Advances in Selectable Marker Genes for Plant Transformation

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 SakthivelÃ

Department of Biotechnology, School of Life Sciences, Pondicherry University, Kalapet, Puducherry 605 014, India

Received 1 March 2008; accepted 4 August 2008

KEYWORDS Summary Biosafety; Plant transformation systems for creating transgenics require separate process for Plant introducing cloned DNA into living plant cells. Identiﬁcation or selection of those transformation; cells that have integrated DNA into appropriate plant genome is a vital step to Positive selection; regenerate fully developed plants from the transformed cells. Selectable marker Selectable marker; genes are pivotal for the development of plant transformation technologies because Transgenic plants 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 ...... 1699 Classical selectable marker genes ...... 1700 Arabidopsis thaliana ATP-binding cassette (ABC) transporter ...... 1702 Neomycin phosphotransferase II (NPT II) (nptII)...... 1702

ÃCorresponding author. Tel.: +91 413 2654430; fax: +91 413 2655715. E-mail address: [email protected] (N. Sakthivel).

Advances in selectable marker genes for plant transformation 1699

Hygromycin phosphotransferase (HPT) (hpt)...... 1702 Phosphinothricin N-acetyltransferase (PAT) (pat and bar) ...... 1703 Methionine sulfoximine (MSO) ...... 1703 5-Enolpyruvylshikimate-3-phosphate (EPSP) synthase (epsps) ...... 1703 Cyanamide hydratase (cah) ...... 1703 Cytochrome P450 monooxygenases...... 1703 Phytoene desaturase (PDS)...... 1704 a-Tubulin gene ...... 1704 Protoporphyrinogen oxidase (PPO) ...... 1705 Anthranilate synthase (AS) (ASA2)...... 1705 Tryptophan (Trp) synthase beta 1 (TSB1) ...... 1706 Threonine deaminase (TD) ...... 1706 Cytosine deaminase (codA) ...... 1707 Xylose isomerase (xylA)...... 1707 Phosphomanose isomerase (PMI) (manA)...... 1707 Isopentyl transferase (IPT) (ipt) ...... 1708 Indole acetic acid (iaaM and iaaH) and hairy root inducing (rolABC) ...... 1708 D-Amino acid oxidase (DAAO) (dao1)...... 1708 Arabitol dehydrogenase...... 1709 D-Serine ammonia lyase (DSD) ...... 1709 Trehalose-6-phosphate synthase ...... 1709 Knotted1 (kn1) homeobox gene...... 1710 Future prospects and conclusion ...... 1710 Acknowledgments ...... 1711 References ...... 1711

Introduction generate substantial number of non-chimeric transformants, genes conferring resistance to selective The advent of recombinant DNA technology agents such as antibiotics or herbicides were widely provided an immense potential in the field of plant employed to select transformants. These select- transformation. In order to achieve high food able marker genes enable the transformed cells to productivity and to ensure nutritional quality, survive on medium containing the selective agent, genetic engineering methods in generating trans- while non-transformed cells and tissues die. The genics with useful agronomic traits are assuming low efficiencies of most plant transformation significance. Plant biotechnology is based on the methods necessitate the use of selectable marker delivery, integration and expression of defined genes to identify those cells that successfully genes into plant cells, which can be grown to integrate and express transferred DNA. Genes generate transformed plants. Efficiency of stable encoding resistance to specific antibiotics or gene transfer is not high even in the most herbicides have proved particularly effective for successful transfer systems and only a fraction of selection and provide a means of rapidly identifying the cells exposed integrate the DNA construct into transformed cells, tissues and regenerated shoots their genomes. Therefore, systems to select the that have integrated foreign DNA and that express transformed cells, tissues or organisms from the the selectable gene product and by inference, the non-transformed ones are indispensable and se- gene(s) of interest (Goodwin et al., 2004). Differ- lectable marker genes are vital to the plant ent selectable marker genes were used for trans- transformation process. From the development of genic and transplastomic plant research or crop first transgenic plants during early 1980s and development. These marker genes were assessed subsequently after its commercialization world- for efficiency, biosafety, scientific applications as wide over a decade, antibiotic and herbicide well as commercialization (Miki and McHugh, 2004). resistance selectable marker genes were one Selectable marker genes can be divided into among the integral feature of plant genetic several categories depending on whether they modification (Ramessar et al., 2007). Selectable confer positive or negative selection and whether marker genes are introduced into plant genome to selection is conditional or non-conditional in express a protein generally with an enzymatic the presence of external substrates. Positive activity, which allows distinguishing transformed selectable marker genes are defined as those that from non-transformed cells (Brasileiro and Dusi, allow the growth of transformed tissues, whereas 1999). In all plant transformation systems to negative selectable marker genes result in the ARTICLE IN PRESS

1700 I.K. Sundar, N. Sakthivel

death of the transformed tissue (Joersbo et al., tems. Positive selection systems are those that 1998; Miki and McHugh, 2004). Most commonly and allow the growth of transformed plant cells. Based recently developed selectable marker genes, selec- on their functionality the positive selection systems tion agents, enzymes, source of those genes and can be classified into conditional and non-conditional their importance and applications in plant trans- positive selection system. A conditional-positive formation have been discussed in this review. 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 Classical selectable marker genes growth as well as differentiation of the transformed cells alone. This system includes antibiotics, Most of the published scientific literature on herbicides (Table 1a), toxic and non-toxic drugs or transgenic crop plants revealed that antibiotics metabolite analog or a carbon source (Table 1b). (kanamycin or hygromycin) and herbicide (phosphi- Non-conditional-positive selection systems do not nothricin, PPT) was widely used as selection agent require external substrates but promote the selec- since the early days of plant transformation. The tive growth and differentiation of transformed popularity of these selection systems reflects on cells (Miki and McHugh, 2004). Almost all the the efficiency, availability and applicability of their antibiotic resistant selectable marker genes for use across a wide range of plant species and its use in transgenic plants have been identified from regenerative efficacy in plant tissue culture sys- bacterial sources.

