Plant Cell Reports
Plant Cell Reports (1996) 16:133-136
© Springer-Verlag1996
Silicon carbide fiber-mediated DNA delivery into cells of wheat (Triticum aestivum L.) mature embryos Omirulleh Serik 1, 2, Ismagulova Ainur 1, 2, Karabaev Murat 2 Meshi Tetsuo i, and Iwabuchi Masaki 1 I Department of Botany, Faculty of Science, Kyoto University, Sakyo-Ku, Kyoto 606-01, Japan z Institute of Molecular Biology and Biochemistry, Almaty 480012, Kazakhstan Received 6 February 1996/Revised version received 27 May 1996 - Communicated by A. Komamine
We have demonstrated that foreign DNA can be delivered into cells of mature embryos of wheat (Triticum aestivum L.) using silicon carbide fibers (SCF). The highest transient expression of the gusA (GUS) gene was detected when dry embryos were vortexed for 10-30 min in a SCF-DNA solution containing 90-120 g/l of sucrose. Up to 100 (on average 20-40) blue expression units per embryo were observed. Scutellum side and epiblast of the intact wheat embryos are preferentially transformed. When embryos with the coleoptilar tip removed were treated and allowed to germinate, GUS staining was observed in emerging leaf tissues. The potential of this new approach for stable transformation of wheat is under investigation. It has been found that callus tissues induced from the SCF treated embryos contain GUSexpressing sectors one month after treatment. Abstract
Introduction
The microprojectile bombardment technique widely used for cereal genetic transformation resulted in recovery of fertile transgenic plants in several important crop species, including wheat (Vasil et al. 1993; Becket et al. 1994; Nehra et al. 1994; Weeks et al. 1994; Shimada, person, commun.). However, microprojectile bombardment requires expensive equipment and the efficiency of the presently utilized transformation protocols is quite low. Silicon carbide fiber-mediated genetic transformation (Asano et al. 1991; Kaeppler et al. 1990; Kaeppler et al. 1992; Thompson et al. 1995) is a simple and inexpensive alternative system to microprojectile bombardment. Correspondence to." M. Iwabuchi
Recently, Frame et al. (1994) showed that fertile transgenic maize plants can be readily obtained from embryogenic suspension cells treated with silicon carbide whiskers in the presence of the appropriate DNA molecules. In the present paper we demonstrate that this method can be used for foreign DNA delivery into wheat mature embryo cells as well. Materials and methods
Seeds of wheat (T. aestivum L.) cv. Akadamma, Aruana, Basar and Rausin were surface sterilized by immersing in 70% ethanol (v/v) for 5 minutes followed by incubation in 10% sodium hypochlorite (v/v) for 20 min, and finally rinsing five times with sterile distilled water. The seeds were dried in a laminar hood and kept in plastic Petri dishes for one week prior to aseptic embryo isolation. The transformation mixture was prepared by combining 2.5 volumes of water (or sucrose solution) with 1 volume of freshly prepared 10% solution of SCF TWS-100 (Tokai Carbon Co. Ltd, Japan) in water and 0.5 volumes of pAHC25 (gusA and bar genes driven by maize ubiquitin promoters; Christensen et al. 1992) plasmid DNA (lgg/gl) in a 1.5 ml Eppendorf tube. Aliquots (40 gl) of the mixture were distributed into sterile Eppendorf tubes containing 2-10 embryos each. The tubes were vortexed on a Micro tube Mixer MT-360 (Tomy Seiko Co., Ltd, Japan) at a mixing speed 9 for 5-60 min. The embryos were then plated directly onto the surface of gelrite-solidified (0.25% (w/v)) half-strength MS (Murashige and Skoog 1962) medium with 20 g/1 maltose and 2 mg/l
134
GA3. The plates were kept in the dark at 26°C until assayed. GUS transient expression was detected 24 h after treatment (unless otherwise stated) by histochemical staining utilizing the 5-bromo-4chloro-3-indolyl fI-D-glucuronide (X-glcA; Sigma, B-4782) substrate (0.5 mg/ml) dissolved in a small volume of DMSO (ling/ 10 gl) and diluted by a solution containing 0.1 M sodium phosphate buffer (pH 7.0), 10 mM EDTA, 0.1% Triton X-100 and 0.5 mM of potassium ferricyanide and potassium ferrocyanide. Incubation was carried out at 37°C for 6 hours. No blue staining was detected in non-treated control embryos incubated under these conditions. For callus induction, the treated embryos were plated on MS (-NH4NO3) with 2 mg/1 2,4-D and 5 mg/l bialaphos.
