Pmi Gene Research Articles

Exogenous brassinolide improves the antioxidant capacity of Pinellia ternata by enhancing the enzymatic and nonenzymatic defense systems under non-stress conditions.

Brassinolide (BR) improves the antioxidant capacity of plants under various abiotic stresses. However, it is not clear about the effect of BR on the antioxidant capacity in plants under non-stress conditions. In the present study, the antioxidant defense response of Pinellia ternata was to be assessed by applying BR and propiconazole (Pcz) under non-stress conditions. BR treatment enhanced the flavonoid content, peroxidase, and ascorbate peroxidase (APX) activity by 12.31, 30.62, and 25.08% and led to an increase in 2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity by 4.31% and a decrease in malondialdehyde content by 1.04%. Exogenous application of BR improved the expression levels of PAL, CHS, CHI, and DFR genes by 3. 18-, 3. 39-, 2. 21-, and 0.87-fold in flavonoid biosynthesis, PGI, PMI, and GME genes by 6. 60-, 1437. 79-, and 3.11-fold in ascorbic acid (ASA), biosynthesis, and γECs and GSHS genes by 6.08- and 2.61-fold in glutathione (GSH) biosynthesis pathway, and the expression of these genes were inhibited by Pcz treatment. In addition, BR treatment promoted the ASA–GSH cycle by enhancing the expression of APX, DHAR, and MDHAR genes, which were enhanced by 3. 33-, 157. 85-, and 154.91-fold, respectively. These results provided novel insights into the effect of BR on the antioxidant capacity in bulbil of P. ternata under non-stress conditions and useful knowledge of applying BR to enhance the antioxidant capacity of plants.

Food/Feed and Environmental Risk Assessment of Insect-resistant Genetically Modified Maize MIR604 for Food and Feed Uses, Import and Processing under Regulation (EC) No 1829/2003 (EFSA/GMO/UK/2005/11)

Food/Feed and Environmental Risk Assessment of Insect-resistant Genetically Modified Maize MIR604 for Food and Feed Uses, Import and Processing under Regulation (EC) No 1829/2003 (EFSA/GMO/UK/2005/11)

The use of phosphomannose isomerase selection system for Agrobacterium-mediated transformation of tobacco and flax aimed for phytoremediation

ABSTRACTA plant selection system based on the phosphomannose isomerase gene (pmi) as a selectable marker is often used to avoid selection using antibiotic resistance. Nevertheless, pmi gene is endogenous in several plant species and therefore difficult to use in such cases. Here we evaluated and compared Agrobacterium-mediated transformation of Linum usitatissimum breeding line AGT-952 (without endogenous pmi gene) and Nicotiana tabacum var. WSC-38 (with endogenous pmi gene). Transformation was evaluated for vectors bearing transgenes that have the potential to be involved in improved phytoremediation of contaminated environment. Tobacco regenerants selection resulted in 6.8% transformation efficiency when using a medium supplemented with 30 g/L mannose with stepwise decrease of the sucrose concentration. Similar transformation efficiency (5.3%) was achieved in transformation of flax. Relatively low selection efficiency was achieved (12.5% and 34.8%, respectively). The final detection of efficient pmi selection was conducted using PCR and the non-endogenous genes; pmi transgene for flax and todC2 transgene for tobacco plants.

Long-term T-DNA insert stability and transgene expression consistency in field propagated sugarcane

This study addresses T-DNA insert stability and transgene expression consistency in multiple cycles of field propagated sugarcane. T-DNA inserts are stable; no transgene rearrangements were observed. AmCYAN1 and PMI protein accumulation levels were maintained. There was no evidence that production of either protein declined across generations and no transgene silencing was observed in three commercial sugarcane varieties through commercially relevant ratooning, propagation-by-setts, and micro-propagation generation processes over 4years of field testing. Long term transgene expression consistency and T-DNA insert stability can be achieved in sugarcane, suggesting that it is highly probable that transgenic sugarcane can be successfully commercialized. This study addresses T-DNA insert stability and transgene expression consistency in multiple cycles of field propagated sugarcane. These data are critical supporting information needed for successful commercialization of GM sugarcane. Here seventeen transgenic events, containing the AmCYAN1 gene driven by a CMP promoter and the E. coli PMI gene driven by either a CMP or Ubi promoter, were used to monitor T-DNA insert stability and consistency of transgene encoded protein accumulation through commercially relevant ratooning, propagation-by-setts, and micro-propagation generation processes. The experiments were conducted in three commercial sugarcane varieties over 4years of field testing. DNA gel blot analysis showed that the T-DNA inserts are stable; no transgene rearrangements were observed. Quantitative ELISA showed no evidence of decreasing AmCYAN1 and PMI protein levels across generations and no transgene silencing was observed. These results indicate that long term transgene expression consistency and T-DNA insert stability can be achieved in sugarcane, suggesting that it is highly probable that transgenic sugarcane can be successfully commercialized.

