Plant Cell Rep (2006) 25: 198–205DOI 10.1007/s00299-005-0042-0

GENETIC TRANSFORMATION AND HYBRIDIZATION

Hector G. Nunez-Palenius · Daniel J. Cantliffe ·Don J. Huber · Joseph Ciardi · Harry J. Klee

Transformation of a muskmelon ‘Galia’ hybrid parental line(Cucumis melo L. var. reticulatus Ser.) with an antisenseACC oxidase gene

Received: 7 March 2005 / Revised: 23 June 2005 / Accepted: 9 July 2005 / Published online: 16 December 2005C© Springer-Verlag 2005

Abstract ‘Galia’ muskmelon (Cucumis melo L. var.reticulatus Ser.) has been recalcitrant to transformationby Agrobacterium tumefaciens. Transformation of the‘Galia’ male parental line, ‘Krymka’, with an ACC oxi-dase (CMACO-1) gene in antisense orientation is describedherein. Explants were transformed using A. tumefaciensstrain ABI, which contained a vector pCmACO1-AS plas-mid, bearing an antisense gene of CMACO-1 and the CP4syn gene (glyphosate-tolerance). Both CMACO-1 and CP4syn genes were assessed by a polymerase chain reactionmethod. Flow cytometry analysis was performed to de-termine plant ploidy level of primary transformants. Twocompletely diploid independent transgenic plants were ob-tained. Southern blot and segregation analysis in the T1 gen-eration determined that each independent transgenic linehad one single insertion of the transgene. These transgenicmuskmelon male parental lines have potential for use in theproduction of ‘Galia’ F1 hybrids with improved shelf life.

Keywords Ethylene . ‘Galia’ . Organogenesis . Planttissue culture . Agrobacterium . Glyphosate . Fruitripening

Abbreviations ACC: 1-Aminocyclopropane-1-carboxylic acid . ACO-1: ACC oxidase-1 .

Communicated by P. Ozias-Akins

H. G. Nunez-Palenius · D. J. Cantliffe (�) · D. J. Huber ·J. Ciardi · H. J. KleeHorticultural Sciences Department, University of Florida, IFAS,Gainesville, FL 32611, USAe-mail: djc@ifas.ufl.eduTel.: +(352)-392-1928Fax: +(352)-392-6479

H. G. Nunez-PaleniusGenetic Engineering Department,CINVESTAV, Irapuato, Gto., CP 36500, Mexico

Present address:J. CiardiBioRexis Pharmaceutical Corporation,3400 Horizon Drive, King of Prussia, PA 19406, USA

CMACO-1: Cucumis melo ACO-1 .BA: 6-Benzylaminopurine . IAA: Indole-3-acetic acid .MS: Murashige and Skoog medium

Introduction

‘Galia’ muskmelon (Cucumis melo L.) is an Israeli melonF1 hybrid bred by Dr. Zvi Karchi at the Newe Ya’ar researchcenter of the Agricultural Research Organization (A.R.O.,Israel) and released in 1973 (Karchi 2000). This cultivarhas green-flesh characteristics of the ‘Ha’Ogen’ melon cul-tivar, which was used as the female parent, and netted rindfrom ‘Krymka’, which was used as the male parental line.It has exceptional fruit quality with 13–15% total solublesolids (TSS), bold flavor and a distinct aroma, leading torapid adoption in both local and export markets. ‘Galia’type melons have been the mainstay in muskmelon sales inthe European market for more than 30 years (Karchi 2000).One disadvantage of ‘Galia’ is its storage life, which is lim-ited to 2–3 weeks. Timing of harvest is critical for ‘Galia’fruit, because in order to develop peak flavor and aroma themelon should be picked at maturity (Karchi 2000). ‘Galia’F1 hybrid tends to be extremely soft at and past peak fruitmaturity. Traditional breeding methods have helped breed-ers develop a strategy to obtain ‘Galia’ muskmelons with along shelf life; however, this approach can result in a lossof favorable fruit quality characteristics.

