Abstract:
A method of genetic improvement of coffee plants, using technique of molecular bleeding, is disclosed. The method provides a transformant of coffee plants produced from embryogenic calli, using Agrobacterium method.

Description:
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; Menéndez-Yuffá and García, 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&#39;s woody plant medium (WPM) supplemented with 5 μM N 6 -[2-isopentenyl]-adenosine (2-iP). These calli were co-cultured with Agrobacterium tumefaciens EHA101 harboring pIG121-Hm, containing β-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 μM 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 μM N 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 μM 2-iP. Prolonged culture of embryogenic callus induced somatic embryos. Germination of somatic embryos strongly enhanced by GA 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 ½ MS medium containing GA 3 . 
     FIG. 18 shows browned small plantlets cultured on ½ 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 ½ 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&#39;s woody plant salt mixture (Lloyd and McCown, 1981), Gamborg&#39;s B 5  (Gamborg et al. 1968) vitamins, 3% sucrose and 5 μM N 6 -[2-isopentenyl]-adenosine (2-iP). The medium was adjusted to pH 5.7 before autoclaving at 120° C. for 15 min. The culture room was maintained at 25° C. with 16-h light illumination of 24 μmol m −2 s −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 5  vitamin, 3% sucrose and 10 μM 2,4-dichlorophenoxyacetic acid (2,4-D). The CM medium was also adjusted to pH 5.7 before autoclaving at 120° C. for 15 min. Agrobacterium tumefaciens EHA101 harboring pIG121-Hm containing β-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° C. in WPM liquid medium containing 5 μM 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 μM 2-iP at 25° 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 μM 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 μM 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 μM 2-iP in 10×2 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-β-D-glucuronide (X-gluc) solution with a composition modified to 50 mM Na 2 HPO 4 , 10 mM Na 2 EDTA, 0.3% Triton X-100, 0.5 mM K 3 Fe(CN) 6 , 0.5 mM K 4 Fe(CN) 6 , and antioxidants (0.5% caffeine, 1% PVP and 1% sodium ascorbate). After 16 hours at 37° 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 μM 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 μM 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 μM 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 μM 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° C. for 5 min, followed by 30 cycles of 94° C. for 30 sec, 58° C. for 30 sec, and 72° C. for 2 min with a final 5 min extension at 72° 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&#39;s B 5  (Gamborg et al. 1968) vitamins, 3% sucrose and 10 μM N 6 -[2-isopentenyl]-adenosine (2-iP). The medium was adjusted to pH 5.7 before autoclaving at 120° C. for 15 min. The culture room was maintained at 25° C. with 16-h light illumination of 24 μmol m −2 s −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 5  vitamin, 3% sucrose and 10 μM 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° C. in MS liquid medium containing 10 μM 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 μM 2-iP at 25° 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 μM 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 μM 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 μM 2-iP was transferred to MS medium with 3 μM 2-iP. Somatic embryos developed spontaneously from embryogenic callus. After selection of somatic embryos, they were cultured on ½ MS agar medium with 10 μM GA 3  to support germination. After 3 weeks of culture, small plantlets were transferred to ½ 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 ½ 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° C. for 5 min, followed by 30 cycles of 94° C. for 30 sec, 58° C. for 30 sec, and 72° C. for 2 min with a final 5 min extension at 72° 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 μM 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 μM 2-iP and transferred to MS solid medium containing 10 mg/l acetosyringone and 10 μM 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 μM 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 μM 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 ½ MS medium containing 10 μM GA 3 , germination frequency was strongly enhanced. All (100%) the embryos turn to green after 3 weeks of culture on GA 3  containing medium (FIG.  17 ). Whereas, only 37% of somatic embryos were in green colour after 3 weeks of culture on GA 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 ½ 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 
