Plant Cell Rep (2006) 25: 92–99 DOI 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 using non-antibiotic selection marker phosphomannose isomerase

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

Abstract A new selection system for onion transformation that does not require the use of antibiotics or herbicides was developed. The selection system used the Escherichia coli gene that encodes phosphomannose isomerase (pmi). Transgenic plants carrying the manA gene that codes for pmi can detoxify mannose-6-phosphate by conversion to fructose-6-phosphate, an intermediate of glycolysis, via the pmi activity. Six-week-old embryogenic callus initiated from seedling radicle was used for transformation. Transgenic plants were produced efficiently with transformation rates of 27 and 23% using Agrobacterium and biolistic system, respectively. Untransformed shoots were eliminated by 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 the second selection. Integrative transformation was confirmed by PCR, RT-PCR and Southern hybridization. Keywords Onion . Transformation . Phosphomannoseisomerase . 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 planted vegetable crops in the world with a global production of
Communicated by I. S. Chung C. R. Aswath Indian Institute of Horticultural Research, Bangalore, 560089, India e-mail: aswath@iihr.ernet.in S. Y. Mo · D. H. Kim · S. W. Park ( ) Department of Horticultural Science, Konkuk University, Seoul, South Korea e-mail: sewapark@konkuk.ac.kr e-mail: mosy0708@hanmail.net e-mail: kimdh@konkuk.ac.kr

about 105 billion pounds in 6.7 million ha. In previous studies, callus induction, regeneration and transformation of A. cepa by various explants was reported. Among them, some reported direct DNA delivery (Klein et al. 1987; Eady et al. 1996; Barandiaran et al. 1998; Scott et al. 1999), while some others reported Agrobacterium transformation using mature (Joubert et al. 1995; Eady et al. 1996; Zheng et al. 2001) and immature embryos (Eady et al. 2000). However the use of mature embryos is tedious, while immature embryo leads to contamination. Hence seedling radicle was used as an explant in this study. The presence of antibiotic marker genes, seen as an unpredictable hazard to the ecosystem and human health, can be solved by removing the selectable antibiotic marker gene. Recently, a number of markers for positive selection for transformation have been developed (Joersbo and Okkels 1996; Joersbo 2001; Zhang et al. 2000). Among them the Escherichia coli gene-encoding phosphomannose isomerase (pmi) using mannose as the selection indices is useful in the transformation of many plant species (Joersbo et al. 1998; Negrotto et al. 2000; Wang et al. 2000; Lucca et al. 2001). Mannose in plant cells converts into mannose-6-phosphate, an inhibitor of glycolysis, thereby inhibiting germination and development. The pmi activity converts mannose-6-phosphate to fructose-6-phosphate, an intermediate of glycolysis that positively supports growth of transformed cells. Transformed cells are able to utilize mannose as a carbon source and grow either in the absence of or with the addition of only small amounts of other carbon sources such as glucose or sucrose. Cells genetically transformed to express pmi acquire a growth advantage (positive selection) on mannose-containing media, which makes it a useful selection agent for generation of transgenic plants. Since onion is consumed on a large scale, there is a need for the elimination of controversial antibiotic markers. Hence, the study was taken up to evaluate the use of pmi as a selectable marker for the recovery of transgenic onion bulbs following Agrobacterium and biolistic-mediated transformation of embryogenic calli initiated from seedling radicle.

