Plant Cell Rep (2006) 25: 1043–1051DOI 10.1007/s00299-006-0168-8

GENETIC TRANSFORMATION AND CELL HYBRIDIZATION

Yongqin Chen · Litang Lu · Wei Deng · Xingyu Yang ·Richard McAvoy · Degang Zhao · Yan Pei ·Keming Luo · Hui Duan · William Smith ·Chandra Thammina · Xuelian Zheng · Donna Ellis ·Yi Li

In vitro regeneration and Agrobacterium-mediated genetictransformation of Euonymus alatus

Received: 17 October 2005 / Revised: 24 January 2006 / Accepted: 12 March 2006 / Published online: 30 May 2006C© Springer-Verlag 2006

Abstract An in vitro plant regeneration method and anAgrobacterium tumefaciens-mediated genetic transforma-tion protocol were developed for Euonymus alatus. Morethan 60% of cotyledon and 70% of hypocotyl sections from10-day-old seedlings of E. alatus produced 2–4 shoots onwoody plant medium (WPM) supplemented with 5.0 mg/l6-benzylaminopurine (BA) plus 0.2 mg/l α-naphthaleneacetic acid (NAA), and 77% of shoots produced roots onWPM medium with 0.3 mg/l NAA and 0.5 mg/l Indole-3-butyricacid (IBA). On infection with Agrobacterium tume-faciens strain EHA105 harboring a gusplus gene that con-tained a plant recognizable intron from the castor bean cata-lase gene to ensure plant-specific β-glucuronidase (GUS)expression, 16% of cotyledon and 15% of hypocotyl ex-plants produced transgenic shoots using kanamycin as aselection agent, and 67% of these shoots rooted. Stableinsertion of T-DNA into the host genome was determinedwith organ- and tissue-specific expression of the gusplus

Communicated by P. Lakshmanan

Y. Chen (�) · L. Lu · W. Deng · R. McAvoy · K. Luo · H. Duan ·W. Smith · C. Thammina · X. Zheng · D. Ellis · Y. Li (�)Department of Plant Science, University of Connecticut,Storrs, CT 06269, USAe-mail: yi.li@uconn.edu; chenyongqin03@yahoo.com

Y. Chen · X. YangDepartment of Biotechnology, Faculty of Life Sciences, HubeiUniversity,Wuhan, P. R. China

L. Lu · D. ZhaoCollege of Life Sciences, Guizhou University,Guiyang, P. R. China

W. Deng · Y. Pei · K. LuoBiotechnology Center, Southwest University,Chongqing, P. R. China

Present address:H. DuanDepartment of Cell & Structural Biology, University of Illinois,Urbana, IL 61801, USA

gene and further confirmed with a PCR-based molecularanalysis.

Keywords Agrobacterium tumefaciens . Euonymusalatus . Genetic transformation . β-Glucuronidase .Plantlet regeneration

Abbreviations AS: acetosyringone .BA: 6-benzylaminopurine . GUS: glucuronidase . hptII: hygromycin phosphotransferase .IBA: indole-3-butyric acid . NAA: α-naphthaleneaceticacid . nptII: neomycin phosphotransferase II

Introduction

Euonymus. alatus (Thunb.) Sieb., a deciduous shrub na-tive to Asia, was introduced into the United States in the1860s as an ornamental plant. Its green leaves turn to bril-liant purplish red to scarlet in autumn and this spectacularfall foliage makes it an extremely popular landscape plantwell known as “winged euonymus” or “burning bush”. Be-cause of its huge economical value and popularity, E. ala-tus has been widely grown in both urban and rural areas inNorth America and is sold by many commercial nurseriesthroughout the US.

E. alatus grows well in sun or shade and thrives un-der a wide range of soil types and pH levels, and it hasno serious pest problems in North America. E. alatus isusually propagated by cutting but in nature it propagateseasily via seeds. A single mature plant can produce hun-dreds of seeds each year and seeds can be dispersed bywater and birds to distant areas. Because of these reproduc-tive and growth characteristics, E. alatus spreads rapidlyin North America in an uncontrolled fashion. The prob-lem caused by E. alatus is that its fibrous roots usuallyform a dense mat below the soil surface that makes theestablishment of other native flora extremely difficult. Ithas been observed that E. alatus has been replacing na-

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0.0

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2412

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1.7

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4617

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2725

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5024

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2532

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2.5

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4727

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3036

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5.0

5259

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2673

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5062

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2669

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2740

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tive shrubs in the eastern U.S. and Midwest, notably in thestates of Connecticut, Virginia, Pennsylvania, and Illinois(http://tncweeds.ucdavis.edu/index.html), which poses athreat to ecosystems (Randall and Marinelli 1996).