     Barton CR, Adams TL, Zarowitz MA (1991) Stable transformation of foreign DNA into  Coffea arabica  plants. In: 14 ème  Colloq Sci Int Café, ASIC, Paris, pp 460-464 
     Gamborg OL, Miller RA, Ojima K (1968) Plant cell cultures. (1) Nutrient requirements of suspension cultures of soybean root cells. Exp Cell Res 50: 151-158 
     Hatanaka T, Arakawa 0, Yasuda T, Uchida N, Yamaguchi T (1991) Effect of plant growth regulators on somatic embryogenesis in leaf cultures of Coffea canephora. Plant Cell Rep 10: 179-182 
     Hiei Y, Ohta S, Komari T, Kumashiro T (1994) Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA. Plant J 6: 271-282 
     Hood EE, Halmer GL, Fraley RT, Chilton MD (1986) The hypervirulence of Agrobacterium tumefaciens A281 is encoded in a region pTIB0542 outside of T-DNA. J Bacteriol 168: 1291-1301 
     James DJ, Uratsu S, Cheng J, Negri P, Viss P, Dandekar AM (1993) Acetosyringone and osmoprotectants like betaine or proline synergistically enhance Agrobacterium-mediated transformation of apple. Plant Cell Rep 12: 559-563 
     Kikuchi K, Niwa Y, Yamaguchi T, Sunohara H, Hirano H-U, Umeda M (1998) A rapid and easy-handling procedure for isolation of DNA from rice, Arabidopsis and tobacco. Plant Biotechnology 15: 45-48 
     Lloyd G, McCown, B (1981) Commercially-feasible micropropagation of mountain laurel, Kalmia latiforia, by use of shoot tip culture. Comb Proc Int Plant Propagator&#39;s Soc. 30: 421-427 
     Menéndez-Yuffá A, García E de (1996) Coffea species (coffee) In: Bajai YPS (eds) Biotechnology in Agriculture and Forestry, Vol 35, Springer-Verlag, Berlin Heidelberg, pp 95-119 
     Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue. Physiol Plant 15: 473-497 
     Ocampo C, Manzanera L (1991) Advances in genetic manipulation of the coffee plant. In: 14 ème  Colloq Sci Int Café, ASIC, Paris, pp 378-382 
     Ohta S, Mita S, Hattori T, Nakamura K (1990) Construction and expression in tobacco of a β-glucuronidase (GUS) reporter gene containing an intron within the coding sequence. Plant Cell Physiol 31: 805-813 
     Spiral J, Thierry C, Paillard M, Pétiard V (1993) Obtention de plantules de Coffea canephora Pierre (Robusta) transformées par Agrobacterium rhizogenes. C R Acad Sci Paris Serff Sci Vie 316: 1-6 
     Staritsky G (1970) Embryoid formation in callus tissues of coffee. Acta Bot Neerl 19: 509-514 
     Van Boxtel J, Berthouly M, Carasco C, Dufour M, Eskes A (1995) Transient expression of β-glucuronidase following biolistic delivery of foreign DNA into coffee tissues. Plant Cell Rep 14: 748-752 
     Yokoi S, Toriyama K, Hinata K (1996) Protocol for production of transgenic rice plants mediated by Agrobacterium. Plant Tissue Culture Letters 13: 81-84 
     
       
         
           
             6 
           
           
             1 
             19 
             DNA 
             primer used for amplifying GUS gene 
           
            1
aattgatcag cgttggtgg                                                  19
 
           
             2 
             20 
             DNA 
             primer used for amplifying GUS gene 
           
            2
acgcgtggtt acagtcttgc                                                 20
 
           
             3 
             20 
             DNA 
             primer used for amplifying the HPT gene 
           
            3
gcgtgaccta ttgcatctcc                                                 20
 
           
             4 
             20 
             DNA 
             primer used for amplifying the HPT gene 
           
            4
ttctacacag ccatcggtcc                                                 20
 
           
             5 
             21 
             DNA 
             primer used for amplifying the bar gene 
           
            5
atgagcccag aacgacgccc g                                               21
 
           
             6 
             22 
             DNA 
             primer used for amplifying the bar gene 
           
            6
gctcttgaag ccctgtgcct cc                                              22