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Agrobacterium Transformation of Eggplant
J.PlantPhysiol. Vol. 133.pp. 52-55 (1988)
Agrobacterium Transformation of Eggplane A.
GURI
and K. C.
SINK
Department of Horticulture, Michigan State University, East Lansing, MI 48824 U.S.A. Received November 19, 1987 . Accepted February 7,1988
Summary Leaf sections of in vitro cultured seedlings of Solanum melongena L., eggplant cv. Black Beauty, were inoculated with two disarmed binary strains of Agrobacterium, 715 and pCIB10, and with the cointegrated, disarmed plasmid pMON200. Eggplants resistant to kanamycin were only obtained using the pMON200 plasmid. Three plants, selected randomly among the 28 kanamycin resistant plants, were analysed using slot blot DNA hybridization and found to contain a sequence homologous to that of the NOS-NPT chimeric gene. Conversely, a control plant did not have the NOS-NPT homologous sequence. The 3 transformed plants produced nopaline in the presence of arginine. The kanamycin resistant trait was stable and expressed in 12 of the transformed plants after a 6 month culture period in kanamycin-free medium. Preliminary results also indicated that the resistance to kanamycin was transferred as a dominant Mendelian trait.
Key words: Agrobacterium, eggplant, Solanum melongena, trans/ormation, kanamycin. Abbreviations: LB-Luria broth; MS-Murashige and Skoog; NAA-a-naphthaleneactic acid; NPT neomycin phosphotransferase.
Introduction Agrobacterium tume/aciens is a useful tool for plant transformation. This vector has been applied to a broad range of dicot species and has been especially successful to date in transforming several Solanaceous species including: tobacco (Chilton, 1983), petunia (Shah et aI., 1986), tomato (McCormick et aI., 1986) and potato (Shahin and Simpson, 1986). Agrobacterium tume/aciens bacteria carry a Ti (tumor-inducing) plasmid, part of which, the T-DNA is transferred and integrated into the plant nuclear genome (Chilton, 1983; Hooykaas and Schilperoort, 1984). The ability to use TDNA as a vector for engineering the plant genome has been greatly facilitated by the use of bacterial antibiotic resistance genes as selectable markers (Horsch et aI., 1985). For example, the bacterial neomycin phosphotransferase (NPT) gene, when integrated as a chimeric gene in combination with the promoter and terminator sequences of the T-DNA borne nopaline synthase (NOS) gene, endows the plant with 1 Michigan Agricultural Experiment Station Journal Article No. 12459.
© 1988 by Gustav Fischer Verlag, Stuttgart
resistance to kanamycin (Bevan, 1984). Thus, the chimeric gene NOS-NPT can be used as a selectable marker at the cell level in tissue culture studies. In this paper we demonstrate the successful A. tume/aciens-mediated transformation of the crop species eggplant (Solanum melongena L.) by the expression of kanamycin resistance in regenerated whole plants, slot-blot analysis for the presence of the NOS-NPT sequence, and Mendelian inheritance of the kanamycin resistance trait.
Material and Methods Plant material Seeds of eggplant (Solanum melongena L.) cv. Black Beauty were obtained from A. H. Hummert Seed Co., St. Louis, MO. They were sterilized by soaking for 30 min in an aqueous solution containing 20 % commercial bleach (5.25% sodium hypochlorite) and 0.1 % Tween-20 and then washing 4 times with sterile water. The seeds were placed in 840 ml glass jars containing MS salts and vitamins (Murashige and Skoog, 1962) medium supplemented with 3 % sucrose; 0.01 mg .1- 1 NAA and 0.9 % agar and placed under
Agrobacterium transformation of eggplant 30 - 50 JLEm - 2 s - 1 cool white fluorescent tubes at 27°C. Leaves from 2-week old in vitro grown seedlings were used for Agrobacte· rium transfection.
