The use of the phosphomannose isomerase gene as alternative selectable marker for Agrobacterium-mediated transformation of flax (Linum usitatissimum)

In order to meet the future requirement of using non-antibiotic resistance genes for the production of transgenic plants, we have adapted the selectable marker system PMI/mannose to be used in Agrobacterium-mediated transformation of flax (Linum usitatissimum L.) cv. Barbara. The Escherichia coli pmi gene encodes a phosphomannose isomerase (E.C. 5.1.3.8) that converts mannose-6-phosphate, an inhibitor of glycolysis, into fructose-6-phosphate (glycolysis intermediate). Its expression in transformed cells allows them to grow on mannose-selective medium. The Agrobacterium tumefaciens strain GV3101 (pGV2260) harbouring the binary vector pNOV2819 that carries the pmi gene under the control of the Cestrum yellow leaf curling virus constitutive promoter was used for transformation experiments. Transgenic flax plants able to root on mannose-containing medium were obtained from hypocotyl-derived calli that had been selected on a combination of 20 g L⁻¹ sucrose and 10 g L⁻¹ mannose. Their transgenic state was confirmed by PCR and Southern blotting. Transgene expression was detected by RT-PCR in leaves, stems and roots of in vitro grown primary transformants. The mean transformation efficiency of 3.6%, that reached 6.4% in one experiment was comparable to that obtained when using the nptII selectable marker on the same cultivar. The ability of T1 seeds to germinate on mannose-containing medium confirmed the Mendelian inheritance of the pmi gene in the progeny of primary transformants. These results indicate that the PMI/mannose selection system can be successfully used for the recovery of flax transgenic plants under safe conditions for human health and the environment.     

The use of phosphomannose-isomerase as a selectable marker to recover transgenic maize plants (Zea mays L.) via Agrobacterium transformation

A new selectable marker system has been adapted for use in Agrobacterium-mediated transformation of maize. This selection system utilizes the pmi gene encoding for phosphomannose-isomerase that converts mannose-6-phosphate to fructose-6-phosphate. Only transformed cells are capable of utilizing mannose as a carbon source. Agrobacterium-mediated transformation of immature embryos followed by a pre-selection of 10–14 days prior to selection at a level of 1% mannose and 0.5% sucrose led to the recovery of trangenic lines of a frequency of as high as 30% in about 12 weeks. Molecular and genetic analysis showed that selected plants contained the pmi gene and that the gene was transmitted to the progeny in a Mendelian fashion. Received: 24 August 1999 / Revision received: 27 September 1999 / Accepted: 9 November 1999     

Improved methods in Agrobacterium–mediated transformation of almond using positive (mannose/pmi) or negative (kanamycin resistance) selection-based protocols

A protocol for Agrobacterium-mediated transformation with either kanamycin or mannose selection was developed for leaf explants of the cultivar Prunus dulcis cv. Ne Plus Ultra. Regenerating shoots were selected on medium containing 15 μM kanamycin (negative selection), while in the positive selection strategy, shoots were selected on 2.5 g/l mannose supplemented with 15 g/l sucrose. Transformation efficiencies based on PCR analysis of individual putative transformed shoots from independent lines relative to the initial numbers of leaf explants tested were 5.6% for kanamycin/nptII and 6.8% for mannose/pmi selection, respectively. Southern blot analysis on six randomly chosen PCR-positive shoots confirmed the presence of the nptII transgene in each, and five randomly chosen lines identified to contain the pmi transgene by PCR showed positive hybridisation to a pmi DNA probe. The positive (mannose/pmi) and the negative (kanamycin) selection protocols used in this study have greatly improved transformation efficiency in almond, which were confirmed with PCR and Southern blot. This study also demonstrates that in almond the mannose/pmi selection protocol is appropriate and can result in higher transformation efficiencies over that of kanamycin/nptII selection protocols.     

The use of the phosphomannose-isomerase/mannose selection system to recover transgenic apple plants

