Plant Cell Reports
Plant Cell Reports (1995) 15:301 - 304
,~ Springer-Verlag 1995
Transgenic barley (Hordeum vulgate L.) by eleetroporation of protoplasts M. Salmenkallio-Marttila 1, K. Aspegren 2 S. ~,kerman 3 U. Kurt6n 1, L. Mannonen 1, A. Ritala t, T.H. Teeri z, and V. Kauppinen t 1 VTT Biotechnology and F o o d Research, P.O. Box 1505, FIN-02044 VTT, Espoo, Finland 2 Institute of Biotechnology, P.O. Box 45 (Karvaamokt~ja 3), FIN-00014 University of Helsinki, Finland 3 VTT Biotechnology and F o o d Research, P.O. Box 1505, FIN-70211 Kuopio, Finland
Received 2 December 1994/Revisedversion received20 March 1995 - Communicatedby H. Lrrz
Summary. Protoplasts isolated from calli derived from cultured microspores of barley (Hordeum vulgare L. cv. Kymppi, an elite eultivar) were transformed with the neomycin phosphotransferase marker gene (nptII) by electroporation. Screening of the regenerated plants for the NFI'II activity by gel assay resulted in three positive signals. Southern blot analysis and NPTII assays of second and third generation plants confirmed the genomic integration of the transferred gene and that the new trait was inherited by the progeny.
derived from embryogenic suspension cultures (Yah et al. 1990, J~ihne et al, 1991, Funatsuki et al. 1992) and from microspore culture-derived protoplasts (SalmenkallioMarttila and Kauppinen 1995). Transgenic callus lines of barley, but no plants, have been obtained using PEGinduced direct DNA uptake into protoplasts (Lazzeri et al. 1991). To our knowledge there are no reports of successful production of transgenic barley plants by elcctroporation. In the present study microspore cultures were used for the isolation of protoplasts which were transformed by electroporation and regenerated to mature fertile plants.
Introduction Materials and methods Advances in tissue cultm'e and molecular biology techniques have made possible the genetic improvement of a variety of plants including cereals. Most of the important dicotyledonous crops have been shown to be susceptible to Agrobacterium-mediated transformation, monocotyledonous species, including the Poaceae, are resistant to Agrobacterium infection (Potrykus 1991, Karp and Lazzeri 1992). Therefore, alternative transformation methods are required for the transformation of cereal and grass crops. Different methods have been proposed and tested with cereals, but hitherto only direct DNA uptake into protoplasts and tissues and particle bombardment have yielded transformed plants whose progeny inherit the transferred genes. For example transgenic barley plants have been produced by particle bombardment of immature embryos (Ritala et al. 1994, Wan and Lemaux 1994). Protoplasts are of great value for the genetic modification of plants. They can be transformed by direct gene transfer, i.e. uptake of DNA through the plasma membrane stimulated either chemically or electrically. Transient gene expression in protoplasts has been used to optimize gene transfer methods and to study the function of regulatory elements of plant genes. Transgenic rice (Toriyama et al. 1988, Zhang and Wu 1988, Zhang et al. 1988) and maize (Rhodes et al. 1988) plants have been obtained following direct DNA transfer into protoplasts. Barley plants have been regenerated from protoplasts Correspondence to." M. Salmenkallio
Plant material. Barley grains (Hordeum vulgare L. cv. Kymppi, a tworowed spring barley) were obtained from Kesko Agronomic Station, Hauho. Plants were grown in a growth room (22/13~ day/night, 16 h light 4000 Nnol m"2s-t). For microspore culture the fillers were harvested when the sheath of the flag leaf had emerged 5 - 10 cm, which correlates with the late uninucleate to early binucleate stage of microspore development. The tillers were wrapped in aluminium foil and placed in beakers in a cold room at 7~ for 3 - 4 weeks. Microsporeisolation and culture. Mierospores were isolated and cultured in 108-medium, which is the N6 basal medium (Chu et al. 1975) supplemented with 1.1 mM glutamine, 2.8 mM m-inositol, 0.175 M maltose, 7.9 pM 2,4-D and 1.2 laM kinetin. For microspore isolation ten s p i k e s were cut into 3 - 4 pieces and macerated with a teflon rod in the 108-medium. The crude microspore preparation was then filtered through an 80 jam nylon sieve and the microspores were collected by centrifugation for 5 rain at 300 x g. The microspores were resuspended in 9 ml of the same medium and the total number of microspores and the number of embryogenie microspores were counted in a Fuchs-Rosenthal haemocytometer. The microspores were cultured in 108-medium at a density of 0.8 - 1.0 x 10~ ml "1 in 5 cm Petfi dishes in the dark at 23~ The cultures were incubated stationary for the first 1.5 weeks and then on a rotary shaker (65 rpm, stroke radius 2.5 cm). Protoplast isolation and culture. Three- to four week old cultures of microspores were used for protoplast isolation. Protoplasts were isolated and cultured according to the protocol of Lazzeri et al. (1991) for barley suspension cultures. 2 - 3 g of microspore m a s s w a s incubated in 20 ml of enzyme solution containing 1.0% cellulase Ouozuka RS, 0.5% Macerozyme R10 and 0.05% pectolyase Y23 in washing solution (LW). After 2 - 3 h incubation the suspension was diluted with an equal volume