Plant biotechnology has the potential to geneticallytransform plants and transfer novel characteristics. Inorder for plant transformation to be successful a reliableplant in vitro regeneration system must first be developed(Guis et al. 1998). Using Agrobacterium and gun-particlebombardment methods, several transgenes, which providedifferent phenotypic characteristics, have been transferredinto C. melo explants. These have included selectablemarker and reporter genes (Akasaka-Kennedy et al. 2004;Dong et al. 1991; Fang and Grumet 1990; Gaba et al. 1992;Gray et al. 1995; Valles and Lasa 1994), virus-resistancegenes (Clough and Hamm 1995; Fang and Grumet 1993;

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Gonsalves et al. 1994; Yoshioka et al. 1992, 1993),halotolerance genes (Bordas et al. 1997), and genes toimprove fruit quality (Ayub et al. 1996; Clendennen et al.1999; Ezura et al. 1997; Guis et al. 2000; Shellie 2001;Silva et al. 2004). Improved quality of Charentais typemuskmelon has been achieved by inserting ACC oxidasegenes in antisense orientation in order to reduce fruitethylene biosynthesis (Ayub et al. 1996; Guis et al. 1997;Silva et al. 2004).

Increasing the shelf life of ‘Galia’ F1 muskmelon by planttransformation of parental lines is a feasible alternative.Unlike other melon types, such as Cantaloupe Charentais(Akasaka-Kennedy et al. 2004; Ayub et al. 1996; Silva et al.2004), ‘Galia’ muskmelon is not easily cultivated in vitroand complete regenerated wild-type plants are especiallydifficult to obtain (Edriss et al. 1996; Gaba et al. 1994,1996; Galperin et al. 2003; Kintzios and Taravira 1997;Leshem 1989; Leshem et al. 1994a,b). Moreover, Gabaet al. (1999) reported ‘Galia’ muskmelon to be recalcitrantto transformation by Agrobacterium tumefaciens.

The goal of this research was to transform the maleparental line (cv. ‘Krimka’) of ‘Galia’ muskmelon withthe ACC oxidase (CMACO-1) antisense gene in an attemptto delay fruit ripening.

Materials and methods

Plant material

Coats of 50 male parental line ‘Galia’ (C. melo L. var. retic-ulatus Ser.) cv. ‘Krimka’ seeds were removed, and the em-bryos’ surface sterilized in 1.2% sodium hypochlorite (20%commercial bleach solution containing two drops of Tween20TM per 100 ml) for 15 min in a sterile Erlenmeyer flask(50 ml), and washed three times with sterilized distilled wa-ter. Embryos were then cultivated for 2 days in regenerationmedium (RM), which consisted of Murashige and Skoogsalts (MS) (Murashige and Skoog 1962), supplementedwith 30 g l−1 sucrose, 0.1 g l−1 myo-inositol, 0.001 g l−1

thiamine-HCl, 0.05 mg l−1 pyridoxine-HCl, 0.05 mg l−1

nicotinic acid, 2 mg l−1 glycine, 1 mg l−1 benzyladenine(BA), 0.001 mg l−1 α-naphthaleneacetic acid (NAA), and7.0 g l−1 phytagar. Seedling growth and plant regenerationwere conducted in a growth chamber (Lab-Line Instru-ments, Inc., Melrose Park, IL) under 100 µmol m−2 s−1

light and a 16 h photoperiod provided by cool-white fluo-rescent lamps and constant 25◦C temperature.