Table 2. Influence of the sucrose concentration on the level of GUS transient expression
Sucrose concentrataion
Expression units/embryo
No sucrose
23.2 + 4.2
60 g/1
30.2 + 3.8
90 g/1
32.3 + 3.7
120 g/1
33.4 + 4.1
150 g/1
30.9 + 3.1
Embryos of cv. Akadaruma were treated for 20 min,
Results and discussion Optimization of some of the parameters for foreign DNA delivery has been carried out utilizing the GUS histochemical assay system (Jefferson et al. 1987). When 2, 5 or 10 intact embryos per tube were treated, the efficiency of transient expression calculated as the number of blue GUS expressing loci per embryo (Fig. la) was comparable (Table 1), therefore we subsequently used 10 embryos/tube. Tables 2 and 3 show that the highest transient expression of the gusA gene occured when dry embryos were vortexed for 10-30 rain in a SCF-DNA solution containing 90-120 g/l of sucrose. Up to 100 blue (on average 20-40) expression units per embryo were observed. Table 1. Dependence of the level of GUS expression on the number of treated embryos.
Number of embryos/tube
Expression unitsl/embryo
2
29.5 + 2.1
5
30.2 + 3.4
10
30.2 + 4.1
'One unit = one blue cell or group of adjacent blue ceils. Embryos of cv. Akadaruma were treated for 20 min in a SCF-DNA mixture with 120 g/1 sucrose.
Figure l a . Wheat mature embryos expressing the GUS gene 24 h after SCF treatment (Note: remainders of SCF are visible as a light-green pellet on the surface of the embryos); Fig. 1 b. Mature wheat embryos transformed by: 1. Microprojectile bombardment ; 2. SCF treatment; Fig. l c . Strong GUS expression in damaged tips of SCF treated wheat embryos; Fig. l d. Leaf tissues of a SCF treated wheat seedling with GUS positive areas 120 h after treatment; F i g . le. SCF treated embryo tips exibiting GUS expression; Fig. l f . One month old callus, induced from a SCF treated embryo, displaying GUS expression. Embryos of cv. Akadaruma were SCF treated for 20 min in a solution with 120 g/l sucrose.
135 Table 3. Influence of the duration of treatment on the level of GUS transient expression.
Time of incubation (min)
5 10 20 30 60
Expression units/embryo
18.5 26.7 32.7 33.0 19.5
+ 1.3 _+ 4.3 _+ 2.8 + 4.5 _ 2.4
Embryos of cv. Akadaruma were treated in a SCF-DNA mixture with t20 g/1 sucrose.