A selection system based on the phosphomannose-isomerase gene (pmi) as a selectable marker and mannose as the selective agent was evaluated for the transformation of apple (Malus domestica Borkh.). Mannose is an unusable carbon source for many plant species. After uptake, mannose is phosphorylated by endogenous hexokinases to mannose-6-phosphate. The accumulation of mannose-6-phosphate leads to a block in glycolysis by inhibition of phosphoglucose-isomerase, resulting in severe growth inhibition. The phosphomannose-isomerase is encoded by the manA gene from Escherichia coli and catalyzes the conversion of mannose-6-phosphate to fructose-6-phosphate, an intermediate of glycolysis. Transformed cells expressing the manA gene can therefore utilize mannose as a carbon and survive on media containing mannose. The manA gene along with a β-glucuronidase (GUS) gene was transferred into apple cv. ‘Holsteiner Cox’ via Agrobacterium tumefaciens-mediated transformation. Leaf explants were selected on medium supplemented with different concentrations and combinations of mannose and sorbitol to establish an optimized mannose selection protocol. Transgenic lines were regenerated after an initial selection pressure of 1–2 g l⁻¹ mannose in combination with 30 g l⁻¹ sorbitol followed by a stepwise increase in the mannose concentration up to 10 g l⁻¹ and simultaneous decrease in the sorbitol concentration. Integration of transgenes in the apple genome of selected plants was confirmed by PCR and southern blot analysis. GUS histochemical and chlorophenol red (CPR) assays confirmed activity of both transgenes in regenerated plants. The pmi/mannose selection system is shown to be highly efficient for producing transgenic apple plants without using antibiotics or herbicides.     

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     

Phosphomannose isomerase: A versatile selectable marker forArabidopsis thaliana germ-line transformation

A new selection system using mannose has been evaluated for germ-line transformation ofArabidopsis thaliana. Although mannose itself has no adverse effects on plant cells, it leads to an accumulation of mannose-6-phosphate, which depletes intracellular stores of inorganic phosphate. This results in an inhibition of plant cell growth. The selection system uses theEscherichia coli pmi gene that encodes phosphomannose isomerase (PMI). Transgenic plants carrying thepmi gene can detoxify mannose-6-phosphate by conversion to fructose-6-phosphate, an intermediate of glycolysis, via the PMI activity. Germ-line transformation ofA. thaliana followed by sterile selection on 2–5 mM of mannose resulted in the isolation of mannose-6-phosphate-resistant progeny in about 2.5% of the treated seed, consistent with transformation rates using other selection schemes. Integrative transformation was confirmed by Southern hybridization. Analysis of PMI enzyme activity demonstrated a 5-fold range of activity levels, although these differences had little effect on the ability to select transformed plants or on the growth of transformed plants on mannose. Finally, mannose selection using thepmi gene could be accomplished in sterile plates and in soil, making this an extremely versatile tool forA. thaliana transformation.     

Development of a phosphomannose isomerase-based <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation system for chickpea (<Emphasis Type="Italic">Cicer arietinum</Emphasis> L.)

To develop an alternative genetic transformation system that is not dependent on an antibiotic selection strategy, the phosphomannose isomerase gene (pmi) system was evaluated for producing transgenic plants of chickpea (Cicer arietinum L.). A shoot morphogenesis protocol based on the thidiazuron (TDZ)-induced shoot morphogenesis system was combined with Agrobacterium-mediated transformation of the pmi gene and selection of transgenic plants on mannose. Embryo axis explants of chickpea cv. C-235 were grown on a TDZ-supplemented medium for shoot proliferation. Embryo axis explants from which the first and second flush of shoots were removed were transformed using Agrobacterium carrying the pmi gene, and emerging shoots were allowed to regenerate on a zeatin-supplemented medium with an initial selection pressure of 20 g l⁻¹ mannose. Rooting was induced in the selected shoots on an indole-3-butyric acid (IBA)-supplemented medium with a selection pressure of 15 g l⁻¹ mannose. PCR with marker gene-specific primers and chlorophenol red (CPR) assay of the shoots indicated that shoots had been transformed. RT-PCR and Southern analysis of selected regenerated plants further confirmed integration of the transgene into the chickpea genome. These positive results suggest that the pmi/mannose selection system can be used to produce transgenic plants of chickpea that are free from antibiotic resistance marker genes.     

EfficientAgrobacterium-mediated transformation of rice by phosphomannose isomerase/mannose selection

A positive selection system was developed forAgrobacterium-mediated transformation of rice that does not use toxic compounds such as antibiotics or herbicides. The selection system is based on theEscherichia coli phosphomannose isomerase (pmi) gene as a selectable marker and mannose as the selective agent. Only transgenic plants were able to metabolize mannose into a usable source of carbon, fructose. Selection was achieved using a combination of mannose and sucrose at 10 g/L and 5 g/L, respectively. Transgenic rice plants were produced efficiently injapanica rice variety Zhonghua 8, with transformation frequency of 16.5%, which was slightly lower than that achieved by hygromycin selection.     

