Resistance Gene Bar Research Articles

Optimization of Agrobacterium tumefaciens-Mediated Genetic Transformation of Maize

Immature embryos of inbred maize (Zea mays) lines (H8183, H8184, and H8185) were used for Agrobacterium infection. We used the β-glucuronidase gene (GUS) as the target gene and the glufosinate resistance gene (bar) as the selection marker. We conducted research on several aspects, such as different genotypes, coculture conditions, screening agent concentrations, and concentrations of indole-3-butytric acid (IBA), 6-benzylaminopurine (6-BA), and ascorbic acid (Vc) in the differentiation medium. We optimized the genetic transformation system, and the obtained results indicated that among the three lines studied, the induction rate of H8185 was the highest at 93.2%, followed by H8184, with H8183 having the lowest induction rate (80.1%). The best coculture method was that using the N6 coculture medium layered with a sterile filter paper. Using orthogonal analysis, we found that the optimal combination of the three factors in the differentiation medium was A3 (1 mg mL−1 IBA), B3C1 (1.6 mg mL−1 6-BA), and D3 (1.5 mg mL−1 Vc). Through GUS staining analysis, Bar test-strip analysis, and polymerase chain reaction, five transgenic plants were finally obtained. This study established the optimal conditions for genetic transformation in maize.

Screening and Functional Verification of Selectable Marker Genes for Cordyceps militaris

The selectable marker genes are necessary resistance genes for gene knockout, gene complementation, and gene overexpression in filamentous fungi. Moreover, the more sensitive the filamentous fungi are to antibiotics, the more helpful it is to screen the target transformants. In order to obtain the antibiotic (or herbicide) which can effectively inhibit the growth of Cordyceps militaris and verify the function of the corresponding resistance gene in C. militaris, the sensitivity of C. militaris to hygromycin and glufosinate ammonium was compared to determine the resistance gene that was more suitable for the screening of C. militaris transformants. The binary vector of the selectable marker gene was constructed by combining the double-joint PCR (DJ-PCR) method and the homologous recombination method, and the function of the selectable marker gene in C. militaris was verified by the Agrobacterium tumefaciens-mediated transformation method. The results showed that C. militaris was more sensitive to glufosinate ammonium than hygromycin. The growth of C. militaris could be completely inhibited by 250 μg/mL glufosinate ammonium. The expression cassette of the glufosinate ammonium resistance gene (bar gene) was successfully constructed by DJ-PCR. The binary vector pCAMBIA0390-Bar was successfully constructed by homologous recombination. The bar gene of the vector pCAMBIA0390-Bar was successfully integrated into the C. militaris genome and could be highly expressed in the transformants of C. militaris. This study will promote the identification of C. militaris gene function and reveal the biosynthetic pathways of bioactive components in C. militaris.

Optimization of regeneration and Agrobacterium-mediated transformation of Stevia (Stevia rebaudiana Bertoni): a commercially important natural sweetener plant

Stevia rebaudiana Bertoni is a commercially important zero calorie natural-sweetener herb which produce sweet compounds known as steviol glycosides. Rising demands of steviol glycosides by food and beverage industries has led to an increase in its cultivation in various countries. Unfortunately, stevia cultivation faces 2–25% yield penalty due to weeds which further adds to its cultivation cost. To resolve this major challenge, Agrobacterium-mediated genetic transformation of in vitro derived stevia-nodal explants using herbicide resistance gene (bar) has been optimized, for the production of stable transgenic stevia plants. Several parameters including explant type, pre-incubation duration, acetosyringone (As) concentration, Agrobacterium cell density, Agro-inoculation duration, co-cultivation duration, selection regime and plant growth regulators (PGRs) combination and concentration, have been successfully optimized. Among the two types of explants used, nodal explants showed a higher regeneration response of 82.85%, with an average of 25 shoots/explant. The best PGRs combination and concentration for shoot-induction, shoot-elongation and root-induction was found to be 6-benzyladenine (1.0 mg l−1) + naphthalene acetic acid (0.5 mg l−1), gibberellic acid (1.0 mg l−1), and half-strength MS medium, respectively. The two-step selection (phosphinothricin) regime resulted in an average transformation efficiency of 40.48% with nodal explants. Molecular characterization of putative transformants through PCR, RT-PCR, qRT-PCR and Southern-blot hybridization confirmed the presence, stability, expression as well as copy number of bar gene respectively. Compared to the non-transgenic plants, the T0 transgenic plants successfully tolerated 8 mg l−1 glufosinate ammonium sprays. Thus, the optimized protocol can be useful for the introduction of other genes (inter-kingdom transfer) into stevia genome.