Vector plasmid Three disarmed plasmids were used to inoculate eggplant leaf strips. Two of them, 715 and pCIB10, (provided by D. Rice, CibaGeigy) are 3.2kb plasmids each carrying in the T-DNA two chimeric genes that confer resistance to kanamycin and hygromycin. These binary plasmids are accompanied by the original Ti plasmid that lacks the entire T-DNA region. The third was pMON200 a cointegrated plasmid (provided by S. Rogers, Monsanto Co.) that contains T-DNA with the genes for kanamycin resistance and for nopaline synthesis (See Rogers et aI., 1986).
53
dishes, and placed under 30 - 50 JLEm - 2 s - 1 light (cool white fluorescent tubes) at 27°C. Three weeks later, when callus initiated at a few regions along the cut edges, the leaves were subcultured. Shoot regeneration from the primary calluses started 2 weeks later, and exised shoots (2.0 em) were inserted into plant hormone-free MS medium containing 10-fold thiamine HCI (1 mg .1- 1) (MSOT), 2 % sucrose, 0.9 % agar (Sigma), and 0.1 mg· ml- 1 kanamycin. Some of the kanamycin resistant plants were transferred to kanamycin-free MSOT medium. Six months later, leaves of kanamycin resistant plants that grew on MSOT with or without kanamycin, and leaves taken from non-transformed (control) plants were sliced and placed on MS2Z + 0.3 mg· ml- 1 kanamycin. This was done to determine expression of the NOS-NPT gene in plants grown with or without selection. Two kanamycin resistant plants were subsequently grown in the greenhouse. Seeds obtained by self-pollination were surface sterilized, as stated previously, and placed on MS medium supplemented with 2 % sucrose, 0.01 mg .1- 1 NAA, 0.05 mg· ml- 1 kanamycin and 0.9 % agar.
Inoculation and shoot regeneration The bacteria were cultured on solid (2 % Difco agar) Luria Broth (LB) medium (10 g .1- 1 yeast extract, Difco; 5 g .1- 1 trypton, Difco; 5 g .1- 1 NaCl) containing 0.05 mg· ml- 1 kanamycin sulfate (Sigma) at 28°C for 24 h. Inoculum from a single colony was transferred into sterile 25 ml Erlenmeyer flasks containing 10 ml of liquid LB plus 0.05 mg· ml- 1 kanamycin and placed on a gyratory shaker (180rpm) at 28°C for 12-16h. Subsequently, the bacterial culture was transferred to 100 x 10 mm sterile Petri dishes. Eggplant leaves (3.5 x 2.0 em) were sliced transversely (3.5 x 0.5 em), dipped in the bacterial culture a few times, and placed on plant hormone-free MS medium supplemented with 5% glucose and 0.8% agar (Difco) at 26°C for 48 h. Afterward, the inoculated leaves were rinsed with sterile water to remove excess bacteria and placed on MS medium supplemented with 2 mg ·1- 1 zeatin, 3 % sucrose, (MS2Z), 0.1 mg· ml- 1 kanamycin or 0.025 mg· ml- 1 hygromycin (Calibiochem Co.) depending on the plasmid, 0.3 mg· ml- 1 cefotaxime (Calbiochem Co.) and 0.8 % agar (Sigma) in 100 x 10 mm Petri
Opine assay To determine the presence of the nopaline synthase gene in the kanamycin resistant plants we used the method described by Otten and Schilperoort (1978) except that the samples ran for 1.5 h at 240 V and 180 rnA.
DNA extraction and hybridization Ten grams of leaves from 3 kanamycin resistant plants or a kanamycin sensitive (control) plant, all grown in vitro, were ground in a mortar in 20 ml of warm (38°C) CTAB extraction buffer (0.1 M Tris-CI pH8.D; 104M NaCI; D.D2M EDTA, 2% hexadecyltrimethylammonium bromide, Sigma). Immediately before the extraction 1 % v/v of 2-mercaptoethanol was added to the extraction buffer. The slurry was passed through 4 layers of cheese cloth, in-
Fig. 1: Leaf explants from non-transformed (left) and transformed (right) eggplants cultured for three weeks on MS medium + 2 mg .1- 1 zeatin + D.3 mg· ml- 1 kanamycin.