Table 1

Selection agent Genes Enzymes Sources References

(a) Antibiotics and herbicides used as selection agent in plant transformation Antibiotics Neomycin, kanamycin neo nptII Neomycin Escherichia coli Tn5 Fraley et al. (1983) Bevan et al. (1983) Kanamycin (aphA2), Phosphotransferases Escherichia coli Tn601 Carrer et al. (1993) Atwbc19 ATP-binding cassette Arabidopsis thaliana Mentewab and Stewart (2005) Paramomycin, G418 nptI, (aphA1) Aminoglycosides aaC3 Aminoglycoside-N- Serratia marcesens Hayford et al. (1988) (kanamycin, neomycin, acetyltransferase geneticin, paramomycin, gentamycin, tobramycin, apramycin) aaC4 Klebsiella pneumoniae 6’gat Shigella sp. Gossele et al. (1994) Spectinomycin aadA Aminoglycoside-300-adenyl Shigella sp. Svab and Maliga (1993) transferase Streptomycin SPT Streptomycin Tn5 Maliga et al. (1988) phosphotransferase Hygromycin B hph, (aphIV) Hygromycin Escherichia coli Waldron et al. (1985) phosphotransferase Bleomycin Ble Bleomycin resistance Escherichia coli Tn5 Hille et al. (1986) Phleomycin Streptoalloteichus Perez et al. (1989) hindustanus Sulphonamides sulI Dihydropteroate synthase Escherichia coli pR46 Guerineau et al. (1990) Streptothricin sat3 Acetyl transferase Streptomyces sp. Jelenska et al. (2000) Chloramphenicol cat Chloramphenicol acetyl Escherichia coli Tn5, De Block et al. (1984, transferase Phage p1cm 1985) ARTICLE IN PRESS

Advances in selectable marker genes for plant transformation 1701

Table 1. (continued )

Selection agent Genes Enzymes Sources References

Herbicides Phosphinothricin pat, bar Phosphinothricin acetyl Streptomyces De Block et al. (1989) transferase hygroscopicus Methionine sulfoximine Phosphinothricin acetyl Chai et al. (2007) transferase Glyphosate EPSP 5-Enolpyruvylshikimate-3- Petunia hybrida, Zhou et al. 1995 synthase phosphate synthase Zea mays Howe et al. (2002) aroA Salmonella typhimurium Comai et al. (1988) Escherichia coli Della-Cioppa et al. (1987) cp4 epsps Agrobacterium Barry et al. (1992) tumefaciens gox Glyphosate Ochrobactrum anthropi Barry et al. (1992) oxidoreductase Sulfonylueras csr1-1 Acetolactate synthase Arabidopsis thaliana Olszewski et al. (1988) Imidazolinones csr1-2 Acetolactate synthase Arabidopsis thaliana Aragao et al. (2000) Oxynils bnx Bromoxynil nitrilase Klebsiella pneumoniae sub Freyssinet et al. (1996) sp. Ozanaenae Gabaculine hemL Glutamate-1- Synechococcus PCC6301 Gough et al. (2001) semialdehyde aminotransferase Cyanamide cah Cyanamide hydratase Myrothecium verrucaria Weeks et al. (2000) Acetochlor, chlorpropham P450 Cytochrome P450 Human Inui et al. (2005) amiprophos-methyl, chlorsulfuon, norflurazon and pendimethalin Norflurazon and fluridone pds Phytoene desaturase Hydrilla verticillata Arias et al. (2006) Trifluralin TUAm a-Tubulin Eleusine indica Yemets et al. (2008) Butafenacil Protoporphyrinogen Myxococcus xanthus Lee et al. (2007) oxidase (b) Toxic and non-toxic agents, drug and metabolite analogs used as selection agent in plant transformation Drugs and analogs 2-Deoxyglucose DOG 1 2-Deoxyglucose-6- Saccharomyces cerevisiae Kunze et al. (2001) phosphate phosphatase Betaine aldehyde BADH Betaine aldehyde Spinacia oleracea Daniell et al. (2001) dehydrogenase S-Aminoethyl DHPS Dihydropicolinate Escherichia coli Perl et al. (1993) synthase L-Cysteine (AEC) ocs Octopine synthase Agrobacterium Koziel et al. (1984) tumefaciens 4-Methyltryptophan (4- TDC Tryptophan decarboxylase Catharanthus roseus Goddijn et al. (1993) mT) Methotrexate DHFR Dihydrofolate reductase Escherichia coli, mouse, Herrera-Estrella et al. Candida albicans (1983) Eichholtz et al. (1987) Irdani et al. (1998) L-Methionine sulfoximine bar Phosphinothricin acetyl Streptomyces Chai et al. (2007) transferase hygroscopicus 5-Methyltryptophan ASA2 Anthranilate synthase Tobacco Cho et al. (2004) 5-Methyltryptophan (5MT) OASA1D Mutant anthranilate Rice Kobayashi et al. (2005) synthase 5MT/Cadmium cholride TSB1 Tryptophan synthase beta Arabidopsis thaliana Hsiao et al. (2007) L-O-Methylthreonine ilvA or ilvA- Threonine deaminase Escherichia coli Ebmeier et al. (2004) 466 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) codA Cytosine deaminase Escherichia coli Kobayashi et al. (1995) D-Xylose xylA Xylose isomerase Streptomyces rubignosus Haldrup et al. (1998b) Thermoanaerobacterium Haldrup et al. (1998b) sulfurogenes D-Mannose manA (pmi) Phosphomannose Escherichia coli Joersbo et al. (1998) isomerase Benzyladenine-N-3- uidA (gusA) b-Glucuronidase Escherichia coli Joersbo and Okkels glucuronide (1996) None ipt Isopentyl transferase Agrobacterium Endo et al. (2001) tumefaciens None iaaM, iaaH Indole acetic acid Agrobacterium Tuominen et al. (1995) tumefaciens None rolC ‘Hairy root’ phenotype Agrobacterium rhizogenes Ebinuma and Komamine, (2001) D-Amino acids (D-alanine dao1 D-Amino acid oxidase Rhodotorula gracilis Erikson et al. (2004) and D-serine) Arabitol atlD Arabitol dehydrogenase Escherichia coli strain C LaFayette et al. 2005 D-Serine dsdA D-Serine ammonia lyase Escherichia coli Erikson et al. (2005) Glucose AtTPS1 Trehalose-6-phosphate Arabidopsis thaliana Leyman et al. (2006) synthase Hormone-free medium kn1 Maize Luo et al. (2006)

Arabidopsis thaliana ATP-binding cassette well as forest tree species including angiosperms (ABC) transporter (e.g., polar and elms) and gymnosperms (e.g., pine and spruce). ABC proteins are ubiquitous proteins that share 30–40% identity between family members and are Neomycin phosphotransferase II (NPT II) characterized by the presence of an ABC (Higgins, (nptII) 1992). Arabidopsis thaliana ABC transporters have been classiﬁed on the basis of their domain The nptII (or neo) gene was isolated from the organization and their homology to orthologous transposon Tn5 of Escherichia coli and it encodes genes, but their functions remain largely unknown NPT II (E.C. 2.7.1.95), also known as aminoglyco- (Sanchez-Fernandez et al., 2001). The AtWBC side 30phosphotransferase II (Fraley et al., 1983; family (White-Brown Complex homologs) is the Herrera-Estrella et al., 1983). The active amino- largest group of all Arabidopsis thaliana ABC glycoside antibiotic inhibits the protein synthesis transporters, with 29 members. A plant gene, in prokaryote cells, by binding to the 30S subunit Atwbc19 (2178 bp) that encodes an Arabidopsis of the ribosome, blocking the formation of thaliana ABC transporter was characterized and it initiation complexes and decreasing the ﬁdelity of confers antibiotic (kanamycin) resistance to trans- translation. genic plants. The mechanism of resistance is novel, and the levels of resistance achieved are comparable to those attained through expression of Hygromycin phosphotransferase (HPT) (hpt) bacterial antibiotic-resistance genes in transgenic tobacco using CaMV 35S promoter. Because ABC The hpt (or aph IV) gene of Escherichia coli codes transporters are endogenous to plants, the use of for the enzyme HPT (E.C. 2.7.1.119) and confers Atwbc19 as a selectable marker in transgenic plants resistance to the antibiotic hygromycin B (Waldron may provide a practical alternative to current et al., 1985). When hygromycin occupies the bacterial marker genes in terms of the risk for ribosomal binding site of the elongation factor 2 horizontal transfer of resistance genes (Mentewab (EF-2) in prokaryotic cells, consequently the elon- and Stewart, 2005). Hence this marker gene may be gation of polypeptide chain is inhibited and protein valuable for transformation of agriculturally im- synthesis interrupted, causing the same symptoms portant dicot species such as soybean, cotton, described for the other aminoglycoside antibiotics. Brassica crops, tomato and other Solanaceae as In plant cells, these antibiotics exert its effect on ARTICLE IN PRESS

