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ARTICLE IN PRESS Journal of Plant Physiology 165 (2008) 1698—1716 www.elsevier.de/jplph Advances in selectable marker genes for plant transformation Isaac Kirubakaran Sundar, Natarajan Sakthivel Department of Biotechnology, School of Life Sciences, Pondicherry University, Kalapet, Puducherry 605 014, India Received 1 March 2008; accepted 4 August 2008 KEYWORDS Biosafety; Plant transformation; Positive selection; Selectable marker; Transgenic plants Summary Plant transformation systems for creating transgenics require separate process for introducing cloned DNA into living plant cells. Identification or selection of those cells that have integrated DNA into appropriate plant genome is a vital step to regenerate fully developed plants from the transformed cells. Selectable marker genes are pivotal for the development of plant transformation technologies because marker genes allow researchers to identify or isolate the cells that are expressing the cloned DNA, to monitor and select the transformed progeny. As only a very small portion of cells are transformed in most experiments, the chances of recovering transgenic lines without selection are usually low. Since the selectable marker gene is expected to function in a range of cell types it is usually constructed as a chimeric gene using regulatory sequences that ensure constitutive expression throughout the plant. Advent of recombinant DNA technology and progress in plant molecular biology had led to a desire to introduce several genes into single transgenic plant line, necessitating the development of various types of selectable markers. This review article describes the developments made in the recent past on plant transformation systems using different selection methods adding a note on their importance as marker genes in transgenic crop plants. & 2008 Elsevier GmbH. All rights reserved. Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Classical selectable marker genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Arabidopsis thaliana ATP-binding cassette (ABC) transporter . . . . . . . . . . . . . . . . . . . . . . . . . Neomycin phosphotransferase II (NPT II) (nptII). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Corresponding author. Tel.: +91 413 2654430; fax: +91 413 2655715. E-mail address: [email protected] (N. Sakthivel). 0176-1617/$ - see front matter & 2008 Elsevier GmbH. All rights reserved. doi:10.1016/j.jplph.2008.08.002 1699 1700 1702 1702 ARTICLE IN PRESS Advances in selectable marker genes for plant transformation Hygromycin phosphotransferase (HPT) (hpt) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Phosphinothricin N-acetyltransferase (PAT) (pat and bar) . . . . . . . . . . . . . . . . . . . . . . . . . . . Methionine sulfoximine (MSO) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-Enolpyruvylshikimate-3-phosphate (EPSP) synthase (epsps) . . . . . . . . . . . . . . . . . . . . . . . . . Cyanamide hydratase (cah) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cytochrome P450 monooxygenases. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Phytoene desaturase (PDS). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . a-Tubulin gene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Protoporphyrinogen oxidase (PPO) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Anthranilate synthase (AS) (ASA2) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Tryptophan (Trp) synthase beta 1 (TSB1) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Threonine deaminase (TD) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cytosine deaminase (codA) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Xylose isomerase (xylA). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Phosphomanose isomerase (PMI) (manA) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Isopentyl transferase (IPT) (ipt) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Indole acetic acid (iaaM and iaaH) and hairy root inducing (rolABC) . . . . . . . . . . . . . . . . . . . . D-Amino acid oxidase (DAAO) (dao1) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Arabitol dehydrogenase. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D-Serine ammonia lyase (DSD) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Trehalose-6-phosphate synthase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Knotted1 (kn1) homeobox gene. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Future prospects and conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Introduction The advent of recombinant DNA technology provided an immense potential in the field of plant transformation. In order to achieve high food productivity and to ensure nutritional quality, genetic engineering methods in generating transgenics with useful agronomic traits are assuming significance. Plant biotechnology is based on the delivery, integration and expression of defined genes into plant cells, which can be grown to generate transformed plants. Efficiency of stable gene transfer is not high even in the most successful transfer systems and only a fraction of the cells exposed integrate the DNA construct into their genomes. Therefore, systems to select the transformed cells, tissues or organisms from the non-transformed ones are indispensable and selectable marker genes are vital to the plant transformation process. From the development of first transgenic plants during early 1980s and subsequently after its commercialization worldwide over a decade, antibiotic and herbicide resistance selectable marker genes were one among the integral feature of plant genetic modification (Ramessar et al., 2007). Selectable marker genes are introduced into plant genome to express a protein generally with an enzymatic activity, which allows distinguishing transformed from non-transformed cells (Brasileiro and Dusi, 1999). In all plant transformation systems to 1699 1702 1703 1703 1703 1703 1703 1704 1704 1705 1705 1706 1706 1707 1707 1707 1708 1708 1708 1709 1709 1709 1710 1710 1711 1711 generate substantial number of non-chimeric transformants, genes conferring resistance to selective agents such as antibiotics or herbicides were widely employed to select transformants. These selectable marker genes enable the transformed cells to survive on medium containing the selective agent, while non-transformed cells and tissues die. The low efficiencies of most plant transformation methods necessitate the use of selectable marker genes to identify those cells that successfully integrate and express transferred DNA. Genes encoding resistance to specific antibiotics or herbicides have proved particularly effective for selection and provide a means of rapidly identifying transformed cells, tissues and regenerated shoots that have integrated foreign DNA and that express the selectable gene product and by inference, the gene(s) of interest (Goodwin et al., 2004). Different selectable marker genes were used for transgenic and transplastomic plant research or crop development. These marker genes were assessed for efficiency, biosafety, scientific applications as well as commercialization (Miki and McHugh, 2004). Selectable marker genes can be divided into several categories depending on whether they confer positive or negative selection and whether selection is conditional or non-conditional in the presence of external substrates. Positive selectable marker genes are defined as those that allow the growth of transformed tissues, whereas negative selectable marker genes result in the ARTICLE IN PRESS 1700 I.K. Sundar, N. Sakthivel death of the transformed tissue (Joersbo et al., 1998; Miki and McHugh, 2004). Most commonly and recently developed selectable marker genes, selection agents, enzymes, source of those genes and their importance and applications in plant transformation have been discussed in this review. Classical selectable marker genes Most of the published scientific literature on transgenic crop plants revealed that antibiotics (kanamycin or hygromycin) and herbicide (phosphinothricin, PPT) was widely used as selection agent since the early days of plant transformation. The popularity of these selection systems reflects on the efficiency, availability and applicability of their use across a wide range of plant species and its regenerative efficacy in plant tissue culture sys- tems. Positive selection systems are those that allow the growth of transformed plant cells. Based on their functionality the positive selection systems can be classified into conditional and non-conditional positive selection system. A conditional-positive selection system consists of a gene encoding for a protein, usually an enzyme that confers