54
A.
GURI
and K. C. SINK
t
•
,
t
1
2
3
, I 4
cubated for 1 h at 60 °e, set at 25 °e for 1 h with 2 mg ribonuclease A, (Sigma) then extracted 3 times with 24: 1 chloroform: isoamyl alcohol. DNA was precipitated twice with - 20 °e isopropyl alcohol and dissolved in TE buffer (10 mM Tris. el pH 8.0; 1 mM EDTA, pH 8.0) to a final concentration of 1 p,g' p,l-l DNA. The plasmid pMON200 was cut with EcoRI and BamHI and the 1.3 kb insert that contained the NOS-NPT gene was 32p labelled (approx. 1 x 108 cpm specific activity) by the oligonucleotide primer extension method of Feinberg and Vogelstein (1983) using a Pharmacia kit. Total DNA extracted from putatively transformed plants and a control plant was bound to nitrocellulose filters (5 p,g per slot) using the slot blot technique described by Rivin (1986) and probed as described in Maniatis et al. (1982).
Results and discussion
The inoculation procedure used to transform eggplant was successful only with the cointegrated plasmid pMON200. Twenty-eight kanamycin resistant plants, from separate transformation events, were obtained. Following inoculation with the two binary plasmids pCIBI0 and 715 we did not detect callus initiation on MS2Z medium supplemented with 0.1 mg' ml- 1 kanamycin or 0.025 mg' ml- 1 hygromycin. However, on the same medium lacking these two antibiotics, callus and regenerated plants were obtained. Similar results were reported by McCormick et ai. (1986) on tomato. The success of the cointegrated versus the binary plasmids could result from: 1) the lack of an overdrive sequence in the binary plasmids or 2) instability of the binary plasmid in Agrobacterium in the absence of drug selection (Klee et aI., 1987). Kanamycin resistant shoots that were transferred to MSOT with 0.1 mg' ml- I kanamycin grew normally and developed roots while control plants died 3 - 4 weeks after insertion in the same medium. Explants (3.5 x 1.0 em) taken from kanamycin resistant plants that grew constantly in the presence of kanamycin, and those taken from kanamycin resistant plants that grew 6 months without kanamycin, formed callus and shoots when placed on MS2Z with 0.3 mg . ml- 1 kanamycin. Conversely, leaves from control plants did not produce callus or shoots (Fig. 1). The results of the blot hybridization showed that only the three selected kanamycin resistant plants contained the
5
Fig. 2: Slot Dot DNA hybridization of 32p labelled NOS-NPT gene (1 x 108 cpm specific activity) to: Lane 1 - total DNA of pMON200; Lanes 2-4 - transformed eggplant plants 1-3; Lane 5 - non-transformed eggplant plant. Five p,g of total DNA loaded in each slot. The Ne filter with the 32p hybridized probe was exposed to Kodak diagnostic film X-OMAT AR for 8 hours.
chimeric NOS-NPT gene sequence (Fig. 2). According to the hybridization intensities, it appeared that the 3 plants, each taken from a different experimental Petri dish, varied with regard to the number of NOS-NPT copies (Fig. 2, plant 2 vs. 3). However, at a dose of 0.3 mg' ml- 1 kanamycin they all exhibited the same degree of resistance. The nopaline assay (Fig. 3) showed that with the 3 tested transformants the nopaline synthase gene co-transformed with the NOS-NPT gene; therefore, they could synthesize nopaline in the presence of arginine and a-ketoglutaric acid. Preliminary results
A
N NS
1
2
3
4
Fig. 3: Analysis of nopaline synthase activity in: Lane 1 - non-transformed; Lanes 2-4 - three transformed plants; A = arginine, N = nopaline, NS = neutral sugar.