Advances in selectable marker genes for plant transformation 1703 mitochondria and chloroplast, acting in the same 5-Enolpyruvylshikimate-3-phosphate (EPSP) manner by impairing protein synthesis. These synthase (epsps) organelles have ribosomes that are similar to those found in bacteria and are also susceptible to The aroA (or epsps) gene was isolated from aminoglycoside antibiotics. Therefore, in the pre- Salmonella typhimurium treated with mutagenic sence of antibiotic, the plant tissue will show a agent and selected for resistance to the herbicide chlorosis, caused by the lack of chlorophyll synth- glyphosate. The herbicide glyphosate inhibits, by esis and inhibition of growth (Benveniste and competition, the enzyme EPSP synthase (E.C. Davies, 1973; Brasileiro, 1998). 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 Phosphinothricin N-acetyltransferase (PAT) in shikimate accumulation, inhibition of synthesis (pat and bar) of aromatic amino acids and secondary metabolites causing cell death. An excellent and comprehensive Since 1987, the bialaphos resistance genes bar review on many other selectable marker genes in from Streptomyces hygroscopicus and pat from transgenic crop plants has been published (Miki and Streptomyces viridochromogenes encoding the en- McHugh, 2004). zyme PAT have been used as selectable marker in plants (De Block et al., 1989). The PPT (ammonium Cyanamide hydratase (cah) glufosinate) is analogous to glutamate, the substrate of glutamate synthetase (GS), and acts as a The cah gene coding for the enzyme cyanamide competitive inhibitor of GS. The enzyme GS hydratase (urea hydrolase; E.C. 4.2.1.69) has been catalyzes the conversion of glutamate to gluta- isolated from the soil fungus Myrothecium verru- mine, removing the toxic ammonia from the cell. caria (Maier-Greiner et al., 1991a). Cyanamide This enzyme plays an essential role in the regula- hydratase catalyzes the hydration of the nitrile tion of nitrogen metabolism and ammonia assimila- group of cyanamide to form urea, which can be tion. When the GS is inhibited, it results in used for plant growth. Cyanamide is a nitrile ammonia accumulation and consequently disrup- derivative that in its aqueous or calcium salt forms tion of chloroplast that leads to inhibition of can be used as a fertilizer. It has the additional photosynthesis and to death of the plant cell characteristics of acting as a non-persistant herbi- (Lindsey, 1992). cide when applied prior to seed germination. The enzyme has extremely narrow substrate specificity. Methionine sulfoximine (MSO) The use of cyanamide hydratase as a selectable marker has been demonstrated in wheat (Weeks et al., 2000), tobacco (Maier-Greiner et al., 1991b; An alternative selection agent, L-MSO was used Kirubakaran and Sakthivel, 2008), potato, tomato, for selection of the bar marker, which also confers rice, Arabidopsis (Damm, 1998) and soybean (Zhang resistance to PPT. MSO like PPT is a glutamate et al., 2005; Ulanov and Widholm, 2007). In a analog that inhibits growth of dicotleydonous plant recent study, the heterologous expression of cah Arabidopsis due to its action on GS. Maughan and marker gene was reported. Due to its innate ability Cobbett (2003) demonstrated the utility of MSO as an effective and economical alternative to PPT for to convert calcium cyanamide to urea and the broad-spectrum antimicrobial activity of cyana- selection and regeneration of plants. MSO was mide, the cah gene can also be used to facilitate 40-fold more potent, inhibits growth of wild-type plant growth promotion and biocontrol of phyto- plants and detoxified in the similar manner as pathogens (Kirubakaran and Sakthivel, 2008). PPT by PPT acetyl transferase. In a recent study, MSO was employed as a novel selection agent for genetic transformation of orchids. The success- Cytochrome P450 monooxygenases ful application of MSO in orchid transformation opens up new avenues to study functional P450 monooxygenases are heme proteins that use orchid genes as well as genetic engineering of electrons from NADPH to catalyze the activation of orchids with commercially valuable traits. This molecular oxygen. The catalyzed reaction is usually readily available selection agent also has great a mono-oxygenation, with the formation of a potential for the transformation of economi- molecule of water and an oxygenated product. cally important monocotyledonous plant species Mammalian P450 species show overlapping and (Chai et al., 2007). broad substrate specificity and confer the ability ARTICLE IN PRESS

