Plant Cell Reports
Plant Cell Reports (1994) 14:t92-196
9 Springer-Verlag 1994
Transformation of wheat (Triticum aestivum L.) through electroporation of protoplasts D.G. He, A. Mouradov, Y.M. Yang, E. Mouradova, and K.J. Scott Department of Biochemistry, The University of Queensland, Brisbane, Australia 4072 Received 24 March 1994/Revised version received 30 May 1994 - Communicated by H. L t r z
ABSTRACT Protoplasts isolated from embryogenic suspension cultures of wheat (Triticum aestivum cv. Hartog) were electroporated in the presence of plasmid pEmuGN and/or pEmuPAT, which contained the reporter gene gus and selectable marker gene bar, respectively. Under optimised electroporation conditions, up to 0.9% of viable protoplasts displayed gus activity two days after electroporation. To select for phosphinothricin (PPT) resistant colonies, electroporated protoplasts were incubated for six weeks in a medium containing 10 ktg/ml PPT. The cells surviving the selection were maintained as individual colonies on solid medium or as suspension cultures. More than 60% of these colonies exhibited tolerance to 40 ktg/ml PPT when tested 10 months after initial selection. To date, 57 green plants have been regenerated from these colonies and 24 have been transferred to soil. Southern blot analyses of colonies and plants, using the bar gene sequence as the probe, confirmed transformation of the cells. Positive PAT assays of both regenerated colonies and plants indicated the presence of the bar gene product. These results provide a basis for the establishment of routine procedures for transformation of wheat by direct gene transfer into protoplasts. Key words: gene transfer, selection for phosphinothricin resistance, transgenic plants
Abbreviations: gus: ~-glucuronidase, PAT: phosphinothricin Nacetyltransferase, PPT: phosphinothricin, MS: Murashige and Skoog medium.
INTRODUCTION Recent progress in genetic engineering of cereals and grasses has been achieved primarily through two approaches: firstly particle bombardment of embryogenic cells and secondly direct gene transfer into protoplasts. Transgenic wheat plants have been recovered from callus (Vasil et al. 1992) and immature embryos (Vasil et al. 1993, Weeks et al. 1993, Becker et al. 1994, Nehra et al. Correspondence to: D.G. He
1994) following microprojectile bombardment. Direct gene transfer into protoplasts either by electroporation or in the presence of polyethylene glycol is considered an efficient approach for genetic transformation (Potrykus 1991) and has resulted in transgenic plants of orchardgrass (Horn et al. 1988), Festuca (Ha et al. 1992), maize (Rhodes et al. 1988) and rice (Shimamoto et al. 1989, Peng et al. 1990). In some instances fertile plants were regenerated from electroporated protoplasts (Shimamoto et al. 1989). Since 1985, there have been several reports of transient expression of foreign genes in protoplasts of einkorn wheat (Triticum monococcum) (LOrz et al. 1985, Ou-Lee et al, 1986, Hauptmann et al. 1987, Oard et al. 1989). Analogous reports for bread wheat (T. aestivum) transformation are rare; Lee et al. (1988) observed a low expression of gus gene in electroporated wheat protoplasts and the conditions affecting transient expression of wheat protoplasts were studied by Zaghmout and Wang (1992), and Zaghmout and Trolinder (1993). Recently, several groups (Chamberlain et al. 1994, Marsan et al. 1993, Zhou et al. 1993) have reported the recovery of stably transformed wheat callus from protoplasts but plant regeneration was not obtained. We have previously reported the establishment of embryogenic suspension cultures and plant regeneration from wheat protoplasts (Yang et al. 1991, He et al. 1992, Yang et al. 1994). The regeneration potential of these suspension cultures has been maintained for three years and plants have been regenerated reproducibly from protoplasts isolated from the suspension cultures. We now report transformation of these protoplasts leading to the production of stably transformed embryogenic colonies from which flowering wheat plants have been regenerated. A brief account of this work has been reported previously (Scott et al. 1994).
MATERIALS AND METHODS Isolation of protoplasts
Embryogenic suspension cultures of T.
aestivum cv. Hartog (Yang et al. 1991) were used as the source material for protoplast isolation. Isolation and purification of protoplasts was performed as previously described (He et al. 1992). Protoplast viability was estimated before and after electroporation by staining protoplasts with fluorescein diacetate (FDA) and counting the number of fluorescent protoplasts using a haemocytometer under a fluorescent microscope.
193
Plasmids The plasmids pEmuGN and pEmuPAT, used in this study, both containedthe Emu promoter (Chamberlainet al. 1994)linked either to the reporter gus gene (Jefferson 1987) or to the selectablemarker bar gene. The bar gene confers resistance to phosphinothricin(PPT), the active ingredientin the herbicideBasta (De Block et al. 1989).