The surface was disinfected for the second time with sodium hypochlorite for 10 min followed by rinsing in sterile water. Twelve explants were cultured per petridish and incubated at 25 ± 1◦ C in dark for 4 weeks. They were then washed in sterile water 5 times. the best medium from Zheng et al. sucrose was not added to the mannose containing . and stored in sterile water at 4◦ C for 16 h.93 Fig.4-dichlorophenoxy acetic acid (2. 1 Flow diagram showing the different steps of transformation using callus initiated from seedling radicle in onion Materials and methods Plant material Seeds of onion inbred line KU-31 were rinsed in 70% ethanol for 30 s. the callus was subcultured in the same conditions for another 2 weeks (Fig. 3a) and cultured in a 9 cm petridish containing MS basal medium supplemented with 5 µM 2. To test whether mannose could substitute sucrose in sustaining growth. soaked in detergent for 5 min. 1).4-D. 1998). Six-week-old proliferated callus was then used for transformation. Determination of mannose concentration for selection Six-week-old compact and nodular embryogenic callus was cultured on MS basal medium supplemented with 5 µM 2.4-D. varying concentrations of sucrose (0–20 g l−1 ) and mannose (0–20 g l−1 ) to establish mannose lethality. The seeds were germinated aseptically and the seedling radicle of 1 cm in length growing from the shoot apex was cut (Fig. later. washed for 1 min. rinsed and surface disinfected with 4% sodium hypochlorite for 30 min by agitation.

and 72◦ C for 30 s. Typically. the rooted plantlets were transferred to soil. For negative control. Fragment DNA was precipitated on to gold micro carriers (<1 µm) as described in manual. the callus was transferred to MS basal medium supplemented with 5 µM 2. the primer set 5 ACAGCCACTCTCCATTCA 3 and 5 GTTTGCCATCACTTCCAG 3 .4D. Transformation efficiency After 3 days of co-culture with Agrobacterium or bombardment.com). 1 µM abscisic acid (ABA) and 10 g l−1 mannose for induction of somatic embryos.com) conditions were performed as follows: one cycle at 94◦ C for 5 min followed by 30 cycles at 94◦ C for 1 min. At the end of the 23rd week.4-D and 20 g l−1 sucrose in the dark at 23◦ C. At the end of the 15th week the transformation efficiency was counted as number of somatic embryos formed in each callus clumps.4 µM of each primer. Analysis of transformants Total genomic DNA was isolated from leaf tissues of the control and putatively transformed plants regenerated from mannose resistant calli (Wettasinghe and Peffley 1998). The developed somatic embryos were then transferred to MS basal medium supplemented with 10 g l−1 mannose. The callus was then transferred to only mannose medium without any sucrose in order to reduce the number of escaped untransformed callus and improve the selection efficiency. coli derived manA gene encoding pmi driven by CMPS promoter and NOS terminator sequences was used for transformation.8% agarose gel in TBE buffer. 10 g l−1 mannose. and 100 µM acetosyringone (Sigma Aldrich.sigmaaldrich.25 ± 0. 9 mm diameter) contained nine clumps each of 3 mm2 (0.5 mM). 20 clumps were immersed in bacterial suspension for 5 min every time and blotted on sterile filter papers. was used. www.10 min) and suspended to a final OD 600 of 20 ml liquid MS medium with 5 µM 2. Biolistic transformation A fragment preparation of construct pNOV2820 containing the E. www.jp). www. At the end of the 19th week the transformation efficiency was counted as number of plantlets formed from well-developed somatic embryos and then transferred to half-strength MS medium with 10 g l−1 mannose. The setting on the device were as follows: 6 mm between the rupture disk and macro carrier.94 media in a few treatments. The band containing the fragment DNA was cut from the gel and eluted using kit (Takara. in case of biolistic experiments.takaratoys. the callus was bombarded without DNA. The DNA was then precipitated and resuspended in 100 µl of TE buffer to a final concentration of 1 µg µl−1 . Five microgram DNA was used in each six-shot micro carrier preparation.syngenta. The transformation efficiency was recorded at the end of 7th week as the number of callus clumps survived without browning.co. the callus without co-culture or bombardment was transferred to respective growth hormone medium with 20 g l−1 sucrose. 