Plant Cell Rep (2006) 25: 92–99DOI 10.1007/s00299-005-0022-3

GENETIC TRANSFORMATION AND HYBRIDIZATION

Chenna Reddy Aswath · Sung Youn Mo ·Doo Hwan Kim · S. Won Park

Agrobacterium and biolistic transformation of onion usingnon-antibiotic selection marker phosphomannose isomerase

Received: 20 November 2003 / Revised: 25 January 2005 / Accepted: 26 January 2005 / Published online: 7 October 2005C© Springer-Verlag 2005

Abstract A new selection system for onion transformationthat does not require the use of antibiotics or herbicideswas developed. The selection system used the Escherichiacoli gene that encodes phosphomannose isomerase (pmi).Transgenic plants carrying the manA gene that codes forpmi can detoxify mannose-6-phosphate by conversion tofructose-6-phosphate, an intermediate of glycolysis, viathe pmi activity. Six-week-old embryogenic callus initiatedfrom seedling radicle was used for transformation. Trans-genic plants were produced efficiently with transformationrates of 27 and 23% using Agrobacterium and biolistic sys-tem, respectively. Untransformed shoots were eliminatedby a stepwise increase from 10 g l−1 sucrose with 10 g l−1

mannose in the first selection to only10 g l−1 mannose in thesecond selection. Integrative transformation was confirmedby PCR, RT-PCR and Southern hybridization.

Keywords Onion . Transformation . Phosphomannose-isomerase . Positive selection

Abbreviations pmi: Phosphomannose isomerase . 2,4-D:2,4-Dichlorophenoxy acetic acid . ABA: Abscisic acid

Introduction

Onion (Allium cepa L.) is one of the most widely plantedvegetable crops in the world with a global production of

Communicated by I. S. Chung

C. R. AswathIndian Institute of Horticultural Research,Bangalore, 560089, Indiae-mail: aswath@iihr.ernet.in

S. Y. Mo · D. H. Kim · S. W. Park (�)Department of Horticultural Science, Konkuk University,Seoul, South Koreae-mail: sewapark@konkuk.ac.kre-mail: mosy0708@hanmail.nete-mail: kimdh@konkuk.ac.kr

about 105 billion pounds in 6.7 million ha. In previousstudies, callus induction, regeneration and transformationof A. cepa by various explants was reported. Amongthem, some reported direct DNA delivery (Klein et al.1987; Eady et al. 1996; Barandiaran et al. 1998; Scottet al. 1999), while some others reported Agrobacteriumtransformation using mature (Joubert et al. 1995; Eady etal. 1996; Zheng et al. 2001) and immature embryos (Eadyet al. 2000). However the use of mature embryos is tedious,while immature embryo leads to contamination. Henceseedling radicle was used as an explant in this study.

The presence of antibiotic marker genes, seen as anunpredictable hazard to the ecosystem and human health,can be solved by removing the selectable antibiotic markergene. Recently, a number of markers for positive selectionfor transformation have been developed (Joersbo andOkkels 1996; Joersbo 2001; Zhang et al. 2000). Amongthem the Escherichia coli gene-encoding phosphomannoseisomerase (pmi) using mannose as the selection indices isuseful in the transformation of many plant species (Joersboet al. 1998; Negrotto et al. 2000; Wang et al. 2000;Lucca et al. 2001). Mannose in plant cells converts intomannose-6-phosphate, an inhibitor of glycolysis, therebyinhibiting germination and development. The pmi activityconverts mannose-6-phosphate to fructose-6-phosphate, anintermediate of glycolysis that positively supports growthof transformed cells. Transformed cells are able to utilizemannose as a carbon source and grow either in the absenceof or with the addition of only small amounts of other car-bon sources such as glucose or sucrose. Cells geneticallytransformed to express pmi acquire a growth advantage(positive selection) on mannose-containing media, whichmakes it a useful selection agent for generation oftransgenic plants.

Since onion is consumed on a large scale, there is aneed for the elimination of controversial antibiotic markers.Hence, the study was taken up to evaluate the use of pmi asa selectable marker for the recovery of transgenic onionbulbs following Agrobacterium and biolistic-mediatedtransformation of embryogenic calli initiated from seedlingradicle.

