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Transgenic superroots of Lotus corniculatus can be regenerated from superroot-derived leaves following Agrobacterium-mediated transformation
ARTICLE IN PRESS Journal of Plant Physiology 165 (2008) 1313—1316
www.elsevier.de/jplph
SHORT COMMUNICATION
Transgenic superroots of Lotus corniculatus can be regenerated from superroot-derived leaves following Agrobacterium-mediated transformation Hidenori Tanakaa, Jun Toyamaa, Masatsugu Hashiguchib, Yasuyo Kutsunac, Shin-ichi Tsurutac, Ryo Akashib,, Franz Hoffmannd a
JST Innovation Satellite Miyazaki, Japan Science and Technology Agency, 1-1 Gakuen Kibanadai Nishi, Miyazaki 889-2192, Japan b Frontier Science Research Center, University of Miyazaki, Miyazaki 889-2192, Japan c Department of Biological Production and Environmental Science, University of Miyazaki, Miyazaki 889-2192, Japan d Department of Developmental and Cell Biology, University of California, Irvine, CA 92697-2300, USA Received 15 February 2008; received in revised form 13 March 2008; accepted 13 March 2008
KEYWORDS Agrobacterium tumefaciens; b-Glucuronidase; Lotus corniculatus; Superroots; Transformation
Summary Super-growing roots (superroots; SR), which have been established in the legume species Lotus corniculatus, are a fast-growing root culture that allows continuous root cloning, direct somatic embryogenesis and mass regeneration of plants under entirely growth regulator-free culture conditions. These features are unique for nonhairy root cultures, and they are now stably expressed since the culture was isolated more than 10 years ago (1997). Attempts to achieve direct and stable transformation of SR turned out to be unsuccessful. Making use of the supple regeneration plasticity of SR, we are reporting here an indirect transformation protocol. Leaf explants, derived from plants regenerated from SR, were inoculated with Agrobacterium tumefaciens strain LBA4404 harboring the binary vector pBI121, which contains the neomycin phosphotransferase II (NPTII) and b-glucuronidase (GUS) genes as selectable and visual markers, respectively. After co-cultivation, the explants were selected on solidified MS medium with 0.5 mg/L benzylamino purine (BAP), 100 mg/L kanamycin and 250 mg/L cefotaxime. Kanamycin-resistant calli were transferred to liquid rooting medium. The newly regenerated, kanamycin-resistant roots were harvested and SR cultures re-established, which exhibited all the characteristics of the original SR. Furthermore, kanamycin-resistant roots cultured onto solidified MS medium supplemented with 0.5 mg/L BAP produced plants at the same rate as control SR. Six months after gene transfer, PCR analysis and histochemical locating indicated that the NPTII gene was integrated into the genome and that the GUS gene
Abbreviations: GUS; b-glucuronidase; NBRP; National BioResource Project; SR; Superroots. Corresponding author. Tel./fax: +81 985 58 7257. E-mail address: [email protected] (R. Akashi). 0176-1617/$ - see front matter & 2008 Elsevier GmbH. All rights reserved. doi:10.1016/j.jplph.2008.03.003
ARTICLE IN PRESS 1314
H. Tanaka et al. was regularly expressed in leaves, roots and nodules, respectively. The protocol makes it now possible to produce transformed SR and nodules as well as transgenic plants from transformed SR. & 2008 Elsevier GmbH. All rights reserved.
