Crop Science 40:1749-1754 (2000)
© 2000 Crop Science Society of America
CELL BIOLOGY & MOLECULAR GENETICS
Wheat Transformation Using Cyanamide as a New Selective Agent
J.T. Weeksa,
K.Y. Koshiyamab,
U. Maier-Greinerc,
T. Schäeffnerd and
O.D. Andersone
a ARS, USDA, 344 Keim Hall, Univ. of Nebraska-Lincoln, Lincoln, NE 68583 USA
b School of Public Health, Univ. of California-Berkeley, Berkeley, CA 94720 USA
c Hechingerstr. 12, D-72144 Dusslingen, Germany
d Institute of Biochemical Plant Pathology, GSF Research Center, Xenobiotics, D-85758 Oberschleissheim, Germany
e ARS, USDA, WRRC, 800 Buchanan Street, Albany, CA 94710 USA
tweeks{at}unlserve.unl.edu
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ABSTRACT
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There is a general need for additional selectable marker genes for plant transformation. Only a few have been reported in wheat (Triticum aestivum L.) transformation experiments, some of which are under patent restriction or have other disadvantages. A new selectable marker gene was identified which can be used to select resistant callus in tissue culture and regenerate transgenic wheat plants. A gene from the soil fungus Myrothecium verrucaria (Albertini & Schwein.) Ditmar:Fr., coding for the enzyme cyanamide hydratase which converts cyanamide into urea, was previously described. In our wheat transformation experiments, the gene conferred resistance to cyanamide at a tissue culture level and therefore cyanamide could be used to select for transformants. At the whole plant level, progeny of transformed wheat plants showed resistance to cyanamide, whereas sensitive plants expressed a lethal necrosis and yellowing when cyanamide was applied. This gene has several potential advantages when compared with other selectable marker genes. Transformed wheat plants can be selected at the tissue culture level and may be able to convert cyanamide into a useful nitrogen compound (fertilizer). The selectable marker gene could be introduced with other genes for value-added traits in wheat and might also be applicable in other transformation systems.
Abbreviations: Cah, a gene from the soil fungus Myrothecium verrucaria that encodes the enzyme cyanamide hydratase • MS, Murashige and Skoog • PAT, phosphinothricin acetyl transferase • nos, nopaline synthase transcription termination element
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INTRODUCTION
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THE TRANSFORMATION AND REGENERATION of wheat plants has been reported by a number of laboratories (Vasil et al., 1992; Weeks et al., 1993; Becker et al., 1994; Zhou et al., 1995; Ortiz, et al., 1996; Chen et al., 1998; Stoger et al., 1999). One critical element of these reports is the selectable marker used in the experiments. The appropriate selective marker must be considered when designing experiments because of the low transformation frequencies encountered for wheat (Wilmink and Dons, 1993; Witrzens et al., 1998). Several different selectable marker genes (bar, dfhrr, hph, nptII, CP4, GOX) have been reported in wheat transformation experiments to obtain stable transgenic plants. Each selectable marker has its particular advantages and disadvantages depending on its application.
Of the six selectable marker genes, the bar gene was the most widely used in wheat transformation experiments. The bar gene was isolated from the bacterium Streptomyces hygroscopicus, which encodes the enzyme phosphinothricin acetyl transferase (PAT) which confers resistance to the herbicides bialaphos and Basta, Hoechst, Frankfurt, Germany, [2-amino-4-(hydroxymethylphosphinyl)butanoic acid)]. The use of the bar gene (Thompson et al., 1987) has the advantage of creating stringent selection in culture to produce herbicide-resistant plants. Disadvantages include the cost of the selective agent bialaphos and the concern that the bar gene may be sexually transmitted to wild relatives producing herbicide-resistant weeds (Vasil, 1994; Arriola and Ellstrand, 1996). Two other herbicide resistance genes, CP4 and GOX (Barry et al., 1992; Kishore et al., 1992), have also been used for selection in wheat transformation. When either of these genes are used, they confer resistance to glyphosate, an active ingredient in the herbicide Roundup (Monsanto, St. Louis, MO). Zhou et al. (1995) reported regeneration of transgenic wheat plants using the CP4 and GOX genes as selectable markers. However, the transformation efficiencies for the CP4 and GOX genes were lower than those of nptII and bar under the same experimental conditions. As with the bar gene, there is also the concern for the possibility of transfer of these herbicide resistance genes to related weedy species. Furthermore, many of the selectable marker genes are proprietary and the commercial use of these genes may be limited.
