PlantCeU Reports
Plant Cell Reports (1991) 9:535-538
9 Springer-Verlag1991
Transformation of Vicia narbonensis via Agrobacterium-mediated gene transfer Thomas Pickardt, Martin Meixner, Verena Schade, and Otto Schieder Institut ffir Angewandte Genetik, Freie Universit~t Berlin, Albrecht-Thaer-Weg 6, W-t000 Berlin 33, FRG Received March 27, 1990/Revisedversion received October 15, 1990 - Communicated by H. L6rz
ABSTRACT Shoot tips and epicotyl-segments of Vicia narbonensis were co-cultivated with Agrobacterium tumefaciens strain C58C 1 pGV 3850 Htrl", carrying a plasmid coding for hygmmycinphosphotransferase. On callus-induction medium containing 60 mg/1 hygromycin for selection, approximately 18% of the explants produced hygromycin-resistant callus. After transfer to regeneration-medium these calluses produced hygromycin-resistant and nopaline-positive somatic embryos which could be regenerated to plantlets. The integration of the T-DNA into the plant genome was confirmed by Southern analysis. ABBREVIATIONS: NAA=naphthyl-acetic acid; BAP=6-benzylaminopurine; HPT=hygromycin-phosphotransferase; MS=Murashige and Skoog; CaMV=cauliflower mosaic virus; nos=nopalinersynthase; nop=nopaline; hyg=hygromycin; SDS=sodium dodecyl sulfate
INTRODUCTION The success of any gene transfer method depends on a) the susceptibility of the target cells and b) on the availability of a regeneration procedure for the transformed tissue. In many grain legumes the application of gene transfer techniques has been limited by the inability to regenerate intact plants from transformed cells. Reproducible regeneration systems via somatic embryogenesis or organogenesis were developed for soybean by Lazzeri et al. (1985), Ranch et al. (1985), Barwale et al. (1986) and Wright et al. (1987) by using young tissue like immature cotyledons or cotyledonary nodes as starting material. Though immature cells are considered to have a very low amenability to Agrobacterium-infection (Hildebrand
Offprint requests to: T. Pickardt
1934, Kupila-Ahvenniemi & Therman 1968, Parrot et al. 1988), Hinchee et al. (1988) and Parrot et al. (1989) could show, that some of the above mentioned regeneration procedures could successfully be combined with Agrobacteriummediated gene transfer. Also in pea (Pisum sativum) transgenic plants could be obtained after co-cultivation of young epicotyl- and nodal-segments with A.tumefaciens (Puonti-Kaerlas et al. 1990, De Kathen & Jacobsen 1990). In a previous paper (Pickardt et al. 1989) we described a protocol for the induction of embryogenic cultures from young shoot tips in Vicia narbonensis, a close relative of the faba-bean (Viciafaba). The protocol consists of the following steps: a) excised shoot-tips on MS-medium (Murashige and Skoog 1962) containing picloram in the range of 1-10 mg/1 for callus-induction (auxinPlUS-phase) b) transfer to MS-medium supplemented with 1 mg/1 NAA (auxinminus-phase/somatic embryos arising) c) transfer of somatic embryos to MS-medium with cytokinins ("germination") Our results presented here indicate that this regeneration protocol can sucessfully be combined with Agrobacteriummediated gene transfer.
MATERIALS AND METHODS Plant Material: Seeds of Vicia narbonensis L. var. narbonensis (originally obtained from G. Donn/Hoechst-Frankfurt/FRG) were surface sterilized by immersion for 1 min in 70% (v/v) ethanol and for 10 rain in 3% (v/v) sodium hypochlorite solution with a few drops of Tween 80 added, After three washes seeds were soaked in sterile tap water for 12 hours and subsequently placed on water agar for germination in the dark. The shoot-tips and epicotyls of 2-3 day old etiolated seedlings were used in transformation experiments.