Establishment of a chloroplast transformation system in Tisochrysis lutea

Tisochrysis lutea is a haptophyte microalga commonly used as a commercial feed for juvenile fish and shellfish larvae. Genetic modification is of considerable importance for developing the potential economic value of T. lutea. However, the genetic transformation system of T. lutea has not yet been established, which limits functional genomic studies and strain improvement of this important microalgal species. In the current study, a chloroplast transformation vector harboring the phosphinothricin resistance gene (bar) as a selectable marker was established, and two short peptide-encoding genes (ant1 and ant2) driven by the endogenous psbA and rbcL promoters were cloned in this vector. The recombinant plasmid was transformed using a biolistic method into the trnI/trnA locus of the chloroplast genome via homologous recombination. After continuous selection on phosphinothricin, the integration of foreign genes and the expression of specific products in the transformants were detected using polymerase chain reaction (PCR), Southern blotting, and western blot analysis. This is the first report of establishment of a stable transformation system in T. lutea, which is a prerequisite for functional genomics and applied research on this species.

Biolistic-mediated oil palm transformation with alfalfa glucanase (AGLU1) and rice chitinase (RCH10) genes for increasing oil palm resistance towards Ganoderma boninense

Basal stem rot (BSR) caused by Ganoderma boninense, a white-rot fungus is the most devastating disease in oil palm. In addition to the available methods that have been used to control this disease, genetic engineering was explored to produce fungal resistant transgenic oil palm. In this study, oil palm embryogenic calli were co-bombarded with a construct carrying the AGLU1 and RCH10 genes and a construct carrying the Basta® resistance gene (bar). After selection with the herbicide Basta®, 25 plants from seven events were regenerated, transferred onto soil and grown in the fully contained biosafety screen house. Polymerase chain reaction (PCR) analysis for the plants demonstrated that only eight plants were positive for the presence of three transgenes, the bar, AGLU1, and RCH10 genes. The quantitative reverse transcription PCR (RT-qPCR) demonstrated the expression of both AGLU1 and RCH10 genes in five out of eight plants tested. The infection of these plants with the G. boninense fungus resulted in only two plants exhibited resistance to the fungus. The results from this study demonstrated that fungal resistant oil palm could be produced via biolistic using alfalfa β-1,3-glucanase (AGLU1) and rice chitinase (RCH10) genes.

Targeted expression of a cysteine protease (AdCP) in tapetum induces male sterility in Indian mustard, Brassica juncea.

The development of male sterile plants is a prerequisite to developing hybrid varieties to harness the benefits of hybrid vigor in crops and enhancing crop productivity for sustainable agriculture. In plants, cysteine proteases have been known for their multifaceted roles during programmed cell death, and in ubiquitin- and proteasome-mediated proteolysis. Here, we showed that Arachis diogoi cysteine protease (AdCP) expressed under the TA-29 promoter induced complete male sterility in Indian mustard, Brassica juncea. The herbicide resistance gene bar was used for the selection of transgenic plants. Mustard transgenic plants exhibited male sterile phenotype and failed to produce functional pollen grains. Irregularly shaped aborted pollen grains with groove-like structures were observed in male sterile plants during scanning electron microscopy analysis. The T1 progeny plants obtained from the seed of primary transgenic male sterile plants crossed with the wild-type plants exhibited segregation of the progeny into male sterile and fertile plants with normal seed development. Further, male sterile plants exhibited higher transcript levels of AdCP in anther tissues, which is consistent with its expression under the tapetum-specific promoter. Our results clearly suggest that the targeted expression of AdCP provides a potential tool for developing male sterile lines in crop plants by the malfunction of tapetal cells leading to male sterility as shown earlier in tobacco transgenic plants (Shukla et al.2014, Funct Integr Genomics 14:307-317).

Improved Genetic Transformation of Sugarcane (Saccharum spp.) Embryogenic Callus Mediated by Agrobacterium tumefaciens.

