Development of transformation vectors based upon a modified plant α-tubulin gene as the selectable marker

Cell Biology International 32 (2008) 566e570 www.elsevier.com/locate/cellbi

Development of transformation vectors based upon a modified plant a-tubulin gene as the selectable marker Alla Yemets a,*, Vladimir Radchuk a,c, Oleg Bayer a, Galina Bayer a, Alexey Pakhomov a, W. Vance Baird b, Yaroslav B. Blume a a

Institute of Cell Biology and Genetic Engineering, National Academy of Sciences of Ukraine, Kiev, Ukraine b Department of Horticulture, Clemson University, Clemson, SC, USA c Institute of Plant Genetics and Crop Plant Research (IPK), Gatersleben, Germany

Abstract A plant transformation and selection system has been developed utilizing a modified tubulin gene as a selectable marker. The vector constructs carrying a mutant a-tubulin gene from goosegrass conferring resistance to dinitroaniline herbicides were created for transformation of monocotyledonous and dicotyledonous plants. These constructs contained b- and/or mutant a-tubulin genes driven either by ubiquitin or CaMV 35S promoter. The constructs were used for biolistic transformation of finger millet and soybean or for Agrobacterium-mediated transformation of flax and tobacco. Trifluralin, the main representative of dinitroaniline herbicides, was used as a selective agent in experiments to select transgenic cells, tissues and plantlets. Selective concentrations of trifluralin estimated for each species were as follows: 10 mM for Eleusine coracana, Glycine max, Nicotiana plumbaginifolia and Nicotiana sylvestris; 3 mM for Linum usitatissimum. PCR and Southern blotting analyses of transformed lines with a specific probe to nptII, a-tubulin or b-tubulin genes were performed to confirm the transgenic nature of regenerated plants. Band specific for the mutant a-tubulin gene was identified in transformed plant lines. Results confirmed the stable integration of the mutant tubulin gene into the plant genomes. The present study clearly demonstrates the use of a plant mutant tubulin as a selective gene for plant transformation. Ó 2007 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved. Keywords: Selectable marker gene; Mutant tubulin; Dinitroaniline resistance; Plant transformation

1. Introduction There is substantial public concern about the potential spread of antibiotic resistance genes in soil and intestinal bacteria as a consequence of the use of genetically modified organisms and, in particular, of the agricultural use of transgenic crops. Common sense dictates that marker genes conferring resistance to significant therapeutic antibiotics should not be used. Therefore, development of new selection systems for plant transformation that are based exclusively on genetic information essentially already present in the host plant and that do not require antibiotic resistance genes would avoid such a risk.

* Corresponding author. Tel.: þ380 44 5261467; fax: þ380 44 5267104. E-mail address: [email protected] (A. Yemets).

Anti-microtubule drugs, whose target is tubulin (the principle protein constituent of microtubules), potentially can function as strong selection agents (Baird et al., 2000; Yemets et al., 2000). To facilitate the development of a new generation of transgenic systems, naturally occurring variant tubulin genes conferring anti-microtubule drug resistance may be appropriate for use as selectable markers. Such genes have been identified in a few plant and fungal species (Baird et al., 2000). In goosegrass (Eleusine indica), two alleles of a-tubulin 1 (each is the result of a single unique point mutation) have been described, which conferring either an intermediate or a high level of tolerance to a number of antimicrotubule herbicides e dinitroanilines and phosphoroamidates (Zeng and Baird, 1997; Yamamoto et al., 1998; Yamamoto and Baird, 1999; Anthony et al., 1998; Nyporko et al., 2002). In this work, we describe the development of a new selectable marker system for plant transformation, which is based

1065-6995/$ - see front matter Ó 2007 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.cellbi.2007.11.012