Although the ecological impact of undesirable spread ofE. alatus can be devastating (Marinelli and Hanson 1996),completely banning the use of E. alatus would be politi-cally, socially and economically problematic. Recent ad-vances in plant molecular biology have made it possibleto neutralize invasiveness of E. alatus (Li et al. 2004).To achieve that goal, establishing an efficient regenerationand genetic transformation system is prerequisite. Smithand Jernstedt (1989) reported an in vitro plant regenerationprotocol using hypocotyls of E. alatus, but the efficienciesof both shoot formation and root formation were relativelylow. In this paper, we report highly efficient methods usingboth hypocotyls and cotyledons of E. alatus as explantsfor regeneration and for Agrobacterium-mediated genetictransformation.

Materials and methods

Plant materials

Seeds of E. alatus were purchased from ForestryConsulting-Seed Sales, Winslow, ME, USA and stored at4◦C before use. In vitro plantlets of Nicotiana tabacummcv. Xanthin were used for transformation to verify the ex-pression of Nos promoter-gusplus-Tnos gene.

Seeds of E. alatus were soaked in tap water overnight,washed thoroughly with tap water, and then surface-sterilized in 70% ethanol for 5 min, 10% (v/v) clorox for40 min and then triple rinsed with sterile distilled water.Embryos were isolated aseptically from the seeds and cul-tured on a basal medium that contained the macro saltsof woody plant medium (Lloyd and McCown 1980), themicro salts of MS medium (Murashige and Skooge 1962),100 mg/l myo-inositol, 0.5 mg/l nicotinic acid, 1.0 mg/lpyridoxin-HCl, 1.0 mg/l thiamine-HCl, 2 mg/l glycine,20.0 g/l sucrose, and 8.0 g/l agar.

In vitro plantlet regeneration of E. alatus

Cotyledons and hypocotyls of 10-day-old seedlings derivedfrom isolated E. alatus embryos were cut into segments ofapproximate 5 mm in length and each hypocotyl segmentwas then longitudinally cut into two halves. The explantswere cultured on the basal medium supplemented with 2.0,5.0 or 10.0 mg/l N6-benzylaminopurine (BA) plus 0, 0.2 or0.5 mg/l α-naphthalene acetic acid (NAA) (Table 1) for 50days for shoot regeneration. Adventitious shoots that wereup to 1 cm in length were then excised from the explantsand cultured on the basal medium containing 0.5, 1.0 or2.0 mg/l indole-3-butyric acid (IBA) plus 0 mg/l, 0.3 mg/lor 0.5 mg/l NAA (Table 2) for 45 days. The pH of all mediawas adjusted to 5.8 with KOH or HCl prior to autoclavingat 121◦C for 20 min. All cultures for in vitro plant regen-

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Table 2 Effects of NAA andIBA concentrations on therooting of E. alatus shoots

Plant growth substances (mg/l) Number of shootsused

Rooting rate (%) Average number ofroots/plantletIBA NAA

0.5 0.0 26 15.4 a 1.3 ± 0.3 a1.0 0.0 28 32.1 b 1.4 ± 0.2 a2.0 0.0 25 44.0 c 1.6 ± 0.3 a0.5 0.3 30 77.8 d 4.3 ± 1.6 b1.0 0.3 27 59.2 e 3.5 ± 1.2 b2.0 0.3 30 56.7 e 3.7 ± 07 b0.5 0.5 32 65.6 e 2.7 ± 0.4 c1.0 0.5 32 59.4 e 2.7 ± 0.5 c2.0 0.5 29 65.5 e 2.2 ± 0.4 c

Note. χ2-test and Duncan’smultiple range test were used toevaluate the differences in theroot rates and the averagenumber of roots per plantlet,respectively; data sharing thesame letter in the same columnwere not significantly differentat the 5% level

Table 3 Effect of kanamycinconcentrations on the callusformation and bud induction ofE. alatus

Kanamycin (mg/l) Number ofexplants used

Callus formationrate (%)

Adventitious budsformation rate (%)

Average number ofbuds/explant

0 30 100 a 63.3 a 2.8 ± 0.6 a20 32 84.4 b 46.9 b 1.6 ± 0.2 b30 34 55.9 c 14.7 c 1.0 ± 0.0 c50 36 8.3 d 0.0 d 0.0 ± 0.0 d80 36 0.0 e 0.0 d 0.0 ± 0.0 d100 29 0.0 e 0.0 d 0.0 ± 0.0 d

Note. χ2-test and Duncan’s multiple range test were used to evaluate the differences in the rates of callusformation and bud formation and the average number of roots per plantlet, respectively; data sharing thesame letter in the same column were not significantly different at the 5% level

eration were kept at 24◦C under 30–45 µmol m−2s−1 lightprovided with white cool fluorescent tube lamps over a 14-hphotoperiod.

