Method for producing the transformants of coffee plants and transgenic coffee plants

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to a method for producing the stable transformants of coffee plants.

2. Description of Related Art

Coffee is a commercially important woody shrub planted in a large scale for harvesting its beans. Among more than 80 species, the most economically important are Coffee arabica (2n-44) and C. canephora (2n-22). In C. arabica, genetic diversity is limited by conventional breeding because of its self-pollination characteristic, and the plants are highly sensitive to pests and diseases. C. canephora, used for instant coffee powder products, is a cross-pollinated specie but has low production quality. Conventional breeding of coffee is difficult because of the long duration of cultivation to set seeds. Molecular breeding, therefore, is a desirable technique for the genetic improvement of coffee species, although production of transgenic coffee plants via gene transformation has generally been considered problematic.

Plant regeneration via in vitro tissue culture is a basic system for achieving genetic transformation, and there have been many reports involving somatic embryogenesis in coffee plants (Staritsky, 1970; Hatanaka et al. 1991; Menendez-Yuffa and Garcia, 1996). However, data for genetic transformation of coffee are limited. Barton et al. (1991) obtained transformants from electroporated protoplasts of C. arabica, but the cultured protoplasts did not develop into whole plants. Spiral et al. (1993) reported the transformation of coffee (C. canephora) by co-cultivation of Agrobacterium rhizogenes with microcut-somatic embryos. However, the efficiency of transformation was very low. Van Boxtel et al. (1995) reported only transient expression of GUS genes on the surfaces of coffee leaf tissues following biolistic delivery.

SUMMARY OF THE INVENTION

Agrobacterium tumefaciens-mediated transformation is considered to be best for plant transformation because of the availability of vectors. Despite such advantage, no report has been presented of successful coffee transformation using Agrobacterium tumefaciens strains, except for GUS positive transgenic callus induction at a low frequency reported from Ocampo and Manzanera (1991).

This invention provides the successful genetic transformation of Coffea canephora using Agrobacterium tumefaciens EHA101 harboring pIG121-Hm from embryogenic calli.

Embryogenic calli were induced from leaf explants of Coffea canephora on McCown's woody plant medium (WPM) supplemented with 5 .mu.M N.sup.6 -[2-isopentenyl]-adenosine (2-iP). These calli were co-cultured with Agrobacterium tumefaciens EHA101 harboring pIG121-Hm, containing .beta.-glucuronidase (GUS)-, hygromycin phosphotransferase (HPT)- and neomycin phosphotransferase II (NPT II) genes. Selection of putative transgenic callus was performed by gradual increase in hygromycin concentrations (5, 50, 100 mg/l). The embryogenic calli surviving on a medium containing 100 mg/l hygromycin showed a strong GUS positive reaction with X-gluc solution. Somatic embryos were formed and germinated from these putative transgenic calli on WPM medium with 5 .mu.M 2-iP. Regenerated small plantlets with shoots and roots were transferred to a medium containing both 100 mg/l hygromycin and 100 mg/l kanamycin for final selection of transgenic plants. The selected plantlets exhibited strong GUS activity in leaves and roots as indicated by a deep blue color. GUS and HPT genes were confirmed to be stably integrated into the genome of the coffee plants by the polymerase chain reaction (PCR).

Moreover, the inventors have succeeded in production of a transgenic plant of Coffea arabica, wherein phosphinothricin acetyl transferase (BAR) gene was incorporated to render resistance against herbicide. Commercially, Coffea arabica is more valuable than Coffea canephora. Embryogenic calli derived from Coffea arabica were induced from leaf explants of coffee on Murashige and Skoog (MS) medium supplemented with 10 .mu.M N.sup.6 -[2-isopentenyl]-adenosine (2-iP). These calli were co-cultured with Agrobacterium tumefaciens EHA101 harboring pSMBuba, containing herbicide resistant BAR gene and hygromycin phosphotransferase (HPT) gene. Selection of putative transgenic callus was performed by gradual increase in hygromycin concentrations (25, 50 mg/l). The embryogenic calli maintained on MS medium with 50 mg/l hygromycin and 10 .mu.M 2-iP. Prolonged culture of embryogenic callus induced somatic embryos. Germination of somatic embryos strongly enhanced by GA.sub.3 treatment and developed into transgenic plantlets after 2 months of culture. Transgenic embryogenic callus, somatic embryos and small plantlets were tolerant to 2 mg/l Bialaphos. Whereas non-transformed ones were dead after 1 month. Prescence of HPT and BAR genes in those transgenic plantlets was confirmed by the genomic PCR and Northern assays.

