Plant Cell Reports
Plant Cell Reports (1992) 11:567-570
9 Springer-Verlag1992
Electroporation and PEG delivery of DNA into maize microspores Anne Fennell a, 2 and Randai Hauptmann 1, 3 1 Plant Molecular Biology Center, Northern Illinois University, DeKalb, IL 60115, USA 2 Current address: Dept. Horticulture, Forestry, Landscape and Parks, South Dakota State University, Brookings, SD 57007, USA 3 Current address: Amoco Technology Company, Naperville, IL 60566, USA
Received March 27, 1992/Revised version received July 6, 1992 Communicated by J. M. Widholm
S u m m a r y . The ability to deliver and detect reporter gene activity in maize microspores was tested. Tested expression vectors contained the chloramphenicol acetyl transferase (CAT) gene and one of the following promoter-intron combinations: 1) cauliflower mosaic virus (CaMV 35S), 2) CaMV 35S + maize alcohol dehydrogenase 1 intron 6 (Adhl-I6), 3) maize alcohol dehydrogenase 1 + intron 1 (Adhl-I1), or 4) maize ubiquitin 1 + intron 1 (Ubiq 1-I1) promoter + intron. The expression vectors were delivered into maize microspores using electroporation or polyethylene glycol (PEG). Both methods were effective for delivering free DNA into microspores. Although all four promoters were active in maize protoplasts, only two promoters were active in maize microspores. The CaMV 35S and the Adhl promoters did not promote gene expression in maize microspore. The CaMV 35S + Adhl-I6 and Ubiql-I1 promoters produced high levels of CAT activity in maize microspores.
Keywords: Microspore-Electroporation-Transformation-
continual initiation, selection and maintenance of embryogenic cell or callus cultures. Transforming plant microspores with free DNA could bypass some of the problems associated with current procedures. Upon release from the tetrad the microspore is uninucleate and thin walled. It begins to enlarge and develops a germpore before the exine forms (Chang and Nueffer 1989). A microspore at this stage is potentially more amenable to transformation with exogenous DNA than other plant cells. In addition, microspore development can be altered in vitro to produce either haploid embryos or embryogenic callus that can be regenerated into plants (Coumans et al. 1989, Datta et al. 1990, Maheshwari et al. 1982, Schaeffer 1989, Swanson et al. 1987). Thus, transformed microspores could be regenerated directly into haploid plants or dihaploid fertile plants upon chromosome doubling (Wan et al. 1989). The purpose of this study was to determine if it is possible to deliver free DNA into maize microspores using electroporation or polyethylene glycol (PEG) transformation techniques.
Zea mays
Materials and Methods
Introduction Tassels were collected at the uninueleateto binucleate stage from B73XPa91, (H99XFR16)XPa91 or Seneca 60-1I plants and stored at 8~ for two weeks. Floretswere removedfromthe tassel and surfaced sterilized with 70% ethanol for 60 seconds, 10% bleach + 0.05% Tween20 for 15 min and then rinsed three times with sterile water. The florets were homogenizedfor 10 seconds in sterile 4~ HBS electroporationbuffer (10 mM HepesBuffer, 150 mM NaCI, 5 mM CaCI*2H:0,0.2 M mannitol, pH 7.2), in a sterile stainless steel blender (Waring Commercial Blendor). The resulting slurry was filtered through sterile cheese cloth. The mierosporeswere collected, resuspended in 20 % sucrose and centrifuged in sterile Babcockbottles at 100xg for 10 rain at 4~ Viable mlcrosporesbanded in the bottle neck. They were collected, rinsed twice with 4~ HBS, resuspended at lxl0 n microspores/mlHBS and placed on ice until used. Microsporepreparation.