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upon the utilization of an altered a-tubulin from the highly resistant-biotype of E. indica. Transgenic plants carrying the TUAm gene (TUA1-239T/I; Fig. 1) were resistant to the herbicide after direct selection. We tested the possibility to use the mutant a-tubulin as a selective marker gene for transformation of finger millet (Eleusine coracana, a tetraploid relative of goosegrass), soybean (Glycine max), flax (Linum usitatissimum) and tobacco (Nicotiana plumbaginifolia and Nicotiana sylvestris). Effective selection of transgenic plant lines, using trifluralin (dinitroaniline herbicide) as a selective agent, and analysis of the lines obtained will be discussed. 2. Material and methods 2.1. Plant material N. plumbaginifolia and N. sylvestris sterile plants as well as soybean (G. max), flax (L. usitatissimum) and finger millet (E. coracana) seedlings were grown in vitro at 23e25  C on corresponding nutrient media described early for tobacco (Yemets et al., 2000), soybean (Pakhomov et al., 2004), flax (Bayer et al., 2004) and finger millet (Yemets et al., 2003a,b). 2.2. Herbicide Trifluralin, TFL [2,6-dinitro-N,N-dipropyl-4-(trifluoromethyl)benzenamine] was obtained from Dr. Guse (DowElanco, Greenfield, USA). Stock solutions of herbicide (10 mM) were prepared in dimethylsulfoxide, stored at 20  C and added in appropriate concentrations directly into cooled autoclave sterilized media. 2.3. Plant transformation vectors Two constructs, pAHTUAm and pAHTUB1, were developed for biolistic transformation based on the pAHC25 vector (Christensen and Quail, 1996). The pAHTUAm construct contained a mutant a-tubulin 1 cDNA (TUAm) isolated from goosegrass (Yamamoto et al., 1998) driven by a maize ubiquitin promoter and the nopaline synthase (NOS) terminator. To create pAHTUB1, the barley b-tubulin (HvTUB1) cDNA was cloned between the same promoter and the terminator sequences as in pAHTUAm (Radchuk et al., 2007). In addition, each plasmid carried the bar gene, which confers resistance to phosphinothricin, also driven by a ubiquitin promoter and terminated with the NOS 30 polyadenylation signal. The bar gene

Fig. 1. Alignment of the deduced amino acid sequence of the three alleles of a1-tubulin gene from goosegrass (E. indica). S ¼ wild type susceptible, R ¼ highly resistant, and I ¼ intermediately resistant. The Isoleusine for Threonine substitution at amino acid residue (codon) 239 is shown in the white box.

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was included in these constructs to additionally ensure selection of transformed cells. Integrity of the vector constructs was confirmed by restriction fragment analysis and sequencing. These two constructs were used for plant co-transformation to ensure co-expression of the a- and b-tubulin genes in an attempt to achieve a balance of both microtubule subunits (Anthony et al., 1998). The construct pBITUBA8 was created for Agrobacterium-mediated transformation and is a derivate of the binary vector pBINAR (Ho¨fgen and Willmitzer, 1990). pBITUBA8 contains the mutant TUAm gene from E. indica and the barley HvTUB1 gene, both driven by 35S CaMV promoter and terminated by 30 -octopine synthase (OCS ) terminator. For cloning, the TUB1 gene was first amplified by PCR with the following primers: 50 TCGCCCGGGATGAGGGAGATCCTGCAC-30 (SmaI restriction site is italicised, as are further restriction sites) and 50 -GCACCATCTAGACCTCCCCCTCCTTAC-30 (XbaI) and cloned between the promoter and terminator into SmaI/XbaI restriction sites of pRT101 plasmid to create the vector pRTUB1. The cassette consisting of 35S promoter, TUB1 and OCS terminator was cut out from the pRTUB1 with HindIII and cloned into the appropriate restriction site of the dephosphorylated pBINAR plasmid in order to create the vector pBITUB1. pBITUB1 was used to clone the TUAm gene between the existing 35S promoter and OCS terminator. For this, the TUAm sequence was amplified by PCR with the following primers: 50 -GAAGTCGACTTATGAGGGAGTG CATCTCG-30 (SalI and 50 -CTGGAATTCGGCTTCTAGTC GACGTCAC-30 SalI) and cloned into the appropriate restriction site of the dephosphorylated pBITUB1. The correct insertion was proved by restriction analysis and sequencing. The construction also carried nptII gene for kanamycin resistance selectability. 2.4. Plant transformation and selection Biolistic transformation of soybean callus and finger millet hypocotyl explants was carried out using the protocol of Abumhadi et al. (2001) with modifications. Two micrograms of each pAHTUB1 and pAHTUAm plasmid DNA, in 25 ml 2.5 M CaCl2 and 10 ml 0.1 M spermidine, was precipitated onto tungsten particles. The particles were washed once with 180 ml ethanol and resuspended in 40 ml ethanol. The suspension of coated particles was spread on a syringe filter and dried for 10 min. Small tissue explants (2e3 cm) were centered in Petri dishes containing osmotic medium (Abumhadi et al., 2001) for 4 h before and for 16 h after bombardment. The main parameters used for particle bombardment of finger millet or soybean, respectively, were as follows: 0.9 or 0.7 MPa of helium pressure, 0.9 bar of vacuum pressure and 12 or 7 cm of particle flight distance. One week after particle bombardment, the explants were transferred to appropriate nutrient media containing selective concentration of TFL for selection and regeneration of transgenic plants. In parallel, transformants were selected on media supplemented with 5 mg/l phosphinothricin (PPT/BastaÒ, Agro Evo) instead of TFL to test the effectiveness of biolistic transformation using previously

