Title of Invention	AN IMPROVED MEDIUM FOR REGENERATIONOF TRANSGENIC SECONDARY EMBRYOS OF COFFEA CANEPHORA.P.EX.FR
Abstract	The present invention relates to an improved culture medium for regeneration of transgenic secondary embryos of Coffea Canephora.P.ex.Fr The invention is aimed at development of improved culture media for regeneration of plants through secondary embryos from transgenic tissue to obtain complete plantlets,.

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The present invention relates to an improved culture medium for regeneration of transgenic secondary embryos of Coffea Canephora.P.ex.Fr The invention is aimed at development of improved culture media for regeneration of plants through secondary embryos from transgenic tissue to obtain complete plantlets. Coffea is an extremely important perennial agricultural crop in tropical areas with more than 6.5 million tons of green beans produced every year on about 11 million hectares . The genus Coffea (Rubiaceae) consists of about 80 species in which only Coffea arabica (arabica) and Coffea canephora (robusta ) are important for the production of Coffee beans . C.arabica contributes nearly 70% of coffee consumed world wide due to its superior quality and C.canephora accounts for the rest 30% The most economically important species are Coffee arabica (2n-44) and C. canephora (2n-22). C. canephora, used for instant coffee powder products, is a cross-pollinated specis and more resistant to pest and diseases compared to C.arabica 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. Plant regeneration through in vitro tissue culture is a basic system for achieving genetic transformation, and there have been many reports involving somatic embryogenesis in coffee plants. Plant regeneration via various tissue culture methods should be very effective for propagation and improvement of coffee plants (Staritsky G 1970, Embryoid formation in callus tissues of Coffee. Acta Botanica. Neerl. 18: 509-514, Sondahl MR , Sharp WR 1977 High frequency induction of somatic embryos in cultured leaf explants of Coffea arabica L. Z.Pflanzenphysiology 81395-408., Yasuda T, Fujii Y.Yamaguchi T 1985, Embryogenic callus induction from Coffea arabica leaf explants by benzyladenine. Plant & Cell Physiology 26: 595-597., Hatanaka T, Arawaka O, Yasuda T, Uchida N, Yamaguch T 1991, Effect of plant growth regulators on somatic embryogenesis in leaf cultures of Coffea canephora. Plant Cell Rep 10: 179-182.Hatanaka T, Choi YE, Kusano T , Sano H 1999). Somatic embryogenesis and secondary embryogenesis from primary somatic embryo is a highly useful method for a large-scale propagation and for genetic transformation of species of economic interest. Somatic embryos are widely considered to be of single cell origin, hence is advantageous for transformation studies. Moreover, the process of somatic embryogenesis offers a mean to propagate large number of transgenic plants over a short period of time. In spite of many reports on somatic embryogenesis of Coffea, the induction of somatic embryogenesis and plantlets formation are not applicable easily to get plants from genetic transformation of coffee. References may be made to Barton et al. (1991) who obtained transformants from electroporated protoplasts of C. arabica.The drawbacks are cultured protoplasts did not develop into whole plants. Van Boxtel et al. (1995) reported expression of GUS genes on the surfaces of coffee leaf tissues following biolistic delivery. The drawbacks are it includes only transient