PlantCeil Reports
Plant Cell Reports (1990) 9:245-248
9 Springer-Verlag1990
Transformation of Cucumis sativus tissue by Agrobacterium tumefaciens and the regeneration of transformed plants Paula P. Chee Molecular Biology Division, The Upjohn Co., 301 Henrietta Street, Kalamazoo, MI 49007, USA Received May 2, 1990/Revised version received June 14, 1990 - Communicated by J.M. Widholm
ABSTRACT: Cotyledons of cucumber seedlings (Cucumis sativus L. cv. Poinsett 76) were co-cultivated with disarmed AErobacterium strain C58Z707. The Agrobaeterium strain contained the AErobacteriumderived binary vector plasmid pGA482, its T-DNA region contains a plant expressible bacterial derived neomycin phosphotransferase II (NPT II) gene which upon transfer, genome integration, and expression in plant tissues confers resistance to the antibiotic kanamycin. After growth of inoculated cotyledon sections on selective medium containing i00 mg/l kanamycin, transformed embryogenic calli were obtained followed by the development of embryos and plant regeneration. Transformed R 0 and R 1 cucumber plants appeared normal and tested positive for NPT II enzyme activity. Genomic DNAs isolated from the NPT II positive plants all showed hybridization to the characteristic 2.0 kb (BamHI to HindIII) NPT II gene-containing fragment. These results show that the AErobacterium-mediated gene transfer system and regeneration via somatic embryogenesis is an effective method for the transfer of genetic material into plant species belonging to the family
Cucurbitaceae. INTRODUCTION Many of the species belonging to the family Cucurbitaeeae are known to be susceptible to infection by Agrobacterium pathogens (Anderson and Moore, 1979; Smarrelli et al., 1986). In addition, procedures for regenerating many of these species have already been established (Jelaska, 1986; Chee, 1990). The combination of these two facts suggests that Agrobacterium-mediated transfer of genetic information into the genome of eucurbit species should be readily achievable. The development of a system for the transfer and stable integration of genetic material into the genome of eueurbit species will be useful for the transfer of genetic traits which are difficult or impossible to transfer via
Abbreviation: Cb, carbenicillin; 2,4-D, 2,4dichlorophenoxyacetic acid; Km, kanamycin; KN, kinetin; MS, Murashige and Skoog; NAA, naphthaleneacetic acid; NPT II, neomycin phosphotransferase II.
Conventional plant breeding techniques. The development of gene transfer technology for cucurbit Species can be used to transfer engineered genes which will confer resistance to: viral infection (Powell-Able et al., 1986; Cuozzo et al., 1988), herbicides (Comai et al., 1985; Stalker et al., 1985; della-Cioppa et al., 1987; Lee et al., 1988;), or other useful traits as they become available. The transformation and regeneration of transformed cucumber plants which express the NPT II gene has been previously reported by Trulson et al. (1986). This transformation was achieved using AErobacterium rhizogenes to transfer the T-DNA region of the binary plasmid pARC8, which contains a Nos-NPT II gene, by infecting cucumber hypocotyl segments. The resulting transformed roots were regenerated into whole plants (Trulson et al., 1986). The main limitation of this approach is its low frequency of plant regeneration from root tissues. Hence, only a few transformed plants were obtained. The present report describes the use of disarmed strains of AErobacteriu m ~umefaciens to mediate T-DNA transfers into cucumber cotyledon tissues and the use of somatic embryogenesis route to obtain a large number of morphologically normal transformed plants. MATERIALS AND METHODs P~ant materials Seeds of cucumber (Cucumis sativus L. Poinsett 76, Asgrow Seed Co., Kalamazoo, MI) were soaked in tap water for approximately 15 minutes. The seed coats were removed manually. The de-coated seeds Were surface sterilized with 70% alcohol for 1 minute. A 25 minute treatment with 25% (v/v) solution of Clorox (commercial bleach, 5.25% sodium hypochlorite) followed. The seeds were then rinsed four times with sterile distilled water. Sterilized seeds were germinated at 28~ on 0.8% water agar (Difco Laboratories) for three to five days in darkness. Unless otherwise stated, all media were supplemented with 3% sucrose and solidified with 0.8% phytagar (Gibco). The pH of all media was adjusted to 5.8 before being autoclaved. All media were autoclaved at 121~ for 20 minutes. T2ansformatio_____~n Transformation of cucumber was performed by a method similar to that described by Horsch et al.
