PlantCell Reports
Plant Cell Reports (1988) 7:512-516
© Springer-Verlag1988
Parameters affecting the frequency of kanamycin resistant alfalfa obtained by Agrobacterium tumefaciens mediated transformation Mireille Chabaud, Joan E. Passiatore, Frank Cannon, and Vicky Buchanan-WoHaston BioTechnica International, Inc., 85 Bolton Street, Cambridge, Massachusetts 02140, USA Received July 20, 1988/Revised version received October 17, 1988 - Communicated by J. M. Widholm
ABSTRACT Kanamycin resistant plants of Medicago varia A2 were obtained by an optimized procedure for high frequency transformation using Agrobacterium tumefaciens infection of leaf and petiole tissue. Parameters which affected the frequency were explant type, the Agrobacterium strain used and the time allowed for cocultivation. Under optimum conditions, i.e., using the Agrobacterium strain A281 and a 4 day cocultivation period, the frequency of transformed leaflets obtained was greater than 70%.
INTRODUCTION A. tumefaciens-mediated transformation has been reported with a growing number of plant species including Medicago. Deak et al. (1986) used the strain A281 containing a binary transformation vector and obtained kanamycin resistant calli from stem segments of M. varia which were regenerated to embryos through cell suspension culture. Shahin et al. (1986) have reported the transformation of a cultivar of M. sativa from stem segments using the strain LBA4404 with a binary vector. In this paper we study different parameters of the transformation method with the aim of improving the transformation efficiency as measured by the percentage of transformed explants. This work was done with a Medicago varia line known for its high regeneration potential via embryogenesis in vitro. We report a transformation protocol which enabled us to obtain kanamycin resistant plants at a high frequency,
MATERIALS AND METHODS Plant material The genotype used in this study is Medicago varia (M. sativa x M. falcata) A2, It is an autotetraploid Hungarian genotype selected by A. Atanassov for its embryogenic potential in vitro. Media and culture conditions
Offprint requests to." V. Buchanan-Wollaston
PetioLe or stem segments (0.5 to 1.0 cm long) and leaflets from axenic plants were wounded with a scalpel and used for in vitro culture. Callus was induced on a modified B5h medium (Atanassov and Brown 1984) and embryos began to appear after 6-8 weeks. Embryos at the globular to torpedo stage were transferred to B0i2Y medium for maturation (Bingham et al. 1975, Blaydes 1966). The healthiest looking embryos were then transferred to a modified Sh medium (Shbl0) (Stavarek et al. 1980, Schenk and Hilderbrandt 1972) for rooting and further growth into plantlets. All media above were modified by the addition of 3 mm MES buffer (2(N-Morpholino)ethanesulfonic acid) pH 5.8, and 4 g/L agarose (International Biotechnologies Inc., New Haven, CT) was used instead of agar. The explants were cultured in sterile petri dishes (90 x 15 mm) under 15 to 30 ~E m -2 s -1, with a 16h photoperiod at 25°C. The plantlets were transferred to Shbl0 medium in PlantCon sterile boxes (Flow Laboratories, McLean, VA) under the same light and temperature conditions. Minimum inhibitory concentration
of kanamycin
Leaves and petioles of A2 were cultured on B5h with 25, 50, i00 or 250 ~g/mL of kanamycin to test the sensitivity of the tissue to the antibiotic and determine a suitable concentration for selection of transformed cells. Bacterial
strains and plasmid
A supervirulent agropine strain A281 (An et al. 1986, Hood et al. 1987) and a disarmed octopine strain LBA4404 (Hoekema et al. 1983) were the Agrobacterium tumefaciens strains used for transformation. The plasmid pVWI30 (Buchanan-Wollaston et al. 1987) was introduced into the Agrobacterium strains by triparental crosses using pRK2013 as a helper plasmid (Ditta et al. 1980). Agrobacterium transconjugants containing the plasmid were selected with rifampicin at 25 Dg/mL and chloramphenicol at 20 ~g/mL or 300 ~g/mL for the strains LBA4404 and A281 respectively. The plasmid pVW130 (Figure i) contains the T-DNA borders flanking the APHII gene for kanamycin resistance which is fused to the nopaline synthase promoter and nopaline synthase polyadenylation site.