Phosphomannose isomerase: An efficient selectable marker for plant transformation

Summary Phosphomannose isomerase (PMI) catalyzes the reversible interconversion of mannose 6-phosphate and fructose 6-phosphate. Plant cells lacking this enzyme are incapable of surviving on synthetic medium containing mannose as a carbon source. Maize, wheat and barley plants, genetically modified to express the Escherichia coli manA gene (pmi) under the control of a plant promoter, were able to survive selection on mannose-containing medium. Transformation frequencies averaged 45% for maize transformation via Biolistics^™ 35% for maize Agrobacterium-mediated transformation, 20% for wheat, 3% for barley, and 2% for watermelon transformation. Moreover, the frequencies exceeded those obtained for maize and wheat using the pat or bar gene with Basa^? selection. A preliminary safety assessment has been conducted for PMI. Purified PMI protein demonstrates no adverse, effects in an acute mouse toxicity test. Purified PMI protein was readily digested in simulated mammalian gastric and intestinal fluids. Plants derived from surgar beet and corn cells that had been genetically modified to express the E. coli manA gene were evaluated for biochemical changes in mannose-associated pathways. No detectable changes in glycoprotein profiles were detected in PMI-transformed plants as compared to nontransgenic controls. The yield and nutritional composition of grain from PMI-transformed corn plants compared to their non-transformed isogenic counterparts were also determined and no statistically significant differences were found. The inherent safety of a system based on simple sugar metabolism coupled with high transformation frequencies for monocots make pmi and ideal selectable marker for plant transformation.     

Successful genetic transformation of Chinese cabbage using phosphomannose isomerase as a selection marker

A mannose selection system was adapted for use in the Agrobacterium-mediated transformation of Chinese cabbage. This system makes use of the pmi gene that encodes phosphomannose isomerase, which converts mannose-6-phosphate to fructose-6-phosphate. Hypocotyl explants from 4–5-day-old seedlings of Chinese cabbage inbred lines were pre-cultured for 2–3 days and then infected with Agrobacterium. Two genes (l-guluno-γ-lactone oxidase, GLOase, and jasmonic methyl transferase, JMT) were transformed into Chinese cabbage using the transformation procedure developed in this study. We found that supplementing the media with 7 g l⁻¹ mannose and 2% sucrose provides the necessary conditions for the selection of transformed plants from nontransformed plants. The transformation rates were 1.4% for GLOase and 3.0% for JMT, respectively. The Southern blot analysis revealed that several independent transformants (T ₀) were obtained from each transgene. Three different inbred lines were transformed, and most of the T ₁ plants had normal phenotypes. The transformation method presented here for Chinese cabbage using mannose selection is efficient and reproducible, and it can be useful to introduce a desirable gene(s) into commercially useful inbred lines of Chinese cabbage.     

RNA virus accumulation is inhibited by ribonuclease activity of 3D8 scFv in transgenic Nicotiana tabacum

A mannose selection system was adapted for Agrobacterium-mediated transformation of plum (Prunus domestica L.) hypocotyl explants and the recovery of transgenic plants. Adventitious regeneration from non-transformed hypocotyl sections was inhibited when 3 mg/l mannose, combined with 10 mg/l sucrose, was added to the medium. Mature seed hypocotyl slices from the cultivar ‘Claudia Verde’ were infected with A. tumefaciens AGL1, carrying the pNOVgus vector, and placed onto different selective media with mannose. A low mannose selection (1.5 g/l, regeneration below the inhibitory concentration) applied for 16 weeks led to the regeneration of escapes. However, when mannose at 1.5 g/l or at 3 g/l (the regeneration-inhibiting concentration) was applied for 6 weeks from the beginning of the experiments and, after that, was increased to 5 g/l, several independent transgenic lines were obtained. The transformation events were monitored by detection of the GUS enzymatic activity at different stages of the process. Nevertheless, stable integration of transgenes into the genome of the plum plants was confirmed by PCR and Southern blot analysis. The transformed shoots were rooted on a medium supplemented with 10 g/l sucrose and 4 g/l mannose. The transformation procedure described here, using the pmi/mannose system for selection of transgenic plum plants, represents an alternative for the production of transgenic plum plants under conditions that are safe regarding human health and the environment, and would permit the insertion of more transgene/s in a pre-existing transgenic line.     

Phosphomannose-isomerase as a selectable marker to recover transgenic orchid plants (<Emphasis Type="Italic">Oncidium</Emphasis> 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.     