0. A single colony of bacteria was inoculated into a liquid YEP medium containing 50 mg l−1 spectinomycin and incubated for more than 24 h at 28◦ C with reciprocal shaking (150 cycles min−1 ). At the end of 9th week transformation efficiency was counted as the number of callus clumps proliferated. tissues were incubated in MS basal medium supplemented with 5 µM 2. . For PCR analysis of manA gene. It carries pmi gene with constitutive promoter CMPS (Cestrum yellow leaf curling virus Promoter-Shorter version).jp) in a 20 µl final volume. 1× Taq DNA polymerase reaction buffer and one unit Taq DNA polymerase (Takara.4-D. The callus weight was measured after 4 weeks of selection culture. Six-week-old callus was segmented into small pieces of 3 mm2 . and 10 g l−1 sucrose (with 300 mg l−1 cefotaxime in case of Agrobacterium) (Fig. PCR amplification reaction contained 20 ng of template DNA.05 g) callus. Agrobacterium-mediated transformation Plasmid pNOV2819 conferring streptomycin resistance harbored in Agrobacterium tumefaciens strain LBA4404 was obtained from seed company (Syngenta Seeds AG. Cultured bacterial cells were collected by centrifugation (2.5-cm diameter in the center of the plate.com). 0. 1). Genes were delivered to the target tissue cells using the PDS-1000HE BiolisticsTM device. 20 g l−1 sucrose. The target callus were shot twice with 1 µg DNA using 1.25 g from the final weight of the callus. 10 mm between the macro carrier and the stopping screen and 6 cm between the stopping screen and the target. followed by the NOS terminator sequences. Each treatment contained three replications. Twenty explants were placed onto 25 ml aliquots of above medium with 7 g l−1 agar in 9-cm petridishes. each replication (one petridish. DNA was extracted using the Qiagen mega preparation kit.100-psi rupture disks (Eady et al. After the gene delivery.5 µl dNTP mixture (2. www. which amplifies 514 bp. PCR (Perkin Elmer. 1996). 60◦ C for 30 s. Amplified products were resolved in 1% (w/v) agarose gel.perkinelmer. then the plates were sealed with serene wrap and incubated at 25◦ C in dark for 3 days.takaratoys. the callus was inoculated with an Agrobacterium strain free of any plant selectable marker. www. 250 µg of plasmid DNA was digested in a final volume of 500 µl and then run on 0. For positive control. The proliferated sectors from resistant callus were then transferred to the regeneration medium containing MS medium with 5 µM Kinetin.000×g.co. The target plate was composed of six-week-old actively growing embryogenic callus of nine clumps of 3 mm2 arranged in concentric circles of 2. Increase in weight was calculated by subtracting the initial weight of 0.

. ReddyMixTM (Tamar. which readily formed roots upon transfer to half-strength MS medium without growth regulators (Fig. After the 15th week. 3f). Three morphologically different callus types could be easily distinguished: a compact type. Plants continued to grow after transfer to the glasshouse under short-day conditions. they turned green and formed roots. the regenerated plants produced bulbs. During initial selection at the 7th week (Table 1). •. Though many of these embryos failed to form somatic embryos. the callus growth decreases as the percentage of mannose increases (Fig. callus became swollen and enlarged considerably.amershambiosciences. www1.il) according to the manufacturer’s recommendations. total RNA was conducted according to Manuel et al. proliferation was 33% in Agrobacterium and 27% in biolistic transformation. as compared to the control. 