Plant regeneration

Cotyledons from 2-day-old seedlings, grown asepticallyin RM, were dissected and cut tranversely in four equalpieces, then placed with their abaxial side on the surface ofRM. Each cotyledon slice constituted an explant. Com-plete shoots were obtained de novo from these cotyle-don explants after 4 weeks of culture. Regeneration effi-ciency was calculated as BFC index, BFC index= (aver-

age number of shoots per explant) × (% explants formingshoots)/100 (Martinez-Pulido et al. 1992). Shoots were ex-cised from cotyledon explants and incubated on elongationmedium (EM), which consisted of MS salts, supplementedwith 30 g l−1 sucrose, 0.1 g l−1 myo-inositol, 0.001 g l−1

thiamine-HCl, 0.05 mg l−1 pyridoxine-HCl, 0.05 mg l−1

nicotinic acid, 2 mg l−1 glycine, 0.025 mg l−1 BA and8.0 g l−1 phytagar, for three more weeks. Elongated shootswere then transferred to rooting media consisting of half-strength MS medium supplemented with 1 mg l−1 indole-3-acetic acid (IAA). After 3 weeks of culture, shoots de-veloped a complete and normal root system. Rooted shootswere hardened using common practices and then trans-ferred to glasshouse. Plants were grown in an evaporative-cooled fan and pad glasshouse, which maintained temper-atures of 28◦C day and 20◦C night, in plastic pots (11.3 l)filled with soil-less media and following common growingpractices recommended by Rodriguez and Cantliffe (2001).A complementary light regime was supplied by Metalarclamps with a light intensity of 350–530 µmol m−2 s−1. Thelight period was set to 18 h per day.

Agrobacterium inoculation and plant transformation

Agrobacterium tumefaciens strain ABI containing abinary vector, pCmACO1-AS, harboring a selectableglyphosate resistance marker [CP4 syn gene encoding for5-enolpyruvylshikimate-3-phosphate synthase (EPSPS)],and the ACC oxidase (CMACO-1) gene in antisense orien-tation was used (Fig. 1a). Both genes were under controlof the Figwort Mosaic Virus promoter (Richins et al. 1987)(Fig. 1a). Agrobacterium was transferred to 5 ml of Luria-Bertani Broth (LB) medium (pH 7.5) supplemented with25 mg l−1 chloramphenicol, 50 mg l−1 kanamycin, and100 mg l−1 spectinomycin, and then incubated on an or-bital shaker (200 rpm) for 12 h at 25◦C. Afterward, theculture (5 ml aliquot) was then transferred to 250 ml baf-fled culture flask containing 50 ml liquid LB medium withthe same antibiotics and concentrations previously used,and incubated at 25◦C on an orbital shaker (150 rpm) foran additional 14–20 h until an A600=0.7–1.0 was reached.

Cotyledonary explants were immersed in Agrobacteriumsuspension for 20–30 min with orbital shaking (50 rpm),and then were blotted three times onto sterile filter paper(Whatman No. 1) to remove the excess bacterial suspen-sion. Explants were then cultured and de novo shoots wereobtained as previously described in “Plant regeneration”section, except that RM was supplemented with 50 µMGlyphosate and 150 mg l−1 Timentin (GlaxoSmithKline,Research Triangle Park, NC). Glyphosate selection wascarried out only on RM.

Flow cytometry analysis

Plant nuclei were freshly isolated from the third leaf belowthe shoot apex of acclimatized plants grown in a green-house. Briefly, plant material (4 cm2) was chopped for 3 min

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Fig. 1 Map of the T-DNA in pCmACO1-AS, PCR and South-ern blot hybridization assay of male muskmelon transgenic plants.(a) T-DNA region of binary vector pCmACO1-AS. The underlinedfragment of CP4syn gene was used as a probe for Southern blot hy-bridization assay. RB: right border of T-DNA, FMV: figwort mosaicvirus promoter, CP4syn: 5-enolpyruvylshikimate-3-phosphate syn-thase (EPSPS) gene, E9: 3′end of pea rbcSE9 terminator, CMACO-1:melon ACC oxidase gene in antisense orientation, NOS: nopalinesynthase terminator, LB: left border of T-DNA. (b) PCR assay forputative transgenic ‘Galia’ muskmelon male line plants. The ampli-fication product for CP4syn, native ACO-1, and engineered ACO-1genes were 1.4, 0.5, and 0.3 Kb, respectively. M: HyperLadder (Bio-line), 1–6: putative transgenic plants, 7: DNA from positive plant, 8:DNA from negative plant, 9: PCR reaction mixture. (c) Total genomicDNA (20 µg) extracted from leaves of T1 plants was digested andelectrophoresed according to “Material and methods” section. M:HyperLadder (Bioline), WT: wild type ‘Galia’ male plant. 1–4: T1plants from T0 TGM-AS-2 line. 5–8: T1 plants from T0 TGM-1 line.(d) Southern blot hybridization assay for BamHI-digested genomicDNA. 1, 2, and 4: PCR-positive T1 plants of T0 TGM-AS-2 line. 3:PCR-negative T1 plant of T0 TGM-AS-2 line. 5–7: PCR-positive T1plants of T0 TGM-AS-1 line, 8: PCR-negative T1 plant of T0 TGM-1line