Introduction
Materials and methods
Super-growing roots (superroots; SR) of the allopolyploid legume Lotus corniculatus (bird’s-foot trefoil) are a fast-growing root culture that allows continuous root cloning, direct somatic embryogenesis and mass regeneration of plants under entirely hormone-free culture conditions (Akashi et al., 1998a, 2003). The complete regeneration cycle, from primary root culture to plant formation and re-initiation of SR from regenerated plants, works on a single culture medium. The developmental switch is environmentally controlled, primarily by light. Furthermore, plants can also be obtained from isolated SR protoplasts (Akashi et al., 2000). The SR features are unique for non-hairy root cultures and are now stably expressed since the culture was isolated more than 10 years ago (1997) without showing signs of genetic or epigenetic instability. Sustained growth of cultured roots can also be achieved through genetic manipulation with Agrobacterium rhizogenes (Chilton et al., 1982; reviewed by Veena and Taylor, 2007). However, the resulting hairy roots are developmentally altered and represent a pathological state. SR do not show developmental aberrations. They are distributed world-wide through legumebase, the National BioResource Project (NBRP), University of Miyazaki, Japan (http://www.legumebase.agr.miyazaki-u.ac.jp/index.jsp), together with seeds of the diploid Lotus model species L. japonicus (Japanese trefoil, Ma ´rquez, 2005; review by Sato and Tabata, 2006). Highly efficient transformation protocols for L. japonicus are available (Stiller et al., 1997; Lohar et al., 2001), forming the basis for the successful application of this species in legume research. Direct stable transformation of SR turned out to be unsuccessful in numerous attempts with multiple strategies, leaving the SR system severely incomplete. Making use of the supple regeneration plasticity of SR, we established an indirect root transformation system via leaf explants. The efficiency of the successful protocol suggests its suitability for the production of gain-of-function genotypes (Ichikawa et al., 2006; Fujita et al., 2007).
The original SR culture (Akashi et al., 1998a) of bird’sfoot trefoil (L. corniculatus L.) cv. Viking is routinely maintained in 100-mL Erlenmeyer flasks containing 20 mL hormone-free MS medium (Murashige and Skoog, 1962) of pH 6.8 on a rotary shaker (110 rpm) at 27 1C in the dark. Periodically, deteriorated tissues are mechanically removed. Subcultures are started from 4-week-old cultures by harvesting the apical 1–2 cm of secondary roots and placing 20 such root explants into fresh hormone-free MS medium. For the production of target tissue, 4-week-old SR cultures were transferred to light under stationary conditions. Regenerated shoots were separated from the roots and placed into plastic containers with 30 mL half-strength hormone-free MS medium solidified with 0.7% agar. After 4 weeks on this medium, leaves, stems and roots of regenerated plants were separately tested for best regeneration on solidified MS medium with 0.3% gellan gum (Wako, Osaka, Japan) and 0.5 mg/L of benzylamino purine (BAP; Sigma, St. Louis, MO, USA). For transformation, Agrobacterium tumefaciens strain LBA4404, harboring the binary vector pBI121 (Clontech, Palo Alto, CA, USA), was used. The vector contains the bglucuronidase (GUS) gene linked to the cauliflower mosaic virus 35S promoter and the nopaline synthase (NOS) terminator. A neomycin phosphotransferase II gene (NPTII), conferring kanamycin tolerance, is linked to the NOS promoter and NOS terminator. Agrobacterium was grown for 24 h in LB medium, consisting of 1.0% tryptone, 1.0% NaCl and 0.5% yeast extract with 300 mg/L streptomycin (Wako) and 25 mg/L kanamycin (Wako). Petiolules were removed from leaflets (1 cm long) of regenerated plants and 2 mm explants cut off from both ends of the leaflets. In total, 919 explants were used in three independent experiments. The explants were immersed in the bacterial culture for 30 min at room temperature, briefly dried between blotting paper and transferred to solidified MS medium with 0.5 mg/L of BAP and 100 mg/L acetosyringone, and incubated at room temperature. After 7 d, the explants were rinsed thrice with sterilized distilled water to remove bacteria, blotted and transferred to solidified MS medium with 0.5 mg/L BAP, 100 mg/L kanamycin and 250 mg/L cefotaxime (Sanofi Aventis, Tokyo, Japan). Explants were subcultured to fresh medium every 2–3 weeks. After 90 d, calli were transferred to MS liquid medium with 0.5 mg/L a-naphthylacetic acid (NAA; Sigma) and 10 mg/L kanamycin, and incubated at 27 1C in the dark for 2 weeks to regenerate roots. The resultant roots were transferred to hormone-free MS liquid medium and subcultured as described for SR above. Alternatively, after 4 weeks on
ARTICLE IN PRESS Transgenic superroots of Lotus corniculatus
1315
hormone-free MS, tips of roots (0.5–1 cm) were transferred to solidified MS medium with 0.5 mg/L BAP. After 4 weeks, the regenerated shoots were placed onto halfstrength solidified MS medium for plant regeneration. PCR analysis was performed for confirmation of integration of the foreign genes. DNA was extracted using the ISOPLANT DNA isolation kit (Wako) and PCRs were carried out in a thermal cycler (GeneAmp 9700; Applied Biosystems, Foster City, CA, USA) as described previously (Akashi et al., 1998b).