Real-time and visual loop-mediated isothermal amplification: Efficient GMO screening targeting pat and pmi marker genes

Rapid and efficient screening assays based on loop-mediated isothermal amplification (LAMP) were developed, targeting two marker genes, namely, phosphinothricin-N-acetyltransferase (pat) and phosphomannose isomerase (pmi). These marker genes are being employed in more than 35% of GM events approved globally. Specificity of developed visual and real-time LAMP assays was confirmed using seven GM events of two crops, viz., maize (3272, 59122, Bt11, Bt176, MIR604, TC1507), and cotton (Widestrike™). In visual LAMP, positive amplification can be directly analyzed by the colorimetric change from orange to green, whereas real-time LAMP is based on the monitoring of fluorescence signals as amplification and annealing curves. Visual LAMP was found sensitive enough to detect up to 0.05% GM content for pat and 0.1% for pmi within 40 min. Real-time LAMP efficiently detected up to 0.01% GM content within 30 min. Practical applicability of developed assays was confirmed using proficiency test samples of maize. LAMP assays for pmi gene have been reported for the first time. Due to portability of systems, the developed LAMP assays when combined with a fast DNA extraction method could facilitate on-site GMO screening.

Plant phosphomannose isomerase as a selectable marker for rice transformation.

The E. coli phosphomannose isomerase (EcPMI) gene is widely used as a selectable marker gene (SMG) in mannose (Man) selection-based plant transformation. Although some plant species exhibit significant PMI activity and active PMIs were even identified in Man-sensitive plants, whether plant PMIs can be used as SMGs remains unclear. In this study, we isolated four novel PMI genes from Chlorella variabilis and Oryza sativa. Their isoenzymatic activities were examined in vitro and compared with that of EcPMI. The active plant PMIs were separately constructed into binary vectors as SMGs and then transformed into rice via Agrobacterium. In both Indica and Japonica subspecies, our results indicated that the plant PMIs could select and produce transgenic plants in a pattern similar to that of EcPMI. The transgenic plants exhibited an accumulation of plant PMI transcripts and enhancement of the in vivo PMI activity. Furthermore, a gene of interest was successfully transformed into rice using the plant PMIs as SMGs. Thus, novel SMGs for Man selection were isolated from plants, and our analysis suggested that PMIs encoding active enzymes might be common in plants and could potentially be used as appropriate genetic elements in cisgenesis engineering.

USING MANNOSE AS A POSITIVE SELECTION OF TRANSFORMED CARICA PAPAYA L. VAR ‘EKSOTIKA’

The main objective of this study is to develop marker-free transgenic papaya plants via positive selection using phosphomannose isomerase (pmi) gene. Phosphomannose isomerase (PMI) is an enzyme that converts mannose-6-phosphate to fructose-6-phosphate, a glycolysis intermediate that supports the growth of plant cells. To establish a marker-free positive selection system using this PMI, the effect of mannose on the growth and development of embryogenic ‘Eksotika’ papaya callus was evaluated. One-month old embryogenic calli were cultured on Murashige and Skoog (MS) medium in which 60 g/L sucrose in the original recipe was replaced with different concentrations of mannose and sucrose. Mannose was supplied as the sole carbon source or in combination with sucrose at 0, 5, 10, 15, 20, 25 or 30 g/L. Embryogenic calli cultured on medium supplemented with a ratio of 0:60 g/L mannose: sucrose was used as a control. The results after six sub-cultures showed that most of the embryogenic calli transferred on media containing only mannose turned brown. Higher concentrations of mannose resulted in higher percentage of brown calli (dead). Mannose at 30 g/L mannose was found to be effective for screening transformed embryogenic calli. Evaluation of papaya transformation efficiency using this positive selection system was pursued using 650 one-month-old embryogenic calli Agrobacterium-transformed with pNOV2819 harboring the pmi gene. Only transformed cells are capable of utilizing mannose as a carbon source to grow. After five months on mannose selection, all 67 putative transformants obtained were PCR-positives for the pmi gene.