resistance to a specific substrate that may be toxic to the untransformed plant cells or that facilitates the growth as well as differentiation of the transformed cells alone. This system includes antibiotics, herbicides (Table 1a), toxic and non-toxic drugs or metabolite analog or a carbon source (Table 1b). Non-conditional-positive selection systems do not require external substrates but promote the selective growth and differentiation of transformed cells (Miki and McHugh, 2004). Almost all the antibiotic resistant selectable marker genes for use in transgenic plants have been identified from bacterial sources. Table 1 Selection agent Genes Enzymes Sources (a) Antibiotics and herbicides used as selection agent in plant transformation Antibiotics Neomycin, kanamycin neo nptII Neomycin Escherichia coli Tn5 Kanamycin (aphA2), Atwbc19 sulI sat3 cat Fraley et al. (1983) Bevan et al. (1983) Carrer et al. (1993) Phosphotransferases Escherichia coli Tn601 ATP-binding cassette Arabidopsis thaliana Mentewab and Stewart (2005) Serratia marcesens Hayford et al. (1988) Klebsiella pneumoniae Shigella sp. Shigella sp. Gossele et al. (1994) Svab and Maliga (1993) Tn5 Maliga et al. (1988) Escherichia coli Waldron et al. (1985) Paramomycin, G418 nptI, (aphA1) aaC3 Aminoglycoside-NAminoglycosides acetyltransferase (kanamycin, neomycin, geneticin, paramomycin, gentamycin, tobramycin, apramycin) aaC4 6’gat Spectinomycin aadA Aminoglycoside-300 -adenyl transferase Streptomycin SPT Streptomycin phosphotransferase Hygromycin B hph, (aphIV) Hygromycin phosphotransferase Bleomycin Ble Bleomycin resistance Phleomycin Sulphonamides Streptothricin Chloramphenicol References Escherichia coli Tn5 Streptoalloteichus hindustanus Dihydropteroate synthase Escherichia coli pR46 Acetyl transferase Streptomyces sp. Chloramphenicol acetyl Escherichia coli Tn5, transferase Phage p1cm Hille et al. (1986) Perez et al. (1989) Guerineau et al. (1990) Jelenska et al. (2000) De Block et al. (1984, 1985) ARTICLE IN PRESS Advances in selectable marker genes for plant transformation 1701 Table 1. (continued ) Selection agent Genes Enzymes Herbicides Phosphinothricin pat, bar Phosphinothricin acetyl Streptomyces transferase hygroscopicus Phosphinothricin acetyl transferase 5-Enolpyruvylshikimate-3- Petunia hybrida, phosphate synthase Zea mays Salmonella typhimurium Escherichia coli Methionine sulfoximine Glyphosate EPSP synthase aroA cp4 epsps gox Sulfonylueras Imidazolinones Oxynils csr1-1 csr1-2 bnx Gabaculine hemL Cyanamide Acetochlor, chlorpropham amiprophos-methyl, chlorsulfuon, norflurazon and pendimethalin Norflurazon and fluridone Trifluralin Butafenacil cah P450 pds TUAm Glyphosate oxidoreductase Acetolactate synthase Acetolactate synthase Bromoxynil nitrilase Glutamate-1semialdehyde aminotransferase Cyanamide hydratase Cytochrome P450 Phytoene desaturase a-Tubulin Protoporphyrinogen oxidase (b) Toxic and non-toxic agents, drug and metabolite analogs used Drugs and analogs 2-Deoxyglucose DOG 1 2-Deoxyglucose-6phosphate phosphatase Betaine aldehyde BADH Betaine aldehyde dehydrogenase S-Aminoethyl DHPS Dihydropicolinate synthase L-Cysteine (AEC) ocs Octopine synthase Sources Agrobacterium tumefaciens Ochrobactrum anthropi Chai et al. (2007) Zhou et al. 1995 Howe et al. (2002) Comai et al. (1988) Della-Cioppa et al. (1987) Barry et al. (1992) Barry et al. (1992) Olszewski et al. (1988) Aragao et al. (2000) Freyssinet et al. (1996) Myrothecium verrucaria Human Weeks et al. (2000) Inui et al. (2005) Hydrilla verticillata Eleusine indica Myxococcus xanthus Arias et al. (2006) Yemets et al. (2008) Lee et al. (2007) Gough et al. (2001) as selection agent in plant transformation Saccharomyces cerevisiae Kunze et al. (2001) Spinacia oleracea Daniell et al. (2001) Escherichia coli Perl et al. (1993) 4-Methyltryptophan (4mT) Methotrexate TDC DHFR Dihydrofolate reductase L-Methionine bar Phosphinothricin acetyl transferase 5-Methyltryptophan ASA2 Anthranilate synthase 5-Methyltryptophan (5MT) OASA1D Mutant anthranilate synthase 5MT/Cadmium cholride TSB1 Tryptophan synthase beta L-O-Methylthreonine ilvA or ilvA- Threonine deaminase 466 De Block et al. (1989) Arabidopsis thaliana Arabidopsis thaliana Klebsiella pneumoniae sub sp. Ozanaenae Synechococcus PCC6301 Agrobacterium tumefaciens Tryptophan decarboxylase Catharanthus roseus sulfoximine