Sensitivity of explants of E. alatus to kanamycin

A kanamycin sensitivity test was performed using cotyle-don and hypocotyl explants from 10- day-old seedlings onthe shoot induction medium supplemented with 0.5 mg/lNAA, 5.0 mg/l BA, and various concentrations ofkanamycin (Table 3).

Plasmid and bacterial strain for transformation

The binary vector of pCAMBIA1305.1 (seehttp://www.cambia.org; Genbank accession number:AF354045) is derived from pCAMBIA1300, in which E.coli gusA has been replaced by gusplus. pCAMBIA1305.1is a compact binary vector with a pBR322 ori for highcopy replication in E. coli and a broad host range ori forlow copy, stable replication in Agrobacterium. It containsthe hygromycin resistance gene (hptII) for selection oftransgenic plants. The gusplus gene contains an intronfrom the castor bean catalase gene to prevent bacterialexpression but ensure plant-specific expression of glu-curonidase. We modified pCAMBIA1305.1 as following:the original CaMV 35S promoter-hptII-3′ CaMV 35Swas replaced with the Nos promoter-nptII-Tnos (Nospromoter-nptII-Tnos) gene that was from pBin19 (Bevan1984). Nos promoter sequence used to drive the nptII

gene was from the Agrobacterium nopaline synthasegene. The 3′ untranslated sequence of nopaline synthasegene (Tnos) is also from the nopaline synthase gene. Thekanamycin resistance gene is the nptII gene that encodesfor the neomycin phosphotransferase II. The gusplusgene in pCAMBIA-gusplus-nptII was under the controlof the Nos promoter. We named the modified versionof pCAMBIA1305.1 as pCAMBIA-gusplus-nptII. ThepCAMBIA-gusplus-nptII plasmid was then introducedinto A. tumefaciens strain EHA105 (Hood et al. 1993) forplant transformation. This bacterial strain was culturedand selected on a YEP (10 g/l yeast extract, 10 g/lbactopeptone, 5 g/l NaCl, pH 7.2) agar plate containing50 mg/l kanamycin and 50 mg/ml rifampicin.

Agrobacterium-mediated transformation of E. alatusand N. tabacumm

Single colonies of A. tumefaciens strain EHA 105 har-boring pCAMBIA-gusplus-nptII were cultured in 3 ml ofYEP medium containing 50 mg/l kanamycin and 50 mg/lrifampicin at 28◦C on a shaker at 250 rpm for 24 h. TheAgrobacterium cultures were diluted to 30 times with freshYEP medium containing 50 mg/l kanamycin and 50 mg/lrifampicin and then cultured at 28◦C on the shaker untilthey reach an O.D.600 value of 0.6–0.7. The bacterial cul-tures were collected and centrifuged at 4◦C at 3600 × gfor 10 min and the pellets were resuspended in the basalmedium supplemented with 0.5 mg/l NAA, 10 mg/l BA and100 µM acetosyringone (AS). The explants prepared fromcotyledons and hypocotyls of 10-day-old sterile seedlings

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of E. alatus as described above were immersed in theAgrobacterium culture for 30 min, then blotted on steriletissue paper, transferred onto co-culture medium (the basalmedium supplemented with 0.5 mg/l NAA, 10 mg/l BA and200 µM AS). Three days later, the infected explants weretransferred onto the selection and regeneration medium (thebasal medium supplemented with 0.5 mg/l NAA, 5.0 mg/lBA, 35 mg/l kanamycin and 150 mg/l timentin). The ex-plants were transferred to fresh medium every 2 weeks.Shoots regenerated from the infected explants were ex-cised and transferred on basal medium supplemented with0.3 mg/l NAA, 0.5 mg/l indole-3-butyric acid (IBA) and100 mg/l timentin for rooting. The Agrobacterium infec-tion and all subsequent steps involved in production andselection of transgenic plants were done at 24◦C with a 14-h photoperiod of 30–45 µmol m−2s−1 light provided withwhite cool fluorescent tube lamps.