1704 I.K. Sundar, N. Sakthivel to metabolize a number of chemicals, including acid 304 of PDS in aquatic weed Hydrilla verticilla- herbicide. Most classes of herbicides are aryl- or ta have been reported to impart herbicide resis- alkyl-hydroxylated or N-, S-orO-dealkylated by tance (Michel et al., 2004). Based on the P450 species. The phenylurea herbicide chlortolur- aforementioned studies, Arias et al. (2005) has on is detoxified either via hydroxylation of the ring- reported for the first time the evolved resistance to methyl or via di-N-demethylation (Gonneau et al., PDS inhibitors in higher plants. Factors that may 1988). The introduction of highly active mammalian have contributed to this unique case include: the P450 into plant species metabolizes herbicide thus unusual growth habits, multiple means of vegeta- providing not only herbicide tolerance, but also tive reproduction of Hydrilla verticillata as well as acts as a selectable marker. Human P450 species the methods adopted to control aquatic environ- have been used to generate herbicide-tolerant ments using fluridone. Arias et al. (2006) identified tobacco, potato and rice plants (Inui et al., 1999, the potential use of this herbicide-resistant gene 2001) Interestingly, Kawahigashi et al. (2002) from higher plant as a selectable marker. The reported that the CYP2B6 gene could also be used amino acid 304 of Hydrilla verticillata PDS was as a negative selectable marker gene, due to its substituted with the other 19 amino acids and the ability to metabolize the herbicides benfuresate activity of the enzymes was tested in vitro against and ethofumesate to more toxic compounds. Hu- fluridone. Four of these mutations (Threonine – Thr, man P450 species are much more effective at Cysteine – Cys, Alanine – Ala and Glutamine – Gln), herbicide metabolism than plant P450 species in addition to the wild-type PDS were selected for (Ohkawa et al., 1998). In a study, the human P450 expression in Escherichia coli and Arabidopsis genes were employed for Arabidopsis thaliana by thaliana for further characterization. Results re- Agrobacterium-mediated transformation method. vealed that Arabidopsis plants transformed with Herbicide-tolerant seedlings transformed with Hydrilla pds containing mutations for Cys, Ser, His CYP1A1, CYP2B6, CYP2C9 or CYP2C19 were se- and Thr at position 304 showed increase in lected using acetochlor, amiprophos-methyl, chlor- resistance to fluridone and norflurazon. Hydrilla propham, chlorsulfuon, norflurazon and pendi- PDS with Thr304 was proposed as a selectable methalin. Results revealed that herbicide-tolerant marker based on two main advantages. Firstly, plants transformed with CYP1A1, CYP2B6 and consumers may better accept a genetically mod- CYP2C19 expressed the corresponding P450 cDNAs ified (GM) food if transformed with a plant gene. in transgenic Arabidopsis plants coordinately as Secondly, Hydrilla PDS Thr304 confers high resis- well as functioned as selectable marker because of tance to a limited spectrum of herbicides (flur- active metabolism of the herbicides (Inui et al., idone, norflurazon and flurtamone), whereas it 2005). renders the GM plants more susceptible than the wild-type to other PDS inhibitors, such as diflufe- nican, picolinafen and beflubutamid. These fea- Phytoene desaturase (PDS) tures could be beneficial to eliminate undesirable transgenic plants in the wild, rather than creating The enzyme PDS has been the main target for highly resistant weeds (Arias et al., 2006). herbicides that inhibit the carotenoid biosynthetic pathway. In plants, PDS converts phytoene to z-carotene. Carotenoids are essential components a-Tubulin gene of the photosynthetic apparatus. They participate in light harvesting and protect the chloroplast from To facilitate the development of a new genera- the harmful effect of singlet oxygen formed during tion of plant transformation systems, naturally photosynthesis (Sandmann and Bo¨ger, 1997). PDS- occurring variant of tubulin genes conferring anti- inhibiting herbicides prevent the formation of microtubule drug resistance may be appropriate carotenoids, resulting in the degradation of chlor- (Baird et al., 2000). Recently, a mutant a-tubulin ophyll and destruction of chloroplast membranes, gene from goosegrass that confers resistance to which is characterized by the photobleaching of dinitroaniline herbicide (trifluralin, TFL) was em- green tissues (Boger and Sandmann, 1998). Muta- ployed as selectable marker gene for transformations of the cyanobacterium Synechococcus PDS tion both in monocots and dicots (Yemets et al., have resulted in the herbicide-resistant microbial 2008). In goosegrass (Eleusine indica), two alleles enzymes (Chamovitz et al., 1991) and have of a-tubulin 1 (each is the result of a single unique conferred herbicide resistance when expressed in point mutation) have been identified, which con- transgenic tobacco plants (Wagner et al., 2002). fers either an intermediate or high level tolerance Recently, naturally occurring mutations at amino to a number of anti-microtubule herbicides such as ARTICLE IN PRESS

Advances in selectable marker genes for plant transformation 1705 dinitroanilines (trifluralin) and phosphoroamidates and Grimm, 2000), many attempts have been made (amiprophos-methyl, APM) (Nyporko et al., 2002). to develop transgenic crops resistant to peroxidiz- Yemets et al. (2008) tested the possibility to use ing herbicides through the ectopic expression of the mutant a-tubulin as a selective marker gene for native or mutant forms of PPO. An Arabidopsis transformation of finger millet, soybean, flax and double mutant of PPO and a Myxococcus xanthus tobacco using TFL as selective agent. The efficiency (Mx) PPO were shown to confer a high level of of transgenic plants selection using TFL was resistance to peroxidizing herbicides in transgenic comparable with those using kanamycin or PPT. maize and rice, respectively. Furthermore, the The anti-microtubule herbicides were used as Arabidopsis double mutant of PPO was successfully selective agents for effective and successful selec- used as a selectable marker gene, in combination tion of plant somatic hybrids after symmetric and with the herbicide butafenacil as a selection agent, asymmetric fusions using Nicotiana plumbaginifolia for generating transgenic crops (Li et al., 2003; mutants resistant to TFL or APM. These findings Jung et al., 2004). In a recent report, transgenic have thus furthered the evidence for the effective rice plants were developed using the Mx PPO gene use of TFL as a selection agent in plant transforma- as a selectable marker coupled with the herbicide tion systems. butafenacil as a selection agent. The overexpression of Mx PPO confers a high level of herbicide resistance in rice. Among the peroxidizing herbi- Protoporphyrinogen oxidase (PPO) cides, butafenacil (0.1 mM) has an efficiency 1000- fold that of oxadiazon, as judged by calli suscept- PPO is a key enzyme in the chlorophyll/heme ibility tests upon herbicide treatment (Lee et al., biosynthetic pathway, catalyzing the oxidation of 2007). protoporphyrinogen IX to protoporphyrin IX (Smith et al., 1993). This is the last common step in the production of heme and chlorophyll. Heme is an Anthranilate synthase (AS) (ASA2) essential cofactor in cytochromes, hemoglobin, oxygenases, peroxidases, and catalases, and, ASA2 gene was used as a selectable marker since therefore, is a necessary product for all aerobic it is naturally occurring gene of plant origin organisms. The production of chlorophyll, a light- (Nicotiana tabacum, tobacco) involved in trypto- harvesting pigment, is an essential process for all phan (Trp) biosynthesis (Song et al., 1998) that green photosynthetic organisms. This characteristic should have no detrimental effects when trans- makes PPO an excellent gene target for herbicide ferred to other plants or microbes. AS catalyses the development (Nandihalli and Duke, 1993). When first reaction in the multi-step Trp biosynthesis PPO is inhibited by the PPO family of herbicides, pathway by converting chorismate to anthranilate. protoporphyrin IX accumulates and causes light- AS is feedback inhibited by the end product Trp, dependent membrane damage (Lee et al., 1993). which binds to an allosteric site on the AS catalytic Peroxidizing herbicides target the enzyme PPO and a-subunit. AS is the control point in the Trp branch have been used commercially since 1960 to control in plant cells as indicated by pathway-intermediate annual grasses and dicotyleonous weeds in soybean, feeding studies (Widholm, 1974). Feedback inhibi- peanut, cotton, rice and other crops (Matringe tion of the respective enzyme activities (Singh and et al., 1989; Duke et al., 1991; Scalla and Matringe, Widholm, 1974) and 5-methyltryptophan (5MT) 1994). Diphenyl ethers, triazolinones, N-phenylpyr- resistance yielded selection of lines with altered azoles, oxadiazoles, thiadiazoles, pyrimidindiones, feedback-inhibited AS and higher free Trp (Wakasa N-phenyl-phthalimides and oxazolidinediones are and Widholm, 1987). Siehl et al. (1997) reported major structural families of herbicidal PPO inhibi- that AS is an effective herbicide target based on tors. As partial structural analogs of protopor- experimental evidence linking the herbicidal activ- phyrinogen IX, these herbicides bind to and ity of 6-methylanthranilate or 4-methyltryptophan competitively inhibit PPO activity (Nandihalli with inhibition of this enzyme. Cho et al. (2004) et al., 1992). New potent PPO inhibitors continue demonstrated that the tobacco feedback-insensi- to be developed and are reported to be effective tive ASA2 gene can be inserted into legume hairy peroxidizing herbicides (Hwang et al., 2004). The roots (Agrobacterium sinicus and soybean) and inhibition of PPO results in the massive production expressed to produce feedback-insensitive AS of singlet oxygen, which is followed by peroxidation activity, which leads to increase in free Trp levels of membrane lipids and subsequent cell death (Lee and resistance to Trp analog 5MT. Direct selection et al., 1993). Since the first report of a transgenic with 5MT produced Agrobacterium sinicus hairy tobacco expressing an Arabidopsis PPO (Lermontova root lines that all expressed ASA2 mRNA while nptII ARTICLE IN PRESS