expression and not stable integration of the transgene. No regenerated plants were obtained. Sugiama et al (1995) reported transformation of Coffee with Agrobacterium rhizogenes . The drawback is it consists of mobilization of only the rol genes by transformation and does not include mobilization of transgenes using a binary vector in Agrobacterium rtiizogenes. A method for producing a transformant of Coffea arabica, by infecting an embryogenic callus of Coffea arabica with Agrobacterium tumefaciens EHA101 strain that comprises of a vector containing an exogenous gene and a gene available for the selection of transformed embryonic callus to produce a transformed embryogenic callus in a medium containing N.6-[2-isopentenyl]-adenosine and selecting transformed embryonic callus, and producing transgenic somatic embryo thereby regenerating a transformed Coffea arabica by Sano et al United States Patent: 6,392,125. The drawback is it consists of transforming the callus which leads to somaclonal variations in the transformed lines and do not include direct regeneration. So there is a need to develop efficient regeneration protocol which does not involve an intermediate callusing phase. Regeneration by inducing secondary embyogenesis directly from the primary embryo after a transformation event is very much useful in quick regeneration of transformants from primary embryos of Coffea caneptiora. The main object of the present invention is to provide an improved culture medium for regeneration of transgenic secondary embryos of Coffea Caneptiora.P.ex.Fr. which obviates the drawback as detailed above. Another object of the present invention is to develop improved culture medium for regeneration of plantlets from transgenic secondary embryos. Accordingly the present invention provides an improved medium for regeneration of transgenic secondary embryos of Coffea canephora P.ex.Fr. which comprises media 1 comprising NH4NO3 0.08-0.09%, KNO3 0.08-0.095%, H3BO4 0.003-0.004%, KH2PO4 0.008-0.009% Kl 0.00004-0.000045 %, Na2MoO4.2H20 0.0012-0.0013%, C0CI2 2H2O 0.00000121-0.00000129%, CaCl2.2H20 0.021-029% , MgS04.4H20 0.0181-0.0189%, MnS04.4H20 0.001115- 0.00112%, ZnS04.7 H2O 0.00041- 0.00049%, CUSO4.5H2O 0.00000121-0.00000129%, Na2EDTA.2H20 0.001861-0.001869%, FeS04. 7H2O 0.0013921- 0.0013929%, vitamins ,Thiamine HCI 0.00004-0.00006%, Pyridoxine HCI 0.00004-0.00006%l, Nicotinic acid 0.00004-0.00006%, Glycine 0.00015-0.00025%, Myoinositol 0.005-0.015%, Sucrose 1.5-2.0% characterized in that supplemented with thidiazuron (TDZ) 0.0001-0.0002%, Hygromycin 0.0005-0.002%, Cephotaxime 0.025-0.035%, Augmentin 0.01-0.03%, and agar 0.6-0.8% at a pH level of 5.7. for 2-4 months in dark at 22-27°C for the formation of transgenic secondary embryos, and media II comprising NH4NO3 0.08-0.09 %, KNO3 0.08-0.095%, H3BO4 0.0003-0.0004%, KH2PO4 0.008-0.009%!, Kl 0.00004-0.000045 %, Na2MoO4.2H20 0.0012-0.0013%, C0CI2 2H2O 0.00000121- 0.00000129%, CaCl2.2H20 0.021-0.029%l, MgS04.4H20 0.0181- 0.0189%, MnS04.4H20 0.001115-0.00112% ZnS04. 7 H2O 0.00041-0.00049%l, CUSO4.5H2O 0.00000121-0.00000129%, Na2EDTA.2H20 0.001861-0.001869%, FeS04. THzO 0.0013921- 0.0013929%, vitamins ,Thiamine HCI 0.00004-0.0006%, Pyridoxine HCI 0.00004-0.00006 %, Nicotinic acid 0.00004-0.00006%, Glycine 0.00015-0.00025%, Myoinositol 0.005-0.015%, sucrose1-3%, thidiazuron(TDZ) 0.00001-0.0001%, Hygromycin 0.002-0.005%, Cephotaxime 0.025-0.035%, Augmentin 0.01-0.03% and agar 0.6- 0.8% at a pH level of 5.7. for 3-4 months in dark at 22-27°C for the formation of plantlets from transgenic secondary embryos. Obtaining secondary embryos from Coffea canephora primary somatic embryos after transformation using Agrobactehum sp. harboring a binary vector on secondary embryogenic medium (NH4NO3 825 mg/l, KNO3 950 mg/l, H3BO4 3.1 mg/l, KH2PO4 85 mg/l, Kl 0.415 mg/l, Na2MoO4.2H20 12.5 mg/l, C0CI2 2H2O 0.0125 mg/l, CaCl2.2H20 220 mg/l, MgS04.4H20 185 mg/l, MnS04.4H20 11.15mg/i ZnS04. 