This invention provides a method to incorporate an exogenous gene using Agrobacterium tumefaciens mediated method. Embryogenic calli were induced from leaf explants of coffee plants. The embryogenic calli thus obtained were infected by Agrobacterium tumefaciens, harboring a plasmid containing an exogenous gene to be incorporated and hygromycin phosphotransferase (HPT) gene. Putative transformed calli were selected using the hygromycin resistance as an indicator. And then somatic embryos were induced from the putative transformed calli. Transformed plantlets can be regenerated from the somatic embryos thus obtained.

Various species of coffee plants can be transformed using the method of this invention. The coffee plant species may preferably be cultivative coffee species such as Coffea arabica, Coffea canephora, Coffea liberica and Coffea dewevrei.

Theoretically, any exogenous gene can be incorporated into coffee plants by the method of this invention. The exogenous genes to be incorporated may preferably be caffeine synthetase gene, herbicide resistance gene such as phosphinothricin acetyl transferase (BAR) gene, insect injury resistance gene such as Bacillus thuringiensis gene, and disease resistance gene such as chitinase gene and glucanase gene.

Other and further objects, features and advantages of the invention will appear more fully from the following descriptions. It is to be understood that, examples mentioned above and description of detailed embodiments are not to be intended to limit the range of this invention.

BRIEF DESCRIPTION OF THE DRAWINGS

The file of this patent contains at least one drawing executed in color. Copies of this patent, with color drawings, will be provided by the Patent and Trademark Office upon request and payment of the necessary fee.

FIG. 1 shows embryogenic coffee callus surviving on WPM medium containing 100 mg/l hygromycin after co-cultivation with Agrobacterium tumefaciens EHA 101.

FIG. 2 shows GUS activity staining of transformed or non-transformed embryogenic calli.

FIG. 3 shows formation of somatic embryo, derived from transformed embryogenic calli.

FIG. 4 shows GUS activity staining of somatic embryos, derived from transformed or non-transformed embryogenic calli.

FIG. 5 shows small plantlets cultured on WPM medium containing both 100 mg/l hygromycin and 100 mg/l kanamycin.

FIG. 6 shows hygromycin and kanamycin resistant putative transgenic plantlets after transfer to WPM medium supplemented with 3% sucrose.

FIG. 7 shows GUS activity staining of leaves, derived from a non-transformed or transformed coffee plant.

FIG. 8 shows GUS activity staining of roots, derived from a non-transformed or transformed coffee plant.

FIG. 9 shows a transgenic coffee plantlets after transfer to soil.

FIG. 10 shows frequency of GUS positive calli from survived embryogenic calli of coffee on selection media.

FIG. 11 shows detection of GUS gene using PCR, wherein the lanes T1 to T4 indicate results of transformed samples and the lane N indicates that of non-transformed sample.

FIG. 12 shows detection of HPT gene using PCR, wherein the lanes T1 to T4 indicate results of transformed samples and lane N indicates that of non-transformed sample.

FIG. 13 shows embryogenic coffee callus derived from a leaf explant of Coffea arabica.

FIG. 14 shows embryogenic calli maintained in MS medium containing 2-iP.

FIG. 15 shows survived embryogenic callus on the surface of browned non-transformed calli.

FIG. 16 shows somatic embryo formation from a transformed embryogenic callus of Coffea arabica.

FIG. 17 shows somatic embryo on 1/2 MS medium containing GA.sub.3.

FIG. 18 shows browned small plantlets cultured on 1/2 MS medium containing 2 mg/l bialaphos.

FIG. 19 shows the bialaphos resistance of transgenic small plantlets.

FIG. 20 shows a transgenic plantlets of Coffea arabica grew on 1/2 MS medium in flasks.

FIG. 21 shows detection of BAR gene using PCR, wherein the lanes T1 to T4 indicate results of transformed samples and the lane N indicates that of non-transformed sample.

FIG. 22 shows detection of HPT gene using PCR, wherein the lanes T1 to T4 indicate results of transformed samples and the lane N indicates that of non-transformed sample.