513
BamHI
EcoR~1 A P H pSUP104
~
H ~'BgllI
BamH ElcoR1 Figure i. The plasmid pVWI30 used in the transformation experiments (Buchanan-Wollaston et al. 1987). Inoculum preparation
Isolation of plant DNA and Southern blotting
The strains LBA4404 and A281 containing the vector pVWI30 were cultured overnight in i0 mL liquid LB with chloramphenicol at 20 ~g/mL or i00 ~g/mL respectively by shaking at 250 to 300 rpm at 30°C. The overnight culture was pelleted at 2300xg for 15 min, resuspended in the same volume of 0.85% saline solution, and diluted in saline to give 5 x 107 bacteria/mL. Transformation
procedure
The method used was a variation of the explant method of Horsch et al. (1985). The explants (wounded leaflets and petiole segments from sterile plants) were dipped briefly in the diluted bacteria, blotted between sterile filter papers, and placed on B5h medium for 2, 3 or 4 days of cocultivation. A layer of either 4 day old alfalfa cell suspension cultured in liquid B5h or liquid B5h with 50 ~M acetosyringone (Stachel et al. 1985) was used to induce the Agrobacterium Vir genes. This was pipetted onto the solid B5h medium (i mL/plate) and covered with a sterile filter paper onto which the explants were placed, abaxial side up, after infection. After 4 day cocultivation the explants were washed with sterile distilled water to remove excess bacteria, blotted and transferred to selective medium. Explants cocultivated for 2 or 3 days were transferred without washing. Selection of transformed
tissue
After cocultivation the explants were transferred to B5h medium containing carbenicillin (carb) at 300 ~g/mL and kanamycin (kan) at 50 or i00 ~g/mL. Explants were transferred every 3-4 weeks to fresh selective medium. The kanamyein resistant embryos were placed on B0i2Y containing carb 300 ~g/mL and kan 50 ~g/mL for embryo maturation, and these mature embryos were developed further and rooted on Shbl0 containing carb 300 ~g/mL and kan 50 ~g/mL. Recallusing
a slightly modified version of a method previously described (An et al. 1985, Haas and Dowding 1975). Crude extracts of the transformed tissues were prepared by grinding between 0.5 and 1.0 grams (x g) of fresh tissue in an equivalent volume of extraction buffer (x mL) (0.5M sucrose, 0.1M Tris-HCl, 0.1% ascorbic acid, 0.1% cysteine-HCl, pH 7.5) and spun in an Eppendorf centrifuge for 5 min. The reaction mixture contained 20 ~L of the extract, i0 ~L of the assay buffer (67 mM Tris-HCl, 42 mM MgCI2, 400 mM NH4CI, 1.7 mM dithiothreitol, pH 7.5), I0 ~L of [32p]-ATP solution (15 ~L of 50 mM ATP adjusted to pH 7.2 with NaOH plus 50 ~L of [32p]-ATP (30 Ci/mmol and 2 mCi/mL) made up to i mL with deionized water) and 2 ~L of 1 mg/mL kanamycin. After incubation at 37oc for 20 min the reaction was terminated by loading the mixture on 1 cm 2 of Whatman P-81 phosphocellulose paper. The 1 cm2 P-81 papers were washed twice in hot water (80oc) for 5 min and four times in 50 mM sodium phosphate buffer pH 7.4 for 30 min. The papers were then dried and counted in i0 mL ScintiVerse II scintillation fluid (Fisher Scientific, Springfield, NJ).
assay
Leaves and petioles of plantlets derived from kanamycin resistant calli were cultured in vitro on B5h medium containing kan at i00 ~g/mL. Neomycin phosphotransferase
assay
Neomycin phosphotransferase
activity was measured by
Total DNA was isolated from transformed plantlets as described by Dellaporta et al. (1983). The DNA was digested with EcoRI, restriction fragments were separated on a 0.7% agarose gel and transferred by alkaline blotting to a Zeta-Probe membrane (BioRad Laboratories, Rockville Centre, NY) as described by Reed (1986). The BglII-PstI fragment of the NPTII gene of Tn5 was cloned into pSP65 (Melton et al. 1984), and 32p labelled RNA was synthesized from this clone using the Riboprobe SP6 transcription system according to manufacturer's instructions (Promega Biotec, Madison, WI). Hybridization was carried out as follow~ The membrane was hybridized for at least 15 min in 20 mL of prehybridization buffer at 58oc with strong agitation. The prehybridization buffer contained IM Tris HCI pH 7.4, 50% deionized formamide, 20% dextran sulfate, i% SDS. The buffer was mixed by inversion and put in a 58oc water bath for 15 min, NaCI was then added to obtain a final concentration of 0.4M, mixed and put in the same water bath for 15 min, then the buffer was filtered through a 0.45 ~M filter. The labelled Riboprobe and 1.5 mg of denatured salmon sperm DNA were added to the bag containing the membrane and the prehybridization buffer. Hybridization was carried out overnight at 58oc with strong agitation. The next day the membrane was rinsed briefly in IxSSPE, 1% SDS, washed three times in IxSSPE, 1% SDS at 65oc for 15 min, once in 0.1xSSPE, i% SDS at 65°C for 20 min, and briefly in IxSSPE at room temperature. The membrane was then wrapped in Saran Wrap and exposed to X-ray film for 5 to i0 days at -70°C. RESULTS Evidence for alfalfa transformation The A2 genotype was found to be very sensitive to kanamycin and no growth of calli or embryos was observed on media containing kanamycin at 25 ~g/mL or more. Therefore kanamycin at 50 or i00 ~g/mL was used for selection of transformed ealli. Kanamycin resistant calli developed on B5h containing carb at 300 ~g/mL and kan at 50 or i00 ~g/mL after approximately I month of culture and kanamycin resistant embryos appeared after another month on the same medium (Figure 2). The embryos matured to the first leaflet stage on B0i2Y containing earb 300 ~g/mL and kan 50 or i00 ~g/mL. No callus was observed on control explants (explants treated with bacteria containing no plasmid placed on kanamycin 50 or i00 ~g/mL).
514 Transformation and DNA integration were confirmed by Southern blotting of DNA from 4 plantlets derived from individual kanamycin resistant calli (Figure 3). Two plantlets showed two sites of integration of the kanamycin resistance gene; two others showed one. No hybridization was obtained to the control plant DNA.
a
b
c
d
e
f
15.7 Kb
Figure 2. Transformation of leaflets and petioles of kanamycin by Agrobacterium tumefaciens. (a) Kanamycin resistant embryogenic calli from leaflets and petioles infected with A281 (pVWI30) 3 weeks after start of culture on B5h medium. (b) Kanamycin resistant embryos derived from a leaflet infected by A281 (pVWI30) 6 weeks after start of culture on B5h medium. This tissue piece was about 5 mM across. (c) Transgenic plantlets derived from kanamycin resistant calli transformed with LBA4404 (pVWI30).