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Fig. 1 Flow diagram showingthe different steps oftransformation using callusinitiated from seedling radiclein onion

Materials and methods

Plant material

Seeds of onion inbred line KU-31 were rinsed in 70%ethanol for 30 s, washed for 1 min, soaked in detergentfor 5 min, rinsed and surface disinfected with 4% sodiumhypochlorite for 30 min by agitation. They were thenwashed in sterile water 5 times, and stored in sterile waterat 4◦C for 16 h. The surface was disinfected for the sec-ond time with sodium hypochlorite for 10 min followed byrinsing in sterile water. The seeds were germinated asep-tically and the seedling radicle of 1 cm in length growingfrom the shoot apex was cut (Fig. 3a) and cultured in a9 cm petridish containing MS basal medium supplemented

with 5 µM 2,4-dichlorophenoxy acetic acid (2,4-D; thebest medium from Zheng et al. 1998). Twelve explantswere cultured per petridish and incubated at 25 ± 1◦C indark for 4 weeks; later, the callus was subcultured in thesame conditions for another 2 weeks (Fig. 1). Six-week-oldproliferated callus was then used for transformation.

Determination of mannose concentration for selection

Six-week-old compact and nodular embryogenic callus wascultured on MS basal medium supplemented with 5 µM2,4-D, varying concentrations of sucrose (0–20 g l−1) andmannose (0–20 g l−1) to establish mannose lethality. Totest whether mannose could substitute sucrose in sustaininggrowth, sucrose was not added to the mannose containing

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media in a few treatments. Each treatment contained threereplications; each replication (one petridish, 9 mm diame-ter) contained nine clumps each of 3 mm2 (0.25 ± 0.05 g)callus. The callus weight was measured after 4 weeks ofselection culture. Increase in weight was calculated by sub-tracting the initial weight of 0.25 g from the final weight ofthe callus.

Agrobacterium-mediated transformation

Plasmid pNOV2819 conferring streptomycin resistanceharbored in Agrobacterium tumefaciens strain LBA4404was obtained from seed company (Syngenta Seeds AG,www.syngenta.com). It carries pmi gene with constitu-tive promoter CMPS (Cestrum yellow leaf curling virusPromoter-Shorter version), followed by the NOS termina-tor sequences.

A single colony of bacteria was inoculated into a liquidYEP medium containing 50 mg l−1 spectinomycin and in-cubated for more than 24 h at 28◦C with reciprocal shaking(150 cycles min−1). Cultured bacterial cells were collectedby centrifugation (2,000×g,10 min) and suspended to afinal OD 600 of 20 ml liquid MS medium with 5 µM 2,4-D, 20 g l−1 sucrose, and 100 µM acetosyringone (SigmaAldrich, www.sigmaaldrich.com). Six-week-old callus wassegmented into small pieces of 3 mm2, 20 clumps wereimmersed in bacterial suspension for 5 min every time andblotted on sterile filter papers. Twenty explants were placedonto 25 ml aliquots of above medium with 7 g l−1 agar in9-cm petridishes; then the plates were sealed with serenewrap and incubated at 25◦C in dark for 3 days.

Biolistic transformation

A fragment preparation of construct pNOV2820 contain-ing the E. coli derived manA gene encoding pmi driven byCMPS promoter and NOS terminator sequences was usedfor transformation. DNA was extracted using the Qiagenmega preparation kit. Typically, 250 µg of plasmid DNAwas digested in a final volume of 500 µl and then run on0.8% agarose gel in TBE buffer. The band containing thefragment DNA was cut from the gel and eluted using kit(Takara, www.takaratoys.co.jp). The DNA was then pre-cipitated and resuspended in 100 µl of TE buffer to a finalconcentration of 1 µg µl−1.

The target plate was composed of six-week-old activelygrowing embryogenic callus of nine clumps of 3 mm2 ar-ranged in concentric circles of 2.5-cm diameter in the cen-ter of the plate. Fragment DNA was precipitated on togold micro carriers (<1 µm) as described in manual. Fivemicrogram DNA was used in each six-shot micro carrierpreparation. Genes were delivered to the target tissue cellsusing the PDS-1000HE BiolisticsTM device. The setting onthe device were as follows: 6 mm between the rupture diskand macro carrier, 10 mm between the macro carrier and thestopping screen and 6 cm between the stopping screen andthe target. The target callus were shot twice with 1 µg DNA

using 1,100-psi rupture disks (Eady et al. 1996). After thegene delivery, tissues were incubated in MS basal mediumsupplemented with 5 µM 2,4-D and 20 g l−1 sucrose in thedark at 23◦C.