246 (1985). Three to five day-old in vitro grown seedlings were used as donors of explants. The cotyledons were removed ase~tically and cut into pieces of approximately 5 mm ~ in size. The pieces were submerged in a diluted overnight culture (2 x 108 cells/ml) of the disarmed Agrobacterium strain C58Z707 (Hepburn et al., 1985). The AErobacterium strain contains the binary plasmid pGA482 (An et al., 1985; An, 1986) which was grown in LB medium containing 25 mg/l Km. After gentle shaking to ensure that all edges were infected, the cotyledon pieces were blotted dry and cultured abaxial side down in a sterile i00 X 20 Petri plate (20 pieces per plate) containing the initiation medium (MS basal medium + 2.0 mg/l 2,4-D + 0.5 mg/l KN) (Chee, 1990). After 4 days of growth in the dark at 26~ the AgroDacterium-infected cucumber cotyledon pieces were transferred to Petri plates containing the same medium supplemented with 500 mg/l Cb and i00 mg/l Km and cultured as before for five additional weeks in the dark at 26~
(between 5 to i0 ~g) was digested with five fold excess BamHl and Hindlll enzymes, then electrophoresed in a 0.7% agarose gel. The gel was then electroblotted onto nylon filter (Reed and Mann, 1985). Blotted filters were fixed by exposure for 3 min to a UV light source, then pre-hybridized in hybridization solution (Denhardt, 1966) (6X hybridization solution contains 0.9 M NaCI, 0.09 M Na Citrate pH7.2, and 0.02% BSA, PVP-40 [Sigma], and Ficoll-400 [Sigma]) for at least 2 h, at 68~ Filters were hybridized against a 32p-labeled 600 bp BglII-NcoI fragment which contains mostly NPT II coding sequences. DNA labeling was done using a kit purchased from Bethesda Research Laboratories. Hybridizations were done using about 7.5 X 105 cpm of the labeled probe per ml of hybridization buffer containing 2% SDS and incubating at 68~ for at least 18 hours. Hybridized filters were washed in IX hybridization solution (Denhardt, 1966) for 1 hour at 68~ dried and then exposed to film. Film exposures were generally for 12 to 24 hours.
Plant regeneration
RESULTS AND DISCUSSIONS
Regeneration of potentially transformed embryogenic callus tissue was done according to the procedure described by Chee (1990). Briefly, after five to six weeks on initiation medium, the cultures were transferred to MS medium + 1.0 mg/l NAA + 0.5 mg/l KN + I00 mg/l Km + 500 mg/l Cb for somatic embryo development. These cultures were incubated for an additional two weeks at 26~ under diffuse cool white fluorescent lamps (4000 lux) with 16 hour photoperiod. The tissues were then transferred to hormone-free MS medium + 50 mg/l Km for conversion to plantlets under the same conditions. When plantlets developed an extensive root system on the latter medium, they were transplanted to pots containing planting medium and covered with Ziploc (Dow Co.) storage bags 'for hardening-off. Subsequently, the regenerated plants were potted in a mixture of (i:I, v/v) of soil and Perlite and grown in a greenhouse under standard conditions.
Transformation of Cucumber Tissues:
NPT II enzyme assay NPT II enzyme activity was detected by the in
situ gel assay procedure described by Reiss et al. (1984). Briefly, approximately i00 mg of cucumber callus or regenerated fresh leaf tissue was mixed with 20 ~I of extraction buffer in a 1.5 ml Eppendorf tube. The tissue was macerated with a Konte pestle and centrifuged at 10,000 x g for 15 minutes at 4~ to remove cell debris. From each sample tested, an aliquot of 35 ~i of the supernatant solution was electrophoresed on a 10% non-denaturing polyacrylamide gel and the gel was then exposed to kanamycin sulfate and [32p-7]-ATP. Phosphorylated kanamycin, which is produced in those regions of the gel containing the modified bacterial Nos-NPT II enzyme activity is transferred by blotting onto phosphocellulose paper and detected by autoradiography. Genomic DNA Isolation Total nucleic acids were extracted from cucumber leaves using the cetyltrimethyla~onium bromide (CTAB) procedure described by Saghai-Maroof et al. (1984). This DNA was not subjected to any further purification steps as it was readily digestible by the restriction enzymes used for gene mapping. Genomic Blot Hybridization Genomic
DNA
from
individual
cucumber
plants