DETAILED DESCRIPTION OF EMBODIMENTS

EMBODIMENT 1

Production of a Transformant of Coffea canephora

Induction of Embryogenic Calli

Leaf explants of coffee (Coffea canephora) were prepared from leaves of greenhouse-grown trees, according to the method previously described (Hatanaka et al. 1991). The leaf explants were cultured on woody plant agar (0.9%) media (WPM) which consisted of McCown's woody plant salt mixture (Lloyd and McCown, 1981), Gamborg's B.sub.5 (Gamborg et al. 1968) vitamins, 3% sucrose and 5 .mu.M N.sup.6 -[2-isopentenyl]-adenosine (2-iP). The medium was adjusted to pH 5.7 before autoclaving at 120.degree. C. for 15 min. The culture room was maintained at 25.degree. C. with 16-h light illumination of 24 .mu.mol m.sup.-2 s.sup.-1 (white fluorescent tubes).

Agrobacterium Transformation

After 4 months of the above culture, embryogenic calli induced from the leaf explants were transferred to callus proliferation medium (CM) which consisted of MS salts (Murashige and Skoog, 1962), 0.25% Gellan Gum, B.sub.5 vitamin, 3% sucrose and 10 .mu.M 2,4-dichlorophenoxyacetic acid (2,4-D). The CM medium was also adjusted to pH 5.7 before autoclaving at 120.degree. C. for 15 min. Agrobacterium tumefaciens EHA101 harboring pIG121-Hm containing .beta.-glucuronidase (GUS)-, hygromycin phosphotransferase (HPT)- and neomycin phosphotransferase II (NPT II) genes in the T-DNA region of the plasmid was used for the transformation. Freshly subcultured embryogenic calli (3 days after culture) were co-cultivated in bacterial suspension (absorbance of 0.6 at 600 nm) for 30 min at 25.degree. C. in WPM liquid medium containing 5 .mu.M 2-iP and 50 mg/l acetosyringone, then these calli were transferred to WPM agar medium containing 50 mg/l acetosyringone, 3% sucrose and 5 .mu.M 2-iP at 25.degree. C. in the dark for four days. To eliminate bacteria, the calli were washed 5 times with sterilized water, followed by water containing 300 mg/l cefotaxime once. Thereafter the embryogenic calli were cultured on WPM agar medium containing 300 mg/l cefotaxime, 5 mg/l hygromycin, and 5 .mu.M 2-iP, and subcultured on the same medium at 2 week intervals. After 2 months of culture, embryogenic calli were transferred to fresh medium with an increased concentration of hygromycin (50 mg/l). After 2 months of culture, each line of embryogenic callus was maintained by transferring to fresh WPM agar medium containing 5 .mu.M 2-iP and 100 mg/l hygromycin.

Somatic Embryogenesis and Plant Regeneration

After selection at the concentration of 100 mg/l hygromycin, survived embryogenic calli were transferred to WPM medium containing 5 .mu.M 2-iP in 10.times.2 cm plastic Petri dishes. Partially germinated embryos (about 1-2 cm in length) were transferred to phytohormone-free WPM agar medium containing both 100 mg/l hygromycin and 100 mg/l kanamycin for final selection of transgenic plantlets. After selection, they were cultured on WPM agar medium without growth regulators to support continued growth in 300 ml culture bottles. Plantlets with both shoots and roots were transferred to plastic pots containing soil and peat moss (1:1 v/v) in a greenhouse.

Histochemical GUS Assay

Histochemical assays of GUS were performed for hygromycin-resistant embryogenic calli, somatic embryos, and leaves and roots of plantlets, according to the method of Van Boxtel et al. (1995). For staining, the materials were incubated in 5-bromo-4-chloro-3-indolyl-.beta.-D-glucuronide (X-gluc) solution with a composition modified to 50 mM Na.sub.2 HPO.sub.4, 10 mM Na.sub.2 EDTA, 0.3% Triton X-100, 0.5 mM K.sub.3 Fe(CN).sub.6, 0.5 mM K.sub.4 Fe(CN).sub.6, and antioxidants (0.5% caffeine, 1% PVP and 1% sodium ascorbate). After 16 hours at 37.degree. C., these explants were immersed in 99.5% ethanol for chlorophyll bleaching and observed under a dissecting microscope.