Electroporation of protoplasts
An electroporator similar to that described by Larkin et al. (1990) was used in this study; it consists of capacitors of 120-1000 }.tF,delivers 1-10 pulses of up to 400 volts, with pulse length from 10 micro-secondsto 100 milliseconds,and delay from 10 milliseconds to 100 seconds. The distance between the two electrodes is 0.6 cm. The followingsettings were routinely used in this study: 400 v (667 v/cm), 10 pulses, 3 ms pulse length, 100 ms delay, and 120 pF capacitance. The electroporation buffer was modified from Larkin et al. (1990), and contained 150 mM NaCI, 6 mM CaCI2, and 30 mM Tris, pH 9.0. Mannitolwas added to the buffer to give an osmolarity of 750 mOsm/kg. After purification, protoplasts were suspended in the electroporation buffer at densities 1-10 X 106 / ml. Plasmidwas added to the protoplast suspension at a final concentrationof 50 lag/ml, or 50 lag/ml each in co-transformation experiments. The mixture containing plasmids and protoplasts was left on ice for 10 rain before transferto a disposable 1 nfl cuvette (BioRad) for electroporation. After electroporation, the protoplasts were again placed on ice for 10 min and then washed once with a solution containing 20 mM CaCI2 and 0.6 M mannitol(He et al. 1992).
Protoplast culture and selection of transformed cells Pmtoplastswere either resuspended in a liquid medium or incubated in a solid medium (1.2% agarose) at a density of 1-5 X 106 protoplasts/ml(He et al. 1992). The cultures were incubatedin disposable plastic Petri dishes in the dark at 25~ Following 10-14 days incubation,when the coloniesconsisted of 2-8 ceils, fresh liquid medium was added to the cultures and PPT was added to give a final concentrationof I0 lag/ml. After six weeks the viable colonies were transferred either to a differentiationmedium for plant regeneration, or to a PPT-free liquid medium and maintained as suspension cultures. After several weeks, the cells of these new suspension cultures were transferred to a differentiation medium for plant regeneration. The regenerated plants were incubatedin plasticjars and leaves were collected for Southernblot analysis. Those plants with well developedroot systems were transferred to soil and maintainedin a glasshouse. The cells, colonies and plants which were derived from electroporated protoplasts and survived the PPT selection were designated ES (electroporated and selected) cells, ES colonies,and ES plants.
GUS and PAT assay The histologicaland fluorescence assays of gus were performed as describedby Jefferson (1987). To test the tolerance of ES cells to PPT, small pieces of callus (ca lmm in diameter) were cut from 10-month-oldcolonies and transferred to agar medium containing40 gg/ml PPT. Growth of cells was assessed after 4-6 weeks incubation. PAT enzyme activity was assayed as described by De Block et al. (1989). Leavestaken from bar transformed tobacco plants were used as positive controls.
extraction and Southern blot analysis DNA was extracted from ES callus or leaves of ES plants accordingto the method of Doyle and Doyle (1990). The DNA was purified further by CsC1 gradient centrifugation. The concentration of DNA in the extracts was determined and aliquots (20 lag) were digested with a threefold excess of HindlIL A PCR fragment comprising450 bp of the bar coding region was used as the probe in the hybridization. Molecularweight markers were provided using SPPI'EcoRI.
DNA
RESULTS
Electroporation o f protoplasts and transient expression The electroporation was performed as described by Larkin et al. (1990) but modified by increasing the pulse number (10 instead of 6) and using higher concentrations of plasmids (50 gg/ml compared to 10 gg/ml). Fluorescence assay performed 48 hours after
electroporation showed that the higher pulse number resulted in a four-fold increase in transient expression of gus. In addition, when plasmid concentrations were increased from 10 ~tg/ml to 60 gg/ml, transient expression of gus increased about 5 times, and when 100 gg/ml plasmid was applied, expression of gus increased almost 14 times. F D A staining of protoplasts 30 minutes after electroporation showed that 30 - 60% of the protoplasts had survived the electroporation.
Expression o f gus Protoplasts were electroporated using 100 gg/ml plasmid and 10 pulses; the histological assay for gus expression after 48 hours showed that up to 0.9% of protoplasts, which had survived electroporation, were blue, indicating the expression of the gus gene (Fig. 1A). Blue colonies consisting of 2-10 cells were observed following 4 weeks' incubation (Fig. 1B). However, although there were colonies consisting of more than 10 cells in the same Petri-dishes, wholly blue colonies were not observed. After subculture for 10 months, the histological assay for gus showed that 7.8% (20/254) of the co-transformed ES colonies contained a few single blue cells.