To the best of our knowledge, this is the first report on the application of S CF treatment for foreign DNA delivery into cells of mature embryos of cereal species. It has certain advantages over the other existing approaches for wheat transformation: Mature seeds are available throughout the year. Embryo isolation and SCF treatment are simple procedures requiring no sophisticated equipment or highly qualified personnel. Mature embryos are quite rigid, enabling them to be vortexed with SCF more intensively and for longer periods of time to ensure uniform treatment. It is possible to treat and propagate only the regenerable parts of an embryo by dissecting it (see below). SCF treatment of mature embryos may shorten the time between transformation and regeneration of plants, thereby decreasing the frequency of somaclonal variations. Since many samples can be treated readily and uniformly at a time, it is very easy to optimize the DNA delivery parameters. The efficiency of the present protocol is comparable to results obtained by others in bombardment experiments with immature embryos. The mature embryos bombarded by us exibited irregular pattern of transient expression with most of the embryos having only a few GUS expressing loci, while SCF treated embryos displayed rather uniform pattern of GUS expression (Fig. lb). Recent papers on the in vitro culture of wheat mature embryos show that it is possible to increase plant regeneration frequency from embryo-derived calli by manipulating the culture conditions (McKinnon et al. 1987; Racz et al. 1993) or by using dissected embryos. The use of embryo axis and plumula (Elena and Ginzo 1988) allows efficient plant regeneration. Zhang and Seilleur (1987) found that embryos deprived of
their scutellum and hypocotyl displayed regeneration with a frequency of up to 61.5%. It has been observed in our experiments that embryos with damaged coleoptilar tips sometimes display very strong GUS expression in that region (Fig. lc). The seedlings grown up from the treated embryos with the removed or damaged coleoptilar tip exibited GUS staining in emerging leaf tissues (Fig. ld). When dissected embryo tips were used for SCF treatment, it was found that they still show efficient transient expression (Fig. le). Therefore, for stable transformation experiments, we used only the upper half of the embryo with the coleoptilar tip removed. We found that the SCF-mediated DNA incorporation process is different from that by imbibition of mature embryos in DNA solution (Topfer et al. 1989; Senaratna et al. 1991; Yoo and Jung 1995), since after 30-120 min of preimbibition in water followed by SCF treatment the efficiency of transient expression was still high. However, imbibition overnight is detrimental to transformation resulting in a significant drop in the number of GUS expressing loci (data not shown). In our attempts to reproduce the transformation protocol of Topfer et al (1989) we could not detect any GUS expression in imbibed wheat mature embryos by histochemical staining. Rice embryos have been SCF treated as well and GUS expression was detected in leaf tissues of germinating seedlings (data not shown). Since SCF treatment relies on a purely physical process for DNA delivery into cells, it seems apparent that the protocol can be used for a wide variety of plant species. We observed comparable efficiency of DNA delivery in four wheat genotypes (Table 4) tested, as well as in mature embryos of japonica rice cv. Nipponbare. Table 4. GUS transient expression in wheat embryos of different genotypes.
Cultivar
Expression units/embryo
Akadaruma
27.6 _+ 6.4
Aruana
28.3 + 4.2
Basar
26.5 + 5.6
Rausin
24.4 _+ 4.6
Embryos were treated for 10 min with 120 g/1 sucrose.
136 Two potential approaches may be used for stable genetic transformation of mature embryos by SCF treatment: a. callus induction from regenerative tissues (mesocotyl, epicotyl, epiblast) with subsequent selection and regeneration; b. propagation and selectionregeneration of shoot meristems as was demonstrated by Lowe et al. (1995) who were able to obtain transformed plants from several hybrid maize varieties, after bombardment of developing shoot meristems with subsequent selection and enrichment procedures on a cytokinin containing medium. Work is m progress to check the applicability of these approaches to transgenic wheat production after SCF treatment of mature wheat embryos. It has been found that callus tissues induced on a medium with 5 mg/1 bialaphos from the SCF treated embryos contain GUS expressing sectors one month after treatment (Fig. If). We believe that this new approach might become a useful tool in the genetic transformation of wheat and certain other plant species, when the treatment parameters (speed, duration of vortexing, composition of the treatment mixture, geometry and uniformity of the fibers etc.) is optimized further and a reliable system of selection-regeneration of treated cells is established. It might be a valuable alternative to expensive biolistic transformation methods especially for developing countries like Kazakhstan.
Acknowledgments We express our gratitude to Prof. T. Shimada (Research Institute of Agricultural Resources, Ishikawa Agricultural College, Ishikawa, Japan) and Dr. F. Sagi (Cereal Research Institute, Szeged, Hungary) for valuable comments on the manuscript. We thank Prof. P. Quail for providing the pAHC25 construct. We are grateful to Ms. S. Brutsaert in our laboratory for proofreading. O.S. thanks the Inoue Foundation for the fellowship and the members of the laboratory for help and valuable discussions.