The effect of co-cultivation and selection parameters on <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of Chinese soybean varieties

In the present study, an efficient Agrobacterium-mediated gene transformation system was developed for soybean [Glycine max (L.) Merrill] based on the examinations of several factors affecting plant transformation efficiency. Increased transformation efficiencies were obtained when the soybean cotyledonary node were inoculated with the Agrobacterium inoculum added with 0.02% (v/v) surfactant (Silwet L-77). The applications of Silwet L-77 (0.02%) during infection and l-cysteine (600 mg l⁻¹) during co-cultivation resulted in more significantly improved transformation efficiency than each of the two factors alone. The optimized temperature for infected explant co-cultivation was 22°C. Regenerated transgenic shoots were selected and produced more efficiently with the modified selection scheme (initiation on shoot induction medium without hygromycin for 7 days, with 3 mg l⁻¹ hygromycin for 10 days, 5 mg l⁻¹ hygromycin for another 10 days, and elongation on shoot elongation medium with 8 mg l⁻¹ hygromycin). Using the optimized system, we obtained 145 morphologically normal and fertile independent transgenic plants in five important Chinese soybean varieties. The transformation efficacies ranged from 3.8 to 11.7%. Stable integration, expression and inheritance of the transgenes were confirmed by molecular and genetic analysis. T₁ plants were analyzed and transmission of transgenes to the T₁ generation in a Mendelian fashion was verified. This optimized transformation system should be employed for efficient Agrobacterium-mediated soybean gene transformation.     

Effective selection and regeneration of transgenic rice plants with mannose as selective agent

A new method for the selection of transgenic rice plants without the use of antibiotics or herbicides has been developed. The phosphomannose isomerase (PMI) gene from Escherichia coli has been cloned and consitutively expressed in japonica rice variety TP 309. The PMI gene was transferred to immature rice embryos by Agrobacterium-mediated transformation, which allowed the selection of transgenic plants with mannose as selective agent. The integration and expression of the transgene was confirmed by Southern and northern blot analysis and the activity of PMI indirectly proved with the chlorophenol red assay. The results of genetic analysis showed that the transgenes were segregated in a Mendelian fashion in the T₁ generation. The establishment of this selection system in rice provides an efficient way for producing transgenic plants without using antibiotics or herbicides with a transformation frequency of up to 41%.     

Assessment of transgenic maize events produced by particle bombardment or Agrobacterium-mediated transformation

Particle bombardment and Agrobacterium-mediated transformation are two popular methods currently used for producing transgenic maize. Agrobacterium-mediated transformation is expected to produce transformants carrying fewer copies of the transgene and a more predictable pattern of integration. These putative advantages, however, tradeoff with transformation efficiency in maize when a standard binary vector transformation system is used. Using Southern, northern, real-time PCR, and real-time RT-PCR techniques, we compared transgene copy numbers and RNA expression levels in R₁ and R₂ generations of transgenic maize events generated using the above two gene delivery methods. Our results demonstrated that the Agrobacterium-derived maize transformants have lower transgene copies, and higher and more stable gene expression than their bombardment-derived counterparts. In addition, we showed that more than 70% of transgenic events produced from Agrobacterium-mediated transformation contained various lengths of the bacterial plasmid backbone DNA sequence, indicating that the Agrobacterium-mediated transformation was not as precise as previously perceived, using the current binary vector system.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of cotton (<Emphasis Type="Italic">Gossypium hirsutum</Emphasis> L.) via vacuum infiltration

AnAgrobacterium-mediated gene transfer system with recovery of putative transformants was developed for cotton (Gossypium hirsutum L.) cv. Cocker-312. Two-month-old hypocotyl-derived embryogenic calli were infected through agroinfiltration for 10 min at 27 psi in a suspension ofAgrobacterium tumefaciens strain GV3101 carrying tDNA with theGUS gene, encoding β-glucuronidase (GUS), and the neomycin phosphotransferase II (nptII) gene as a kanamycin-resistant plant-selectable marker. Six days after the histochemicalGUS assay was done, 46.6% and 20%GUS activity was noted with the vacuum-infiltration and commonAgrobacterium-mediated transformation methods, respectively. The transformed embryogenic calli were cultured on selection medium (100 mg/L and 50 mg/L kanamycin for 2 wk and 10 wk, respectively) for 3 mo. The putative transgenic plants were developed via somatic embryogenesis (25 mg/L kanamycin). In 4 independent experiments, up to 28.23% transformation efficiency was achieved. PCR amplification and Southern blot analysis fo the transformants were used to confirm the integration of the transgenes. Thus far, this is the only procedure available for cotton that can successfully be used to generate cotton transformants.     