Agrobacterium-mediated Transformation and Somatic Embryogenesis

Friable and yellow (FIG. 1) embryogenic calli were obtained from leaf explants of coffee (Coffea canephora) after 4 months of culture on WPM agar medium with 5 .mu.M 2-iP. Embryogenic calli were co-cultivated with Agrobacterium tumefaciens EHA101, a super-virulent line for rice transformation (Hiei et al. 1994; Yokoi et al. 1996), in WPM medium containing 50 mg/l 2 acetosyringone and 5 .mu.M 2-iP for 30 min. After washing in sterilized water, embryogenic calli were transferred to CM solid medium containing 50 mg/l acetosyringone in the dark for 4 days. It has been reported that acetosyringone treatment is highly effective for increasing the transformation efficiency (James et al. 1993). To eliminate remnant bacteria, embryogenic calli were transferred to WPM medium containing 300 mg/l cefotaxime, 5 mg/l hygromycin and 5 .mu.M 2-ip. After 2 months of culture, about 90% of the calli (267 out of 298 calli) survived on WPM medium with 5 mg/l hygromycin, and 96 calli (36.0%) demonstrated GUS positive blue spots after immersion in X-gluc solution (FIG. 10). Thereafter, the survived calli were transferred to the same medium containing 50 mg/l hygromycin. After 2 months of culture, 81 (43.3%) out of 187 calli that survived on medium with 50 mg/l hygromycin had blue spots by X-gluc reaction (FIG. 10). These 187 calli were transferred to WPM medium containing 100 mg/l hygromycin and 131 calli (70.1%) continued to proliferate even after 2 months of incubation. When the hygromycin resistant embryogenic calli were reacted with X-gluc solution, 90 calli (68.7%) showed a strong GUS positive reaction (FIG. 2, arrows, FIG. 10). However, embryogenic calli without co-cultivation did not show any GUS activity (FIG. 2, arrowhead).

After selection of survived embryogenic calli in the presence of 100 mg/l hygromycin, embryogenic calli were transferred to WPM medium with 5 .mu.M 2-iP. Numerous somatic embryos were formed from the putative transgenic calli after 2 months of culture (FIG. 3). The X-gluc reaction revealed that somatic embryos (FIG. 4, arrows) were formed from hygromycin resistant embryogenic calli to be positive. Somatic embryos from non-transformed embryogenic calli (FIG. 4, arrowhead) were stained negatively except for intrinsic reaction with pale blue color. It had been reported that intrinsic GUS-like activity was observed in immature and mature somatic embryos of coffee (Van Boxtel et al. 1995).

A Production of Transgenic Plantlets

Somatic embryos germinated and regenerated to small plantlets with shoots and roots after transferring to WPM medium lacking growth regulators. To check finally the transgenic plantlets, the small plantlets (1-2 cm in length) were transferred to WPM medium containing both 100 mg/l hygromycin and 100 mg/l kanamycin. In this medium, non-transformed plantlets did not grow at all and rapidly browned (FIG. 5, arrowheads), whereas transformed plantlets grew very well (FIG. 5, arrows). Especially, the roots thrived without showing any growth suppression and browning. Eighty seven % of plantlets survived on this medium. After transfer to a medium without growth regulators in Petri dishes (FIG. 6) or 300 ml culture bottles, these plantlets grew to about 7 cm in height with about 6-10 leaves and formed well-developed roots after 3-5 months of culture. Transgenic plantlets were transferred to a mixture of autoclaved soil in a greenhouse. Most of the plants survived without wilting and the loss of their green color (FIG. 9).

The leaves (FIG. 7, arrow) and roots (FIG. 8, arrow) of the putative transgenic plantlets demonstrated a deep blue color on reaction with X-gluc. Explants from non-transformed plantlets (FIGS. 7-8, arrowheads) did not react with X-gluc. While leaf tissues in the transformed case were not always stained by X-gluc, the roots always showed a strong GUS positive reaction. Furthermore, surgical wounding on leaf surfaces increased their positivity (FIG. 7), suggesting a blocking effect of the well-developed cuticle of the coffee leaf.