Agrobacterium transformation of eggplant
from seeds of two fruits taken from two transformed plants (Nos. 1 and 3) gave 62 and 81 seeds in which the ratio of kanamycin resistant to sensitive seedlings (49: 13 and 57: 27, respectively) statistically fit the 3: 1 segregation ratio expected by a single dominant trait. The ability to introduce the NOS-NPT gene into the vegetable crop plant eggplant is important in somatic cell genetics. Such a gene can serve as an efficient selectable marker for somatic hybrid detection and may facilitate the cloning of useful genes. Introduction of NPT into other Solanaceous crop plants such as tomato and potato was reported by McCormick et al. (1986) and Shahin and Simpson (1986), respectively. Based on the present success with eggplant, it should also be possible to use the plasmid pMON200 as a vector to introduce the NPT gene into other non-tuberous wild Solanum species related to eggplant such as S. gila, S. torvum, S. sisymbrifolium, S. mamosum, S. integrifolium, S. in· canum, S. indicum and S. macrocarpon. According to Yamakawa and Mochizuki (1978), these wild Solanum species are endowed with resistance to several diseases which are lacking in eggplant. Use of this transformation technology could facilitate gene cloning since the T-DNA appears to insert randomly within the plant genome (Yadav et ai., 1982; Chyi et al., 1986). Thus, insertion of disarmed T-DNA carrying the NPT gene at a site nearby the yet uncloned disease resistance gene(s) may allow their integration into the eggplant genome following fragmentation (using X-ray or ),-ray irradiation) of the donor wild species genome and protoplast fusion. Acknowledgement We wish to thank Mrs. Michal Volokita for technical assistance, and Dr. M. F. Thomashow for the pMON200 probe.
55
References BEVAN, M.: Nucleic Acid Res. 12, 8711-8721 (1984). CHILTON, M.-D.: Scientific Amer. 248, 36-45 (1983). CHYI, Y. S., R. A. JORGENSEN, D. GOLDSTEIN, S. D. TANKSELY, and F. LOAIZA-FIGUEROA: Mol. Gen. Genet. 204, 64-69 (1986). FEINBERG, A. and B. VOGELSTEIN: Anal. Biochem. 132, 6-13 (1983). HOOYKAAs, P. J. J. and R. A. SCHILPEROORT: Adv. Genet. 22, 209-271 (1984). HORSCH, R. B., J. E. FRY, N. L. HOFFMAN, D. EICHHOLTZ, S. G. RoGERS, and R. T. FRALEY: Science 227, 1229-1230 (1985). KLEE, H., R. B. HORSCH, and S. G. ROGERS: Ann. Rev. Plant Physiol. 38,467 -486 (1987). MANIATIS, T., E. F. FRITSCH, and J. SAMBROOK: Molecular Cloning, Cold Spring Harbor (1982). MCCORMICK, S., S. J. NIEDERMEYER, J. E. FRY, A. BRANASON, R. B. HORSCH, and R. T. FRALEY: Plant Cell Reports 5, 81-84 (1986). MURASHIGE, T. and F. SKOOG: Physiol. Plant. 15,473-497 (1962). OTTEN, L. A. B. M. and R. A. SCHILPEROORT: Biochem. Biophy. Acta 572, 497 -500 (1978). RIVIN, c.: Methods Enzymol. 118, 75-86 (1986). ROGERS, S. G., R. B. HORSCH, and R. T. FRALEY: Methods Enzymol. 118,627 -640 (1986). SHAH, D., R. B. HORSCH, H. KLEE, G. KISHORE, J. WINTER, N. TURNER, C. HIRONARA, P. SANDERS, C. GASSER, S. AYBENT, N. SIEGEL, S. G. ROGERS, and R. T. FRALEY: Science 233, 478-481 (1986). SHAHIN, E. A. and R. B. SIMPSON: HortScience 21, 1199-1201 (1986). YADAV, N. S., J. VANDERLEYDEN, P. R. BENNET, W. M. BARNES, and M.-D. CHILTON: Proc. Nat!. Acad. Sci. 79, 6322-6326 (1982). YAMARAWA, K. and H. MOCHIZUKI: The 20th International Congress Horticulture Meeting, Sydney, Australia, Abstract No. 1674 (1978).