536
Bacterial strain: Inoculations were made with A. tumefaciens strain C58C1 carrying the disarmed plasmid pGV3850 HPT. This cointegrate vector contains the coding sequence for hygromycin-phosphotransferasedriven by the CaMV 35S promoter and the nopaline synthase-gene with the nos-promoter (A. Kato and F. Kreuzaler, unpublished results). For inoculation of plant cells bacteria were grown overnight at 28~ in YEB medium (Van Larebeke et al. 1977). Inoculation procedure: Small segments (2-4 mm) from epicotyls and shoot tips of 2-3 day old etiolated seedlings were placed into 200 ml glass-jars containing 30 ml of liquid MS-medium with 4 rag/1 picloram (Dow chemical) and precultured on a rotary shaker (120 rpm) at 25~ (preconditioning-phase). After 24 hours 0.3 ml of an overnight culture of A.tumefaciens was added. The cultures were coincubated on a rotary shaker (120 rpm) at 25~ for 48 hours. The segments were then washed three times with sterile distilled water and transferred to solidified MS-medium (0.25% gelrite/Nevco) containing 4 rag/1 picloram and 0.5 g/1 claforan. As controls, segments of shoot-tips and epicotyls were treated as described above, but without A.tumefaciens co-cultivation. Selection of transformed callus: After 48 hours explants were transferred to solidified (gelrite) MS-medium with 4 rag/1 picloram (auxinPlUS-medium),30 mg/l hygromycin, 0.5 g/1 claforan and placed in the dark at 25~ Cultures remained on this medium for a period of 4 weeks. They were then transferred to fresh medium (auxinpluS/as stated above), but with an increased hygromycin-concentration of 60 mg/1 and a reduced claforan-concentration of 0.2 g/1. Cultures were maintained on this medium for a period of 3 months (subculture every 4 weeks). Hygromycin-resistant callus (putative transformants) was subsequently transferred to MS-medium with 1 mg/l NAA and a reduced sucrose level of 1.5% (auxinmmUS-medium),40 mg/l hygromycin, 0.1 g/I claforan, solidified with gelrite. Calluses were continuously subcultured on this medium every 4 weeks (claforan was omitted from the second period on). Somatic embryos arising from hygromycin resistant tissue were placed on MS-medium containing 0.1 mg/l NAA and 0.03 mg/1 each of BAP, kinetin and zeatin (modified BKZNmedium, Lazzeri et al. 1985), 20 mg/1 hygromycin, solidified with gelrite. Somatic embryos from non-transformed callus served as controls. Embryos developing primary leaflets were transferred to MSmedium (solidified with gelrite) containing 2 rag/1 BAP and 20 mg/1 hygromycin resulting in continuous proliferation of shoot primordia under selection pressure. Single shoot buds derived from these cultures were placed on MS without hormones for shoot elongation and root development. With the exception of the auxinpluS-phase (in the dark) all
cultures were kept at 25~ under cool white fluorescent lights (3000 - 5000 lux) with a 16 h photoperiod. All media were adjusted to pH 5.7 prior to autoclaving.
Opine analysis: Nos-activity was determined as described by Reynaerts et al. 1988. Non-transformed callus was used as control and synthetic nopaline/arginine as standards. Southern blot analysis: Total DNA from A.tumefaciens GV 3850 HPT and plant DNA from putative transformants as well as untransformed shoot cukures as control (1-1.5g of tissue) was isolated according to Rogers and Bendich (1985) with modifications. The DNA was digested with HindlII (2 unitsAtgDNA) for 4h at 37~ electrophoretically separated on 0.8% agarose gels (2.5 V/cm) and transferred to compasmembrane (Genofit/Switzerland) by vacuum blotting9 The filters were probed with the 3.2kb/H23-fragment (right border probe) from pGV 3850 HFF (kindly provided by Thomas Altmann/IGF-Berlin). The probe was labeled to a specific activity of 3x109 dpm/I.tgDNA (5x107 cpm/ml) according to Feinberg & Vogelstein (1983). Hybridization was performed for 18h at 60~ in 5x SSC (Maniatis et al. 1982), 5x Denhardtsolution, 0.5% SDS and 100 g/ml sheared salmon sperm DNA. Films were exposed at -70~ C for 72h.