Another reported selective agent is methotrexate, which targets the enzyme dihydrofolate reductase (DHFR). The inhibition of DHFR activity produces a deficiency of thymidylate and rapidly leads to cell cycle arrest (Crosti et al., 1993). A dhfr gene producing DHFR resistance to methotrexate has been identified and used as a selectable marker in wheat transformation experiments (Dhir et al., 1994; Hauptmann et al., 1988). The disadvantage of methotrexate used as a selective agent is that it is a highly toxic compound which should not be used as an aerosol spray (Kemper et al., 1992).
Other selectable markers consist of bacterial drug resistance genes which encode enzymes that inactivate antibiotics by modification to nontoxic forms. There are several bacterial genes that may serve as selectable markers in cereals including the neomycin phosphotransferase II (nptII) gene and the hygromycin phosphotransferase (hph) gene. The nptII gene confers resistance to kanamycin or G418 and the hph gene to hygromycin B. Ortiz et al. (1996) developed a highly efficient protocol for stable wheat transformation using hygromycin resistance as a selectable marker. They reported that the hph gene was as good or better than the bar gene as a selectable marker; however, it has been reported that cereals show higher levels of resistance to antibiotic selection agents than most dicot species, thus requiring high concentrations of the compounds and making the timing and maintenance of selection pressure critical (Hauptmann et al., 1988; Vasil et al., 1991; Vasil, 1994). Another concern in the use of antibiotic resistance selectable markers is public acceptance. Some individuals believe that the antibiotic resistance could be transferred to infectious bacteria, rendering the antibiotic ineffective for animal health applications (Balter, 1997).
In this paper, we report in wheat the introduction and expression of the cyanamide hydratase (Cah) gene from Myrothecium verrucaria, a soil fungus. This gene, which had been isolated and characterized by Maier-Greiner et al. (1991b), produces an enzyme that converts cyanamide into urea. We demonstrate that the Cah gene can be used as a selectable marker for monocot transformation. Analysis of cyanamide-resistant wheat transformants revealed that they contain the Cah gene and showed resistance to cyanamide. The Cah gene has possible whole plant benefits which are not conferred by other selectable marker genes.
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Material and methods
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Callus Culture and Plant Material
Highly embryogenic callus tissue was derived from wheat plants (`Bobwhite') which were maintained in a greenhouse at 21°C (Albany, CA). To establish callus cultures, caryopses 10 to 18 d postanthesis were surface sterilized with 70% (v/v) ethanol for 5 min and 20% (v/v) sodium hypochlorite solution for 15 min, followed by two rinses of sterile distilled water. Immature embryos, 0.5 to 1 mm long, were aseptically removed with a sterilized forceps in a laminar flow hood under a stereo dissecting microscope. The embryos were placed with the scutellum exposed onto MS medium (Murashige and Skoog, 1962) modified for wheat cell culture (Sears and Deckard, 1982) and solidified with 2.5 g L-1 Phytagel (Sigma Chemical Co., St. Louis, MO). Calli were maintained at 25°C with a 16-h photoperiod on MS medium containing 20 g L-1 sucrose and 0.0015 g L-1 2,4-dichlorophenoxyacetic acid, and transferred to fresh medium at 2-wk intervals. For regeneration, embryogenic calli were transferred to MS medium with 0.0005 g L-1 dicamba (Sandoz Crop Protection, Des Plaines, IL) as described by Hunsinger and Schauz (1987). When shoots reached 2 to 3 cm in length, they were transferred with a long forceps to culture tubes (25 by 150 mm) containing 0.018 L of rooting medium composed of half-strength MS maintenance medium without hormones. For selection following bombardment, media plates at each stage were supplemented with 0.0375 g L-1 cyanamide (Sigma Chemical Co.).
Plantlets were transferred from rooting medium to soil pots of Sunshine soil mixture #1 (Fisons Horticulture Co., Mississauga, MB) and acclimated to lower humidity at 21°C with a 16-h photoperiod in an environmental chamber. After 2 wk, plants were transferred to the greenhouse. These primary putative transgenic plants are T0 plants. The first generation progeny of these plants are T1 plants.