Sugarcane (Saccharum spp.) is a monocotyledonous semi-perennial C4 grass of the Poaceae family. Its capacity to accumulate high content of sucrose and biomass makes it one of the most important crops for sugar and biofuel production. Conventional methods of sugarcane breeding have shown several limitations due to its complex polyploid and aneuploid genome. However, improvement by biotechnological engineering is currently the most promising alternative to introduce economically important traits. In this work, we present an improved protocol for Agrobacterium tumefaciens-mediated transformation of commercial sugarcane hybrids using immature top stalk-derived embryogenic callus cultures. The callus cultures are transformed with preconditioned A. tumefaciens carrying a binary vector that encodes expression cassettes for a gene of interest and the bialaphos resistance gene (bar confers resistance to glufosinate-ammonium herbicide). This protocol has been used to successfully transform a commercial sugarcane cultivar, SP80-3280, highlighting: (i) reduced recalcitrance and oxidation; (ii) high yield of embryogenic callus; (iii) improved selection; and (iv) shoot regeneration and rooting of the transformed plants. Altogether, these improvements generated a transformation efficiency of 2.2%. This protocol provides a reliable tool for a routine procedure for sugarcane improvement by genetic engineering. © 2017 by John Wiley & Sons, Inc.

Comparison of transformation frequency using the bar gene regulated by the CaMV 35S or NOS promoter in Agrobacterium-mediated soybean (Glycine max L.) transformation

In Agrobacterium tumefaciens-mediated plant transformation, the promoter chosen to drive a selectable marker gene has an effect on transformation frequency. The objective of this work was to compare the effect on soybean transformation of different promoter and regulator combinations driving a selectable marker gene using mature seeds as explants. Two commonly used promoters, double cauliflower mosaic virus 35S (CaMV 35S) and nopaline synthase (NOS), and one regulator, tobacco etch virus (TEV) translational enhancer, were tested. Four different promoter/enhancer combinations were constructed to drive a bialaphos resistance gene (bar) and used for multiple independent transformation experiments. Herbicide application and PCR analysis were used to confirm the inheritance of the bar gene in the T 1 generation. Quantitative real-time PCR (qRT-PCR) was used to estimate transgene copy number in T 1 transgenic events. The average transformation frequency from combining NOS with the TEV enhancer (3.5% across 12 replications) was significantly higher than the frequencies obtained from the double CaMV 35S without an enhancer (1.6% across 16 replications), double CaMV 35S with an enhancer (1.4% across 8 replications), and NOS without an enhancer (1.0% across 12 replications). The bar transcript levels in T 1 transgenic leaf tissue did not correlate with transformation frequencies achieved by the different constructs. No significant differences were identified between constructs in the average transgene copy number across events. These data show that a selectable marker system comprised of the bar gene regulated by the NOS promoter in combination with the TEV enhancer is preferred in Agrobacterium-mediated soybean transformation.

Genetic transformation of cultivated sesame (Sesamum indicum L. cv Rama) through particle bombardment using 5-day-old apical, meristematic tissues of germinating seedlings

An in vitro plant generation and genetic transformation protocol was established in sesame (Sesamum indicum L. cv Rama) through biolistic particle gun bombardment. 5-day-old apical, meristematic tissues of in vitro-germinating seedlings were used as explants. 10–15 Multiple shoots were generated from each explant using Murashige and Skoog basal medium containing 18.0 µM benzylamino purine and 5.37 µM naphthalene acetic acid. Four independent sets of transformation were carried out and each set consisted of three independent experiments each comprising three replications with 30 explants per replication. A synthetically designed bialaphos resistance gene (bar) was used for transformation. The positive transformants containing the bar gene were selected in growth medium containing 2.5 mg/L bialaphos. Green shoots recovered from bombarded explants were subjected to root development on Murashige and Skoog basal medium containing 5.37 µM naphthalene acetic acid. The rooted shoots were established in soil and grown to maturity in greenhouse. Polymerase chain reaction (PCR), Southern and reverse-transcription PCR, real-time quantitative PCR, western blot and enzymatic assay of four putative transformants from independent sets provided evidence for full-length gene integration as well as high level expression of the transgene. Analysis of the T1 plants revealed a stable inheritance of the transgene through the progenies. This is the first report of biolistic mediated stable transformation of sesame and should pave the way for future genetic engineering strategies to be employed for improvement of this very important oil-seed crop.