Efficient use of the PMI/mannose selection system in Agrobacterium-mediated transformation of tobacco (Nicotiana tabacum)

AbstractA safe and environment friendly selectable marker system using phosphomannose isomerase (pmi) gene was employed for Agrobacterium tumefaciens mediated transformation of tobacco (Nicotinia tabacum L.). Sensitivity of tobacco leaf explants to mannose as selective agent was determined before genetic transformation. Twenty mg/L mannose was chosen for selection to suppress the emergence of untransformed shoots from the explants. Leaf explants from 4-week old seedlings of tobacco cv. Gewone Groene were transformed using Agrobacterium harbouring plasmid pCAMBIA3300-PMI carrying the pmi gene. Regenerating transformed shoots were selected on Murashige & Skoog basal medium supplemented with 1.0 mg/L 6-benzylaminopurine, 0.1 mg/L indole-3-butyric acid, and 20 g/L mannose as carbon source. The mannose-resistant shoots were rooted on a plant growth regulator-free Murashige & Skoog basal medium and the potted transgenic plants were acclimatized successfully in the greenhouse, with a survival rate of 100%. Selection efficiency and transformation efficiency based on PCR analysis of individual putative transformants were achieved as high as 94.56% and 21.75%, respectively, with a lower rate of untransformed plant escapes. Southern blot analysis of selected putative transformed plants confirmed the transgene integration into the tobacco genome. The following reverse transcription PCR analysis showed transcriptional activity of the pmi transgene in all T0 transgenic plants analysed with differences in the level of pmi transcripts, and chlorophenol red assay confirmed the activity of PMI in transgenic plants. The PMI/mannose-based transformation system presented here is efficient and reproducible, and can be used for the mass production of transgenic tobacco plants to produce novel products with industrial and medicinal values.

Effective selection and regeneration of transgenic sugarcane plants using positive selection system

We compared the transformation frequency of a positive selection system, which employs an Escherichia coli-derived pmi (phosphomannose isomerase) gene as the selectable marker and mannose as the selective agent, to commonly used negative selection systems of bar (phosphinothricin acetyltransferase) /phosphinothricin (PPT), npt II (neomycin phosphotransferase) /G418 (geneticin), and hpt (hygromycin phosphotransferase) /Hygromycin. Transgenic sugarcane plants were obtained following biolistic transformation of callus with four different selectable marker genes. Under different selective agents, transformed callus was selectively proliferated on MS medium containing 13.57 μM of 2,4-D, regenerated on MS medium with 4.44 μM of BA, 9.29 μM of KT, and 0.89 μM of NAA, and rooted on half of MS medium with 13.38 μM of NAA and 0.44 μM of 6-BA. The results demonstrated that the transformation frequency of 4.2% scored in the pmi/mannose positive system was significantly higher than those in the negative selection systems (0.47% for bar/PPT, 1.38% for npt II/G418, and 0.63% for hpt/Hygromycin, respectively). Transgenic sugarcane plants using the pmi/mannose system were confirmed by polymerase chain reaction (PCR) analysis and chlorophenol red (CPR) assay. The CPR assay was found to efficiently and qualitatively detect the transgenic sugarcane from the nontransformed plants because medium color changes in the CPR assay were highly responsive to pmi gene expression and enzyme activity. Thus, a pmi/mannose positive selection system for sugarcane transformation was developed to obviate the use of negative selection systems based on herbicides and antibiotics.

Retraction : The Use of Phosphomannose-isomerase as a Selectable Marker to Recover Transgenic Cauliflower

We attempted to establish a new selection marker system from cauliflower without antibiotics or herbicide. This new selection marker system was based on the principle that only the transformed plants could convert mannose to fructose. A transformed plant could then use the fructose as carbon source by using pmi gene that can recognize phosphomannose-isomerase that converts mannose-6-phosphate into fructose-6-phosphate. Agrobacterium tumefaciens LBA4404 contained pNWB-HSP101 vector linked with HSP101. Five days after germination, the heat tolerant gene was inoculated into the cauliflower's hypocotyl. After culturing, selection was performed in MS medium that contained BAP, NAA, 0.4% (w/v) mannose and 2.0% (w/v) sucrose. PCR, Southern blot and Northern blot analyses were performed to determine whether HSP101 gene was inserted in the transformed plant. After acclimation, seeds were successfully collected from the transformed plants in the greenhouse. This is the first report for successful transformation of cauliflower plant using the mannose selection system. Moreover, the stable selection system established in this study could be applied an alternative reducing antibiotics for human health and environmental safety during the genetic transformation of higher plants.