Transforming DNA
The vector (Fig. 1)
used for wheat transformation consisted of the Cah gene under the control of the maize ubiquitin Ubi1 promoter (Christensen et al., 1992; Christensen and Quail, 1996). The Cah gene encodes the enzyme cyanamide hydratase, which hydrates the nitrile group of cyanamide to form urea. Plasmid DNA was purified by an alkaline lysis method using a Qiagen kit (Chatsworth, CA) and stored at a concentration of 1 g L-1 in Tris-EDTA buffer, pH 8.0 (Sambrook et al., 1989).
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Fig. 1 Schematic representation of the plasmid used for selection and transformation. Cah+ indicates the coding sequence which is under the control of the maize ubiquitin (Ubi1) promoter and the transcription termination region nos of Agrobacterium tumefaciens. A plasmid pUC8 was employed as the cloning vector
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Microprojectile Bombardment Preparation
Prior to bombardment, 1-µm gold particles were coated with transforming DNA by the procedure of Daines (1990). A stock suspension of gold particles was suspended at 60 g L-1 in absolute ethanol. Thirty-five microliters of the suspension was transferred into a 1.5-mL microcentrifuge tube, centrifuged at 14 000 g for 3 min, and the pellet suspended in 200 µL of sterile distilled water. Following a second centrifugation, the pellet was suspended in 25 µL of Tris-EDTA buffer, pH 8.0 containing 25 µg of the transforming plasmid DNA. The following chilled sterile solutions were added in order: 220 µL of water, 250 µL of 2.5 M CaCl2, and 50 µL of 0.1 M spermidine (0.2-µm filter sterilized). The microcentrifuge tubes were shaken with a Tomy microtube shaker at 4°C for 15 min and centrifuged at 16 000 g for 5 min. The supernatant was removed, the pellet washed with 200 µL of ethanol and the DNA-coated gold particles suspended in 36 µL of ethanol.
Four hours prior to bombardment, approximately 50 embryo-derived calli (5 d in culture) were placed in a circle (4-cm diam) in the center of a Petri dish (15 by 100 mm) containing 0.4 M mannitol in MS maintenance medium solidified with 3.5 g Phytagel. A petri dish containing the target callus tissue was placed in the helium driven Bio-Rad Biolistic Delivery System (Model PDS-1000/He, Bio-Rad Laboratories, Hercules, CA) and 10 µL of the DNA–gold suspension was pipetted onto the center of a macroprojectile. The distance between the stopping plate and the target callus tissue was adjusted to 13 cm and calli were bombarded under vacuum with the rupture disk strength at 7.59 MPa. Sixteen hours following bombardment, calli were transferred onto a Petri dish (20 by 100 mm) containing MS selection medium (0.0375 g cyanamide L-1 maintenance medium). Calli were maintained at 25°C with a 16-h photoperiod and transferred onto fresh MS selection medium at 2-wk intervals.
Cyanamide Hydratase Colorimetric Assay
The cyanamide hydratase assay is based on the decrease in cyanamide concentration during incubation in the presence of the cyanamide hydratase enzyme (Maier-Greiner et al., 1991b). Wheat leaf plant tissue (100 mg) was frozen in liquid nitrogen, homogenized with a pestle and mortar, diluted with 100 µL of 5 mM sodium phosphate buffer (pH 8.0), vortexed for 5 min, and then centrifuged at 8160 g for 5 min. The supernatant was decanted and 2 µL of 1 M cyanamide was added to the solution (20 mM final concentration). The solution was incubated at 37°C. After 12 h, 60 µL of the incubation mixture was added to 40 µL of sodium carbonate buffer solution (pH 10.4) and 5 µL of color reagent consisting of a 4% (w/v) solution of trisodium pentacyanoammineferroate (Eastern Chemical, Smithtown, NY). The solution was incubated for 10 min at room temperature in the dark. Absorbance at 530 nm (Steller et al., 1965) was measured to detect reduced cyanamide concentration indicative of the presence of cyanamide hydratase.
