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Vector B contained a phosphorus-related gene172,a small RNA DBE#172from Arabidopsis thaliana ecotype Columbia,under the control of CaMV35S,used as a control; while,vectors C and D carried the phosphorus-related gene ath-microRNA399d,a microRNA from A.thaliana ecotype Columbia under the control of CaMV35S and rd29A pro-moter from A.thaliana ecotype Columbia,respectively,for transformation experiments.

Agrobacterium culture and cocultivation

A single colony of A.tumefaciens carrying the binary vector was inoculated in10ml Luria-Bertani(LB)broth containing100mg l-1kanamycin,25mg l-1rifampicin, and50mg l-1streptomycin,and grown overnight on a rotary shaker at28°C.Overnight-grown bacterial precul-tures were incubated to OD600nm=1.0,unless otherwise stated,diluted tenfold with L

B liquid medium,and grown for another4h.Cultures were collected by centrifugation at4,0009g for10min at room temperature,and resus-pended in a liquid cocultivation medium-I(Table1),and adjusted to OD600nm=0.1.Precultured cotyledons were submerged in the bacterial solution for15min.Agrobac-terium-infected explants were blotted onto a sterile paper towel,and cultured on cocultivation medium-II(Table1, unless otherwise stated)for3days at22–24°

C in the dark.

To investigate the in?uence of Agrobacterium precul-ture density on transformation ef?ciency(TE)and transient GUS expression,the overnight Agrobacterium preculture was cultured to OD600nm=0.1,0.5,1.0,1.5,and2.0,and then diluted with LB liquid medium to OD600nm=0.1and grown for another4h.Apart from Agrobacterium precul-ture densities,all other parameters remained the same as described above.To investigate the effects of Agrobacte-rium preculture density in combination with acetosyrin-gone(AS),zeatin(ZT),and pH of cocultivation medium-II on transient GUS expression,an overnight Agrobacterium preculture was cultured to OD600nm=0.1,1.0,and2.0, and then diluted with LB liquid medium to OD600nm=0.1 and grown for another4h.Apart from Agrobacterium culture densities,all other parameters remained the same as described above.

To investigate whether Agrobacterium density effects on transient GUS expression levels were in?uenced by the concentration of AS in cocultivation medium-II,0,100, and200l M AS were added to cocultivation medium-II (apart from AS concentration,other parameters used in this study are listed in Table1).To investigate whether Agro-bacterium density effects on transient GUS expression level were in?uenced by ZT in cocultivation medium,0 and2mg l-1ZT were added to medium-II(apart from the ZT concentration,other parameters used in this study are listed in Table1).To investigate whether Agrobacterium density effects on transient GUS expression levels were in?uenced by the pH of the cocultivation medium,med-ium-II was adjusted to pH5.2,5.5or5.8using diluted NaOH solution(apart from pH,other parameters used in this study are listed in Table1).

TE was calculated as:number of cotyledons with putative transformants/the total number of cotyledons per treatment,and the transient GUS levels were evaluated as: number of cotyledons with blue-stained/the total number of cotyledons per treatment.

Selection of transformants

Agrobacterium-infected explants cocultivated for3days were then transferred to selection medium(Table1),and subcultured onto fresh medium once every3weeks until shoot buds were observed.When shoots where5mm in length,they were excised from callus and transferred to rooting medium-I(Table1)for2weeks.Rooted shoots were then transferred to rooting medium-II(Table1)for an additional week.Finally,putative transformed plants were transferred to100%sterilized vermiculite and watered with 1/50MS basal medium(Murashige and Skoog1962).Pots with plants were covered with a plastic bag to maintain high relative humidity.The plastic bag was opened for1h daily to add fresh air,and the plants were lightly watered. About7days later,the plastic bags were removed and plants were grown under greenhouse conditions.Plants later were transferred to an agriculture soil into larger pots for maturation and seed collection of self-pollinated T1 generation.

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GUS assays

For transient histochemical GUS assay,3-day-old co-cul-tured cotyledons were immersed in the staining solution (50mM sodium phosphate buffer,pH7.0,0.1%Triton X-100,1mM5-bromo-4-chloro-3-indole-b-D-glucuronide) overnight(16h)at37°C as described by Jefferson et al. (1987).These were rinsed successively with70%ethanol and30%acetic acid for24h,and numbers of blue-stained explants were counted.For stable histochemical GUS assay,plantlets were incubated in the staining solution,as described above.After16h at37°C,plantlets were rinsed successively with70%ethanol and30%acetic acid for 24h.