1706 I.K. Sundar, N. Sakthivel expression was variable. The expression of ASA2 in presumably may allow detoxification of excess plant tissues leads to increased levels of free Trp, Cd2+. These observations imply that accumulated which could be desirable since Trp is an essential levels of Trp in AtTSB1 transgenic plants may amino acid required in the diets of humans and non- restrict the uptake of Cd2+ and other heavy metals ruminant animals. Since soybean hairy roots ex- or may affect the activity of metal transporters. pressing ASA2 selected with Kan were also resistant Therefore, the use of AtTSB1 gene as selection to 5MT, direct selection will be effective in this marker in agronomically important crop species case. Recently, rice OASA1D a mutant anthranilate transformation has generated transgenic plants synthase a subunit gene was used as selectable that may also be successfully cultivated in heavy marker for effective transformation in Arabidopsis metal polluted soils. thaliana (Kobayashi et al., 2005), rice and potato (Yamada et al., 2005). As demonstrated, ASA2 can be used as a selectable marker gene to select Threonine deaminase (TD) transformants in different plant species with the Trp analog 5MT as the selection agent. TD is one of the key enzymes downstream in the aspartate family biosynthetic pathway, involved in the first step of isoleucine (Ile) biosynthesis. TD Tryptophan (Trp) synthase beta 1 (TSB1) converts threonine to a-ketobutyrate, and itself is feedback-regulated by Ile. Inhibition of TD is Trp, one of the essential amino acids present in herbicidal to plant cells, and the herbicidal plants, is not synthesized by animals and is the activity of TD inhibition can be circumvented with major contributor of the indole ring for the the supplements of a-ketobutyrate and Ile itself synthesis of auxins, glucosinolates, nicotinic acid, (Szamosi et al., 1994), reflecting the depletion of phytoalexins and alkaloids (Pruitt and Last, 1993). Ile as the cause of cell death. An alternative Biosynthesis of Trp in plants is not constitutive and approach to mimic Ile depravation is the use of hence, it is produced when required, presumably structural analog of the amino acid that can under different stress conditions such as wounding. effectively compete during translation to induce Although the Trp biosynthetic pathway is primarily cell death. One such example is O-methylthreonine derived from bacteria and fungi, the real biochem- (OMT) (Rabinovitz et al., 1955). As part of large istry and mechanism of Trp synthesis and its mutant screening in Arabidopsis lines having regulation in plants became well understood only altered biosynthesis of branched-chain amino after the innovation of Arabidopsis mutants trp1-1, acids, Mourad and King (1995) characterized a set trp2-1 (Last et al., 1991; Pruitt and Last, 1993), of mutants resistant to OMT. The ability of these yucca (Zhao et al., 2001) and yeast cDNA screening mutant lines to develop on OMT was attributed to a (Hull et al., 2000). Two genes, Arabidopsis thaliana single nuclear gene omr1, a mutant allele of TD, tryptophan synthase beta 1 (AtTSB 1) and AtTSB2, which was mapped to chromosome 3. The OMT- encode the Trp beta subunit in Arabidopsis. Even tolerant Arabidopsis lines possessed substantially though both are highly conserved, AtTSB1 mRNA enhanced intracellular levels of Ile compared with transcript is more abundant than that of AtTSB2in wild-type plants. Moreover, there was a 50-fold leaf tissues (Pruitt and Last, 1993). Hence, the increase in the Ile feedback insensitivity of TD AtTSB1 gene in plants plays a key role in the Trp activity in the OMT-tolerant lines compared with biosynthesis pathway in converting indole and that in wild-type. The omr1 allele of Arabidopsis serine into Trp (Last et al., 1991). Recently, Hsiao thaliana may serve as an effective plant selectable et al. (2007) explored the practical applicability of marker gene, coupled with OMT, for use in plant the AtTSB1 gene as a novel non-antibiotic selection genetic engineering studies (Mourad and King, marker in plant transformation using a simple, 1995). Slater et al. (1999) introduced into Arabi- efficient, 5MT or heavy metal-resistant selection dopsis thaliana and Brassica napus with the procedure. Transgenic plants were efficiently se- Escherichia coli wild-type TD gene, ilvA,ora lected on MS medium supplemented with 75 mM feedback-insensitive mutant ilvA-466 transformant 5MT or 300 mM CdCl2 devoid of antibiotics. Engi- expressing either of these transgenes displayed neering the AtTSB1 gene to overexpress and elevated intracellular levels of Ile. Ebmeier et al. accumulate Trp for its greater availability and (2004) designed two binary plasmids that harbored multi-flux distribution is an important step in an nptII cassette and either the wild-type ilvA or metabolomics. Results demonstrate that AtTSB1 mutant ilvA-466. The ilvA gene was effectively overexpression in Arabidopsis increases Trp content utilized as a selectable marker gene to identify and enhances Cd2+ tolerance. The high level of Trp tobacco transformants when coupled with OMT as ARTICLE IN PRESS

Advances in selectable marker genes for plant transformation 1707 the selection agent. However, the transformation which can then be used as carbon source. The efficiency was substantially lower than that ob- efficiency of selection in this case was much served with nptII using kanamycin as a selection greater than for the nptII gene and the regenera- agent. Moreover, in the subset of the ilvA transfor- tion of shoots was significantly faster. It was mants and in majority of ilvA-466 transgenic lines, suggested that the enzyme from Streptomyces a severe off-type was observed under greenhouse rubiginosus posed no biosafety issues as it is used conditions that correlated with increased level in the food industry and is considered safe (Haldrup expression of the ilvA transgene. et al., 1998a).