Regeneration of transformed plants is routine in many dicot species and some monocot species. Transformation of cereal crop plants however is not common. To date only two methods have yielded fertile transgenic cereal crop plants. Stable fertile transformants of corn have been produced by microprojectile bombardment of embryogenic callus (Fromm et al. 1990, Gordon-Kamm et al. 1990). Fertile transformed rice has been produced using electroporation or polyethylene glycol-mediated transformation of protoplasts (Hayashimoto et al. 1990, Toriyama et al. 1988). These procedures require Correspondence to: A. Fennell
568 Cell suspension preparation. Cell suspension cultures derived from callus o f immature embryos o f the Pioneer hybrid t:)3377 (P3377 eulture obtained from J. M. Widholm, Univ. of Illinois, Urbana, IL) were subcultured on a 7 day interval in liquid D/G medium (50:50 vol:vol) (Duncan et al. 1985). Four days after subculture, cells were strained through a 10 mesh sieve. Cells passing through the mesh were collected and rinsed twice with HBS. These cells were resuspended at 200 #1 packed cell volume (PCV)/ml HBS and placed on ice until used. Protoplast Preparation. Protoplasts were isolated from the Pioneer hybrid P3377 suspension cells. Fifty ml of a filter sterilized enzyme solution (2.0% Cellulase RS CtGnki Yakmlt, Japan), 0.05% Pectolyase (Seishin, Japan), 0.1% macerozyme in 0.45 M mannltol, 7 mM CaCIz.2H20, pH 5.7) was added to a 5 ml PCV. The cells were incubated in the enzyme solution at room temperature for 4-12 hours and then collected by centrifugation. Viable protoplastswere separated from cells and cellular debris by centrifugation at 100xg in 20% sucrose. The protoplasts were collected, rinsed twice with HBS, resuspended at lxl06 protoplasts/ml of HBS and placed on ice until used.
Expression vectors.
Pour expression vectors containing different promoter + intron combinations fused to the coding region of the ehloramphenicol acetyl transferase (CAT) gene and terminated by the polyadenylation signals of the nopaline synthase (NOS) gene were used for transformation expe,'iments. The expression vectors are: 1) pNIU22, containing die CaMV 35S promoter, this vector was constructed by replacing the B-glueuronldase gene in the plasmid pBI221 (Clonetech) with the coding region of the CAT gene from pNCN (Clonetech). 2) pNIU24, this vector contains the sixth intron from maize alcohol dehydrogenase 1 (Adhl) inserted 5' of the CAT gene. 3) pAI1CN (Callis et al. 1987), this vector contains the maize Adhl promoter + Adh intron 1 (Adhl-ll) 4) pUBI-CAT, this vector contains the maize ubiquitin 1 promoter + intron 1 (Ubiql-ll) (obtained from A. H. Christensen, George Mason Univ., Fairfax, VI). All expression vectors are referred to by the promoter designation rather than plasmid name.
analyzed using analysis of variance and means were separated using a Student-Newman-Keuls test at p = 0.05.
Results and Discussion The ability to deliver free DNA to microspores by electroporation or PEG was demonstrated with transient expression of the reporter gene CAT. Four expression vectors containing different promoters showed differing activity in maize microspores. The four promoters tested were chosen because they are not microspore or pollen specific and are active at different levels in maize protoplasts (Fig. 1). CaMV 35S produced an average of four times greater acetylated chloramphenicol than the background levels of the -DNA control. Soaking the maize protoplasts with DNA did not produce any CAT activity above the background levels of the -DNA controls, therefore only the relative activity of the four expression vectors at 200 V is presented. The activity of CaMV 35S + Adhl-I6 was 7 times, Adhl-I1 was 2.5 times and Ubiql-I1 was 13 times higher than that of CaMV 35S. a
.> O
< I.-
b
< O
O
.> Electroporation and PEG treaonents. Equilmolar amounts of cesium chloride purified plasmid DNA (equivalent to 20 #g pUBI-CAT ) were added to one ml HBS containing microspores, protoplasts or cell suspensions. They were immediately subjected to electroporation or PEG-mediated transformation. The microspores and protoplasts were electroporated on ice in sterile electroporation cuvettes using a BRL Cell Porator (Bethesda Research Laboratories Life Technologies Inc., Gaithersburg, MD). Microspore electroporation conditions were 0, 100, 200 or 400 volts (V) at 1180 #Farads (#F). Protoplasts and cells were electroporated at 0 and 200 V. All cell types were placed on ice for 10 rain prior to plating. PEG mediated transformation was performed as previously described (Vasil et al. 1988). After the PEG treatment, the microspores were collected by ceutrifugation, rinsed with HBS and then plated. Both the microspores and protoplasts were plated at a density of 100,000 microspores/ml in MC liquid medium (Pescitelli et al. 1990) plus 8 % maltose instead of sucrose. Suspension cells were plated in 10 ml of the above medium. The samples were harvested 16 to 20tl later, rinsed wlth fresh medium and stored at -SC0C for CAT assays. Transient expression of the introduced DNA was monitored by assaying for CAT activity using standard procedures (Herrera-Estrella et al. 1990). The protoplast data is expressed as a relative activity with CaMV 35S (pAF1) activity set to 100%. Results are from two separate experiments with three samples for each treatment in each experiment. The percent o f 14C chloramphenicol converted to acetylated chloramphenicol (%conversion) was determined for microspores and cell suspensions. Microspore results are from three separate experiments with duplicate samples in each experiment. Cell suspension data are the result of two separate experiments with three samples for each treatment in each experiment. The data were
10
5
(9
n-
35S
35S + Adhl-16
Adhl-I1
Ubiql-I1
Fig.1. Transient expression of CAT in protoplasts. Protoplasts (lxl06hnl HBS) were mixed with equilmolar amounts (equivalent to 20 #g pUBI-CAT DNA) of DNA and electroporated at 200V. Means with different letters were determined to be different with a SNK test at p = 0.05 level.