References
Asano Y, Otsuki Y, Ugaki M (1991) Plant Sci 79:247-252 Becker D., Brettschneider R, Lorz H (1994) Plant J 5:299-307 Christensen AH, Sharrock RA and Quail PH (1992) Plant Mol Biol 18:675-689 Elena EB and Ginzo HD (1988) J Plant Physiol 132:600-603 Frame BR, Drayton PR, Bagnall SV, Lewnau C J, Bullock WP, Wilson HM, Dunwell JM, Thompson JA, Wang K (1994) Plant J 6: 941948 Jefferson RA (1987) Plant Mol Biol Rep 5: 387405 Kaeppler HF, Gu W, Somers DA, Rines HW, Cockburn AF (1990) Plant Cell Rep 9:415-418 Kaeppler HF, Somers DA, Rines HW, Cockburn AF (1992) Theor Appl Genet 84:560-566 Lowe K, Bowen B, Hoester G, Ross M, Bond D, Pierce D, Gordon-Kamm B (1995) Bio/Technology. 13:677-682 McKinnon C, Gunderson G, Nabors MW (1987) In vitro 23:443-448 Murashige T, Skoog F (1962) Physiol Plant 15: 473-497 Nehra NS, Chibbar RN, Leung N, Caswell K, Mallard C, Steinhauer L, Baga M, Kartha KK (1994) Plant J 5:285-297 Racz I, Paldi E, Lasztity D, Buzas B, Aczel M (1993) Acta Agronom Hungarica 42:255-260 Senaratna T, McKersie BD, Kasha KJ (1991) Plant Sci 79:223-228 Thompson JA, Drayton PR, Dunwell JM (1995) Euphytica 85:75-80 Topfer R, Gronenborn B, Schell J, Steinbiss HH (1989) Plant Cell 1:133-139 Vasil V, Srivastava V, Castillo AM, Fromm ME, Vasil IK (1993) Bio/Technology 11:1553-1558 Weeks IT, Anderson OD, Blechl AE (1993) Plant Physiol 102:1077-1084 Yoo J, Jung G (1995) Physiol Plant 94:453-459 Zhang LG, Seilleur P (1987) Bull.Rech.Agron.Gembloux 22:187-197
Plant Cell Reports (1996) 16:133-136
© Springer-Verlag1996
Silicon carbide fiber-mediated DNA delivery into cells of wheat (Triticum aestivum L.) mature embryos Omirulleh Serik 1, 2, Ismagulova Ainur 1, 2, Karabaev Murat 2 Meshi Tetsuo i, and Iwabuchi Masaki 1 I Department of Botany, Faculty of Science, Kyoto University, Sakyo-Ku, Kyoto 606-01, Japan z Institute of Molecular Biology and Biochemistry, Almaty 480012, Kazakhstan Received 6 February 1996/Revised version received 27 May 1996 - Communicated by A. Komamine
We have demonstrated that foreign DNA can be delivered into cells of mature embryos of wheat (Triticum aestivum L.) using silicon carbide fibers (SCF). The highest transient expression of the gusA (GUS) gene was detected when dry embryos were vortexed for 10-30 min in a SCF-DNA solution containing 90-120 g/l of sucrose. Up to 100 (on average 20-40) blue expression units per embryo were observed. Scutellum side and epiblast of the intact wheat embryos are preferentially transformed. When embryos with the coleoptilar tip removed were treated and allowed to germinate, GUS staining was observed in emerging leaf tissues. The potential of this new approach for stable transformation of wheat is under investigation. It has been found that callus tissues induced from the SCF treated embryos contain GUSexpressing sectors one month after treatment. Abstract
Introduction
The microprojectile bombardment technique widely used for cereal genetic transformation resulted in recovery of fertile transgenic plants in several important crop species, including wheat (Vasil et al. 1993; Becket et al. 1994; Nehra et al. 1994; Weeks et al. 1994; Shimada, person, commun.). However, microprojectile bombardment requires expensive equipment and the efficiency of the presently utilized transformation protocols is quite low. Silicon carbide fiber-mediated genetic transformation (Asano et al. 1991; Kaeppler et al. 1990; Kaeppler et al. 1992; Thompson et al. 1995) is a simple and inexpensive alternative system to microprojectile bombardment. Correspondence to." M. Iwabuchi