PCR Analysis of GUS and HPT Genes

DNA extraction from leaves of coffee plantlets having positive GUS activity was carried out according to the described procedure (Kikuchi et al. 1998) using the modified (addition of 3% 2-mercaptoethanol in solution 1) benzyl chloride method (ISOPLANT kit, Wako Co.). The primers [SEQ ID NOS.: 1-4] used for amplifying the GUS gene were 5'-AATTGATCAGCGTTGGTGG-3' and 5'-ACGCGTGGTTACAGTCTTGC-3' and those for the HPT gene were 5'-GCGTGACCTATTGCATCTCC-3' and 5'-TTCTACACAGCCATCGGTCC-3'. The reaction mixture for PCR was incubated in a DNA thermal cycler (Perkin Elmer Cetus, 9700) under the following conditions: 96.degree. C. for 5 min, followed by 30 cycles of 94.degree. C. for 30 sec, 58.degree. C. for 30 sec, and 72.degree. C. for 2 min with a final 5 min extension at 72.degree. C.

Examination of the leaves of GUS positive transgenic plantlets (T) by PCR revealed amplified fragments coinciding with the GUS (515 bp band in FIG. 11) and HPR (713 bp band in FIG. 12) genes. In non-transformed plantlets (N), neither GUS nor HPR genes were detectable.

EMBODIMENT 2

Production of a Tranformant of Coffea arabica with Herbicide Resistance

Induction of Embryogenic Calli

Leaf explants of coffee (Coffea arabica) were prepared from leaves of greenhouse-grown trees, according to the method described previously (Hatanaka et al. 1991). Leaf explants were cultured on Murashige and Skoog agar (0.9%) medium (Murashige and Skoog, 1962) containing Gamborg's B.sub.5 (Gamborg et al. 1968) vitamins, 3% sucrose and 10 .mu.M N.sup.6 -[2-isopentenyl]-adenosine (2-iP). The medium was adjusted to pH 5.7 before autoclaving at 120.degree. C. for 15 min. The culture room was maintained at 25.degree. C. with 16-h light illumination of 24 .mu.mol m.sup.-2 s.sup.-1 (white fluorescent tubes).

Agrobacterium Transformation

After selection of embryogenic callus, these calli were serially subcultured by two-week intervals onto MS medium supplemented with 0.9% agar, B.sub.5 vitamin, 3% sucrose and 10 .mu.M 2-iP to induce friably embryogenic callus. Agrobacterium tumefaciens EHA101 harboring pSMBuba containing BAR and hygromycin phosphotransferase (HPT) genes in the T-DNA region of the plasmid was used for the transformation. Freshly subcultured embryogenic calli (3 days after culture) were co-cultivated in bacterial suspension (absorbance of 0.6 at 600 nm) for 30 min at 25.degree. C. in MS liquid medium containing 10 .mu.M 2-iP and 10 mg/l acetosyringone, then these calli were transferred to MS agar medium containing 10 mg/l acetosyringone, 3% sucrose and 10 .mu.M 2-iP at 25.degree. C. in the dark for four days. To eliminate bacteria, the calli were washed for 5 times with sterilized water, followed by water containing 300 mg/l cefotaxime once. Thereafter the embryogenic calli were cultured on MS agar medium containing 300 mg/l cefotaxime and 10 .mu.M 2-iP, and subcultured on the same medium at 2 week intervals. After 2 months of culture, embryogenic calli were transferred to fresh MS medium containing hygromycin (25 mg/l) for one month. Thereafter, each line of embryogenic callus was maintained by transferring to fresh MS agar medium containing 10 .mu.M 2-iP and 50 mg/l hygromycin by three weeks of culture cycle.

Somatic Embryogenesis and Plant Regeneration

To induce somatic embryos, embryogenic callus maintained on MS medium with 10 .mu.M 2-iP was transferred to MS medium with 3 .mu.M 2-iP. Somatic embryos developed spontaneously from embryogenic callus. After selection of somatic embryos, they were cultured on 1/2 MS agar medium with 10 .mu.M GA.sub.3 to support germination. After 3 weeks of culture, small plantlets were transferred to 1/2 MS agar medium in 300 ml Erlenmeyer flasks to support the further growth.

Observation of Bialaphos Resistance

Hygromycin-resistant embryogenic calli, somatic embryos, and small plantlets survived on medium containing 50 mg/l hygromycin were transferred to 1/2 MS medium containing 2 mg/l bialaphos. After one month of culture, survival rate was examined.