7 H2O 4.30 mg/l, CUSO4.5H2O 0.0125 mg/l, Na2EDTA.2H20 18.65 mg/l, FeS04. 7H2O 13.925mg/l, Thiamine HCI 0.5 mg/l, Pyridoxine HCI 0.5 mg/l. Nicotinic acid 0.5 mg/l. Glycine 2.0 mg/l, Myoinositol 100.0 mg/l, sucrose 15,000- 20,000 mg/l, thidiazuron 1.0 -2.0mg/l, hygromycin 5-20 mg/l, Cephotaxime 200-350mg/l & Augmentin200-350mg/l, agar 6000 - 8000 mg at pH levels of 5.4-6.2. Further regeneration of plantlets from transgenic secondary embryo on improved medium (NH4NO3 825 mg/l, KNO3 950 mg/l, H3BO4 3.1 mg/l, KH2PO4 85 mg/l, Kl 0.415 mg/l, Na2MoO4.2H20 12.5 mg/l, C0CI2 2H2O 0.0125 mg/l, CaCl2.2H20 220 mg/l, MgS04.4H20 185 mg/l, MnS04.4H20 11.15mg/i ZnS04. 7 H2O 4.30 mg/l, CUSO4.5H2O 0.0125 mg/l, Na2EDTA.2H20 18.65 mg/l, FeS04. 7H2O 13.925mg/l, Thiamine HCI 0.5 mg/l, Pyridoxine HCI 0.5 mg/l. Nicotinic acid 0.5 mg/l. Glycine 2.0 mg/l, Myoinositol 100.0 mg/l, sucrose 15,000- 20,000 mg/l, thidiazuron 0.05-0.2mg/l, hygromycin 5-20 mg/l, Cephotaxime 200-350mg/l & Augmentin200-350mg/l, agar 6000 - 8000 mg at pH levels of 5.4-6.2. In an embodiment of the present invention regeneration of transgenic Coffea canephora plantlets obtained by inducing secondary embryogenesis from primary somatic embryos after transforming with Agrobacterium sp. harboring a binary vector with selection marker as hygromycine on improved MS medium supplemented with growth regulators and selection marker. In another embodiment of the present invention the transgenic secondary embryos obtained from primary somatic embryos after transforming with Agrobactehum sp. harboring a binary vector with selection maker as hygromycin may be further cultured on improved plantlet regeneration (embryo germination) medium to get regenerated plantlets expressing the transgene. In yet another embodiment of the present invention an improved media may be used for regeneration of transgenic plants from primary somatic embryos comprises: The novelty of the present invention is it provides for the first time an improved culture media for regeneration of plants by secondary embryogenesis from primary somatic embryos of Coffea canephora after transformation using Agrobacterium sp. harboring a binary vector with a marker gene for the selection of transgenic secondary embryos . This is potentially very useful in plant biotechnology for micro propagation and for rapid selection of transformants in Coffea canephora. In the present invention : Leaf explants of coffee {Coffea canephora ) were prepared from leaves of In vitro germinated seedlings according to the method previously described (Hatanaka et al. 1991). The leaf explants were cultured on primary embryogenic media according to the method previously described (Van Boxtel and Berthouly 1996) After 4 months of the culture, embryogenic calli(Figl) induced from the leaf gave rise to embryos ( Fig 2 ) in embryogenic media according to the method previously described. For Agrobacterium mediated Transformation, Agrobacterium strains harboring a binary vector containing P -glucuronidase {gus) as a reporter gene and hygromycin phosphotransferase (hpt) as a marker gene in the T-DNA region of the vector was