Cucumber cotyledon pieces were subjected to antibiotic selection by transfering onto initiation medium which supplemented with different levels (25 to 200 mg/l) of kanamycin. The lowest level of kanamycin found to be useable for selection of the transformed callus tissues and not to allow escapes, was determined to be i00 mg/l. At I00 mg/l, nontransformed tissues were not capable of growth while the putative transformed calli showed little if any growth inhibition. After five to six weeks all putative transformed calli obtained from cotyledon pieces cultured on initiation medium supplemented with I00 mg/l Km were tested for NPT II activity and all of these were found to be positive. Fig. i shows the results in NPT II enzyme assay for some of the Kinr cucumber callus tissues. These results indicate that at least the modified Nos-NPT II gene, contained within the T-DNA region of pGA482, had been transferred into the cucumber tissues by using the disarmed Agrobacterium strain. Res of Cucumber Plantlets from Transformed Embryogenic Callus: After about six weeks on initiation medium supplemented with 500 mg/l Cb and i00 mg/l Km, a characteristic gel-like callus was formed on the surface of the explants and at the site of tissue contact with the medium. The upper part of the gelatinous tissue contained small sectors of putative embryogenic tissue, which upon microscopic examination, consisted of cytoplasmically dense, multicellular aggregates, resembling the proembryonic masses. The sectors with proembryonic masses were selected for transfer to the secondary medium supplemented with I00 mg/l Km. Upon transfer, the putative transformed proembryonic structures developed into embryo-like structures. After two weeks, the embryos were transferred to hormone-free MS medium supplemented with 50 mg/l Km. Ten percent of the explants produced plantlets and all followed normal development upon transfer to the latter medium. Transgenic plantlets were visually identifiable by their ability to form roots on MS medium supplemented with 50 mg/l Km (Fig. 2). In contrast, non-transgenic, control plantlets were inhibited from root development on medium supplemented with 50 mg/l Km. All transgenic plantS flowered and set seeds normally (Fig. 3).
247 Detection of NPT II Enzyme Activity ~0 Cucumber Plants:
in Transformed
Chee (1990) for the regeneration of cucumber plants from cotyledon explants and transformation of leaf pieces described by Horsch et al. (1985). The work presented here also shows that the selection of transgenic Km r cucumber tissues can be accomplished by supplementing culture media with i00 mg/l Km.
A total of more than I00 transformed kan r R 0 plants were obtained. In order to determine if this group of plants contained any escapes during the regeneration process, each plant was tested for NPT I~ enzyme activity. All of the the putative transgenic R 0 plants showed the presence of NPT II enzyme activity in their protein extracts which comigrated with the bacterial-derived NPT II enzyme. Fig. 4 shows the analysis of NPT II enzyme activity in the protein extracts of some transformed R 0 plants. In contrast, the negative control plants, which resulted from co-cultivation with C58Z707 minus the binary plasmid showed no co-migrating NPT II enzyme activity in leaf extracts.
An G (1986)
Analysis of Transgenic R 0 Cucumber Presence of Nos-NPT II Gene:
Beck E, Ludwig G, Auerswald EA, Reiss B, Schaller, H (1982) Gene 19:327-336.
Plants
for the
REFERENCES Plant Physiol. 81:86-91.
An G, Watson BD, Stachel (1985) EMBO ~. 4:277-284. Anderson 323.
AR,
Moore,
LW
S, Gordon MP,
(1979)
Chee PP, Fober KA, Slightom Physiol. 91:1212-1218.
Nester
Phytopath.
JL
EW
69:320-
(1989)
Plant
Total nucleic acids were extracted from leaves of the NPT II positive R 0 plants. This DNA was subjected to restriction endonuclease digestion with both BamHl and Hindlll. The restriction map of the Nos-NPT II fusion gene of pGA482 (An, 1986) predicts a fragment of about 2.0 kb in length with Hindlll and BamHl digestion. Digested cucumber DNAs were attached to nylon filters which were hybridized against nick-translated 32p-labeled Bglll-Ncol 600 bp fragment isolated from the bacterial NPT II gene. This fragment contains only the bacterial NPT II gene portion of Tn5 (Beck et al., 1982) and was selected because of its low background hybridization against various plant genomic DNA (Chee et al., 1989). The predicted 2 kb Nos-NPT II hybridizing fragment was observed for DNAs isolated from all NPT II positive plants. Fig. 5 shows the genomic blots for Hindlll and BamHl enzymes digestions on transgenic R 0 plants. Copy number reconstructions suggest that each of these plants contain one gene copy of the NPT II gene.
Hepburn AG, White J, Pearson L, Maunders MJ, Clarke LE, Prescott AG, Blundy KS (1985) ~. General Microbio. 131: 2961-2969.
Analysis of Transgenic Nos-NPT II Gene:
the
Horsch RB, Fry JE, Hoffmann NL, Eichholtz D, Rogers SG, Fraley RT (1985) Science 227:1229-1231.