Selection of regeneration
transformed
colonies
and
plant
The kill concentration of PPT for non-transformed protoplast-derived colonies of cv. Hartog, was investigated by adding varying concentrations of PPT to 14-day-old protoplast cultures. In stationary liquid medium, most colonies died on exposure to 2.5 gg/ml PPT; however, a few viable colonies were observed in medium containing PPT as high as 10 gg/ml when the incubation density of protoplasts was high. The kill concentration of PPT for protoplasts in agarose medium was investigated by cutting the agarose into small blocks which were transferred to Petri dishes containing fresh liquid medium. PPT was added to the m e d i u m and the cultures were placed on a shaker. The m i n i m u m kill concentration of PPT for protocolonies embedded in agarose m e d i u m was lower than for those incubated in liquid medium. Most of the colonies in the agarose blocks died in the presence of 1 p . g / m l PPT and only rarely were viable colonies observed in medium containing 5 gg/ml PPT. To select bar-transformed colonies, electroporated protoplasts were incubated in either liquid or agarose m e d i u m and 10-14 days after electroporation PPT was added at a final concentration of 10 gg/ml. Following six weeks' incubation in m e d i u m containing PPT, on average 20 viable colonies were obtained from a 3.5 cm Petri dish where initially I-5 X 106 protoplasts had been incubated. A n accurate calculation of the transformation frequency was difficult as some colonies were very friable and tended to break up, especially when the colonies were maintained on a shaker which may have resulted in different cell clumps having a c o m m o n origin. To date, embryogenic ES colonies have been obtained in 24 independent experiments. A total of 57 green plants have been regenerated from 7 independent experiments. Nine of those plants were regenerated directly from protoplast-derived callus and remaining 48 plants were regenerated from ES protocolony-derived suspension cultures. Due to the breaking up of protocolonies during selection and propagation as suspension cultures, it was not possible to determine if the plants obtained in a
194 single experiment were independently transformed or if they were of common origin. Twenty-four plants have been transferred to soil and although 6 plants flowered none set seeds.
Confirmation of the transformation After subculture for 10 months, the tolerance of ES colonies to PPT was tested. Small pieces (
Plant Cell Reports (1994) 14:t92-196
9 Springer-Verlag 1994
Transformation of wheat (Triticum aestivum L.) through electroporation of protoplasts D.G. He, A. Mouradov, Y.M. Yang, E. Mouradova, and K.J. Scott Department of Biochemistry, The University of Queensland, Brisbane, Australia 4072 Received 24 March 1994/Revised version received 30 May 1994 - Communicated by H. L t r z
ABSTRACT Protoplasts isolated from embryogenic suspension cultures of wheat (Triticum aestivum cv. Hartog) were electroporated in the presence of plasmid pEmuGN and/or pEmuPAT, which contained the reporter gene gus and selectable marker gene bar, respectively. Under optimised electroporation conditions, up to 0.9% of viable protoplasts displayed gus activity two days after electroporation. To select for phosphinothricin (PPT) resistant colonies, electroporated protoplasts were incubated for six weeks in a medium containing 10 ktg/ml PPT. The cells surviving the selection were maintained as individual colonies on solid medium or as suspension cultures. More than 60% of these colonies exhibited tolerance to 40 ktg/ml PPT when tested 10 months after initial selection. To date, 57 green plants have been regenerated from these colonies and 24 have been transferred to soil. Southern blot analyses of colonies and plants, using the bar gene sequence as the probe, confirmed transformation of the cells. Positive PAT assays of both regenerated colonies and plants indicated the presence of the bar gene product. These results provide a basis for the establishment of routine procedures for transformation of wheat by direct gene transfer into protoplasts. Key words: gene transfer, selection for phosphinothricin resistance, transgenic plants
Abbreviations: gus: ~-glucuronidase, PAT: phosphinothricin Nacetyltransferase, PPT: phosphinothricin, MS: Murashige and Skoog medium.