Tobacco transformation was done in the same way asdescribed above, but leaf explants were excised from the invitro plantlets, co-culture medium was MS supplementedwith 0.1 mg/l NAA, 2.0 mg/l BA and 100 µM AS, selectionand shoot induction medium was MS supplemented with0.1 mg/l NAA, 2 mg/l BA, 50 mg/l kanamycin and 150 mg/ltimentin, and rooting medium was MS supplemented with1 mg/l IBA and 100 mg/l timentin.

All antibiotics (kanamycin, rifampicin and timentin) usedin this study were filter-sterilized before adding to auto-claved media. The conditions for selection and regenera-tion were the same as those for in vitro plantlet regenerationdescribed as above.

Histochemical staining for GUS activity

A. tumefaciens strains harboring pCAMBIA-gusplus-nptII(containing the Nos promoter- gusplus-Tnos), pBin19 (con-taining no gusA gene), and pBin19 (containing the Nospromoter-gusA-Tnos) were cultured in 3 ml of liquidYEP medium supplemented with 50 mg/l kanamycin and50 mg/l rifampicin at 28◦C on the shaker at 250 rpm for30 h, and 800 µl of the cultures were collected in 1.5-mlmicro-tubes and centrifuged at 5,000 rpm for 2 min. Thepellets were resuspended in 800 µl of GUS-staining solu-tion (100 mM potassium phosphate buffer, pH 7, 10 mMNaEDTA, 0.5 mM K3Fe(CN)6, 0.5 mM K4Fe(CN)6, 0.1%Triton X-100, 1 mg/l X-Gluc (5-bromo-4-chloro-3-indolyl-β-d-glucuronic acid) and incubated at 37◦C for 12 h.Shoots, leaves or roots regenerated from noninfected ex-plants and infected explants were cut into segments andincubated in a GUS-staining solution at 37◦C for 12 h. Theplant tissues were destained in ethanol gradually to removechlorophylls and other pigments prior to visual analysisand photographing.

PCR reaction

Genomic DNA was isolated from leaves of 5 GUS posi-tive plants and a wild type plant of E. alatus with a mod-

ified CTAB method (Doyle and Doyle 1990) and thenfractioned on 0.8% (w/v) agarose gel with pCAMBIA-gusplus-nptII Ti-plasmid DNA loaded on the side as a ref-erence. Large-sized genomic DNA molecules (about 20–25 kb that was much larger than the Ti-plasmid DNA)were recovered. The recovered plant genomic DNA (500 ngper PCR reaction) was used as template. Three primers asmarked on Fig. 2A: primer 1 (5′- acggatggtacttcgatggc-3′),primer 2 (5′- tgacaccgcgcgcgataatt-3′), and primer 3 (5′-tcaagcatcctggccagctc-3′) were synthesized according to theDNA sequence of pCAMBIA-gusplus-nptII. As shown inFig. 2A, primers 1 and 2 were used to amplify a 665 bpfragment containing a partial gusplus gene within the T-DNA region while primers 1 and 3 were used to amplifya 1212 bp fragment (665 bp within the T-DNA region anda 547 bp outside of the T-DNA right border sequence).For other PCR reactions, pCAMBIA-gusplus-nptII plasmidDNA (10 pg) was also added to each 500 ng of plant ge-nomic DNA as templates. Ten picro-grams of pCAMBIA-gusplus-nptII plasmid DNA in 500 ng of E. alatus genomicDNA are equivalent to one copy of transgene per E. alatusgenome. PCR was performed under the following condi-tions: DNA was denatured in 20 ul reaction buffer at 95◦Cfor 5 min, followed by 40 cycles of amplification (94◦C for1 min, 56◦C for 45 s and 72◦C for 90 s) and 7 min at 72◦C.

Statistical analysis

Data on shoot number per explant and root number perplantlet were analyzed with Duncan’s multiple range testat p ≤ 0.05; data on callus formation rates, bud induc-tion rates and rooting rates were subjected to χ2-test atp ≤ 0.05.