Results and discussion Leaf, stem and root explants derived from regenerated shoots (Figure 1a) and plants (Figure 1b) were tested for their regeneration capability. Frequency of shoot formation and elongation (Figure 1c–e) was highest (87% and 8.2 shoots/explant) from leaf-derived explants (Table 1). Based on this preliminary experiment, we used leaves as explant source for the following transformation study. After 7 d of co-cultivation of leaf explants with Agrobacterium, explants were transferred to selection
Figure 1. Production of transgenic superroots (SR) and plants of Lotus corniculatus through Agrobacterium-mediated transformation. (a–b) Plant regeneration from SR. (c–d) Callus induced from leaf explants cultured for 3 and 6 weeks, respectively, on shooting medium. (e) Petri plate with shoot-producing callus after 8 weeks. (f) Resistant callus on MS medium containing 100 mg/L kanamycin. (g) Stable GUS expression in kanamycin-resistant callus. (h) Root regeneration from kanamycin-resistant calli in liquid medium supplemented with 10 mg/L kanamycin. (i) Transgenic superroots displaying GUS expression. (j) GUS activity in regenerated plants, (k) leaflets, (l) roots and (m) nodules. (n) Detection of NPTII gene in transgenic plants by PCR (M, DNA marker; Co, non-transformed plant; 1–8, transgenic plants; P, plasmid pBI121). Table 1.
Shoot regeneration from different explants taken from superroot-derived plantlets of Lotus corniculatus
Explant type
Number of explants cultureda (A)
Number of explants with shoots (B)
Percent of explants with shoots (B/A, %)
Number of regenerated shoots (C)
Number of regenerated shoots per explant (C/B)
Leaf Stem Root
46 42 42
40 36 20
87.0 85.7 47.6
328 264 86
8.2 7.3 4.3
a
Approximately 90% of all explants showed callus formation.
ARTICLE IN PRESS 1316 medium. Callus became visible at the cuts of 56 leaf segments (sample size 919) 50 d after transfer (Figure 1f). Regenerated calli showed the same regeneration vigor as the controls, and 18 tolerant callus lines expressed the GUS gene (Figure 1g). The first kanamycin-tolerant shoots emerged after 30 additional days. This is similar to previously reported data on Agrobacterium-mediated transformation in wild-type (non-SR) bird’s-foot trefoil (Akashi et al., 1998b). Kanamycin-resistant calli were transferred to liquid MS medium with 0.5 mg/L NAA and 10 mg/L kanamycin for root formation. After 10–14 d in culture, roots formed on small calli (Figure 1h) and grew vigorously. Cultures initiated from callus-derived root explants showed all characteristics of SR again and remained GUS-positive (Figure 1i). These cultures are true transgenic SR, which could not be generated through direct root transformation. By making use of the supple regeneration plasticity of SR, we established an indirect SR transformation system. As typical for SR (Figure 1a), the newly established transgenic SR cultures also showed multiple shoot formation (Figure 1j) after transfer to light. To induce caulogenesis, hormone-free medium is sufficient (Akashi et al., 1998a). The addition of BAP speeds up the procedure. Histochemical localizing of GUS activity in randomly selected plants indicated that the gene is regularly expressed in leaves including petiolules, leafstalk and stems (Figure 1k), roots (Figure 1l) and nodules (Figure 1m). Control plants did not display any GUS activity under microscopical analysis. Leaves of transgenics showed consistently a high activity, which is in agreement with results obtained with wild-type plants (Akashi et al., 1998b). Stable integration of the NPTII gene was confirmed by PCR. A 1.4 kbp fragment was amplified and identified as NPTII (Figure 1n). Six months after Agrobacteriummediated transformation (co-culture), PCR-confirmed GUS and NPTII double transformants were potted in soil, acclimatized and transferred to the greenhouse. The transgenic plants looked normal and flowered. The A. tumefaciens-based transformation protocol reported here for SR of L. corniculatus takes advantage of the simplicity and efficiency of the SR regeneration system, allowing the production of transgenic SR, nodules and plants. Transformation rate and regenerability
H. Tanaka et al. suggest that the system should be suitable for the production of gain-of-function genotypes using the FOX hunting system (Full-length cDNA Over-eXpressing gene hunting system) described by Ichikawa et al. (2006) for Arabidopsis. Attempts to produce FOX lines of SR are in progress. The lines will be added to the NBRP collection and made available through legumebase. The transgenic SR system could also be useful as a plant expression factory (Lie ´nard et al., 2007) and could be further improved through the use of bioreactor culture (Eibl and Eibl, 2007).