RESULTS AND DISCUSSION In two independent experiments approximately 2500 epicotyl/shoot tip-segments were cocultivated with A.tumefaciensstrain GV3850 HPT and transferred to hygromycin-mediafor selection. As control, segments were treated as above without bacterial inoculation. The in vitro protocol was generally maintained as described above (see Introduction), except for the following changes: For better selection the 4-week auxinPlUS-phasewas prolonged to a total of 4 x 4 weeks. It was assumed that this treatment leads to a reduction in number of the non-transformed, embryogenic cells in the following auxinr'UnUs-phase,where regeneration of somatic embryos occurs. 9 plus Initially the hygromycin concentration in the auxin -phase was kept lower (30 mg/1) in order to prevent excessive death of non-transformed cells, which is known to lead to a high toxicity level for the developing transformed cells within the callus. In the following subculture the hygromycin concentration was increased to 60 mg/l and kept at this level for the whole auxinpluS-phase, During the auxinplus -phase the control tissue on hygromycinmedium exhibited extremely poor proliferation and browning of the tissue. In approximately 20% of the A.tumefaciens-treated explants, however, the development of a whitish, slowgrowing callus could be observed (Fig. 1). After a total of 4 months in the auxinPlUS-phasethese calluses were transferred
537
9
r
+
Fig.5 Southem analyses of 5 hyg/hop -clones. Total DNA from GV 3850 HPT (lane 1), untransformed shoot-cultures (lane 2) and putative transformed clones (lanes 3-7) was digested with HindKI and probed with the H23-fragment (3.2 kb-fight hand probe) from pGV 3850 HPT. The H23probe is expected to hybridize to HindIII-fragments extending across the T-DNA borderinto theplant genome. In all 5 transformants the hybridizing fragments are of different size, indicating that the T-DNA is integrated at different locations in the genome. All tested transformants contain single inserts. A weak signalat 2.6kb is present in lanes 2-7, obviously due to the presence of loci in V.narbonensis with partial homology to the probe.
Fig.1 Selection on auxin+medium: treated (a) and untreated (control/b) material on 60 mg/1 hygromycin Fig.2 Shoot bud proliferating cultures (derived from a single embryo) on MS + 2mg/1 BAP and 20 rag/1 hygromycin Fig.3 nop+-shoot on root induction medium Fig.4 Nopaline-production of shoot bud proliferating clones (s = nopaline/arginine standard, c = control, no. 1-12 = hygr-clones)
to auxinmmUS-medium.In order to improve the conditions for the regeneration of somatic embryos, the selection pressure was reduced by lowering the hygromycin concentration to 40 mg/1. The cultures were scored after three weeks in the auxinminusphase (somatic embryos had not yet started to arise from the tissue macroscopically). A total of 438 of the treated explants (approx. 18%) formed well-growing callus; from 44 of these putative transformants 41 proved to be nopaline-positive (Table 1). After two subcultures on auxinminUS-medium somatic embryos developed from the calluses. For "germination" they were transferred to MS-medium supplemented with 0.1 mg/l NAA and 0.03 mg/1 each of BAP, kinetin and zeatin, and 20 mg/1 hygromycin. Within 2 - 3 weeks several embryos