Identi?cation and analysis of putative transformants

with polymerase chain reaction(PCR),Southern blot, and reverse transcription PCR(RT-PCR)analysis Genomic DNA was extracted from leaves of greenhouse-grown tomato plants,and the presence of a492-bp nptII gene-speci?c fragment was used to identify transformants. Approximately10ng of genomic DNA was used as tem-plate for PCR analysis.The nptII gene-speci?c primers used in this analysis were NptII F1-252(50-CACTGAA GCGGGAAGGGACT-30)and NptII R1-743(50-GCGGC GATACCGTAAAGCAC-30).

To con?rm integration of the transgene,Southern blot-ting was conducted.Genomic DNA from PCR-positive transformants was subjected to enzymatic digestion.About 30l g of genomic DNA was completely digested with Eco RI or Bam HI,separated on a1.0%agarose gel,and transferred to a Hybond H?membrane(Amersham Bio-sciences,Buckinghamshire,UK).The membrane was hybridized with a nptII Dig-labeled probe(Roche Applied Science,Mannheim,Germany),following the manufac-turer’s protocol.For the nptII probe,an internal fragment of the gene was obtained by PCR ampli?cation.

For expression analysis,total RNA was extracted from leaves of greenhouse-grown tomato seedlings using RNAsio Reagent(TaKaRa,Tokyo,Japan)according to the manufacturer’s protocol.To ensure the absence of genomic DNA contamination,RNA preparations were tested for the ampli?cation of an a-tubulin gene fragment using the fol-lowing primer pairs:tub F(50-TGAACAACTCATAA GTGGCAAAG-30)and tub R(50-TCCAGCAGAAGTGA CCCAAGAC-30).Only RNAs without DNA contamination (negative a-tubulin gene fragment ampli?cation)were used for subsequent preparation in cDNA synthesis.One microgram of total RNA was used to synthesize cDNA using reverse transcriptase XL(AMV)(TaKaRa)following the manufacturer’s protocol.The cDNA samples were used as templates for RT-PCR analysis.Primers NptII F2-61(50-GGCTATGACTGGGCACAACA-30)and NptII R2-329(50-GCAGGAGCAAGGTGAGATGAC-30)were used to amplify the269-bp nptII gene fragment.PCR and RT-PCR reactions were performed in a10-l l total volume consisting of19reaction buffer,1.5mM MgCl2,0.2mM dNTPs,0.2l M of each primer,and0.25U Taq DNA polymerase(TaKaRa).The PCR cycles included an initial 5min denaturation at94°C,followed by30cycles each of 30s at94°C,30s at55°C,30s at72°C,and a?nal7min extension at72°C.

Results

In?uence of Agrobacterium preculture density on TE

As it was important to optimize the recovery of putative transformants of tomato to increase the likelihood of stable transformation,we evaluated several Agrobacterium pre-culture densities for their ability to recovery of putative transformants in cotyledon.TE was calculated as:number of cotyledons with putative transformants/the total number of cotyledons per treatment.An Agrobacterium preculture absorption OD600nm of1.0produced the highest TE of 44.7%of tomato cotyledonary explants.TE decreased dramatically when the Agrobacterium preculture OD600nm was either0.1or2.0(Fig.2).As the concentration of Agrobacterium solution used for infection was the same, this indicated that the density of Agrobacterium preculture signi?cantly affected TE

values.

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Effect of Agrobacterium preculture density on transient GUS expression

When the preculture OD 600nm was 0.1,transient GUS expression in cotyledons was 78.8%;while,when the preculture OD 600nm was 2.0,transient GUS expression in cotyledons was 65.0%.The highest transient GUS expression in cotyledons was obtained at OD 600nm of 1.0(Fig.3).Using an optimized Agrobacterium preculture density (OD 600nm =1.0),the transient GUS expression ef?ciency reached 90%.A strong correlation between

transient GUS expression and TE (R 2=0.7073;Fig.4)was observed,indicating that a transient GUS expression level was critical for achieving high rates of TE.Effects of Agrobacterium preculture density in combination with AS,ZT,and the pH

of cocultivation media-II on transient GUS expression To investigate whether bacterial density effects on transient GUS expression are in?uenced by the presence of a phe-nolic compound such as AS,cytokinin such as ZT,or pH of the cocultivation medium,the effects of Agrobacterium preculture density on TE was further investigated using a transient GUS expression system.An Agrobacterium pre-culture OD 600nm of 1.0resulted in the highest transient GUS expression with various phenolic compound,pH,and composition of the cocultivation medium-II.