References Akashi R, Hoffmann-Tsay S-S, Hoffmann F. Theor Appl Genet 1998a;96:758–64. Akashi R, Uchiyama T, Sakamoto A, Kawamura O, Hoffmann F. J Plant Physiol 1998b;152:84–91. Akashi R, Harris S, Hoffmann-Tsay S-S, Hoffmann F. J Plant Physiol 2000;157:215–21. Akashi R, Kawano T, Hashiguchi M, Kutsuna Y, HoffmannTsay S-S, Hoffmann F. Plant Soil 2003;255:27–33. Chilton MD, Tepfer DA, Petit A, David C, Casse-Delbart, Tempe´ J. Nature 1982;295:432–4. Eibl R., Eibl D. Phytochem Rev 2007; in press, doi:10.1007/s11101-007-9083-z. Fujita M, Mizukado S, Fujita Y, Ichikawa T, Nakazawa M, Seki M, et al. Biochem Biophys Res Commun 2007;364:250–7. Ichikawa T, Nakazawa M, Kawashima M, Iizumi H, Kuroda H, Kondou Y, et al. Plant J 2006;48:974–85. Lie ´nard D, Sourrouille C, Gomord V, Faye L. Biotechnol Annu Rev 2007;13:115–47. Lohar DP, Schuller K, Buzas DM, Gresshoff PM, Stiller J. J Exp Bot 2001;52:1697–702. Ma ´rquez AJ, editor. Lotus japonicus handbook. Dordrecht: Springer; 2005. Murashige T, Skoog F. Physiol Plant 1962;15:473–97. Sato S, Tabata S. Curr Opin Plant Biol 2006;9:128–32. Stiller J, Martirani L, Tuppale S, Chian R-J, Chiurazzi M, Gresshoff PM. J Exp Bot 1997;48:1357–65. Veena V, Taylor CG. In Vitro Cell Dev Biol Plant 2007;43:383–403.
www.elsevier.de/jplph
SHORT COMMUNICATION
Transgenic superroots of Lotus corniculatus can be regenerated from superroot-derived leaves following Agrobacterium-mediated transformation Hidenori Tanakaa, Jun Toyamaa, Masatsugu Hashiguchib, Yasuyo Kutsunac, Shin-ichi Tsurutac, Ryo Akashib,, Franz Hoffmannd a
JST Innovation Satellite Miyazaki, Japan Science and Technology Agency, 1-1 Gakuen Kibanadai Nishi, Miyazaki 889-2192, Japan b Frontier Science Research Center, University of Miyazaki, Miyazaki 889-2192, Japan c Department of Biological Production and Environmental Science, University of Miyazaki, Miyazaki 889-2192, Japan d Department of Developmental and Cell Biology, University of California, Irvine, CA 92697-2300, USA Received 15 February 2008; received in revised form 13 March 2008; accepted 13 March 2008
KEYWORDS Agrobacterium tumefaciens; b-Glucuronidase; Lotus corniculatus; Superroots; Transformation
Summary Super-growing roots (superroots; SR), which have been established in the legume species Lotus corniculatus, are a fast-growing root culture that allows continuous root cloning, direct somatic embryogenesis and mass regeneration of plants under entirely growth regulator-free culture conditions. These features are unique for nonhairy root cultures, and they are now stably expressed since the culture was isolated more than 10 years ago (1997). Attempts to achieve direct and stable transformation of SR turned out to be unsuccessful. Making use of the supple regeneration plasticity of SR, we are reporting here an indirect transformation protocol. Leaf explants, derived from plants regenerated from SR, were inoculated with Agrobacterium tumefaciens strain LBA4404 harboring the binary vector pBI121, which contains the neomycin phosphotransferase II (NPTII) and b-glucuronidase (GUS) genes as selectable and visual markers, respectively. After co-cultivation, the explants were selected on solidified MS medium with 0.5 mg/L benzylamino purine (BAP), 100 mg/L kanamycin and 250 mg/L cefotaxime. Kanamycin-resistant calli were transferred to liquid rooting medium. The newly regenerated, kanamycin-resistant roots were harvested and SR cultures re-established, which exhibited all the characteristics of the original SR. Furthermore, kanamycin-resistant roots cultured onto solidified MS medium supplemented with 0.5 mg/L BAP produced plants at the same rate as control SR. Six months after gene transfer, PCR analysis and histochemical locating indicated that the NPTII gene was integrated into the genome and that the GUS gene
Abbreviations: GUS; b-glucuronidase; NBRP; National BioResource Project; SR; Superroots. Corresponding author. Tel./fax: +81 985 58 7257. E-mail address: [email protected] (R. Akashi). 0176-1617/$ - see front matter & 2008 Elsevier GmbH. All rights reserved. doi:10.1016/j.jplph.2008.03.003
ARTICLE IN PRESS 1314
H. Tanaka et al. was regularly expressed in leaves, roots and nodules, respectively. The protocol makes it now possible to produce transformed SR and nodules as well as transgenic plants from transformed SR. & 2008 Elsevier GmbH. All rights reserved.
