Plant Cell Reports
Plant Cell Reports (1996) 16:6-11
© Springer-Verlag1996
Rapid transformation of Medicago truncatula " regeneration via shoot organogenesis Anthony T. Trieu and Maria J. Harrison
The Samuel Roberts Noble Foundation, Plant Biology Division, 2510 Sam Noble Parkway, Ardmore, Oklahoma 73401, USA Received 14 May t996/Revised version received 12 June 1996 - Communicated by J. M. Widholm
Summary. A rapid transformation and regeneration system has been developed for M. truncatula cv Jemalong (barrel medic) by which it is possible to obtain transgenic plants within 2.5 months. The procedure involves Agrobacterium-mediated transformation of cotyledon exptants coupled with the regeneration of transformed plants via direct organogenesis. To develop the procedure, M. truncatuIa explants were transformed with the binary plasmid pSLJ525 which carries the bar gene. The bar gene encodes phosphinothricin acetyl transferase, and transformed plants were selected on media containing phosphinothricin (Ignite, AgrEvo). Transformed plants show phosphinothricin acetyl transferase activity and Southern blot analysis indicates that they carry the /~czr gene integrated into their genomes. The resistance to phosphinothricin is stable and is inherited by the R1 progeny as a single dominant Mendelian trait. The transgenic plants are highly resistant to the broad spectrum herbicide, Ignite and therefore may also have commercial applications. Key words: PAT, Phosphinothricin acetyl transferase; PPT, phosphinothricin Introduction
Annual species of the genus Medicago, including Medicago truncatula Gaertn. (barrel medic) are grown as pasture legumes in a number of regions throughout the world, including mediterranean areas, South Africa and Australia (Crawford et al. 1989; Lamprecht and Knox-Davies 1984). In Australia, the annual medics are the main legume found on over 50 million hectares of agricultural land and a variety of species and ecotypes have been developed. The first commercial cultivar of M. truncatula was sown in 1938 and this species has been favoured due to its ability to tolerate both low rainfall and high lime soils (Crawford et al. 1989). Breeding and selection programs for the annual medics are ongoing and the development of genetic
Correspondence to: A. T. Trieu
transformation and regeneration methods provides an additional means to introduce agronomically important traits (Crawford et al. 1989). In addition to its agricultural importance, M. truncatula is also emerging as a useful model for the molecular-genetic investigation of two important plant-microbe associations, the Rhizobium-legume symbiosis (Pichon et al. 1992; B6naben et al. 1995; Cook et al. 1995) and the vesicular-arbuscular mycorrhizal symbiosis (Harrison and Dixon 1994; Harrison and van Buuren 1995). The diploid, selffertilising nature of this legume, coupled with a small genome, fairly rapid generation time and ability to be genetically transformed by Agrobacterium (Thomas et al.1992; Chabaud et al. 1996), make it an attractive model legume for molecular-genetic studies (Barker et al. 1990). Two protocols for transformation and regeneration of M. truncatula have been reported previously. Both of the procedures involve Agrobacterium-mediated transformation coupled with regeneration of transformed plants via somatic embryogenesis. The first procedure is extremely cultivar specific and is useful only with highly regenerable lines developed for this purpose (Thomas et al. 1992). The second method was developed for the lines more frequently used in molecular genetic studies (Chabaud et al. 1996), however the process is still lengthy and labour intensive, taking between 4 and 10 months to obtain transgenic plants. As rapid transformation procedures are essential for molecular genetic analyses we sought to improve transformation of M. truncatula and investigated the possibility o f transformation coupled with regeneration via direct organogenesis. Such protocols have recently been described for white clover (Voisey et al. 1994) and pea (Schroeder et al. 1993). Here we report a new, rapid method to transform and regenerate M. truncanda. The procedure uses Agrobacterium-mediated transformation coupled with regeneration via direct shoot organogenesis and
7 tranformed plants can be obtained in 2.5 months. We demonstrate stable transformation, expression of the introduced bar gene and inheritance of bar in a typical 3:1 Mendelian pattern. In addition, as the bar gene was used as the selectable marker, the transformed plants show resistance to the herbicide Ignite at levels significantly higher than those used in field conditions. Consequently, the transgenic plants resulting from the development of the tranformation protocol may also have commercial applications.
Materials and Methods Plant material. Medicago truncatula cv. Jemalong (line A17, T. Huguet INRA) seeds were collected from greenhouse-grown plants. Plant culture media. All media below contained PDM salts and vitamins (Chaband et al. 1996) and were adjusted to pH 5.8. Each medium also contained 0.8% agar-agar (Sigma Chemical Co., St. Louis, MO, USA) except M2A medium. M2A, M2C, and M2R all contained 15 11M BAP (6-Benzylaminopurine) and 0.5 11M NAA (ct-Napthaleneacetic acid). M2R, M3 and M4 all contained 5.0 mg/l PPT (Ignite, AgrEvo Co., Wilmington, Delaware, U.S.A.), 500rag/1 carbenicillin (Sigma Chemical Co.) and 2% sucrose. Additional components for each medium are listed below: M1 (Germination medium): 5 11M BAP and 1% (w/v) sucrose. M2A (Agrobacterium resuspension medium): 1% sucrose and 100 11M acetosyringone (3',5'-dimethoxy-4'-hydroxy acetophenone, Aldrich Chemical Co., Milwaukee, WI, U.S.A.). M2C (Co-cultivation medium): 2% sucrose and 100 11M acetosyringone. M2R (Regeneration medium): no additional components. M3 (Shoot development medium): 0.5 gM NAA. M4 (Rooting medium): 0.2mg/1 IBA (Indole-3-butyric acid). Seed sterilisation and germination. Seeds were selected and scarified by rubbing with sand paper. The seeds were then immersed in 70% (v/v) ethanol for 30 seconds, followed by gentle agitation in 20% (v/v) Clorox (commercial bleach, 5.25% NaOC1) solution plus 0.1% Tween-20 for 10 rain. Seeds were then washed 3 times with sterile water and incubated in sterile water for 1 hr at 42°C. This was followed by incubation in 111M Giberellic acid (GA 3) at room temperature for 1 hour. Seeds were then blotted dry on sterile Whatman filter paper and spread on MI seed germination medium in 15x100 nun petri dishes (Fisher Scientific Co., PA.,U.S.A.). The seeds were incubated overnight in the dark at 2325°C and then for two days under fluorescent light with a 16-h photo period (23-25°C) to allow germination to proceed. Plasmid and bacterial strains. The binary plasmid pSLJ525 (Jones et al. 1992) carries both an NPT II gene and a bar gene. The bar gene encodes phosphinothrcin acetyl transferase (PAT) which confers resistance to phosphinothricin (PPT), a component of the herbicide Ignite (AgrEvo). pSLJ525 was mobilized