DNA Isolation and Analysis
Wheat genomic DNA was isolated as described by D'Ovidio et al. (1992) and quantitated by absorbance at 260 nm. Fifty micrograms of each DNA sample were digested overnight with EcoRI in 400 µL of the manufacturer's (GIBCO BRL, Grand Island, NY) buffer. Twenty-five micrograms of digested or undigested DNA were separated by electrophoresis through 0.8% (w/v) agarose (FMC Corp., Rockland, ME) gels in TBE (Sambrook et al., 1989) buffer. To reconstruct a single copy of plasmid per wheat hexaploid genome, 25 µg of EcoRI-digested nontransformed wheat DNA was mixed with 0.592 pg of a 0.8-kb PstI fragment of the pCAM cah coding region. Southern blot analysis was performed as previously described (Weeks et al., 1993).
Cyanamide Application
Three grams of Perlka (SKW, Trostberg, Germany), which is granulated calcium cyanamide, was applied to 1-wk-old wheat seedling plants grown in soil in 15-cm standard pots. A 10% (v/v) solution of Dormex (SKW), which is a 50% (v/v) liquid solution of hydrogen cyanamide, was sprayed as a mist sufficient to wet leaf surface onto 1-wk-old wheat seedling plants. The plants were observed over a 2-wk period.
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Results
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Regeneration of Transgenic Plants
Apparent stable wheat transformation was carried out and achieved by following the wheat transformation procedure described by Weeks (1995). Embryo-derived callus was transferred to plates containing the selective agent cyanamide 16 h following bombardment. A concentration of 0.0375 g L-1 cyanamide was used throughout the study as it had been previously determined as optimal in primary kill-curve experiments (data not shown). At that concentration, callus growth was inhibited about 70% compared with normal or control callus growth. Cyanamide as the selective agent caused more necrosis of plant cells than did bialaphos as a selective agent. Calli were transferred to fresh selection medium every 2 wk. At the end of 8 wk (fourth transfer), resistant calli could be distinguished from nonresistant calli (Fig. 2A)
. Nonresistant calli had a mucous appearance with development of brown and necrotic tissue. Resistant callus proliferated and formed green sectors followed by primary shoots (Fig. 2B). In previous experiments in which bialaphos was used as a selection agent, the selected resistant callus was of a friable type (Weeks et al., 1993). In contrast, resistant callus formed from cyanamide selection was more compact in form.
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Fig. 2 Selection and regeneration of transgenic wheat. A, Calli on selective medium containing 0.0375 g L-1 of cyanamide 8 wk following bombardment. B, Shoot formation from cyanamide-resistant callus tissue. C, A series of dilutions for the colorimetric assay (left to right); 0, 40, 20, 10, 5, 2, and 1 mM of cyanamide. D, Use of the colorimetric assay to test for the presence of cyanamide hydratase (left to right); nonplant sample containing no cyanamide (cuvette no. 1), nonplant sample containing 5 mM cyanamide (cuvette no. 2), transformed plant sample containing 5 mM cyanamide (cuvette no. 3), and a nontransformed plant sample containing 5 mM cyanamide (cuvette no. 4). E, Wheat plantlets sprayed with a Dormex solution (10%) 2 wk after planting; sprayed nontransformed control plant (right), sprayed 1722 plant (left). F, Wheat plants growing in the presence (3 g) of Perlka 2 wk after application, control wheat plant (left) and a plant of the 1722 line (right)
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Resistant callus lines were transferred to regeneration medium which consisted of MS medium with 0.0005 g L-1 dicamba and 0.0375 g L-1 of cyanamide. Callus proliferation was decreased on this medium while shoot differentiation from green sectors was increased. Shoots were transferred to culture tubes containing rooting medium (half-strength MS) with 0.0625 g L-1 of cyanamide. Only about 3% of the shoots from the regeneration medium were able to form roots in the medium containing the cyanamide. Sensitive shoots did not form roots and after 1 wk exhibited yellow necrosis and died. Resistant plantlets that remained on the rooting medium longer than 3 wk showed signs of reduced vigor and yellowing necrosis.
After roots had been established (2 wk), plantlets were transferred to soil and placed in a growth chamber under high humidity where they were allowed to acclimate before transfer to a greenhouse. Most of the putative transgenic plants matured with a normal appearance and flowered; however, there were degrees of variation in fertility and seed set among independent plant lines. The two independent transgenic plant lines discussed in this report were normal in appearance and growth and were fully fertile. The time frame from bombardment of explant material to flowering of transgenic plants was 7 to 8 mo.