0. the transgenics and control plants were transferred to pots during the 24th week.co. untransformed callus clumps were starved and did not develop further (Fig. where both recorded 23–27% positive callus for the mannose selection. 5. only compact and friable callus were used. www. Amplified products were resolved in 1% (w/v) agarose gel. (1999). separated on a 0. For Southern blotting. and 50 mM) were dissolved in water and sprayed daily for 25 days. Therefore. . Hybridization. even in the presence of sucrose. the percentage of callus forming primaries was more in biolistic than Agrobacterium (Fig. To ensure that the amplified bands were not from trace genomic DNA in the RNA solution.10. RT-PCR was performed using Reverse-iTTM one step RT-PCR Kit.amershambiosciences. At the end of the 9th week.95 For RT-PCR. genomic DNA (about 10 µg) was restricted with EcoRI.25 gFW 1 0. is an effective selection agent for plants grown in soil under nonsterile conditions. 3d). washing and detection were performed according to the instruction manual of Alkphos Direct Labeling and Detection System with CDP-Star (Amersham Biosciences. was the minimum amount of carbohydrate used in the experiments for the maintenance of onion cultures. 3b).8% agarose gel and transferred onto Hybond-N nylon membranes as per manufacturer’s instructions (Amersham Biosciences. the osmotic equivalent of 20 g l−1 sucrose. 2). 3c). There was no significant difference between Agrobacterium and biolistic transformation. ◦. since most of the untransformed callus was filtered earlier. Upon transfer to long-day conditions.tamar. Different concentration of mannose (2. Filters were hybridized with probe as template using Alkphos Direct Labeling Reagent (Amersham Biosciences. Results Response of callus to mannose concentration A mannose dose response curve revealed that. www1. 2 1.5 0. Ten grams per liter mannose. In some callus clumps. Selection for pmi positive plants Mannose. mannose selection on soil-grown onion was investigated. 20) on average gram fresh weight (gFW) when cultured with different sucrose concentration. The toxic effect of mannose decreases with increasing sucrose concentrations in the medium.75 0. 10. the percentage somatic embryos forming plantlets was much less with no significant difference between Agrobacterium and biolistic. At the end of 19th week. somatic embryos and embryo-like structures were formed (Fig. The mix of 10 g l−1 mannose with 10 g l−1 sucrose decreased the tissue growth by 42% as compared to normal 20 g l−1 sucrose. later they were hardened in green house for 1 week.com).com). For selection of transformants.75 1. www1. In further subcultures. a white and nodular type with no apparent structures and a watery and transparent type. For transformation.amershambiosciences. the RNA solutions were used without reverse transcriptase. 2 The effect of mannose concentration (g l−1 ) (•. and 72◦ C for 30 s. ManA gene was amplified using the above indicated primers according to the following cyclic conditions: one cycle at 47◦ C for 30 min and 94◦ C for 5 min followed by 30 cycles at 94◦ C for 1 min. 60◦ C for 30 s. Compact callus was prominent at the site of the apex whereas friable callus was abundantly produced at the other end.25 0 0 5 10 15 20 sucrose g/l Mannose 0 Mannose 5 Mannose 10 Mannose 15 Mannose 20 Fig. As early as 1 week after the transfer of callus to regeneration medium. multiple embryos were formed (Fig. 3e). like phosphinothricin. 5.5 1. after which necrosis and mortality were recorded. 15. . Value represents means±SE Callus multiplication and regeneration Callus formation from seedling radicle could be observed after 4–5 days of culture. Ten grams per liter mannose was found to starve the tissue within the first 2 weeks of exposure. Both plasmids were restricted with HindIII and Kpn1 and isolated using the QIAquick Gel Extraction Kit. These somatic embryos germinated and developed into plantlets.com).