302 of LW and filtered through 160/am, 55 tam and 20 ~m nylon sieves. The protoplasts were washed twice with LW (100 x g, 5 min) and suspended in protoplast culture medium at a density of 2 - 3 x 106 ml a. Protoplasts were cultured in L1 medium (Lazzeri et al. 1991) supplemented with 2.3 pM 2,4-D, 0.5 M maltose and 1.2% agarose (Sea-Plaque, FMC Corporation). One ml aliquots of the protoplast suspension were plated on Millicell-CM culture plate inserts (MiUipore), which were placed in 9 cm Petri dishes containing 8 ml of nurse culture. The nurse cultures were prepared by suspending approximately 0.5 g of the same microspore mass as was used for protoplast isolation in 8 ml of protoplast medium without agarose. Cultures were incubated on a rotary shaker (65 rpm, stroke radius 2.5 cm) at 230C in the dark. After one week of culture the feeder cells were removed and 2 ml of fresh 108 medium (0.14 M maltose, 9.0 ~M 2,4-D) was added to the plates. After another week of culture the old medium was replaced with fresh 108 medium. After one more week the agarose pieces were transferred to L2 medium supplemented with 11.3/aM 2,4-D and solidified with 0.3% geUan gum (Gelrite, Scott laboratories). The plating efficiencies were determined as the number of protoplasts producing microcalli in four weeks.
Plant regeneration. The regeneration media II and III were the modified MS-media II and III respectively used by Olsen (1987) for barley anther culture. The medium II contained maltose instead of sucrose (Hunter 1987) and the media were solidified with 0.3% gellan gum. Embryogenic structures developing from the dividing protoplasts were transferred to regeneration medium II for further development. The plates were incubated with illumination (50 lumol m'2s-1). Green plantlets were transferred to medium 11I for further growth and potted in soil when about 10 cm tall. Plasmid constructions, pHTT303 (5.3 kb) contains the CaMV 35S promoter, the nptll gene coding for neomycine phosphotransfemse II and the T-DNA gene 7 polyadenylation signal. Electroporation. Protoplasts were suspended in electroporation buffer (buffer 1: 0.55 M mannitol, 35 mM aspartic acid monopotassium salt, 35 mM glutamic acid monopotassium salt, 5 mM calcium gluconate, 5 mM MES, pH 7.0 or buffer 2:0.55 M mannitol; 130 mM KC1, 10 mM NaC1, 4 mM CaCI~ and 10 mM HEPES, pH 7.0) at a density of 3 - 5 x 104 ml-k For electroporation 300 lal samples of the protoplast suspension were mixed with 30/ag of plasmid DNA in multi-well dishes (Sterilin 33F24L) and chilled on ice for 10 min before electroporation. An electrical field of 670 "v'/cm was applied by discharge of a 200 laF capacitor. The protoplasts were kept on ice for 10 min, after which the protoplast suspension was mixed with the protoplast medium and plated for culturing. Neomycin_ phosphotransferase H assays. The protoplasts were collected by centrifugation (110 x g, 5 min). The samples were extracted with 50 mM Tris-HCl-buffer, pH 6.8, containing 130 /ag/ml leupeptine and 1% [3mercaptoethanol. After centrifugation (13 000 x g, 15 min) the supematant was assayed for NPTII activity with the method of Reiss el al. (1984) as modified by Van den Broeck et al. (1985). Samples were separated on a polyacrylamide gel. After electrophoresis the gel was overlaid with 1% agarose gel containing kanamycin sulphate and ['~zP]ATP. After blotting to Whatman PS1 the phosphorylated kanamycin was localized by autoradiography. Leaves from a tobacco plant transformed with the nptlI geue were used as positive control, non-transformed barley was used as negative control. Neomycin phosphotransfemse II was assayed by dot blot at rooting stage of the plantlets according to McDonnell et al. (1987) with the following modifications: Extraction buffer with 0.13 mg ml 4 lenpeptine and a small amount of purified sea sand (Merck 7712) was added to the leaf material which was then ground thoroughly in an Eppendorf tube and centrifuged. The supematant was collected and the protein concentration determined (Bradford 1976). The amount of extract used in the enzyme assay corresponded to 20 lag of total protein. The reaction mixture was blotted after 5 minutes centrifugation via a dot blot apparatus (Milliblot-D, Millipore #MBBD D0960) onto P81 paper (Whatman 3698 915). Screening of plants by PCR technique. The plant material was screened by the polymerase chain reaction (PCR) carried out in a Perkin-Elmer cetus 9600 thermocycler. The complete PCR mixture contained 100-500 ng