into Agrobacterium tumefaciens strain LBA4404 (Clontech, USA) by direct transformation (An et al. 1988). To prepare A. tumefaciens LBA4404 for transformation a single colony was used to inoculate 2mi of liquid TY medium (5g/1 tryptone, 3g/l yeast extract and 6mM CaC12.6H20 ) plus antibiotics (10mg/1 rifampicin and 5mg/l tetracycline). This culture was grown for 18 hrs at 28°C with shaking (250 RPM). The 2 ml culture was then used to inoculate 25ml of liquid TY medium with antibiotics and the resulting culture was grown under the same conditions until the OD600 reached 0.5-0.7. The cells were collected by centrifugation at 8000 RPM for 10 min at 4°C, and the bacterial pellet resuspended in equal volume of M2A liquid medium. This suspension was then used for transformation. Plant transformation and regeneration. The protocol below was developed using the bar gene as a selectable marker. Approximately 60 seedlings (3 days old) were transferred to a sterile 15x100 man p e ~ dish containing 2-3ml of A. tumefaciens LBA4404 suspension in M2A media. Seedlings were bisected at the embryonic axis and the hypocotyl was removed 1-2 mm below the cotyledonary node. Thus each explant has 1 cotyledon and 1-2 mm of the split embryonic axis. Explants were briefly blotted dry with sterile Whatman filter paper and transferred to 15xl00mm petri plates containing M2C co-cultivation medium. The explants were
oriented so that the adaxial side of the cotyledon faced up and the abaxial side was in contact with the media (25 explants/petri plate). All explants were maintained on this medium at 23-25°C for 5 days under a 16-h photo period. Following co-cultivation, explants were transferred to M2R regeneration medium in 25x100 mm petri plates (10 explants per dish). Explants were maintained on this medium for 4 weeks and were transferred to fresh medium every 10 days. At this time, resistant shoots developed and the explants were subsequently transferred to M3 shoot development medium in Magenta boxes (Magenta Corporation, Chicago, IL, USA). Resistant shoots 0.5 cm or taller were excised and transferred to M4 rooting medium. Shoots were laid horizontally on the surface of the agar. Resistant shoots developed roots within 10-15 days and had extensive root development after an additional 10 days. These plants were then transferred to soil in the greenhouse and maintained initially at high humidity. The plants flowered approximately two months after transfer to the greenhouse. M. truncatula is self-fertile and the plants were allowed to self pollinate and set seed. This seed was collected for inheritance studies.
Histology. Cotyledon explants were collected for 24 days and prepared for sectioning by standard histological procedures (Berlyn and Miksche, 1976). Sections were collected on glass slides coated with Hanpt's adhesive (Berlyn and Miksche 1976), air dried overnight, and stained with 1% toluidine blue for 30 s without removing the parafilm. After 3 rinses in distilled water, slides were air-dried overnight. The slides were rinsed twice (10 rain each time) in xylene to remove parafilm and mounted with Permount (Fisher Scientific Co., N.J., USA). Chlorophenol red (CR) assay. The CR assay was carried out according to Kramer et al. (1993) with modification for leaf tissue instead of callus. 1-3 leaflets from PPT-resistant shoots were incubated in 250 111 of CR medium in a 24-well microtitre plate (Becton Dickson Labware, NJ, USA) under fluorescent light with a 16 hr photo period for 1-4 days. CR medium contained PDM salts and vitamins, 15gM BAP, 0.5 11M NAA, 50mg/l CR (Sigma Chemical Co.,USA), 5.0 mg/1 PPT and the pH was adjusted to 6.0 at which point the medium is red in color. Untransformed leaflets and a leaf segment from a transformed tobacco plant carrying the bar gene were included as controls. Phosphinothricin acetyl transferase (PAT) assay. PAT activity was assayed as described by DeBlock et al. (1987). Four partially open trifoliates (approx 150mg of tissue) were collected in a 1.5 ml eppendorf tube and placed on ice. The tissue was ground in liquid nitrogen with a plastic pestle using an electric handrill. 150 111of extraction buffer (100mM sodium phosphate, pH 7.5, 20raM sodium chloride, ImM phenylmethylsulfonylfluoride (PMSF), and 1 mg/1 bovine serum albumin (BSA) was added to the ground tissue. The tube was briefly vortexed at high speed, then centrifuged at 13,000 RPM for 15 min at 4°C. The extract was transferred to a new tube and recentrifuged at the same speed for l0 rain. The concentration of protein in 5 111 extracts was quantified (Bio-Rad protein assay) using BSA as a standard. To prepare the reaction mixture, 2gl of i00 I.tg/111 PPT (diluted in extraction buffer) and 3tll 14C acetyl-CoA (6OmCi/inrnol, Amersham, USA) was added to 25 gg of protein in 20pl of extraction buffer. The reaction mixture was incubated 30 min at 37°C and then 12 pl was applied to a TLC plate (J.T. Baker, Inc., Phillipsburg, NJ, USA) in two 6111 applications. The spot was dried briefly with a hair dryer between applications. Ascending chromatography was carried out in 1-propanol / NH4OH (3:2) for 3 h. The TLC plate was air-dried overnight and exposed to x-ray film (Kodak X-Omat AR film, Kodak, Rochester, USA). The 14C signal was visible after 2 days. Southern blots. Genomic DNA was isolated from leaf tissue of putative transformants and untransformed M. truncatula plants, using the protocol of Dellaporta et al. (1983). For a fight border analysis DNA (10pg) was digested with HindIII, separated by gel electrophoresis and blotted to nylon membranes (GeneScreen Plus, NEN Research Products, MA, USA) according to standard procedures (Sambrook et al. 1989). The blot was hybridised with an internal 423bp fragment of the bar gene which had been labelled with 32p cc-dATP (3,000 Ci/rnmol, Dupont, NEN, USA) by random-priming (Feinberg and Vogelstein, 1983). Hybridisation was according to the protocol of Church and Gilbert (1984). The membranes were exposed to Kodak X-Omatic AR f'tim with intensifying screen for 2.5 h at -80°C. The vector pSLJ525 contains a HindlII site 1886bp from the T-DNA fight border. The bar gene is contained between the fight border and the HindIII
Fig. 1. Regeneration of M. truncatula plants. (A) Cotyledon explant, bar = lmm. (B) Shoots (s) developed primary leaves after 15 days on M2R medium, bar = lmm. This example was not on selective media. The shoots emerging from explants on selection appear similar but the explant itself begins to turn yellow and die. (C) Phosphinothricin-resistant plantlets following I0 days growth on M3 shoot development medium containing phosphinothricin, bar = lcm (D) Phosphinothricin-resistant shoots develop roots (r) after 18 days on M4 rooting medium containing phosphinothficin, bar = lcm (E) Light micrograph of longitudinal section through an explant (8 days old). Arrows indicate the developing shoot meristems which are clearly distinct from the primary shoot meristem. The section is stained with toluidine blue, bar = 200~tm.