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reported procedures (Yemets et al., 2000, 2003a; Pakhomov et al., 2004). The A. tumefaciens mediated transformation was carried out accordingly to An et al. (1986) using Nicotiana leaf disks and flax hypocotyl segments as explants. After co-cultivation for 2 days, bacterial cells were washed off and the plant explants were grown on nutrient agar medium containing 250 mg/l carbenicillin, 250 mg/l cefatoxime and 3 mM TFL for flax or 10 mM TFL for tobacco. In parallel, transformants were selected on media containing 100 mg/l kanamycin as the selective agent instead of TFL. 2.5. PCR analysis For polymerase chain reaction (PCR) and Southern blot hybridization, total DNA was isolated from young shoots of putative transformants and controls using the DNeasy Plant Mini Kit (Qiagen, Germany). Initially, all plants regenerated after Agrobacterium-mediated transformation was tested by PCR for the presence of the nptII gene. Amplification reactions were performed in a total volume of 25 ml containing 500 ng of genomic DNA, 0.5 U Taq-DNA-polymerase (Roche Diagnostics, Germany), 1 reaction buffer, 0.2 mM of each dNTP and 1 mM of each primer. The primers used for amplification of a 791 base pair fragment of the nptII gene were 50 -ATGATTGAACAAGATGGATTG-30 and 50 -GAAGAACT CGTCAAGAAGCGA-30 . Amplification was carried out in the Perkin Elmer thermocycler (Perkin Elmer, USA) under the following conditions: initial denaturation for 4 min at 94  C; followed by 25 cycles of 40 s at 94  C, 40 s at 56  C and 1 min at 72  C; and a final stage of 7 min at 72  C. The amplification products were separated in 1% agarose gels and visualized under UV light after staining with ethidium bromide. 2.6. Southern blotting For Southern blots, about 5 mg of total genomic DNA from transformed plants and from untransformed control plants was digested with HindIII endonuclease (Roche Diagnostics, Germany) overnight at 37  C. Restricted DNAs were separated on 1% agarose gel and blotted to Hybond NX membrane (Amersham, USA). Hybridizations were performed overnight at 65  C essentially as described by Radchuk et al. (2005). PCR amplified fragments of nptII, HvTUB1 or TUAm genes served as probes and were labeled with 32P-dCTP using the Rediprime II kit (Amersham, UK).

Fig. 2. Results of selection of transformed plants: N. plumbaginifolia embryogenic callus formation (a) and plant regeneration (b) in the presence of 10 mM TFL; view of flax callus (c) with regeneration structures (d) and plantlets (e) grown on the selective medium with 3 mM TFL; rooted transgenic flax plant in greenhouse (f); finger millet embryogenic callus selection (g) and plantlets regeneration (h) in the presence of 10 mM TFL; normal growth of finger millet transgenic plant (left) and suppression of control E. coracana plant growth (right) on media with 10 mM TFL (i) seed formation in finger millet transgenic plant (j) growing on 10 mM TFL.