Cytosine deaminase (codA) Phosphomanose isomerase (PMI) (manA) codA enzyme in bacteria catalyzes the conversion of cytosine to uracil. This enzyme is also The manA gene codes for the enzyme PMI (E.C. capable of converting non-toxic 5-fluorocytosine 5.3.1.8) isolated from Escherichia coli (Miles and (5-FC) to 5-fluorouracil (5-FU), a toxic compound Guest, 1984). In the presence of mannose in for plant growth (Kobayashi et al., 1995). In plants, transformed cells, the PMI converts mannose- a chimeric codA gene under the control of CaMV 35S 6-phosphate into fructose-6-phosphate that can be promoter was introduced into tobacco and Lotus immediately incorporated in the plant metabolic japonicus. The results revealed that the codA pathway (Privalle et al., 1999). Thus, the mannose marker gene is useful for substrate-dependent can be used as sole source of carbohydrate for the negative selection and segregates as a dominant transformed cells. This selection system is immedi- marker. The lack of cytosine deaminase activity in ate and extremely efficient (Joersbo et al., 1998). many plants species including Arabidopsis, pea, Mannose cannot be usually metabolized by non- barley, soyabean and sugar beet indicates the fact transformed cells and is converted into mannose- that this gene can be employed widely as negative 6-phosphate by endogenous hexokinase. Therefore, selective marker (Stougaard, 1993; Koprek et al., when mannose is added to the culture medium, it 1999). Perera et al. (1993) also reported that codA could minimize the plant growth due to mannose- gene works as a negative selectable marker in 6-phosphate accumulation. Even though most of Arabidopsis. The effectiveness of the negative the plant species are sensitive to mannose, some selection with the codA gene, however, would be species, especially dicotyledonous have shown a dependent on the permeability and transport of the considerable insensibility to this sugar, including substrate 5-FC in plant tissues and on the intensity carrot, tobacco, sweet potato and leguminous of expression, tissue specificity and temporal crops. Other species are extremely sensitive and specificity of the promoter fused to the codA gene. were successfully transformed using mannose as Since CaMV 35 S promoter is sufficiently active in selective agent such as sugar beet (Joersbo et al., root cells (Benfey et al., 1989) and the root tissues 1998), maize (Negrotto et al., 2000; Wang et al., can be in contact directly with the substrate in 2000), wheat (Wright et al., 2001), rice (Lucca vitro, the P35S should be a suitable promoter in the et al., 2001), sweet orange (Boscariol et al., 2003), codA negative selection system. Therefore, cyto- oat, barley, tomato, potato, sunflower, oilseed rape sine deaminase gene from Escherichia coli is and pea (Joersbo et al., 1999, 2000). The support- functional and useful for negative selection in ing principle in this approach is the inability of Arabidopsis and sensitivity to 5-FC, which makes some plants to use mannose as carbon source. The codA gene a suitable conditional negative select- major difference from selection based on antibio- able marker gene in plants (Kobayashi et al., 1995). tics or herbicides, which kill the non-transformed cells, whereas the mannose selection system Xylose isomerase (xylA) arrests the growth and development of non- transformed cells by carbohydrate starvation (Wang The xylA genes were isolated from Streptomyces et al. 2000) but still survive (Haldrup et al., rubiginosus (Haldrup et al., 1998a) and Thermo- 1998a, b). Owing to this growth advantage of anaerobacterium thermosulfurogenes (Haldrup transformed cells, this strategy is called ‘positive et al., 1998b). Plant cells from species such as selection’ (Joersbo and Okkels, 1996). Some plant tobacco, potato and tomato cannot use D-xylose transformation protocols that utilize the positive as a sole carbon source. This enzyme xylose selection system with PMI have shown at least 10 isomerase (D-xylose ketol-isomerase; E.C. 5.3.1.5) times more efficiency than the traditional protocols catalyzes the isomerization of xylose to D-xylulose, based on the use of kanamycin as selection agent. ARTICLE IN PRESS

1708 I.K. Sundar, N. Sakthivel

Isopentyl transferase (IPT) (ipt) these changes seem undesirable for the improve- ment of wood quality, other changes, such as a The enzyme IPT, which is encoded by the T-DNA reduction in the number of side shoots following of Agrobacterium tumefaciens Ti plasmids, con- decapitation and changes in the xylem structure tributes to crown gall formation in infected plants. and composition show some potential for trans- This enzyme catalyzes the synthesis of isopentyl- genic trees expressing these genes. ademosine-50-monophosphate, which is the first The rolC gene from Agrobacterium rhizogenes step in cytokinin biosynthesis. Studies using the has also been shown to alter the growth and ipt gene under the control of constitutive promo- development of transgenic forest trees. Overex- ters showed that ipt overexpression leads to pression of this gene leads to dwarfing, breaking of elevated cytokinin levels in transgenic plants apical dominance, reduced growth rate and de- (Medford et al., 1989) resulting in poor internode creased stem-internode length in transgenic hybrid elongation, altered leaf morphology and delayed aspen trees (Nilsson et al., 1996). It is hard to leaf senescence. Moreover, in most of the trans- predict the specific silvicultural advantages of the genic regenerants, root growth is delayed or absent rolC gene in woody plants from their phenotypic and the plants are often sterile (Smigocki and appearance, but transgenic aspen plants expressing Owens, 1989; Ebinuma et al., 1997). The selection the rolABC native fragment from Agrobacterium system using ipt gene is a positive selection that rhizogenes increased the growth rate in vitro,as does not stress the transformants with a low-level well as giving improved rooting characteristics and expression of transgene. Endo et al. (2001) sug- stem:root production index (Tzfira et al., 1997). gested that overexpression of ipt gene from These unique characteristics can be related to the Agrobacterium tumefaciens favors plant regenera- combination of several rol genes and their specific, tion from transformed cells and the transformation low expression pattern, as they are driven by their frequency of the ipt plus kanamycin selection native promoters. It should be noted that undesir- resulted in 1.6-fold higher transformation effi- able alterations, such as reduced apical dominance ciency than kanamycin selection alone. The reason and the breaking of axillary shoot buds, were for the increase in transformation frequency was also observed. Agrobacterium rhizogenes-mediated the increase in endogenous hormone with the transformation generates plants with altered mor- introduction of ipt gene. Endogenous zeatin and phology (i.e. the hairy root phenotype) and the zeatin riboside concentrations in transformed cells responsible rol genes have been used in certain increased 100-fold compared with non-transformed plant transformation vectors as a selectable mar- cells (Smigocki and Owens, 1989). Several research- ker. In general, this selection system is not ers have observed that ipt gene induces abnormal extensively used except to monitor the transposi- shoots and cell proliferation in the medium without tion or excision or marker genes in the develop- a supplementary hormone in potato (Ooms et al., ment of marker-free transformation technologies 1983), cucumber (Smigocki and Owens, 1988) and (Ebinuma and Komamine, 2001). other Nicotiana species (Smigocki and Owens, 1989). These physiological abnormalities prevent the development of ipt as a selectable transforma- D-Amino acid oxidase (DAAO) (dao1) tion marker. D-Amino acids are naturally present in many higher plant species including the dicots and monocots Indole acetic acid (iaaM and iaaH) and hairy (Bru¨ckner and Hausch, 1989). The marker gene dao1 root inducing (rolABC) encoding DAAO (E.C. 1.4.3.3) was used as a conditional marker in Arabidopsis thaliana and proven to be These genes have been extensively employed in versatile allowing either positive or negative selection forest tree biotechnology for genetic transforma- depending on the substrate (Erikson et al., 2004). tion of tree species, including the possible applica- DAAO catalyzes the oxidative deamination of a range tions for improving and introducing novel traits into of D-amino acids (Alonso et al. 1998). Selection is forest tree-species. The use of the iaaM and iaaH based on the toxicity of different D-amino acids auxin-biosynthetic genes from Agrobacterium tu- and their metabolites to plants. Thus, D-alanine and mefaciens significantly affected several wood char- D-serine are toxic to plants, but are metabolized by acteristics in transgenic hybrid aspen (Tuominen DAAO into non-toxic products, whereas D-isoleucine et al., 1995). The transgenic trees were generally and D-valine have low toxicity, but are metabolized smaller than the controls, exhibiting reduced to toxic keto acids 3-methyl-2-oxopentanoate and growth rate, leaf size and stem diameter. Although 3-methyl-2oxobutanoate, respectively. The toxicity of ARTICLE IN PRESS