0c 0. The percentage was calculated based on total number of callus clumps. For Agrobacterium and biolistic transformation. hatched area represents the explant. respectively. The experiment was repeated 20 times. Note the untransformed callus starving.5a 17.5c 95. Mean separation in column by Duncan’s multiple range tests. In the negative controls of both Agrobacterium and biolistic transformation. it was frequently observed that as the structures enlarged.5a SE’s forming plantlets at the end of 19th week (%) 22.47 shoots per callus clump was observed in both transformed and non-transformed treatments.2c 90. c Somatic embryos under MS with 5 µM kinetin.5a Callus proliferated Callus forming at the end of 9th somatic embryos week (%) (SE’s) at the end 15th week (%) 33b 27b 2. while in the 9th week 1–2% of the untransformed callus starved but .4b 23. f Selection at the end of the 15th week under MS with 5 µM 2.0c 45. d Plantlets developed from somatic embryos. Plantlets could be readily induced to form roots by transfer to half-strength MS medium without growth regulators. 1 µM ABA and 10 g l−1 mannose.0c 65. proliferation and regeneration of onion callus under different periods of selections Callus survived at the end of 7th week (%) Agrobacterium Biolistic Negative control (Agrobacterium) Negative control (biolistic) Positive control 27b 23b 3. additional shoot meristem regions were produced raising the number of shoots than would be expected from the initial number of embryogenic structures within the culture (data not shown).1c 2.5a Note. 3 Plant regeneration via somatic embryogenesis in onion.7c 4.0c 0.5b 23.4-D and 10 g l−1 mannose In positive control. 20 and 9 callus clumps were taken each time. e Co-cultured callus at the end of the 7th week. a Germinated seed showing radicle. An average of 3.96 Table 1 Effect of Agrobacterium and biolistic-mediated transformation on survival. b Compact callus derived from seedling radicle used for transformation.4b 0.7b 0. 3–4% callus proliferated at the end of the 7th week. Within the column figure followed by same letter do not differ significantly at P≤0.05 Fig.

lanes 1–6 transformed plants through Agrobacterium (lanes 1–3) and biolistic (lanes 4–6) mediated transformation still survived. Selection on plates requires purified and expensive phosphinothricin. Consequently. Discussion A reproducible and stable transformation system for onion has been developed using Agrobacterium tumefaciens and biolistic approach with 6-week-old callus induced from seedling radical. Lane C control plant. Callus growth continued when mannose was added with sucrose at higher concentrations. which is a carbon source. A common feature among transformation studies in onion was the use of immature embryos as target explants due to their excellent morphogenetic competence (Eady et al. b RT-PCR analysis for the leaf tissue of transgenic onion plants for pmi gene. (2001) used mature embryos. which is consistent with the findings of Wright et al. 1992). (1987) and Scott et al. Though hygromycin is moderately expensive and works well both in media and soil it is not recommended because of its high toxicity to humans (Altmann et al. In addition it does not lead to necrotic cells. lanes 1–12 transformed plants through Agrobacterium (lanes 1–6) and biolistic (lanes 7–12) mediated transformation. while control shoots failed to produce any roots. Selection for pmi-positive onion in soil We observed negligible effect with the spray of the lowest concentrations (2. 4b).97 ever 50 mM of mannose effectively killed non-transformed plants within 10 days of spray. Klein et al. ranged from 40 to 100%. Phosphinothricin is an excellent marker for soil-grown plants. whereas the transgenic pmi-expressing plants developed a normal root system. (2001) and Negrotto et al. pmi confers a positive advantage to plants grown in tissue culture using mannose. In onion Eady and Lister (1998) demonstrated that kanamycin was ineffective up to 200 mg l−1 and used 50–100 mg l−1 geneticin. Unlike most selectable markers. 4 Molecular analysis of transgenics. Lane P1 plasmid from pNOV 2819 used for Agrobacterium transformation. 1996. 1992). and is very cheap. which is in no way inferior to other explants with an added advantage of easy extraction and zero contamination. 