(at 4◦C) with a razor blade in a Petri dish containing 5 ml ofQuesenberry (1995) extraction buffer. The nuclei suspen-sion was filtered through Spectra r©/Mesh nylon filter (60-µm mesh size) to remove cell debris. The nuclei-filteredsuspension was stained with propidium iodide by adding1 ml of a propidium iodide stock solution (1 mg ml−1) to

2 ml of nuclei suspension. After gently stirring, the nucleimixture was incubated for 5 min and then analyzed by flowcytometry. DNA content of the isolated plant nuclei wasanalyzed with a flow cytometry apparatus (FACScan. BD-Biosciences, San Jose, Cal). It was calibrated using the 2Cpeak from nuclei of young leaves of diploid plants derivedfrom seed. A minimum of 10,000 nuclei were measured foreach sample.

Detection of transgenes

Total DNA isolation from wild-type and putative trans-genic melon leaflets (0.25–0.5 g) was performed using amodified CTAB protocol (Doyle and Doyle 1987, 1990).The forward and reverse primers for CP4 syn gene were5′-CGG TGC AAG CAG CCG TCC AGC-3′ and 5′-CCTTAG TGT CGG AGA GTT CG-3′, respectively, ampli-fying a fragment of 1,400 bp (1.4 Kb). The forward andreverse primers for ACC oxidase gene were 5′-GCA ATTATC CGC CGT GTC-3′ and 5′-TCT TCA AAC ACA AACTTG GGG-3′, respectively. These primers will produce a503 bp fragment from the native (genomic) ACC oxidasegene and a 378 bp product from the transgene. The ACC ox-idase and CP4 syn fragments in total DNA were amplifiedunder the following conditions: a pre-incubation period at94◦C for 7 min followed by 40 cycles of 94◦C for 1 min fordenaturation, 60◦C for 1 min for annealing, and 72◦C for1 min for extension, and a final extension period at 72◦Cfor 10 min. The amplified PCR products (25 µl) were sub-jected to electrophoresis on a 1% agarose gel and visualizedby UV light.

ACO activity in vivo assay

ACO activity was measured in vivo in melon mesocarptissue from wild type and T0 transgenic antisense fruitsbased on the conversion of exogenous ACC to ethylene(Amor et al. 1998; Smith et al. 1994). This assay was carriedout on three fruit developmental stages, i.e. zero-, half- andfull-slip stage. Samples of mesocarp tissue were taken intriplicate with a No. 5 cork-borer from the equatorial regionof the fruit. After removing the peel, 1 g of tissue wasincubated for 3 h at 25◦C in 3 ml reaction buffer, containing50 mM Tris–HCl (pH 7), 100 mM sucrose, 250 µM ACC,and 100 µM cycloheximide (Amor et al. 1998). Boiled(95◦C for 10 min) mesocarp tissue and reaction buffer alonewere used as controls. After the incubation period, a 1-mlgas sample was taken from the head space and analyzed bygas chromatography (Ciardi et al. 2000). Controls alwayshad non-detectable ACO activity.