Agrobacterium Transformation of Eggplane A.
GURI
and K. C.
SINK
Department of Horticulture, Michigan State University, East Lansing, MI 48824 U.S.A. Received November 19, 1987 . Accepted February 7,1988
Summary Leaf sections of in vitro cultured seedlings of Solanum melongena L., eggplant cv. Black Beauty, were inoculated with two disarmed binary strains of Agrobacterium, 715 and pCIB10, and with the cointegrated, disarmed plasmid pMON200. Eggplants resistant to kanamycin were only obtained using the pMON200 plasmid. Three plants, selected randomly among the 28 kanamycin resistant plants, were analysed using slot blot DNA hybridization and found to contain a sequence homologous to that of the NOS-NPT chimeric gene. Conversely, a control plant did not have the NOS-NPT homologous sequence. The 3 transformed plants produced nopaline in the presence of arginine. The kanamycin resistant trait was stable and expressed in 12 of the transformed plants after a 6 month culture period in kanamycin-free medium. Preliminary results also indicated that the resistance to kanamycin was transferred as a dominant Mendelian trait.
Key words: Agrobacterium, eggplant, Solanum melongena, trans/ormation, kanamycin. Abbreviations: LB-Luria broth; MS-Murashige and Skoog; NAA-a-naphthaleneactic acid; NPT neomycin phosphotransferase.
Introduction Agrobacterium tume/aciens is a useful tool for plant transformation. This vector has been applied to a broad range of dicot species and has been especially successful to date in transforming several Solanaceous species including: tobacco (Chilton, 1983), petunia (Shah et aI., 1986), tomato (McCormick et aI., 1986) and potato (Shahin and Simpson, 1986). Agrobacterium tume/aciens bacteria carry a Ti (tumor-inducing) plasmid, part of which, the T-DNA is transferred and integrated into the plant nuclear genome (Chilton, 1983; Hooykaas and Schilperoort, 1984). The ability to use TDNA as a vector for engineering the plant genome has been greatly facilitated by the use of bacterial antibiotic resistance genes as selectable markers (Horsch et aI., 1985). For example, the bacterial neomycin phosphotransferase (NPT) gene, when integrated as a chimeric gene in combination with the promoter and terminator sequences of the T-DNA borne nopaline synthase (NOS) gene, endows the plant with 1 Michigan Agricultural Experiment Station Journal Article No. 12459.
© 1988 by Gustav Fischer Verlag, Stuttgart
resistance to kanamycin (Bevan, 1984). Thus, the chimeric gene NOS-NPT can be used as a selectable marker at the cell level in tissue culture studies. In this paper we demonstrate the successful A. tume/aciens-mediated transformation of the crop species eggplant (Solanum melongena L.) by the expression of kanamycin resistance in regenerated whole plants, slot-blot analysis for the presence of the NOS-NPT sequence, and Mendelian inheritance of the kanamycin resistance trait.
Material and Methods Plant material Seeds of eggplant (Solanum melongena L.) cv. Black Beauty were obtained from A. H. Hummert Seed Co., St. Louis, MO. They were sterilized by soaking for 30 min in an aqueous solution containing 20 % commercial bleach (5.25% sodium hypochlorite) and 0.1 % Tween-20 and then washing 4 times with sterile water. The seeds were placed in 840 ml glass jars containing MS salts and vitamins (Murashige and Skoog, 1962) medium supplemented with 3 % sucrose; 0.01 mg .1- 1 NAA and 0.9 % agar and placed under
Agrobacterium transformation of eggplant 30 - 50 JLEm - 2 s - 1 cool white fluorescent tubes at 27°C. Leaves from 2-week old in vitro grown seedlings were used for Agrobacte· rium transfection.