Introduction
Materials and methods
Super-growing roots (superroots; SR) of the allopolyploid legume Lotus corniculatus (bird’s-foot trefoil) are a fast-growing root culture that allows continuous root cloning, direct somatic embryogenesis and mass regeneration of plants under entirely hormone-free culture conditions (Akashi et al., 1998a, 2003). The complete regeneration cycle, from primary root culture to plant formation and re-initiation of SR from regenerated plants, works on a single culture medium. The developmental switch is environmentally controlled, primarily by light. Furthermore, plants can also be obtained from isolated SR protoplasts (Akashi et al., 2000). The SR features are unique for non-hairy root cultures and are now stably expressed since the culture was isolated more than 10 years ago (1997) without showing signs of genetic or epigenetic instability. Sustained growth of cultured roots can also be achieved through genetic manipulation with Agrobacterium rhizogenes (Chilton et al., 1982; reviewed by Veena and Taylor, 2007). However, the resulting hairy roots are developmentally altered and represent a pathological state. SR do not show developmental aberrations. They are distributed world-wide through legumebase, the National BioResource Project (NBRP), University of Miyazaki, Japan (http://www.legumebase.agr.miyazaki-u.ac.jp/index.jsp), together with seeds of the diploid Lotus model species L. japonicus (Japanese trefoil, Ma ´rquez, 2005; review by Sato and Tabata, 2006). Highly efficient transformation protocols for L. japonicus are available (Stiller et al., 1997; Lohar et al., 2001), forming the basis for the successful application of this species in legume research. Direct stable transformation of SR turned out to be unsuccessful in numerous attempts with multiple strategies, leaving the SR system severely incomplete. Making use of the supple regeneration plasticity of SR, we established an indirect root transformation system via leaf explants. The efficiency of the successful protocol suggests its suitability for the production of gain-of-function genotypes (Ichikawa et al., 2006; Fujita et al., 2007).
The original SR culture (Akashi et al., 1998a) of bird’sfoot trefoil (L. corniculatus L.) cv. Viking is routinely maintained in 100-mL Erlenmeyer flasks containing 20 mL hormone-free MS medium (Murashige and Skoog, 1962) of pH 6.8 on a rotary shaker (110 rpm) at 27 1C in the dark. Periodically, deteriorated tissues are mechanically removed. Subcultures are started from 4-week-old cultures by harvesting the apical 1–2 cm of secondary roots and placing 20 such root explants into fresh hormone-free MS medium. For the production of target tissue, 4-week-old SR cultures were transferred to light under stationary conditions. Regenerated shoots were separated from the roots and placed into plastic containers with 30 mL half-strength hormone-free MS medium solidified with 0.7% agar. After 4 weeks on this medium, leaves, stems and roots of regenerated plants were separately tested for best regeneration on solidified MS medium with 0.3% gellan gum (Wako, Osaka, Japan) and 0.5 mg/L of benzylamino purine (BAP; Sigma, St. Louis, MO, USA). For transformation, Agrobacterium tumefaciens strain LBA4404, harboring the binary vector pBI121 (Clontech, Palo Alto, CA, USA), was used. The vector contains the bglucuronidase (GUS) gene linked to the cauliflower mosaic virus 35S promoter and the nopaline synthase (NOS) terminator. A neomycin phosphotransferase II gene (NPTII), conferring kanamycin tolerance, is linked to the NOS promoter and NOS terminator. Agrobacterium