Southern blot analysis

Total melon DNA was isolated from young leaves of wildtype and transgenic T1, TGM-AS-1 and TGM-AS-2 lines(TGM-AS stands for Transgenic Galia Male AntiSense

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line), using a modified CTAB protocol from Doyle andDoyle (1987, 1990). Twenty micrograms of this DNA weredigested overnight at 37◦ C with BamHI and separatedby electrophoresis in a 1.3% agarose gel. Because BamHIhas one cut-site within T-DNA, a fragment bigger than0.398 Kb is expected for each insertion event. DNA wasthen denatured and transferred to N+ Hybond membrane.The nylon membrane was prehybridized 4 h at 42◦C withconstant shaking at 60 rpm, in a solution containing 50%(v/v) formamide, 5× Denhardt’s, 1% sodium dodecyl sul-fate (SDS), 5× SSPE (1× SSPE is 15 mM NaCl, 10 mMNaH2PO4

.H2O and 1 mM EDTA), and 100 µg ml−1 de-natured salmon sperm DNA. Hybridization was carriedovernight at 42◦C with constant shaking at 60 rpm, in a so-lution containing 50% (v/v) formamide, 5× Denhardt’s, 1%sodium dodecyl sulfate (SDS), 5× SSPE, and 100 µg ml−1

denatured salmon sperm DNA plus 1 × 106 cpm ml−1 dena-tured 32P-labeled 1.4 Kb PCR-product from CP4 syn gene.All further washes were performed with constant shakingat 60 rpm. Membranes were first washed in a solution con-taining 2× SSPE, 0.05% sarkosyl and 0.01% sodium py-rophosphate at 42◦C during 20 min. The second wash wasperformed in a solution containing 2× SSPE, 0.05% sarko-syl and 0.01% sodium pyrophosphate at 65◦C for 20 min.The third and last (fourth) washes were carried out in a so-lution containing 0.1× SSPE, 0.05% sarkosyl and 0.01%sodium pyrophosphate for 20 min at 65◦C. Membraneswere exposed to Kodak BIOMAX MR film at −80◦C for3–5 days.

Segregation analysis of transgenes in primarytransformants

In order to analyze the segregation pattern of transgenes, 62randomly selected T1 seedlings of TGM-AS-1 and TGM-AS-2 transgenic lines of ‘Galia’ male parental line wereevaluated. DNA was extracted at least three times fromeach individual seedling according to the modified CTABprotocol (Doyle and Doyle 1987, 1990). A PCR assay withspecific primers for CMACO-1 and CP4 syn genes wasperformed for each DNA sample as described earlier fortransgene detection.

Results and discussion

Transformation efficiency

A total of six experiments of transformation were com-pleted, under a completely randomized design, using thevector pCmACO1-AS. Glyphosate selection was not com-pletely efficient, because several “escapes” were detectedby PCR assays. In addition, these PCR assays aided in de-tecting the presence of CP4 syn and CMACO-1 antisensegenes (Fig. 1b). We were able to observe the presence of1.4, 0.5, and 0.3 Kb fragments that belong to the CP4 syn,the native ACC oxidase and the engineered ACC oxidasegenes, respectively. False positives due to residual Agrobac-

Fig. 2 Stable expression of β-D-glucuronidase (GUS) gene in T0shoots (A and B) and roots (C and D) of ‘Galia’ muskmelon maleparental line

terium in the primary transformed shoots appeared to be in-frequent. Using the cotyledon-protocol and the CMACO-1 construct, the individual transformation efficiency foreach experiment, assessed by PCR, ranged between 7.5 and12.5%. Similar transformation efficiency rates (9.5%) wereobtained for muskmelon cv. ‘Krymka’ using another inde-pendent transformation system, such as the GUS reportergene (Fig. 2). This transformation efficiency obtained for‘Galia’ male parental line is one of the highest reported forany C. melo transformation protocol (Akasaka-Kennedyet al. 2004; Bordas et al. 1997; Dong et al. 1991; Fang andGrumet 1990; Gaba et al. 1992; Gonsalves et al. 1994; Guiset al. 2000). This high transformation efficiency could bethe result of a combination of factors such as A. tumefa-ciens strain, selectable marker, and construct used (Febreset al. 2003; Hellens and Mullineaux 2000), which may haveprovided the right conditions to accomplish these positiveresults.