used. Freshly subcultured primary emgryos (3 days after culture) were co-cultivated in bacterial suspension (absorbance of 0.6 OD at 600 nm) in MS liquid medium containing lOOuM acetosyringone for 30 min at 25°C, ultrasonicated at frequency of 50Hz for a period of 30 seconds in order to make micro wounding on the surface of somatic embryos to facilitate infection of Agrobactehum for T-DNA transfer. These somatic embryos were transferred to MS agar medium containing 200 uM acetosyringone, 3% sucrose at 25°C. in the dark for two days. To eliminate bacteria, the somatic embryos were washed 5 times with sterilized water, followed by water containing 500 mg/l cefotaxim once. Thereafter the somatic embryos were cultured on half strength MS medium supplimented wth 15,000-20,000 mg/l of sucrose; half strength vitamins selected from MS medium; 80-120 mg/l inositol, growth hormones selected from 1-2 mg/l Thidiazuron (TDZ); antibiotics for selection of putative transgenic secondary embryos-hygromycin 5-20 mg/l, antibiotic for killing the Agrobactehum -Cephotaxim 350mg/l & Augmentin300 mg/l, agar 6000 - 8000 mg at pH levels of 5.4-6.2. After 2 months of culture, initiation of putative transgenic secondary embryos (Fig 3) were found. These secondary embryos were transferred to fresh medium with an increased concentration of hygromycin (50 mg/l) and low concentration of Thidiazuron. After 2 months of culture, each line of secondary embryogenic mass was maintained by transferring to fresh medium containing 50 mg/l hygromycin. These secondary embryos gave rise to small plantlets which are resistant to hygromycin (Fig 4). Histochemical GUS Assay Histochemical assays of GUS were performed for hygromycin-resistant secondary somatic embryos according to the method of Van Boxtel et al. (1995). Hygromycin resistant secondary embryos were reacted with X-gluc solution showed a strong GUS positive reaction (FIG. 5 ) However, secondary embryos without co-cultivation did not show any GUS activity . PCR for hpt gene: DNA extraction from leaves of coffee plantlets showing hygromycine resistance was carried out using genomic DNA isolation kit (Sigma USA) according to the manufacturers instruction. The primers used for amplifying the 497 bp hpt gene were 5'-gatgttggcgacctcgtatt-3' and 5'-gtgtcacgttgcaagacctg-3' . The reaction mixture for PCR was incubated in a DNA thermal cycler Primus-MWG Biotech, under the following conditions: 96°C for 5 min, followed by 30 cycles of 94°C for 30 sec, 50°C for 30 sec, and 72°C for 2 min with a final 5 min extension at 72°C PCR revealed amplification of 497 bp fragment of the hpt gene ( FIG 6). Further confirmation of the transformants were carried out by southern hybridization using a labeled probe to hybridize the 497 bp PCR fragment (Fig 7). In untransformed platelets, neither GUS staining nor hpt fragment amplification was detected. Leaf explants ↓ Somatic embryos ↓ Transformation using Agrobacterium sp. ↓ harboring a binary vector with gus reporter gene and hygronnycin marker ↓ Co cultivation for two days without any antibiotics in MS medium ↓ Culturing in secondary embryogenic selection medium ↓ Selection of putatively transgenic secondary somatic embryos for gus assay and PCR detection ↓ Confirmation of the transgene integration by Southern blot Example. 