Similar treatment of cell suspensions did not produce any CAT activity (Fig.2). No activity was found when cells were electroporated at 0 or 200 V. However a slight amount of activity was obtained with the ubiquitin promoter when cells were electroporated at 400 V. This result was obtained with Ubiql-I1 only. Increasing DNA concentration and electroporation at 400 V or the use of PEG did not promote CAT expression above background levels of the -DNA control with any of the other DNA expression vectors. These results indicate a very small amount of DNA can be delivered through the cell walls.
569 a
A
O I B
20 >, 0~ O
0v
j
"1
200 V 400V
I~ J ~ J
b .b. b
b
Fig.2. Traaslent expression of CAT in maize suspension cells. Suspension cells (200/xl PCV/ml HBS) were mixed with equimolar amounts (equivalent to 20 #g of pUBI-CAT DNA) of plasmid DNA and eleetroporated at 0, 200, or 400 V, 1180/,tF. Means with different letters were determined to be different with a SNK test at p = 0.05 level.
o~ ,m
>
10
O
I-,,:r U
- DNA
.b b 35S
b .b b
b .b. b
35S + Adhl-16
Adhl-I1
In spite of the presence of a cell wall and developing exine in the maize microspore, DNA could be delivered to the microspore using conditions sufficient to deliver DNA to maize protoplasts (Fig.3). There was a slight amount of activity in microspores which were soaked on ice with DNA (0 V). This could be a result of low temperature effects on the microspore plasma membranes as maize is a chilling sensitive plant. r-
.2 (g
w
a
6O a
50
(I) O ,~
40
30 ._>
20
O
.< I--
,
Plant Cell Reports (1992) 11:567-570
9 Springer-Verlag1992
Electroporation and PEG delivery of DNA into maize microspores Anne Fennell a, 2 and Randai Hauptmann 1, 3 1 Plant Molecular Biology Center, Northern Illinois University, DeKalb, IL 60115, USA 2 Current address: Dept. Horticulture, Forestry, Landscape and Parks, South Dakota State University, Brookings, SD 57007, USA 3 Current address: Amoco Technology Company, Naperville, IL 60566, USA
Received March 27, 1992/Revised version received July 6, 1992 Communicated by J. M. Widholm
S u m m a r y . The ability to deliver and detect reporter gene activity in maize microspores was tested. Tested expression vectors contained the chloramphenicol acetyl transferase (CAT) gene and one of the following promoter-intron combinations: 1) cauliflower mosaic virus (CaMV 35S), 2) CaMV 35S + maize alcohol dehydrogenase 1 intron 6 (Adhl-I6), 3) maize alcohol dehydrogenase 1 + intron 1 (Adhl-I1), or 4) maize ubiquitin 1 + intron 1 (Ubiq 1-I1) promoter + intron. The expression vectors were delivered into maize microspores using electroporation or polyethylene glycol (PEG). Both methods were effective for delivering free DNA into microspores. Although all four promoters were active in maize protoplasts, only two promoters were active in maize microspores. The CaMV 35S and the Adhl promoters did not promote gene expression in maize microspore. The CaMV 35S + Adhl-I6 and Ubiql-I1 promoters produced high levels of CAT activity in maize microspores.
Keywords: Microspore-Electroporation-Transformation-
continual initiation, selection and maintenance of embryogenic cell or callus cultures. Transforming plant microspores with free DNA could bypass some of the problems associated with current procedures. Upon release from the tetrad the microspore is uninucleate and thin walled. It begins to enlarge and develops a germpore before the exine forms (Chang and Nueffer 1989). A microspore at this stage is potentially more amenable to transformation with exogenous DNA than other plant cells. In addition, microspore development can be altered in vitro to produce either haploid embryos or embryogenic callus that can be regenerated into plants (Coumans et al. 1989, Datta et al. 1990, Maheshwari et al. 1982, Schaeffer 1989, Swanson et al. 1987). Thus, transformed microspores could be regenerated directly into haploid plants or dihaploid fertile plants upon chromosome doubling (Wan et al. 1989). The purpose of this study was to determine if it is possible to deliver free DNA into maize microspores using electroporation or polyethylene glycol (PEG) transformation techniques.