Transgenic explants had an epinastic response while theywere growing in vitro on Glyphosate selection (Fig. 3).However, this abnormality was not observed once trans-genic shoots were rooted and transferred to ex vitro con-ditions. Glyphosate is a strong herbicide. It is able to in-duce ‘stress’ responses even in Glyphosate-tolerant plants(Raymer and Grey 2003; Saroha et al. 1998). Also, it is wellknown that ethylene is a plant hormone related to ‘stressresponses’ (Klee and Clark 2002). Therefore, it is plausi-ble that epinastic phenotype was induced by Glyphosate.A similar response was observed in positive explants forβ-D-glucuronidase gene (GUS) growing on Glyphosate aswell (Nunez-Palenius et al. unpublished results).

In summary, transgenic shoots were obtained by us-ing Glyphosate as a selectable agent and were putativelyidentified by means of PCR assay. This PCR system

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Fig. 3 Transgenic (left) and non-transgenic (right) in vitro ‘Galia’male line explants. Transgenic explants, growing on 50 µMglyphosate, had curled leaves and shorter internodes than wild type

also allows us to identify those shoots which were non-transgenic.

Ploidy level of primary regenerants

Melon plants regenerated through plant tissue culturemethodologies are very susceptible to increases in ploidylevel while they are in vitro cultivated (Bouabdallah andBranchard 1986; Debeaujon and Branchard 1992; Ezuraand Oosawa 1994; Ezura et al. 1992a,b, 1994; Fassuliotisand Nelson 1992; Guis et al. 1998; Kathal et al. 1992).Therefore, flow cytometry analyses were performed to de-termine the ploidy level of primary regenerants. Youngleaves from all in vitro transgenic regenerated plants andalso from wild-type plants obtained from seed were used.This was done with the aim of discarding any triploid,aneuploid, and tetraploid or mixoploid plants. Based onthese analyses, only diploid plants were used in subsequentexperiments. Unexpectedly, some leaves from plants ob-tained from seeds resulted as tetraploids. This observationhas been reported in melon plants under field conditions aswell (Nugent and Ray 1992). In Arabidopsis it has beendescribed that ethylene and gibberellins might have animportant role in inducing the cellular endoreduplicationprocess, which leads to an increase in ploidy level (Kon-dorosi et al. 2000). Figure 4 depicts charts for wild-typediploid and tetraploid plants, as well as transgenic diploidand tetraploid plants. In Fig. 4a, the nuclei sample was ob-tained from the third leaf below the shoot apex (diploid),whereas nuclei in Fig. 4b were obtained from shoot re-growth from the plant base (fourth and fifth node distal tothe cotyledonary leaf). For subsequent experiments usingflow cytometry analysis, all leaf samples were harvestedfrom the third leaf below the shoot apex. In Fig. 4c andd, a transgenic diploid and tetraploid plant is shown. Us-ing flow cytometry analysis it was found that only 20% ofthe transgenic plants were diploid. This number of diploidmelon plants attained through in vitro culture is lower com-pared with results previously reported (Curuk et al. 2003;

Guis et al. 2000). This is because our system is based onusing cotyledons as explants, which is a tissue with highpropensity to bear tetraploid cells since the mature seedstage in cucurbits (Colijn-Hooymans et al. 1994). Afteran entire flow cytometry evaluation (at least seven leavesfrom different nodes) of all transgenic regenerants, twocompletely diploid-independent transgenic plants were se-lected, which were named as TGM-AS-1 and TGM-AS-2.Plants that were PCR positive and completely diploid wereadvanced to the next generation by selfing.

