Cross-protection and selectable marker genes in plant transformation

Summary Selectable marker genes play an important role in plant transformation. The level of selection pressure is generally established by generating a kill curve for the selectable marker. In most cases, the lowest concentration which kills all explants is used. This study examined two selectable marker genes, phosphinothricin acetyl transferase (PAT) and hygromycin phosphotransferase (HPT), in transformation of tobacco leaf disks. Experiments to determine the lethal level of the herbicide, glufosinate-ammonium (phosphinothricin) (PPT) using a leaf-disk regeneration assay established that no shoots regenerated at 2 to 4 mg PPT per 1. Likewise with the antibiotic, hygromycin (HYG), no plants regenerated at 50 mg hygromycin per 1. In contrast, after cocultivation of the leaf disks withAgrobacterium tumefaciens containing either the PAT or HPT gene in combination with a Bt gene for insect resistance, plants were successfully regenerated from leaf disks at 2 to 4 mg PPT per 1 and 50 mg hygromycin per 1. However, most plants regenerated at 2 and 3 mg PPT per 1 were found to be nontransformed (95–100% escapes) by i) Southern-blot analysis, ii) herbicide application test, and iii) insect feeding bioassay. On the other hand, plants that regenerated on 50 mg hygromycin per 1 and 4 mg PPT per 1 were transgenic as determined by Southern analysis, leaf assay for PPT or HYG resistance, and death of tobacco budworms feeding on these leaves. This study showed a significant level of cross-protection and/or transient expression of the PAT selectable marker gene allowing escapes (95–100%) at selection levels of 2 and 3 mg PPT per 1 which completely kill controls. On the other hand, the HPT gene at 50 mg is efficient in selecting for T-DNA integration.     

Advances in selectable marker genes for plant transformation

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.     

Positive selectable marker genes for routine plant transformation

Summary Plant genetic transformation technologies rely upon the selection and recovery of transformed cells. Selectable marker genes used so far have been either antibiotic resistance genes or herbicide tolerance genes. There is a need to apply alternative principles of selection, as more transgenic traits have to be incorporated into a transgenic crop and because of concern that the use of conventional marker genes may pose a threat to humans and the environment. New classes of marker genes are now available, conferring metabolic advantage of the transgenic cells over the non-transformed cells. The new selection systems, as described in this review, are being used with success and superior performance over the traditional marker systems.     

Glyphosate-tolerant CP4 and GOX genes as a selectable marker in wheat transformation

The lack of alternative selectable markers in crop transformation has been a substantial barrier for commercial application of agricultural biotechnology. We have developed an efficient selection system for wheat transformation using glyphosate-tolerant CP4 and GOX genes as a selectable marker. Immature embryos of the wheat cultivar Bobwhite were bombarded with two separate plasmids harboring the CP4/GOX and GUS genes. After a 1 week delay, the bombarded embryos were transferred to a selection medium containing 2 mM glyphosate. Embryo-derived calli were subcultured onto the same selection medium every 3 weeks consecutively for 9–12 weeks, and were then regenerated and rooted on selection media with lower glyphosate concentrations. Transgenic plants tolerant to glyphosate were recovered. ELISA assay confirmed expression of the CP4 and GOX genes in R₀ plants. Southern blot analysis demonstrated that the transgenes were integrated into the wheat genomes and transmitted to the following generation. The use of CP4 and GOX genes as a selectable marker provides an efficient, effective, and alternative transformation selection system for wheat.     

High molecular weight glutenin subunit in durum wheat (T. durum)

Summary The diversity of high molecular weight (HMW) glutenin subunits of 502 varieties of durum wheat (Triticum durum) from 23 countries was studied using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Twenty-nine types of patterns were observed with 18 mobility bands. A total of 18 alleles were identified by comparing the mobilities of their subunits to those previously found in hexaploid wheat (T. aestivum) and in Triticum turgidum var. dicoccum. Five new alleles were detected: two on the Glu A1 and three on the Glu B1 locus. Comparison of the frequency of alleles in the three species T. aestivum, T. dicoccum and T. durum was investigated. Significant differences exist between each of these species on the basis of the frequency distributions of their three and four common alleles at the Glu A1 and Glu B1 locus, respectively. The Glu B1c allele occuring very frequently in hexaploid wheats was not found in the two tetraploid species. More than 83% of the T. durum analysed were found to have the Glu A1c (null) allele.     