Wounded leaves and petioles from plantlets derived from kanamycin resistant calli were cultured on B5h medium containing kanamycin at 50 or i00 Hg/mL for a recallusing assay. After two months embryos developed from the transformed explants. Control explants from a non-transformed plantlet showed no callus or embryo development on the same medium. The calli and embryos from the recallusing assay were tested for neomycin phosphotransferase activity. Recallused explants from medium containing kanamycin at i00 ~g/mL showed high levels of NPTII activity (Table i). Untransformed callus showed low background activity.
Table i. NPTII activity in the embryogenic calli developed from explants from three different kanamycin resistant plantlets on medium containing kanamycin at i00 ~g/mL.
c.p.m./20 DL extract control a kanamycin resistant calli
394 3795 5055 2102
aCalli from leaves and petioles of an untransformed plantlet regenerated on medium without kanamycin.
3.2
Kb
1.5
Kb
Figure 3. Southern analysis of DNA from kanamycin resistant plantlets transformed with LBA4404 (pVWI30). Plant DNA was digested with EcoRI. Lane a: control plant DNA (untransformed). Lanes b, c, d, e: DNA of 4 different transformed plants. Lane f: molecular size markers.
Parameters
affecting
transformation
frequency
Several parameters were studied in order to obtain the highest efficiency of transformation. These were explant type, different Agrobacterium strains, number of days of cocultivation and concentration of kanamycin used for selection. The results are shown in Table 2 and are expressed as percentage of explants transformed. Observations were made after at least 3 months of culture and only the embryogenic calli were counted. Results indicated that several of the parameters tested had a significant effect on the transformation frequencies obtained (Table 2). The wounded leaves gave a transformation frequency four times that obtained with petioles, indicating that leaves are a better source of tissue for transformation. The A281 strain does not produce tumors on the A2 genotype and the kanamycin resistant calli formed after infection with A281 were embryogenic, indicating that integration of the kanamycin resistance gene had occurred and that the presence of the T-DNA in this strain did not give rise to tumor induction. The transformation efficiency of this strain was at least twice that of LBA4404. The number of days of cocultivation had a significant effect on transformation efficiency. A 4 day cocultivation resulted in a transformation frequency at least twice as high as that obtained after 2 or 3 days cocultivation. After a 4 day cocultivation, control explants appeared more resistant to kanamycin, swelling slightly, but they never developed embryogenic calli. Selection on kanamycin at 50 or i00 ~g/mL did not affect the transformation frequency. The maximum frequency obtained under optimum conditions when wounded leaflets were cocultivated with A281 (pVWI30) for 4 days on an alfalfa cell suspension layer and selected on kanamycin at 50 or I00 ~g/mL was 72% (data from two independent experiments).
515 Table 2. Percentage of transformed explants (explants giving rise to one or more kanamycin resistant embryogenic calli compared to the total number of explants tested) obtained under various conditions of transformation.
Explants transformed/Total
Parameters Explants a
Leaves
99/272
Petioles .
.
.
.
.
.
.
.
.
.
.
.
.
Bacteria b
.
.
.
.
.
.
.
.
.
.
.
.
.
Cocultivation time c
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
Selection on kan d
.
.
.
.
.
.
.
.
9 .
.
.
.
.
.
.
.
.
LBA4404
18/127
14
A281
40/119
34
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
2 days
6/60
i0
3 days
35/183
19
4 days .
36
23/256 .
.
.
.
.
.
.
.
31/71 .
.
.
.
.
.
.
.
.
.
.
.
.
.
Significant difference e
%
yes .
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
50 Ng/mL
18/90
20
i00 Ng/mL
20/88
23
.
yes .
.
no
43 .
.
yes .
.
.
.
.
.
.
.
almost impossible to wound them longitudinally). Therefore wounded leaves may offer more sites for infection by A. tumefaciens. Virulence of the Agrobacterium strain had a significant effect on the transformation frequency. A281 (pVWI30) does not form tumors on the A2 genotype, and all the calli obtained with A281 (pVWI30) when cultured in vitro gave embryos after 3 or 4 months. The observations made with A281 confirmed the increased virulence of this strain as was reported previously for tobacco (An et al. 1986). Duration of cocultivation was an important parameter and differences observed could be due to several factors. The bacterial concentration was higher after 4 days than after 2 or 3 days and this may increase the probability of DNA transfer. However it was important to control bacterial growth since overgrowth of bacteria can kill the tissue. The 4 day cocultivation period allowed selection on kanamycin for an extra day, allowing further expression of the kanamycin resistance gene which could give the transformed cells a better chance of survival once selection is applied.