Fig. 5. Resistance to phosphinothricin. One control plant (left) and two transformed plants (transformant 2.1 and 3.3) (right) 7 days after being sprayed with 400mg/1 phosphinothricin (Ignite). Fig. 2. Chlorophenol red assay. Wells 1-4A and 1-5B contain leaf pieces from transformed plants. Transformants 3.7, 5.1, 5.2, and 5.3 are in wells Al-4 respectively. Transformants 1.2, 3.1, 3.4, and 3.7 are in wells B1-4 respectively. A6 and B6 contain leaf pieces from non-transformed control plants. Well A5 contains chlorophenol red medium only. The plate was incubated for 4 days.
site. ThereforeDNA fromtransformedplants would be expected to contain hybridisingfragmentslargerthan 1886 bp. Results
Transformation and regeneration of PPT- resistant M. truncatula Cotyledonary explants (Fig. 1A) were co-cultivated with A. tumefaciens LBA4404 carrying the binary vector pSLJ525 and then transferred to regeneration medium containing phosphinothricin (PPT) to select for transformed shoots. Shoots began to emerge from the cut face of the cotyledon within 12 days of transfer to regeneration medium (Fig. IB). The combination of the bar gene, and the selection agent PPT, proved to be extremely efficient for the selection of transformed M. truncatula shoots. Shoots from control explants (lacking Agrobaeteria) stopped growing within 3 days of incubation on selective medium and became necrotic within 5 days. Resistant shoots were allowed to develop further on an additional shoot development medium for 15 days (Fig. 1C) and were then transferred to rooting medium. Rooting medium contains PPT and resistant roots developed within 10-15 days (Fig. 1D). The plants were subsequently transferred to the greenhouse. The TO transformed plants exhibited the normal phenotypic characteristics of wild type M. truncatuIa plants. They continued to grow and develop, and flowered approximately 2 months after transfer to the greenhouse. Fourteen plants, determined to be transformed on the basis of Southern blot and/or PAT assay, were obtained from 450 cotyledonary explants. In order to confirm the origin of the shoot meristems we prepared and stained thin sections of the cotyledon explants. Microscopic examination of the sections indicated that the shoot meristems developed de novo from the epidermal cell layer in the regions adjacent to the primary apical meristem and embryonic axis and clearly indicated that the shoots were distinct from the primary apical meristem (Fig. 1E).
alive and the medium changed colour from red to yellow. The colour change occurred within 1 to 4 days and probably indicates differences in the levels of PAT expression. The cuttings were grown without carbenicillin and did not show Agrobacterial growth. Therefore the detoxification of PPT is not due to Agrobacterium harboured in the leaf pieces. Nontransformed leaves (Fig. 2, wells 6A and 6B) were killed by exposure to PPT and the medium changed colour from red to purple.
Phosphinothricin aeetyl transferase assays. PAT assays were performed on leaf tissue from transformed plants after transfer to soil and 15 days of growth in the greenhouse. Leaf tissue from two nontransformed control plants and nine transformed plants was assayed for PAT activity. The control plants did not show any PAT activity while all nine of the transformed plants displayed PAT activity (Fig. 3). The level of expression varied significantly between the nine plants. Transformants 3.1, 3.3, 3.4, 5.1, 5.2 and 5.3 (lanes 7, 8, 9, 12, 13 and 14 respectively) showed strong expression while 3.5 and 3.7 (lanes 10 and 11) were weaker expressors and transformant 2.3 (lane 6) displayed very low expression. These results correlate well with the chlorophenol red test and support the efficacy of this test for an early indication of transformation. The transformants showing the highest levels of expression as determined by the PAT activity assay had previously given a rapid yellow 'positive' result in the chlorophenol red assay.
Expression of phosphinothricin acetyl transferase (PAT) Chlorophenol red assay. The chlorophenol red assay is a pH indicator-based assay that is useful for confirming the resistance of transformed shoots at an early stage in the transformation procedure when the amount of tissue available may be quite low. Leaf pieces from transformed shoots were incubated in medium containing PPT and the indicator dye chlorophenol red. The assay is based on the observation that healthy, live tissue acidifies the medium which results in a colour change from red to yellow (Kramer et al. 1993), while tissue that is unable to detoxify PPT dies due to the accumulation of ammonia, and the increase in the pH of the medium results in a colour change from red to purple (Droge-Laser et al. 1994; Kramer et al. 1993). Leaflets from four clonally propagated cuttings from putative transformed shoots (Fig. 2, wells 1-4A and 15B) were able to detoxify PPT and therefore remained
Fig. 3. Phosphinothricinacetyl transferase assay. Autoradiograph of TLC plate. The acetylated phosphinothricin (PPT) product is present in extractsof transformedplants 1.1, 1.2, 2.2, 2.3, 3.1, 3.3, 3.4, 3.5, 3.7, 5.1, 5.2, 5.3 (lanes 3,-14 respectively) but is not present in extractsfrom controlplants (lanes 1 and 2). The origin at which the sample was applied, the acetyl-CoA substrate and acetylatedPPT productare indicatedto the left of the figure.
