Plant Molecular Biology 8 : 4 6 1 - 4 6 9 (1987) © Martinus Nijhoff Publishers, Dordrecht - Printed in the Netherlands
461
Transformation of the forage legume Trifolium repens L. using binary Agrobacterium vectors Derek W. R. White and Denise Greenwood
Grasslands Division, Department of Scientific and Industrial Research, Palmerston North, New Zealand Received 4 November 1986; in revised form 16 February 1987; accepted 20 February 1987
Key words: Agrobacterium tumefaciens, binary vectors, gene transfer, kanamycin resistance, legume transformation, Trifolium repens Abstract
A system was established for introducing cloned genes into white clover (Trifolium repens L.). A high regeneration white clover genotype was transformed with binary Agrobacterium vectors containing a chimaeric gene which confers kanamycin resistance. Transformed kanamycin resistant callus was obtained by culturing Agrobacterium inoculated stolon internode segments on selective medium. The kanamycin resistance phenotype was stable in cells and in regenerated shoots. Transformation was confirmed by the expression of an unselected gene, nopaline synthase in selected cells and transgenic shoots and by the detection of neomycin phosphotransferase II enzymatic activity in kanamycin resistant cells. Integration of vector DNA sequences into plant DNA was demonstrated by Southern blot hybridisation.
Introduction
Efficient methods have been developed for introducing cloned genes into some plant species (5, 11). Hence, defined additions to the plant genome can now be made using a combination of gene manipulation and gene transfer techniques. These developments are important for studying the regulation of plant gene expression and for engineering improvements in crop plant performance. Unfortunately, progress in developing systems for the transformation of economically important legume species has been very limited. The natural gene transfer capacity of Agrobacterium tumefaciens has been modified to provide a method for the transformation of dicotyledonous plants. Virulent strains of A. tumefaciens cause tumorous growths when inoculated on to wounded tissue of susceptible plants. A specific segment (TDNA) of the large Ti-plasmid is integrated into the
plant nuclear genome (6). This T-DNA contains genes for the synthesis of novel compounds called opines and genes conferring hormone independent growth. To allow plant regeneration from transformed cells the tumour-inducing genes have been deleted from T-DNA. Transformed cells can be identified by incorporating a chimaeric gene, constructed using regulatory regions from the nopaline synthase gene and the coding sequence of a bacterial neomycin phosphotransferase which confers kanamycin resistance, into the non-oncogenic TDNA (2, 9). Transfer of the T-DNA into plant cells is mediated by trans acting products from several genes located in a separate virulence (vir) region of the Ti-plasmid. The vir and T-DNA components can be placed on separate plasmids to form a binary system of T-DNA transfer (10). Consequently, efficient binary Agrobacterium vectors have been constructed by placing a T-DNA, containing a selectable gene, into a broad host range plasmid
462 which will replicate in both Escherichia coli and A. tumefaciens (1, 3). Plant regeneration from cultured cells is required for current transformation methods. Unfortunately, plant regeneration from cultured cells of many legumes is inefficient. However, an efficient method has been developed for regenerating plants from cells of white clover (Trifolium repens) by selecting a high-regeneration genotype (18). Here we describe the development of a system for introducing cloned genes into this white clover genotype using binary Agrobacterium vectors containing a selectable kanamycin resistance gene.
Materials and methods
Plant material Three-year-old vegetatively propagated plants of a "high-regeneration" white clover genotype, WR8 (18), were grown in 42 cm x 30 cm x 6 cm plastic trays in a potting mix of 60% peat and 40°70 sand containing Osmocote controlled release fertiliser (18% N, 4.8% P, 8.3% K, 3.4% S) (Sierra Chemical Europe B.V.). Plants were grown in glasshouse conditions of 1 5 ° C - 2 6 ° C , natural daylight and photoperiod and were trimmed at 4 month intervals. New stolon outgrowths containing 5 - 8 internodes were used during a 14-month period for transformation experiments. There was no indication that the time of the year when stolons were collected influenced transformation frequency.
Bacterial strains and plasmids White clover transformation Two non-oncogenic A. tumefaciens strains, LBA4404 (10) and GV3850 (19) (containing modified Ti-plasmids pAL4404 and pGV3850 respectively), were used in transformation experiments together with four different binary vectors (pBin6, pBinl9 (3); pGA470, pGA472 (1)) containing chimaeric kanamycin resistance genes. One of the vectors, pBin6, also has an unselected marker gene encoding nopaline synthase. The binary vectors pGA470 and pGA472 were mobilised from E. coli strain MC1000 into A. tumefaciens strain GV3850 using a triparental mating (8) with E. coli strain HB101 containing pRK2013. Transconjugants GV3850(pGA470) and GV3850(pGA472) were selected on minimal medium containing tetracycline (10/zg/ml) and kanamycin (10/xg/ml). Plasmid isolation and restriction endonuclease mapping was used to confirm the presence of vectors in A. tumefaciens. A. tumefaciens strains were cultured on TY medium containing 5 g / L Difco bacto tryptone, 3 g / L Difco yeast extract, 1.3 g / L CaClz.6H20 and 15 g / L Difco bacto agar, at 28 °C. E. coli strains were grown on LB agar medium at 37 °C.
Stolon internode segments, 1 - 2 cm in length, were surface sterilised for 30 min in 2.5°7o (w/v) sodium hypochlorite containing 1 - 2 drops of "Tween 80" wetting agent and washed five times in sterile distilled water. After the bleached ends were cut off inoculation was effected by touching both the basal and apical fresh wound sites on to A. tumefaciens cultured on agar solidified medium. Segments were incubated for 1 - 3 days in a moist petri dish at 28 °C in continuous light (10 W/m2). Then 1 mm terminal slices were cut off and placed inoculated side uppermost on callusing medium containing cefotaxime (Calbiochem) (500 t~g/ml) to stop bacterial growth and kanamycin (100 ~g/ml) for the selection of transformed plant cells. Callus that developed on selection medium was subcultured at 21-day intervals on to the same medium. Some kanamycin-resistant cell lines were grown in suspension culture in the absence of antibiotics. Shoot formation was initiated from callus both in the presence and in the absence of kanamycin (100 ~g/ml). Media and conditions for callus initiation, maintenance, suspension culture and shoot regeneration were as previously described (18). To test for the expression of kanamycin resistance in transgenic shoots, shoots regenerated from both transformed and non-transformed cell lines in the
463 absence of selection were placed on 1/2 strength MS medium (12) containing kanamycin (100/~g/ml) and cultured for 30 days.