Advances in selectable marker genes for plant transformation 1709 some D-amino acids on organisms is not well known and corresponding keto acids, ammonium and water. is very rarely studied in plants (Gamburg and D-Serine is toxic to Escherichia coli strains lacking Rekoslavskaya, 1991). Erikson et al. (2004) apart from dsdA as a result of the inhibition of L-serine and Arabidopsis thaliana, they have also tested the pantothenate synthesis, and the recognized func- susceptibility D-serine in other plant species (tobacco, tion of DSD is detoxification of D-serine, which also barley,maize,tomatoandspruce)andfoundsuscep- enables the bacteria to use D-serine as a carbon and tible at millimolar range similar to that shown to be nitrogen source (Cosloy and McFall, 1973). Further- toxic for Arabidopsis thaliana. Therefore, this new more, the dsdA gene has proven useful as a plant selectable marker gene using the genes involved selectable marker in transformation of Escherichia in D-amino acid metabolism of plants can be widely coli, by using strains naturally lacking DSD activity employed both as positive and negative selection in (Maas et al., 1995). Plants are sensitive to D-serine, plants. but functional expression of the dsdA gene, encoding D-serine ammonia lyase from Escherichia coli can alleviate this toxicity. Plants in contrast to Arabitol dehydrogenase many other organisms lack the common pathway for oxidative deamination of D-amino acids. This Different Escherichia coli strains can use a difference in metabolism has major consequence multitude of carbohydrate sources, due to a series for plant responses to D-amino acids, since several of operons that are located within the Escherichia D-amino acids are toxic to plants even at relatively coli genome (Reiner, 1975). One of these, in low concentrations. In a study, Erikson et al. (2005) particular is the ability of Escherichia coli strain C, has explored the use of dsdA gene encoding D-serine but not the laboratory K12 strains to grow on ammonia lyase from Escherichia coli as a selectable D-arabitol (Scangos and Reiner, 1978), Since most marker in the model plant Arabidopsis thaliana. plants cannot metabolize most sugar alcohols, D-Serine ammonia lyase catalyzes the deamination of including D-arabitol (Stein et al., 1997), there is D-serine into pyruvate, water and ammonium. dsdA an opportunity to develop positive selection system transgenic seedlings can be clearly distinguished based on sugar alcohols. In Escherichia coli strain C, from wild type, having an unambiguous phenotype the arabitol genes are located in the atl operon and immediately following germination when selected include atlT, atlD, atlK and atlR(Reiner, 1975). on D-serine containing medium. The dsdA marker AtlD encodes D-arabitol dehydrogenase (EC allows flexibility in application of selective agent, 1.1.1.11), which converts arabitol into xylulose. it can be applied in sterile plates, as foliar sprays or Xylulose is an intermediate of the oxidative in liquid culture. pentose phosphate pathway (Kruger and Von Schaewen, 2003). Plants can grow on D-xylulose (Haldrup et al., 1998b), so if a plant cell could Trehalose-6-phosphate synthase express arabitol dehydrogenase, then such a cell would be able to grow in a medium containing Trehalose is non-reducing disaccharide that is D-arabitol, whereas an untransformed plant cell abundantly present as stress protectant in micro- would not proliferate. LaFayette et al. (2005) used organisms and some desert plants such as Selagi- Escherichia coli arabitol dehydrogenase gene as a nella lepidophilla. In higher plants such as plant selectable marker in rice crop. The enzyme Arabidopsis thaliana, trehalose is only detected in converts the non-plant-metabolizable sugar arabi- small quantities (Vogel et al., 2001). In yeast tol into xylose, which is metabolized by plant cells. Saccharomyces cerevisiae, trehalose is synthesized Selection on 2.75% arabitol and 0.25% sucrose from UDP-glucose and glucose-6-phosphate by yielded a transformation efficiency (9.3%) equal trehalose-6-phosphate (T6P) synthase (Tps1) and to that obtained with hygromycin (9.2%). Thus, T6P phosphatase (Tps2). Deletion of TPS1 elimi- arabitol could serve as an effective means for plant nates growth on glucose because both Tps1 and T6P selection. are indispensable for the regulation of glucose influx into glycolysis (Thevelein and Hohmann, D-Serine ammonia lyase (DSD) 1995). The Arabidopsis genome encodes 11 TPS1 homologs that fall into 2 subfamilies displaying DSD (EC 4.2.1.14, formerly D-serine dehydra- most similarity either to yeast TPS1 (Class I, tase), encoded by the dsdA gene from Escherichia AtTPS1-4)orTPS2 (Class II, AtTPS5-11)(Leyman coli is a pyridoxal 50-phosphate-requiring enzyme, et al., 2001). AtTPS1 complements the yeast tps1 which catalyzes the deamination of the D-enantio- strain for its growth defect on glucose, which mers of serine, threonine and allothreonine to the suggests a possible regulatory role for AtTPS1 in ARTICLE IN PRESS