Genetic transformation of cultivated jute (Corchorus capsularis L.) by particle bombardment using apical meristem tissue and development of stable transgenic plant

An in vitro plant regeneration and genetic transformation protocol was established in jute (Corchorus capsularis L. var JRC321). One-day-old apical, meristematic tissues of germinating seedlings were used as explants. Multiple shoots were regenerated from each explant using Murashige and Skoog basal medium containing 1.78 µM benzylamino purine and 4.92 µM indole-3-butyric acid. Transformation was carried out in three independent sets (each set comprising of three independent experiments each comprising three replications with 35 explants per replication) using the bialaphos resistance gene (bar), synthetically designed for high level plant expression. The positive transformants containing the bar gene were selected in growth medium containing 2.5 mg/l bialaphos. Polymerase chain reaction (PCR), Southern and northern blots, real-time quantitative PCR, western blot and enzymatic assay of five putative transformants from three independent sets provided evidence for full-length gene integration into the genomic DNA of transformed jute, as well as high level expression of the transgene. Analysis of the T1 plants revealed a stable inheritance of the transgene through the progenies. The data presented in this report showed considerable advancement in jute transformation and should improve future genetic engineering strategies to be employed for improvement of this very important fibre crop.

Development of some transgenic rice lines containing the herbicide resistance gene BAR and study on inheritance of this BAR gene up to the T2 generation

Development of some transgenic rice lines containing the herbicide resistance gene BAR and study on inheritance of this BAR gene up to the T2 generation

Carrot (Daucus carota L.).

Plants are susceptible to infection by a broad range of fungal pathogens. A range of proteins have been evaluated that can enhance tolerance to these pathogens by heterologous expression in transgenic carrot tissues. The protocols for carrot transformation with Arabidopsis NPR1 (Non-Expressor of Pathogenesis-Related Proteins 1) are described in this chapter, using the herbicide resistance gene bar, which encodes phosphinothricin acetyltransferase, as a selectable marker. In this protocol, petiole segments (0.5-1.0 cm long) from aseptically grown carrot seedlings are exposed to Agrobacterium tumefaciens strain LBA4404 for 10-30 min and cocultivated for 2-3 days. Herbicide selection is then imposed for 8-12 weeks on a series of different tissue culture media until embryogenic calli are produced. The transfer of the embryogenic calli to hormone-free medium results in embryo development which eventually gives rise to transgenic plantlets. Embryogenic calli can also be propagated in suspension cultures. This protocol has yielded transgenic carrot plants with defined T-DNA inserts at the rate of between 1 and 3 Southern-positive independent events out of 100.

Fertile transgenic Lotus corniculatus resistant to the non‐selective herbicide phosphinothricin

AbstractResistance to the non‐selective herbicide dl‐phosphinothricin (PPT) was introduced into commercial Lotus corniculatus cv. Bokor by co‐cultivation of cotyledons with Agrobacterium tumefaciens AGL1 harbouring the binary vector pDM805 which contains the bialaphos resistance gene (bar) from Streptomyces hygroscopicus encoding phosphinothricin acetyltransferase (PAT) and the uidA gene encoding β‐glucuronidase. The half‐cotyledon explants were precultured on regeneration Murashige and Skoog's (MS) medium supplemented with 6‐benzyladenine (BA) and 1‐naphthaleneacetic acid (NAA) at 0.5 mg L−1 each, 3 days prior to infection. Upon co‐cultivation, the explants were cultured on PPT‐free regeneration medium for 10 days, and then subcultured on regeneration/selection media with increasing PPT concentrations (5–7 mg L−1) for about 18 weeks. Out of 480 initially co‐cultivated explants, 272 regenerated shoots survived the entire PPT selection procedure. Resistant shoots were grown further, multiplied by tillering that was additionally promoted by PPT and rooted on hormone‐free MS medium containing 5 mg L−1 PPT. Established shoot cultures, continuously maintained on the same medium, have preserved PPT resistance up to now (more than 2 years). Transformed plants assessed in vitro and in a greenhouse were tolerant to the herbicide PPT at 300 mg L−1 equivalent to more than twofold the recommended field dosage for weed eradication. Applied PPT treatment did not affect the activities of glutamine synthetase (GS; EC 6.3.1.2) and NADH‐dependent glutamate dehydrogenase (NADH‐GDH; EC 1.4.1.2) in transformed plants. However, PPT did increase the mobility of glutamine synthetase isoforms GS1 and GS2 as well as the inhibition of an additional high mobility GS (hmGS) activity. In untransformed plants, PPT treatment reduced total GS activity by 4.4‐fold while contrary the activity of NADH‐GDH was increased by ninefold. All transformed herbicide‐resistant plants were phenotypically normal and exhibited genomic stability, as were the untransformed plants analysed by flow cytometry. Under greenhouse conditions, they grew to maturity, flowered and set seeds. Stable integration and expression of the bar gene in T0 and T1 plants were confirmed by Southern and Western blot analysis, while integration of the reporter uidA gene did not occur. The bar gene was inherited in a Mendelian fashion by the progeny, as detected by PPT resistance. The production of PPT‐resistant plants may have significant practical applications in weed control in fields of L. corniculatus.