Results

Plant regeneration from cotyledons and hypocotyls ofE. alatus

Mature embryos of E. alatus easily germinated when cul-tured on the basal medium without any plant hormonesadded. Embryos started to increase their size 2 days af-ter culture and fully expanded their cotyledons within 10days. At Day 10, both cotyledons and hypocotyls were ex-cised and cultured on the bud induction media with variouscombinations of plant hormones (Table 1). Bud formationfrom some explants was observed after 25 days of cul-ture on BA-containing media and these buds developedinto shoots during subsequent culture (Fig. 1A and B). Wenoticed that increasing BA concentrations from 2 mg/l to5 mg/l or 10 mg/l BA led to increases in number of budsand shoots per explant and the presence of NAA (either0.2 mg/l or 0.5 mg/l) further enhanced bud induction (Table1). Under our experimental conditions, both hypocotyls andcotyledons exhibited similar responses to BA and NAA, al-though hypocotyls produced more buds than cotyledons onthe same induction media. The high rates of bud induction,

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Fig. 1 Plant regeneration and genetic transformation of E. alatus.A A hypocotyl explant forming buds and shoots on basal mediumwith 0.5 mg/l NAA plus 10 mg/l BA after 50 days. B a cotyledonexplant forming buds and shoots on basal medium with 0.2 mg/lNAA plus 5 mg/l BA after 50 days. C a plantlet regenerated onbasal medium with 0.3 mg/l NAA plus 0.5 mg/l IBA after 45 days.D a transgenic plantlet 5 months after transfer to the greenhouse.E histochemical staining of GUS activity of A. tumefaciens strainEHA105 harboring pBin19 without a gusA gene (left), pCAMBIA-gusplus-nptII with Nos promoter-gusplus-Tnos gene (middle), andpBin19 with the Nos promoter-gusA-Tnos gene (right), indicatingthat Nos promoter-gusplus-Tnos gene is not expressed in Agrobac-

terium cells. F histochemical staining of GUS activity of leaf crosssections of non-transgenic (top) and transgenic (bottom) E. alatusplants. G histochemical staining of GUS activity of leaf sections oftransgenic tobacco plants carrying Nos promoter-gusplus-Tnos gene(top) and CaMV 35S promoter-gusplus-Tnos gene (bottom). H his-tochemical staining of GUS activity of cross-sections of stems ofnon-transgenic (top) and transgenic (bottom) E. alatus plants. I his-tochemical staining of GUS activity of roots of non-transgenic (left)and transgenic (right) E. alatus plants. The tissue specific GUS ex-pression in E. alatus demonstrates that Nos promoter-gusplus-Tnosgene is expressed in plant cells, not in Agrobacterium cells

60–70% of cotyledons and hypocotyls produced 2–4 shootsper explant, were observed on the media supplemented with5.0 or 10.0 mg/l BA plus 0.2 or 0.5 mg/l NAA.

As shown in Table 2, concentrations of NAA and IBAsignificantly affected root production. On NAA free media,increases in IBA concentration from 0.5 mg/l to 2 mg/l in-creased the rates of rooting but at the same time loose callialso formed on the basal regions of shoots as IBA levelreached 1 mg/l or higher. Under the same experimentalconditions, addition of NAA (0.3 mg/l or 0.5 mg/l) furtherenhanced the callus formation. The best rooting mediumwas the one with 0.3 mg/l NAA plus 0.5 mg/l IBA. Underthis condition, the rooted shoots produced little callus tis-sues although they swelled a little. Also, on this medium,as much as 77% of the shoots produced four roots per shooton average after 45 days of culture (Table 2 and Fig. 1C).

Regeneration and verification of transgenic E. alatusplants

A kanamycin test showed that E. alatus was sensitive tokanamycin. Both callus induction and bud formation wereseverely affected at 30 mg/l kanamycin (Table 3). Cotyle-dons and hypocotyls cultured on the media with 50 mg/lkanamycin or higher became pale within 30 days and noneof them initiated any buds within 50 days of culture.

Both cotyledons and hypocotyls of E. alatus were usedas explants for Agrobacterium infection. As we producedfew shoots after Agrobacterium infection with 40 mg/lkanamycin, we used 35 mg/l kanamycin for selection oftransformants. In the presence of 35 mg/l kanamycin, 30%explants initiated buds and the average shoot number perexplant was much lower than that on kanamycin-free me-