.
no
aResults from 14 independent experiments with the 2 bacterial strains, a 2 or 3 or 4 day cocultivation, selected on kanamycin at 50 or i00 Dg/mL. Petioles and leaves were tested in the same experiment. bResults from 4 experiments in which the 2 bacterial strains were compared, with a 3 day cocultivation and selection on kanamycin at 50 or i00 ~g/mL. Petioles and leaflets were included. CResults from 2, 8 and 4 experiments with a 2 or 3 or 4 day cocultivation respectively, with LBA4404 (pVWI30) and selection on kanamycin at 50 or i00 ~g/mL. After 4 days coculture the explants were washed before being transferred to selective medium. The three durations were tested in different experiments. Petioles and leaflets were included. dResults from 4 experiments using the 2 bacterial strains with a 2 or 3 or 4 day cocultivation in which the 2 concentrations of kanamycin were compared. Petioles and leaflets were included. e Slgnlflcance . " " is based on the X 2 test with a probability of less than 5% that the difference is due to sampling error. DISCUSSION Kanamycin resistant plantlets of alfalfa were obtained at a high frequency and we have presented evidence to show that they were transformed. All kanamycin resistant calli tested were positive for NPTII or in the Southern hybridization and since no resistant calli ever appeared on control plates it was assumed that all kanamycin resistant calli obtained were transformed. The plantlets tested had either one or two sites of integration of the kanamycin resistance gene. In this study three factors appeared to affect the transformation efficiency of the Medicago varia A2 genotype: the type of explant, virulence of the A. tumefaciens strain used, and duration of the cocultivation. Leaf tissue was more amenable to transformation than stem or petiole tissue, perhaps due to the fact that wounding was easier on leaves than on petioles (petioles were so thin that it was
These results allow us to describe a suitable protocol for transformation of the A2 genotype of Medicago varia based on the parameters that have been studied. This protocol is itemized in Table 3. Table 3.
Protocol for A2 transformation.
Agrobacterium tumefaciens strain A281 containing a suitable plasmid is grown overnight in liquid LB with selective antibiotic, at 30°C, shaken at 300 rpm. The culture is pelleted by centrifugation at 2300xg, 15 min, resuspended in an equal volume of 0.85% saline and then diluted to give 5 x 107 bacteria/mL. - Wounded leaves are dipped briefly into the bacterial suspension and blotted on sterile filter paper. One mL of alfalfa cell suspension is spread over solidified B5h medium. A filter paper disc is placed on top of the cell suspension. - Dipped tissue is placed on the filter paper. - Tissue is cocultivated with bacteria for 4 days. - The tissue is then washed in sterile distilled water and blotted. - The tissue is placed on solid B5h + carb 300 ~g/mL + kan i00 ~g/mL. - Every 3-4 weeks the tissue is transferred to the same fresh selective medium. By this protocol a transformation efficiency of more than 70% has been obtained in several experiments.
For each variety of alfalfa and, more generally, for each variety of any species, these three parameters could be optimized to increase transformation frequencies. The tissue most amenable to A. tumefaciens infection and the most virulent bacterial strain may vary from one species to another and should be determined in each case. Optimum cocultivation time could vary from one genotype to another in the same genus, depending on resistance of the
516 genotype to the antibiotic and sensitivity of the genotype to overgrowth of the A. tumefaciens. Now that the transformation protocol has been optimized with a highly embryogenic line of Medicago, transformation of embryogenic genotypes from commercial cultivars of alfalfa should be possible. Introduction of useful foreign genes such as those for herbicide resistance, disease resistance, improved protein content and other desirable traits by this method will allow development of new alfalfa varieties. ACKNOWLEDGMENTS We thank G. An for kindly providing the strain A281, P. McLean and G. Hernandez for helpful discussion, and A. Naser and C. Cornell for technical assistance. REFERENCES An G, Watson BD, Stachel S, Gordon MP, Nester EW (1985) EMBO J. 4: 277-284. An G, Watson BD, Chiang CC (1986) Plant Physiol. 83: 529-534. Atanassov A, Brown DCW (1984) Plant Cell, Tissue and Organ Culture 3: 149-162. Bingham ET, Hurley LV, Kaatz DM, Saunders JW (1975) Crop Science 15: 719-721. Blaydes DF (1966) Plant Physiol. 19: 748-753.
Buchanan-Wollaston V, Passiatore J, Cannon F (1987) Nature 328: 172-175. Deak M, Kiss GB, Koncz C, Dudits D (1986) Plant Cell Reports 5: 97-100. Dellaporta 8L, Wood J, Hicks JB (1983) Plant Mol. Biol. Reporter i: 19-21. Ditta D, Stanfield S, Corbin D, Helinski DR (1980) Proc. Natl. Acad. Sci. USA 77: 7347-7351. Haas M, Dowding JE (1975) Methods in Enzymol. 43: 611-633. Hoekema A, Hirsch PR, Hooykaas PJJ (1983) Nature 303: 179-180. Hood EE, Fraley RJ, Chilton MD (1987) Plant Physiol. 83: 529-534. Horsch RB, Fry JE, Hoffmann NL, Eichholz D, Rogers SG, Fraley RJ (1985) Science 227: 1229-1231. Melton DA, Krieg PA, Rebagliati MR, Maniatis T, Zinn K, Green ~fR (1984) Nucl. Acids Res. 12: 7035-7056. Reed KC (1986) BioRad Bulletin 1233: 1-5. Schenk RU, Hilderbrandt AC (1972) Can. J. Bot. 50: 199-204. Shahin EA, Spielman A, Sukapinda K, Simpson RB, Yasher M (1986) Crop Science 26: 1235-1239. Stachel SE, Messens E, Van Montagu M, Zambryski P (1985) Nature 318: 624-629. Stavarek ST, Croughan TP, Rains DW (1980) Plant Sci. Lett. 19: 253-261.