Zea mays
Materials and Methods
Introduction Tassels were collected at the uninueleateto binucleate stage from B73XPa91, (H99XFR16)XPa91 or Seneca 60-1I plants and stored at 8~ for two weeks. Floretswere removedfromthe tassel and surfaced sterilized with 70% ethanol for 60 seconds, 10% bleach + 0.05% Tween20 for 15 min and then rinsed three times with sterile water. The florets were homogenizedfor 10 seconds in sterile 4~ HBS electroporationbuffer (10 mM HepesBuffer, 150 mM NaCI, 5 mM CaCI*2H:0,0.2 M mannitol, pH 7.2), in a sterile stainless steel blender (Waring Commercial Blendor). The resulting slurry was filtered through sterile cheese cloth. The mierosporeswere collected, resuspended in 20 % sucrose and centrifuged in sterile Babcockbottles at 100xg for 10 rain at 4~ Viable mlcrosporesbanded in the bottle neck. They were collected, rinsed twice with 4~ HBS, resuspended at lxl0 n microspores/mlHBS and placed on ice until used. Microsporepreparation.
Regeneration of transformed plants is routine in many dicot species and some monocot species. Transformation of cereal crop plants however is not common. To date only two methods have yielded fertile transgenic cereal crop plants. Stable fertile transformants of corn have been produced by microprojectile bombardment of embryogenic callus (Fromm et al. 1990, Gordon-Kamm et al. 1990). Fertile transformed rice has been produced using electroporation or polyethylene glycol-mediated transformation of protoplasts (Hayashimoto et al. 1990, Toriyama et al. 1988). These procedures require Correspondence to: A. Fennell
568 Cell suspension preparation. Cell suspension cultures derived from callus o f immature embryos o f the Pioneer hybrid t:)3377 (P3377 eulture obtained from J. M. Widholm, Univ. of Illinois, Urbana, IL) were subcultured on a 7 day interval in liquid D/G medium (50:50 vol:vol) (Duncan et al. 1985). Four days after subculture, cells were strained through a 10 mesh sieve. Cells passing through the mesh were collected and rinsed twice with HBS. These cells were resuspended at 200 #1 packed cell volume (PCV)/ml HBS and placed on ice until used. Protoplast Preparation. Protoplasts were isolated from the Pioneer hybrid P3377 suspension cells. Fifty ml of a filter sterilized enzyme solution (2.0% Cellulase RS CtGnki Yakmlt, Japan), 0.05% Pectolyase (Seishin, Japan), 0.1% macerozyme in 0.45 M mannltol, 7 mM CaCIz.2H20, pH 5.7) was added to a 5 ml PCV. The cells were incubated in the enzyme solution at room temperature for 4-12 hours and then collected by centrifugation. Viable protoplastswere separated from cells and cellular debris by centrifugation at 100xg in 20% sucrose. The protoplasts were collected, rinsed twice with HBS, resuspended at lxl06 protoplasts/ml of HBS and placed on ice until used.
Expression vectors.
Pour expression vectors containing different promoter + intron combinations fused to the coding region of the ehloramphenicol acetyl transferase (CAT) gene and terminated by the polyadenylation signals of the nopaline synthase (NOS) gene were used for transformation expe,'iments. The expression vectors are: 1) pNIU22, containing die CaMV 35S promoter, this vector was constructed by replacing the B-glueuronldase gene in the plasmid pBI221 (Clonetech) with the coding region of the CAT gene from pNCN (Clonetech). 2) pNIU24, this vector contains the sixth intron from maize alcohol dehydrogenase 1 (Adhl) inserted 5' of the CAT gene. 3) pAI1CN (Callis et al. 1987), this vector contains the maize Adhl promoter + Adh intron 1 (Adhl-ll) 4) pUBI-CAT, this vector contains the maize ubiquitin 1 promoter + intron 1 (Ubiql-ll) (obtained from A. H. Christensen, George Mason Univ., Fairfax, VI). All expression vectors are referred to by the promoter designation rather than plasmid name.
analyzed using analysis of variance and means were separated using a Student-Newman-Keuls test at p = 0.05.