2-Deoxyglucose resistance: a novel selection marker for plant transformation

A novel selection marker for plant transformation alternative to antibiotic and herbicide resistance is described. The selective agent applied is 2-deoxyglucose (2-DOG) which in the cytosol of plant cells is phosphorylated by hexokinase yielding 2-DOG-6-phosphate (2-DOG-6-P). 2-DOG-6-P exerts toxic effects on overall cellular metabolism leading to cell death. We observed that constitutive expression of the yeast DOG ^R1 gene encoding a 2-DOG-6-P phosphatase resulted in resistance towards 2-DOG in transgenic tobacco plants. This finding was exploited to develop a selection system during transformation of tobacco and potato plants. The lowest concentration of 2-DOG leading to nearly complete inhibition of regeneration of wild-type explants was found to range between 400 and 600 mg/l 2-DOG for tobacco, potato and tomato plants. After Agrobacterium tumefaciens-mediated transformation cells expressing the DOG ^R1 gene were selected by resistance to 2-DOG. More than 50% of tobacco explants formed shoots and on average 50% of these shoots harboured the DOG ^R1 gene. Similar results were obtained for potato cv. Solara. The acceptability of the resistance gene derived from baker's yeast, the unobjectionable toxicological data of 2-DOG as well as the normal phenotype of DOG ^R1-expressing plants support the use of this selection system in crop plant transformation.     

Development of a nuclear transformation system with a codon-optimized selection marker and reporter genes in Pyropia yezoensis (Rhodophyta)

Genetic transformation systems using reporter genes in whole plants have a wide variety of applications for molecular biological study including the visualization of expression patterns of particular genes and intracellular biological phenomena as well as the identification of novel genes. In this study, we assessed co-expression of each three codon-optimized reporter genes and a selectable marker in the nuclear transformation system of whole Pyropia yezoensis, a red marine alga. With the use of an endogenous promoter, both the codon-optimized hygromycin resistance gene and ß-glucuronidase gene (PyGUS) were co-expressed in P. yezoensis cells. A high level of GUS activity was observed in 60 % of the individuals in hygromycin-resistant lines. A histochemical GUS assay revealed that the PyGUS reporter gene was stably introduced and expressed throughout the algae's life cycle. In addition, two live cell reporters, humanized cyan fluorescent protein from Anemonia majano and luciferase from Gaussia princeps, were successfully expressed in whole P. yezoensis. The development of this transformation system involving three types of reporter genes provides opportunities for monitoring temporal changes in gene expression and for genetic screening in red marine algae.     

Trehalose-6-phosphate synthase as an intrinsic selection marker for plant transformation

Insertion of foreign DNA into plant genomes occurs randomly and with low frequency. Hence, a selectable marker is generally required to identify transgenic plants. Until now, all selection systems have been based on the use of non-plant genes, derived from microorganisms and usually conferring antibiotic or herbicide resistance. The use of microorganism-derived genes however has raised biosafety concerns. We have developed a novel selection system based on enhancing the expression of a plant-intrinsic gene and the use of a harmless selection agent. Selection takes advantage of the reduced glucose sensitivity of seedlings with enhanced expression of AtTPS1, a gene encoding trehalose-6-P synthase. As a result, transformants can be identified as developing green seedlings amongst the background of small, pale non-transformed plantlets on high glucose medium. In addition, vegetative regeneration of tobacco leaf explants is very sensitive to high external glucose. Overexpression of AtTPS1 in tobacco allows selecting glucose insensitive transgenic shoots.     

Genomic selection for grain yield and quality traits in durum wheat

The prediction accuracies of genomic selection depend on several factors, including the genetic architecture of target traits, the number of traits considered at a given time, and the statistical models. Here, we assessed the potential of single-trait (ST) and multi-trait (MT) genomic prediction models for durum wheat on yield and quality traits using a breeding panel (BP) of 170 varieties and advanced breeding lines, and a doubled-haploid (DH) population of 154 lines. The two populations were genotyped with the Infinium iSelect 90K SNP assay and phenotyped for various traits. Six ST-GS models (RR-BLUP, G-BLUP, BayesA, BayesB, Bayesian LASSO, and RKHS) and three MT prediction approaches (MT-BayesA, MT-Matrix, and MT-SI approaches which use economic selection index as a trait value) were applied for predicting yield, protein content, gluten index, and alveograph measures. The ST prediction accuracies ranged from 0.5 to 0.8 for the various traits and models and revealed comparable prediction accuracies for most of the traits in both populations, except BayesA and BayesB, which better predicted gluten index, tenacity, and strength in the DH population. The MT-GS models were more accurate than the ST-GS models only for grain yield in the BP. Using BP as a training set to predict the DH population resulted in poor predictions. Overall, all the six ST-GS models appear to be applicable for GS of yield and gluten strength traits in durum wheat, but we recommend the simple computational models RR-BLUP or G-BLUP for predicating single trait and MT-SI for predicting yield and protein simultaneously.     