Transformation efficiency

After 3 days of co-culture with Agrobacterium or bom-bardment, the callus was transferred to MS basal mediumsupplemented with 5 µM 2,4-D, 10 g l−1 mannose, and10 g l−1 sucrose (with 300 mg l−1 cefotaxime in case ofAgrobacterium) (Fig. 1). The transformation efficiency wasrecorded at the end of 7th week as the number of callusclumps survived without browning. The callus was thentransferred to only mannose medium without any sucrosein order to reduce the number of escaped untransformedcallus and improve the selection efficiency. At the end of9th week transformation efficiency was counted as the num-ber of callus clumps proliferated. The proliferated sectorsfrom resistant callus were then transferred to the regenera-tion medium containing MS medium with 5 µM Kinetin,1 µM abscisic acid (ABA) and 10 g l−1 mannose for in-duction of somatic embryos. At the end of the 15th weekthe transformation efficiency was counted as number ofsomatic embryos formed in each callus clumps. The devel-oped somatic embryos were then transferred to MS basalmedium supplemented with 10 g l−1 mannose. At the end ofthe 19th week the transformation efficiency was counted asnumber of plantlets formed from well-developed somaticembryos and then transferred to half-strength MS mediumwith 10 g l−1 mannose. At the end of the 23rd week, therooted plantlets were transferred to soil.

For negative control, the callus was inoculated with anAgrobacterium strain free of any plant selectable marker,in case of biolistic experiments, the callus was bombardedwithout DNA. For positive control, the callus withoutco-culture or bombardment was transferred to respectivegrowth hormone medium with 20 g l−1 sucrose.

Analysis of transformants

Total genomic DNA was isolated from leaf tissues ofthe control and putatively transformed plants regeneratedfrom mannose resistant calli (Wettasinghe and Peffley1998). For PCR analysis of manA gene, the primer set5′ ACAGCCACTCTCCATTCA 3′and 5′ GTTTGCCAT-CACTTCCAG 3′, which amplifies 514 bp, was used. PCRamplification reaction contained 20 ng of template DNA,0.4 µM of each primer, 0.5 µl dNTP mixture (2.5 mM),1× Taq DNA polymerase reaction buffer and one unit TaqDNA polymerase (Takara, www.takaratoys.co.jp) in a 20 µlfinal volume. PCR (Perkin Elmer, www.perkinelmer.com)conditions were performed as follows: one cycle at 94◦Cfor 5 min followed by 30 cycles at 94◦C for 1 min, 60◦C for30 s, and 72◦C for 30 s. Amplified products were resolvedin 1% (w/v) agarose gel.

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For RT-PCR, total RNA was conducted according toManuel et al. (1999). RT-PCR was performed usingReverse-iTTM one step RT-PCR Kit, ReddyMixTM (Tamar,www.tamar.co.il) according to the manufacturer’s recom-mendations. To ensure that the amplified bands were notfrom trace genomic DNA in the RNA solution, the RNA so-lutions were used without reverse transcriptase. ManA genewas amplified using the above indicated primers accordingto the following cyclic conditions: one cycle at 47◦C for30 min and 94◦C for 5 min followed by 30 cycles at 94◦Cfor 1 min, 60◦C for 30 s, and 72◦C for 30 s. Amplifiedproducts were resolved in 1% (w/v) agarose gel.

For Southern blotting, genomic DNA (about 10 µg)was restricted with EcoRI, separated on a 0.8% agarosegel and transferred onto Hybond-N nylon membranesas per manufacturer’s instructions (Amersham Bio-sciences, www1.amershambiosciences.com). Bothplasmids were restricted with HindIII and Kpn1 andisolated using the QIAquick Gel Extraction Kit. Filterswere hybridized with probe as template using AlkphosDirect Labeling Reagent (Amersham Biosciences,www1.amershambiosciences.com). Hybridization, wash-ing and detection were performed according to theinstruction manual of Alkphos Direct Labeling andDetection System with CDP-Star (Amersham Biosciences,www1.amershambiosciences.com).

Selection for pmi positive plants

Mannose, like phosphinothricin, is an effective selectionagent for plants grown in soil under nonsterile conditions.Therefore, mannose selection on soil-grown onion was in-vestigated. For selection of transformants, the transgenicsand control plants were transferred to pots during the 24thweek; later they were hardened in green house for 1 week.Different concentration of mannose (2, 5, 10, and 50 mM)were dissolved in water and sprayed daily for 25 days, afterwhich necrosis and mortality were recorded.