Nopaline analyses For the detection of nopaline in callus or shoot material, 100-200 mg fresh weight of tissue was transferred to a sterile Eppendorf tube and macerated with a plastic rod. After centrifugation for 15 rain in a MSE micro-centrifuge, 5 >1 aliquots of clear supernatant were spotted, 2 cm apart, on Whatman 3MM chromatography paper and dried. A mixture of 0.02% nopaline, 0.02% octopine and 30 mM arginine was used as a standard. Electrophoresis was at 45 V/cm for 30 min in formic acid-acetic acid-water (1:3:16, v/v) at pH 2.1. The dried papers were stained by spraying with a freshly prepared 1:1 solution of 0.2% ( w / v ) p h e n a n threnequinone in 100070 ethanol and 10% (w/v) NaOH in 60% ethanol (14). The dried papers were viewed and photographed under U.V. light (254 nm).
Neomycin phosphotransferase H assay Tissue samples (100 mg fresh weight) taken from calli grown in the presence of kanamycin (100/~g/ml) were macerated with a plastic rod in Eppendorf tubes in an equal volume of ice-cold extraction buffer containing 62.5 mM Tris-HC1 pH 6.8, 10% glycerol, 5O7o /5-mercaptoethanol, 0.1°70 sodium dodecyl sulphate (SDS) and 100 #g/ml phenylmethylsulphonyl fluoride. After centrifugation for 5 min in a MSE micro-centrifuge aliquots (50/~g protein as determined by the method of Bradford (4)) of extracts containing bromophenol blue were electrophoresed through a 10°70 polyacrylamide gel at 25 mA for 3 h in a cold room and assayed for neomycin phosphotransferase II activity in situ as reported by Reiss et al. (16). The gel was overlayed with an agarose slab containing 33.5 mM Tris maleate, 21 mM MgC1, 200 mM NH4C1 , 1.5% low melting point agarose (Seaplaque, FMC), 30/,g/ml kanamycin and 25 /xCi [3,-32p]ATP,
2 000 Ci/mmol, incubated for 30 min at room temperature and blotted on to Whatman P81 phosphocellulose paper overnight. To remove excess background radioactivity the P81 paper was incubated for 30 rain in a solution containing 1°70 SDS and 1 mg/ml proteinase K (Boehringer Mannheim) at 60°C and washed 3× in 10 mM phosphate buffer (pH 7.5) at 80°C. The dried paper was exposed to X-ray film in the presence of intensifying screens at - 7 0 ° C for 1 - 4 h.
DNA preparation and Southern blot analysis Plant DNA was isolated from suspension cultured cells of both transformed and non-transformed cell lines using the procedure of Sutton (17). Samples (10 >g) of total plant DNA were digested with approximately I00 units of each of the restriction endonucleases used for 1 6 - 2 4 h. DNA samples were electrophoresed in 7-mm-thick, horizontal, 0.8% agarose (BRL, ultrapure) gels in Tris acetate buffer (pH 8.0) using 1.25 V/cm. The DNA was then depurinated and transferred to Zeta-Probe nylon membrane (Bio-Rad) in a flow of 0.4 M NaOH as described by Reed and Mann (15). To determine molecular weights directly from subsequent autoradiograms of the filters, parallel lanes of the gel contained a Hind III plus EcoRI, Hind III, digest of bacteriophage X DNA. To estimate integrated DNA copy number, 1 and 5 copy reconstructions were made by adding 15 ng and 75 ng respectively of the DNA to be used as a probe to nontransformed plant DNA prior to restriction endonuclease digestion. Specific probes were prepared from bacteriophage ), and pKan 2 (pBR322 with a 3.4 kilobase pair insert encoding a neomycin phosphotransferase II gene from Tn5) DNA by labelling with 32p in modified nick translation reactions (Reed and Mann, pers. comm.). For one reaction 500 ng of DNA was incubated with 1 ng of DNase I (bovine pancreas, Sigma) in a total volume of 35 /A containing 50 mM Tris-HC1 (pH 7.5), 7.5 mM magnesium acetate, 4 mM dithiothreitol, 2.5 /~M each of dATP, dGTP, d T T P (Boehringer), 100 t,g/ml of nuclease-free BSA (Boehringer) and 20/~Ci of [c~-32p]dCTP (3000 Ci/mmol; Amer-
464 sham), at 14°C for 15 min. DNase I was then inactivated by heating at 70 °C for 5 min and placing on ice. Subsequently, 8 units o f D N A polymerase I (Boehringer) were added and incubation extended for a further 15 rain at 14 °C for repair synthesis. The reaction was stopped by the addition of 20 mM EDTA and 0.5% SDS. With this procedure more than 90O7o o f the [c¢-32P]dCTP was incorporated into DNA, giving probes labelled at a specific activity of 2 × 108 dpm//zg DNA. Labelled DNA was denatured by adding 2/xl of 4 M N a O H to the reaction mixture. Zeta-Probe filters were prehybridised in a polyethylene bag containing 10 ml of 10 × Denhart's solution (0.2 070 each of ficoll, polyvinyl pyrrolidone and BSA) containing 50 mM H E P E S buffer (pH 7.0), 3 x SSC (1 × SSC = 0.15 M sodium chloride, 0.015 M sodium citrate), 17/xg/ml herring DNA, 20/~g/ml E. coli t R N A and 0.1°70 SDS for 16 h at 68°C. All but 2 ml of the solution was then removed from the bag, denatured probes (k 106 dpm, pKan 2 2 - 4 × 106 dpm) were added and the filters were incubated for a further 16 h at 68°C. Filters were washed for 15 min each in 2 × SSC, 0.1070 SDS; 0.5 × SSC, 0.1070 SDS; 0.1 x SSC, 0.1% SDS; all at room temperature and finally in 0.1 x SSC, 0.1% SDS at 50°C for 30 rain. The dried filters were placed between Cronex Lighting Plus (Du Pont) intensifying screens with X-ray films for 4 - 7 days at - 7 0 ° C .
Results
Selection of kanamycin-resistant white clover transforman ts Preliminary experiments established that the white clover genotype WR8 will form tumours when stolon internode wounds are inoculated with virulent Agrobacterium tumefaciens strains. Also callus initiation from white clover stolon internode explants is inhibited by 100/zg/ml kanamycin. This allowed the development of a A. tumefaciens mediated transformation system which utilises a chimaeric gene that confers kanamycin resistance to plant cells. Two non-oncogenic A. tumefaciens strains harbouring different helper plasmids, which provide virulence functions, were used to mobilise the TDNA sited kanamycin resistance gene from four different binary vectors into white clover (Table 1). Since both pBin6 and pBinl9 are rapidly lost from A. tumefaciens LBA4404 in the absence of selection (3), the wounded ends of internode segments were inoculated by contact with bacterial colonies cultured on selective medium. After a short period of incubation, to allow transformation to occur, callus initiation, bacterial elimination and transformant selection were combined in one step by transferring terminal segments to callusing medium containing cefotaxime and kanamycin.