The most convincing evidence for the integration of the Nos-NPT II gene of pGA482 would be the transfer of the transgenic phenotype from the R 0 plants (self-pollinated) to their progenies. Assay for NPT II enzyme activity in forty progeny plants obtained from four randomly selected R 0 cucumber lines indicated that 75% of the progenies expressed NPT II aetivities (data not shown). In order to confirm that the progenies have inherited the Nos-NPT II gene, total nucleic acids were extracted from leaves of five NPT II positive R I cucumber plants obtained from R 0 plant #36 and subjected to restriction endonuelease digestion with either BamHl and Hindlll (which is expected to show the characteristic 2.0 kb hybridizing fragment) or with only Hindlll which should yield a hybridizing fragment characteristic to the chromosomal location of the integrated T-DNA within the genome of R 0 cucumber plant. The results of the genomic blots for both enzyme digestions are shown in Fig. 6. The Hindlll and BamHl digest show the expected 2.0 kb band, while digestion with Hindlll alone yields a hybridizing band of about 5 kb for each of the progeny plants. The presence of the identical size band for the Hindlll digest suggests, as expected, that all of these progeny plants were derived from a single transformation event which gave rise to the parent R 0 plant. The results of these experiments clearly show that transformed cucumber plants can be obtained by using both the established procedure described by
Jelaska S (1986) Cucurbits, In: Biotechnology in Agriculture and Forestry (ed. by Y.P.S. Bajai) 2:371-386.
R I Cucumber
Plants
for
Chee, PP (1990) Hort. Sci. in press Comai L, Facciotti D, Hiatt WR, Thompson G, Rose RE, Stalker DM (1985) Nature 317:741-744. Cuozzo M, O'Connell KM, Kaniewski W, Fang R-X, Chua N-H, Tumer N (1988) Bio/tech. 6:549-557. della-Cioppa G, Bauer C, Taylor ML, Rochester DE, Klein BK, Shah DM, Fraley RT, Kishore G (1987) Bio/tech. 5:579-584. Denhardt DT, 23:641-646.
(1966)
Bioehem.
~iophu
Res.
Comm.
Lee KY, Townsend J, Tepperman J, Black M, Chui CF, Mazur B, Dunsmuir p, Bedbrook, J (1988) EMBO ~. 7:1241-1248. Powell-Abel P, Nelson RS, De B, Hoffmann N, Rogers SG, Fraley RT, Beachy, RN (1986) Science 232:738743.
Reed KC, Mann DA (1985) 7221 Reiss B, Sprengel Gene 30:211-218.
R,
Nuclic acids Res. 13:7207-
Will
H,
Schaller,
H
(1984)
Saghai-Maroof MA, Soliman KM, Jorgensen RA, Allard RW (1984) Proc. Nat !. 6cad. Sci. USA 81:8014-8018. Smarrelli Jr. J, Watters MT, Diba, L (1986) Physiol. 82:622-624. Stalker DM, Hiatt WR, Comai L (1985) 260:4724-4728. Trulson AJ, Simpson RB, A_p~l. Genet. 73:11-15.
Shahin
EA
Plant.
~. Biol. Chem.
(1986)
Theor.
248 Acknowledgments I thank Krystal A. Fober for plant care in the greenhouse. I also thank G. An (Washington State University) for his gift of the binary plasmid pGA482 and A. Hepburn for his gift of the disarmed Agrobacterium strain C58Z707.
Figure 3. Mature greenhouse.
transgenic
cucumber
plants
in the
Figure I. Analysis of NPT II activity in a series of cucumber calli which has been selected on kanamycin containing medium. Lane i contains the bacterial derived NPT II protein control; Lane 2 contains extract from non-transformed cucumber callus; Lanes 3 to 22 contain extracts from cucumber callus selected on kanamycin medium'.
Figure 4. Analysis of NPT II activity in transgenic R 0 cucumber plants. Lane i contains the bacterial derived NPT II protein control; Lanes 5 and 14 contains extract from leaf material of nontransformed cucumber plants. Lanes 2 to 4 and 6 to 13 contain extract from leaf material of independent transgenic R 0 cucumber plants.
Figure 2. Cucumber plantlets cultured on MS medium supplemented with 50 mg/l Km. Left: control plantlet, Right: transgenic plantlet containing NPT II gene.
Figure 5. Detection of the NPT II gene in R 0 cucumber plants which showed NPT II activity. Total DNA was isolated from plants, digested with BamHl and Hindlll. Lanes i to 7 contain a digest of DNA isolated from independent transformed cucumber plants. Lane 8 contains a digest of pGA482 DNA. Lane 9 contains a digest of DNA isolated from a nontransformed cucumber plant.