was grown for 24 h in LB medium, consisting of 1.0% tryptone, 1.0% NaCl and 0.5% yeast extract with 300 mg/L streptomycin (Wako) and 25 mg/L kanamycin (Wako). Petiolules were removed from leaflets (1 cm long) of regenerated plants and 2 mm explants cut off from both ends of the leaflets. In total, 919 explants were used in three independent experiments. The explants were immersed in the bacterial culture for 30 min at room temperature, briefly dried between blotting paper and transferred to solidified MS medium with 0.5 mg/L of BAP and 100 mg/L acetosyringone, and incubated at room temperature. After 7 d, the explants were rinsed thrice with sterilized distilled water to remove bacteria, blotted and transferred to solidified MS medium with 0.5 mg/L BAP, 100 mg/L kanamycin and 250 mg/L cefotaxime (Sanofi Aventis, Tokyo, Japan). Explants were subcultured to fresh medium every 2–3 weeks. After 90 d, calli were transferred to MS liquid medium with 0.5 mg/L a-naphthylacetic acid (NAA; Sigma) and 10 mg/L kanamycin, and incubated at 27 1C in the dark for 2 weeks to regenerate roots. The resultant roots were transferred to hormone-free MS liquid medium and subcultured as described for SR above. Alternatively, after 4 weeks on
ARTICLE IN PRESS Transgenic superroots of Lotus corniculatus
1315
hormone-free MS, tips of roots (0.5–1 cm) were transferred to solidified MS medium with 0.5 mg/L BAP. After 4 weeks, the regenerated shoots were placed onto halfstrength solidified MS medium for plant regeneration. PCR analysis was performed for confirmation of integration of the foreign genes. DNA was extracted using the ISOPLANT DNA isolation kit (Wako) and PCRs were carried out in a thermal cycler (GeneAmp 9700; Applied Biosystems, Foster City, CA, USA) as described previously (Akashi et al., 1998b).
Results and discussion Leaf, stem and root explants derived from regenerated shoots (Figure 1a) and plants (Figure 1b) were tested for their regeneration capability. Frequency of shoot formation and elongation (Figure 1c–e) was highest (87% and 8.2 shoots/explant) from leaf-derived explants (Table 1). Based on this preliminary experiment, we used leaves as explant source for the following transformation study. After 7 d of co-cultivation of leaf explants with Agrobacterium, explants were transferred to selection
Figure 1. Production of transgenic superroots (SR) and plants of Lotus corniculatus through Agrobacterium-mediated transformation. (a–b) Plant regeneration from SR. (c–d) Callus induced from leaf explants cultured for 3 and 6 weeks, respectively, on shooting medium. (e) Petri plate with shoot-producing callus after 8 weeks. (f) Resistant callus on MS medium containing 100 mg/L kanamycin. (g) Stable GUS expression in kanamycin-resistant callus. (h) Root regeneration from kanamycin-resistant calli in liquid medium supplemented with 10 mg/L kanamycin. (i) Transgenic superroots displaying GUS expression. (j) GUS activity in regenerated plants, (k) leaflets, (l) roots and (m) nodules. (n) Detection of NPTII gene in transgenic plants by PCR (M, DNA marker; Co, non-transformed plant; 1–8, transgenic plants; P, plasmid pBI121). Table 1.
Shoot regeneration from different explants taken from superroot-derived plantlets of Lotus corniculatus
Explant type
Number of explants cultureda (A)
Number of explants with shoots (B)
Percent of explants with shoots (B/A, %)
Number of regenerated shoots (C)
Number of regenerated shoots per explant (C/B)
Leaf Stem Root
46 42 42
40 36 20
87.0 85.7 47.6
328 264 86
8.2 7.3 4.3
a
Approximately 90% of all explants showed callus formation.