Table 1. Transformation of white clover using A. tumefaciens containing binary vectors. A. tumefaciens
Plasmid(s)
Number of segments treateda
07oof segments kanamycin resistant b
pAL4404 pAL4404, pBinl9 pAL4404, pBin6 pGV3850 pGV3850, pGA470 pGV3850, pGA472
208 99 50 75 72 24
0 41 44 0 24 33
strain
LBA4404 LBA4404(pBin19) LBA4404(pBin6) GV3850 GV3850(pGA470) GV3850(pGA472)
a WR8 stolon segments taken from the 3rd - 8th subapical internodes were inoculated, incubated for 3 days, then placed on callusing medium containing 100/~g/ml kanamycin. b Segments were scored for the presence of kanamycin resistant growth after 3 weeks on selective medium.
465
Fig. 1. Selection of transformed kanamycin resistant white clover cells. Stolon internode segments were inoculated with A. tumefaciens strains, incubated for 3 days and transferred to callusing medium containing 100 #g/ml kanamycin. Segments transformed with pBin6 are shown on the right and segments inoculated with strain LBA4404 are shown on the left. The photograph was taken after 35 days of culture.
Kanamycin resistant outgrowths developed on segments inoculated with A. tumefaciens containing both a helper plasmid and binary vector (Fig. 1 and Table 1). These outgrowths were first visible 12-18 days after placing the segments on selective medium and the resistant callus grew rapidly when transferred to fresh medium. No kanamycin resistant callus was obtained from segments inoculated with A. tumefaciens harbouring only a helper plasmid (Fig. 1 and Table 1) even after prolonged (5 weeks) culture. Approximately 40°7o of the segments inoculated with LBA4404(pBin6) or LBA4404(pBin19) produced kanamycin-resistant callus, often from a number of different sites on the surface of the segment (Fig. 1). The proportion of segments that produced kanamycin resistant callus was lower when inoculation was with GV3850(pGA470) or GV3850(pGA472) than when inoculation was with LBA4404(pBin6) or LBA4404(pBin19). By contrast, kanamycin resistant callus appeared more rapidly when segments were transformed with GV3850(pGA470) or GV3850(pGA472). To refine the transformation method both the incubation period required and the effect of the position of internodes on the plant were examined. Two days of incubation in the presence of
LBA4404(pBin6) or LBA4404(pBin19) was required to obtain kanamycin resistant callus and higher frequencies resulted from a 3-day incubation period (Table 2). The proportion of segments producing kanamycin resistant callus varied according to the position of the internode on the plant. Segments taken from internodes near the shoot apex produced fewer kanamycin resistant calli than those taken from internodes in a more basal position (Table 3). After 3 - 4 subcultures on selective medium some Table 2. Effect of incubation period on transformation of white clover by binary Agrobacterium vectors. Incubation period (days)
A. tumefaciens
1
LBA4404 LBA4404(pBinI9) LBA4404(pBin6)
46 38 53
0 0 0
2
LBA4404(pBinl 9)
37
14
3
LBA4404 LBA4404(pBin19) LBA4404(pBin6)
208 99 198
0 41 37
strain
Number of segments treated
% of segments kanamycin resistant a
a Kanamycin resistant growth was scored after 3 weeks of culture.
466 Table 3. Influence of explant position on the frequency of transformation of white clover internode segments. Internode segment a
N u m b e r of segments treated b
°70 of segments kanamycin resistant c
1
41
5
2 3 4 5
42 42 38 26
24 31 47 42
in extracts from white clover cells transformed with pBin6, but not in extracts from cells transformed with pBinl9, a vector which does not have a nopaline synthase gene (Fig. 2A). Expression of nopaline synthase was detected in pBin6 transgenic shoots. Extracts taken from shoots regenerated in the absence of kanamycin selection were nopaline positive, whereas non-transformed shoots of white clover (WR8) did not accumulate nopaline (Fig. 2B).
a Segment 1 is closest to the apical meristem. b Inoculated with A. tumefaciens strain LBA4404(pBin6) for 3 days. c Kanamycin resistant growth was scored after 3 weeks of culture.
of the kanamycin resistant callus was grown in the absence of antibiotics either as callus or in suspension culture. Even after 3 - 5 months of culture in non-selective conditions these cells retained their kanamycin resistance phenotype. Approximately 3 months after initial selection kanamycin resistant callus obtained from segments inoculated with LBA4404(pBin6), LBA4404(pBin19) or GV3850(pGA470) was transferred to 1/2 strength MS medium without growth regulators to promote shoot formation. Shoot initiation occurred in both the presence and in the absence of 100 #g/ml kanamycin and was similar to that obtained on non-transformed callus from the WR8 genotype. Furthermore, shoots regenerated in the absence of selection from cells transformed with pBin6 or pBinl9 remained green and grew in the presence of 100 ~g/ml kanamycin, whereas shoots regenerated from non-transformed WR8 cells turned white after 14 days and died (data not shown).
Expression of an unselected marker genenopaline synthase To confirm that selected kanamycin resistant calli of white clover were transformed, the expression of an unselected gene within the T-DNA of pBin6, nopaline synthase, was analysed. Nopaline synthase activity in transformed plant cells leads to the accumulation of nopaline. Nopaline was detected
Fig. 2. Nopaline accumulation in cells transformed by A. tumefaciens containing pBin6 and in transgenic shoots. Extracts were assayed by paper electrophoresis with octopine (OCT) and nopaline (NOP) standards (lanes) and stained with phenanthrenequinone. (A) Extracts from cells transformed with pBin6 (Lanes 1 - 5 ) or pBinl9 (Lane 6). (B) Extracts from pBin6 transgenic shoots regenerated in the absence of kanamycin (Lanes 1 - 5 ) or from a non-transformed shoot (Lane 6).
467 The introduced neomycin phosphotransferase H gene was expressed
To demonstrate that the selected kanamycin resistance phenotype was due to the expression of an introduced neomycin phosphotransferase II (NPT I/) gene and the synthesis of the expected bacterial protein, NPT-H enzyme activity was assayed in situ after electrophoretic separation on a nondenaturing polyacrylamide gel. The activity was absent in extracts from non-transformed white clover callus and present in extracts from pBin6, pBinl9 and pGA470 transformants (Fig. 3). The activity of extracts from pGA472 transformants was not assayed. While NP-IZ-H activity from pBin6 and pBinl9 transformants had the same mobility as the bacterial enzyme, activity from pGA470 transformants had a slower mobility. Four different pGA470 transformants all had a higher NPT-H activity when compared with four pBinl9 transformed cell lines (data not shown).
lysed by Southern blot hybridisation. Separated restriction endonuclease enzyme fragments were blotted on to Zeta-Probe nylon filters and hybridised with a 32p labelled probe specific to the coding sequence of the introduced NPT-H gene. The probe hybridised with DNA from the transformed cell line (Fig. 4). While DNA from non-
D N A analysis o f a transformed white clover cell line
To confirm chromosomal integration of the chimaeric NPT-H gene, genomic DNA was isolated from a cell line transformed with pBinl9 and aria-
Fig. 3. Autoradiograph showing NPT-H activity of extracts from kanamycin resistant calli separated on a 10% polyacrylamide gel and assayed in situ. Lane 1 non-transformed. Lane 2 pBinl9 transformant. Lane 3 pBin6 transformant. Lane 4 pGA470 transformant. The arrow indicates the position of bacterial NPT-II activity.