1710 I.K. Sundar, N. Sakthivel plant carbon metabolism (Van Dijck et al., 2002). transformation efficiency. Based on the results, kn1 Sugar homeostasis is tightly regulated in plants; gene could be used as an effective alternative plantlets germinated in the presence of high selection marker with a potential to enhance plant glucose concentrations remain white and petite transformation efficiency in many plant species. because the external sugar switches off the With kn1 gene as a selection marker, no antibiotic- photosynthetic machinery. Previous report states resistance or herbicide-resistance genes are that AtTPS1 plays a regulatory role in this phenom- needed, so that potential risks associated with enon as ectopic expression of AtTPS1 in Arabidopsis the use of these traditional selection marker genes rendering plants significantly less sensitive to can be eliminated (Luo et al., 2006). glucose compared with wild-type plants (Avonce et al., 2004). Based on this trait, researches started to exploit AtTPS1 as a selection marker for plant transformation in combination with glucose as Future prospects and conclusion selection agent. In order to develop a purely plant-based and environment-friendly selection Plant transformation technology offers an array system Leyman et al. (2006) explored the useful- of opportunities for basic scientific research and for ness of the trehalose-6-phosphate synthase gene genetic modification of crops. The generation of AtTPS1. This novel selection system was based on transgenic plants requires the use of different enhancing the expression of plant intrinsic gene selectable marker genes that are introduced using a harmless selection agent. Overexpression of together with the exogenous gene of interest. AtTPS1 in tobacco allows selecting glucose-insensi- Recent progress in plant molecular biology and tive transgenic shoots. Selection takes advantage genome research has lead to a desire to introduce of the reduced glucose sensitivity of seedlings with several genes into a single transgenic plant, enhanced expression of AtTPS1. As a result, necessitating the importance of various types of transformants can be identified as developing green selectable marker genes. Among the selectable seedlings amongst the background of small, pale marker genes applied to plant transformation, the non-transformed plantlets on high glucose medium. most popular markers are bacterial genes that confer resistance to the antibiotics (kanamycin and hygromycin) or to the herbicide (glufosinate am- Knotted1 (kn1) homeobox gene monium). As these have been the target of concern among environmental authorities, scientists have The maize homeobox gene kn1 and its homologs been encouraged to develop alternative selection from other plant species are expressed normally in strategies (ACNFP, 1994), although such concerns shoot meristems (Smith et al., 1992; Jackson et al., may prove unfounded (Flavell et al., 1992). 1994) and are essential for meristem initiation and Development of environment-friendly marker- maintenance (Long et al., 1996). Transgenic plants assisted selection system involving natural plant overexpressing kn1 gene exhibit morphological materials is gaining momentum. Novel selection alterations, including changes in leaf shape, loss procedures for orchid transformation have been of apical dominance and production of ectopic developed by introducing a native plant gene, meristems on leaves (Tamaoki et al., 1997). sweet pepper ferredoxin-like protein (pflp), as a Homeobox gene knotted1 (kn1) from maize was selection marker into the Oncidium genome via used as a selectable marker gene for plant Agrobacterium-mediated and particle bombard- transformation in Nicotiana tabacum cv. Xanthi ment. Putatively transformed plants were screened via Agrobacterium-mediated transformation. Un- by use of the natural bacterial pathogen Erwinia der non-selective conditions (without antibiotic carotovora as a selection agent (You et al., 2003; selection) on a hormone-free Murashige Skoog Chan et al., 2005). In the recent years, use of (MS) medium, a large number of transgenic calli conditional-positive selection system using the non- and shoots were obtained from explants that were toxic metabolic intermediates such as xylose and infected with Agrobacterium tumefaciens LBA 4404 mannose has gained importance due to its ease and harboring the 35SHkn1 gene. On the other hand, no scientific merit. Lai et al. (2007) demonstrated the calli or shoots were produced from explants that use of D-amino acids using dsdA (D-serine dehydra- were infected with an Agrobacterium strain har- tase) gene from Escherichia coli and the dao1 boring pBI121 (nptII selection) or from uninfected (D-amino acid oxidase) gene from Rhodotorula controls cultured under identical conditions. Re- gracilis as a new set of effective selectable markers lative to kanamycin selection conferred by nptII, for maize transformation. Utility of selectable the use of kn1 resulted in a 3-fold increase in markers in crop plants fulfils the demand for ARTICLE IN PRESS

Advances in selectable marker genes for plant transformation 1711 alternative selection markers in the development Baird V, Blume YaB, Wick SM. Microtubular and cytoske- of genetically modified organisms. Earlier studies letal mutants. In: Nick P, editor. Plant microtubules – have supported the safety of the use of marker potential targets for biotechnological applications. gene systems in plant transformation (Ramessar Frankfurt-Berlin: Springer; 2000. p. 155–87. et al. 2007). Barry G, Kishore G, Padgette S, Talor M, Kolacz K, Weldon In summary, selectable marker genes play a vital M, et al. Inhibitors of amino acid biosynthesis: role to stimulate regeneration of transformed plant strategies for imparting glyphosate tolerance to plants. In: Singh BK, Flores HE, Shannon JC, editors. cells, thus supporting transgenic research of future Biosynthesis and molecular regulation of amino acids plant biotechnologists and geneticists. Plant trans- in plants. American Society of Plant Physiology; 1992. formation works in monocot and dicot crops have p. 139–45. proved that a wide range of selectable marker Benfey PN, Ren L, Chua N-H. The CaMV 35S enhancer genes can be employed for successful genetic contains at least two domains which can confer transformations. Selection systems do not pose different developmental and tissue-specific expres- any threat to mankind as evidenced by several sion patterns. EMBO J 1989;8:2195–202. experimental reports, thus leading to accelerated Benveniste R, Davies J. Mechanisms of antibiotic resis- research towards the identification of new and tance in bacteria. Annu Rev Biochem 1973;42: novel marker gene systems from different sources. 471–506. Bevan MW, Flavell RB, Chilton M-D. A chimaeric antibiotic resistance gene as a selectable marker for plant cell transformation. Nature 1983;304:184–7. Acknowledgments Boger P, Sandmann G. Carotenoid biosynthesis inhibitor herbicides – mode of action and resistance mechan- The authors thank the Department of Science and isms. Pesticide Outlook 1998;9:29–35. Technology (DST), Government of India for the Boscariol RL, Almeida WA, Derbyshire MT, Mouraõ Filho FA, financial support through a research project Mendes BM. The use of the PMI/mannose selection awarded to N. Sakthivel. system to recover transgenic sweet orange plants (Citrus sinensis L. Osbeck). Plant Cell Rep 2003;22:122–8. Brasileiro ACM. Neomicina Fosfotransferase II (NPT II). In: Brasileiro ACM, Carneiro VTC, editors. Manual de References transformac-aõ gene´tica de plantas. Brası´lia, Brazil: Embrapa-SPI/Embrapa-Cenargen; 1998. p. 143–54. Brasileiro ACM, Dusi DMA. Transformac-aõ gene´tica de ACNFP. Report on the use of antibiotic resistance markers plantas. In: Torres AC, Caldas LS, Buso JA, editors. in genetically modified food organisms. 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Plant Biotechnol J 2006;4:263–73. (ASA2) as a selectable marker for legume hairy root Avonce N, Leyman B, Mascorro-Gallardo JO, Van Dijck P, transformation. Plant Cell Rep 2004;23:104–13. Thevelein JM, Iturriaga G. The Arabidopsis trehalose- Comai L, Larson-Kelly N, Kiser J, Mau CJD, Pokalsky AR, 6-p synthase AtTPS1 gene is a regulator of glucose, Shewmaker CK, et al. Chloroplast transport of abscisic acid, and stress signaling. Plant Physiol a ribulose bisphosphate carboxylase small subunit- 2004;136:3649–59. 5-enolpyruvyl 3-phosphoshikimate synthase chimeric ARTICLE IN PRESS

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