4a). lanes 1–5 transformed plants through Agrobacterium (lanes 1–3) and biolistic (lanes 4–5) mediated transformation. perhaps the least expensive among all agents (Todd and Tague 2001). No band was produced in the negative control (lane N in Fig. (2002) who observed reduction in shoot regeneration capacity in Fig. However. which is tedious to remove and requires stereomicroscope to identify the shoot apex portion. This is the first report on the use of mannose as a selection index in onion. 10 g l−1 mannose was used to screen for transgenic plants which can root normally. 4c). However. However contamination rate was also high in epidermis. lane P2 plasmid pNOV2820 used for biolistic transformation. RT-PCR transgenic plants yielded the expected 514-bp band while untransformed control did not have the band (Fig. lane N negative control without reverse transcriptase. it was in contrast with Kim et al. and 10 mM) of mannose. c Southern hybridization analysis of genomic DNA derived from putative transgenic plants. even 20–50 mg l−1 hygromycin in onion resulted in few escapes (Eady and Lister 1998). 5. while the transformed plants were completely resistant. Lane C untransformed control. Mannose selection of resistant plants works equally well in soil and in plates. How- . Zheng et al. which may release toxic compounds and is non-toxic to humans. whereas previously only kanamycin and hygromycin were used. Southern hybridization following EcoRI digestion produced a single band for DNA from the transformed plantlets and none for the control (Fig. the starving of tissue was observed within 2 weeks. respectively. Confirmation of transformation PCR amplification of manA gene showed that all the plants except untransformed control produced bands of an expected size of 514 bp for the pmi fragment at the same position as the binary vector positive control (Fig. they reported that the contamination rate. and phosphinothricin. which probably arose from infected embryos. 2000). (2000). 2000). When onion callus was exposed to 10 g l−1 mannose without sucrose. hygromycin. (1999) used epidermal tissues in onion with high velocity microprojectiles resulting in transient expression of chloromphenicol acetyl transferase and green fluorescent protein gene. 4b). We have used seedling radicle. The most common selective protocols for plant transformation are the use of kanamycin. a PCR analysis of transgenics for pmi gene. a large number of escapes were noticed during selection on media containing 10–30 mg l−1 phosphinothricin (Eady et al. Additionally. Kanamycin is not recommended for spray. In the rooting test (data not shown) rooting of wild type shoots was totally inhibited by 10 g l−1 mannose. as it is very expensive (Altmann et al.

In our studies. indicating the integration of one copy of the pmi gene. Negrotto et al. the mannose selection system using Agrobacterium was reported to result in 10-fold higher transformation frequencies as compared to kanamycin selection (Joersbo et al. The average transformation frequency was 25. 4c) showed integration of gene at one locus while polymorphism of hybridization pattern among plants bombarded by gene gun (plants 4. Kim HS.0%. Beaupere D. Choi SH.. Plant Cell Rep 18:117–121 Eady CC. (2001) also observed mean frequencies of 45% for maize and 20% for wheat using biolistic transformation. Phytochemistry 40:1623–1628 Kim JY. (2000) recovered frequency of 30% in maize plants using Agrobacterium transformation. Further spray of 50 mM mannose to plants grown in pots perfected the selection without any escapes. Harn CH (2002) A new selection system for pepper regeneration by mannose. Plant Cell Rep 17:734–741 Eady CC. Switzerland for sparing the pmi construct. In sugar beet. Wolf ED. Negrotto et al. Hence for onion transformation both approaches can be used. Physiol Plant 111:269–272 Joersbo M. Lister CE (1998) A comparison of four selective agents for use with Allium Cepa L. The lower frequency by earlier workers was also due to large contamination of explants. Schaper D. if the right gene constructs are delivered into the right tissue and selected with a non-controversial selection marker like mannose. Plant Cell Rep 16:219–221 Joubert P. Snagwan Norereel BS (1995) Influence of phenolic compounds on Agrobacterium vir gene induction and onion gene transfer. while 3 g l−1 inhibited regeneration in sugar beet totally (Joersbo et al. where RNA solutions was used without reverse transcriptase indicating that the RT-PCR bands from transgenic lines were produced from the mRNA of pmi.98 pepper with the addition of sucrose to mannose at higher concentration. Jung M. while Kim et al. Willmitzer L. We would like to acknowledge Dr Tim Conner for critically reviewing the manuscript. 