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3. Results and discussion Because TFL, as the main representative of dinitroaniline herbicides, demonstrates a very high specificity for plant tubulins compared to animal tubulins (Baird et al., 2000; Blume et al., 2003; Yemets and Blume, 1999), it was chosen for use as the selective agent in experiments on transgenic cell and tissue cultures. The effective TFL concentration (EC50) was estimated for each plant species by in vitro tests for dicotyledonous (Yemets et al., 2000) and for monocotyledonous (Yemets et al., 2003b) plant lines. It was found that the TFL EC50 for E. coracana, G. max, N. plumbaginifolia and N. sylvestris is 10 mM; and for L. usitatissimum it is 3 mM. These concentrations were then used to select transgenic lines after both biolistic and Agrobacterium-mediated transformation. Suppression of growth and proliferation of untransformed plant cells were clearly visible on agar nutrient medium supplemented with TFL (Fig. 2a, d, and g). Cells, transformed with the mutant a-tubulin gene, produced friable white or green-coloured organogenic and embryogenic calli on media with selective concentrations of herbicide (Fig. 2a, c and g). Later, the callus lines of tobacco, soybean, flax and finger millet were able to regenerate into plantlets under the selective conditions (Fig. 2b, d, e and h). Control untransformed plants could not survive under the same selective conditions (Fig. 2i). Moreover, transgenic finger millet flowered and produced viable seeds in the presence of TFL (Fig. 2j). All regenerated and rooted plants were transferred to soil, acclimatized and transferred to the greenhouse for further investigations (Fig. 2f). Some of these lines were taken for molecular biological analyses. In parallel, tobacco and flax transformants were selected on nutrient media containing 100 mg/l kanamycin as the selective agent instead of TFL in order to compare the effectiveness of kanamycin and TFL selection for the routine production of transgenic plants. Similar experiments were done for finger millet and soybean, where transformants were selected on media supplemented with 5 mg/l phosphinothricin instead of TFL. The efficiency of transgenic plant selection using TFL was comparable with those using kanamycin or phosphinothricin. These results are consistent with earlier reports on the production of interspecific and intertribal somatic hybrids with resistance to the dinitroanilinine herbicide TFL (Yemets et al., 1997) or the phosphorothioamidate herbicide amiprophosmethyl, APM (Yemets et al., 2000). In these studies, the anti-microtubule herbicides were used as selective agents for effective and successful selection of plant somatic hybrids after symmetric and asymmetric fusions using N. plumbaginifolia mutants resistant to TFL or APM. The present work provides further evidence of the effective use of TFL for different plant selection programmes. To confirm the transgenic nature of regenerated plants, the molecular genetic analysis was carried out. The presence of transferred nptII gene in transgenic shoots was first investigated by the PCR analysis (Fig. 3). The 791 bp length fragment of the nptII gene was amplified from most regenerated plants. The stable insertion of the nptII, TUAm and HvTUB

Fig. 3. PCR analysis of transformed Linum lines with a probe to nptII. L1, L2, and L3 e different transgenic lines of Linum, Co e non-transformed control line, P1 e plasmid pBITUBA8. Molecular size markers are shown at right (in bp). Specific bands for transferred nptII gene are arrowed.

genes into genomic DNA was further investigated by Southern analysis. DNAs from two regenerated flax plants, six soybean plants and the corresponding untransformed control plants were digested with HindIII. Hybridization of the restricted DNAs with the nptII gene probe revealed the presence of one to three bands (Fig. 4a) indicating one or more T-DNA integrations. All of them had a different molecular size than the corresponding plasmid. Hybridization with the probes to TUAm and HvTUB genes resulted in the detection of several bands, indicating cross-hybridization with endogenous a- or b-tubulin sequences. The integrated TUAm and HvTUB genes were visible as additional single bands, not present in genomic DNA of control plants (Figs. 4b, c, and 5) and one soybean transformed but not transgenic, as it was revealed, line S2 (Fig. 5). The present work has demonstrated the effective transformation of monocotyledonous and dicotyledonous plants (e.g., finger millet, flax, soybean and tobacco) with the constructs carrying the mutant a-tubulin sequence as a selective

Fig. 4. Southern analysis of transformed Linum lines, using a probe to nptII (a), alpha-tubulin (b) and beta-tubulin (c) genes. L1 and L3 e different transgenic lines of Linum; Lc e control plant line; Pl e plasmid pBITUBA8. Molecular size markers are shown at right (in bp). Specific bands for transferred alphaand beta-tubulin genes are arrrowed.

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Fig. 5. Transformed soybean lines (S1eS6) possess one additional band (with the exception of line S2) for alpha-tubulin (arrow) in comparison to the control (SC). The band size corresponds to the integrated mutant alpha-tubulin (TUAm).

marker gene. The transformed plant lines resistant to antimicrotubule drugs were established through in vitro selection on TFL. Thus, we conclude that a general plant transformation and selection system has been developed utilizing a modified tubulin gene from a natural biotype of E. indica, which confers resistance to the dinitroanilines, as a selectable marker gene. The selection system is based exclusively on genetic information of plant origin and presents a gene-based selection system as an alternative to traditional antibiotic resistance. The transformation/selection method involves the production of altered tubulin isotypes that result from a mutation(s) in the herbicidebinding site. Acknowledgments This work was supported by a grant from NATO e Life Sciences and Technology division (CLG #979536), and grants 17K and II-7-07 from National Academy of Sciences of Ukraine. References Abumhadi N, Trifonova A, Takumi S, Nakamura C, Todorovska E, Getov L, et al. Development of the particle inflow gun and optimizing the particle bombardment method for efficient genetic transformation in mature embryos of cereals. Biotech Equip 2001;15(2):87e96.