Integration of the bar gene Genomic DNA was extracted from eight of the transformed plants and used in Southern blot analyses to confirm the integration of the bar gene. A right border analysis, using the bar gene as a probe, indicated that all of the transformed plants contained
10 one or multiple copies of the T-DNA (Fig. 4). The hybridising bands were 1886bp or larger as was expected. Non-transformed controls showed no cross hybridisation with the bar gene. The banding patterns indicate that a number of the plants are vegetative clones, for example plants 1.1 and 1.2 (Fig. 4, lanes 4 and 5 respectively) have probably originated from a single transformation event. This was expected as multiple shoots had developed on some of the explants; however, 5 shoots developed from explant 3 (plants 3.1, 3.3, 3.4, 3.5, 3.7) and the Southern blot data (Fig. 4 lanes 7-11) suggests that in this case there were 3 separate transformation events. Therefore the multiple shoots arising from a single cotyledon explant may also be the result of a number of independent transformation events. Most of the plants contained a single T-DNA insert; however transformants 1.1 and 1.2 (vegetative clones) and transformant 2.2 contained multiple copies (Fig. 4). The plasmid pSLJ525 also carries the NptlI gene and the integration patterns observed using the bar gene as a probe were confmned by probing the same Southem blot with an NptlI probe (data not shown). To demonstrate that the integrated bar gene was inherited by the subsequent generation we allowed the transformed plants to self-pollinate and set seed. The progeny of transformant 3.3 which contains a single TDNA insert, were assayed for resistance to PPT. 298 seedlings (progeny of 3.3) were sprayed with 40 mg/1 PPT. 223 plants were resistant to the herbicide, while
Fig. 4. Southern blot indicating the presence of the
bar gene in the genome of the transformed plants. Right border analysis, HindlII digested DNA (10].tg) from 2 control plants (lanes 2 and 3) and transformed plaats 1.1, 1.2, 2.2, 3.1, 3.3, 3.4, 3.5 and 3.7 (lanes 4-11, respectively) probed with the bar gene. Only the transformed plants contain the bar gene. Lane 1 contains 0.5ng of pSLJ525 plasmid DNA digested with HindlII. The vector pSLJ525 contains a HindlII site 1886bp from the T-DNA right border, therefore hybridising bands are expected to be larger than 1886bp. The ba'r gene is contained between the right border and the HindlII site.
75 were susceptible and died within 7 days. The ratio of
resistant to susceptible plants is close to 3:1, which is the value expected for inheritance of a single dominant gene.
Transgenic plants show high levels of resistance to PPT. One non-transformed M. truncatula plant and nine transgenic plants were sprayed with 20ml PPT (40mg/1) 13 days after transfer to the greenhouse. The control plant became severely necrotic within 7 days while the transformants remained healthy. 17 days later the same plants and a new non-transfomled control were sprayed with a higher concentration o f PPT (400mg/1, 50ml per plant). The non-transformed plant died within 5 days while the transformants remained healthy. Transformant 3.7, which had showed the lowest levels of PAT expression as determined by the PAT assays, showed some minor leaf damage following this elevated level of spraying (data not shown). This level of PPT is higher than that used in agricultural field conditions.
Discussion We have developed a rapid transformation and regeneration system for M. truncatula which is significantly faster than previous methods, and will be of considerable benefit for researchers using M. truncatuIa as a model system for molecular genetic analyses. Previous protocols were based on transformation coupled with regeneration via somatic embryogenesis and took between 4 and 10 months to regenerate transformed plants. The protocol described here is based on Agrobacterium-mediated transformation of cotyledon explants of M. truncatula and shoots are regenerated via direct organogenesis. To our knowledge, this type of regeneration system has not been used previously with Medicago species. Part of the success of the protocol can be attributed to the use of the bar gene as a selectable marker, coupled with PPT (Ignite) as the selection agent. We have also tested this procedure using the NptlI gene as a selectable marker and kanamycin as a selectable agent and found that it was considerably less efficient. The level of kanamycin required to kill developing M. truncatula shoots was very high, 250mg/1, and nontransformed shoots were able to persist on selection for up to 10 days making it more difficult to select genuine resistant shoots (ATT and MJH unpublished results). The protocol that we developed was based on a procedure used for white clover (Trifolium repens L.) (Voisey et al. 1994) to which we made a number of modifications. We used single cotyledons with 12mm of the embryonic axis as the explant source rather than the whole shoot tip as used in the orginal white dover protocol. By splitting the embryonic axis we aimed to create a direct wound surface on the
11 basal area of the cotyledon to assist infection by Agrobacterium. This is the surface from which the shoot meristems would regenerate. The seedlings were dissected during immersion in a suspension of Agrobacteria to ensure that Agrobacteria were present on the freshly wounded tissue. The media that we used contained the PDM salts and vitamins developed by Chabaud et al. 1996 specifically for M. truncatula instead of those used for white clover. We included 5gM BAP in the germination medium as it had been previously reported that it was required for multiple shoot formation from soybean nodal explants (Wright et al. 1986). We observed that BAP increased the rate of germination and shoot production and we increased the BAP concentration in the regeneration medium to 15gM to stimulate shoot l~roduction and suppress the growth of the apical shoot. To avoid all possibility of escapes and to decrease the chance of survival of chimaeras, PPT was included in all of the media throughout the regeneration procedure. The Southern blot data indicate that multiple transformation events can occur on a single cotyledon explant. Therefore by increasing the number of shoots regenerating from the explant it should be possible to further improve the efficiency of this procedure. We have tested a number of different media for their ability to promote shoot regeneration and observed that inclusion of 0,5gM thidiazon (TDZ) in the M2 medium results in 3-4 times more shoots than the original M2 medium used in the initial transformation protocol (ATT and MJI-I unpublished results), TDZ has previously been shown to exhibit strong cytokinin activity (Mok et al. 1982) and has been reported to be more successful than BAP for the regeneration of shoots from both peanut (Kanyand et al. 1994) and bellpepper (Sz~isz et al. 1995). These improved media can now be tested in future transformation experiments. In addition to the regeneration frequency it may also be possible to improve the transformation efficiency by using different strains of Agrobacterium. In this protocol we used LBA 4404 however it has been reported that, this may not be the most efficient Agrobacterium strain (Schroeder et al. 1993; Voisey et al. 1994) and it would be worth investigating some of the more virulent strains such as EHA101 (Hood et al. 1986), EHA 105 (Hood et al. 1993) or AGL1 (Lazo et al. 1991). de Jong et al. (1994) reported that the virulent strain AGLO was significantly more efficient than LBA4404 for the production of transformed shoots in chrysanthemum. In addition to improving the transformation and regeneration efficiency of M. truncatula, our procedure has also resulted in the development of herbicide resistant M. truncatula plants. In greenhouse spraying tests the transgenic plants showed high levels of resistance to Ignite, a commercial herbicide developed by AgrEvo, Field trials will determine whether high level resistance is expressed under field conditions.
Acknowledgements. We thank J Jones for providing pSLJ525, AgrEvo for the kind gift of the Ignite and M. Chabaud and T. Huguet (INRA, France) for sharing the details of their transformation/regeneration media prior to publication. We thank Pon S. Jayakumar for helpful discussions, K. Korth and M. Bhattacharyya for critical review of the manuscript and Allyson Wilkins for help with the final preparation of the manuscript. The work was supported by the Samuel Roberts Noble Foundation.