genomic or 0.15 pg pHTT303 DNA, 50 pmol of each oligonueleotide primer, 200/aM dNTPs, 2 U DynazymeTM and buffer supplied by the enzyme manufacturer (Finnzymes OY) in a total volume of 100 ~L. Thirty cycles were performed under following conditions: 75 s at 94~ 2 min at 55~ and 3 min at 72~ Primers were designed to amplify a 5' sequence of nptll in pHTr303. The forward primer used was 5'-ACACGCTGAAA TCACCAGTCTC-3' (+1 from start of transcription) and the reverse primer 5'-CTCGTCCTGCAGTFCAqTC-3" or 5'-TCGCCCAATAGCAGCCA GTC-3' (+281 and +417 from start of transcription, respectively). The PCR performance was controlled by including a primer pair amplifying the promoter fragment of one of the ct-amylase genes. Tne amplified samples were analyzed by electroforesis in a 2 % agarose gel.
Extraction of DNA and Southern analysis. Total DNA was isolated from leaves according to Dellaporta et al. (1983). For Southem blots, 5/ag undigested DNA or 10 tag Barn HI and Eco RI digested DNA were separated by electrophoresis in 0.5% and 0.8% agarose gels, respectively. The gels were blotted onto MagnaGraph (Micron Separation Inc.) nylon membrane (Southern 1975). A 32p-dCTP-labelled 1.1 kb BamHI-SalI fragment containing the nptll gene from pHTr303 was used as probe. Hybridization (at 42~ overnight in 50% formamide) and washing of the filters were carried out according to Sambrook et al. (1989). After washing the filters were exposed to X-ray film using intensifying screens at -70~
Results and discussion
Protoplasts isolated from cultured microspores were electroporated and cultured to produce transgenic plants. From 16.5 x 106 protoplasts treated, 42 green plants were regenerated (Table 1). The regenerated plants were screened for expression of the transferred nptlI gene by gel assay. Three plants showed NPTII activity (Fig. 1). Southern blot hybridizations performed with genomic DNA from leaves of two of the positive plants and from three plants of the progeny of each three parent plant confirmed the incorporation of several copies of the transferred gene (data not shown). The integration fragments of the progeny plants analyzed were identical indicating that the three parent plants originated from the same transformed cell. To study the inheritance of the transferred gene, 100 randomly selected seeds of the positive plants were germinated and NFFII activity was assayed from the leaves by dot blot test (Fig. 2). 69 of the plantlets were positive. PCR analysis of the samples confirmed the result (data not shown). However, the frequency of segregation of the npt II gene of one of the three sets of progeny was significantly different from the other two (Table 2). Further analysis of the progenies is needed to explain this apparent discrepancy. Table 1. Regeneration of control and transfomaed protoplast cultures after treatment by electroporation.
Treatment
No. of No. of No. of green Transgenic protoplasts microcalli plants green plants
buffer 1 Control Sample
5 x 106 5 x 106
20 55
2 37
:
buffer 2 Control Sample
3.25 x fly 3.25 x 10~
18 20
3
3
303
Table 2. NPTII activity in second generation transgenic plants.
Plant
NPTII activity of progeny positive
negative
l 2 3
28 36 5
26 5 0
Total
69
31
To prove the stable inheritance of the transferred nptll gene in the NPTII positive plants, genomic DNA from three plants of the T2 progeny of two parent plants (plants 1 and 2 in Table 2.) was analyzed by Southern analysis (Fig. 3). Undigested and Barn HI- and Eco RI-digested DNA samples were hybridized to a 3~-labelled nptlI probe. In undigested DNA samples hybridization of the probe was associated with high molecular weight DNA (Fig. 3A), providing evidence that the transferred nptll gene integrated into genomic DNA. In digested samples the probe recognized the characteristic 1.7 kb nptlI fragment and several (border) fragments (Fig 3B). Comparison of the intensities of the hybridization signals in digested total DNA samples to those of the digested plasmid controls gives an estimate of less than 10 copies of pHTI?303 with an intact nptlI gene and at least 3 such copies of pHTT303, in which the nptll gene is disrupted by integration to the barley genome. Although the analyzed DNA samples were isolated from the progeny of two different parent plants, the digested DNA samples showed an identical general hybridization pattern as was also observed in the analysis of the earlier generations. This indicates that the parent plants originated from the same transformed cell.