Fig. 4. Southern blot analysis of D N A from white clover transformed by A. tumefaciens containing pBinl9. Total DNA extracted from transformed and non-transformed white clover cells was digested with restriction endonucteases and the fragments were separated by electrophoresis and transferred to ZetaProbe membrane. The radioactive probe was a nick-translated Hind Ill digest of a pBR322 derived plasmid containing a 3.4 kb Hind IIl NPT-II specific sequence from Tn5, Lane 1 is a EcoRI digest of non-transformed DNA and lanes 2 - 4 digests of transformed DNA. Lane 2, EcoRl; Lane 3, Hind III; Lane 4, Hind III/Pst!. A X specific probe was used to determine the D N A fragment sizes given from a X Hind I I I + [Hind Ill + EcoRI] digest.
468 transformed cells did not show any hybridisation signal. The pattern of NPT-H specific probe hybridisation to DNA fragments from EcoRI and Hind III digests (lanes 2 and 3, Fig. 4), suggests that the chimaeric NPT-H gene had been integrated into the white clover genome. The intensity of the hybridisation signal compared with 1 and 5 copy reconstructions (not shown) suggests that only one or 2 copies of pBinl9 T-DNA have been integrated into the genome.
Discussion
We demonstrate here that a combination of A. tumefaciens mediated DNA transfer, binary vectors containing a selectable chimaeric gene conferring kanamycin resistance and a genotype capable of regeneration from cultured cells can be used to establish a system for introducing foreign genes into white clover. Genetic transformation of white clover has been established on the basis of the following criteria: (i) the stable kanamycin resistant phenotype of selected calli and shoots derived from them; (ii) the NPT-H enzymatic activity found in transformed lines; (iii) the integration of an introduced gene sequence into plant genomic DNA; and (iv) expression of an unselected gene - nopaline synthase - in transformed cells and transgenic shoots. Thus the Agrobacterium-binary vector approach previously shown to function for introducing genes into tobacco (1, 3) and Medicago varia (7), is also an effective means of transforming white clover, an economically important legume. To date all the methods devised for introducing cloned genes into plants rely on a capability for regenerating plants from cultured cells or protoplasts. At present only a portion of white clover genotypes will give satisfactory plant regeneration from cell culture (18). Therefore, foreign DNA can be incorporated into only a limited number of white clover genotypes. However, since white clover is an outcrossing species, a foreign gene could be introduced into a single recipient genol~ype and the gene could subsequently be moved into an appropriate genetic background by conventional breeding practices.
The results presented in Table 1 indicate that transformation of WR8 stolon internode segments may be more effective with A. tumefaciens strains LBA4404(pBin6) and LBA4404(pBin19) than with strains GV3850(pGA470) and GV3850(pGA472). Interpretation of this result is complicated by differences in bacterial genetic backgrounds, virulence helper plasmids, and binary vectors. The earlier appearance of kanamycin resistant growth on segments inoculated with GV3850(pGA470) or GV3850(pGA472) suggests that the chimaeric kanamycin resistance gene in pGA470 and pGA472 may be more effective than the corresponding gene in pBin6 and pBinl9. The higher NPT-H activity levels of extracts from pGA470 transformants, when compared with pBin transformants, supports this conclusion. The N t ~ - H enzymatic activity of extracts from pGA470 transformants migrated slower on polyacrylamide gels than extracts from pBin transformants or bacteria containing the NPT-H gene. This alteration in the migration of NPT-H enzymatic activity is due to the method used to construct the chimaeric gene in pGA470. The gene in pGA470 was constructed by making an in-frame translational fusion between the NPT-H coding region and the amino terminus of the nos gene (1), whereas the corresponding gene in the pBin vectors has an intact NPT-H coding sequence. This results in the addition of positively charged amino acids to the amino terminus of the protein, the reason for its altered migration. The proportion of internode segments which produced kanamycin resistant callus was affected by both the length of the incubation period with A. tumefaciens and the position on the plant from which the explants were taken. Other reports (3, 13) have also commented on the influence of the position on the plant or the axial orientation of an explant upon transformation "competence". In white clover a combination of 3 days incubation with A. tumefaciens and the use of explants from the 4th internode gave the highest transformation frequency. The availability of a transformation system for white clover will enable the introduction of cloned foreign genes into this important forage legume.
469 This development has implications both for the improvement of white clover agronomic performance and for studying the regulation of legume specific genes, e.g. plant genes involved in nodule formation and function.
Acknowledgements The authors wish to thank Drs M. Bevan and G. An for generously supplying the binary vectors used in this study.
References 1. An G, Watson BD, Stachel S, Gordon MP, Nester EW: New cloning vehicles for transformation of higher plants. EMBO Journal 4:277-284 (1985). 2. Bevan MW, Flavell RB, Chilton MD: A chimaeric antibiotic resistance gene as a selectable marker for plant cell transformation. Nature 304:184 187 (1983). 3. Bevan MW: Binary Agrobacterium vectors for plant transformation. Nucleic Acids Research 12:8711 8721 (1984). 4. Bradford MM: A rapid and sensitive method for quantification of microgram quantities of protein utilizing the principle of protein dye binding. Anal Biochem 72:248-254 (1976). 5. Caplan A, Herrera-Estrella L, lnze D, Van Haute E, Van Montagu M, Schell J, Zambryski P: Introduction of genetic material into plant cells. Science 222:815-821 (1983). 6. Chilton MD, Drummond MH, Merlo DJ, Sciaky D, Montoya A, Gordon MP, Nester EW: Stable incorporation of plasmid DNA into higher plant cells: the molecular basis of crowngall tumorigenesis. Cell 11:263-271 (1977). 7. Deak M, Kiss GB, Koncz C, Dudits D: Transformation of Medicago by Agrobacteriurn mediated gene transfer. Plant Cell Reports 5: 97-100 (1986).