1 Leaf explants of coffee {Coffea canephora) were cultured on primary embryogenic callus induction media according to the method previously described (Van Boxtel and Berthouly 1996). After 4 months of the culture, embryogenic calli induced from the leaf explants were transferred to primary embryogenic media according to the method previously described. Afer 4 months primary embryos were obtained. Agrobacteiium rhizogenes A4 harboring pCAMBIA1301 containing. (3 glucuronidase (gus) gene and hygromycin phosphotransferase (hpt) genes in the T-DNA region of the plasmid was used for the transformation. Freshly subcultured primary embryos were co-cultivated in bacterial suspension (absorbance of 0.6 at 600 nm) in MS liquid medium (NH4NO3 1650 mg/l, KNO3 1900 mg/l, H3BO4 6.2 mg/l, KH2PO4 170 mg/l, Kl 0.83 mg/l, Na2MoO4.2H20 0.25 mg/l, C0CI2 2H2O 0.025 mg/l, CaCl2.2H20 440 mg/l, MgS04.4H20 22.3 mg/l, MnS04. 4 H2O 22.30 mg/l. ZnS04. 7 H2O 8.60 mg/l, CUSO4.5H2O 0.025mg/l, Na2EDTA.2H20 37.30 mg/l, FeS04. 7H2O 27.850 mg/l, Thiamine HCI 1.0 mg/l, Pyridoxine HCI 1.0 mg/l. Nicotinic acid 1.0 mg/l. Glycine 4.0 mg/l. Myoinositol 100.0 mg/l, sucrose 30,000 mg/l, pH adjusted to 5.7)containing lOOuM acetosyringone for 30 min at 25°C ultrasonicated at frequency of 50Hz for a period of 30 seconds in order to make micro woundings on the surface of somatic embryos to facilitate infection of A.rhizogenes for T DNA transfer. These somatic embryos were transferred to MS medium (NH4NO3 1650 mg/l, KNO3 1900 mg/l, H3BO4 6.2 mg/l, KH2PO4 170 mg/l, Kl 0.83 mg/l, Na2MoO4.2H20 0.25 mg/l, C0CI2 2H2O 0.025 mg/l, CaCl2.2H20 440 mg/l, MgS04.4H20 22.3 mg/l, MnS04. 4 H2O 22.30 mg/l, ZnS04. 7 H2O 8.60 mg/l, CUSO4.5H2O 0.025mg/l, Na2EDTA.2H20 37.30 mg/l, FeS04. 7H2O 27.850 mg/l. Thiamine HCI 1.0 mg/l, Pyridoxine HCI 1.0 mg/l. Nicotinic acid 1.0 mg/l. Glycine 4.0 mg/l. Myoinositol 100.0 mg/l, sucrose 30,000 mg/l, pH adjusted to 5.7 before adding agar, (agar for gelling 8000 mg/l) containing 200 ^M acetosyringone at 25°C. in the dark for two days. To eliminate bacteria, the somatic embryos were washed 5 times with sterilized water, followed by water containing 500 mg/l cefotaxime once. Thereafter the somatic embryos were cultured on secondary embryo induction medium (NH4NO3 825 mg/l, KNO3 950 mg/l, H3BO4 3.1 mg/l, KH2PO4 85 mg/l, Kl 0.415 mg/l, Na2MoO4.2H20 12.5 mg/l, C0CI2 2H2O 0.0125 mg/l, CaCl2.2H20 220 mg/l, MgS04.4H20 185 mg/l, MnS04.4H20 11.15mg/l ZnS04. 7 H2O 4.30 mg/l, CUSO4.5H2O 0.0125 mg/l, Na2EDTA.2H20 18.65 mg/l, FeS04. 7H2O 13.925mg/l, Thiamine HCI 0.5 mg/l, Pyridoxine HCI 0.5 mg/l, Nicotinic acid 0.5 mg/l, Glycine 2.0 mg/l. Myoinositol 100.0 mg/l, sucrose 15,000- 20,000 mg/l, thidiazuron 1.5mg/l, hygromycin 5-20 mg/l, Cephotaxime 350mg/l & Augmentin300mg/I,. pH adjusted to 5.7 before adding agar, (agar for gelling 8000 mg/l ) After 3 months of culture in the dark, initiation of putatively transgenic secondary embryos were obtained. 3-6 percent transformation and regeneration frequency was obtained, more than 60% explants responded and gave rise to secondary embryos in control experiments where transformation and selection was not done. The above said medium was found to be very efficient in inducing secondary embryogenesis. These secondary embryos were transferred to fresh medium (NH4NO3 825 mg/l, KNO3 950 mg/l, H3BO4 3.1 mg/l, KH2PO4 85 mg/l, Kl 0.415 mg/l, Na2MoO4.2H20 12.5 mg/l, C0CI2 2H2O 0.0125 mg/l, CaCl2.2H20 220 mg/l, MgS04.4H20 185 mg/l, MnS04.4H20 11.15mg/i ZnS04. 7 H2O 4.30 mg/l, CUSO4.5H2O 0.0125mg/l, Na2EDTA.2H20 18.65 mg/l, FeS04. 