Results

Response of callus to mannose concentration

A mannose dose response curve revealed that, even in thepresence of sucrose, the callus growth decreases as thepercentage of mannose increases (Fig. 2). Ten grams perliter mannose was found to starve the tissue within the first2 weeks of exposure. Ten grams per liter mannose, theosmotic equivalent of 20 g l−1 sucrose, was the minimumamount of carbohydrate used in the experiments for themaintenance of onion cultures. The mix of 10 g l−1 mannosewith 10 g l−1 sucrose decreased the tissue growth by 42%as compared to normal 20 g l−1 sucrose. The toxic effect ofmannose decreases with increasing sucrose concentrationsin the medium.

0

0.25

0.5

0.75

1

1.25

1.5

1.75

2

0 5 10 15 20

sucrose g/l

gF

W

Mannose 0

Mannose 5

Mannose 10

Mannose 15

Mannose 20

Fig. 2 The effect of mannose concentration (g l−1) (•, 0; �, 5; �,10;•, 15; ◦, 20) on average gram fresh weight (gFW) when cultured withdifferent sucrose concentration. Value represents means±SE

Callus multiplication and regeneration

Callus formation from seedling radicle could be observedafter 4–5 days of culture. Three morphologically differ-ent callus types could be easily distinguished: a compacttype, a white and nodular type with no apparent structuresand a watery and transparent type. Compact callus wasprominent at the site of the apex whereas friable calluswas abundantly produced at the other end. In further sub-cultures, callus became swollen and enlarged considerably.For transformation, only compact and friable callus wereused.

As early as 1 week after the transfer of callus to regen-eration medium, multiple embryos were formed (Fig. 3b).Though many of these embryos failed to form somatic em-bryos, they turned green and formed roots. In some callusclumps, somatic embryos and embryo-like structures wereformed (Fig. 3c). These somatic embryos germinated anddeveloped into plantlets, which readily formed roots upontransfer to half-strength MS medium without growth reg-ulators (Fig. 3d). Plants continued to grow after transferto the glasshouse under short-day conditions. Upon trans-fer to long-day conditions, the regenerated plants producedbulbs.

During initial selection at the 7th week (Table 1), untrans-formed callus clumps were starved and did not developfurther (Fig. 3e). There was no significant difference be-tween Agrobacterium and biolistic transformation, whereboth recorded 23–27% positive callus for the mannose se-lection. At the end of the 9th week, since most of theuntransformed callus was filtered earlier, proliferation was33% in Agrobacterium and 27% in biolistic transformation.After the 15th week, the percentage of callus forming pri-maries was more in biolistic than Agrobacterium (Fig. 3f).At the end of 19th week, as compared to the control, thepercentage somatic embryos forming plantlets was muchless with no significant difference between Agrobacteriumand biolistic.

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Table 1 Effect ofAgrobacterium andbiolistic-mediatedtransformation on survival,proliferation and regenerationof onion callus under differentperiods of selections

Callus survived atthe end of 7thweek (%)

Callus proliferatedat the end of 9thweek (%)

Callus formingsomatic embryos(SE’s) at the end15th week (%)

SE’s formingplantlets at the endof 19th week (%)

Agrobacterium 27b 33b 17.4b 22.5bBiolistic 23b 27b 23.4b 23.7bNegative control(Agrobacterium)

3.7c 2.1c 0.0c 0.0c

Negative control(biolistic)

4.5c 2.2c 0.0c 0.0c

Positive control 95.5a 90.5a 45.5a 65.5a

Note. For Agrobacterium and biolistic transformation, 20 and 9 callus clumps were taken each time,respectively. The experiment was repeated 20 times. The percentage was calculated based on totalnumber of callus clumps. Mean separation in column by Duncan’s multiple range tests. Within thecolumn figure followed by same letter do not differ significantly at P≤0.05

Fig. 3 Plant regeneration viasomatic embryogenesis in onion.a Germinated seed showingradicle, hatched area representsthe explant. b Compact callusderived from seedling radicleused for transformation. cSomatic embryos under MS with5 µM kinetin, 1 µM ABA and10 g l−1 mannose. d Plantletsdeveloped from somaticembryos. e Co-cultured callus atthe end of the 7th week. Note theuntransformed callus starving.f Selection at the end of the15th week under MS with 5 µM2,4-D and 10 g l−1 mannose

In positive control, it was frequently observed that as thestructures enlarged, additional shoot meristem regions wereproduced raising the number of shoots than would be ex-pected from the initial number of embryogenic structureswithin the culture (data not shown). An average of 3.47shoots per callus clump was observed in both transformed

and non-transformed treatments. Plantlets could be read-ily induced to form roots by transfer to half-strength MSmedium without growth regulators. In the negative controlsof both Agrobacterium and biolistic transformation, 3–4%callus proliferated at the end of the 7th week, while inthe 9th week 1–2% of the untransformed callus starved but