An G, Watson BD, Chiang CC. Transformation of tobacco, tomato, potato, Arabidopsis thaliana using a binary Ti vector system. Plant Physiol 1986;81:301e5. Anthony R, Waldin T, Ray J, Bright S, Hussey P. Herbicide resistance caused by spontaneous mutation of the cytoskeletal protein tubulin. Nature 1998;393:260e3. Baird V, Blume YaB, Wick SM. Microtubular and cytoskeletal mutants. In: Nick P, editor. Plant microtubules e potential targets for biotechnological applications. Frankfurt-Berlin: Springer Verlag; 2000. p. 155e87. Bayer OA, Bayer GYa, Yemets AI, Blume YaB. In vitro tissue culture establishment and regeneration capacity of fiber flax genotypes with different wind resistance. Physiol Biochem Cult Plants 2004;36(1):48e54. Blume YaB, Yemets AI, Nyporko AYu, Baird WV. Structural modelling of plant a-tubulin interaction with dinitroanilines and phosphoroamidates. Cell Biol Int 2003;27:171e4. Christensen AH, Quail PH. Ubiquitin promoter-based vectors for high level expression of selectable and/or screenable marker genes in monocotyledonous plants. Transgenic Res 1996;5:213e8. Ho¨fgen R, Willmitzer L. Biochemical and genetic analysis of different patatin isoforms expressed in various organs of potato (Solanum tuberosum). Plant Sci 1990;66:221e30. Nyporko AYu, Yemets AI, Klimkina LA, Blume YaB. Correlation of Eleusine indica callus susceptibility to trifluralin and amiprophosmethyl and peculiarities of interaction these compounds with tubulin. Rus J Plant Physiol 2002;49(3):1e7. Pakhomov AV, Yemets AI, Hu CY, Blume YaB. Evaluation of embryological potential of soybean cultivars zoned in forest, steppe, marshy woodlands of Ukraine as essential stage for the further transformation. Cytol Genet 2004;38(1):49e54. Radchuk VV, Van DT, Klocke E. Multiple gene co-integration in Arabidopsis thaliana predominantly occurs in the same genetic locus after simultaneous in planta transformation with distinct Agrobacterium tumefaciens strains. Plant Sci 2005;168(6):1515e23. Radchuk VV, Sreenivasulu N, Blume Ya, Weschke W. Distinct tubulin genes are differentially expressed during barley grain development. Physiol Plant 2007;131(4):571e80. Yamamoto E, Baird WV. Molecular characterization of four beta-tubulin genes from dinitroaniline susceptible and resistant biotypes of Eleusine indica. Plant Mol Biol 1999;39:45e69. Yamamoto E, Zeng L, Baird WV. Alpha-tubulin missense mutations correlate with antimicrotubule drug resistance in Eleusine indica. Plant Cell 1998;10:297e308. Yemets AI, Bayer GYa, Klimkina LA, Stadnichuk NA, Abramov AA, Blume YaB. Establishment in vitro callus culture and plant regeneration of dagussa Eleusine coracana (L.) Gaertn. cv Tropicanka. Physiol Biochem Cult Plants 2003a;35(2):152e9. Yemets AI, Blume YaB. Resistance to herbicides with antimicrotubular activity: from natural mutants to transgenic plants. Rus J Plant Physiol 1999;46(6):789e96. Yemets AI, Klimkina LA, Tarassenko LV, Blume YaB. Efficient callus formation and plant regeneration from dinitroaniline-resistant and susceptible biotypes of Eleusine indica (L.). Plant Cell Rep 2003b;21:503e10. Yemets AI, Kundel’chuk OP, Smertenko AP, Solodushko VA, Rudas VA, Gleba YY, et al. Transfer of amiprophosmethyl-resistance from a Nicotiana plumbaginifolia mutant by somatic hybridisation. Theor Appl Genet 2000;100:847e57. Yemets AI, Strashnyuk NM, Blume YaB. Plant mutants and somatic hybrids with resistance to trifluralin. Cell Biol Int 1997;21:912e4. Zeng L, Baird WV. Genetic basis of dinitroaniline herbicide resistance in a highly resistant biotype of goosegrass (Eleusine indica). J Heredity 1997;88:427e32.