Agrobacterium-mediated large-scale transformation of wheat (Triticum aestivum L.) using glyphosate selection

An Agrobacterium-mediated transformation system with glyphosate selection has been developed for the large-scale production of transgenic plants. The system uses 4-day precultured immature embryos as explants. A total of 30 vectors containing the 5-enol-pyruvylshikimate-3-phosphate synthase gene from Agrobacterium strain CP4 (aroA:CP4), which confers resistance to glyphosate, were introduced into wheat using this system. The aroA:CP4 gene served two roles in this study-selectable marker and gene of interest. More than 3,000 transgenic events were produced with an average transformation efficiency of 4.4%. The entire process from isolation of immature embryos to production of transgenic plantlets was 50-80 days. Transgenic events were evaluated over several generations based on genetic, agronomic and molecular criteria. Forty-six percent of the transgenic events fit a 3:1 segregation ratio. Molecular analysis confirmed that four of six lead transgenic events selected from Agrobacterium transformation contained a single insert and a single copy of the transgene. Stable expression of theAROA:CP4 gene was confirmed by ELISA through nine generations. A comparison of Agrobacterium-mediated transformation to a particle bombardment system demonstrated that the Agrobacterium system is reproducible, has a higher transformation efficiency with glyphosate selection and produces higher quality transgenic events in wheat. One of the lead events from this study, no. 33391, has been identified as a Roundup Ready wheat commercial candidate.     

Advances in the selection of transgenic plants using non-antibiotic marker genes

Production of transgenic plants started more than a decade ago, but it is still a time-consuming operation. One of the critical points is the selection procedure used for the recovery of transgenic shoots after transformation. Moreover, as more transgenic traits are to be incorporated into crops that already have been transformed, it is clear that there is a need for new methods with higher efficiencies. In this article, recently developed selection systems are reviewed. They differ from conventional selection techniques as they are based on supplementing the transgenic cells with a metabolic advantage rather than killing the non-transgenic cells. In many cases, these new selection systems have been found to be superior to conventional methods.     

Stay-green trait-antioxidant status interrelationship in durum wheat (Triticum durum) flag leaf during post-flowering

Three independent durum wheat mutant lines that show delayed leaf senescence or stay-green (SG) phenotype, SG196, SG310 and SG504, were compared to the parental genotype, cv. Trinakria, with respect to the photosynthetic parameters and the cellular redox state of the flag leaf in the period from flowering to senescence. The SG mutants maintained their chlorophyll content and net photosynthetic rate for longer than Trinakria, thus revealing a functional SG phenotype. They also showed a better redox state as demonstrated by: (1) a lower rate of superoxide anion production due to generally higher activity of the antioxidant enzymes superoxide dismutase and catalase in all of the SG mutants and also of the total peroxidase in SG196; (2) a higher thiol content that can be ascribed to a higher activity of the NADPH-providing enzyme glucose-6-phosphate dehydrogenase in all of the SG mutants and also of the NADP⁺-dependent malic enzyme in SG196; (3) a lower pro-oxidant activity of lipoxygenase that characterises SG196 and SG504 mutants close to leaf senescence. Overall, these results show a general relationship in durum wheat between the SG phenotype and a better redox state. This relationship differs across the different SG mutants, probably as a consequence of the different set of altered genes underlying the SG trait in these independent mutant lines.     

Agrobacterium and biolistic transformation of onion using non-antibiotic selection marker phosphomannose isomerase

A new selection system for onion transformation that does not require the use of antibiotics or herbicides was developed. The selection system used the Escherichia coli gene that encodes phosphomannose isomerase (pmi). Transgenic plants carrying the manA gene that codes for pmi can detoxify mannose-6-phosphate by conversion to fructose-6-phosphate, an intermediate of glycolysis, via the pmi activity. Six-week-old embryogenic callus initiated from seedling radicle was used for transformation. Transgenic plants were produced efficiently with transformation rates of 27 and 23% using Agrobacterium and biolistic system, respectively. Untransformed shoots were eliminated by a stepwise increase from 10 g l⁻¹ sucrose with 10 g l⁻¹ mannose in the first selection to only10 g l⁻¹ mannose in the second selection. Integrative transformation was confirmed by PCR, RT-PCR and Southern hybridization. An erratum to this article can be found at     

Maize transformation using xylose isomerase gene as a selection marker]

The xylA gene, encoding xylose isomerase, was cloned as a 1342-bp BamHI/SacI fragment from the E. coli. As a selection marker, the xylA gene was fused between the enhanced CaMV 35S promoter (E35S) and terminator (35St) in pBAC413 (Fig.2). pBAC413 was constructed to prevent the expression of sbeIIb in maize. PDS1000/He was used to bombard maize calli, which were induced to form by the elite inbred lines. The selection was carried out on the media containing concentrations of xylose from 0 to 100%. The results showed that the media containing 50% to 100% D-xylose were better, but differed with the genotype of maize (Tables 1 and 2). Successful integration of xylA gene into the maize genome was confirmed by DNA dot blotting, PCR and PCR-Southern hybridization (Figs.4 to 6). A method was established in which transformed maize cells were successively screened on a medium containing xylose instead of antibiotic and herbicide for bio-safety.     