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Fig. 4 Molecular analysis of transgenics. a PCR analysis of trans-genics for pmi gene. Lane P1 plasmid from pNOV 2819 used forAgrobacterium transformation, lane P2 plasmid pNOV2820 usedfor biolistic transformation, lanes 1–12 transformed plants throughAgrobacterium (lanes 1–6) and biolistic (lanes 7–12) mediated trans-formation. b RT-PCR analysis for the leaf tissue of transgenic onionplants for pmi gene. Lane C untransformed control, lanes 1–5 trans-formed plants through Agrobacterium (lanes 1–3) and biolistic (lanes4–5) mediated transformation, lane N negative control without re-verse transcriptase. c Southern hybridization analysis of genomicDNA derived from putative transgenic plants. Lane C control plant,lanes 1–6 transformed plants through Agrobacterium (lanes 1–3) andbiolistic (lanes 4–6) mediated transformation

still survived. In the rooting test (data not shown) rooting ofwild type shoots was totally inhibited by 10 g l−1 mannose,whereas the transgenic pmi-expressing plants developed anormal root system. Consequently, 10 g l−1 mannose wasused to screen for transgenic plants which can root nor-mally, while control shoots failed to produce any roots.

Confirmation of transformation

PCR amplification of manA gene showed that all the plantsexcept untransformed control produced bands of an ex-pected size of 514 bp for the pmi fragment at the same posi-tion as the binary vector positive control (Fig. 4a). RT-PCRtransgenic plants yielded the expected 514-bp band whileuntransformed control did not have the band (Fig. 4b).No band was produced in the negative control (lane N inFig. 4b). Southern hybridization following EcoRI diges-tion produced a single band for DNA from the transformedplantlets and none for the control (Fig. 4c).

Selection for pmi-positive onion in soil

We observed negligible effect with the spray of the low-est concentrations (2, 5, and 10 mM) of mannose. How-

ever 50 mM of mannose effectively killed non-transformedplants within 10 days of spray, while the transformed plantswere completely resistant.

Discussion

A reproducible and stable transformation system for onionhas been developed using Agrobacterium tumefaciens andbiolistic approach with 6-week-old callus induced fromseedling radical. This is the first report on the use of man-nose as a selection index in onion, whereas previously onlykanamycin and hygromycin were used.

A common feature among transformation studies inonion was the use of immature embryos as target explantsdue to their excellent morphogenetic competence (Eadyet al. 1996, 2000). However, they reported that the contam-ination rate, which probably arose from infected embryos,ranged from 40 to 100%. Zheng et al. (2001) used matureembryos, which is tedious to remove and requires stere-omicroscope to identify the shoot apex portion. Klein etal. (1987) and Scott et al. (1999) used epidermal tissuesin onion with high velocity microprojectiles resulting intransient expression of chloromphenicol acetyl transferaseand green fluorescent protein gene, respectively. Howevercontamination rate was also high in epidermis. We haveused seedling radicle, which is in no way inferior to otherexplants with an added advantage of easy extraction andzero contamination.

The most common selective protocols for plant trans-formation are the use of kanamycin, hygromycin, andphosphinothricin. In onion Eady and Lister (1998) demon-strated that kanamycin was ineffective up to 200 mg l−1

and used 50–100 mg l−1 geneticin. Kanamycin is notrecommended for spray, as it is very expensive (Altmannet al. 1992). Though hygromycin is moderately expensiveand works well both in media and soil it is not recom-mended because of its high toxicity to humans (Altmannet al. 1992), even 20–50 mg l−1 hygromycin in onionresulted in few escapes (Eady and Lister 1998). Phos-phinothricin is an excellent marker for soil-grown plants.Selection on plates requires purified and expensive phos-phinothricin. Additionally, a large number of escapes werenoticed during selection on media containing 10–30 mg l−1

phosphinothricin (Eady et al. 2000). Unlike most selectablemarkers, pmi confers a positive advantage to plants grownin tissue culture using mannose, which is a carbon source.In addition it does not lead to necrotic cells, which mayrelease toxic compounds and is non-toxic to humans.Mannose selection of resistant plants works equally wellin soil and in plates, and is very cheap, perhaps the leastexpensive among all agents (Todd and Tague 2001). Whenonion callus was exposed to 10 g l−1 mannose withoutsucrose, the starving of tissue was observed within 2weeks. Callus growth continued when mannose was addedwith sucrose at higher concentrations, which is consistentwith the findings of Wright et al. (2001) and Negrotto et al.(2000). However, it was in contrast with Kim et al. (2002)who observed reduction in shoot regeneration capacity in

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pepper with the addition of sucrose to mannose at higherconcentration.