8. Ditta G, Stanfiel S, Corbin D, Helinski DR: Broad host range DNA cloning system for Gram-negative bacteria: Construction of a gene bank of Rhizobium meliloti. Proc Natl Acad Sci USA 77:7347 7351 (1980). 9. Fraley RT, Rogers SG, Horsch RB, Sanders PR, Flick JS, Adams SP, Binner ML, Brand LA, Fink CL, Fry JS, Gallup GR, Goldberg SB, Hoffmann NL, Woo SC: Expression of bacterial genes in plant cells. Proc Natl Acad Sci USA 80: 4803-4807 (1983). 10. Hoekema A, Hirsch PR, Hooykaas PJJ, Schilperoort RA: A binary plant vector strategy based on separation of virand T-region of the Agrobacterium tumefaciens TJplasmid. Nature 303:179-180 (1983). 11. Horsch RB, Fry JE, Hoffmann NL, Eichholtz D, Rogers SG, Fraley RT: A simple and general method for transferring genes into plants. Science 227:1229-1231 (1985). 12. Murashige T, Skoog F: A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15: 473- 497 (1962). 13. Otten L: kysopine dehydrogenase activity as an early marker in crown gall transformation. Plant Sci Lett 25:15 27 (1982). 14. Otten LABM, Schilperoort RA: A rapid micro scale method for the detection of lysopine and nopaline dehydrogenase activities. Biochim Biophys Acta 275:497-500 (1978). 15. Reed KC, Mann DA: Rapid transfer of DNA from agarose gels to nylon membranes. Nucleic Acids Res 13:7207 7221 (1985). 16. Reiss B, Sprengel R, Willi M, Schaller H: A new, sensitive method for qualitative and quantitative assay of the neomycin phosphotransferase in crude cell extracts. Gene 30: 211-218 (1984). 17. Sutton W: Some features of the DNA of Rhizobium bacteroids and bacteria. Biochim Biophys Acta 3 6 6 : 1 - 1 0 (1974). 18. White DWR: Plant regeneration from long-term suspension cultures of white clover. Planta 162:1-7 (1984). 19. Zambryski P, Joos H, Genetello C, Van Montagu M, Schell J: Ti-plasmid vector for the introduction of DNA into plant cells without altering their normal regeneration capacity. EMBO Journal 12:2143-2150 (1983).
461
Transformation of the forage legume Trifolium repens L. using binary Agrobacterium vectors Derek W. R. White and Denise Greenwood
Grasslands Division, Department of Scientific and Industrial Research, Palmerston North, New Zealand Received 4 November 1986; in revised form 16 February 1987; accepted 20 February 1987
Key words: Agrobacterium tumefaciens, binary vectors, gene transfer, kanamycin resistance, legume transformation, Trifolium repens Abstract
A system was established for introducing cloned genes into white clover (Trifolium repens L.). A high regeneration white clover genotype was transformed with binary Agrobacterium vectors containing a chimaeric gene which confers kanamycin resistance. Transformed kanamycin resistant callus was obtained by culturing Agrobacterium inoculated stolon internode segments on selective medium. The kanamycin resistance phenotype was stable in cells and in regenerated shoots. Transformation was confirmed by the expression of an unselected gene, nopaline synthase in selected cells and transgenic shoots and by the detection of neomycin phosphotransferase II enzymatic activity in kanamycin resistant cells. Integration of vector DNA sequences into plant DNA was demonstrated by Southern blot hybridisation.
Introduction
Efficient methods have been developed for introducing cloned genes into some plant species (5, 11). Hence, defined additions to the plant genome can now be made using a combination of gene manipulation and gene transfer techniques. These developments are important for studying the regulation of plant gene expression and for engineering improvements in crop plant performance. Unfortunately, progress in developing systems for the transformation of economically important legume species has been very limited. The natural gene transfer capacity of Agrobacterium tumefaciens has been modified to provide a method for the transformation of dicotyledonous plants. Virulent strains of A. tumefaciens cause tumorous growths when inoculated on to wounded tissue of susceptible plants. A specific segment (TDNA) of the large Ti-plasmid is integrated into the
plant nuclear genome (6). This T-DNA contains genes for the synthesis of novel compounds called opines and genes conferring hormone independent growth. To allow plant regeneration from transformed cells the tumour-inducing genes have been deleted from T-DNA. Transformed cells can be identified by incorporating a chimaeric gene, constructed using regulatory regions from the nopaline synthase gene and the coding sequence of a bacterial neomycin phosphotransferase which confers kanamycin resistance, into the non-oncogenic TDNA (2, 9). Transfer of the T-DNA into plant cells is mediated by trans acting products from several genes located in a separate virulence (vir) region of the Ti-plasmid. The vir and T-DNA components can be placed on separate plasmids to form a binary system of T-DNA transfer (10). Consequently, efficient binary Agrobacterium vectors have been constructed by placing a T-DNA, containing a selectable gene, into a broad host range plasmid
462 which will replicate in both Escherichia coli and A. tumefaciens (1, 3). Plant regeneration from cultured cells is required for current transformation methods. Unfortunately, plant regeneration from cultured cells of many legumes is inefficient. However, an efficient method has been developed for regenerating plants from cells of white clover (Trifolium repens) by selecting a high-regeneration genotype (18). Here we describe the development of a system for introducing cloned genes into this white clover genotype using binary Agrobacterium vectors containing a selectable kanamycin resistance gene.
Materials and methods
Plant material Three-year-old vegetatively propagated plants of a "high-regeneration" white clover genotype, WR8 (18), were grown in 42 cm x 30 cm x 6 cm plastic trays in a potting mix of 60% peat and 40°70 sand containing Osmocote controlled release fertiliser (18% N, 4.8% P, 8.3% K, 3.4% S) (Sierra Chemical Europe B.V.). Plants were grown in glasshouse conditions of 1 5 ° C - 2 6 ° C , natural daylight and photoperiod and were trimmed at 4 month intervals. New stolon outgrowths containing 5 - 8 internodes were used during a 14-month period for transformation experiments. There was no indication that the time of the year when stolons were collected influenced transformation frequency.
Bacterial strains and plasmids White clover transformation Two non-oncogenic A. tumefaciens strains, LBA4404 (10) and GV3850 (19) (containing modified Ti-plasmids pAL4404 and pGV3850 respectively), were used in transformation experiments together with four different binary vectors (pBin6, pBinl9 (3); pGA470, pGA472 (1)) containing chimaeric kanamycin resistance genes. One of the vectors, pBin6, also has an unselected marker gene encoding nopaline synthase. The binary vectors pGA470 and pGA472 were mobilised from E. coli strain MC1000 into A. tumefaciens strain GV3850 using a triparental mating (8) with E. coli strain HB101 containing pRK2013. Transconjugants GV3850(pGA470) and GV3850(pGA472) were selected on minimal medium containing tetracycline (10/zg/ml) and kanamycin (10/xg/ml). Plasmid isolation and restriction endonuclease mapping was used to confirm the presence of vectors in A. tumefaciens. A. tumefaciens strains were cultured on TY medium containing 5 g / L Difco bacto tryptone, 3 g / L Difco yeast extract, 1.3 g / L CaClz.6H20 and 15 g / L Difco bacto agar, at 28 °C. E. coli strains were grown on LB agar medium at 37 °C.