Results and Discussion The ability to deliver free DNA to microspores by electroporation or PEG was demonstrated with transient expression of the reporter gene CAT. Four expression vectors containing different promoters showed differing activity in maize microspores. The four promoters tested were chosen because they are not microspore or pollen specific and are active at different levels in maize protoplasts (Fig. 1). CaMV 35S produced an average of four times greater acetylated chloramphenicol than the background levels of the -DNA control. Soaking the maize protoplasts with DNA did not produce any CAT activity above the background levels of the -DNA controls, therefore only the relative activity of the four expression vectors at 200 V is presented. The activity of CaMV 35S + Adhl-I6 was 7 times, Adhl-I1 was 2.5 times and Ubiql-I1 was 13 times higher than that of CaMV 35S. a
.> O
< I.-
b
< O
O
.> Electroporation and PEG treaonents. Equilmolar amounts of cesium chloride purified plasmid DNA (equivalent to 20 #g pUBI-CAT ) were added to one ml HBS containing microspores, protoplasts or cell suspensions. They were immediately subjected to electroporation or PEG-mediated transformation. The microspores and protoplasts were electroporated on ice in sterile electroporation cuvettes using a BRL Cell Porator (Bethesda Research Laboratories Life Technologies Inc., Gaithersburg, MD). Microspore electroporation conditions were 0, 100, 200 or 400 volts (V) at 1180 #Farads (#F). Protoplasts and cells were electroporated at 0 and 200 V. All cell types were placed on ice for 10 rain prior to plating. PEG mediated transformation was performed as previously described (Vasil et al. 1988). After the PEG treatment, the microspores were collected by ceutrifugation, rinsed with HBS and then plated. Both the microspores and protoplasts were plated at a density of 100,000 microspores/ml in MC liquid medium (Pescitelli et al. 1990) plus 8 % maltose instead of sucrose. Suspension cells were plated in 10 ml of the above medium. The samples were harvested 16 to 20tl later, rinsed wlth fresh medium and stored at -SC0C for CAT assays. Transient expression of the introduced DNA was monitored by assaying for CAT activity using standard procedures (Herrera-Estrella et al. 1990). The protoplast data is expressed as a relative activity with CaMV 35S (pAF1) activity set to 100%. Results are from two separate experiments with three samples for each treatment in each experiment. The percent o f 14C chloramphenicol converted to acetylated chloramphenicol (%conversion) was determined for microspores and cell suspensions. Microspore results are from three separate experiments with duplicate samples in each experiment. Cell suspension data are the result of two separate experiments with three samples for each treatment in each experiment. The data were
10
5
(9
n-
35S
35S + Adhl-16
Adhl-I1
Ubiql-I1
Fig.1. Transient expression of CAT in protoplasts. Protoplasts (lxl06hnl HBS) were mixed with equilmolar amounts (equivalent to 20 #g pUBI-CAT DNA) of DNA and electroporated at 200V. Means with different letters were determined to be different with a SNK test at p = 0.05 level.
Similar treatment of cell suspensions did not produce any CAT activity (Fig.2). No activity was found when cells were electroporated at 0 or 200 V. However a slight amount of activity was obtained with the ubiquitin promoter when cells were electroporated at 400 V. This result was obtained with Ubiql-I1 only. Increasing DNA concentration and electroporation at 400 V or the use of PEG did not promote CAT expression above background levels of the -DNA control with any of the other DNA expression vectors. These results indicate a very small amount of DNA can be delivered through the cell walls.
569 a
A
O I B
20 >, 0~ O
0v
j
"1
200 V 400V
I~ J ~ J
b .b. b
b
Fig.2. Traaslent expression of CAT in maize suspension cells. Suspension cells (200/xl PCV/ml HBS) were mixed with equimolar amounts (equivalent to 20 #g of pUBI-CAT DNA) of plasmid DNA and eleetroporated at 0, 200, or 400 V, 1180/,tF. Means with different letters were determined to be different with a SNK test at p = 0.05 level.
o~ ,m
>
10
O
I-,,:r U
- DNA
.b b 35S
b .b b
b .b. b
35S + Adhl-16
Adhl-I1
In spite of the presence of a cell wall and developing exine in the maize microspore, DNA could be delivered to the microspore using conditions sufficient to deliver DNA to maize protoplasts (Fig.3). There was a slight amount of activity in microspores which were soaked on ice with DNA (0 V). This could be a result of low temperature effects on the microspore plasma membranes as maize is a chilling sensitive plant. r-
.2 (g
w
a
6O a
50
(I) O ,~
40
30 ._>
20
O
.< I--
,