Recently, Frame et al. (1994) showed that fertile transgenic maize plants can be readily obtained from embryogenic suspension cells treated with silicon carbide whiskers in the presence of the appropriate DNA molecules. In the present paper we demonstrate that this method can be used for foreign DNA delivery into wheat mature embryo cells as well. Materials and methods
Seeds of wheat (T. aestivum L.) cv. Akadamma, Aruana, Basar and Rausin were surface sterilized by immersing in 70% ethanol (v/v) for 5 minutes followed by incubation in 10% sodium hypochlorite (v/v) for 20 min, and finally rinsing five times with sterile distilled water. The seeds were dried in a laminar hood and kept in plastic Petri dishes for one week prior to aseptic embryo isolation. The transformation mixture was prepared by combining 2.5 volumes of water (or sucrose solution) with 1 volume of freshly prepared 10% solution of SCF TWS-100 (Tokai Carbon Co. Ltd, Japan) in water and 0.5 volumes of pAHC25 (gusA and bar genes driven by maize ubiquitin promoters; Christensen et al. 1992) plasmid DNA (lgg/gl) in a 1.5 ml Eppendorf tube. Aliquots (40 gl) of the mixture were distributed into sterile Eppendorf tubes containing 2-10 embryos each. The tubes were vortexed on a Micro tube Mixer MT-360 (Tomy Seiko Co., Ltd, Japan) at a mixing speed 9 for 5-60 min. The embryos were then plated directly onto the surface of gelrite-solidified (0.25% (w/v)) half-strength MS (Murashige and Skoog 1962) medium with 20 g/1 maltose and 2 mg/l
134
GA3. The plates were kept in the dark at 26°C until assayed. GUS transient expression was detected 24 h after treatment (unless otherwise stated) by histochemical staining utilizing the 5-bromo-4chloro-3-indolyl fI-D-glucuronide (X-glcA; Sigma, B-4782) substrate (0.5 mg/ml) dissolved in a small volume of DMSO (ling/ 10 gl) and diluted by a solution containing 0.1 M sodium phosphate buffer (pH 7.0), 10 mM EDTA, 0.1% Triton X-100 and 0.5 mM of potassium ferricyanide and potassium ferrocyanide. Incubation was carried out at 37°C for 6 hours. No blue staining was detected in non-treated control embryos incubated under these conditions. For callus induction, the treated embryos were plated on MS (-NH4NO3) with 2 mg/1 2,4-D and 5 mg/l bialaphos.
Table 2. Influence of the sucrose concentration on the level of GUS transient expression
Sucrose concentrataion
Expression units/embryo
No sucrose
23.2 + 4.2
60 g/1
30.2 + 3.8
90 g/1
32.3 + 3.7
120 g/1
33.4 + 4.1
150 g/1
30.9 + 3.1
Embryos of cv. Akadaruma were treated for 20 min,
Results and discussion Optimization of some of the parameters for foreign DNA delivery has been carried out utilizing the GUS histochemical assay system (Jefferson et al. 1987). When 2, 5 or 10 intact embryos per tube were treated, the efficiency of transient expression calculated as the number of blue GUS expressing loci per embryo (Fig. la) was comparable (Table 1), therefore we subsequently used 10 embryos/tube. Tables 2 and 3 show that the highest transient expression of the gusA gene occured when dry embryos were vortexed for 10-30 rain in a SCF-DNA solution containing 90-120 g/l of sucrose. Up to 100 blue (on average 20-40) expression units per embryo were observed. Table 1. Dependence of the level of GUS expression on the number of treated embryos.
Number of embryos/tube
Expression unitsl/embryo
2
29.5 + 2.1
5
30.2 + 3.4
10
30.2 + 4.1
'One unit = one blue cell or group of adjacent blue ceils. Embryos of cv. Akadaruma were treated for 20 min in a SCF-DNA mixture with 120 g/1 sucrose.