Plant Cell Reports (1988) 7:512-516
© Springer-Verlag1988
Parameters affecting the frequency of kanamycin resistant alfalfa obtained by Agrobacterium tumefaciens mediated transformation Mireille Chabaud, Joan E. Passiatore, Frank Cannon, and Vicky Buchanan-WoHaston BioTechnica International, Inc., 85 Bolton Street, Cambridge, Massachusetts 02140, USA Received July 20, 1988/Revised version received October 17, 1988 - Communicated by J. M. Widholm
ABSTRACT Kanamycin resistant plants of Medicago varia A2 were obtained by an optimized procedure for high frequency transformation using Agrobacterium tumefaciens infection of leaf and petiole tissue. Parameters which affected the frequency were explant type, the Agrobacterium strain used and the time allowed for cocultivation. Under optimum conditions, i.e., using the Agrobacterium strain A281 and a 4 day cocultivation period, the frequency of transformed leaflets obtained was greater than 70%.
INTRODUCTION A. tumefaciens-mediated transformation has been reported with a growing number of plant species including Medicago. Deak et al. (1986) used the strain A281 containing a binary transformation vector and obtained kanamycin resistant calli from stem segments of M. varia which were regenerated to embryos through cell suspension culture. Shahin et al. (1986) have reported the transformation of a cultivar of M. sativa from stem segments using the strain LBA4404 with a binary vector. In this paper we study different parameters of the transformation method with the aim of improving the transformation efficiency as measured by the percentage of transformed explants. This work was done with a Medicago varia line known for its high regeneration potential via embryogenesis in vitro. We report a transformation protocol which enabled us to obtain kanamycin resistant plants at a high frequency,
MATERIALS AND METHODS Plant material The genotype used in this study is Medicago varia (M. sativa x M. falcata) A2, It is an autotetraploid Hungarian genotype selected by A. Atanassov for its embryogenic potential in vitro. Media and culture conditions
Offprint requests to." V. Buchanan-Wollaston
PetioLe or stem segments (0.5 to 1.0 cm long) and leaflets from axenic plants were wounded with a scalpel and used for in vitro culture. Callus was induced on a modified B5h medium (Atanassov and Brown 1984) and embryos began to appear after 6-8 weeks. Embryos at the globular to torpedo stage were transferred to B0i2Y medium for maturation (Bingham et al. 1975, Blaydes 1966). The healthiest looking embryos were then transferred to a modified Sh medium (Shbl0) (Stavarek et al. 1980, Schenk and Hilderbrandt 1972) for rooting and further growth into plantlets. All media above were modified by the addition of 3 mm MES buffer (2(N-Morpholino)ethanesulfonic acid) pH 5.8, and 4 g/L agarose (International Biotechnologies Inc., New Haven, CT) was used instead of agar. The explants were cultured in sterile petri dishes (90 x 15 mm) under 15 to 30 ~E m -2 s -1, with a 16h photoperiod at 25°C. The plantlets were transferred to Shbl0 medium in PlantCon sterile boxes (Flow Laboratories, McLean, VA) under the same light and temperature conditions. Minimum inhibitory concentration
of kanamycin
Leaves and petioles of A2 were cultured on B5h with 25, 50, i00 or 250 ~g/mL of kanamycin to test the sensitivity of the tissue to the antibiotic and determine a suitable concentration for selection of transformed cells. Bacterial
strains and plasmid
A supervirulent agropine strain A281 (An et al. 1986, Hood et al. 1987) and a disarmed octopine strain LBA4404 (Hoekema et al. 1983) were the Agrobacterium tumefaciens strains used for transformation. The plasmid pVWI30 (Buchanan-Wollaston et al. 1987) was introduced into the Agrobacterium strains by triparental crosses using pRK2013 as a helper plasmid (Ditta et al. 1980). Agrobacterium transconjugants containing the plasmid were selected with rifampicin at 25 Dg/mL and chloramphenicol at 20 ~g/mL or 300 ~g/mL for the strains LBA4404 and A281 respectively. The plasmid pVW130 (Figure i) contains the T-DNA borders flanking the APHII gene for kanamycin resistance which is fused to the nopaline synthase promoter and nopaline synthase polyadenylation site.