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Fig. 2 Molecular characterization of stable insertion of T-DNA intogenome of E. alatus. A A schematic diagram of the Ti plasmid,pCAMBIA-gusplus-nptII, used for plant transformation. The threeprimers used for PCR reactions to verify the stable insertion of trans-genes into the E. alatus genome are indicated as Primer 1, 2 and 3on the diagram. B Two micro-grams of genomic DNA isolated formGUS positive E. alatus plants were fractioned on an agarose gel.DNA in the marked region was recovered for PCR reactions. Lanes1–6 are genomic DNA isolated from transgenic E. alatus plants-3, 4,7, 15 and 17, respectively. Lane 7 is purified pCambia-gusplus-nptIIplasmid DNA. Lane 8: Molecular weight mark of lambda DNA di-gested with EcoRI and Hind III. C PCR amplifications of a T-DNAsegment (primers 1 and 2) and a non-TDNA fragment (primers 1and 3) using 500 ng plant genomic DNA and/or 10 pg pCAMBIA-gusplus-nptII DNA as templates. Lanes 1 and 2: Genomic DNA ofwild type E. alatus as template with either primers 1 and 2 (Lane1) or primers 1 and 3 (Lane 2) for PCR reactions. Lanes 3 and 19:Molecular weight marks of lambda DNA digested with EcoRI and

Hind III. Lanes 4 to 8: Genomic DNA of transgenic E. alatus plant-3as template with primers 1 and 2 (Lane 4), primers 1 and 3 (Lane 5),and single primers (Lane 6 for primer 1; Lane 7 for primer 2; Lane 8for primer 3). Lanes 9 and 10: Genomic DNA of transgenic E. alatusplant-4 as templates with primers 1 and 2 (Lane 9) and primers 1 and3 (Lane 10). Lanes 11 and 12: Genomic DNA of transgenic E. alatusplant-7 as templates with primers 1 and 2 (Lane 11) and primers 1and 3 (Lane 12). Lanes 13 and 14: Genomic DNA of transgenic E.alatus plants-15 as templates with primers 1 and 2 (Lane 13) andprimers 1 and 3 (Lane 14). Lanes 15 and 16: Genomic DNA of trans-genic E. alatus plant-17 as templates with primers 1 and 2 (Lane 15)and primers 1 and 3 (Lane 16). Lanes 17 and 18: Genomic DNA oftransgenic E. alatus plant-17 with 10 pg pCAMBIA-gusplus-nptIIDNA added as templates with primers 1 and 2 (Lane 17) and primers1 and 3 (Lane 18). Lanes 20 and 21: Ten pg pCAMBIA-gusplus-nptIIDNA as templates with primers 1 and 2 (Lane 20) and primers 1 and3 (Lane 21)

dia (Tables 1 and 4). Using both hypocotyls and cotyledonsas explants for Agrobacterium infection, we obtained 35plantlets (rooted) in total from 52 GUS positive shoots.The rooted plants were then transferred into soil and grewhealthily in the greenhouse (Fig. 1D).

Detailed histochemical staining for GUS activity was car-ried out for Agrobacterium cells containing pBin19 (with-out the gusA gene), pBin19-Nos promoter-gusA-Tnos (withthe gusA gene) or pCAMBIA-gusplus-nptII. As shown inFig. 1E, there was no GUS activity observed in Agrobac-terium cells that contained pBin19 (the tube on the left)while high GUS activity was observed in Agrobacteriumcells containing pBin19-Nos promoter-gusA-Tnos gene(the tube on right). However, there was no GUS activ-ity detected in Agrobacterium cells containing pCAMBIA-gusplus-nptII (the tube in the middle) as expected. Theseresults demonstrate that the gusplus gene is not expressedin Agrobacterium cells.

Histochemical staining of GUS activity revealed that 46%of kanamycin resistant plants of E. alatus were GUS pos-itive (Table 4). To confirm the stable incorporation of T-DNA in the host genome of GUS positive E. alatus plants,we conducted a detailed histochemical staining for GUSactivity in leaf, stem, and root tissues. As shown in Fig. 1F,a leaf section from a wild type E. alatus plant had no de-tectable GUS activity while a leaf section from transgenicE. alatus plant showed strong GUS activity in vascular tis-sues. The vascular tissue expression pattern of the gusplus

gene that was under the control of the Nos gene promoteris consistent with its expression pattern in tobacco plants.Fig. 1G shows that the Nos promoter-gusplus-Tnos washighly expressed in vascular tissues of tobacco leaf whilethe CaMV 35S promoter-gusA-Tnos gene was expressedin all tissue types, which is consistent with the expressionpattern observed in E. alatus plants. Also, a distinct ex-pression pattern of the gusplus gene was observed in stemtissues (Fig. 1H) and in roots (Fig. 1I) of transgenic E. ala-tus plants. The patterns of GUS expression in leaf, stem androot tissues of E. alatus confirmed that the GUS activitydetected was not from Agrobacterium contamination.