INTRODUCTION Recent progress in genetic engineering of cereals and grasses has been achieved primarily through two approaches: firstly particle bombardment of embryogenic cells and secondly direct gene transfer into protoplasts. Transgenic wheat plants have been recovered from callus (Vasil et al. 1992) and immature embryos (Vasil et al. 1993, Weeks et al. 1993, Becker et al. 1994, Nehra et al. Correspondence to: D.G. He
1994) following microprojectile bombardment. Direct gene transfer into protoplasts either by electroporation or in the presence of polyethylene glycol is considered an efficient approach for genetic transformation (Potrykus 1991) and has resulted in transgenic plants of orchardgrass (Horn et al. 1988), Festuca (Ha et al. 1992), maize (Rhodes et al. 1988) and rice (Shimamoto et al. 1989, Peng et al. 1990). In some instances fertile plants were regenerated from electroporated protoplasts (Shimamoto et al. 1989). Since 1985, there have been several reports of transient expression of foreign genes in protoplasts of einkorn wheat (Triticum monococcum) (LOrz et al. 1985, Ou-Lee et al, 1986, Hauptmann et al. 1987, Oard et al. 1989). Analogous reports for bread wheat (T. aestivum) transformation are rare; Lee et al. (1988) observed a low expression of gus gene in electroporated wheat protoplasts and the conditions affecting transient expression of wheat protoplasts were studied by Zaghmout and Wang (1992), and Zaghmout and Trolinder (1993). Recently, several groups (Chamberlain et al. 1994, Marsan et al. 1993, Zhou et al. 1993) have reported the recovery of stably transformed wheat callus from protoplasts but plant regeneration was not obtained. We have previously reported the establishment of embryogenic suspension cultures and plant regeneration from wheat protoplasts (Yang et al. 1991, He et al. 1992, Yang et al. 1994). The regeneration potential of these suspension cultures has been maintained for three years and plants have been regenerated reproducibly from protoplasts isolated from the suspension cultures. We now report transformation of these protoplasts leading to the production of stably transformed embryogenic colonies from which flowering wheat plants have been regenerated. A brief account of this work has been reported previously (Scott et al. 1994).
MATERIALS AND METHODS Isolation of protoplasts
Embryogenic suspension cultures of T.
aestivum cv. Hartog (Yang et al. 1991) were used as the source material for protoplast isolation. Isolation and purification of protoplasts was performed as previously described (He et al. 1992). Protoplast viability was estimated before and after electroporation by staining protoplasts with fluorescein diacetate (FDA) and counting the number of fluorescent protoplasts using a haemocytometer under a fluorescent microscope.
193
Plasmids The plasmids pEmuGN and pEmuPAT, used in this study, both containedthe Emu promoter (Chamberlainet al. 1994)linked either to the reporter gus gene (Jefferson 1987) or to the selectablemarker bar gene. The bar gene confers resistance to phosphinothricin(PPT), the active ingredientin the herbicideBasta (De Block et al. 1989).
Electroporation of protoplasts
An electroporator similar to that described by Larkin et al. (1990) was used in this study; it consists of capacitors of 120-1000 }.tF,delivers 1-10 pulses of up to 400 volts, with pulse length from 10 micro-secondsto 100 milliseconds,and delay from 10 milliseconds to 100 seconds. The distance between the two electrodes is 0.6 cm. The followingsettings were routinely used in this study: 400 v (667 v/cm), 10 pulses, 3 ms pulse length, 100 ms delay, and 120 pF capacitance. The electroporation buffer was modified from Larkin et al. (1990), and contained 150 mM NaCI, 6 mM CaCI2, and 30 mM Tris, pH 9.0. Mannitolwas added to the buffer to give an osmolarity of 750 mOsm/kg. After purification, protoplasts were suspended in the electroporation buffer at densities 1-10 X 106 / ml. Plasmidwas added to the protoplast suspension at a final concentrationof 50 lag/ml, or 50 lag/ml each in co-transformation experiments. The mixture containing plasmids and protoplasts was left on ice for 10 rain before transferto a disposable 1 nfl cuvette (BioRad) for electroporation. After electroporation, the protoplasts were again placed on ice for 10 min and then washed once with a solution containing 20 mM CaCI2 and 0.6 M mannitol(He et al. 1992).
Protoplast culture and selection of transformed cells Pmtoplastswere either resuspended in a liquid medium or incubated in a solid medium (1.2% agarose) at a density of 1-5 X 106 protoplasts/ml(He et al. 1992). The cultures were incubatedin disposable plastic Petri dishes in the dark at 25~ Following 10-14 days incubation,when the coloniesconsisted of 2-8 ceils, fresh liquid medium was added to the cultures and PPT was added to give a final concentrationof I0 lag/ml. After six weeks the viable colonies were transferred either to a differentiationmedium for plant regeneration, or to a PPT-free liquid medium and maintained as suspension cultures. After several weeks, the cells of these new suspension cultures were transferred to a differentiation medium for plant regeneration. The regenerated plants were incubatedin plasticjars and leaves were collected for Southernblot analysis. Those plants with well developedroot systems were transferred to soil and maintainedin a glasshouse. The cells, colonies and plants which were derived from electroporated protoplasts and survived the PPT selection were designated ES (electroporated and selected) cells, ES colonies,and ES plants.