513
BamHI
EcoR~1 A P H pSUP104
~
H ~'BgllI
BamH ElcoR1 Figure i. The plasmid pVWI30 used in the transformation experiments (Buchanan-Wollaston et al. 1987). Inoculum preparation
Isolation of plant DNA and Southern blotting
The strains LBA4404 and A281 containing the vector pVWI30 were cultured overnight in i0 mL liquid LB with chloramphenicol at 20 ~g/mL or i00 ~g/mL respectively by shaking at 250 to 300 rpm at 30°C. The overnight culture was pelleted at 2300xg for 15 min, resuspended in the same volume of 0.85% saline solution, and diluted in saline to give 5 x 107 bacteria/mL. Transformation
procedure
The method used was a variation of the explant method of Horsch et al. (1985). The explants (wounded leaflets and petiole segments from sterile plants) were dipped briefly in the diluted bacteria, blotted between sterile filter papers, and placed on B5h medium for 2, 3 or 4 days of cocultivation. A layer of either 4 day old alfalfa cell suspension cultured in liquid B5h or liquid B5h with 50 ~M acetosyringone (Stachel et al. 1985) was used to induce the Agrobacterium Vir genes. This was pipetted onto the solid B5h medium (i mL/plate) and covered with a sterile filter paper onto which the explants were placed, abaxial side up, after infection. After 4 day cocultivation the explants were washed with sterile distilled water to remove excess bacteria, blotted and transferred to selective medium. Explants cocultivated for 2 or 3 days were transferred without washing. Selection of transformed
tissue
After cocultivation the explants were transferred to B5h medium containing carbenicillin (carb) at 300 ~g/mL and kanamycin (kan) at 50 or i00 ~g/mL. Explants were transferred every 3-4 weeks to fresh selective medium. The kanamyein resistant embryos were placed on B0i2Y containing carb 300 ~g/mL and kan 50 ~g/mL for embryo maturation, and these mature embryos were developed further and rooted on Shbl0 containing carb 300 ~g/mL and kan 50 ~g/mL. Recallusing
a slightly modified version of a method previously described (An et al. 1985, Haas and Dowding 1975). Crude extracts of the transformed tissues were prepared by grinding between 0.5 and 1.0 grams (x g) of fresh tissue in an equivalent volume of extraction buffer (x mL) (0.5M sucrose, 0.1M Tris-HCl, 0.1% ascorbic acid, 0.1% cysteine-HCl, pH 7.5) and spun in an Eppendorf centrifuge for 5 min. The reaction mixture contained 20 ~L of the extract, i0 ~L of the assay buffer (67 mM Tris-HCl, 42 mM MgCI2, 400 mM NH4CI, 1.7 mM dithiothreitol, pH 7.5), I0 ~L of [32p]-ATP solution (15 ~L of 50 mM ATP adjusted to pH 7.2 with NaOH plus 50 ~L of [32p]-ATP (30 Ci/mmol and 2 mCi/mL) made up to i mL with deionized water) and 2 ~L of 1 mg/mL kanamycin. After incubation at 37oc for 20 min the reaction was terminated by loading the mixture on 1 cm 2 of Whatman P-81 phosphocellulose paper. The 1 cm2 P-81 papers were washed twice in hot water (80oc) for 5 min and four times in 50 mM sodium phosphate buffer pH 7.4 for 30 min. The papers were then dried and counted in i0 mL ScintiVerse II scintillation fluid (Fisher Scientific, Springfield, NJ).
assay
Leaves and petioles of plantlets derived from kanamycin resistant calli were cultured in vitro on B5h medium containing kan at i00 ~g/mL. Neomycin phosphotransferase
assay
Neomycin phosphotransferase
activity was measured by
Total DNA was isolated from transformed plantlets as described by Dellaporta et al. (1983). The DNA was digested with EcoRI, restriction fragments were separated on a 0.7% agarose gel and transferred by alkaline blotting to a Zeta-Probe membrane (BioRad Laboratories, Rockville Centre, NY) as described by Reed (1986). The BglII-PstI fragment of the NPTII gene of Tn5 was cloned into pSP65 (Melton et al. 1984), and 32p labelled RNA was synthesized from this clone using the Riboprobe SP6 transcription system according to manufacturer's instructions (Promega Biotec, Madison, WI). Hybridization was carried out as follow~ The membrane was hybridized for at least 15 min in 20 mL of prehybridization buffer at 58oc with strong agitation. The prehybridization buffer contained IM Tris HCI pH 7.4, 50% deionized formamide, 20% dextran sulfate, i% SDS. The buffer was mixed by inversion and put in a 58oc water bath for 15 min, NaCI was then added to obtain a final concentration of 0.4M, mixed and put in the same water bath for 15 min, then the buffer was filtered through a 0.45 ~M filter. The labelled Riboprobe and 1.5 mg of denatured salmon sperm DNA were added to the bag containing the membrane and the prehybridization buffer. Hybridization was carried out overnight at 58oc with strong agitation. The next day the membrane was rinsed briefly in IxSSPE, 1% SDS, washed three times in IxSSPE, 1% SDS at 65oc for 15 min, once in 0.1xSSPE, i% SDS at 65°C for 20 min, and briefly in IxSSPE at room temperature. The membrane was then wrapped in Saran Wrap and exposed to X-ray film for 5 to i0 days at -70°C. RESULTS Evidence for alfalfa transformation The A2 genotype was found to be very sensitive to kanamycin and no growth of calli or embryos was observed on media containing kanamycin at 25 ~g/mL or more. Therefore kanamycin at 50 or i00 ~g/mL was used for selection of transformed ealli. Kanamycin resistant calli developed on B5h containing carb at 300 ~g/mL and kan at 50 or i00 ~g/mL after approximately I month of culture and kanamycin resistant embryos appeared after another month on the same medium (Figure 2). The embryos matured to the first leaflet stage on B0i2Y containing earb 300 ~g/mL and kan 50 or i00 ~g/mL. No callus was observed on control explants (explants treated with bacteria containing no plasmid placed on kanamycin 50 or i00 ~g/mL).
514 Transformation and DNA integration were confirmed by Southern blotting of DNA from 4 plantlets derived from individual kanamycin resistant calli (Figure 3). Two plantlets showed two sites of integration of the kanamycin resistance gene; two others showed one. No hybridization was obtained to the control plant DNA.
a
b
c
d
e
f
15.7 Kb
Figure 2. Transformation of leaflets and petioles of kanamycin by Agrobacterium tumefaciens. (a) Kanamycin resistant embryogenic calli from leaflets and petioles infected with A281 (pVWI30) 3 weeks after start of culture on B5h medium. (b) Kanamycin resistant embryos derived from a leaflet infected by A281 (pVWI30) 6 weeks after start of culture on B5h medium. This tissue piece was about 5 mM across. (c) Transgenic plantlets derived from kanamycin resistant calli transformed with LBA4404 (pVWI30).