ARTICLE IN PRESS 1316 medium. Callus became visible at the cuts of 56 leaf segments (sample size 919) 50 d after transfer (Figure 1f). Regenerated calli showed the same regeneration vigor as the controls, and 18 tolerant callus lines expressed the GUS gene (Figure 1g). The first kanamycin-tolerant shoots emerged after 30 additional days. This is similar to previously reported data on Agrobacterium-mediated transformation in wild-type (non-SR) bird’s-foot trefoil (Akashi et al., 1998b). Kanamycin-resistant calli were transferred to liquid MS medium with 0.5 mg/L NAA and 10 mg/L kanamycin for root formation. After 10–14 d in culture, roots formed on small calli (Figure 1h) and grew vigorously. Cultures initiated from callus-derived root explants showed all characteristics of SR again and remained GUS-positive (Figure 1i). These cultures are true transgenic SR, which could not be generated through direct root transformation. By making use of the supple regeneration plasticity of SR, we established an indirect SR transformation system. As typical for SR (Figure 1a), the newly established transgenic SR cultures also showed multiple shoot formation (Figure 1j) after transfer to light. To induce caulogenesis, hormone-free medium is sufficient (Akashi et al., 1998a). The addition of BAP speeds up the procedure. Histochemical localizing of GUS activity in randomly selected plants indicated that the gene is regularly expressed in leaves including petiolules, leafstalk and stems (Figure 1k), roots (Figure 1l) and nodules (Figure 1m). Control plants did not display any GUS activity under microscopical analysis. Leaves of transgenics showed consistently a high activity, which is in agreement with results obtained with wild-type plants (Akashi et al., 1998b). Stable integration of the NPTII gene was confirmed by PCR. A 1.4 kbp fragment was amplified and identified as NPTII (Figure 1n). Six months after Agrobacteriummediated transformation (co-culture), PCR-confirmed GUS and NPTII double transformants were potted in soil, acclimatized and transferred to the greenhouse. The transgenic plants looked normal and flowered. The A. tumefaciens-based transformation protocol reported here for SR of L. corniculatus takes advantage of the simplicity and efficiency of the SR regeneration system, allowing the production of transgenic SR, nodules and plants. Transformation rate and regenerability
H. Tanaka et al. suggest that the system should be suitable for the production of gain-of-function genotypes using the FOX hunting system (Full-length cDNA Over-eXpressing gene hunting system) described by Ichikawa et al. (2006) for Arabidopsis. Attempts to produce FOX lines of SR are in progress. The lines will be added to the NBRP collection and made available through legumebase. The transgenic SR system could also be useful as a plant expression factory (Lie ´nard et al., 2007) and could be further improved through the use of bioreactor culture (Eibl and Eibl, 2007).
References Akashi R, Hoffmann-Tsay S-S, Hoffmann F. Theor Appl Genet 1998a;96:758–64. Akashi R, Uchiyama T, Sakamoto A, Kawamura O, Hoffmann F. J Plant Physiol 1998b;152:84–91. Akashi R, Harris S, Hoffmann-Tsay S-S, Hoffmann F. J Plant Physiol 2000;157:215–21. Akashi R, Kawano T, Hashiguchi M, Kutsuna Y, HoffmannTsay S-S, Hoffmann F. Plant Soil 2003;255:27–33. Chilton MD, Tepfer DA, Petit A, David C, Casse-Delbart, Tempe´ J. Nature 1982;295:432–4. Eibl R., Eibl D. Phytochem Rev 2007; in press, doi:10.1007/s11101-007-9083-z. Fujita M, Mizukado S, Fujita Y, Ichikawa T, Nakazawa M, Seki M, et al. Biochem Biophys Res Commun 2007;364:250–7. Ichikawa T, Nakazawa M, Kawashima M, Iizumi H, Kuroda H, Kondou Y, et al. Plant J 2006;48:974–85. Lie ´nard D, Sourrouille C, Gomord V, Faye L. Biotechnol Annu Rev 2007;13:115–47. Lohar DP, Schuller K, Buzas DM, Gresshoff PM, Stiller J. J Exp Bot 2001;52:1697–702. Ma ´rquez AJ, editor. Lotus japonicus handbook. Dordrecht: Springer; 2005. Murashige T, Skoog F. Physiol Plant 1962;15:473–97. Sato S, Tabata S. Curr Opin Plant Biol 2006;9:128–32. Stiller J, Martirani L, Tuppale S, Chian R-J, Chiurazzi M, Gresshoff PM. J Exp Bot 1997;48:1357–65. Veena V, Taylor CG. In Vitro Cell Dev Biol Plant 2007;43:383–403.