Figure 6. Genomir blot analysis of DNAs isolated from the progeny plants (RI) obtained from transformed R 0 eucumber plant number 36. DNAs from progeny plants, numbered 36-1, 36-2, 36-3, 36-4, 365, were subjected to restriction enzyme digests using both Hindlll and BamHl, lanes 6 to i0; and only Hindlll, lanes i to 5. Lane ii contains a digest (Hindlll and BamHl) of DNA isolated from a non-transformed cucumber plant, to which pGA482 DNA had been added at a concentration which represents a reconstruction of one copy equivalence per haploid cucumber genome. DNA fragment sizes indicated on the right correspond to two fragments from the BRL DNA size standard mix. The observed hybridizing bands were measured to be 2 kb for the Hindlll and BamHI digestions and about 5.0 kb when only the Hindlll enzyme was used.
Plant Cell Reports (1990) 9:245-248
9 Springer-Verlag1990
Transformation of Cucumis sativus tissue by Agrobacterium tumefaciens and the regeneration of transformed plants Paula P. Chee Molecular Biology Division, The Upjohn Co., 301 Henrietta Street, Kalamazoo, MI 49007, USA Received May 2, 1990/Revised version received June 14, 1990 - Communicated by J.M. Widholm
ABSTRACT: Cotyledons of cucumber seedlings (Cucumis sativus L. cv. Poinsett 76) were co-cultivated with disarmed AErobacterium strain C58Z707. The Agrobaeterium strain contained the AErobacteriumderived binary vector plasmid pGA482, its T-DNA region contains a plant expressible bacterial derived neomycin phosphotransferase II (NPT II) gene which upon transfer, genome integration, and expression in plant tissues confers resistance to the antibiotic kanamycin. After growth of inoculated cotyledon sections on selective medium containing i00 mg/l kanamycin, transformed embryogenic calli were obtained followed by the development of embryos and plant regeneration. Transformed R 0 and R 1 cucumber plants appeared normal and tested positive for NPT II enzyme activity. Genomic DNAs isolated from the NPT II positive plants all showed hybridization to the characteristic 2.0 kb (BamHI to HindIII) NPT II gene-containing fragment. These results show that the AErobacterium-mediated gene transfer system and regeneration via somatic embryogenesis is an effective method for the transfer of genetic material into plant species belonging to the family
Cucurbitaceae. INTRODUCTION Many of the species belonging to the family Cucurbitaeeae are known to be susceptible to infection by Agrobacterium pathogens (Anderson and Moore, 1979; Smarrelli et al., 1986). In addition, procedures for regenerating many of these species have already been established (Jelaska, 1986; Chee, 1990). The combination of these two facts suggests that Agrobacterium-mediated transfer of genetic information into the genome of eucurbit species should be readily achievable. The development of a system for the transfer and stable integration of genetic material into the genome of eueurbit species will be useful for the transfer of genetic traits which are difficult or impossible to transfer via
Abbreviation: Cb, carbenicillin; 2,4-D, 2,4dichlorophenoxyacetic acid; Km, kanamycin; KN, kinetin; MS, Murashige and Skoog; NAA, naphthaleneacetic acid; NPT II, neomycin phosphotransferase II.
Conventional plant breeding techniques. The development of gene transfer technology for cucurbit Species can be used to transfer engineered genes which will confer resistance to: viral infection (Powell-Able et al., 1986; Cuozzo et al., 1988), herbicides (Comai et al., 1985; Stalker et al., 1985; della-Cioppa et al., 1987; Lee et al., 1988;), or other useful traits as they become available. The transformation and regeneration of transformed cucumber plants which express the NPT II gene has been previously reported by Trulson et al. (1986). This transformation was achieved using AErobacterium rhizogenes to transfer the T-DNA region of the binary plasmid pARC8, which contains a Nos-NPT II gene, by infecting cucumber hypocotyl segments. The resulting transformed roots were regenerated into whole plants (Trulson et al., 1986). The main limitation of this approach is its low frequency of plant regeneration from root tissues. Hence, only a few transformed plants were obtained. The present report describes the use of disarmed strains of AErobacteriu m ~umefaciens to mediate T-DNA transfers into cucumber cotyledon tissues and the use of somatic embryogenesis route to obtain a large number of morphologically normal transformed plants. MATERIALS AND METHODs P~ant materials Seeds of cucumber (Cucumis sativus L. Poinsett 76, Asgrow Seed Co., Kalamazoo, MI) were soaked in tap water for approximately 15 minutes. The seed coats were removed manually. The de-coated seeds Were surface sterilized with 70% alcohol for 1 minute. A 25 minute treatment with 25% (v/v) solution of Clorox (commercial bleach, 5.25% sodium hypochlorite) followed. The seeds were then rinsed four times with sterile distilled water. Sterilized seeds were germinated at 28~ on 0.8% water agar (Difco Laboratories) for three to five days in darkness. Unless otherwise stated, all media were supplemented with 3% sucrose and solidified with 0.8% phytagar (Gibco). The pH of all media was adjusted to 5.8 before being autoclaved. All media were autoclaved at 121~ for 20 minutes. T2ansformatio_____~n Transformation of cucumber was performed by a method similar to that described by Horsch et al.