Wounded leaves and petioles from plantlets derived from kanamycin resistant calli were cultured on B5h medium containing kanamycin at 50 or i00 Hg/mL for a recallusing assay. After two months embryos developed from the transformed explants. Control explants from a non-transformed plantlet showed no callus or embryo development on the same medium. The calli and embryos from the recallusing assay were tested for neomycin phosphotransferase activity. Recallused explants from medium containing kanamycin at i00 ~g/mL showed high levels of NPTII activity (Table i). Untransformed callus showed low background activity.
Table i. NPTII activity in the embryogenic calli developed from explants from three different kanamycin resistant plantlets on medium containing kanamycin at i00 ~g/mL.
c.p.m./20 DL extract control a kanamycin resistant calli
394 3795 5055 2102
aCalli from leaves and petioles of an untransformed plantlet regenerated on medium without kanamycin.
3.2
Kb
1.5
Kb
Figure 3. Southern analysis of DNA from kanamycin resistant plantlets transformed with LBA4404 (pVWI30). Plant DNA was digested with EcoRI. Lane a: control plant DNA (untransformed). Lanes b, c, d, e: DNA of 4 different transformed plants. Lane f: molecular size markers.
Parameters
affecting
transformation
frequency
Several parameters were studied in order to obtain the highest efficiency of transformation. These were explant type, different Agrobacterium strains, number of days of cocultivation and concentration of kanamycin used for selection. The results are shown in Table 2 and are expressed as percentage of explants transformed. Observations were made after at least 3 months of culture and only the embryogenic calli were counted. Results indicated that several of the parameters tested had a significant effect on the transformation frequencies obtained (Table 2). The wounded leaves gave a transformation frequency four times that obtained with petioles, indicating that leaves are a better source of tissue for transformation. The A281 strain does not produce tumors on the A2 genotype and the kanamycin resistant calli formed after infection with A281 were embryogenic, indicating that integration of the kanamycin resistance gene had occurred and that the presence of the T-DNA in this strain did not give rise to tumor induction. The transformation efficiency of this strain was at least twice that of LBA4404. The number of days of cocultivation had a significant effect on transformation efficiency. A 4 day cocultivation resulted in a transformation frequency at least twice as high as that obtained after 2 or 3 days cocultivation. After a 4 day cocultivation, control explants appeared more resistant to kanamycin, swelling slightly, but they never developed embryogenic calli. Selection on kanamycin at 50 or i00 ~g/mL did not affect the transformation frequency. The maximum frequency obtained under optimum conditions when wounded leaflets were cocultivated with A281 (pVWI30) for 4 days on an alfalfa cell suspension layer and selected on kanamycin at 50 or I00 ~g/mL was 72% (data from two independent experiments).
515 Table 2. Percentage of transformed explants (explants giving rise to one or more kanamycin resistant embryogenic calli compared to the total number of explants tested) obtained under various conditions of transformation.
Explants transformed/Total
Parameters Explants a
Leaves
99/272
Petioles .
.
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.
.
.
.
Bacteria b
.
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.
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.
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.
Cocultivation time c
.
.
.
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.
.
.
.
.
.
.
.
.
.
.
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.
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.
.
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.
Selection on kan d
.
.
.
.
.
.
.
.
9 .
.
.
.
.
.
.
.
.
LBA4404
18/127
14
A281
40/119
34
.
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.
2 days
6/60
i0
3 days
35/183
19
4 days .
36
23/256 .
.
.
.
.
.
.
.
31/71 .
.
.
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.
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Significant difference e
%
yes .
.
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.
50 Ng/mL
18/90
20
i00 Ng/mL
20/88
23
.
yes .
.
no
43 .
.
yes .
.
.
.
.
.
.
.
almost impossible to wound them longitudinally). Therefore wounded leaves may offer more sites for infection by A. tumefaciens. Virulence of the Agrobacterium strain had a significant effect on the transformation frequency. A281 (pVWI30) does not form tumors on the A2 genotype, and all the calli obtained with A281 (pVWI30) when cultured in vitro gave embryos after 3 or 4 months. The observations made with A281 confirmed the increased virulence of this strain as was reported previously for tobacco (An et al. 1986). Duration of cocultivation was an important parameter and differences observed could be due to several factors. The bacterial concentration was higher after 4 days than after 2 or 3 days and this may increase the probability of DNA transfer. However it was important to control bacterial growth since overgrowth of bacteria can kill the tissue. The 4 day cocultivation period allowed selection on kanamycin for an extra day, allowing further expression of the kanamycin resistance gene which could give the transformed cells a better chance of survival once selection is applied.