PCR Analysis of BAR and HPT Genes

DNA extraction from small coffee plantlets resistant to hygromycine was carried out according to a described procedure (Kikuchi et al. 1998) using a modified (addition of 3% 2-mercaptoethanol in solution 1) benzyl chloride method (ISOPLANT kit, Wako Co.). The primers [SEQ ID NOS.: 5-6 and 3-4, respectively] used for amplifying the bar gene were 5'-ATGAGCCCAGAACGACGCCCG-3' (forward) and 5'-GCTCTTGAAGCCCTGTGCCTCC-3' (reverse), and those for the BPT gene were 5'-GCGTGACCTATTGCATCTCC-3' (forward) and 5'-TTCTACACAGCCATCGGTCC-3' (reverse). The reaction mixture for PCR was incubated in a DNA thermal cycler (Perkin Elmer Cetus, 9700) under the following conditions: 96.degree. C. for 5 min, followed by 30 cycles of 94.degree. C. for 30 sec, 58.degree. C. for 30 sec, and 72.degree. C. for 2 min with a final 5 min extension at 72.degree. C.

Agrobacterium-mediated Transformation and Somatic Embryogenesis

Yellow (FIG. 13) embryogenic calli were obtained from excised margins of leaf explants of coffee (Coffea arabica) after 4 months of culture on MS agar medium with 10 .mu.M 2-iP. These calli were selected and maintained on that medium by 3 weeks of subculture cycle (FIG. 14). Embryogenic calli were co-cultivated with Agrobacterium tumefaciens EHA101 in MS liquid medium containing 10 mg/l acetosyringone and 10 .mu.M 2-iP and transferred to MS solid medium containing 10 mg/l acetosyringone and 10 .mu.M 2-iP in the dark for 4 days. To eliminate remnant bacteria, the co-cultivated embryogenic calli were transferred to MS medium containing 300 mg/l cefotaxime and 10 .mu.M 2-iP. After 2 months of culture, these calli were transferred to the same medium containing 25 mg/l hygromycin. After 2 months of culture, survived embryogenic calli were transferred to MS medium containing 50 mg/l hygromycin.

After selection of survived embryogenic calli (FIG. 15) in the presence of 50 mg/l hygromycin, these calli were transferred to MS agar medium containing 2 mg/l bialaphos. In 33% of embryogenic callus, proliferation and colour was not influenced by the bialaphos treatment. Whereas, in non-transformed callus, colour of callus rapidly turn to brown and did not proliferlated further after 2 weeks of culture.

To induce somatic embryos from embryogenic callus, embryogenic calli were transferred to MS medium containing 3 .mu.M 2-iP. Prolonged culture of embryogenic callus stimulated somatic embryo formation from embryogenic cells. Over one month of subculture cycle was efficient for somatic embryo induction from callus. Numerous somatic embryos were formed from the putative transgenic calli after 2 months of culture (FIG. 16).

Germination of Transgenic Plantlets

When somatic embryos were transferred to 1/2 MS medium containing 10 .mu.M GA.sub.3, germination frequency was strongly enhanced. All (100%) the embryos turn to green after 3 weeks of culture on GA.sub.3 containing medium (FIG. 17). Whereas, only 37% of somatic embryos were in green colour after 3 weeks of culture on GA.sub.3 -free medium and germination speed of somatic embryos was very slow. Somatic embryo and small plantlets survived on 50 mg/l hygromycin also tolerant to the 2 mg/l bialaphos. Eighty three percent of somatic embryos and 92% of small plantlets were grew normally in 2 mg/l bilaphos without change of colour and growth ability (FIG. 19). While, in non-transformed somatic embryos and plantlets, most of them were browned and eventually dead after one to two months of culture (FIG. 18). These survived plantlets were transferred to 1/2 strength MS medium in 300 ml culture bottles for further growth (FIG. 20).

Examination of transgenic small plantlets (T) by genomic PCR revealed amplified fragments coinciding with the bar (362 bp band in FIG. 21) and HPT (713 bp band in FIG. 22) genes. In non-transformed plantlets (N), neither bar nor HPT genes were detectable (FIG. 21, FIG. 22).

References

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