Fig. 1. NPTII gel assay of plants regenerated from electroporated protoplasts. Lane i positive controI (transgenic tobacco), lane 2 negative control, lanes 3 to 10 badey plants regenerated after electroporation treatment.
The experiments described show that it is possible to establish barley microspore cultures from which regenerable protoplasls can be isolated reproducibly. Further, transgenic barley plants can be produced from these protoplasts byelectroporation. However, the plating efficiencies obtained are quite low and the number of green plants regenerated is less than one tenth of the regeneration capacity of the microspore cultures. Improvement of the protoplast culture efficiency is required to make the system competitive with particle bombardment for the production of transgenic barley. With our transformation protocol no selectable marker genes are used. The transformed protoplasts are regenerated without any selection and the plants regenerated are screened for the transferred gene. Regeneration without any selective agents offers several advantages. Kanamycin selection has been found to reduce the number of transformed calli recovered by 50% (Shillito et al. 1985) as well as reduce the number of plants regenerated (Zhang et al. 1988), Because with all transformation methods only a relatively small percentage of cells become stably transformed, a selectable marker gene is usually included in the transformation system. However, when transforming crop plants selectable marker genes might give rise to problems concerning consumer acceptance of the product (Yoder and Goldsbrough 1994). Therefore transformation methods which allow the production of transgenic plants containing gene constructs of interest without any additional selectable markers are of particular interest. Electroporation is a new method for the production of transgenic barley. The results of NPT II enzyme assays and DNA gel blot hybridization analysis of transformed plants show that several copies of the nptll gene can be integrated in the barley genome using electroporation and that the introduced gene is stably inherited and expressed by transformant progeny.
Fig. 2. NPTII dot blot test of the second generation pIanflets. PC = positive control, tobacco transformed with the nptlI gene; NC = negative control, non-transformed barley. All other spots are second generation planflets, 36 positive and 7 negative in this analysis.
304
Figure 3. Southern blot analysis of DNA isolated from transgenic T 2 progeny. Three T2 plants (Lanes 1 - 3 and 4 - 6) representing progeny of two NPTII positive TOplants (I and II) were analyzed. 5 lag undigested DNA samples (Fig. 3A) or 10 lag Barn HI and Eco RI digested DNA samples (Pig. 3B) were hybridized to the 32P-labelled nptll probe. The negative controls (NC) contain undigested or Barn HI and Eco RI digested DNA isolated from nontransformed barley leaves. The positive controls (PC) are undigested or Barn HI and Eco RI digested pH'I'F303, mixed with same DNA as in the negative controls. Digestion of pHTT303 with Barn HI and Eco RI releases the nptII gene in a 1.7 kb fragment, which in addition to several border fragments is recognized by the probe in digested DNA samples of all analyzed T2 plants. In the blot with digested DNA samples 57 pg and 5.7 pg of the plasmid control correspond to 10 copies and 1 copy of plasmid per barley genome, respectively. MW shows the molecular weight markers (Pst I digested ~, DNA) in kb.
Acknowledgements. The skillful technical assistance of Jaana Juvonen and Tuuli Teikari is gratefully acknowledged.