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Plant Cell Reports (1996) 16:6-11
© Springer-Verlag1996
Rapid transformation of Medicago truncatula " regeneration via shoot organogenesis Anthony T. Trieu and Maria J. Harrison
The Samuel Roberts Noble Foundation, Plant Biology Division, 2510 Sam Noble Parkway, Ardmore, Oklahoma 73401, USA Received 14 May t996/Revised version received 12 June 1996 - Communicated by J. M. Widholm
Summary. A rapid transformation and regeneration system has been developed for M. truncatula cv Jemalong (barrel medic) by which it is possible to obtain transgenic plants within 2.5 months. The procedure involves Agrobacterium-mediated transformation of cotyledon exptants coupled with the regeneration of transformed plants via direct organogenesis. To develop the procedure, M. truncatuIa explants were transformed with the binary plasmid pSLJ525 which carries the bar gene. The bar gene encodes phosphinothricin acetyl transferase, and transformed plants were selected on media containing phosphinothricin (Ignite, AgrEvo). Transformed plants show phosphinothricin acetyl transferase activity and Southern blot analysis indicates that they carry the /~czr gene integrated into their genomes. The resistance to phosphinothricin is stable and is inherited by the R1 progeny as a single dominant Mendelian trait. The transgenic plants are highly resistant to the broad spectrum herbicide, Ignite and therefore may also have commercial applications. Key words: PAT, Phosphinothricin acetyl transferase; PPT, phosphinothricin Introduction
Annual species of the genus Medicago, including Medicago truncatula Gaertn. (barrel medic) are grown as pasture legumes in a number of regions throughout the world, including mediterranean areas, South Africa and Australia (Crawford et al. 1989; Lamprecht and Knox-Davies 1984). In Australia, the annual medics are the main legume found on over 50 million hectares of agricultural land and a variety of species and ecotypes have been developed. The first commercial cultivar of M. truncatula was sown in 1938 and this species has been favoured due to its ability to tolerate both low rainfall and high lime soils (Crawford et al. 1989). Breeding and selection programs for the annual medics are ongoing and the development of genetic
Correspondence to: A. T. Trieu
transformation and regeneration methods provides an additional means to introduce agronomically important traits (Crawford et al. 1989). In addition to its agricultural importance, M. truncatula is also emerging as a useful model for the molecular-genetic investigation of two important plant-microbe associations, the Rhizobium-legume symbiosis (Pichon et al. 1992; B6naben et al. 1995; Cook et al. 1995) and the vesicular-arbuscular mycorrhizal symbiosis (Harrison and Dixon 1994; Harrison and van Buuren 1995). The diploid, selffertilising nature of this legume, coupled with a small genome, fairly rapid generation time and ability to be genetically transformed by Agrobacterium (Thomas et al.1992; Chabaud et al. 1996), make it an attractive model legume for molecular-genetic studies (Barker et al. 1990). Two protocols for transformation and regeneration of M. truncatula have been reported previously. Both of the procedures involve Agrobacterium-mediated transformation coupled with regeneration of transformed plants via somatic embryogenesis. The first procedure is extremely cultivar specific and is useful only with highly regenerable lines developed for this purpose (Thomas et al. 1992). The second method was developed for the lines more frequently used in molecular genetic studies (Chabaud et al. 1996), however the process is still lengthy and labour intensive, taking between 4 and 10 months to obtain transgenic plants. As rapid transformation procedures are essential for molecular genetic analyses we sought to improve transformation of M. truncatula and investigated the possibility o f transformation coupled with regeneration via direct organogenesis. Such protocols have recently been described for white clover (Voisey et al. 1994) and pea (Schroeder et al. 1993). Here we report a new, rapid method to transform and regenerate M. truncanda. The procedure uses Agrobacterium-mediated transformation coupled with regeneration via direct shoot organogenesis and
7 tranformed plants can be obtained in 2.5 months. We demonstrate stable transformation, expression of the introduced bar gene and inheritance of bar in a typical 3:1 Mendelian pattern. In addition, as the bar gene was used as the selectable marker, the transformed plants show resistance to the herbicide Ignite at levels significantly higher than those used in field conditions. Consequently, the transgenic plants resulting from the development of the tranformation protocol may also have commercial applications.
Materials and Methods Plant material. Medicago truncatula cv. Jemalong (line A17, T. Huguet INRA) seeds were collected from greenhouse-grown plants. Plant culture media. All media below contained PDM salts and vitamins (Chaband et al. 1996) and were adjusted to pH 5.8. Each medium also contained 0.8% agar-agar (Sigma Chemical Co., St. Louis, MO, USA) except M2A medium. M2A, M2C, and M2R all contained 15 11M BAP (6-Benzylaminopurine) and 0.5 11M NAA (ct-Napthaleneacetic acid). M2R, M3 and M4 all contained 5.0 mg/l PPT (Ignite, AgrEvo Co., Wilmington, Delaware, U.S.A.), 500rag/1 carbenicillin (Sigma Chemical Co.) and 2% sucrose. Additional components for each medium are listed below: M1 (Germination medium): 5 11M BAP and 1% (w/v) sucrose. M2A (Agrobacterium resuspension medium): 1% sucrose and 100 11M acetosyringone (3',5'-dimethoxy-4'-hydroxy acetophenone, Aldrich Chemical Co., Milwaukee, WI, U.S.A.). M2C (Co-cultivation medium): 2% sucrose and 100 11M acetosyringone. M2R (Regeneration medium): no additional components. M3 (Shoot development medium): 0.5 gM NAA. M4 (Rooting medium): 0.2mg/1 IBA (Indole-3-butyric acid). Seed sterilisation and germination. Seeds were selected and scarified by rubbing with sand paper. The seeds were then immersed in 70% (v/v) ethanol for 30 seconds, followed by gentle agitation in 20% (v/v) Clorox (commercial bleach, 5.25% NaOC1) solution plus 0.1% Tween-20 for 10 rain. Seeds were then washed 3 times with sterile water and incubated in sterile water for 1 hr at 42°C. This was followed by incubation in 111M Giberellic acid (GA 3) at room temperature for 1 hour. Seeds were then blotted dry on sterile Whatman filter paper and spread on MI seed germination medium in 15x100 nun petri dishes (Fisher Scientific Co., PA.,U.S.A.). The seeds were incubated overnight in the dark at 2325°C and then for two days under fluorescent light with a 16-h photo period (23-25°C) to allow germination to proceed. Plasmid and bacterial strains. The binary plasmid pSLJ525 (Jones et al. 1992) carries both an NPT II gene and a bar gene. The bar gene encodes phosphinothrcin acetyl transferase (PAT) which confers resistance to phosphinothricin (PPT), a component of the herbicide Ignite (AgrEvo). pSLJ525 