7H2O 13.925mg/l, Thiamine HCI 0.5 mg/l, Pyridoxine HCI 0.5 mg/l, Nicotinic acid 0.5 mg/l, Glycine 2.0 mg/l. Myoinositol 100.0 mg/l, sucrose 15,000- 20,000 mg/l, thidiazuron 0.2mg/l, hygromycin 50 mg/l, Cephotaxim 350mg/l & Augmentin300mg/I,. pH adjusted to 5.7 before adding agar, agar for gelling 8000 mg/l )with an increased concentration of hygromycin (50 mg/l) and decreased concentration of thidiazuron. After 2 months of culture, each line of secondary embryogenic mass gave rise to small plantlets on fresh medium containing 50 mg/l hygromycin. The transgenic nature of the plants were confirmed by PCR and PCR southern Example. 2 Leaf explants of coffee {Coffea canephora) were cultured on primary embryogenic callus induction media according to the method previously described (Van Boxtel and Berthouly 1996). After 4 months of the culture, embryogenic calli induced from the leaf explants were transferred to primary embryogenic media according to the method previously described. Afer 4 months primary embryos were obtained. Agrobacterium rhizogenes A4 harboring pCAMBIA1301 containing. 3 glucuronidase (GUS)gene and hygromycin phosphotransferase (HPT) genes in the T-DNA region of the plasmid was used for the transformation. Freshly subcultured primary emgryos were co-cultivated in bacterial suspension (absorbance of 0.6 at 600 nm) in MS liquid medium (NH4NO3 1650 mg/l, KNO3 1900 mg/l, H3BO4 6.2 mg/l, KH2PO4 170 mg/l, Kl 0.83 mg/l, Na2MoO4.2H20 0.25 mg/l, C0CI2 2H2O 0.025 mg/l, CaCl2.2H20 440 mg/l, MgS04.4H20 22.3 mg/l, MnS04. 4 H2O 22.30 mg/l, ZnS04. 7 H2O 8.60 mg/l, CUSO4.5H2O 0.025mg/l, Na2EDTA.2H20 37.30 mg/l, FeS04. 7H2O 27.850 mg/l, Thiamine HCI 1.0 mg/l, Pyridoxine HCI 1.0 mg/l. Nicotinic acid 1.0 mg/l, Glycine 4.0 mg/l. Myoinositol 100.0 mg/l, sucrose 30,000 mg/l, pH adjusted to 5.7)containing lOOuM acetosyringone for 30 min at 25°C ultrasonicated at frequency of 50Hz for a period of 30 seconds in order to make micro woundings on the surface of somatic embryos to facilitate infection of A.rhizogenes for T DNA transfer. These somatic embryos were transferred to MS medium (NH4NO3 1650 mg/l, KNO3 1900 mg/l, H3BO4 6.2 mg/l, KH2PO4 170 mg/l, Kl 0.83 mg/l, Na2MoO4.2H20 0.25 mg/l, C0CI2 2H2O 0.025 mg/l, CaCl2.2H20 440 mg/l, MgS04.4H20 22.3 mg/l, MnS04. 4 H2O 22.30 mg/l, ZnS04. 7 H2O 8.60 mg/l, CUSO4.5H2O 0.025mg/l, Na2EDTA.2H20 37.30 mg/l, FeS04. 7H2O 27.850 mg/l. Thiamine HCI 1.0 mg/l, Pyridoxine HCI 1.0 mg/l. Nicotinic acid 1.0 mg/l, Glycine 4.0 mg/l, Myoinositol 100.0 mg/l, sucrose 30,000 mg/l, pH adjusted to 5.7 before adding agar, (agar for gelling 8000 mg/l) containing 200 jxM acetosyringone at 25°C. in the dark for two days. To eliminate bacteria, the somatic embryos were washed 5 times with sterilized water, followed by water containing 500 mg/l Cefotaxime once. Thereafter the somatic embryos were cultured on secondary embryo induction medium (NH4NO3 825 mg/l, KNO3 950 mg/l, H3BO4 3.1 mg/l, KH2PO4 85 mg/l, Kl 0.415 mg/l, Na2MoO4.2H20 12.5 mg/l, C0CI2 2H2O 0.0125 mg/l, CaCl2.2H20 220 mg/l, MgS04.4H20 185 mg/l, MnS04.4H20 11.15mg/i ZnS04. 7 H2O 4.30 mg/l, CUSO4.5H2O 0.0125 mg/l, Na2EDTA.2H20 18.65 mg/l, FeS04. 7H2O 13.925mg/l, Thiamine HCI 0.5 mg/l, Pyridoxine HCI 0.5 mg/l, Nicotinic acid 0.5 mg/l. Glycine 2.0 mg/l. Myoinositol 100.0 mg/l, sucrose 15,000- 20,000 mg/l, thidiazuron 0.5mg/l, hygromycin 5-20 mg/l, Cephotaxime 350mg/l & Augmentin 300mg/l,. pH adjusted to 5.7 before adding agar, (agar for gelling 8000 mg/l ) After 3 months of culture in the dark, initiation of putatively transgenic secondary embryos were obtained. Less than one percent transformation and regeneration frequency was obtained. The above said medium was found to be comparitively less efficient in inducing secondary embryogenesis. These secondary embryos were transferred to fresh medium (NH4NO3 825 mg/l, KNO3 950 mg/l, H3BO4 3.1 mg/l, KH2PO4 85 mg/l, Kl 0.415 mg/l, Na2MoO4.2H20 12.5 mg/l, C0CI2 2H2O 0.0125 mg/l, CaCl2.2H20 220 mg/l, MgS04.4H20 185 mg/l, MnS04.4H20 11.15mg/i ZnS04. 