Vector plasmid Three disarmed plasmids were used to inoculate eggplant leaf strips. Two of them, 715 and pCIB10, (provided by D. Rice, CibaGeigy) are 3.2kb plasmids each carrying in the T-DNA two chimeric genes that confer resistance to kanamycin and hygromycin. These binary plasmids are accompanied by the original Ti plasmid that lacks the entire T-DNA region. The third was pMON200 a cointegrated plasmid (provided by S. Rogers, Monsanto Co.) that contains T-DNA with the genes for kanamycin resistance and for nopaline synthesis (See Rogers et aI., 1986).
53
dishes, and placed under 30 - 50 JLEm - 2 s - 1 light (cool white fluorescent tubes) at 27°C. Three weeks later, when callus initiated at a few regions along the cut edges, the leaves were subcultured. Shoot regeneration from the primary calluses started 2 weeks later, and exised shoots (2.0 em) were inserted into plant hormone-free MS medium containing 10-fold thiamine HCI (1 mg .1- 1) (MSOT), 2 % sucrose, 0.9 % agar (Sigma), and 0.1 mg· ml- 1 kanamycin. Some of the kanamycin resistant plants were transferred to kanamycin-free MSOT medium. Six months later, leaves of kanamycin resistant plants that grew on MSOT with or without kanamycin, and leaves taken from non-transformed (control) plants were sliced and placed on MS2Z + 0.3 mg· ml- 1 kanamycin. This was done to determine expression of the NOS-NPT gene in plants grown with or without selection. Two kanamycin resistant plants were subsequently grown in the greenhouse. Seeds obtained by self-pollination were surface sterilized, as stated previously, and placed on MS medium supplemented with 2 % sucrose, 0.01 mg .1- 1 NAA, 0.05 mg· ml- 1 kanamycin and 0.9 % agar.
Inoculation and shoot regeneration The bacteria were cultured on solid (2 % Difco agar) Luria Broth (LB) medium (10 g .1- 1 yeast extract, Difco; 5 g .1- 1 trypton, Difco; 5 g .1- 1 NaCl) containing 0.05 mg· ml- 1 kanamycin sulfate (Sigma) at 28°C for 24 h. Inoculum from a single colony was transferred into sterile 25 ml Erlenmeyer flasks containing 10 ml of liquid LB plus 0.05 mg· ml- 1 kanamycin and placed on a gyratory shaker (180rpm) at 28°C for 12-16h. Subsequently, the bacterial culture was transferred to 100 x 10 mm sterile Petri dishes. Eggplant leaves (3.5 x 2.0 em) were sliced transversely (3.5 x 0.5 em), dipped in the bacterial culture a few times, and placed on plant hormone-free MS medium supplemented with 5% glucose and 0.8% agar (Difco) at 26°C for 48 h. Afterward, the inoculated leaves were rinsed with sterile water to remove excess bacteria and placed on MS medium supplemented with 2 mg ·1- 1 zeatin, 3 % sucrose, (MS2Z), 0.1 mg· ml- 1 kanamycin or 0.025 mg· ml- 1 hygromycin (Calibiochem Co.) depending on the plasmid, 0.3 mg· ml- 1 cefotaxime (Calbiochem Co.) and 0.8 % agar (Sigma) in 100 x 10 mm Petri
Opine assay To determine the presence of the nopaline synthase gene in the kanamycin resistant plants we used the method described by Otten and Schilperoort (1978) except that the samples ran for 1.5 h at 240 V and 180 rnA.
DNA extraction and hybridization Ten grams of leaves from 3 kanamycin resistant plants or a kanamycin sensitive (control) plant, all grown in vitro, were ground in a mortar in 20 ml of warm (38°C) CTAB extraction buffer (0.1 M Tris-CI pH8.D; 104M NaCI; D.D2M EDTA, 2% hexadecyltrimethylammonium bromide, Sigma). Immediately before the extraction 1 % v/v of 2-mercaptoethanol was added to the extraction buffer. The slurry was passed through 4 layers of cheese cloth, in-
Fig. 1: Leaf explants from non-transformed (left) and transformed (right) eggplants cultured for three weeks on MS medium + 2 mg .1- 1 zeatin + D.3 mg· ml- 1 kanamycin.