was mobilized into Agrobacterium tumefaciens strain LBA4404 (Clontech, USA) by direct transformation (An et al. 1988). To prepare A. tumefaciens LBA4404 for transformation a single colony was used to inoculate 2mi of liquid TY medium (5g/1 tryptone, 3g/l yeast extract and 6mM CaC12.6H20 ) plus antibiotics (10mg/1 rifampicin and 5mg/l tetracycline). This culture was grown for 18 hrs at 28°C with shaking (250 RPM). The 2 ml culture was then used to inoculate 25ml of liquid TY medium with antibiotics and the resulting culture was grown under the same conditions until the OD600 reached 0.5-0.7. The cells were collected by centrifugation at 8000 RPM for 10 min at 4°C, and the bacterial pellet resuspended in equal volume of M2A liquid medium. This suspension was then used for transformation. Plant transformation and regeneration. The protocol below was developed using the bar gene as a selectable marker. Approximately 60 seedlings (3 days old) were transferred to a sterile 15x100 man p e ~ dish containing 2-3ml of A. tumefaciens LBA4404 suspension in M2A media. Seedlings were bisected at the embryonic axis and the hypocotyl was removed 1-2 mm below the cotyledonary node. Thus each explant has 1 cotyledon and 1-2 mm of the split embryonic axis. Explants were briefly blotted dry with sterile Whatman filter paper and transferred to 15xl00mm petri plates containing M2C co-cultivation medium. The explants were
oriented so that the adaxial side of the cotyledon faced up and the abaxial side was in contact with the media (25 explants/petri plate). All explants were maintained on this medium at 23-25°C for 5 days under a 16-h photo period. Following co-cultivation, explants were transferred to M2R regeneration medium in 25x100 mm petri plates (10 explants per dish). Explants were maintained on this medium for 4 weeks and were transferred to fresh medium every 10 days. At this time, resistant shoots developed and the explants were subsequently transferred to M3 shoot development medium in Magenta boxes (Magenta Corporation, Chicago, IL, USA). Resistant shoots 0.5 cm or taller were excised and transferred to M4 rooting medium. Shoots were laid horizontally on the surface of the agar. Resistant shoots developed roots within 10-15 days and had extensive root development after an additional 10 days. These plants were then transferred to soil in the greenhouse and maintained initially at high humidity. The plants flowered approximately two months after transfer to the greenhouse. M. truncatula is self-fertile and the plants were allowed to self pollinate and set seed. This seed was collected for inheritance studies.
Histology. Cotyledon explants were collected for 24 days and prepared for sectioning by standard histological procedures (Berlyn and Miksche, 1976). Sections were collected on glass slides coated with Hanpt's adhesive (Berlyn and Miksche 1976), air dried overnight, and stained with 1% toluidine blue for 30 s without removing the parafilm. After 3 rinses in distilled water, slides were air-dried overnight. The slides were rinsed twice (10 rain each time) in xylene to remove parafilm and mounted with Permount (Fisher Scientific Co., N.J., USA). Chlorophenol red (CR) assay. The CR assay was carried out according to Kramer et al. (1993) with modification for leaf tissue instead of callus. 1-3 leaflets from PPT-resistant shoots were incubated in 250 111 of CR medium in a 24-well microtitre plate (Becton Dickson Labware, NJ, USA) under fluorescent light with a 16 hr photo period for 1-4 days. CR medium contained PDM salts and vitamins, 15gM BAP, 0.5 11M NAA, 50mg/l CR (Sigma Chemical Co.,USA), 5.0 mg/1 PPT and the pH was adjusted to 6.0 at which point the medium is red in color. Untransformed leaflets and a leaf segment from a transformed tobacco plant carrying the bar gene were included as controls. Phosphinothricin acetyl transferase (PAT) assay. PAT activity was assayed as described by DeBlock et al. (1987). Four partially open trifoliates (approx 150mg of tissue) were collected in a 1.5 ml eppendorf tube and placed on ice. The tissue was ground in liquid nitrogen with a plastic pestle using an electric handrill. 150 111of extraction buffer (100mM sodium phosphate, pH 7.5, 20raM sodium chloride, ImM phenylmethylsulfonylfluoride (PMSF), and 1 mg/1 bovine serum albumin (BSA) was added to the ground tissue. The tube was briefly vortexed at high speed, then centrifuged at 13,000 RPM for 15 min at 4°C. The extract was transferred to a new tube and recentrifuged at the same speed for l0 rain. The concentration of protein in 5 111 extracts was quantified (Bio-Rad protein assay) using BSA as a standard. To prepare the reaction mixture, 2gl of i00 I.tg/111 PPT (diluted in extraction buffer) and 3tll 14C acetyl-CoA (6OmCi/inrnol, Amersham, USA) was added to 25 gg of protein in 20pl of extraction buffer. The reaction mixture was incubated 30 min at 37°C and then 12 pl was applied to a TLC plate (J.T. Baker, Inc., Phillipsburg, NJ, USA) in two 6111 applications. The spot was dried briefly with a hair dryer between applications. Ascending chromatography was carried out in 1-propanol / NH4OH (3:2) for 3 h. The TLC plate was air-dried overnight and exposed to x-ray film (Kodak X-Omat AR film, Kodak, Rochester, USA). The 14C signal was visible after 2 days. Southern blots. Genomic DNA was isolated from leaf tissue of putative transformants and untransformed M. truncatula plants, using the protocol of Dellaporta et al. (1983). For a fight border analysis DNA (10pg) was digested with HindIII, separated by gel electrophoresis and blotted to nylon membranes (GeneScreen Plus, NEN Research Products, MA, USA) according to standard procedures (Sambrook et al. 1989). The blot was hybridised with an internal 423bp fragment of the bar gene which had been labelled with 32p cc-dATP (3,000 Ci/rnmol, Dupont, NEN, USA) by random-priming (Feinberg and Vogelstein, 1983). Hybridisation was according to the protocol of Church and Gilbert (1984). The membranes were exposed to Kodak X-Omatic AR f'tim with intensifying screen for 2.5 h at -80°C. The vector pSLJ525 contains a HindlII site 1886bp from the T-DNA fight border. The bar gene is contained between the fight border and the HindIII
Fig. 1. Regeneration of M. truncatula plants. (A) Cotyledon explant, bar = lmm. (B) Shoots (s) developed primary leaves after 15 days on M2R medium, bar = lmm. This example was not on selective media. The shoots emerging from explants on selection appear similar but the explant itself begins to turn yellow and die. (C) Phosphinothricin-resistant plantlets following I0 days growth on M3 shoot development medium containing phosphinothricin, bar = lcm (D) Phosphinothricin-resistant shoots develop roots (r) after 18 days on M4 rooting medium containing phosphinothficin, bar = lcm (E) Light micrograph of longitudinal section through an explant (8 days old). Arrows indicate the developing shoot meristems which are clearly distinct from the primary shoot meristem. The section is stained with toluidine blue, bar = 200~tm.