developed green primary leaflets, whereas somatic embryos derived from non-transformed tissue failed to undergo any further development. A total number of 142 embryos were collected from the putative transformexl calluses during the auxinrmnUs-phase, from which 26 were able to grow in the presence of hygromycin. The low number of regenerants as compared to the results previously published (Pickardt et al. 1989) could be possibly explained by a) the difference in the length of the auxinpluS-phase(4 weeks vs. 16 weeks) and b) the presence of two antibiotics (claforan and hygromycin). The low number of hygromycin-resistant embryos (26 from 142) can be explained through the occurrence of crossfeeding between transformed and non-transformed embryogenic tissue. Also the inactivation of information during the redifferentiation process would be another explanation. The direct regeneration of intact plants from somatic embryos of V. narbonensis can not yet be done with high efficiency (approximately 1%). Therefore we first stimulated shoot primordia proliferation with a majority of embryos on MS-medium supplemented with 1 mg/1BAP under selection pressure (20 mg/1 hygromycin), which led to large numbers of shootsystems per embryonic clone (Fig.2). Excised shoot buds derived from these cultures can be regenerated to plantlets via shoot elongation on MS-medium without hormones and subsequent rooting (Figo3).The efficiencyis not higher compared to the above mentioned "direct" regeneration from somatic Table 1 Response of explants after co-cultivation with A.tumefaciens GV
3850 HPT no. of no. of co-cultivated hygr explants calli app.2500
438
no. of no. of no. of no. of nop* nop*calli/ somatic hygr embryos/ total tested embryos embryos total tested 41/44
142
26
7/12
538 embryos, but the material is not lost after unsucessful regeneration and the procedure can be repeated several times. At present 12 shoot bud proliferating clones (i.e. each derived from one single embryo) are being cultivated on 20 mg/1 hygromycin. From these clones 7 are nopaline-positive (Fig.4). A southem analysis of 5 nopalin-positive clones is shown in Fig.5. The H23-probe is expected to hybridize to HindlII-fragments extending across the T-DNA border into the plant genome. In all tested transformants the hybridizing fragments are of different size, indicating that the T-DNA is integrated at different locations in the genome. The absence of signals at 3.2kb in all transformants shows that the tissue does not contain any surviving Agrobacteria. Unfortunately, up to now all rooted shoots from these clones failed to undergo further development. Experiments are in progress to optimize the conditions for regeneration under selection pressure. At the moment the outlined experimental protocol will not yield high numbers of transformedplants in a short time. The protocol, however, will possibly provide transgenic V.narbonensis Plants which are a prerequisite for gene transfer studies between Vicia-species (via protoplast fusion) in future breeding programs.
Feinberg AP, Vogelstein B (1983) Anal. Biochem. 132:6-13 Hildebrand EM (1934) J. Agric. Res.48:857-885 Hinchee MAW, Connor-Ward DV, Newell CA, McDonnell RE, Sato SJ, Gasser CS, Fischhoff DA, Re DB, Fraley RT, Horsch RB (1988) Biotechnology 6:915-921 Kupila-Ahvenniemi S, Therman E (1968) Adv. Morphogenesis 7:45-78 Lazzeri PA, Hildebrand DF, Collins GB (1985) Plant Molec.Biol.Reporter 3:160-167 Maniatis T, Fritsch EF, Sambrook J (1982) in: Molecular Cloning, a Laboratory Manual. Cold Spring Harbour, NY Murashige T, Skoog F (1962) Physiol. Plant. 15:473-497 Parrott WA, Hoffman LM, Hildebrand DF, Williams EG, Collins GB (1989) Plant Cell Rep. 7:615-617 Parrott WA, Williams EG, Hildebrand DF, Collins GB (1988) Newsletter IAPTC 54:10-19 Pickardt T, Huancaruna Perales E, Schieder O (1989) Protoplasma 149:5-10 Puonti-Kaerlas J, Eriksson T, Engstr0m P (1990) Theor. Appl. Genet. 80:246-252
Acknowledgements This work was supported by the Bundesministerium fiir Forschung und Technologie/FRG. The authors wish to thank Lothar Willmitzer for providing GV 3850 Ht~ and Thomas Altmann for providing the H23-fragment.