246 (1985). Three to five day-old in vitro grown seedlings were used as donors of explants. The cotyledons were removed ase~tically and cut into pieces of approximately 5 mm ~ in size. The pieces were submerged in a diluted overnight culture (2 x 108 cells/ml) of the disarmed Agrobacterium strain C58Z707 (Hepburn et al., 1985). The AErobacterium strain contains the binary plasmid pGA482 (An et al., 1985; An, 1986) which was grown in LB medium containing 25 mg/l Km. After gentle shaking to ensure that all edges were infected, the cotyledon pieces were blotted dry and cultured abaxial side down in a sterile i00 X 20 Petri plate (20 pieces per plate) containing the initiation medium (MS basal medium + 2.0 mg/l 2,4-D + 0.5 mg/l KN) (Chee, 1990). After 4 days of growth in the dark at 26~ the AgroDacterium-infected cucumber cotyledon pieces were transferred to Petri plates containing the same medium supplemented with 500 mg/l Cb and i00 mg/l Km and cultured as before for five additional weeks in the dark at 26~
(between 5 to i0 ~g) was digested with five fold excess BamHl and Hindlll enzymes, then electrophoresed in a 0.7% agarose gel. The gel was then electroblotted onto nylon filter (Reed and Mann, 1985). Blotted filters were fixed by exposure for 3 min to a UV light source, then pre-hybridized in hybridization solution (Denhardt, 1966) (6X hybridization solution contains 0.9 M NaCI, 0.09 M Na Citrate pH7.2, and 0.02% BSA, PVP-40 [Sigma], and Ficoll-400 [Sigma]) for at least 2 h, at 68~ Filters were hybridized against a 32p-labeled 600 bp BglII-NcoI fragment which contains mostly NPT II coding sequences. DNA labeling was done using a kit purchased from Bethesda Research Laboratories. Hybridizations were done using about 7.5 X 105 cpm of the labeled probe per ml of hybridization buffer containing 2% SDS and incubating at 68~ for at least 18 hours. Hybridized filters were washed in IX hybridization solution (Denhardt, 1966) for 1 hour at 68~ dried and then exposed to film. Film exposures were generally for 12 to 24 hours.
Plant regeneration
RESULTS AND DISCUSSIONS
Regeneration of potentially transformed embryogenic callus tissue was done according to the procedure described by Chee (1990). Briefly, after five to six weeks on initiation medium, the cultures were transferred to MS medium + 1.0 mg/l NAA + 0.5 mg/l KN + I00 mg/l Km + 500 mg/l Cb for somatic embryo development. These cultures were incubated for an additional two weeks at 26~ under diffuse cool white fluorescent lamps (4000 lux) with 16 hour photoperiod. The tissues were then transferred to hormone-free MS medium + 50 mg/l Km for conversion to plantlets under the same conditions. When plantlets developed an extensive root system on the latter medium, they were transplanted to pots containing planting medium and covered with Ziploc (Dow Co.) storage bags 'for hardening-off. Subsequently, the regenerated plants were potted in a mixture of (i:I, v/v) of soil and Perlite and grown in a greenhouse under standard conditions.