Fig. 5. Resistance to phosphinothricin. One control plant (left) and two transformed plants (transformant 2.1 and 3.3) (right) 7 days after being sprayed with 400mg/1 phosphinothricin (Ignite). Fig. 2. Chlorophenol red assay. Wells 1-4A and 1-5B contain leaf pieces from transformed plants. Transformants 3.7, 5.1, 5.2, and 5.3 are in wells Al-4 respectively. Transformants 1.2, 3.1, 3.4, and 3.7 are in wells B1-4 respectively. A6 and B6 contain leaf pieces from non-transformed control plants. Well A5 contains chlorophenol red medium only. The plate was incubated for 4 days.
site. ThereforeDNA fromtransformedplants would be expected to contain hybridisingfragmentslargerthan 1886 bp. Results
Transformation and regeneration of PPT- resistant M. truncatula Cotyledonary explants (Fig. 1A) were co-cultivated with A. tumefaciens LBA4404 carrying the binary vector pSLJ525 and then transferred to regeneration medium containing phosphinothricin (PPT) to select for transformed shoots. Shoots began to emerge from the cut face of the cotyledon within 12 days of transfer to regeneration medium (Fig. IB). The combination of the bar gene, and the selection agent PPT, proved to be extremely efficient for the selection of transformed M. truncatula shoots. Shoots from control explants (lacking Agrobaeteria) stopped growing within 3 days of incubation on selective medium and became necrotic within 5 days. Resistant shoots were allowed to develop further on an additional shoot development medium for 15 days (Fig. 1C) and were then transferred to rooting medium. Rooting medium contains PPT and resistant roots developed within 10-15 days (Fig. 1D). The plants were subsequently transferred to the greenhouse. The TO transformed plants exhibited the normal phenotypic characteristics of wild type M. truncatuIa plants. They continued to grow and develop, and flowered approximately 2 months after transfer to the greenhouse. Fourteen plants, determined to be transformed on the basis of Southern blot and/or PAT assay, were obtained from 450 cotyledonary explants. In order to confirm the origin of the shoot meristems we prepared and stained thin sections of the cotyledon explants. Microscopic examination of the sections indicated that the shoot meristems developed de novo from the epidermal cell layer in the regions adjacent to the primary apical meristem and embryonic axis and clearly indicated that the shoots were distinct from the primary apical meristem (Fig. 1E).
alive and the medium changed colour from red to yellow. The colour change occurred within 1 to 4 days and probably indicates differences in the levels of PAT expression. The cuttings were grown without carbenicillin and did not show Agrobacterial growth. Therefore the detoxification of PPT is not due to Agrobacterium harboured in the leaf pieces. Nontransformed leaves (Fig. 2, wells 6A and 6B) were killed by exposure to PPT and the medium changed colour from red to purple.
Phosphinothricin aeetyl transferase assays. PAT assays were performed on leaf tissue from transformed plants after transfer to soil and 15 days of growth in the greenhouse. Leaf tissue from two nontransformed control plants and nine transformed plants was assayed for PAT activity. The control plants did not show any PAT activity while all nine of the transformed plants displayed PAT activity (Fig. 3). The level of expression varied significantly between the nine plants. Transformants 3.1, 3.3, 3.4, 5.1, 5.2 and 5.3 (lanes 7, 8, 9, 12, 13 and 14 respectively) showed strong expression while 3.5 and 3.7 (lanes 10 and 11) were weaker expressors and transformant 2.3 (lane 6) displayed very low expression. These results correlate well with the chlorophenol red test and support the efficacy of this test for an early indication of transformation. The transformants showing the highest levels of expression as determined by the PAT activity assay had previously given a rapid yellow 'positive' result in the chlorophenol red assay.
Expression of phosphinothricin acetyl transferase (PAT) Chlorophenol red assay. The chlorophenol red assay is a pH indicator-based assay that is useful for confirming the resistance of transformed shoots at an early stage in the transformation procedure when the amount of tissue available may be quite low. Leaf pieces from transformed shoots were incubated in medium containing PPT and the indicator dye chlorophenol red. The assay is based on the observation that healthy, live tissue acidifies the medium which results in a colour change from red to yellow (Kramer et al. 1993), while tissue that is unable to detoxify PPT dies due to the accumulation of ammonia, and the increase in the pH of the medium results in a colour change from red to purple (Droge-Laser et al. 1994; Kramer et al. 1993). Leaflets from four clonally propagated cuttings from putative transformed shoots (Fig. 2, wells 1-4A and 15B) were able to detoxify PPT and therefore remained
Fig. 3. Phosphinothricinacetyl transferase assay. Autoradiograph of TLC plate. The acetylated phosphinothricin (PPT) product is present in extractsof transformedplants 1.1, 1.2, 2.2, 2.3, 3.1, 3.3, 3.4, 3.5, 3.7, 5.1, 5.2, 5.3 (lanes 3,-14 respectively) but is not present in extractsfrom controlplants (lanes 1 and 2). The origin at which the sample was applied, the acetyl-CoA substrate and acetylatedPPT productare indicatedto the left of the figure.