To investigate whether effects of bacterial density on transient GUS expression levels were in?uenced by the concentration of AS in the cocultivation medium-II,dif-ferent levels of AS were added to cocultivation medium-II.As noted above,with the same AS concentration,highest transient GUS expression levels of cotyledons were obtained at bacterial density of OD 600nm =1.0.At this optimal bacterial density,100l M AS in the cocultivation medium yielded the highest transient GUS expression in cotyledonary explants.At both optimal bacterial density and AS concentration in the cocultivation medium-II,the transient GUS expression ef?ciency was over 90%(Fig.5a).

As the effect of bacterial density on transient GUS expression might be in?uenced by ZT in the cocultivation medium,ZT was either added to medium-II or left out.At the same ZT concentration,the highest transient GUS levels in cotyledonary explants were obtained at bacterial density of OD 600nm of 1.0during preculture.In contrast to AS,the presence of ZT in cocultivation medium-II did not signi?cantly improve transient GUS aadc476f011ca300a6c390f3ing optimal Agrobacterium densities (OD 600nm =1.0),the transient GUS expression ef?ciency of cotyledon explants was over 90%(Fig.5b).

To investigate whether Agrobacterium density effects on transient GUS expression levels were in?uenced by the pH of the cocultivation medium,medium-II was adjusted to different pH value before autoclaved.At the same pH,the highest transient GUS levels of cotyledonary explants were obtained with an Agrobacterium LB4404precul-ture OD 600nm of 1.0.Under optimal tenfold bacterial cell dilution,the best pH for the cocultivation medium was 5.2(Fig.5c).Using an optimal bacterial density (OD 600nm =1.0)and pH of cocultivation medium-II,transient GUS expression ef?ciency of cotyledonary explants was over

90%.

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Effects of different constructs on TE

When three different constructs,carrying phosphorus-related genes,were used for transformation,in conjunction with Agrobacterium LB4404preculture density of OD600nm=1.0,no signi?cant differences in TEs were observed.TEs of constructs B,C,and D were47.4±11.9%, 44.9±5.7%,and35.0±2.4%,respectively.

PCR,Southern blot,and RT-PCR analysis

All of putative transformed shoots were successfully rooted on Rooting medium-I.The putative transgenic plants were identi?ed by PCR ampli?cation for presence of the nptII transgene(Fig.6a).The expected490-bp band was detected in61out of70randomly selected plantlets,but it was absent from the negative control(non-transformed) plant(Fig.6a).Putative transgenic plants were further con?rmed by Southern blot analysis(Fig.6b).Three ran-domly selected genomic samples were completely digested with Eco RI or Bam HI,and hybridized with an nptII Dig-labeled probe(Roche Applied Science),following the manufacturer’s protocol.All candidate transformants were detected with the nptII gene fragment.Southern blot analysis of transformant plants con?rmed the integration of the T-DNA into the tomato genome.Positive hybridization signals were observed in all transformants,whereas,the signal was absent in untransformed plant.Southern blot hybridization has revealed multiple insertion copy numbers in genomic DNA of tomato plants.Approximately one to three copies of nptII gene,ranging between3.3and9.4kb in size,are observed in these transgenic plants(Fig.6b).

Con?rmed transformants were further analysis for levels of gene expression by RT-PCR using primers for the nptII gene.The expected270-bp band was found in the positive control plasmid and in44out of48randomly selected plantlets,but it was absent from the negative control (Fig.6c).We also found that the nptII gene expression level was not signi?cantly changed(data not shown).

Genetic analysis

To investigate inheritance of the kanamycin-resistance nptII transgene,six independent transgenic lines were random selected.T0seeds were harvested and germinated on germination medium containing25mg l-1kanamycin. Fig.5Effect of Agrobacterium LB4404preculture density on the transient GUS expression levels at different cocultivation medium-II concentrations.Values of0.1,1.0,and2.0correspond to Agrobac-terium LB4404preculture absorption at OD600nm=0.1,1.0,and2.0, respectively;the transient GUS expression levels were evaluated as: number of cotyledons with blue-stained/the total number of cotyle-dons per treatment in a total of80explants pooled from two independent experiments;differences in transient GUS expression levels were tested at the5%signi?cance level using least signi?cance tests(small letters).a Effect of Agrobacterium LB4404preculture density on the transient GUS expression level with different acetosyringone(AS)concentrations in cocultivation medium-II;b Effect of Agrobacterium LB4404preculture density on the transient GUS expression level at different zeatin(ZT)concentrations in cocultivation medium-II;c Effect of Agrobacterium LB4404precul-ture density on the transient GUS expression level at different pH values of cocultivation medium-II

b

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After10days,kanamycin-resistant seedlings with more than5-cm long primary roots and green leaves were clearly distinguished from sensitive seedlings of less than3-cm primary roots and yellow leaves.Segregation analysis of the T1generation gave a Mendelian ratio of3:1(Table2).