Transformation of Cucumber Tissues:
NPT II enzyme assay NPT II enzyme activity was detected by the in
situ gel assay procedure described by Reiss et al. (1984). Briefly, approximately i00 mg of cucumber callus or regenerated fresh leaf tissue was mixed with 20 ~I of extraction buffer in a 1.5 ml Eppendorf tube. The tissue was macerated with a Konte pestle and centrifuged at 10,000 x g for 15 minutes at 4~ to remove cell debris. From each sample tested, an aliquot of 35 ~i of the supernatant solution was electrophoresed on a 10% non-denaturing polyacrylamide gel and the gel was then exposed to kanamycin sulfate and [32p-7]-ATP. Phosphorylated kanamycin, which is produced in those regions of the gel containing the modified bacterial Nos-NPT II enzyme activity is transferred by blotting onto phosphocellulose paper and detected by autoradiography. Genomic DNA Isolation Total nucleic acids were extracted from cucumber leaves using the cetyltrimethyla~onium bromide (CTAB) procedure described by Saghai-Maroof et al. (1984). This DNA was not subjected to any further purification steps as it was readily digestible by the restriction enzymes used for gene mapping. Genomic Blot Hybridization Genomic
DNA
from
individual
cucumber
plants
Cucumber cotyledon pieces were subjected to antibiotic selection by transfering onto initiation medium which supplemented with different levels (25 to 200 mg/l) of kanamycin. The lowest level of kanamycin found to be useable for selection of the transformed callus tissues and not to allow escapes, was determined to be i00 mg/l. At I00 mg/l, nontransformed tissues were not capable of growth while the putative transformed calli showed little if any growth inhibition. After five to six weeks all putative transformed calli obtained from cotyledon pieces cultured on initiation medium supplemented with I00 mg/l Km were tested for NPT II activity and all of these were found to be positive. Fig. i shows the results in NPT II enzyme assay for some of the Kinr cucumber callus tissues. These results indicate that at least the modified Nos-NPT II gene, contained within the T-DNA region of pGA482, had been transferred into the cucumber tissues by using the disarmed Agrobacterium strain. Res of Cucumber Plantlets from Transformed Embryogenic Callus: After about six weeks on initiation medium supplemented with 500 mg/l Cb and i00 mg/l Km, a characteristic gel-like callus was formed on the surface of the explants and at the site of tissue contact with the medium. The upper part of the gelatinous tissue contained small sectors of putative embryogenic tissue, which upon microscopic examination, consisted of cytoplasmically dense, multicellular aggregates, resembling the proembryonic masses. The sectors with proembryonic masses were selected for transfer to the secondary medium supplemented with I00 mg/l Km. Upon transfer, the putative transformed proembryonic structures developed into embryo-like structures. After two weeks, the embryos were transferred to hormone-free MS medium supplemented with 50 mg/l Km. Ten percent of the explants produced plantlets and all followed normal development upon transfer to the latter medium. Transgenic plantlets were visually identifiable by their ability to form roots on MS medium supplemented with 50 mg/l Km (Fig. 2). In contrast, non-transgenic, control plantlets were inhibited from root development on medium supplemented with 50 mg/l Km. All transgenic plantS flowered and set seeds normally (Fig. 3).
247 Detection of NPT II Enzyme Activity ~0 Cucumber Plants:
in Transformed
Chee (1990) for the regeneration of cucumber plants from cotyledon explants and transformation of leaf pieces described by Horsch et al. (1985). The work presented here also shows that the selection of transgenic Km r cucumber tissues can be accomplished by supplementing culture media with i00 mg/l Km.
A total of more than I00 transformed kan r R 0 plants were obtained. In order to determine if this group of plants contained any escapes during the regeneration process, each plant was tested for NPT I~ enzyme activity. All of the the putative transgenic R 0 plants showed the presence of NPT II enzyme activity in their protein extracts which comigrated with the bacterial-derived NPT II enzyme. Fig. 4 shows the analysis of NPT II enzyme activity in the protein extracts of some transformed R 0 plants. In contrast, the negative control plants, which resulted from co-cultivation with C58Z707 minus the binary plasmid showed no co-migrating NPT II enzyme activity in leaf extracts.
An G (1986)
Analysis of Transgenic R 0 Cucumber Presence of Nos-NPT II Gene:
Beck E, Ludwig G, Auerswald EA, Reiss B, Schaller, H (1982) Gene 19:327-336.
Plants
for the
REFERENCES Plant Physiol. 81:86-91.
An G, Watson BD, Stachel (1985) EMBO ~. 4:277-284. Anderson 323.
AR,
Moore,
LW
S, Gordon MP,
(1979)
Chee PP, Fober KA, Slightom Physiol. 91:1212-1218.
Nester
Phytopath.