References Bradford MM (1976) Anal Biochem 76:248-254 Chu CC, Wang CC, Sun CS, Hsii C, Yin KC, Chu CY, Bi FY (1975) Sci Sin 18:659-668 Dellaporta SL, Wood J, Hicks JB (1983) Plant Mol Biol Rep 1:19-21 Funatsuki H, L6rz H, Lazzeri PA (1992) Plant Sci 85:179-187 Hunter CP (1987) European patent application No. 87200773.7 J/ihne A, Lazzeri PA, L6rz H (1991) Plant Cell Rep 10:1-6 Karp A, Lazzeri PA (1992) In: Shewry PR (ed) Barley: genetics, biochemistry, molecular biology and biotechnology. C.A.B. International, WaUingford, pp 549-571 Lazzeri PA, Brettschneider R, Liihrs R, L6rz H (1991) Theor Appl Genet 81:437-444 McDonnell RE, Clark RD, Smith WA, Hinchee MA (1987) Plant Mol Biol Rep 5:380-386 Olsen FL (1987) Cartsberg Res Commun 52:393-404 Potrykns I (1991) Ann Rev Plant Physiol Plant Mol Biol 42:205-225 Reiss B, Sprengel R, Will H, Schaller H (1984) Gene 30:211-218 Rhodes CA, Pierce DA, Metfler IJ. Mascarenhas D, Detmer JJ (1988) Science 240:204-207 Ritala A, Aspegren K, Kurt6n U, Salmenkallio-Marttila M. Marmonen L, Hannns R, Kauppinen V, Tecri TH, Enari T-M (1994) Plant Mol Biol 24:317-325 Salmenkallio-Marttila M, Kauppinen V (1995) Plant Cell Rep 14:253-256 Sambrook J, Frisch EF, Maniatis T (1989) Molecular cloning, a laboratory manual, 2nd ed., Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press Shillito RD, Saul MW, Paszkowski J, Muller M, Potrykus 1 (1985) Bio/Technol 3:1099-1105
Southem EM (1975) J Mol Biol 98:503-517 Toriyama K, Arimoto Y, Uchimiya H, Hinata K (1988) Bio/Technol 6:1072-1074 Van den Broeck G, Timko MP, Kausch AP, Cashmore AR, Van Montagu M, Herrera-Estrella L (1985) Nature 313:358-363 Wan Y, Lemaux PL (1994) Plant Physiol 104:37-48 Yan Q, Zhang X, Shi J, Li J (1990) Kexue Tongbao 35:1581-1583 Yoder JI, Goldsbrough AP (1994) Bio/Technol 12:263-267 Zhang W, Wu R (1988) Theor Appl Genet 76:835-840 Zhang HM, Yang H, Rech EL, Golds TJ, Davis AS, Mulligan BJ, Cocking EC, Davey MR (1988) Plant Cell Rep 7:379-84
Plant Cell Reports (1995) 15:301 - 304
,~ Springer-Verlag 1995
Transgenic barley (Hordeum vulgate L.) by eleetroporation of protoplasts M. Salmenkallio-Marttila 1, K. Aspegren 2 S. ~,kerman 3 U. Kurt6n 1, L. Mannonen 1, A. Ritala t, T.H. Teeri z, and V. Kauppinen t 1 VTT Biotechnology and F o o d Research, P.O. Box 1505, FIN-02044 VTT, Espoo, Finland 2 Institute of Biotechnology, P.O. Box 45 (Karvaamokt~ja 3), FIN-00014 University of Helsinki, Finland 3 VTT Biotechnology and F o o d Research, P.O. Box 1505, FIN-70211 Kuopio, Finland
Received 2 December 1994/Revisedversion received20 March 1995 - Communicatedby H. Lrrz
Summary. Protoplasts isolated from calli derived from cultured microspores of barley (Hordeum vulgare L. cv. Kymppi, an elite eultivar) were transformed with the neomycin phosphotransferase marker gene (nptII) by electroporation. Screening of the regenerated plants for the NFI'II activity by gel assay resulted in three positive signals. Southern blot analysis and NPTII assays of second and third generation plants confirmed the genomic integration of the transferred gene and that the new trait was inherited by the progeny.
derived from embryogenic suspension cultures (Yah et al. 1990, J~ihne et al, 1991, Funatsuki et al. 1992) and from microspore culture-derived protoplasts (SalmenkallioMarttila and Kauppinen 1995). Transgenic callus lines of barley, but no plants, have been obtained using PEGinduced direct DNA uptake into protoplasts (Lazzeri et al. 1991). To our knowledge there are no reports of successful production of transgenic barley plants by elcctroporation. In the present study microspore cultures were used for the isolation of protoplasts which were transformed by electroporation and regenerated to mature fertile plants.