Figure l a . Wheat mature embryos expressing the GUS gene 24 h after SCF treatment (Note: remainders of SCF are visible as a light-green pellet on the surface of the embryos); Fig. 1 b. Mature wheat embryos transformed by: 1. Microprojectile bombardment ; 2. SCF treatment; Fig. l c . Strong GUS expression in damaged tips of SCF treated wheat embryos; Fig. l d. Leaf tissues of a SCF treated wheat seedling with GUS positive areas 120 h after treatment; F i g . le. SCF treated embryo tips exibiting GUS expression; Fig. l f . One month old callus, induced from a SCF treated embryo, displaying GUS expression. Embryos of cv. Akadaruma were SCF treated for 20 min in a solution with 120 g/l sucrose.
135 Table 3. Influence of the duration of treatment on the level of GUS transient expression.
Time of incubation (min)
5 10 20 30 60
Expression units/embryo
18.5 26.7 32.7 33.0 19.5
+ 1.3 _+ 4.3 _+ 2.8 + 4.5 _ 2.4
Embryos of cv. Akadaruma were treated in a SCF-DNA mixture with t20 g/1 sucrose.
To the best of our knowledge, this is the first report on the application of S CF treatment for foreign DNA delivery into cells of mature embryos of cereal species. It has certain advantages over the other existing approaches for wheat transformation: Mature seeds are available throughout the year. Embryo isolation and SCF treatment are simple procedures requiring no sophisticated equipment or highly qualified personnel. Mature embryos are quite rigid, enabling them to be vortexed with SCF more intensively and for longer periods of time to ensure uniform treatment. It is possible to treat and propagate only the regenerable parts of an embryo by dissecting it (see below). SCF treatment of mature embryos may shorten the time between transformation and regeneration of plants, thereby decreasing the frequency of somaclonal variations. Since many samples can be treated readily and uniformly at a time, it is very easy to optimize the DNA delivery parameters. The efficiency of the present protocol is comparable to results obtained by others in bombardment experiments with immature embryos. The mature embryos bombarded by us exibited irregular pattern of transient expression with most of the embryos having only a few GUS expressing loci, while SCF treated embryos displayed rather uniform pattern of GUS expression (Fig. lb). Recent papers on the in vitro culture of wheat mature embryos show that it is possible to increase plant regeneration frequency from embryo-derived calli by manipulating the culture conditions (McKinnon et al. 1987; Racz et al. 1993) or by using dissected embryos. The use of embryo axis and plumula (Elena and Ginzo 1988) allows efficient plant regeneration. Zhang and Seilleur (1987) found that embryos deprived of
their scutellum and hypocotyl displayed regeneration with a frequency of up to 61.5%. It has been observed in our experiments that embryos with damaged coleoptilar tips sometimes display very strong GUS expression in that region (Fig. lc). The seedlings grown up from the treated embryos with the removed or damaged coleoptilar tip exibited GUS staining in emerging leaf tissues (Fig. ld). When dissected embryo tips were used for SCF treatment, it was found that they still show efficient transient expression (Fig. le). Therefore, for stable transformation experiments, we used only the upper half of the embryo with the coleoptilar tip removed. We found that the SCF-mediated DNA incorporation process is different from that by imbibition of mature embryos in DNA solution (Topfer et al. 1989; Senaratna et al. 1991; Yoo and Jung 1995), since after 30-120 min of preimbibition in water followed by SCF treatment the efficiency of transient expression was still high. However, imbibition overnight is detrimental to transformation resulting in a significant drop in the number of GUS expressing loci (data not shown). In our attempts to reproduce the transformation protocol of Topfer et al (1989) we could not detect any GUS expression in imbibed wheat mature embryos by histochemical staining. Rice embryos have been SCF treated as well and GUS expression was detected in leaf tissues of germinating seedlings (data not shown). Since SCF treatment relies on a purely physical process for DNA delivery into cells, it seems apparent that the protocol can be used for a wide variety of plant species. We observed comparable efficiency of DNA delivery in four wheat genotypes (Table 4) tested, as well as in mature embryos of japonica rice cv. Nipponbare. Table 4. GUS transient expression in wheat embryos of different genotypes.