Comparison of previous and current protocols

of tomato transformation

To compare earlier protocols with the current protocol of tomato transformation,we performed six transient GUS expression experiments according to the previous protocol (overnight Agrobacterium culture diluted with LB liquid medium to OD600nm=0.1,grown for another4h,with other procedures as described in‘‘Materials and methods’’) (McCormick et al.1986;Lipp Joa?o and Brown1993;Frary and Earle1996;Raj et al.2005;Sun et al.2006;Qiu et al. 2007).We also conducted six transient GUS expression experiments according to the improved protocol(overnight Agrobacterium LB4404preculture was cultured to OD600nm=1.0,then diluted with LB liquid medium to OD600nm=0.1,and grown for another4h,with other procedures as described in‘‘Materials and methods’’).The results(Table3)showed that the current protocol yielded a higher ef?ciency of transformation and resulting in a higher frequency of transformation(Fig.7).

Fig.6PCR,Southern blot,and RT-PCR analysis of putative transformants.a PCR ampli?cation of the nptII fragment from putative transformants DNA(491bp):lane M DL2000-bp marker (TaKaRa),lanes1–12putative transformants,lane P positive control (plasmid pBI121DNA),lane N negative control(wild-type tomato);

b Southern blot analysis of genomi

c DNA from three randomly selecte

d tomato plants(T0).Thirty micrograms of DNA,following digestion with Eco RI(E)or Bam HI(B),wer

e loaded in each lane. Lane P positive control,pMD18vector containing the nptII gene fragment(total length=3,375bp);Lane N negative control, untransformed tomato plants;Lanes1–3transformed tomato plants. For the nptII probe,an internal fragment o

f the gene was obtained by PCR ampli?cation;c PCR ampli?cation of nptII fragment from putative transformants RNA(269bp):lane M DL2000-bp marker (TaKaRa),lanes1–12putative transformants,lane P positive control (plasmid pBI121DNA),lane N negative control(wild-type tomato DNA)

b

Table2Inheritance of kanamycin resistance in T1generation of independent lines

Line Segregation of T1seedlings for kanamycin response V2(3:1) Resistant Sensitive

WT0160476

L182330.34* L2107440.35* L363260.63* L42816 2.45* L55310 2.33* L62580.01* *Differences at the5%signi?cance level obtained using a V2test

M 1 2 3 4 5 6 7 8 9 10 11 12 P N

E B E B E B

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Discussion

Agrobacterium -mediated transformation is a common approach for introducing elite genes into plant genotypes.Several factors are taken into consideration to develop highly ef?cient transformation protocols,and among those,the concentration of Agrobacterium in?ltration solution is an important factor in these protocols.

Levels of Agrobacterium in?ltration solutions have been optimized,with OD 600nm ranging between 0.01and 1.0

(Lipp Joa

?o and Brown 1993;Frary and Earle 1996;Ling et al.1998;Krasnyanski et al.2001;Pozueta-Romero et al.2001;Park et al.2003;Dan et al.2006;Qiu et al.2007;Wu et al.2006).Bacterial cells are usually cultured overnight and then diluted 2–50times to achieve an appropriate

infection concentration (McCormick et al.1986;Lipp Joa

?o and Brown 1993;Frary and Earle 1996;Raj et al.2005;Sun et al.2006;Qiu et al.2007).In this study,bacterial cell density during preculture has a signi?cantly effect on both transient GUS expression and TE (Figs.2,3).For strain LB4404,the optimal density is OD 600nm =1.0.

In this study,the in?uence of the bacterial density dur-ing preculture under different AS and ZT concentrations,and different pH,was investigated revealing that regardless of all these factors,the level of transient GUS expression was highest when OD 600nm of bacterial cells was 1.0during preculture.This further con?rmed the importance of the density of bacterial cells in the preculture medium on successful transformation of tomato.Moreover,this is not unique to Agrobacterium strain LB4404.In this study,an agropine/succinamopine-type Agrobacterium strain EHA101harboring pUbi,carrying the GUS gene,and a nopaline-type Agrobacterium strain C58carrying pBI121,also carrying the GUS gene ,were investigated.For Agrobacterium EHA101,bacterial density of OD 600-nm =1.5or 3.0during preculture resulted in highest frequencies of transient GUS expression,71.3±1.8%and 71.3±5.3%,respectively.However,for the nopaline