REFERENCES Barwale UB, Kerns HR, Widholm JM (1986) Planta 167:473481 De Kathen A, Jacobsen HJ (1990) Plant Cell Rep.9:276-279
Ranch JP, Oglesby L, Zielinski AC (1985) In Vitro Cell.& Dev.Biol. 21:653 -657 Rogers SO, Bendich AJ (1985) Plant Mol. Biol.5:69-76 Reynaerts A, De Block M, Hernalsteens JP, Van Montagu M (1988) Plant Mol. Biol. Manual A9:1-16 Van Larebeke N, Genetello CH, Hemalsteens JP, De Picker A, Zaenen I, Messens E, Van Montagu M, ScheU J (1977) Mol. Gen. Genet. 152:119-124 Wright MS, Ward DV, Hinchee MA, Cames MG, Kaufman RJ (1987) Plant Cell Rep. 6:83-89
Plant Cell Reports (1991) 9:535-538
9 Springer-Verlag1991
Transformation of Vicia narbonensis via Agrobacterium-mediated gene transfer Thomas Pickardt, Martin Meixner, Verena Schade, and Otto Schieder Institut ffir Angewandte Genetik, Freie Universit~t Berlin, Albrecht-Thaer-Weg 6, W-t000 Berlin 33, FRG Received March 27, 1990/Revisedversion received October 15, 1990 - Communicated by H. L6rz
ABSTRACT Shoot tips and epicotyl-segments of Vicia narbonensis were co-cultivated with Agrobacterium tumefaciens strain C58C 1 pGV 3850 Htrl", carrying a plasmid coding for hygmmycinphosphotransferase. On callus-induction medium containing 60 mg/1 hygromycin for selection, approximately 18% of the explants produced hygromycin-resistant callus. After transfer to regeneration-medium these calluses produced hygromycin-resistant and nopaline-positive somatic embryos which could be regenerated to plantlets. The integration of the T-DNA into the plant genome was confirmed by Southern analysis. ABBREVIATIONS: NAA=naphthyl-acetic acid; BAP=6-benzylaminopurine; HPT=hygromycin-phosphotransferase; MS=Murashige and Skoog; CaMV=cauliflower mosaic virus; nos=nopalinersynthase; nop=nopaline; hyg=hygromycin; SDS=sodium dodecyl sulfate
INTRODUCTION The success of any gene transfer method depends on a) the susceptibility of the target cells and b) on the availability of a regeneration procedure for the transformed tissue. In many grain legumes the application of gene transfer techniques has been limited by the inability to regenerate intact plants from transformed cells. Reproducible regeneration systems via somatic embryogenesis or organogenesis were developed for soybean by Lazzeri et al. (1985), Ranch et al. (1985), Barwale et al. (1986) and Wright et al. (1987) by using young tissue like immature cotyledons or cotyledonary nodes as starting material. Though immature cells are considered to have a very low amenability to Agrobacterium-infection (Hildebrand
Offprint requests to: T. Pickardt
1934, Kupila-Ahvenniemi & Therman 1968, Parrot et al. 1988), Hinchee et al. (1988) and Parrot et al. (1989) could show, that some of the above mentioned regeneration procedures could successfully be combined with Agrobacteriummediated gene transfer. Also in pea (Pisum sativum) transgenic plants could be obtained after co-cultivation of young epicotyl- and nodal-segments with A.tumefaciens (Puonti-Kaerlas et al. 1990, De Kathen & Jacobsen 1990). In a previous paper (Pickardt et al. 1989) we described a protocol for the induction of embryogenic cultures from young shoot tips in Vicia narbonensis, a close relative of the faba-bean (Viciafaba). The protocol consists of the following steps: a) excised shoot-tips on MS-medium (Murashige and Skoog 1962) containing picloram in the range of 1-10 mg/1 for callus-induction (auxinPlUS-phase) b) transfer to MS-medium supplemented with 1 mg/1 NAA (auxinminus-phase/somatic embryos arising) c) transfer of somatic embryos to MS-medium with cytokinins ("germination") Our results presented here indicate that this regeneration protocol can sucessfully be combined with Agrobacteriummediated gene transfer.
MATERIALS AND METHODS Plant Material: Seeds of Vicia narbonensis L. var. narbonensis (originally obtained from G. Donn/Hoechst-Frankfurt/FRG) were surface sterilized by immersion for 1 min in 70% (v/v) ethanol and for 10 rain in 3% (v/v) sodium hypochlorite solution with a few drops of Tween 80 added, After three washes seeds were soaked in sterile tap water for 12 hours and subsequently placed on water agar for germination in the dark. The shoot-tips and epicotyls of 2-3 day old etiolated seedlings were used in transformation experiments.