Comparative analysis of transgenic rice plants obtained by Agrobacterium-mediated transformation and particle bombardment

We compared rice transgenic plants obtained by Agrobacterium-mediated and particle bombardment transformation by carrying out molecular analyses of the T₀, T₁ and T₂ transgenic plants. Oryza sativa japonica rice (c.v. Taipei 309) was transformed with a construct (pWNHG) that carried genes coding for neomycin phosphotransferase (nptII), hygromycin phosphotransferase (Hyg^r), and -glucuronidase (GUS). Thirteen and fourteen transgenic lines produced via either method were selected and subjected to molecular analysis. Based on our data, we could draw the following conclusions. Average gene copy numbers of the three transgenes were 1.8 and 2.7 for transgenic plants obtained by Agrobacterium and by particle bombardment, respectively. The percentage of transgenic plants containing intact copies of foreign genes, especially non-selection genes, was higher for Agrobacterium-mediated transformation. GUS gene expression level in transgenic plants obtained from Agrobacterium-mediated transformation was more stable overall the transgenic plant lines obtained by particle bombardment. Most of the transgenic plants obtained from the two transformation systems gave a Mendelian segregation pattern of foreign genes in T₁ and T₂ generations. Co-segregation was observed for lines obtained from particle bombardment, however, that was not always the case for T₁ lines obtained from Agrobacterium-mediated transformation. Fertility of transgenic plants obtained from Agrobacterium-mediated transformation was better. In summary, the Agrobacterium-mediated transformation is a good system to obtain transgenic plants with lower copy number, intact foreign gene and stable gene expression, while particle bombardment is a high efficiency system to produce large number of transgenic plants with a wide range of gene expression.     

The Novel Use of a Combination of Sonication and Vacuum Infiltration in <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated Transformation of Kidney Bean (<Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> L.) with <Emphasis Type="Italic">lea</Emphasis> Gene

An efficient gene transfer system without tissue culture steps was developed for kidney bean by using sonication and vacuum infiltration assisted, Agrobacterium-mediated transformation. Transgenic kidney bean with a group 3 lea (late embryogenesis abundant) protein gene from Brassica napus was produced through this approach. Among 18 combinations of transformation methods, Agrobacterium-mediated transformation combined with 5 min sonication and 5 min vacuum infiltration turned to be optimal, resulting in the highest transformation efficiency. Transgenic kidney bean plants demonstrated enhanced growth ability under salt and water deficit stress conditions. The increased tolerance was also reflected by delayed development of damage symptoms caused by drought stress. Transgenic lines with high level of lea gene expression showed higher stress tolerance than lines with lower expression level. Stress tolerance of transgenic kidney bean correlated much better with lea gene expression levels than with gene integration results. There is no prior report on the production of transgenic kidney bean using both ultrasonic and vacuum infiltration assisted, Agrobacterium-mediated transformation.     

Production of herbicide-resistant transgenic sweet potato plants through <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis> method

Transgenic herbicide-resistant sweet potato plants [Ipomoea batatas (L.) Lam.] were produced through Agrobacterium-mediated transformation system. Embryogenic calli derived from shoot apical meristems were infected with Agrobacterium tumefaciens strain EHA105 harboring the pCAMBIA3301 vector containing the bar gene encoding phosphinothricin N-acetyltransferase (PAT) and the gusA gene encoding β-glucuronidase (GUS). The PPT-resistant calli and plants were selected with 5 and 2.5 mg l⁻¹ PPT, respectively. Soil-grown plants were obtained 28–36 weeks after Agrobacterium-mediated transformation. Genetic transformation of the regenerated plants growing under selection was demonstrated by PCR, and Southern blot analysis revealed that one to three copies of the transgene were integrated into the plant genome of each transgenic plant. Expression of the bar gene in transgenic plants was confirmed by RT-PCR and application of herbicide. Transgenic plants sprayed with Basta containing 900 mg l⁻¹ of glufosinate ammonium remained green and healthy. The transformation frequency was 2.8% determined by herbicide application which was high when compared to our previous biolistic method. In addition, possible problems with multiple copies of transgene were also discussed. We therefore report here a successful and reliable Agrobacterium-mediated transformation of the bar gene conferring herbicide-resistance and this method may be useful for routine transformation and has the potential to develop new varieties of sweet potato with several important genes for value-added traits such as enhanced tolerance to the herbicide Basta.     

Genetic transformation of Coffea canephora by vacuum infiltration

Agrobacterium-mediated plant transformation protocol was evaluated as a fast method to obtain genetically modified Coffea canephora plantlets. Leaf explants were used as source material for Agrobacterium tumefaciens-mediated transformation involving a vacuum infiltration protocol, followed by a step of somatic embryogenesis induction and a final selection of the transformed plants. A. tumefaciens strain C58CI containing the binary vector pER10W-35SRed was used. PCR amplification of DsRFP gene and visual detection of the red fluorescent protein demonstrated 33% transformed embryos. The protocol presented here produces reliable transgenic coffee embryos in two months.