54
A.
GURI
and K. C. SINK
t
•
,
t
1
2
3
, I 4
cubated for 1 h at 60 °e, set at 25 °e for 1 h with 2 mg ribonuclease A, (Sigma) then extracted 3 times with 24: 1 chloroform: isoamyl alcohol. DNA was precipitated twice with - 20 °e isopropyl alcohol and dissolved in TE buffer (10 mM Tris. el pH 8.0; 1 mM EDTA, pH 8.0) to a final concentration of 1 p,g' p,l-l DNA. The plasmid pMON200 was cut with EcoRI and BamHI and the 1.3 kb insert that contained the NOS-NPT gene was 32p labelled (approx. 1 x 108 cpm specific activity) by the oligonucleotide primer extension method of Feinberg and Vogelstein (1983) using a Pharmacia kit. Total DNA extracted from putatively transformed plants and a control plant was bound to nitrocellulose filters (5 p,g per slot) using the slot blot technique described by Rivin (1986) and probed as described in Maniatis et al. (1982).
Results and discussion
The inoculation procedure used to transform eggplant was successful only with the cointegrated plasmid pMON200. Twenty-eight kanamycin resistant plants, from separate transformation events, were obtained. Following inoculation with the two binary plasmids pCIBI0 and 715 we did not detect callus initiation on MS2Z medium supplemented with 0.1 mg' ml- 1 kanamycin or 0.025 mg' ml- 1 hygromycin. However, on the same medium lacking these two antibiotics, callus and regenerated plants were obtained. Similar results were reported by McCormick et ai. (1986) on tomato. The success of the cointegrated versus the binary plasmids could result from: 1) the lack of an overdrive sequence in the binary plasmids or 2) instability of the binary plasmid in Agrobacterium in the absence of drug selection (Klee et aI., 1987). Kanamycin resistant shoots that were transferred to MSOT with 0.1 mg' ml- I kanamycin grew normally and developed roots while control plants died 3 - 4 weeks after insertion in the same medium. Explants (3.5 x 1.0 em) taken from kanamycin resistant plants that grew constantly in the presence of kanamycin, and those taken from kanamycin resistant plants that grew 6 months without kanamycin, formed callus and shoots when placed on MS2Z with 0.3 mg . ml- 1 kanamycin. Conversely, leaves from control plants did not produce callus or shoots (Fig. 1). The results of the blot hybridization showed that only the three selected kanamycin resistant plants contained the
5
Fig. 2: Slot Dot DNA hybridization of 32p labelled NOS-NPT gene (1 x 108 cpm specific activity) to: Lane 1 - total DNA of pMON200; Lanes 2-4 - transformed eggplant plants 1-3; Lane 5 - non-transformed eggplant plant. Five p,g of total DNA loaded in each slot. The Ne filter with the 32p hybridized probe was exposed to Kodak diagnostic film X-OMAT AR for 8 hours.
chimeric NOS-NPT gene sequence (Fig. 2). According to the hybridization intensities, it appeared that the 3 plants, each taken from a different experimental Petri dish, varied with regard to the number of NOS-NPT copies (Fig. 2, plant 2 vs. 3). However, at a dose of 0.3 mg' ml- 1 kanamycin they all exhibited the same degree of resistance. The nopaline assay (Fig. 3) showed that with the 3 tested transformants the nopaline synthase gene co-transformed with the NOS-NPT gene; therefore, they could synthesize nopaline in the presence of arginine and a-ketoglutaric acid. Preliminary results
A
N NS
1
2
3
4
Fig. 3: Analysis of nopaline synthase activity in: Lane 1 - non-transformed; Lanes 2-4 - three transformed plants; A = arginine, N = nopaline, NS = neutral sugar.