Construction of plant expression vectors with PMI gene as selection marker and their utilization in transformation of Salvia miltiorrhiza f.alba

To construct plant expression pCAMBIA1301-PMI by substituting PMI for hygromycin resistance gene in pCAMBIA1301 and obtain transgenic Salvia miltiorrhiza f. alba using PMI-mannose selection system. The 6-phosphomannose isomerase gene (PMI) of Escherichia coli was amplified by PCR. Sequence analysis showed that it shared 100% amino acids identities with the sequences of PMI genes isolates reported in the NCBI. Based on pCAMBIA1305, the plant expression pCAMBIA1305-PMI was constructed successfully by substituting PMI for hygromycin resistance gene in pCAMBIA1305. pCAMBIA1305-PMI was transformed into Agrobacterium tumefaciens LBA4404, and then the leaves of S. miltiorrhiza f. alba were inoculated in LBA4404 with pCAMBIA1305-PMI. Plant expression pCAMBIA1301-PMI was successfully constructed and the leaves of S. miltiorrhiza f. alba inoculated in LBA4404 with pCAMBIA1305-PMI were selected on medium supplemented with a combination of 20 g x L(-1) mannose and 10 g x L(-1) sucrose as a carbon source. The transformation efficiency rate was 23.7%. Genetic transformation was confirmed by PCR, indicating that a new method for obtaining transgenic S. miltiorrhiza f. alba plants was developed using PMI-mannose selection system.

The relationship between PMI (manA) gene expression and optimal selection pressure in Indica rice transformation

An efficient mannose selection system was established for transformation of Indica cultivar IR58025B . Different selection pressures were required to achieve optimum transformation frequency for different PMI selectable marker cassettes. This study was conducted to establish an efficient transformation system for Indica rice, cultivar IR58025B. Four combinations of two promoters, rice Actin 1 and maize Ubiquitin 1, and two manA genes, native gene from E. coli (PMI-01) and synthetic maize codon-optimized gene (PMI-09) were compared under various concentrations of mannose. Different selection pressures were required for different gene cassettes to achieve corresponding optimum transformation frequency (TF). Higher TFs as 54 and 53% were obtained when 5 g/L mannose was used for selection of prActin-PMI-01 cassette and 7.5 g/L mannose used for selection of prActin-PMI-09, respectively. TFs as 67 and 56% were obtained when 7.5 and 15 g/L mannose were used for selection of prUbi-PMI-01 and prUbi-PMI-09, respectively. We conclude that higher TFs can be achieved for different gene cassettes when an optimum selection pressure is applied. By investigating the PMI expression level in transgenic calli and leaves, we found there was a significant positive correlation between the protein expression level and the optimal selection pressure. Higher optimal selection pressure is required for those constructs which confer higher expression of PMI protein. The single copy rate of those transgenic events for prActin-PMI-01 cassette is lower than that for other three cassettes. We speculate some of low copy events with low protein expression levels might not have been able to survive in the mannose selection.

Biolistic transformation of oil palm using the phosphomannose isomerase (pmi) gene as a positive selectable marker

The selectable marker system based on the Escherichia coli phosphomannose isomerase (pmi) gene was evaluated for the genetic transformation of oil palm. Four transformation vectors were constructed for transforming oil palm. The transformation vectors both carried the pmi gene alone or in combination with the β-glucuronidase (gusA) gene, and were driven by either the maize ubiquitin promoter (pMI11 and pMI11G) or the CaMV35S promoter (pMI3 and pMI3G). The four transformation vectors were transformed into oil palm embryogenic calli via biolistic-mediated transformation. For stable transformation experiments, bombarded oil palm embryogenic calli were selected on a medium supplemented with mannose as the only carbon source (without sucrose) one month after bombardment. Transformed embryogenic calli that survived selection on mannose were later isolated, proliferated and regenerated into whole plantlets on a regeneration medium containing mannose. The status of the regenerated transgenic plantlets was confirmed by polymerase chain reaction (PCR) and Southern hybridization. Transgene expression was detected by reverse transcription (RT)-PCR analysis. The results of this study indicate that a mannose-based selection system can be successfully used in oil palm transformation.