In our studies, the prolonged exposure of escaped callito high mannose without any sucrose for 2 weeks, furtherexposure of 8 weeks in regeneration media, and continuingof the mannose during rooting might have caused distortionof untransformed cells and virtually eliminated all escapes.Further spray of 50 mM mannose to plants grown in potsperfected the selection without any escapes. Wright et al.(2001) utilized only mannose (10 g l−1) and a mannose(7 g l−1) +sucrose (3 g l−1) combination during callusselection regeneration in maize and wheat transformation,respectively. Negrotto et al. (2000) set a combination of5 g l−1 sucrose and 10 g l−1 mannose as the selection indexfor maize transformation, while Kim et al. (2002) recom-mended 20 g l−1 sucrose and 15 g l−1 or more of mannose(up to 50 g l−1) in pepper transformation. The mannoseconcentration of 20 g l−1 but not 30 g l−1, still alloweda low frequency of organogenesis in cassava (Zhang andPuonti-Kaerlas 2000), while 3 g l−1 inhibited regenerationin sugar beet totally (Joersbo et al. 1998).

The average transformation frequency was 25.0%, whichwas lower than that reported for non-transformed calli us-ing similar plant growth regulators (95.0%). Shoot regener-ation from resistance calli initiated from mature embryo ofonion following Agrobacterium-mediated transformationoccurred at a lower frequency of 1.95% using hygromycin(Zheng et al. 2001) and 2.5% using immature embryo withselection agent geneticin (Eady et al. 2000). The lower fre-quency by earlier workers was also due to large contamina-tion of explants. Negrotto et al. (2000) recovered frequencyof 30% in maize plants using Agrobacterium transforma-tion. Wright et al. (2001) also observed mean frequenciesof 45% for maize and 20% for wheat using biolistic trans-formation, which is a 10-fold higher than the herbicidal se-lection of previous workers. In sugar beet, the mannose se-lection system using Agrobacterium was reported to resultin 10-fold higher transformation frequencies as comparedto kanamycin selection (Joersbo et al. 1998); however incassava, the efficiency of hygromycin selection was about2-fold higher than that of mannose selection-using PEG-mediated particle bombardment (Zhang and Puonti-Kaer-las 2000). This indicated that transformation frequency onmannose selection varies with each crop.

Southern blot analysis of EcoRI restriction digests ofgenomic DNA from transgenic plants using pmi proberevealed one band in the transgenic lines, indicating theintegration of one copy of the pmi gene. Agrobacterium-mediated transformed plants (plants 1, 2, and 3 of Fig. 4c)showed integration of gene at one locus while polymor-phism of hybridization pattern among plants bombardedby gene gun (plants 4, 5, and 6) indicated random integra-tion. RT-PCR transgenic plants yielded the expected 514-bpfragment while untransformed control did not have the frag-ment. No fragment was produced in the negative control(lane N in Fig. 4b), where RNA solutions was used with-out reverse transcriptase indicating that the RT-PCR bandsfrom transgenic lines were produced from the mRNA ofpmi.

Even though bombarded callus recorded better transfor-mation than Agrobacterium there was no statistical differ-ence between the two methods. Hence for onion transfor-mation both approaches can be used.

Conclusion

Callus induction using MS basal medium supplementedwith 5 µM 2,4-D from seedling radicle, and shoot regener-ation via somatic embryogenesis using MS medium with5 µM Kinetin and 1 µM ABA has been demonstrated tobe a very effective procedure for both micro propagationand transformation studies. Initial selection of callus inMS medium with 10 g l−1 mannose and 10 g l−1 sucroseand the subsequent selection with only 10 g l−1 mannoseprevented the production of escapes. The results indicatedthat, if the right gene constructs are delivered into theright tissue and selected with a non-controversial selectionmarker like mannose, it should be possible to producesafer transgenic onions.

Acknowledgements We thank the Korean Federation of Scienceand Technology, Republic of Korea, for the financial support under‘Brain pool project’ and ICAR, Government of India, for the studyleave for C. Aswath. We also thank Syngenta seeds, Switzerland forsparing the pmi construct. We would like to acknowledge Dr TimConner for critically reviewing the manuscript.

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