JL
EW
69:320-
(1989)
Plant
Total nucleic acids were extracted from leaves of the NPT II positive R 0 plants. This DNA was subjected to restriction endonuclease digestion with both BamHl and Hindlll. The restriction map of the Nos-NPT II fusion gene of pGA482 (An, 1986) predicts a fragment of about 2.0 kb in length with Hindlll and BamHl digestion. Digested cucumber DNAs were attached to nylon filters which were hybridized against nick-translated 32p-labeled Bglll-Ncol 600 bp fragment isolated from the bacterial NPT II gene. This fragment contains only the bacterial NPT II gene portion of Tn5 (Beck et al., 1982) and was selected because of its low background hybridization against various plant genomic DNA (Chee et al., 1989). The predicted 2 kb Nos-NPT II hybridizing fragment was observed for DNAs isolated from all NPT II positive plants. Fig. 5 shows the genomic blots for Hindlll and BamHl enzymes digestions on transgenic R 0 plants. Copy number reconstructions suggest that each of these plants contain one gene copy of the NPT II gene.
Hepburn AG, White J, Pearson L, Maunders MJ, Clarke LE, Prescott AG, Blundy KS (1985) ~. General Microbio. 131: 2961-2969.
Analysis of Transgenic Nos-NPT II Gene:
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The most convincing evidence for the integration of the Nos-NPT II gene of pGA482 would be the transfer of the transgenic phenotype from the R 0 plants (self-pollinated) to their progenies. Assay for NPT II enzyme activity in forty progeny plants obtained from four randomly selected R 0 cucumber lines indicated that 75% of the progenies expressed NPT II aetivities (data not shown). In order to confirm that the progenies have inherited the Nos-NPT II gene, total nucleic acids were extracted from leaves of five NPT II positive R I cucumber plants obtained from R 0 plant #36 and subjected to restriction endonuelease digestion with either BamHl and Hindlll (which is expected to show the characteristic 2.0 kb hybridizing fragment) or with only Hindlll which should yield a hybridizing fragment characteristic to the chromosomal location of the integrated T-DNA within the genome of R 0 cucumber plant. The results of the genomic blots for both enzyme digestions are shown in Fig. 6. The Hindlll and BamHl digest show the expected 2.0 kb band, while digestion with Hindlll alone yields a hybridizing band of about 5 kb for each of the progeny plants. The presence of the identical size band for the Hindlll digest suggests, as expected, that all of these progeny plants were derived from a single transformation event which gave rise to the parent R 0 plant. The results of these experiments clearly show that transformed cucumber plants can be obtained by using both the established procedure described by
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248 Acknowledgments I thank Krystal A. Fober for plant care in the greenhouse. I also thank G. An (Washington State University) for his gift of the binary plasmid pGA482 and A. Hepburn for his gift of the disarmed Agrobacterium strain C58Z707.
Figure 3. Mature greenhouse.
transgenic
cucumber
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in the
Figure I. Analysis of NPT II activity in a series of cucumber calli which has been selected on kanamycin containing medium. Lane i contains the bacterial derived NPT II protein control; Lane 2 contains extract from non-transformed cucumber callus; Lanes 3 to 22 contain extracts from cucumber callus selected on kanamycin medium'.
Figure 4. Analysis of NPT II activity in transgenic R 0 cucumber plants. Lane i contains the bacterial derived NPT II protein control; Lanes 5 and 14 contains extract from leaf material of nontransformed cucumber plants. Lanes 2 to 4 and 6 to 13 contain extract from leaf material of independent transgenic R 0 cucumber plants.
Figure 2. Cucumber plantlets cultured on MS medium supplemented with 50 mg/l Km. Left: control plantlet, Right: transgenic plantlet containing NPT II gene.
Figure 5. Detection of the NPT II gene in R 0 cucumber plants which showed NPT II activity. Total DNA was isolated from plants, digested with BamHl and Hindlll. Lanes i to 7 contain a digest of DNA isolated from independent transformed cucumber plants. Lane 8 contains a digest of pGA482 DNA. Lane 9 contains a digest of DNA isolated from a nontransformed cucumber plant.
Figure 6. Genomir blot analysis of DNAs isolated from the progeny plants (RI) obtained from transformed R 0 eucumber plant number 36. DNAs from progeny plants, numbered 36-1, 36-2, 36-3, 36-4, 365, were subjected to restriction enzyme digests using both Hindlll and BamHl, lanes 6 to i0; and only Hindlll, lanes i to 5. Lane ii contains a digest (Hindlll and BamHl) of DNA isolated from a non-transformed cucumber plant, to which pGA482 DNA had been added at a concentration which represents a reconstruction of one copy equivalence per haploid cucumber genome. DNA fragment sizes indicated on the right correspond to two fragments from the BRL DNA size standard mix. The observed hybridizing bands were measured to be 2 kb for the Hindlll and BamHI digestions and about 5.0 kb when only the Hindlll enzyme was used.