Integration of the bar gene Genomic DNA was extracted from eight of the transformed plants and used in Southern blot analyses to confirm the integration of the bar gene. A right border analysis, using the bar gene as a probe, indicated that all of the transformed plants contained
10 one or multiple copies of the T-DNA (Fig. 4). The hybridising bands were 1886bp or larger as was expected. Non-transformed controls showed no cross hybridisation with the bar gene. The banding patterns indicate that a number of the plants are vegetative clones, for example plants 1.1 and 1.2 (Fig. 4, lanes 4 and 5 respectively) have probably originated from a single transformation event. This was expected as multiple shoots had developed on some of the explants; however, 5 shoots developed from explant 3 (plants 3.1, 3.3, 3.4, 3.5, 3.7) and the Southern blot data (Fig. 4 lanes 7-11) suggests that in this case there were 3 separate transformation events. Therefore the multiple shoots arising from a single cotyledon explant may also be the result of a number of independent transformation events. Most of the plants contained a single T-DNA insert; however transformants 1.1 and 1.2 (vegetative clones) and transformant 2.2 contained multiple copies (Fig. 4). The plasmid pSLJ525 also carries the NptlI gene and the integration patterns observed using the bar gene as a probe were confmned by probing the same Southem blot with an NptlI probe (data not shown). To demonstrate that the integrated bar gene was inherited by the subsequent generation we allowed the transformed plants to self-pollinate and set seed. The progeny of transformant 3.3 which contains a single TDNA insert, were assayed for resistance to PPT. 298 seedlings (progeny of 3.3) were sprayed with 40 mg/1 PPT. 223 plants were resistant to the herbicide, while
Fig. 4. Southern blot indicating the presence of the
bar gene in the genome of the transformed plants. Right border analysis, HindlII digested DNA (10].tg) from 2 control plants (lanes 2 and 3) and transformed plaats 1.1, 1.2, 2.2, 3.1, 3.3, 3.4, 3.5 and 3.7 (lanes 4-11, respectively) probed with the bar gene. Only the transformed plants contain the bar gene. Lane 1 contains 0.5ng of pSLJ525 plasmid DNA digested with HindlII. The vector pSLJ525 contains a HindlII site 1886bp from the T-DNA right border, therefore hybridising bands are expected to be larger than 1886bp. The ba'r gene is contained between the right border and the HindlII site.
75 were susceptible and died within 7 days. The ratio of
resistant to susceptible plants is close to 3:1, which is the value expected for inheritance of a single dominant gene.
Transgenic plants show high levels of resistance to PPT. One non-transformed M. truncatula plant and nine transgenic plants were sprayed with 20ml PPT (40mg/1) 13 days after transfer to the greenhouse. The control plant became severely necrotic within 7 days while the transformants remained healthy. 17 days later the same plants and a new non-transfomled control were sprayed with a higher concentration o f PPT (400mg/1, 50ml per plant). The non-transformed plant died within 5 days while the transformants remained healthy. Transformant 3.7, which had showed the lowest levels of PAT expression as determined by the PAT assays, showed some minor leaf damage following this elevated level of spraying (data not shown). This level of PPT is higher than that used in agricultural field conditions.
Discussion We have developed a rapid transformation and regeneration system for M. truncatula which is significantly faster than previous methods, and will be of considerable benefit for researchers using M. truncatuIa as a model system for molecular genetic analyses. Previous protocols were based on transformation coupled with regeneration via somatic embryogenesis and took between 4 and 10 months to regenerate transformed plants. The protocol described here is based on Agrobacterium-mediated transformation of cotyledon explants of M. truncatula and shoots are regenerated via direct organogenesis. To our knowledge, this type of regeneration system has not been used previously with Medicago species. Part of the success of the protocol can be attributed to the use of the bar gene as a selectable marker, coupled with PPT (Ignite) as the selection agent. We have also tested this procedure using the NptlI gene as a selectable marker and kanamycin as a selectable agent and found that it was considerably less efficient. The level of kanamycin required to kill developing M. truncatula shoots was very high, 250mg/1, and nontransformed shoots were able to persist on selection for up to 10 days making it more difficult to select genuine resistant shoots (ATT and MJH unpublished results). The protocol that we developed was based on a procedure used for white clover (Trifolium repens L.) (Voisey et al. 1994) to which we made a number of modifications. We used single cotyledons with 12mm of the embryonic axis as the explant source rather than the whole shoot tip as used in the orginal white dover protocol. By splitting the embryonic axis we aimed to create a direct wound surface on the
11 basal area of the cotyledon to assist infection by Agrobacterium. This is the surface from which the shoot meristems would regenerate. The seedlings were dissected during immersion in a suspension of Agrobacteria to ensure that Agrobacteria were present on the freshly wounded tissue. The media that we used contained the PDM salts and vitamins developed by Chabaud et al. 1996 specifically for M. truncatula instead of those used for white clover. We included 5gM BAP in the germination medium as it had been previously reported that it was required for multiple shoot formation from soybean nodal explants (Wright et al. 1986). We observed that BAP increased the rate of germination and shoot production and we increased the BAP concentration in the regeneration medium to 15gM to stimulate shoot l~roduction and suppress the growth of the apical shoot. To avoid all possibility of escapes and to decrease the chance of survival of chimaeras, PPT was included in all of the media throughout the regeneration procedure. The Southern blot data indicate that multiple transformation events can occur on a single cotyledon explant. Therefore by increasing the number of shoots regenerating from the explant it should be possible to further improve the efficiency of this procedure. We have tested a number of different media for their ability to promote shoot regeneration and observed that inclusion of 0,5gM thidiazon (TDZ) in the M2 medium results in 3-4 times more shoots than the original M2 medium used in the initial transformation protocol (ATT and MJI-I unpublished results), TDZ has previously been shown to exhibit strong cytokinin activity (Mok et al. 1982) and has been reported to be more successful than BAP for the regeneration of shoots from both peanut (Kanyand et al. 1994) and bellpepper (Sz~isz et al. 1995). These improved media can now be tested in future transformation experiments. In addition to the regeneration frequency it may also be possible to improve the transformation efficiency by using different strains of Agrobacterium. In this protocol we used LBA 4404 however it has been reported that, this may not be the most efficient Agrobacterium strain (Schroeder et al. 1993; Voisey et al. 1994) and it would be worth investigating some of the more virulent strains such as EHA101 (Hood et al. 1986), EHA 105 (Hood et al. 1993) or AGL1 (Lazo et al. 1991). de Jong et al. (1994) reported that the virulent strain AGLO was significantly more efficient than LBA4404 for the production of transformed shoots in chrysanthemum. In addition to improving the transformation and regeneration efficiency of M. truncatula, our procedure has also resulted in the development of herbicide resistant M. truncatula plants. In greenhouse spraying tests the transgenic plants showed high levels of resistance to Ignite, a commercial herbicide developed by AgrEvo, Field trials will determine whether high level resistance is expressed under field conditions.
Acknowledgements. We thank J Jones for providing pSLJ525, AgrEvo for the kind gift of the Ignite and M. Chabaud and T. Huguet (INRA, France) for sharing the details of their transformation/regeneration media prior to publication. We thank Pon S. Jayakumar for helpful discussions, K. Korth and M. Bhattacharyya for critical review of the manuscript and Allyson Wilkins for help with the final preparation of the manuscript. The work was supported by the Samuel Roberts Noble Foundation.
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