Agrobacterium transformation of eggplant
from seeds of two fruits taken from two transformed plants (Nos. 1 and 3) gave 62 and 81 seeds in which the ratio of kanamycin resistant to sensitive seedlings (49: 13 and 57: 27, respectively) statistically fit the 3: 1 segregation ratio expected by a single dominant trait. The ability to introduce the NOS-NPT gene into the vegetable crop plant eggplant is important in somatic cell genetics. Such a gene can serve as an efficient selectable marker for somatic hybrid detection and may facilitate the cloning of useful genes. Introduction of NPT into other Solanaceous crop plants such as tomato and potato was reported by McCormick et al. (1986) and Shahin and Simpson (1986), respectively. Based on the present success with eggplant, it should also be possible to use the plasmid pMON200 as a vector to introduce the NPT gene into other non-tuberous wild Solanum species related to eggplant such as S. gila, S. torvum, S. sisymbrifolium, S. mamosum, S. integrifolium, S. in· canum, S. indicum and S. macrocarpon. According to Yamakawa and Mochizuki (1978), these wild Solanum species are endowed with resistance to several diseases which are lacking in eggplant. Use of this transformation technology could facilitate gene cloning since the T-DNA appears to insert randomly within the plant genome (Yadav et ai., 1982; Chyi et al., 1986). Thus, insertion of disarmed T-DNA carrying the NPT gene at a site nearby the yet uncloned disease resistance gene(s) may allow their integration into the eggplant genome following fragmentation (using X-ray or ),-ray irradiation) of the donor wild species genome and protoplast fusion. Acknowledgement We wish to thank Mrs. Michal Volokita for technical assistance, and Dr. M. F. Thomashow for the pMON200 probe.
55
References BEVAN, M.: Nucleic Acid Res. 12, 8711-8721 (1984). CHILTON, M.-D.: Scientific Amer. 248, 36-45 (1983). CHYI, Y. S., R. A. JORGENSEN, D. GOLDSTEIN, S. D. TANKSELY, and F. LOAIZA-FIGUEROA: Mol. Gen. Genet. 204, 64-69 (1986). FEINBERG, A. and B. VOGELSTEIN: Anal. Biochem. 132, 6-13 (1983). HOOYKAAs, P. J. J. and R. A. SCHILPEROORT: Adv. Genet. 22, 209-271 (1984). HORSCH, R. B., J. E. FRY, N. L. HOFFMAN, D. EICHHOLTZ, S. G. RoGERS, and R. T. FRALEY: Science 227, 1229-1230 (1985). KLEE, H., R. B. HORSCH, and S. G. ROGERS: Ann. Rev. Plant Physiol. 38,467 -486 (1987). MANIATIS, T., E. F. FRITSCH, and J. SAMBROOK: Molecular Cloning, Cold Spring Harbor (1982). MCCORMICK, S., S. J. NIEDERMEYER, J. E. FRY, A. BRANASON, R. B. HORSCH, and R. T. FRALEY: Plant Cell Reports 5, 81-84 (1986). MURASHIGE, T. and F. SKOOG: Physiol. Plant. 15,473-497 (1962). OTTEN, L. A. B. M. and R. A. SCHILPEROORT: Biochem. Biophy. Acta 572, 497 -500 (1978). RIVIN, c.: Methods Enzymol. 118, 75-86 (1986). ROGERS, S. G., R. B. HORSCH, and R. T. FRALEY: Methods Enzymol. 118,627 -640 (1986). SHAH, D., R. B. HORSCH, H. KLEE, G. KISHORE, J. WINTER, N. TURNER, C. HIRONARA, P. SANDERS, C. GASSER, S. AYBENT, N. SIEGEL, S. G. ROGERS, and R. T. FRALEY: Science 233, 478-481 (1986). SHAHIN, E. A. and R. B. SIMPSON: HortScience 21, 1199-1201 (1986). YADAV, N. S., J. VANDERLEYDEN, P. R. BENNET, W. M. BARNES, and M.-D. CHILTON: Proc. Nat!. Acad. Sci. 79, 6322-6326 (1982). YAMARAWA, K. and H. MOCHIZUKI: The 20th International Congress Horticulture Meeting, Sydney, Australia, Abstract No. 1674 (1978).