Stolon internode segments, 1 - 2 cm in length, were surface sterilised for 30 min in 2.5°7o (w/v) sodium hypochlorite containing 1 - 2 drops of "Tween 80" wetting agent and washed five times in sterile distilled water. After the bleached ends were cut off inoculation was effected by touching both the basal and apical fresh wound sites on to A. tumefaciens cultured on agar solidified medium. Segments were incubated for 1 - 3 days in a moist petri dish at 28 °C in continuous light (10 W/m2). Then 1 mm terminal slices were cut off and placed inoculated side uppermost on callusing medium containing cefotaxime (Calbiochem) (500 t~g/ml) to stop bacterial growth and kanamycin (100 ~g/ml) for the selection of transformed plant cells. Callus that developed on selection medium was subcultured at 21-day intervals on to the same medium. Some kanamycin-resistant cell lines were grown in suspension culture in the absence of antibiotics. Shoot formation was initiated from callus both in the presence and in the absence of kanamycin (100 ~g/ml). Media and conditions for callus initiation, maintenance, suspension culture and shoot regeneration were as previously described (18). To test for the expression of kanamycin resistance in transgenic shoots, shoots regenerated from both transformed and non-transformed cell lines in the
463 absence of selection were placed on 1/2 strength MS medium (12) containing kanamycin (100/~g/ml) and cultured for 30 days.
Nopaline analyses For the detection of nopaline in callus or shoot material, 100-200 mg fresh weight of tissue was transferred to a sterile Eppendorf tube and macerated with a plastic rod. After centrifugation for 15 rain in a MSE micro-centrifuge, 5 >1 aliquots of clear supernatant were spotted, 2 cm apart, on Whatman 3MM chromatography paper and dried. A mixture of 0.02% nopaline, 0.02% octopine and 30 mM arginine was used as a standard. Electrophoresis was at 45 V/cm for 30 min in formic acid-acetic acid-water (1:3:16, v/v) at pH 2.1. The dried papers were stained by spraying with a freshly prepared 1:1 solution of 0.2% ( w / v ) p h e n a n threnequinone in 100070 ethanol and 10% (w/v) NaOH in 60% ethanol (14). The dried papers were viewed and photographed under U.V. light (254 nm).
Neomycin phosphotransferase H assay Tissue samples (100 mg fresh weight) taken from calli grown in the presence of kanamycin (100/~g/ml) were macerated with a plastic rod in Eppendorf tubes in an equal volume of ice-cold extraction buffer containing 62.5 mM Tris-HC1 pH 6.8, 10% glycerol, 5O7o /5-mercaptoethanol, 0.1°70 sodium dodecyl sulphate (SDS) and 100 #g/ml phenylmethylsulphonyl fluoride. After centrifugation for 5 min in a MSE micro-centrifuge aliquots (50/~g protein as determined by the method of Bradford (4)) of extracts containing bromophenol blue were electrophoresed through a 10°70 polyacrylamide gel at 25 mA for 3 h in a cold room and assayed for neomycin phosphotransferase II activity in situ as reported by Reiss et al. (16). The gel was overlayed with an agarose slab containing 33.5 mM Tris maleate, 21 mM MgC1, 200 mM NH4C1 , 1.5% low melting point agarose (Seaplaque, FMC), 30/,g/ml kanamycin and 25 /xCi [3,-32p]ATP,
2 000 Ci/mmol, incubated for 30 min at room temperature and blotted on to Whatman P81 phosphocellulose paper overnight. To remove excess background radioactivity the P81 paper was incubated for 30 rain in a solution containing 1°70 SDS and 1 mg/ml proteinase K (Boehringer Mannheim) at 60°C and washed 3× in 10 mM phosphate buffer (pH 7.5) at 80°C. The dried paper was exposed to X-ray film in the presence of intensifying screens at - 7 0 ° C for 1 - 4 h.
DNA preparation and Southern blot analysis Plant DNA was isolated from suspension cultured cells of both transformed and non-transformed cell lines using the procedure of Sutton (17). Samples (10 >g) of total plant DNA were digested with approximately I00 units of each of the restriction endonucleases used for 1 6 - 2 4 h. DNA samples were electrophoresed in 7-mm-thick, horizontal, 0.8% agarose (BRL, ultrapure) gels in Tris acetate buffer (pH 8.0) using 1.25 V/cm. The DNA was then depurinated and transferred to Zeta-Probe nylon membrane (Bio-Rad) in a flow of 0.4 M NaOH as described by Reed and Mann (15). To determine molecular weights directly from subsequent autoradiograms of the filters, parallel lanes of the gel contained a Hind III plus EcoRI, Hind III, digest of bacteriophage X DNA. To estimate integrated DNA copy number, 1 and 5 copy reconstructions were made by adding 15 ng and 75 ng respectively of the DNA to be used as a probe to nontransformed plant DNA prior to restriction endonuclease digestion. Specific probes were prepared from bacteriophage ), and pKan 2 (pBR322 with a 3.4 kilobase pair insert encoding a neomycin phosphotransferase II gene from Tn5) DNA by labelling with 32p in modified nick translation reactions (Reed and Mann, pers. comm.). For one reaction 500 ng of DNA was incubated with 1 ng of DNase I (bovine pancreas, Sigma) in a total volume of 35 /A containing 50 mM Tris-HC1 (pH 7.5), 7.5 mM magnesium acetate, 4 mM dithiothreitol, 2.5 /~M each of dATP, dGTP, d T T P (Boehringer), 100 t,g/ml of nuclease-free BSA (Boehringer) and 20/~Ci of [c~-32p]dCTP (3000 Ci/mmol; Amer-
464 sham), at 14°C for 15 min. DNase I was then inactivated by heating at 70 °C for 5 min and placing on ice. Subsequently, 8 units o f D N A polymerase I (Boehringer) were added and incubation extended for a further 15 rain at 14 °C for repair synthesis. The reaction was stopped by the addition of 20 mM EDTA and 0.5% SDS. With this procedure more than 90O7o o f the [c¢-32P]dCTP was incorporated into DNA, giving probes labelled at a specific activity of 2 × 108 dpm//zg DNA. Labelled DNA was denatured by adding 2/xl of 4 M N a O H to the reaction mixture. Zeta-Probe filters were prehybridised in a polyethylene bag containing 10 ml of 10 × Denhart's solution (0.2 070 each of ficoll, polyvinyl pyrrolidone and BSA) containing 50 mM H E P E S buffer (pH 7.0), 3 x SSC (1 × SSC = 0.15 M sodium chloride, 0.015 M sodium citrate), 17/xg/ml herring DNA, 20/~g/ml E. coli t R N A and 0.1°70 SDS for 16 h at 68°C. All but 2 ml of the solution was then removed from the bag, denatured probes (k 106 dpm, pKan 2 2 - 4 × 106 dpm) were added and the filters were incubated for a further 16 h at 68°C. Filters were washed for 15 min each in 2 × SSC, 0.1070 SDS; 0.5 × SSC, 0.1070 SDS; 0.1 x SSC, 0.1% SDS; all at room temperature and finally in 0.1 x SSC, 0.1% SDS at 50°C for 30 rain. The dried filters were placed between Cronex Lighting Plus (Du Pont) intensifying screens with X-ray films for 4 - 7 days at - 7 0 ° C .