Interactions between the scs and Vi genes in alloplasmic durum wheat.

Two nuclear genes, vitality (Vi) on an A- or B-genome chromosome and species cytoplasm specific (scs) on a 1DL telosome from Triticum aestivum L. or a telosome from Aegilops uniaristata Vis. (un telosome), improved compatibility between the nucleus of Triticum turgidum L. var. durum and the cytoplasm of Ae. longissima S. &M. or Ae. uniaristata. To study interactions between Vi and scs and to determine the chromosomal location of Vi, 29-chromosome fertile plants were crossed with 13 D-genome disomic-substitution (d-sub) lines [except 5D(5A)] of 'Langdon' durum. F1 and backcross progenies were examined for meiotic chromosome number and pairing, fertility, and plant vigor. In 11 crosses, Vi restored seed viability but produced double-monosomics (d-monos) with greatly reduced growth and vigor. In contrast, crosses involving 1D(1A) and 1D(1B) d-sub lines produced d-monos with normal vigor and anthesis but nonfunctional pollen. A backcross of 1D + 1A d-mono F1 and 1D(1A) d-sub lines produced 11 male steriles; 3 had 13 II + 1 II 1D + 1 I 1A, 2 had 13 II + 2 I, 1 had 13 II + 1 II 1D(1A), and 5 were not examined. Crosses of 1D + 1A d-mono F1 with control durum, lo durum (with 1DL), and un durum (with un telosome) lines produced 16 male-sterile d-monos and 14 fertiles with 14 II + 1 I 1D, showing that 15-chromosome female gametes transmitted monosomes 1A and 1D. However, BC2F1's from 1D + 1B d-mono x fertile line with un telosome included 20 male-sterile d-monos, 6 fertile triple monosomics (13 II + 1 I 1D + 1 I 1B + t I un telosome), and 1 fertile plant with a 1B/1D translocation. Unlike d-mono 1A + 1D, d-mono 1B + 1D did not transmit 15-chromosome female gametes with monosomes 1D and 1B. Additional backcrosses also indicated that homozygous scs caused male sterility in 1D(1A) and 1D(1B) d-subs and that the procedure used was not suitable for the chromosomal location of Vi.     

Inheritance of deeper root length and grain yield in half-diallel durum wheat (Triticum durum) crosses

Inheritance, heterosis and combining ability of deeper root length (DRL) and grain yield (GY) were investigated in durum wheat populations obtained from half‐diallel crossings among five parental lines differing in their DRL and GY. The study was conducted with the final objective of identifying parent lines to be used in a breeding programme to develop drought‐tolerant wheat varieties. General combining ability and specific combining ability effects were significant for both traits; however, additive gene effects were predominant over non‐additive effects. Partial dominance was ambidirectional for DRL and unidirectional for GY. Lines INRAT69 and Omrabia conferred DRL whereas Omrabia and Khiar transmitted high GY to their respective progenies. In the studied material, both characters were controlled mainly by dominant alleles, but they could also be attributed to recessive alleles although less frequently. Both broad‐sense and strict‐sense heritabilities were high for DRL, confirming the importance of additive gene effects, whereas strict‐sense heritability for GY was average, indicating the importance of interaction effects as compared with the additive effects; this could mean reduced selection efficiency for the latter trait. Thus, the expected genetic progress per cycle of selection will be lower for GY compared with DRL. Omrabia should be included in the breeding programme as a parent so that while maintaining high GY, resulting progeny should be better able to resist drought through DRL.     

A cytoplasmic male sterility (CMS) system in durum wheat

Cytoplasmic male sterility (CMS) systems are based in the incompatible interaction between nucleus and cytoplasm and are commonly used for hybrid seed production in many crop species. The msH1 CMS system in common wheat results from the incompatibility between the nuclear genome of wheat and the cytoplasm of Hordeum chilense. Fertility restoration of the CMS phenotype is associated with the addition of the short arm of chromosome 6H^ch from H. chilense. In this work, we attempt to transfer the msH1 system to durum wheat and to evaluate its potential as a new source of CMS for the production of hybrid durum wheat. For that purpose, an alloplasmic durum wheat line was developed by substituting wheat cytoplasm by that from H. chilense. This line was completely male sterile. Also, the double translocation T6H^chS·6DL was transferred from common wheat into durum wheat, to test its potential as a restorer line. Finally, the system was tested by using the double T6H^chS·6DL translocation in durum wheat as pollen donor for the alloplasmic male sterile line, which confirmed the fertility restoration ability of 6H^chS in durum wheat.