Southern Blot Analysis
Stable integration of the Cah gene into the genome of two independently transformed T0 plants (1650 and 1722) is shown (Fig. 3)
. Genomic DNA was digested with the restriction enzyme EcoRI and hybridized with a 0.8-kb probe of the Cah coding sequence released from the pCAM vector. The DNA digests from independent explants showed different hybridization patterns for the two transformants with variations in copy number and size. Both lines contained the expected size DNA fragment band (arrow) when digested with EcoRI. Plant line 1650 had a more complex band pattern and a higher copy number then plant line 1722. The number of copies per genome were estimated from the band in the single-copy reconstruction (RC) lane. Variation in DNA fragment integration patterns between lines is a common result associated when using particle bombardment as a transformation system (Zhou et al., 1995). In undigested genomic DNA from the transformed plants, the Cah coding region hybridized to a DNA band of high molecular weight indicating integration into wheat chromosomal DNA. No hybridization of the Cah coding region probe was observed to digested DNA from nontransformed plants.
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Fig. 3 Southern blot analysis for T0 transgenic plants and a nontransformed control plant. Gel blot lanes contained 25 µg of genomic DNA. Hybridization was carried out with a radio-labeled Cah gene. The arrow indicates the expected position of the 1661 bp Cah gene fragment. The migration position of the molecular weight markers are shown to the left of the autoradiogram and labeled with sizes in kilobases. The left three lanes contain undigested DNA and the next three lanes contain DNA digested with EcoRI. The lanes marked TA and TB represent genomic DNA from two independently transformed lines (plant lines 1650 and 1722, respectively). Nontransformed control DNA is designated as NT and lanes labeled RC contain 25 µg of genomic nontransformed DNA plus 0.592 pg of a 0.8-kb PstI digest sequence from the pCAM plasmid which should correspond to a single copy reconstruction
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T0 plants were allowed to grow, self-fertilize, and set seed in the greenhouse. T1 seed was collected, planted, and grown in the greenhouse. T1 progeny plants exhibited resistance to application of cyanamide (Fig. 2E and 2F) indicating that the Cah transgene was transferred to the progeny. To determine the segregation ratio of the resistance trait, 40 seedlings from plant line 1722 were germinated from mature T1 seeds and sprayed 14 d later with a 10% solution of Dormex. Thirty-two of the seedlings survived. The 
2 analysis of resistant to sensitive T1 seedlings was consistent (2 = 2.2, 0.2 < P < 0.1, with 1 degree of freedom and the Yates correction factor) with the 3:1 segregation ratio of a dominant single copy gene indicating a Mendelian inheritance of the transgene. Twenty-nine plant line 1650 seedlings survived from 40 seedlings sprayed which also indicates Mendelian inheritance for that line.
Enzyme Assay
Cyanamide hydratase activity was assessed by a colorimetric assay. Lower absorption readings for both the 1650 and 1722 plant lines (Table 1)
indicated a decrease in cyanamide concentration and thus the presence of cyanamide hydratase activity. Plant line 1722 appeared to have more enzyme activity than plant line 1650. Similar results could be seen by looking visually at the solutions. Figure 2C shows a series of different concentrations of cyanamide with the addition of the color reagent. Figure 2D shows the results from assays with a solution without cyanamide and three with 0.5 mM cyanamide solution and no plant extract, extract of line 1722, and extract of a non-transformed plant, respectively. The cuvette from line 1722 shows a reduced color intensity compared with the cuvettes containing either no plant sample or the nontransformed sample, indicating a decrease in cyanamide concentration.
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Table 1 Reduction of absorbance at 530 nm in colorimetric assays containing 0.5 mM cyanamide with extracts from wheat plants nontransformed and transformed with a gene for cyanamide hydratase
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Cyanamide Resistance of Transgenic Plants
Transgenic progeny plants exhibited resistance to cyanamide when it was applied as a solution (Dormex) or in a granule form (Perlka). Transgenic plants survived the application of the Dormex solution whereas control plants perished in 1 wk (Fig. 2E). The resistant plants showed some signs of burning at the leaf tips when the solution was applied but matured and set seed comparable to that of a non-treated control wheat plant. When Perlka was applied, resistant transgenic plants survived and nonresistant control plants died (Fig. 2F) in 2 wk; however, there was little to no leaf burning of the resistant transgenic plants with the Perlka treatment. These plants also grew to maturity and set seed without any apparent side effects of the Perlka treatment. Results from these studies indicate that the application of either Dormex or Perlka would be an effective means of identifying transgenic plants from nontransgenic plants in a population of segregating progeny plants.