Results
Selection of kanamycin-resistant white clover transforman ts Preliminary experiments established that the white clover genotype WR8 will form tumours when stolon internode wounds are inoculated with virulent Agrobacterium tumefaciens strains. Also callus initiation from white clover stolon internode explants is inhibited by 100/zg/ml kanamycin. This allowed the development of a A. tumefaciens mediated transformation system which utilises a chimaeric gene that confers kanamycin resistance to plant cells. Two non-oncogenic A. tumefaciens strains harbouring different helper plasmids, which provide virulence functions, were used to mobilise the TDNA sited kanamycin resistance gene from four different binary vectors into white clover (Table 1). Since both pBin6 and pBinl9 are rapidly lost from A. tumefaciens LBA4404 in the absence of selection (3), the wounded ends of internode segments were inoculated by contact with bacterial colonies cultured on selective medium. After a short period of incubation, to allow transformation to occur, callus initiation, bacterial elimination and transformant selection were combined in one step by transferring terminal segments to callusing medium containing cefotaxime and kanamycin.
Table 1. Transformation of white clover using A. tumefaciens containing binary vectors. A. tumefaciens
Plasmid(s)
Number of segments treateda
07oof segments kanamycin resistant b
pAL4404 pAL4404, pBinl9 pAL4404, pBin6 pGV3850 pGV3850, pGA470 pGV3850, pGA472
208 99 50 75 72 24
0 41 44 0 24 33
strain
LBA4404 LBA4404(pBin19) LBA4404(pBin6) GV3850 GV3850(pGA470) GV3850(pGA472)
a WR8 stolon segments taken from the 3rd - 8th subapical internodes were inoculated, incubated for 3 days, then placed on callusing medium containing 100/~g/ml kanamycin. b Segments were scored for the presence of kanamycin resistant growth after 3 weeks on selective medium.
465
Fig. 1. Selection of transformed kanamycin resistant white clover cells. Stolon internode segments were inoculated with A. tumefaciens strains, incubated for 3 days and transferred to callusing medium containing 100 #g/ml kanamycin. Segments transformed with pBin6 are shown on the right and segments inoculated with strain LBA4404 are shown on the left. The photograph was taken after 35 days of culture.
Kanamycin resistant outgrowths developed on segments inoculated with A. tumefaciens containing both a helper plasmid and binary vector (Fig. 1 and Table 1). These outgrowths were first visible 12-18 days after placing the segments on selective medium and the resistant callus grew rapidly when transferred to fresh medium. No kanamycin resistant callus was obtained from segments inoculated with A. tumefaciens harbouring only a helper plasmid (Fig. 1 and Table 1) even after prolonged (5 weeks) culture. Approximately 40°7o of the segments inoculated with LBA4404(pBin6) or LBA4404(pBin19) produced kanamycin-resistant callus, often from a number of different sites on the surface of the segment (Fig. 1). The proportion of segments that produced kanamycin resistant callus was lower when inoculation was with GV3850(pGA470) or GV3850(pGA472) than when inoculation was with LBA4404(pBin6) or LBA4404(pBin19). By contrast, kanamycin resistant callus appeared more rapidly when segments were transformed with GV3850(pGA470) or GV3850(pGA472). To refine the transformation method both the incubation period required and the effect of the position of internodes on the plant were examined. Two days of incubation in the presence of
LBA4404(pBin6) or LBA4404(pBin19) was required to obtain kanamycin resistant callus and higher frequencies resulted from a 3-day incubation period (Table 2). The proportion of segments producing kanamycin resistant callus varied according to the position of the internode on the plant. Segments taken from internodes near the shoot apex produced fewer kanamycin resistant calli than those taken from internodes in a more basal position (Table 3). After 3 - 4 subcultures on selective medium some Table 2. Effect of incubation period on transformation of white clover by binary Agrobacterium vectors. Incubation period (days)
A. tumefaciens
1
LBA4404 LBA4404(pBinI9) LBA4404(pBin6)
46 38 53
0 0 0
2
LBA4404(pBinl 9)
37
14
3
LBA4404 LBA4404(pBin19) LBA4404(pBin6)
208 99 198
0 41 37
strain
Number of segments treated
% of segments kanamycin resistant a
a Kanamycin resistant growth was scored after 3 weeks of culture.
466 Table 3. Influence of explant position on the frequency of transformation of white clover internode segments. Internode segment a
N u m b e r of segments treated b
°70 of segments kanamycin resistant c
1
41
5
2 3 4 5
42 42 38 26
24 31 47 42
in extracts from white clover cells transformed with pBin6, but not in extracts from cells transformed with pBinl9, a vector which does not have a nopaline synthase gene (Fig. 2A). Expression of nopaline synthase was detected in pBin6 transgenic shoots. Extracts taken from shoots regenerated in the absence of kanamycin selection were nopaline positive, whereas non-transformed shoots of white clover (WR8) did not accumulate nopaline (Fig. 2B).
a Segment 1 is closest to the apical meristem. b Inoculated with A. tumefaciens strain LBA4404(pBin6) for 3 days. c Kanamycin resistant growth was scored after 3 weeks of culture.
of the kanamycin resistant callus was grown in the absence of antibiotics either as callus or in suspension culture. Even after 3 - 5 months of culture in non-selective conditions these cells retained their kanamycin resistance phenotype. Approximately 3 months after initial selection kanamycin resistant callus obtained from segments inoculated with LBA4404(pBin6), LBA4404(pBin19) or GV3850(pGA470) was transferred to 1/2 strength MS medium without growth regulators to promote shoot formation. Shoot initiation occurred in both the presence and in the absence of 100 #g/ml kanamycin and was similar to that obtained on non-transformed callus from the WR8 genotype. Furthermore, shoots regenerated in the absence of selection from cells transformed with pBin6 or pBinl9 remained green and grew in the presence of 100 ~g/ml kanamycin, whereas shoots regenerated from non-transformed WR8 cells turned white after 14 days and died (data not shown).