536
Bacterial strain: Inoculations were made with A. tumefaciens strain C58C1 carrying the disarmed plasmid pGV3850 HPT. This cointegrate vector contains the coding sequence for hygromycin-phosphotransferasedriven by the CaMV 35S promoter and the nopaline synthase-gene with the nos-promoter (A. Kato and F. Kreuzaler, unpublished results). For inoculation of plant cells bacteria were grown overnight at 28~ in YEB medium (Van Larebeke et al. 1977). Inoculation procedure: Small segments (2-4 mm) from epicotyls and shoot tips of 2-3 day old etiolated seedlings were placed into 200 ml glass-jars containing 30 ml of liquid MS-medium with 4 rag/1 picloram (Dow chemical) and precultured on a rotary shaker (120 rpm) at 25~ (preconditioning-phase). After 24 hours 0.3 ml of an overnight culture of A.tumefaciens was added. The cultures were coincubated on a rotary shaker (120 rpm) at 25~ for 48 hours. The segments were then washed three times with sterile distilled water and transferred to solidified MS-medium (0.25% gelrite/Nevco) containing 4 rag/1 picloram and 0.5 g/1 claforan. As controls, segments of shoot-tips and epicotyls were treated as described above, but without A.tumefaciens co-cultivation. Selection of transformed callus: After 48 hours explants were transferred to solidified (gelrite) MS-medium with 4 rag/1 picloram (auxinPlUS-medium),30 mg/l hygromycin, 0.5 g/1 claforan and placed in the dark at 25~ Cultures remained on this medium for a period of 4 weeks. They were then transferred to fresh medium (auxinpluS/as stated above), but with an increased hygromycin-concentration of 60 mg/1 and a reduced claforan-concentration of 0.2 g/1. Cultures were maintained on this medium for a period of 3 months (subculture every 4 weeks). Hygromycin-resistant callus (putative transformants) was subsequently transferred to MS-medium with 1 mg/l NAA and a reduced sucrose level of 1.5% (auxinmmUS-medium),40 mg/l hygromycin, 0.1 g/I claforan, solidified with gelrite. Calluses were continuously subcultured on this medium every 4 weeks (claforan was omitted from the second period on). Somatic embryos arising from hygromycin resistant tissue were placed on MS-medium containing 0.1 mg/l NAA and 0.03 mg/1 each of BAP, kinetin and zeatin (modified BKZNmedium, Lazzeri et al. 1985), 20 mg/1 hygromycin, solidified with gelrite. Somatic embryos from non-transformed callus served as controls. Embryos developing primary leaflets were transferred to MSmedium (solidified with gelrite) containing 2 rag/1 BAP and 20 mg/1 hygromycin resulting in continuous proliferation of shoot primordia under selection pressure. Single shoot buds derived from these cultures were placed on MS without hormones for shoot elongation and root development. With the exception of the auxinpluS-phase (in the dark) all
cultures were kept at 25~ under cool white fluorescent lights (3000 - 5000 lux) with a 16 h photoperiod. All media were adjusted to pH 5.7 prior to autoclaving.
Opine analysis: Nos-activity was determined as described by Reynaerts et al. 1988. Non-transformed callus was used as control and synthetic nopaline/arginine as standards. Southern blot analysis: Total DNA from A.tumefaciens GV 3850 HPT and plant DNA from putative transformants as well as untransformed shoot cukures as control (1-1.5g of tissue) was isolated according to Rogers and Bendich (1985) with modifications. The DNA was digested with HindlII (2 unitsAtgDNA) for 4h at 37~ electrophoretically separated on 0.8% agarose gels (2.5 V/cm) and transferred to compasmembrane (Genofit/Switzerland) by vacuum blotting9 The filters were probed with the 3.2kb/H23-fragment (right border probe) from pGV 3850 HFF (kindly provided by Thomas Altmann/IGF-Berlin). The probe was labeled to a specific activity of 3x109 dpm/I.tgDNA (5x107 cpm/ml) according to Feinberg & Vogelstein (1983). Hybridization was performed for 18h at 60~ in 5x SSC (Maniatis et al. 1982), 5x Denhardtsolution, 0.5% SDS and 100 g/ml sheared salmon sperm DNA. Films were exposed at -70~ C for 72h.