Cultivar
Expression units/embryo
Akadaruma
27.6 _+ 6.4
Aruana
28.3 + 4.2
Basar
26.5 + 5.6
Rausin
24.4 _+ 4.6
Embryos were treated for 10 min with 120 g/1 sucrose.
136 Two potential approaches may be used for stable genetic transformation of mature embryos by SCF treatment: a. callus induction from regenerative tissues (mesocotyl, epicotyl, epiblast) with subsequent selection and regeneration; b. propagation and selectionregeneration of shoot meristems as was demonstrated by Lowe et al. (1995) who were able to obtain transformed plants from several hybrid maize varieties, after bombardment of developing shoot meristems with subsequent selection and enrichment procedures on a cytokinin containing medium. Work is m progress to check the applicability of these approaches to transgenic wheat production after SCF treatment of mature wheat embryos. It has been found that callus tissues induced on a medium with 5 mg/1 bialaphos from the SCF treated embryos contain GUS expressing sectors one month after treatment (Fig. If). We believe that this new approach might become a useful tool in the genetic transformation of wheat and certain other plant species, when the treatment parameters (speed, duration of vortexing, composition of the treatment mixture, geometry and uniformity of the fibers etc.) is optimized further and a reliable system of selection-regeneration of treated cells is established. It might be a valuable alternative to expensive biolistic transformation methods especially for developing countries like Kazakhstan.
Acknowledgments We express our gratitude to Prof. T. Shimada (Research Institute of Agricultural Resources, Ishikawa Agricultural College, Ishikawa, Japan) and Dr. F. Sagi (Cereal Research Institute, Szeged, Hungary) for valuable comments on the manuscript. We thank Prof. P. Quail for providing the pAHC25 construct. We are grateful to Ms. S. Brutsaert in our laboratory for proofreading. O.S. thanks the Inoue Foundation for the fellowship and the members of the laboratory for help and valuable discussions.
References
Asano Y, Otsuki Y, Ugaki M (1991) Plant Sci 79:247-252 Becker D., Brettschneider R, Lorz H (1994) Plant J 5:299-307 Christensen AH, Sharrock RA and Quail PH (1992) Plant Mol Biol 18:675-689 Elena EB and Ginzo HD (1988) J Plant Physiol 132:600-603 Frame BR, Drayton PR, Bagnall SV, Lewnau C J, Bullock WP, Wilson HM, Dunwell JM, Thompson JA, Wang K (1994) Plant J 6: 941948 Jefferson RA (1987) Plant Mol Biol Rep 5: 387405 Kaeppler HF, Gu W, Somers DA, Rines HW, Cockburn AF (1990) Plant Cell Rep 9:415-418 Kaeppler HF, Somers DA, Rines HW, Cockburn AF (1992) Theor Appl Genet 84:560-566 Lowe K, Bowen B, Hoester G, Ross M, Bond D, Pierce D, Gordon-Kamm B (1995) Bio/Technology. 13:677-682 McKinnon C, Gunderson G, Nabors MW (1987) In vitro 23:443-448 Murashige T, Skoog F (1962) Physiol Plant 15: 473-497 Nehra NS, Chibbar RN, Leung N, Caswell K, Mallard C, Steinhauer L, Baga M, Kartha KK (1994) Plant J 5:285-297 Racz I, Paldi E, Lasztity D, Buzas B, Aczel M (1993) Acta Agronom Hungarica 42:255-260 Senaratna T, McKersie BD, Kasha KJ (1991) Plant Sci 79:223-228 Thompson JA, Drayton PR, Dunwell JM (1995) Euphytica 85:75-80 Topfer R, Gronenborn B, Schell J, Steinbiss HH (1989) Plant Cell 1:133-139 Vasil V, Srivastava V, Castillo AM, Fromm ME, Vasil IK (1993) Bio/Technology 11:1553-1558 Weeks IT, Anderson OD, Blechl AE (1993) Plant Physiol 102:1077-1084 Yoo J, Jung G (1995) Physiol Plant 94:453-459 Zhang LG, Seilleur P (1987) Bull.Rech.Agron.Gembloux 22:187-197