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Discussion
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The availability of different selectable markers and marker systems that are superior and more versatile for developing gene-transfer techniques is greatly needed. Several obstacles are encountered with the current selectable markers, including inefficient selection of transformed cells, regulatory approval, proprietary status, and public acceptance. In addition, there is an ongoing concern of crop-to-weed gene flow and hybridization of weedy plants (Arriola and Ellstrand, 1996).
This study describes the development of a new selection system for wheat transformation that may overcome some of the disadvantages mentioned above. Summarizing the transformation frequency (0.2%) of two independent transformation experiments, two transgenic wheat plants were recovered from 852 immature embryos bombarded. Although the transformation frequency is relatively low, it is still in the range of other previously reported wheat transformation frequencies of different selectable marker genes (Ortiz et al., 1996). Because these results reflect initial selection experiments, further refinements and optimization of the protocol are expected to result in higher transformation frequencies.
A colorimetric assay for decrease in cyanamide levels upon incubation with tissue culture or plant extracts provide a means to verify putative transformants expressing cyanamide hydratase enzyme. This assay is inexpensive, provides rapid results, and is easy to use. Moreover, it does not require the use of radioisotopes [14C] as required by other selectable marker assays such as the PAT assay. The selective agent, cyanamide, is commercially available and is inexpensive compared with some of the other selective agents. Cyanamide, when used in accordance with the manufacturer's guidelines and information, can be safely used in the laboratory in the protocols associated with media preparation, autoclaving, and tissue culture.
At the whole plant level, the presence of the Cah gene could offer a number of benefits and contribute to plant improvement. For agricultural purposes, calcium cyanamide (Perlka) is marketed as a fertilizer and has several other advantages. Rieder (1981) reported that calcium cyanamide is superior to urea or ammonium nitrate-based fertilizers because it (i) releases nitrogen gradually not exceeding plant needs; (ii) provides an efficient plant nitrogen source by reducing losses to leaching, runoff, and denitrification; (iii) lowers application cost through reduction in frequency of application; and (iv) reduces environmental concern because there is no nitrate pollution of ground water, streams, and lakes. Calcium cyanamide has low residual activity in the soil and does not accumulate in crop plants.
Besides its usefulness as a fertilizer, calcium cyanamide has properties of a fungicide, controlling soil-borne diseases and can be used for pest control. In addition, calcium cyanamide also functions as a herbicide by controlling weed germination and reducing weed growth below the economic threshold (Rieder, 1981). Maier-Greiner et al. (1991a) produced herbicide-resistant transgenic tobacco plants that were transformed with the Cah gene. They reported that the expression of cyanamide hydratase confers tolerance to cyanamide in transgenic tobacco by its degradation to urea which may be used by the plant as a nitrogen source (Bradley, 1992).
The transformation selection system described in this study demonstrates an effective and alternative transformation selection system for wheat. Current efforts are now focused on optimizing the cyanamide selection protocol and testing transgenic wheat plants for fertilizer response properties. In addition, we are extending this selection technology to other important agronomic crops. We believe that this new technology has the possibility of becoming a routine selection system for transfer of important genes for traits such as disease resistance, insect resistance, and seed quality into wheat. In addition, this research and technology should have a major impact on the application of molecular genetics to solve production problems in agriculture. The USDA-ARS is currently seeking patent protection on this selection technology for transforming wheat and other cereal crops.
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ACKNOWLEDGMENTS
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We thank J. Lin for excellent technical and DNA hybridization assistance and Dr. A. Blechl for helpful discussion and advice. We thank Dr. T. Clemente, Dr. P. Staswick, and Dr. J. Anderson for critical reading of the manuscript. Perlka and Dormex were provided through the generosity of SKW Trostberg AG and Dr. S. Siemer of Siemer & Associates, Inc.
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NOTES
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The use of a brand name by the USDA implies no approval of the product to the exclusion of others that may also be suitable.
Received for publication November 9, 1998.
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