ESTABLISHMENT OF A REGENERATION SYSTEM FOR THE GENETIC TRANSFORMATION OF GROUND-COVER CHRYSANTHEMUM 'JINDINGHONGXIN'

Ground-cover Chrysanthemum has been used in gardens due to its rich colors, excellent ornamental value. Too much precipitation in summer of north China limits its further use. In order to create chrysanthemum with high resistance to waterlogging, the Vgb (Vitreoscilla hemoglobin) and pmi (phosphomannose isomerase) genes are transferred into the genome of ground-cover chrysanthemum ‘Jindinghongxin’ by Agrobacterium tumefaciens mediated transformation in our research. This research is of profound importance to broaden the usage of ground-cover chrysanthemum in gardens. This research optimized the regeneration system of ‘Jindinghongxin’ including choosing young leaves as explants and the best regeneration culture medium (MS+2.0 mg/L 6-BA+0.5 mg/L NAA), Transformation system for ‘Jindinghongxin’ was established by pre-culturing leaf explants for 2 days. The suitable concentration of Agrobacterium tumefaciens and the suitable dipping time for transformation was OD600=0.4 for 5 minutes. Co-cultivation was done for 2 days at 28°C in the dark after infection and the explants were then put in delayed culture for 4 days. Then the explants were cultured in first selection medium with 6 g/L mannose for 15 days, and then cultured in second selection medium with 7 g/L mannose. Once the regeneration shoot was 1 cm long it was transformed into MS with 350 mg/L carbenicillin for root induction.

Selection of genetically transformed Brassica juncea L. cv. Varuna (Indian mustard) based on Positech system

The use of antibiotic and herbicide resistance based negative selection in plant transformation experiments remains a major impediment in the acceptance of transgenic crops. To overcome this, Positech selection system involving the use of phosphomannose isomerase (pmi) gene from Escherichia coli and mannose as selection agent was exploited for the selection of transgenic Brassica juncea L. cv. Varuna. The transgenic plants were generated by transformation with Agrobacterium tumefaciens harbouring the pmi gene driven by a constitutive Cestrum leaf curling viral promoter. Supplementing the selection medium with 0.09 gl-1 mannose and 5 gl-1 glucose provided the optimal condition for the selection of transformed explants. Stable integration and expression of pmi gene was confirmed by Southern and northern blot analysis, respectively. Our results show that the pmi gene driven by the constitutive Cestrum leaf curling viral promoter can be successfully used for positive selection in transformation of B.juncea, an important agronomic oil-seed crop, and that a combination of mannose and glucose rather than mannose alone is more suitable for the selection. To the best of our knowledge, Positech system has not been used so far in transformation of Brassica juncea.

Construction of phosphomannose isomerase (PMI) transformation vectors and evaluation of the effectiveness of vectors in tobacco (Nicotiana tabacum L).

Phosphomannose isomerase (pmi) gene isolated from Escherichia coli allows transgenic plants carrying it to convert mannose-6- phosphate (from mannose), a carbon source that could not be naturally utilized by plants into fructose-6-phosphate which can be utilized by plants as a carbon source. This conversion ability provides energy source to allow the transformed cells to survive on the medium containing mannose. In this study, four transformation vectors carrying the pmi gene alone or in combination with the β-glucuronidase (gusA) gene were constructed and driven by either the maize ubiquitin (Ubi1) or the cauliflower mosaic virus (CaMV35S) promoter. Restriction digestion, PCR amplification and sequencing were carried out to ensure sequence integrity and orientation. Tobacco was used as a model system to study the effectiveness of the constructs and selection system. PMI11G and pMI3G, which carry gusA gene, were used to study the gene transient expression in tobacco. PMI3 construct, which only carries the pmi gene driven by CaMV35S promoter, was stably transformed into tobacco using biolistics after selection on 30 g 1(-1) mannose without sucrose. Transgenic plants were verified using PCR analysis. PMI/pmi - Phosphomannose isomerase, Ubi1 - Maize ubiquitin promoter, CaMV35S - Cauliflower mosaic virus 35S promoter, gusA - β-glucuronidase GUS reporter gene.