Expression of an unselected marker genenopaline synthase To confirm that selected kanamycin resistant calli of white clover were transformed, the expression of an unselected gene within the T-DNA of pBin6, nopaline synthase, was analysed. Nopaline synthase activity in transformed plant cells leads to the accumulation of nopaline. Nopaline was detected
Fig. 2. Nopaline accumulation in cells transformed by A. tumefaciens containing pBin6 and in transgenic shoots. Extracts were assayed by paper electrophoresis with octopine (OCT) and nopaline (NOP) standards (lanes) and stained with phenanthrenequinone. (A) Extracts from cells transformed with pBin6 (Lanes 1 - 5 ) or pBinl9 (Lane 6). (B) Extracts from pBin6 transgenic shoots regenerated in the absence of kanamycin (Lanes 1 - 5 ) or from a non-transformed shoot (Lane 6).
467 The introduced neomycin phosphotransferase H gene was expressed
To demonstrate that the selected kanamycin resistance phenotype was due to the expression of an introduced neomycin phosphotransferase II (NPT I/) gene and the synthesis of the expected bacterial protein, NPT-H enzyme activity was assayed in situ after electrophoretic separation on a nondenaturing polyacrylamide gel. The activity was absent in extracts from non-transformed white clover callus and present in extracts from pBin6, pBinl9 and pGA470 transformants (Fig. 3). The activity of extracts from pGA472 transformants was not assayed. While NP-IZ-H activity from pBin6 and pBinl9 transformants had the same mobility as the bacterial enzyme, activity from pGA470 transformants had a slower mobility. Four different pGA470 transformants all had a higher NPT-H activity when compared with four pBinl9 transformed cell lines (data not shown).
lysed by Southern blot hybridisation. Separated restriction endonuclease enzyme fragments were blotted on to Zeta-Probe nylon filters and hybridised with a 32p labelled probe specific to the coding sequence of the introduced NPT-H gene. The probe hybridised with DNA from the transformed cell line (Fig. 4). While DNA from non-
D N A analysis o f a transformed white clover cell line
To confirm chromosomal integration of the chimaeric NPT-H gene, genomic DNA was isolated from a cell line transformed with pBinl9 and aria-
Fig. 3. Autoradiograph showing NPT-H activity of extracts from kanamycin resistant calli separated on a 10% polyacrylamide gel and assayed in situ. Lane 1 non-transformed. Lane 2 pBinl9 transformant. Lane 3 pBin6 transformant. Lane 4 pGA470 transformant. The arrow indicates the position of bacterial NPT-II activity.
Fig. 4. Southern blot analysis of D N A from white clover transformed by A. tumefaciens containing pBinl9. Total DNA extracted from transformed and non-transformed white clover cells was digested with restriction endonucteases and the fragments were separated by electrophoresis and transferred to ZetaProbe membrane. The radioactive probe was a nick-translated Hind Ill digest of a pBR322 derived plasmid containing a 3.4 kb Hind IIl NPT-II specific sequence from Tn5, Lane 1 is a EcoRI digest of non-transformed DNA and lanes 2 - 4 digests of transformed DNA. Lane 2, EcoRl; Lane 3, Hind III; Lane 4, Hind III/Pst!. A X specific probe was used to determine the D N A fragment sizes given from a X Hind I I I + [Hind Ill + EcoRI] digest.
468 transformed cells did not show any hybridisation signal. The pattern of NPT-H specific probe hybridisation to DNA fragments from EcoRI and Hind III digests (lanes 2 and 3, Fig. 4), suggests that the chimaeric NPT-H gene had been integrated into the white clover genome. The intensity of the hybridisation signal compared with 1 and 5 copy reconstructions (not shown) suggests that only one or 2 copies of pBinl9 T-DNA have been integrated into the genome.
Discussion
We demonstrate here that a combination of A. tumefaciens mediated DNA transfer, binary vectors containing a selectable chimaeric gene conferring kanamycin resistance and a genotype capable of regeneration from cultured cells can be used to establish a system for introducing foreign genes into white clover. Genetic transformation of white clover has been established on the basis of the following criteria: (i) the stable kanamycin resistant phenotype of selected calli and shoots derived from them; (ii) the NPT-H enzymatic activity found in transformed lines; (iii) the integration of an introduced gene sequence into plant genomic DNA; and (iv) expression of an unselected gene - nopaline synthase - in transformed cells and transgenic shoots. Thus the Agrobacterium-binary vector approach previously shown to function for introducing genes into tobacco (1, 3) and Medicago varia (7), is also an effective means of transforming white clover, an economically important legume. To date all the methods devised for introducing cloned genes into plants rely on a capability for regenerating plants from cultured cells or protoplasts. At present only a portion of white clover genotypes will give satisfactory plant regeneration from cell culture (18). Therefore, foreign DNA can be incorporated into only a limited number of white clover genotypes. However, since white clover is an outcrossing species, a foreign gene could be introduced into a single recipient genol~ype and the gene could subsequently be moved into an appropriate genetic background by conventional breeding practices.
The results presented in Table 1 indicate that transformation of WR8 stolon internode segments may be more effective with A. tumefaciens strains LBA4404(pBin6) and LBA4404(pBin19) than with strains GV3850(pGA470) and GV3850(pGA472). Interpretation of this result is complicated by differences in bacterial genetic backgrounds, virulence helper plasmids, and binary vectors. The earlier appearance of kanamycin resistant growth on segments inoculated with GV3850(pGA470) or GV3850(pGA472) suggests that the chimaeric kanamycin resistance gene in pGA470 and pGA472 may be more effective than the corresponding gene in pBin6 and pBinl9. The higher NPT-H activity levels of extracts from pGA470 transformants, when compared with pBin transformants, supports this conclusion. The N t ~ - H enzymatic activity of extracts from pGA470 transformants migrated slower on polyacrylamide gels than extracts from pBin transformants or bacteria containing the NPT-H gene. This alteration in the migration of NPT-H enzymatic activity is due to the method used to construct the chimaeric gene in pGA470. The gene in pGA470 was constructed by making an in-frame translational fusion between the NPT-H coding region and the amino terminus of the nos gene (1), whereas the corresponding gene in the pBin vectors has an intact NPT-H coding sequence. This results in the addition of positively charged amino acids to the amino terminus of the protein, the reason for its altered migration. The proportion of internode segments which produced kanamycin resistant callus was affected by both the length of the incubation period with A. tumefaciens and the position on the plant from which the explants were taken. Other reports (3, 13) have also commented on the influence of the position on the plant or the axial orientation of an explant upon transformation "competence". In white clover a combination of 3 days incubation with A. tumefaciens and the use of explants from the 4th internode gave the highest transformation frequency. The availability of a transformation system for white clover will enable the introduction of cloned foreign genes into this important forage legume.
469 This development has implications both for the improvement of white clover agronomic performance and for studying the regulation of legume specific genes, e.g. plant genes involved in nodule formation and function.
Acknowledgements The authors wish to thank Drs M. Bevan and G. An for generously supplying the binary vectors used in this study.
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