.
no
aResults from 14 independent experiments with the 2 bacterial strains, a 2 or 3 or 4 day cocultivation, selected on kanamycin at 50 or i00 Dg/mL. Petioles and leaves were tested in the same experiment. bResults from 4 experiments in which the 2 bacterial strains were compared, with a 3 day cocultivation and selection on kanamycin at 50 or i00 ~g/mL. Petioles and leaflets were included. CResults from 2, 8 and 4 experiments with a 2 or 3 or 4 day cocultivation respectively, with LBA4404 (pVWI30) and selection on kanamycin at 50 or i00 ~g/mL. After 4 days coculture the explants were washed before being transferred to selective medium. The three durations were tested in different experiments. Petioles and leaflets were included. dResults from 4 experiments using the 2 bacterial strains with a 2 or 3 or 4 day cocultivation in which the 2 concentrations of kanamycin were compared. Petioles and leaflets were included. e Slgnlflcance . " " is based on the X 2 test with a probability of less than 5% that the difference is due to sampling error. DISCUSSION Kanamycin resistant plantlets of alfalfa were obtained at a high frequency and we have presented evidence to show that they were transformed. All kanamycin resistant calli tested were positive for NPTII or in the Southern hybridization and since no resistant calli ever appeared on control plates it was assumed that all kanamycin resistant calli obtained were transformed. The plantlets tested had either one or two sites of integration of the kanamycin resistance gene. In this study three factors appeared to affect the transformation efficiency of the Medicago varia A2 genotype: the type of explant, virulence of the A. tumefaciens strain used, and duration of the cocultivation. Leaf tissue was more amenable to transformation than stem or petiole tissue, perhaps due to the fact that wounding was easier on leaves than on petioles (petioles were so thin that it was
These results allow us to describe a suitable protocol for transformation of the A2 genotype of Medicago varia based on the parameters that have been studied. This protocol is itemized in Table 3. Table 3.
Protocol for A2 transformation.
Agrobacterium tumefaciens strain A281 containing a suitable plasmid is grown overnight in liquid LB with selective antibiotic, at 30°C, shaken at 300 rpm. The culture is pelleted by centrifugation at 2300xg, 15 min, resuspended in an equal volume of 0.85% saline and then diluted to give 5 x 107 bacteria/mL. - Wounded leaves are dipped briefly into the bacterial suspension and blotted on sterile filter paper. One mL of alfalfa cell suspension is spread over solidified B5h medium. A filter paper disc is placed on top of the cell suspension. - Dipped tissue is placed on the filter paper. - Tissue is cocultivated with bacteria for 4 days. - The tissue is then washed in sterile distilled water and blotted. - The tissue is placed on solid B5h + carb 300 ~g/mL + kan i00 ~g/mL. - Every 3-4 weeks the tissue is transferred to the same fresh selective medium. By this protocol a transformation efficiency of more than 70% has been obtained in several experiments.
For each variety of alfalfa and, more generally, for each variety of any species, these three parameters could be optimized to increase transformation frequencies. The tissue most amenable to A. tumefaciens infection and the most virulent bacterial strain may vary from one species to another and should be determined in each case. Optimum cocultivation time could vary from one genotype to another in the same genus, depending on resistance of the
516 genotype to the antibiotic and sensitivity of the genotype to overgrowth of the A. tumefaciens. Now that the transformation protocol has been optimized with a highly embryogenic line of Medicago, transformation of embryogenic genotypes from commercial cultivars of alfalfa should be possible. Introduction of useful foreign genes such as those for herbicide resistance, disease resistance, improved protein content and other desirable traits by this method will allow development of new alfalfa varieties. ACKNOWLEDGMENTS We thank G. An for kindly providing the strain A281, P. McLean and G. Hernandez for helpful discussion, and A. Naser and C. Cornell for technical assistance. REFERENCES An G, Watson BD, Stachel S, Gordon MP, Nester EW (1985) EMBO J. 4: 277-284. An G, Watson BD, Chiang CC (1986) Plant Physiol. 83: 529-534. Atanassov A, Brown DCW (1984) Plant Cell, Tissue and Organ Culture 3: 149-162. Bingham ET, Hurley LV, Kaatz DM, Saunders JW (1975) Crop Science 15: 719-721. Blaydes DF (1966) Plant Physiol. 19: 748-753.
Buchanan-Wollaston V, Passiatore J, Cannon F (1987) Nature 328: 172-175. Deak M, Kiss GB, Koncz C, Dudits D (1986) Plant Cell Reports 5: 97-100. Dellaporta 8L, Wood J, Hicks JB (1983) Plant Mol. Biol. Reporter i: 19-21. Ditta D, Stanfield S, Corbin D, Helinski DR (1980) Proc. Natl. Acad. Sci. USA 77: 7347-7351. Haas M, Dowding JE (1975) Methods in Enzymol. 43: 611-633. Hoekema A, Hirsch PR, Hooykaas PJJ (1983) Nature 303: 179-180. Hood EE, Fraley RJ, Chilton MD (1987) Plant Physiol. 83: 529-534. Horsch RB, Fry JE, Hoffmann NL, Eichholz D, Rogers SG, Fraley RJ (1985) Science 227: 1229-1231. Melton DA, Krieg PA, Rebagliati MR, Maniatis T, Zinn K, Green ~fR (1984) Nucl. Acids Res. 12: 7035-7056. Reed KC (1986) BioRad Bulletin 1233: 1-5. Schenk RU, Hilderbrandt AC (1972) Can. J. Bot. 50: 199-204. Shahin EA, Spielman A, Sukapinda K, Simpson RB, Yasher M (1986) Crop Science 26: 1235-1239. Stachel SE, Messens E, Van Montagu M, Zambryski P (1985) Nature 318: 624-629. Stavarek ST, Croughan TP, Rains DW (1980) Plant Sci. Lett. 19: 253-261.