RESULTS AND DISCUSSION In two independent experiments approximately 2500 epicotyl/shoot tip-segments were cocultivated with A.tumefaciensstrain GV3850 HPT and transferred to hygromycin-mediafor selection. As control, segments were treated as above without bacterial inoculation. The in vitro protocol was generally maintained as described above (see Introduction), except for the following changes: For better selection the 4-week auxinPlUS-phasewas prolonged to a total of 4 x 4 weeks. It was assumed that this treatment leads to a reduction in number of the non-transformed, embryogenic cells in the following auxinr'UnUs-phase,where regeneration of somatic embryos occurs. 9 plus Initially the hygromycin concentration in the auxin -phase was kept lower (30 mg/1) in order to prevent excessive death of non-transformed cells, which is known to lead to a high toxicity level for the developing transformed cells within the callus. In the following subculture the hygromycin concentration was increased to 60 mg/l and kept at this level for the whole auxinpluS-phase, During the auxinplus -phase the control tissue on hygromycinmedium exhibited extremely poor proliferation and browning of the tissue. In approximately 20% of the A.tumefaciens-treated explants, however, the development of a whitish, slowgrowing callus could be observed (Fig. 1). After a total of 4 months in the auxinPlUS-phasethese calluses were transferred
537
9
r
+
Fig.5 Southem analyses of 5 hyg/hop -clones. Total DNA from GV 3850 HPT (lane 1), untransformed shoot-cultures (lane 2) and putative transformed clones (lanes 3-7) was digested with HindKI and probed with the H23-fragment (3.2 kb-fight hand probe) from pGV 3850 HPT. The H23probe is expected to hybridize to HindIII-fragments extending across the T-DNA borderinto theplant genome. In all 5 transformants the hybridizing fragments are of different size, indicating that the T-DNA is integrated at different locations in the genome. All tested transformants contain single inserts. A weak signalat 2.6kb is present in lanes 2-7, obviously due to the presence of loci in V.narbonensis with partial homology to the probe.
Fig.1 Selection on auxin+medium: treated (a) and untreated (control/b) material on 60 mg/1 hygromycin Fig.2 Shoot bud proliferating cultures (derived from a single embryo) on MS + 2mg/1 BAP and 20 rag/1 hygromycin Fig.3 nop+-shoot on root induction medium Fig.4 Nopaline-production of shoot bud proliferating clones (s = nopaline/arginine standard, c = control, no. 1-12 = hygr-clones)
to auxinmmUS-medium.In order to improve the conditions for the regeneration of somatic embryos, the selection pressure was reduced by lowering the hygromycin concentration to 40 mg/1. The cultures were scored after three weeks in the auxinminusphase (somatic embryos had not yet started to arise from the tissue macroscopically). A total of 438 of the treated explants (approx. 18%) formed well-growing callus; from 44 of these putative transformants 41 proved to be nopaline-positive (Table 1). After two subcultures on auxinminUS-medium somatic embryos developed from the calluses. For "germination" they were transferred to MS-medium supplemented with 0.1 mg/l NAA and 0.03 mg/1 each of BAP, kinetin and zeatin, and 20 mg/1 hygromycin. Within 2 - 3 weeks several embryos