Development of Gateway Binary Vectors R4L1pGWB Possessing the Bialaphos Resistance Gene (bar) and the Tunicamycin Resistance Gene as Markers for Promoter Analysis in Plants

We have developed a new series of R4L1 Gateway binary vectors (R4L1pGWB), which carry the bialaphos resistance gene (bar) or the UDP-N-acetylglucosamine:dolichol phosphate N-acetylglucosamine-1-P transferase (GPT) gene as selection markers that confer BASTA® and tunicamycin resistance on plants respectively. R4L1pGWBs have an attR4-attL1-reporter and can accept an attL4-promoter-attR1 entry clone for easy construction of an attB4-promoter-attB1-reporter clone. The new R4L1pGWBs facilitate promoter:reporter analysis in pre-existing transgenic plants that are resistant to kanamycin or hygromycin.

The UDP-N-acetylglucosamine:Dolichol Phosphate-N-acetylglucosamine-phosphotransferase Gene as a New Selection Marker for Potato Transformation

Here we describe the generation of potato plants that constitutively overexpressed, UDP-N-acetylglucosamine:dolichol phosphate-N-acetylglucosamine-phosphotransferase (GPT). Such transgenic plants can be formed in a medium with tunicamycin at 9.8 ± 0.28% efficiency, similar to the 9.4 ± 1.10 for the bialaphos resistance gene (Bar) gene. This study indicated that GPT transformation was very stable with high reproducibility, and that growth and tuber production in the GPT-transformed plants were stronger than in the wild-type plants.

Fertile transgenic Brachiaria ruziziensis (ruzigrass) plants by particle bombardment of tetraploidized callus

We have produced transgenic plants of the tropical forage crop Brachiaria ruziziensis (ruzigrass) by particle bombardment-mediated transformation of multiple-shoot clumps and embryogenic calli. Cultures of multiple-shoot clumps and embryogenic calli were induced on solidified MS medium supplemented with 0.5mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) and 2mg/L 6-benzylaminopurine (BAP) or 4mg/L 2,4-D and 0.2mg/L BAP, respectively. Both cultures were bombarded with a vector containing an herbicide resistance gene (bar) as a selectable marker and the β-glucuronidase (GUS) reporter gene. Sixteen hours after bombardment, embryogenic calli showed a significantly higher number of transient GUS expression spots per plate and callus than multiple-shoot clumps, suggesting that embryogenic callus is the more suitable target tissue. Following bombardment and selection with 10mg/L bialaphos, herbicide-resistant embryogenic calli regenerated shoots and roots in vitro, and mature transgenic plants have been raised in the greenhouse. Polymerase chain reaction (PCR) and DNA gel blot analysis verified that the GUS gene was integrated into the genome of the two regenerated lines. In SacI digests, the two transgenic lines showed two or five copies of GUS gene fragments, respectively, and integration at different sites. Histochemical analysis revealed stable expression in roots, shoots and inflorescences. Transgenic plants derived from diploid target callus turned out to be sterile, while transgenics from colchicine-tetraploidized callus were fertile.

Transgenic tetraploid Isatis indigotica expressing Bt Cry1Ac and Pinellia ternata agglutinin showed enhanced resistance to moths and aphids

Co-expression of multiple genes encoding different kinds of insect resistant proteins has been developed to confer a broader spectrum of pest control. Tetraploid Isatis indigotica Fort was transformed with a plasmid, p3300BP, containing Bacillus thuringiensis Cry1Ac gene (Bt) and Pinellia ternata agglutinin gene (Pta) and the selectable marker herbicide resistance gene (Bar) driven by the CaMV35S promoter via Agrobacterium tumefaciens-mediated transformation. The integration and expression of introduced genes in regenerated transgenic plants were confirmed by PCR and Western blot assays. Insect bioassay test demonstrated transgenic lines had significant inhibition to diamondback moths (Plutella xylostella L.) and peach potato aphids (Myzus persicae Sulzer) simultaneously. Our study reported here would be a great motivation for field culture of tetraploid I. indigotica, also providing an efficient molecular breeding strategy to provide insect tolerant plants.