In addition, we verified the stable incorporation of trans-genes into the host genome using a PCR technique. As the

Table 4 Transformation of cotyledon explants and hypocotyl ex-plants of E. alatus

Cotyledons Hypocotyls

Number of explants infected 268 62Bud formation rate (%) 30 32.3Total number of shoots regenerated 94 21Number of shoots GUS positive 43 9Total number of rooted plants GUS

positive28 7

Transformation efficiency (%)a 10.4 11.3

aTransformation efficiency (%) = Number of rooted transgenicplants/total number of explants infected with Agrobacterium

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plants analyzed were T0 transformants, it was likely thatthese tissues contained Agrobacterium cells and thereforethe Ti-plasmid DNA. The presence of the Ti-plasmid DNAin plant genomic DNA extracts makes it impossible to use aconventional PCR method to verify the stable incorporationof transgenes. To circumvent the problem, we isolated thegenomic DNA from each of GUS positive transgenic plantlines with a special care so that high molecular length ge-nomic DNA could be obtained. To remove the Ti-plasmidDNA in plant genomic DNA samples, we fractionized thegenomic DNA on agarose gels with pCAMBIA-gusplus-nptII plasmid DNA as a reference. As shown in Fig. 2B, wecould easily separate high molecular length genomic DNAwith the Ti-plasmid DNA. We recovered high molecularlength genomic DNAs from gels and used them as PCRtemplates. To confirm that we had no Ti-plasmid DNA inour recovered plant genomic DNA samples, we had a con-trol PCR reaction done using primers 1 and 3. As primer 3is located outside of the T-DNA region, no DNA fragmentshould be amplified if the Ti plasmid DNA is not present inthe genomic DNA. However, an amplification of a 1212p bpDNA segment should indicate that the plant genomic DNAsamples were contaminated with the Ti-plasmid DNA.

Representative PCR analyses of transgenic plants areshown in Fig. 2C. With the genomic DNA isolated fromwild type plants as templates, primers 1 and 2 (Lane 1) orprimers 1 and 3 (Lane 2) produced no DNA products. Withthe genomic DNA from GUS positive plants of E. alatusrecovered from agarose gels (i.e., shown on Fig. 2B) astemplates, PCR reactions using primers 1 and 2 (Lane 1)produced 665 bp products (Lanes 4, 9, 11, 13, and 15) butno product was amplified when primers 1 and 3 were used(Lanes 5, 10, 12, 14 and 16). On the other hand, if 10 pgof pCAMBIA-gusplus-nptII plasmid DNA was added togenomic DNA samples (e.g., Transgenic plant-5) in whichTi-plasmid DNA was previously removed, primers 1 and2 produced the 665 bp fragment and primers 1 and 3 pro-duced the 1212 bp fragment, respectively (Fig. 2C, Lanes17 and 18). These results demonstrate that the techniquewe used to remove the Ti-plasmid DNA molecules fromplant genomic DNA extracts worked effectively. The PCRresults are consistent with the conclusion obtained fromthe histochemical staining of GUS activities and confirmthe stable insertion of transgenes into the host genome.Upon the confirmation of the insertion of transgenes intothe genome of E. alatus, we calculated the efficiencies ofthe Agrobacterium-mediated transformation of E. alatus.We have a 10% transformation efficiency for cotyledonexplants and a 11% for hypocotyl explants (Table 4).

Discussion

Woody plants have a tendency to be recalcitrant regardingtissue culture and transformation (Schuerman and Dan-dekar 1993). It is thought that maturation and aging aremain factors causing the explant regenerative potential de-cline found in tissue culture of most woody species (Durzan1990). For this reason, juvenile materials have been exten-

sively used for regeneration and genetic transformation ofwoody species (Bandyopadhyay et al. 1999; Fitch et al.1993; Kim et al. 2004; McGranahan et al. 1990, 1993;Mullins et al. 1990; Scorza et al. 1995; Smigocki et al.1991). Our results together with that of Smith and Jernst-edt’s (1989) demonstrated that the in vitro plant regenera-tion of E. alatus could be achieved when young seedlingswere used as explants.