ACO activity in vivo assay

When ‘Galia’ male parental mesocarp tissue was incubatedin the presence of reaction buffer mixture, ACO activity wasdetected via quantification of ethylene evolution. At zero-slip developmental stage, the enzyme activity in vivo wasbelow 1 nl g−1 h−1 for all genotypes (Fig. 5). An increase inACO activity was observed in WT mesocarp tissue at thehalf-slip stage. By contrast, TGM-AS-1 and TGM-AS-2fruit did not exhibit increased ACO activity at the half-slipstage. Indeed, expression of the ACO antisense gene intransgenic fruits resulted in a reduction in ACO activity,which was four times less compared to WT fruit at thehalf-slip stage (Fig. 5). Therefore, significant differences inACO activity in vivo were found between ACO antisensefruit and WT at the half-slip developmental stage (datanot shown). At the full-slip stage, the lowest ACO activitywas detected in TGM-AS-2 mesocarp tissue, which wassignificantly different from the other genotypes (Fig. 5).The lower ACO activity found in transgenic antisense fruitcompared to wild type is the result of transgene (CMACO-1) novel expression.

Southern blot analysis

The presence of the introduced CP4 syn gene in T1 plants,both TGM-1 and TGM-2 lines, was established by genomicSouthern blot analysis (Fig. 1c and d). Likewise, this as-say allowed determining the copy number of the transgeneinsertion. The 32P-labeled 1.4 Kb PCR-product from CP4syn gene used as a probe hybridized with a single BamHI-released fragment of genomic DNA isolated from three in-dependent T1 plants from TGM-1 (Fig. 1d, lanes 5–7) andthree independent T1 plants from TGM-2 lines (Fig. 1d,lanes 1, 2, and 4). These data indicated that a single copy ofthe T-DNA was incorporated into the ‘Galia’ male parentalline genome in TGM-AS-1 and TGM-AS-2 lines. Therewas no hybridization signal in any non-PCR positive T1plants (Fig. 1d, lanes 3 and 8).

Transgene inheritance in the T1 progeniesof primary transformants

On a population of 62 T1 plants randomly chosen fromeach transgenic line (TGM-AS-1 and TGM-AS-2), DNA

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was extracted individually, at least three times, to deter-mine the transgene distribution inheritance pattern. PCRanalysis of the progeny of TGM-AS-1 and TGM-AS-2 us-ing CP4 syn- and ACO-antisense-specific primers revealedsegregation for the presence and absence of both CP4 synand ACO-antisense fragments. This observed distributionof the ACO-antisense and CP4 syn genes was consistentwith a 3:1 ratio (χ2 value for TGM-AS-1 and TGM-AS-2was 0.193 and 0.021, with a probability of 66 and 88%, re-spectively), which corresponded to the segregation of onesingle copy insertion of T-DNA. This conclusion is sup-ported by the Southern blot analysis results as well.

Conclusion

We have successfully transformed a parental line of ‘Galia’hybrid muskmelon with a gene of interest and obtained twocompletely diploid transgenic regenerants. Analysis of T1progenies from TGM-AS-1 and TGM-AS-2 by segregationand Southern blot revealed that both transgenic lines had asingle T-DNA insertion.

Analysis of the transgenic fruits from ACO-1 antisensemale line plants has revealed that these fruits have a lowerACC oxidase activity in vivo than their wild-type counter-part.

Fig. 4 Flow cytometry analysis of propidium iodide-stained nu-clei from wild type (a and b) and transgenic (c and d) leaf tissueof ‘Galia’ male parental line. Ten thousand individual nuclei weremeasured for each sample. (a) Wild-type plant having most of nuclei

(55%) at diploid level (M1). (b) Wild-type plant having most of nu-clei (51%) at tetraploid level (M2). (c) Transgenic plant having mostof nuclei (64%) at diploid level (M1). (d) Transgenic plant havingmost of nuclei (58%) at tetraploid level (M2)

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Fig. 5 ACO activity in vivo in WT (•), TGM-AS-1 (◦), and TGM-AS-2 (�) fruits. Vertical bars represent standard error of the means(n=10 fruits). Each fruit was assayed by triplicate

Acknowledgements This work was supported by a USDA/CBAGPL-89-106 grant. We gratefully acknowledge Dawn Bies for thetechnical help for the PCR assays and Brian Kevany for the Southernblot analysis.

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