Gateway Binary Vectors with the Bialaphos Resistance Gene,bar, as a Selection Marker for Plant Transformation

We constructed two series of Gateway binary vectors, pGWBs and R4pGWBs, possessing the bialaphos resistance gene (bar) as a selection marker for plant transformation. The reporters and tags employed in this system are sGFP, GUS, LUC, EYFP, ECFP, G3GFP, mRFP, TagRFP, 6xHis, FLAG, 3xHA, 4xMyc, 10xMyc, GST, T7 and TAP. Selection of Arabidopsis transformants with BASTA was successfully carried out using both plate-grown and soil-grown seedlings. Transformed rice calli and suspension-cultured tobacco cells were selected on plates containing BASTA or glufosinate-ammonium. These vectors are compatible with existing pGWB and R4pGWB vectors carrying kanamycin and hygromycin B resistance.

A TRANSGENIC APPROACH FOR DETERMINING SEX OF PAPAYA SEEDLINGS

Sex segregation in gynodioecious papayas necessitates expensive and inefficient over-planting of indistinguishable hermaphrodite and female seedlings, followed by subsequent removal of surplus plants at flowering time, to ensure uniform stands of economically valuable hermaphrodite trees. Our objective is to produce transgenic papayas with marker genes linked to the sex-determining region, so that growers can distinguish the sex of seedlings at planting time by marker phenotypes. Transgene insertion is random, but genes controlling papaya sex reside in a relatively large non-recombining region of chromosome 1 amounting to 1-2% of the entire genome, so about 150 independent transformations are needed for 95% probability of sex linkage. We have used both particle bombardment and Agrobacterium inoculation to transform Hawaii cultivars with constructs containing a fluorescent yellow protein gene (Eyfp) and/or glufosinate resistance gene (bar). Sex-linked insertions will permit selection of glufosinate-resistant hermaphrodites or elimination of fluorescent yellow females. The papaya regeneration protocol of Fitch and Manshardt (1990), based on embryogenic callus derived from immature zygotes, was improved to promote more normal development by lowering 2,4-D concentration in the callus-induction medium, by venting headspace gasses during germination of somatic embryos and shoot growth, and by rooting shoots under photo-auxotrophic conditions. Nearly 500 putative transgenic lines representing independent transformation events were selected after 4 to 5 months of culture on glufosinate-containing media. Initial PCR assays on 40 lines obtained by particle bombardment indicated that 72.5% carried the bar transgene, however, no expression of Eyfp was detected by epifluorescence microscopy. Transformation success was independent of the transgene construct used (CAMBIA3300 vs. E3410) or the genotype of targeted papaya tissues (Kapoho, Sunrise, line D), but bombarded tissues responded better to glufosinate selection after a 2-week tissue recovery interval on non-selective medium. Success of Agrobacterium transformation was not affected by strain differences (LBA4404 vs. EHA105), but duration of the inoculation period interacted significantly with duration of co-cultivation. Transgene expression assays to confirm functionality are in progress, and screening for lines with sex-linked insertions will be conducted utilizing inverse PCR to sequence the DNA in regions flanking insertion sites and matching them with sequences in the publicly available papaya genome database to locate the sites on chromosomes.

Selectable marker genes and unintended changes to the plant transcriptome

The intended effect of a selectable marker gene is to confer a novel trait that allows for the selection and recovery of transgenic plants. Unintended effects may also occur as a result of interactions between the selectable marker gene or its regulatory elements and genetic elements at the site of insertion. These are called position effects. Other unintended effects may occur if the selectable marker gene has a range of pleiotropic effects related to the functional and regulatory domains within the coding region or the regulatory elements used to drive expression. Both pleiotropic and position effects may generate unpredictable events depending on the process used for transgenesis and the state of knowledge associated with the selectable marker gene. Although some selectable marker genes, such as the neomycin phosphotransferase type II gene (nptII), have no pleiotropic effects on the transcriptomes of transgenic plants, others, such as the bialaphos resistance gene (bar), have pleiotropic effects. These must be clearly understood and accounted for when evaluating the expression patterns conferred by other co-transforming transgenes under study. The number and kinds of selectable marker genes are large. A detailed understanding of their unintended effects is needed to develop transgenic strategies that will minimize or eliminate unintended and unpredictable changes to plants with newly inserted genes.