developed green primary leaflets, whereas somatic embryos derived from non-transformed tissue failed to undergo any further development. A total number of 142 embryos were collected from the putative transformexl calluses during the auxinrmnUs-phase, from which 26 were able to grow in the presence of hygromycin. The low number of regenerants as compared to the results previously published (Pickardt et al. 1989) could be possibly explained by a) the difference in the length of the auxinpluS-phase(4 weeks vs. 16 weeks) and b) the presence of two antibiotics (claforan and hygromycin). The low number of hygromycin-resistant embryos (26 from 142) can be explained through the occurrence of crossfeeding between transformed and non-transformed embryogenic tissue. Also the inactivation of information during the redifferentiation process would be another explanation. The direct regeneration of intact plants from somatic embryos of V. narbonensis can not yet be done with high efficiency (approximately 1%). Therefore we first stimulated shoot primordia proliferation with a majority of embryos on MS-medium supplemented with 1 mg/1BAP under selection pressure (20 mg/1 hygromycin), which led to large numbers of shootsystems per embryonic clone (Fig.2). Excised shoot buds derived from these cultures can be regenerated to plantlets via shoot elongation on MS-medium without hormones and subsequent rooting (Figo3).The efficiencyis not higher compared to the above mentioned "direct" regeneration from somatic Table 1 Response of explants after co-cultivation with A.tumefaciens GV
3850 HPT no. of no. of co-cultivated hygr explants calli app.2500
438
no. of no. of no. of no. of nop* nop*calli/ somatic hygr embryos/ total tested embryos embryos total tested 41/44
142
26
7/12
538 embryos, but the material is not lost after unsucessful regeneration and the procedure can be repeated several times. At present 12 shoot bud proliferating clones (i.e. each derived from one single embryo) are being cultivated on 20 mg/1 hygromycin. From these clones 7 are nopaline-positive (Fig.4). A southem analysis of 5 nopalin-positive clones is shown in Fig.5. The H23-probe is expected to hybridize to HindlII-fragments extending across the T-DNA border into the plant genome. In all tested transformants the hybridizing fragments are of different size, indicating that the T-DNA is integrated at different locations in the genome. The absence of signals at 3.2kb in all transformants shows that the tissue does not contain any surviving Agrobacteria. Unfortunately, up to now all rooted shoots from these clones failed to undergo further development. Experiments are in progress to optimize the conditions for regeneration under selection pressure. At the moment the outlined experimental protocol will not yield high numbers of transformedplants in a short time. The protocol, however, will possibly provide transgenic V.narbonensis Plants which are a prerequisite for gene transfer studies between Vicia-species (via protoplast fusion) in future breeding programs.
Feinberg AP, Vogelstein B (1983) Anal. Biochem. 132:6-13 Hildebrand EM (1934) J. Agric. Res.48:857-885 Hinchee MAW, Connor-Ward DV, Newell CA, McDonnell RE, Sato SJ, Gasser CS, Fischhoff DA, Re DB, Fraley RT, Horsch RB (1988) Biotechnology 6:915-921 Kupila-Ahvenniemi S, Therman E (1968) Adv. Morphogenesis 7:45-78 Lazzeri PA, Hildebrand DF, Collins GB (1985) Plant Molec.Biol.Reporter 3:160-167 Maniatis T, Fritsch EF, Sambrook J (1982) in: Molecular Cloning, a Laboratory Manual. Cold Spring Harbour, NY Murashige T, Skoog F (1962) Physiol. Plant. 15:473-497 Parrott WA, Hoffman LM, Hildebrand DF, Williams EG, Collins GB (1989) Plant Cell Rep. 7:615-617 Parrott WA, Williams EG, Hildebrand DF, Collins GB (1988) Newsletter IAPTC 54:10-19 Pickardt T, Huancaruna Perales E, Schieder O (1989) Protoplasma 149:5-10 Puonti-Kaerlas J, Eriksson T, Engstr0m P (1990) Theor. Appl. Genet. 80:246-252
Acknowledgements This work was supported by the Bundesministerium fiir Forschung und Technologie/FRG. The authors wish to thank Lothar Willmitzer for providing GV 3850 Ht~ and Thomas Altmann for providing the H23-fragment.
REFERENCES Barwale UB, Kerns HR, Widholm JM (1986) Planta 167:473481 De Kathen A, Jacobsen HJ (1990) Plant Cell Rep.9:276-279
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