GUS and PAT assay The histologicaland fluorescence assays of gus were performed as describedby Jefferson (1987). To test the tolerance of ES cells to PPT, small pieces of callus (ca lmm in diameter) were cut from 10-month-oldcolonies and transferred to agar medium containing40 gg/ml PPT. Growth of cells was assessed after 4-6 weeks incubation. PAT enzyme activity was assayed as described by De Block et al. (1989). Leavestaken from bar transformed tobacco plants were used as positive controls.
extraction and Southern blot analysis DNA was extracted from ES callus or leaves of ES plants accordingto the method of Doyle and Doyle (1990). The DNA was purified further by CsC1 gradient centrifugation. The concentration of DNA in the extracts was determined and aliquots (20 lag) were digested with a threefold excess of HindlIL A PCR fragment comprising450 bp of the bar coding region was used as the probe in the hybridization. Molecularweight markers were provided using SPPI'EcoRI.
DNA
RESULTS
Electroporation o f protoplasts and transient expression The electroporation was performed as described by Larkin et al. (1990) but modified by increasing the pulse number (10 instead of 6) and using higher concentrations of plasmids (50 gg/ml compared to 10 gg/ml). Fluorescence assay performed 48 hours after
electroporation showed that the higher pulse number resulted in a four-fold increase in transient expression of gus. In addition, when plasmid concentrations were increased from 10 ~tg/ml to 60 gg/ml, transient expression of gus increased about 5 times, and when 100 gg/ml plasmid was applied, expression of gus increased almost 14 times. F D A staining of protoplasts 30 minutes after electroporation showed that 30 - 60% of the protoplasts had survived the electroporation.
Expression o f gus Protoplasts were electroporated using 100 gg/ml plasmid and 10 pulses; the histological assay for gus expression after 48 hours showed that up to 0.9% of protoplasts, which had survived electroporation, were blue, indicating the expression of the gus gene (Fig. 1A). Blue colonies consisting of 2-10 cells were observed following 4 weeks' incubation (Fig. 1B). However, although there were colonies consisting of more than 10 cells in the same Petri-dishes, wholly blue colonies were not observed. After subculture for 10 months, the histological assay for gus showed that 7.8% (20/254) of the co-transformed ES colonies contained a few single blue cells.
Selection of regeneration
transformed
colonies
and
plant
The kill concentration of PPT for non-transformed protoplast-derived colonies of cv. Hartog, was investigated by adding varying concentrations of PPT to 14-day-old protoplast cultures. In stationary liquid medium, most colonies died on exposure to 2.5 gg/ml PPT; however, a few viable colonies were observed in medium containing PPT as high as 10 gg/ml when the incubation density of protoplasts was high. The kill concentration of PPT for protoplasts in agarose medium was investigated by cutting the agarose into small blocks which were transferred to Petri dishes containing fresh liquid medium. PPT was added to the m e d i u m and the cultures were placed on a shaker. The m i n i m u m kill concentration of PPT for protocolonies embedded in agarose m e d i u m was lower than for those incubated in liquid medium. Most of the colonies in the agarose blocks died in the presence of 1 p . g / m l PPT and only rarely were viable colonies observed in medium containing 5 gg/ml PPT. To select bar-transformed colonies, electroporated protoplasts were incubated in either liquid or agarose m e d i u m and 10-14 days after electroporation PPT was added at a final concentration of 10 gg/ml. Following six weeks' incubation in m e d i u m containing PPT, on average 20 viable colonies were obtained from a 3.5 cm Petri dish where initially I-5 X 106 protoplasts had been incubated. A n accurate calculation of the transformation frequency was difficult as some colonies were very friable and tended to break up, especially when the colonies were maintained on a shaker which may have resulted in different cell clumps having a c o m m o n origin. To date, embryogenic ES colonies have been obtained in 24 independent experiments. A total of 57 green plants have been regenerated from 7 independent experiments. Nine of those plants were regenerated directly from protoplast-derived callus and remaining 48 plants were regenerated from ES protocolony-derived suspension cultures. Due to the breaking up of protocolonies during selection and propagation as suspension cultures, it was not possible to determine if the plants obtained in a
194 single experiment were independently transformed or if they were of common origin. Twenty-four plants have been transferred to soil and although 6 plants flowered none set seeds.
Confirmation of the transformation After subculture for 10 months, the tolerance of ES colonies to PPT was tested. Small pieces (