5. Plant Cell Rep 19:376–381 Joersbo M (2001) Advances in the selection of transgenic plants using non-antibiotic marker genes. Sangwan RS. Halfter U.5% using immature embryo with selection agent geneticin (Eady et al. Suo Y. pp 310–330 Barandiaran X. Brunstedt J. immature embryos and immature embryo-derived cultures. Morric PC (1992) Protoplast transformation and methods to create specific mutants in Arabidopsis thaliana. for the financial support under ‘Brain pool project’ and ICAR. Suo YY (1996) Transient expression of uidA constructs in in vitro onion (Allium cepa L. still allowed a low frequency of organogenesis in cassava (Zhang and Puonti-Kaerlas 2000). Acknowledgements We thank the Korean Federation of Science and Technology. Min BW. Methods in Arabidopsis research. Lee YH. and 6) indicated random integration. 4b). Mol Breed 4:111–117 Joersbo M. Lim YP. Sanford JC (1987) High velocity microprojectiles for delivering nucleic acids in to living cells. and 3 of Fig. Martin J. Okkels FT (1998) Analysis of mannose selection used for transformation of sugar beet.95% using hygromycin (Zheng et al. The results indicated that. Damm B. Petersen SG. Even though bombarded callus recorded better transformation than Agrobacterium there was no statistical difference between the two methods. We also thank Syngenta seeds. This indicated that transformation frequency on mannose selection varies with each crop. Wright et al. and continuing of the mannose during rooting might have caused distortion of untransformed cells and virtually eliminated all escapes. Nature 327:70–73 . Di Pietro A (1998) Biolistic transfer and expression of a uidA reporter gene in different tissues of Allium sativum L. Schell J (eds). Agrobacteriummediated transformed plants (plants 1. RT-PCR transgenic plants yielded the expected 514-bp fragment while untransformed control did not have the fragment. 2000). Conclusion Callus induction using MS basal medium supplemented with 5 µM 2.4-D from seedling radicle. Initial selection of callus in MS medium with 10 g l−1 mannose and 10 g l−1 sucrose and the subsequent selection with only 10 g l−1 mannose prevented the production of escapes. (2000) set a combination of 5 g l−1 sucrose and 10 g l−1 mannose as the selection index for maize transformation. Weld JJ. References Altmann T. it should be possible to produce safer transgenic onions. the prolonged exposure of escaped calli to high mannose without any sucrose for 2 weeks. Wu R. for the study leave for C. (2001) utilized only mannose (10 g l−1 ) and a mannose (7 g l−1 ) +sucrose (3 g l−1 ) combination during callus selection regeneration in maize and wheat transformation. The mannose concentration of 20 g l−1 but not 30 g l−1 .). Lister CE.) cultures following particle bombardment and Agrobacteriummediated DNA delivery. (2002) recommended 20 g l−1 sucrose and 15 g l−1 or more of mannose (up to 50 g l−1 ) in pepper transformation. further exposure of 8 weeks in regeneration media. 2001) and 2. Republic of Korea. Government of India. In: Koncz C. however in cassava. NJ. Aswath. Lister CE (2000) Agrobacterium tumefaciens mediated transformation and transgenic-plant regeneration of onion (Allium Cepa L. which is a 10-fold higher than the herbicidal selection of previous workers.0%). Donaldson I. and shoot regeneration via somatic embryogenesis using MS medium with 5 µM Kinetin and 1 µM ABA has been demonstrated to be a very effective procedure for both micro propagation and transformation studies. 1998). No fragment was produced in the negative control (lane N in Fig. Pietro AD. Kreiberg J. World Scientific Publishing Co. Shoot regeneration from resistance calli initiated from mature embryo of onion following Agrobacterium-mediated transformation occurred at a lower frequency of 1. Aouad MEA. which was lower than that reported for non-transformed calli using similar plant growth regulators (95. Chua N-H. Yang SG. Okkels T (1996) A novel principle for selection of transgenic plant cells: positive selection. Wright et al. respectively. J Plant Biotechnol 4(3):129–134 Klein TM. the efficiency of hygromycin selection was about 2-fold higher than that of mannose selection-using PEGmediated particle bombardment (Zhang and Puonti-Kaerlas 2000). Southern blot analysis of EcoRI restriction digests of genomic DNA from transgenic plants using pmi probe revealed one band in the transgenic lines. 1998). River Edge. Plant Cell Rep 15:958–962 Eady CC. 2.

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