References Escherichia coli, mouse, Candida albicans Streptomyces hygroscopicus Tobacco Rice Arabidopsis thaliana Escherichia coli Koziel et al. (1984) Herrera-Estrella et al. (1983) Eichholtz et al. (1987) Irdani et al. (1998) Chai et al. (2007) Goddijn et al. (1993) Cho et al. (2004) Kobayashi et al. (2005) Hsiao et al. (2007) Ebmeier et al. (2004) ARTICLE IN PRESS 1702 I.K. Sundar, N. Sakthivel Table 1. (continued ) Selection agent Genes Enzymes Sources References Non-toxic agents 5-Flurocytosine (5-FC) D-Xylose codA xylA Cytosine deaminase Xylose isomerase Escherichia coli Streptomyces rubignosus Thermoanaerobacterium sulfurogenes Escherichia coli Kobayashi et al. (1995) Haldrup et al. (1998b) Haldrup et al. (1998b) Escherichia coli Joersbo and Okkels (1996) Endo et al. (2001) D-Mannose Benzyladenine-N-3glucuronide None manA (pmi) Phosphomannose isomerase uidA (gusA) b-Glucuronidase ipt Isopentyl transferase None iaaM, iaaH Indole acetic acid None rolC ‘Hairy root’ phenotype D-Amino acids (D-alanine and D-serine) Arabitol D-Serine Glucose dao1 D-Amino atlD dsdA AtTPS1 D-Serine Hormone-free medium kn1 acid oxidase Arabitol dehydrogenase ammonia lyase Trehalose-6-phosphate synthase Arabidopsis thaliana ATP-binding cassette (ABC) transporter ABC proteins are ubiquitous proteins that share 30–40% identity between family members and are characterized by the presence of an ABC (Higgins, 1992). Arabidopsis thaliana ABC transporters have been classified on the basis of their domain organization and their homology to orthologous genes, but their functions remain largely unknown (Sanchez-Fernandez et al., 2001). The AtWBC family (White-Brown Complex homologs) is the largest group of all Arabidopsis thaliana ABC transporters, with 29 members. A plant gene, Atwbc19 (2178 bp) that encodes an Arabidopsis thaliana ABC transporter was characterized and it confers antibiotic (kanamycin) resistance to transgenic plants. The mechanism of resistance is novel, and the levels of resistance achieved are comparable to those attained through expression of bacterial antibiotic-resistance genes in transgenic tobacco using CaMV 35S promoter. Because ABC transporters are endogenous to plants, the use of Atwbc19 as a selectable marker in transgenic plants may provide a practical alternative to current bacterial marker genes in terms of the risk for horizontal transfer of resistance genes (Mentewab and Stewart, 2005). Hence this marker gene may be valuable for transformation of agriculturally important dicot species such as soybean, cotton, Brassica crops, tomato and other Solanaceae as Joersbo et al. (1998) Agrobacterium tumefaciens Agrobacterium Tuominen et al. (1995) tumefaciens Agrobacterium rhizogenes Ebinuma and Komamine, (2001) Rhodotorula gracilis Erikson et al. (2004) Escherichia coli strain C Escherichia coli Arabidopsis thaliana LaFayette et al. 2005 Erikson et al. (2005) Leyman et al. (2006) Maize Luo et al. (2006) well as forest tree species including angiosperms (e.g., polar and elms) and gymnosperms (e.g., pine and spruce). Neomycin phosphotransferase II (NPT II) (nptII) The nptII (or neo) gene was isolated from the transposon Tn5 of Escherichia coli and it encodes NPT II (E.C. 2.7.1.95), also known as aminoglycoside 30 phosphotransferase II (Fraley et al., 1983; Herrera-Estrella et al., 1983). The active aminoglycoside antibiotic inhibits the protein synthesis in prokaryote cells, by binding to the 30S subunit of the ribosome, blocking the formation of initiation complexes and decreasing the fidelity of translation. Hygromycin phosphotransferase (HPT) (hpt) The hpt (or aph IV) gene of Escherichia coli codes for the enzyme HPT (E.C. 2.7.1.119) and confers resistance to the antibiotic hygromycin B (Waldron et al., 1985). When hygromycin occupies the ribosomal binding site of the elongation factor 2 (EF-2) in prokaryotic cells, consequently the elongation of polypeptide chain is inhibited and protein synthesis interrupted, causing the same symptoms described for the other aminoglycoside antibiotics. In plant cells, these antibiotics exert its effect on ARTICLE IN PRESS Advances in selectable marker genes for plant transformation mitochondria and chloroplast, acting in the same manner by impairing protein synthesis. These organelles have ribosomes that are similar to those found in bacteria and are also susceptible