Smith and Jernstedt (1989) reported that 80–100% ofhypocotyl segments formed 4–10 tiny buds within 4 weekswhen they were cultured on 1/2 MS media supplementedwith 0.5–1 mg/l BA plus 0.01 mg/l NAA, but these budsoften failed to develop into shoots. Our results showed thatboth hypocotyl and cotyledon explants when cultured onWPM containing 0.2 mg/l or 0.5 mg/l NAA plus 5 mg/l or10 mg/l BA formed buds and these buds further developedinto normal shoots. The induction of shoots from cotyle-dons of E. alatus with appropriate hormonal combinationsuggests that cotyledons can also be used for in vitro clonalpropagation. The differences in rates of bud and shoot for-mation between the Smith and Jernstedt’s protocol (Smithand Jernstedt 1989) and ours are likely due to the differ-ences in media, hormone combinations used, and also theages, and sizes of explants. Our result that hypocotyls of E.alatus are slightly better explants to form buds is consistentwith the observation in Perilla frutescens (Kim et al. 2004).

Rooting is often more difficult for woody species than forherbaceous ones. Basal media and the types and levels ofauxins are the main factors affecting rooting. Though somewoody plants such as Phellodendron amurense (Azad et al.2005) and Vitex negundo (Sahoo and Chand 1998), rootedbest on full strength MS medium, many others rooted betteron the media with lower levels of salts, such as 1/2 strengthto 1/4 strength MS medium, full strength to 1/3 strengthWPM, and Nitsch medium (Babu et al. 2003; Grigoriadouet al. 2002; Gu and Zhang 2005; Kaveriappa et al. 1997;Mala and Bylinsky 2004; Martin 2002; Purohit et al. 2002;Te-chato and Lim 1999). IAA, NAA and IBA are the auxinsfrequently used to induce rooting of woody plant species,and in most cases IBA was the most effective one (Anandet al. 1999; Azad et al. 2005; Babu et al. 2003; Kaveriappaet al. 1997; Mala and Bylinsky 2004; Purohit et al. 2002;Te-chato and Lim 1999). In this study, we observed that theshoots of E. alatus did not root well on WPM with 1.0 mg/lor 2.0 mg/l IBA, having a lower rooting rate and callus for-mation at the basal regions of shoots. The presence of cal-lus tissues often caused root abscission when plantlets werepulled out from agar media. Smith and Jernstedt (1989) re-ported that only 50% of shoots of E. alatus produced rootsor root primordia on 1/2 MS medium containing 3 mg/l IBAand predominantly conspicuous callus formed simultane-ously. The similar problems were reported by Purohit et al.(2002) in Quercus leucotrichophora and Q. glauca andthey overcame the problems by treating microshoots with25–100 µM IBA for 24 or 48 h followed by transferringthem to hormone free 1/2 strength WPM. We also noticedthat a combined use of IBA and NAA at low concentrations(0.5 mg/l and 0.3 mg/l, respectively) significantly improvedrooting of E. alatus. It has been previously reported that a

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combination of IBA with IAA or NAA could improve invitro rooting in Greek olive (Grigoriadou et al. 2002), Vitexnegundo (Sahoo and Chand 1998) and Zizyphus jujuba (Guand Zhang 2005).

Our results demonstrated that both cotyledon explantsand hypocotyl explants could be efficiently transformed byA. tumefaciens. The transformation efficiency, total numberof transgenic plantlets produced based on the total num-ber of explants infected by Agrobacterium, is 10–11% forcotyledons and hypocotyls, which is sufficiently high toproduce transgenic E. alatus plants for research or com-mercial applications. The transformation efficiency for E.alatus reported here is significantly higher than many ofother protocols developed for some of woody species suchas blueberry (Song and Sink 2004) and Citrus paradise(Costa et al. 2002). However, the transformation efficien-cies as reported here may have been underestimated be-cause our molecular characterization of the stable inser-tion of transgenes into the host genome was based onGUS positive plants. It is possible that the GUS genemay not be expressed in some of kanamycin resistant plantlines. Agrobacterium-mediated transformation can be sig-nificantly affected by many factors, such as Agrobacteriumstains (Alvarez et al. 2004; Chabaud et al. 2003; Humaraet al. 1999; Nadolska-Orczyk and Orczyk 2000; Sunilku-mar and Rathore 2001), preculture of explants (Kim et al.2004), incubation time in Agrobacterium cultures (Costaet al. 2002), Agrobacterium concentrations (Humara et al.1999; Costa et al. 2002), co-cultivation periods (Cerveraet al. 1998; Costa et al. 2002; Kim et al. 2004), and cocul-tivation temperature (Sunilkumar and Rathore 2001). ForE. alatus, the efficiency of Agrobacterium-mediated trans-formation may be further improved via optimizing theseparameters.

Acknowledgments This project was supported by a grant from theUSDA/CSREES NRI Competitive Grants Program (2003–2006) toYi Li and Donna Ellis.

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