Introduction Materials and methods Advances in tissue cultm'e and molecular biology techniques have made possible the genetic improvement of a variety of plants including cereals. Most of the important dicotyledonous crops have been shown to be susceptible to Agrobacterium-mediated transformation, monocotyledonous species, including the Poaceae, are resistant to Agrobacterium infection (Potrykus 1991, Karp and Lazzeri 1992). Therefore, alternative transformation methods are required for the transformation of cereal and grass crops. Different methods have been proposed and tested with cereals, but hitherto only direct DNA uptake into protoplasts and tissues and particle bombardment have yielded transformed plants whose progeny inherit the transferred genes. For example transgenic barley plants have been produced by particle bombardment of immature embryos (Ritala et al. 1994, Wan and Lemaux 1994). Protoplasts are of great value for the genetic modification of plants. They can be transformed by direct gene transfer, i.e. uptake of DNA through the plasma membrane stimulated either chemically or electrically. Transient gene expression in protoplasts has been used to optimize gene transfer methods and to study the function of regulatory elements of plant genes. Transgenic rice (Toriyama et al. 1988, Zhang and Wu 1988, Zhang et al. 1988) and maize (Rhodes et al. 1988) plants have been obtained following direct DNA transfer into protoplasts. Barley plants have been regenerated from protoplasts Correspondence to." M. Salmenkallio
Plant material. Barley grains (Hordeum vulgare L. cv. Kymppi, a tworowed spring barley) were obtained from Kesko Agronomic Station, Hauho. Plants were grown in a growth room (22/13~ day/night, 16 h light 4000 Nnol m"2s-t). For microspore culture the fillers were harvested when the sheath of the flag leaf had emerged 5 - 10 cm, which correlates with the late uninucleate to early binucleate stage of microspore development. The tillers were wrapped in aluminium foil and placed in beakers in a cold room at 7~ for 3 - 4 weeks. Microsporeisolation and culture. Mierospores were isolated and cultured in 108-medium, which is the N6 basal medium (Chu et al. 1975) supplemented with 1.1 mM glutamine, 2.8 mM m-inositol, 0.175 M maltose, 7.9 pM 2,4-D and 1.2 laM kinetin. For microspore isolation ten s p i k e s were cut into 3 - 4 pieces and macerated with a teflon rod in the 108-medium. The crude microspore preparation was then filtered through an 80 jam nylon sieve and the microspores were collected by centrifugation for 5 rain at 300 x g. The microspores were resuspended in 9 ml of the same medium and the total number of microspores and the number of embryogenie microspores were counted in a Fuchs-Rosenthal haemocytometer. The microspores were cultured in 108-medium at a density of 0.8 - 1.0 x 10~ ml "1 in 5 cm Petfi dishes in the dark at 23~ The cultures were incubated stationary for the first 1.5 weeks and then on a rotary shaker (65 rpm, stroke radius 2.5 cm). Protoplast isolation and culture. Three- to four week old cultures of microspores were used for protoplast isolation. Protoplasts were isolated and cultured according to the protocol of Lazzeri et al. (1991) for barley suspension cultures. 2 - 3 g of microspore m a s s w a s incubated in 20 ml of enzyme solution containing 1.0% cellulase Ouozuka RS, 0.5% Macerozyme R10 and 0.05% pectolyase Y23 in washing solution (LW). After 2 - 3 h incubation the suspension was diluted with an equal volume
302 of LW and filtered through 160/am, 55 tam and 20 ~m nylon sieves. The protoplasts were washed twice with LW (100 x g, 5 min) and suspended in protoplast culture medium at a density of 2 - 3 x 106 ml a. Protoplasts were cultured in L1 medium (Lazzeri et al. 1991) supplemented with 2.3 pM 2,4-D, 0.5 M maltose and 1.2% agarose (Sea-Plaque, FMC Corporation). One ml aliquots of the protoplast suspension were plated on Millicell-CM culture plate inserts (MiUipore), which were placed in 9 cm Petri dishes containing 8 ml of nurse culture. The nurse cultures were prepared by suspending approximately 0.5 g of the same microspore mass as was used for protoplast isolation in 8 ml of protoplast medium without agarose. Cultures were incubated on a rotary shaker (65 rpm, stroke radius 2.5 cm) at 230C in the dark. After one week of culture the feeder cells were removed and 2 ml of fresh 108 medium (0.14 M maltose, 9.0 ~M 2,4-D) was added to the plates. After another week of culture the old medium was replaced with fresh 108 medium. After one more week the agarose pieces were transferred to L2 medium supplemented with 11.3/aM 2,4-D and solidified with 0.3% geUan gum (Gelrite, Scott laboratories). The plating efficiencies were determined as the number of protoplasts producing microcalli in four weeks.