to aminoglycoside antibiotics. Therefore, in the presence of antibiotic, the plant tissue will show a chlorosis, caused by the lack of chlorophyll synthesis and inhibition of growth (Benveniste and Davies, 1973; Brasileiro, 1998). Phosphinothricin N-acetyltransferase (PAT) (pat and bar) Since 1987, the bialaphos resistance genes bar from Streptomyces hygroscopicus and pat from Streptomyces viridochromogenes encoding the enzyme PAT have been used as selectable marker in plants (De Block et al., 1989). The PPT (ammonium glufosinate) is analogous to glutamate, the substrate of glutamate synthetase (GS), and acts as a competitive inhibitor of GS. The enzyme GS catalyzes the conversion of glutamate to glutamine, removing the toxic ammonia from the cell. This enzyme plays an essential role in the regulation of nitrogen metabolism and ammonia assimilation. When the GS is inhibited, it results in ammonia accumulation and consequently disruption of chloroplast that leads to inhibition of photosynthesis and to death of the plant cell (Lindsey, 1992). Methionine sulfoximine (MSO) An alternative selection agent, L-MSO was used for selection of the bar marker, which also confers resistance to PPT. MSO like PPT is a glutamate analog that inhibits growth of dicotleydonous plant Arabidopsis due to its action on GS. Maughan and Cobbett (2003) demonstrated the utility of MSO as an effective and economical alternative to PPT for selection and regeneration of plants. MSO was 40-fold more potent, inhibits growth of wild-type plants and detoxified in the similar manner as PPT by PPT acetyl transferase. In a recent study, MSO was employed as a novel selection agent for genetic transformation of orchids. The successful application of MSO in orchid transformation opens up new avenues to study functional orchid genes as well as genetic engineering of orchids with commercially valuable traits. This readily available selection agent also has great potential for the transformation of economically important monocotyledonous plant species (Chai et al., 2007). 1703 5-Enolpyruvylshikimate-3-phosphate (EPSP) synthase (epsps) The aroA (or epsps) gene was isolated from Salmonella typhimurium treated with mutagenic agent and selected for resistance to the herbicide glyphosate. The herbicide glyphosate inhibits, by competition, the enzyme EPSP synthase (E.C. 2.5.1.19), which is involved in the enzymatic pathway of aromatic amino acids biosynthesis in bacteria and plants. The inhibition of EPSPS results in shikimate accumulation, inhibition of synthesis of aromatic amino acids and secondary metabolites causing cell death. An excellent and comprehensive review on many other selectable marker genes in transgenic crop plants has been published (Miki and McHugh, 2004). Cyanamide hydratase (cah) The cah gene coding for the enzyme cyanamide hydratase (urea hydrolase; E.C. 4.2.1.69) has been isolated from the soil fungus Myrothecium verrucaria (Maier-Greiner et al., 1991a). Cyanamide hydratase catalyzes the hydration of the nitrile group of cyanamide to form urea, which can be used for plant growth. Cyanamide is a nitrile derivative that in its aqueous or calcium salt forms can be used as a fertilizer. It has the additional characteristics of acting as a non-persistant herbicide when applied prior to seed germination. The enzyme has extremely narrow substrate specificity. The use of cyanamide hydratase as a selectable marker has been demonstrated in wheat (Weeks et al., 2000), tobacco (Maier-Greiner et al., 1991b; Kirubakaran and Sakthivel, 2008), potato, tomato, rice, Arabidopsis (Damm, 1998) and soybean (Zhang et al., 2005; Ulanov and Widholm, 2007). In a recent study, the heterologous expression of cah marker gene was reported. Due to its innate ability to convert calcium cyanamide to urea and the broad-spectrum antimicrobial activity of cyanamide, the cah gene can also be used to facilitate plant growth promotion and biocontrol of phytopathogens (Kirubakaran and Sakthivel, 2008). Cytochrome P450 monooxygenases P450 monooxygenases are heme proteins that use electrons from NADPH to catalyze the activation of molecular oxygen. The catalyzed reaction is usually a mono-oxygenation, with the formation of a molecule of water and an oxygenated product. Mammalian P450 species show overlapping and broad substrate specificity and confer the ability