Table 3Transient GUS expression using a previously reported transformation protocol and this newly reported transformation protocol Transformation protocol

Transient GUS expression (%)a Exp.1

Exp.2Exp.3Exp.4Exp.5Exp.6Total b Improved protocol (this study)97.5100.095.0100.0100.0100.098.8±2.1a Previously reported protocol

c

85.0

82.5

100.0

77.5

90.0

75.0

85.0±9.1b

a

Transient GUS expression levels were evaluated as:number of cotyledons with blue-stained/the total number of cotyledons (n =40)per treatment

b

Total transient GUS expression is expressed as mean ±SD.Differences at the 5%signi?cance level were obtained using Student’s t -test (small letters )

c Overnight Agrobacterium culture is dilute

d with LB liquid medium to OD 600nm =0.1and grown for another 4h.This protocol has been

reported by McCormick et al.(1986),Lipp Joa

?o and Brown (1993),Frary and Earle (1996),Raj et al.(2005),Sun et al.(2006),and Qiu et al.(2007),among

others

Fig.7Tomato transformation.a Transient GUS expression visualized by dark grey spots (in print version)or blue spots (in electronic version)in cotyledons;b rooting of transgenic shoots with

50mg l -1kanamycin;c stable GUS expression of a transgenic tomato plant;d transgenic tomato plant growing in the greenhouse

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Agrobacterium strain C58,no bacterial density effects were detected as100%transient GUS expression levels of cotyledons were obtained regardless of OD600nm.As all infection experiments were performed at similar bacterial densities,it is likely that the effect of bacterial density during preculture is likely attributed to the growth stage of the bacterial preculture.Growth curves of bacterial cells showed that the concentrations of optimal dilutions for LB4404and EHA101bacterial cells?t the early-middle exponential(logarithmic)growth phase(data not shown). This indicated that in addition to the population effect;i.e., proper densities of the infection solution,of Agrobacte-rium,developmental stages of inpidual cells likely con-tributed to the vir gene-mediated gene transfer and incorporation into target plant tissues.

Cotyledons have been commonly used as explants in tomato transformation(Chyi and Phillips1987;Lipp Joa?o and Brown1993;Frary and Earle1996;Krasnyanski et al. 2001;Park et al.2003;Raj et al.2005;Sun et al.2006;Wu et al.2006).Cotyledon explants were wounded and infected with Agrobacterium followed by cocultivation for 3days in the dark.Longer exposure of explants to Agro-bacterium resulted in100%Agrobacterium regrowth after this cocultivation period and made it more dif?cult to eliminate Agrobacterium(data not shown).This phenom-enon was also observed by Krasnyanski et al.(2001).

In this study,presence of AS,and ZT,as well as pH of cocultivation medium are critical factors in transformation and regeneration of transformants from explants.Addition of AS to cocultivation media improves transient GUS expression level(Figs.3,5)and TE(Fig.2;Lipp Joa?o and Brown1993;Uranbey et al.2005;Wu et al.2006).The infection capability of Agrobacterium is dependent on the vir gene located on the Ti plasmid.The vir gene has been reported to be highly expressed at an appropriate low pH (Lipp Joa?o and Brown1993;Ogaki et al.2008).This has been also con?rmed in this study whereby a lower pH(pH 5.2)produced the highest transient GUS expression in cotyledonary explants(Fig.5c).

Conclusions

Taken together,our results clearly show that ef?ciency of transformation in tomato is highly in?uenced by the density of preculture of bacterial cells.The improved protocol pre-sented in this study depends on the use of an overnight-grown Agrobacterium preculture density of OD600nm=1.0, diluted1/10th with LB liquid medium,and grown for an additional4h.Cultures are collected and resuspended in a liquid cocultivation medium-I,adjusted to OD600nm=0.1. With this improved protocol,a transient GUS expression is more than90%and a TE of44.7%is obtained.This transformation protocol is simple,repeatable,does not require a feeder layer,and most importantly,it yields a high frequency of transformants.It will be important to evaluate this protocol with other genotypes of tomato. Acknowledgments The authors thank Dr.Sumei Li from the Institute of Soil Science,Chinese Academy of Sciences,for valuable suggestions on transgenic techniques.The authors also thank the anonymous reviewers for their valuable comments and suggestions that had improved this manuscript greatly.This work was supported by the National Natural Science Foundation of China(40671100)and the National Basic Research Program of China(2007CB109303).

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