Plant regeneration. The regeneration media II and III were the modified MS-media II and III respectively used by Olsen (1987) for barley anther culture. The medium II contained maltose instead of sucrose (Hunter 1987) and the media were solidified with 0.3% gellan gum. Embryogenic structures developing from the dividing protoplasts were transferred to regeneration medium II for further development. The plates were incubated with illumination (50 lumol m'2s-1). Green plantlets were transferred to medium 11I for further growth and potted in soil when about 10 cm tall. Plasmid constructions, pHTT303 (5.3 kb) contains the CaMV 35S promoter, the nptll gene coding for neomycine phosphotransfemse II and the T-DNA gene 7 polyadenylation signal. Electroporation. Protoplasts were suspended in electroporation buffer (buffer 1: 0.55 M mannitol, 35 mM aspartic acid monopotassium salt, 35 mM glutamic acid monopotassium salt, 5 mM calcium gluconate, 5 mM MES, pH 7.0 or buffer 2:0.55 M mannitol; 130 mM KC1, 10 mM NaC1, 4 mM CaCI~ and 10 mM HEPES, pH 7.0) at a density of 3 - 5 x 104 ml-k For electroporation 300 lal samples of the protoplast suspension were mixed with 30/ag of plasmid DNA in multi-well dishes (Sterilin 33F24L) and chilled on ice for 10 min before electroporation. An electrical field of 670 "v'/cm was applied by discharge of a 200 laF capacitor. The protoplasts were kept on ice for 10 min, after which the protoplast suspension was mixed with the protoplast medium and plated for culturing. Neomycin_ phosphotransferase H assays. The protoplasts were collected by centrifugation (110 x g, 5 min). The samples were extracted with 50 mM Tris-HCl-buffer, pH 6.8, containing 130 /ag/ml leupeptine and 1% [3mercaptoethanol. After centrifugation (13 000 x g, 15 min) the supematant was assayed for NPTII activity with the method of Reiss el al. (1984) as modified by Van den Broeck et al. (1985). Samples were separated on a polyacrylamide gel. After electrophoresis the gel was overlaid with 1% agarose gel containing kanamycin sulphate and ['~zP]ATP. After blotting to Whatman PS1 the phosphorylated kanamycin was localized by autoradiography. Leaves from a tobacco plant transformed with the nptlI geue were used as positive control, non-transformed barley was used as negative control. Neomycin phosphotransfemse II was assayed by dot blot at rooting stage of the plantlets according to McDonnell et al. (1987) with the following modifications: Extraction buffer with 0.13 mg ml 4 lenpeptine and a small amount of purified sea sand (Merck 7712) was added to the leaf material which was then ground thoroughly in an Eppendorf tube and centrifuged. The supematant was collected and the protein concentration determined (Bradford 1976). The amount of extract used in the enzyme assay corresponded to 20 lag of total protein. The reaction mixture was blotted after 5 minutes centrifugation via a dot blot apparatus (Milliblot-D, Millipore #MBBD D0960) onto P81 paper (Whatman 3698 915). Screening of plants by PCR technique. The plant material was screened by the polymerase chain reaction (PCR) carried out in a Perkin-Elmer cetus 9600 thermocycler. The complete PCR mixture contained 100-500 ng
genomic or 0.15 pg pHTT303 DNA, 50 pmol of each oligonueleotide primer, 200/aM dNTPs, 2 U DynazymeTM and buffer supplied by the enzyme manufacturer (Finnzymes OY) in a total volume of 100 ~L. Thirty cycles were performed under following conditions: 75 s at 94~ 2 min at 55~ and 3 min at 72~ Primers were designed to amplify a 5' sequence of nptll in pHTr303. The forward primer used was 5'-ACACGCTGAAA TCACCAGTCTC-3' (+1 from start of transcription) and the reverse primer 5'-CTCGTCCTGCAGTFCAqTC-3" or 5'-TCGCCCAATAGCAGCCA GTC-3' (+281 and +417 from start of transcription, respectively). The PCR performance was controlled by including a primer pair amplifying the promoter fragment of one of the ct-amylase genes. Tne amplified samples were analyzed by electroforesis in a 2 % agarose gel.
Extraction of DNA and Southern analysis. Total DNA was isolated from leaves according to Dellaporta et al. (1983). For Southem blots, 5/ag undigested DNA or 10 tag Barn HI and Eco RI digested DNA were separated by electrophoresis in 0.5% and 0.8% agarose gels, respectively. The gels were blotted onto MagnaGraph (Micron Separation Inc.) nylon membrane (Southern 1975). A 32p-dCTP-labelled 1.1 kb BamHI-SalI fragment containing the nptll gene from pHTr303 was used as probe. Hybridization (at 42~ overnight in 50% formamide) and washing of the filters were carried out according to Sambrook et al. (1989). After washing the filters were exposed to X-ray film using intensifying screens at -70~
Results and discussion
Protoplasts isolated from cultured microspores were electroporated and cultured to produce transgenic plants. From 16.5 x 106 protoplasts treated, 42 green plants were regenerated (Table 1). The regenerated plants were screened for expression of the transferred nptlI gene by gel assay. Three plants showed NPTII activity (Fig. 1). Southern blot hybridizations performed with genomic DNA from leaves of two of the positive plants and from three plants of the progeny of each three parent plant confirmed the incorporation of several copies of the transferred gene (data not shown). The integration fragments of the progeny plants analyzed were identical indicating that the three parent plants originated from the same transformed cell. To study the inheritance of the transferred gene, 100 randomly selected seeds of the positive plants were germinated and NFFII activity was assayed from the leaves by dot blot test (Fig. 2). 69 of the plantlets were positive. PCR analysis of the samples confirmed the result (data not shown). However, the frequency of segregation of the npt II gene of one of the three sets of progeny was significantly different from the other two (Table 2). Further analysis of the progenies is needed to explain this apparent discrepancy. Table 1. Regeneration of control and transfomaed protoplast cultures after treatment by electroporation.