Mapmi gene contributes to stress tolerance and virulence of the entomopathogenic fungus, Metarhizium acridum

Phosphomannose isomerase (PMI) catalyzes the reversible interconversion of fructose 6-phosphate (Fru-6-P) and mannose 6-phosphate (Man-6-P), providing a link between glycolysis and the mannose metabolic pathway. In this study, we identified pmi gene ( Mapmi) from the entomopathogenic fungus, Metarhizium acridum, and analyzed its functions using RNA interference (RNAi). Amending the growth medium with cell stress chemicals significantly reduced growth, conidial production and percent germination in Mapmi-RNAi mutant strain, compared to the wild-type strain. Growth of RNAi mutant was lower than the wild type strain with glucose or fructose as sole carbon source. RNAi mutant exhibited a normal growth phenotype with mannose at low concentrations, while trace or high concentration of mannose was more negatively impacted the growth of RNAi mutant than the wild type strain. Infection with Mapmi-RNAi mutant against Locusta migratoria manilensis (Meyen) led to a significantly reduced virulence compared to infection with the wild-type strain. These results suggest that Mapmi plays essential roles in stress tolerance and pathogenicity of M. acridum.

Inner‐membrane proteins PMI/TMEM11 regulate mitochondrial morphogenesis independently of the DRP1/MFN fission/fusion pathways

Mitochondria are highly dynamic organelles that can change in number and morphology during cell cycle, development or in response to extracellular stimuli. These morphological dynamics are controlled by a tight balance between two antagonistic pathways that promote fusion and fission. Genetic approaches have identified a cohort of conserved proteins that form the core of mitochondrial remodelling machineries. Mitofusins (MFNs) and OPA1 proteins are dynamin-related GTPases that are required for outer- and inner-mitochondrial membrane fusion respectively whereas dynamin-related protein 1 (DRP1) is the master regulator of mitochondrial fission. We demonstrate here that the Drosophila PMI gene and its human orthologue TMEM11 encode mitochondrial inner-membrane proteins that regulate mitochondrial morphogenesis. PMI-mutant cells contain a highly condensed mitochondrial network, suggesting that PMI has either a pro-fission or an anti-fusion function. Surprisingly, however, epistatic experiments indicate that PMI shapes the mitochondria through a mechanism that is independent of drp1 and mfn. This shows that mitochondrial networks can be shaped in higher eukaryotes by at least two separate pathways: one PMI-dependent and one DRP1/MFN-dependent.

Over-expression of the Arabidopsis Na+/H+ antiporter gene in Populus deltoides CL × P. euramericana CL “NL895” enhances its salt tolerance

Soil salinity is a serious problem worldwide. It is necessary to improve the salt tolerance of plants to avoid the progressive deterioration of saline soil. We showed that the over-expression of AtNHX1 improves salt tolerance in a transgenic poplar (Populus deltoides CL × P. euramericana CL “NL895”) under mannose selection. Four transgenic poplar plants were obtained. Southern blot analysis showed that the pmi gene had integrated into the genome of the poplar. RT-PCR confirmed that AtNHX1 could be expressed normally in the transgenic plants. When tested for salt tolerance by NaCl stress, we measured a 100% increase in Na+ content in the three transgenic lines (T18, T50, T98) significantly higher than the 33% increase seen in wild-type plants. The chlorophyll content of the transgenic plants was not altered significantly, while the chlorophyll content in the control plants showed a small decrease. MDA content was decreased in the transgenic plants. These results show that the AtNHX1 gene may enhance salt tolerance due to increased vacuolar compartmentalization of sodium ions.

Phosphomannose-isomerase as a selectable marker to recover transgenic orchid plants (Oncidium Gower Ramsey)

A transformation method using the phosphomannose-isomerase (pmi) gene as a selectable marker was developed for orchid Oncidium Gower Ramsey. The pmi-gene, which converts mannose-6-phosphate to fructose-6-phosphate allowing for selection of transgenic plants on mannose selective medium. Genetically transformed plants of Oncidium were regenerated after cocultivating protocorm-like bodies with Agrobacterium tumefaciens strain GV3101 containing the vectors pEPYON-42P and pEPYON-42H with 35S::PMI and 35S::HPTII genes respectively. We observed that 35S::PMI (pEPYON-42P) produced high rate (27 plants) of mannose resistant transgenic plants compared to 35S::HPTII (pEPYON-42H) in which only fourteen hygromycin resistant transgenic plants were obtained. Mannose resistant transgenic plants were confirmed by PCR and Southern blot. The pmi gene expression in 35S::PMI (pEPYON-42P) transgenic plants was confirmed by RT-PCR. Furthermore, the duration of regeneration time of transgenic plants was significantly shorter in mannose selected system (4 months) than in hygromycin selected system (8 months). The pmi/mannose selection system is shown to be highly efficient for producing transgenic O. Gower Ramsey without using antibiotics or herbicides. For the first time, the pmi/mannose-based “positive” selection system has been used to obtain genetically engineered O. Gower Ramsey.