Treatment
No. of No. of No. of green Transgenic protoplasts microcalli plants green plants
buffer 1 Control Sample
5 x 106 5 x 106
20 55
2 37
:
buffer 2 Control Sample
3.25 x fly 3.25 x 10~
18 20
3
3
303
Table 2. NPTII activity in second generation transgenic plants.
Plant
NPTII activity of progeny positive
negative
l 2 3
28 36 5
26 5 0
Total
69
31
To prove the stable inheritance of the transferred nptll gene in the NPTII positive plants, genomic DNA from three plants of the T2 progeny of two parent plants (plants 1 and 2 in Table 2.) was analyzed by Southern analysis (Fig. 3). Undigested and Barn HI- and Eco RI-digested DNA samples were hybridized to a 3~-labelled nptlI probe. In undigested DNA samples hybridization of the probe was associated with high molecular weight DNA (Fig. 3A), providing evidence that the transferred nptll gene integrated into genomic DNA. In digested samples the probe recognized the characteristic 1.7 kb nptlI fragment and several (border) fragments (Fig 3B). Comparison of the intensities of the hybridization signals in digested total DNA samples to those of the digested plasmid controls gives an estimate of less than 10 copies of pHTI?303 with an intact nptlI gene and at least 3 such copies of pHTT303, in which the nptll gene is disrupted by integration to the barley genome. Although the analyzed DNA samples were isolated from the progeny of two different parent plants, the digested DNA samples showed an identical general hybridization pattern as was also observed in the analysis of the earlier generations. This indicates that the parent plants originated from the same transformed cell.
Fig. 1. NPTII gel assay of plants regenerated from electroporated protoplasts. Lane i positive controI (transgenic tobacco), lane 2 negative control, lanes 3 to 10 badey plants regenerated after electroporation treatment.
The experiments described show that it is possible to establish barley microspore cultures from which regenerable protoplasls can be isolated reproducibly. Further, transgenic barley plants can be produced from these protoplasts byelectroporation. However, the plating efficiencies obtained are quite low and the number of green plants regenerated is less than one tenth of the regeneration capacity of the microspore cultures. Improvement of the protoplast culture efficiency is required to make the system competitive with particle bombardment for the production of transgenic barley. With our transformation protocol no selectable marker genes are used. The transformed protoplasts are regenerated without any selection and the plants regenerated are screened for the transferred gene. Regeneration without any selective agents offers several advantages. Kanamycin selection has been found to reduce the number of transformed calli recovered by 50% (Shillito et al. 1985) as well as reduce the number of plants regenerated (Zhang et al. 1988), Because with all transformation methods only a relatively small percentage of cells become stably transformed, a selectable marker gene is usually included in the transformation system. However, when transforming crop plants selectable marker genes might give rise to problems concerning consumer acceptance of the product (Yoder and Goldsbrough 1994). Therefore transformation methods which allow the production of transgenic plants containing gene constructs of interest without any additional selectable markers are of particular interest. Electroporation is a new method for the production of transgenic barley. The results of NPT II enzyme assays and DNA gel blot hybridization analysis of transformed plants show that several copies of the nptll gene can be integrated in the barley genome using electroporation and that the introduced gene is stably inherited and expressed by transformant progeny.
Fig. 2. NPTII dot blot test of the second generation pIanflets. PC = positive control, tobacco transformed with the nptlI gene; NC = negative control, non-transformed barley. All other spots are second generation planflets, 36 positive and 7 negative in this analysis.
304
Figure 3. Southern blot analysis of DNA isolated from transgenic T 2 progeny. Three T2 plants (Lanes 1 - 3 and 4 - 6) representing progeny of two NPTII positive TOplants (I and II) were analyzed. 5 lag undigested DNA samples (Fig. 3A) or 10 lag Barn HI and Eco RI digested DNA samples (Pig. 3B) were hybridized to the 32P-labelled nptll probe. The negative controls (NC) contain undigested or Barn HI and Eco RI digested DNA isolated from nontransformed barley leaves. The positive controls (PC) are undigested or Barn HI and Eco RI digested pH'I'F303, mixed with same DNA as in the negative controls. Digestion of pHTT303 with Barn HI and Eco RI releases the nptII gene in a 1.7 kb fragment, which in addition to several border fragments is recognized by the probe in digested DNA samples of all analyzed T2 plants. In the blot with digested DNA samples 57 pg and 5.7 pg of the plasmid control correspond to 10 copies and 1 copy of plasmid per barley genome, respectively. MW shows the molecular weight markers (Pst I digested ~, DNA) in kb.
Acknowledgements. The skillful technical assistance of Jaana Juvonen and Tuuli Teikari is gratefully acknowledged.
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