7 H2O 4.30 mg/l, CUSO4.5H2O 0.0125mg/l, Na2EDTA.2H20 18.65 mg/l, FeS04. 7H2O 13.925mg/l, Thiamine HCI 0.5 mg/l, Pyridoxine HCI 0.5 mg/l. Nicotinic acid 0.5 mg/l. Glycine 2.0 mg/l, Myoinositol 100.0 mg/l, sucrose 15,000- 20,000 mg/l, thidiazuron 0.2mg/l, hygromycin 50 mg/l, Cephotaxime 350mg/l & Augmentin300mg/I,. pH adjusted to 5.7 before adding agar, agar for gelling 8000 mg/l) with an increased concentration of hygromycin (50 mg/l) and decreased concentration of thidiazuron. After 2 months of culture, each line of secondary embryogenic mass gave rise to small plantlets on fresh medium containing 50 mg/l hygromycin. The transgenic nature of the plants were confirmed by PCR and PCR southern Example. 3 Leaf explants of coffee {Coffea canephora) were cultured on primary embryogenic callus induction media according to the method previously described (Van Boxtel and Berthouly 1996). After 4 months of the culture, embryogenic calli induced from the leaf explants were transferred to primary embryogenic media according to the method previously described. Afer 4 months primary embryos were obtained. Agrobacterium rhizogenes A4 harboring pCAMBIA1301 containing. 3 glucuronidase (GUS)gene and hygromycin phosphotransferase (HPT) genes in the T-DNA region of the plasmid was used for the transformation. Freshly subcultured primary emgryos were co-cultivated in bacterial suspension (absorbance of 0.6 at 600 nm) in MS liquid medium (NH4NO3 1650 mg/l, KNO3 1900 mg/l, H3BO4 6.2 mg/l, KH2PO4 170 mg/l, Kl 0.83 mg/l, Na2MoO4.2H20 0.25 mg/l, C0CI2 2H2O 0.025 mg/l, CaCl2.2H20 440 mg/l, MgS04.4H20 22.3 mg/l, MnS04. 4 H2O 22.30 mg/l, ZnS04. 7 H2O 8.60 mg/l, CUSO4.5H2O 0.025mg/l, Na2EDTA.2H20 37.30 mg/l, FeS04. 7H2O 27.850 mg/l. Thiamine HCI 1.0 mg/l, Pyridoxine HCI 1.0 mg/l. Nicotinic acid 1.0 mg/l. Glycine 4.0 mg/l. Myoinositol 100.0 mg/l, sucrose 30,000 mg/l, pH adjusted to 5.7)containing lOOuM acetosyringone for 30 min at 25°C ultrasonicated at frequency of 50Hz for a period of 30 seconds in order to make micro woundings on the surface of somatic embryos to facilitate infection of A.rhizogenes for T DNA transfer. These somatic embryos were transferred to MS medium (NH4NO3 1650 mg/l, KNO3 1900 mg/l, H3BO4 6.2 mg/l, KH2PO4 170 mg/l, Kl 0.83 mg/l, Na2MoO4.2H20 0.25 mg/l, CoCb 2H2O 0.025 mg/l, CaCl2.2H20 440 mg/l, MgS04.4H20 22.3 mg/l, MnS04. 4 H2O 22.30 mg/l, ZnS04. 7 H2O 8.60 mg/l, CUSO4.5H2O 0.025mg/l, Na2EDTA.2H20 37.30 mg/l, FeS04. 7H2O 27.850 mg/l, Thiamine HCI 1.0 mg/l, Pyridoxine HCI 1.0 mg/l. Nicotinic acid 1.0 mg/l. Glycine 4.0 mg/l. Myoinositol 100.0 mg/l, sucrose 30,000 mg/l, pH adjusted to 5.7 before adding agar, (agar for gelling 8000 mg/l) containing 200 ^M acetosyringone at 25°C. in the dark for two days. To eliminate bacteria, the somatic embryos were washed 5 times with sterilized water, followed by water containing 500 mg/l cefotaxime once. Thereafter the somatic embryos were cultured on secondary embryo induction medium in full streangth MS medeum (NH4NO3 1650 mg/l, KNO3 1900 mg/l, H3BO4 6.2 mg/l, KH2PO4 170 mg/l, Kl 0.83 mg/l, Na2MoO4.2H20 0.25 mg/l, C0CI2 2H2O 0.025 mg/l, CaCl2.2H20 440 mg/l, MgS04.4H20 22.3 mg/l, MnS04. 4 H2O 22.30 mg/l, ZnS04. 7 H2O 8.60 mg/l, CUSO4.5H2O 0.025mg/l, Na2EDTA.2H20 37.30 mg/l, FeS04. 7H2O 27.850 mg/l, Thiamine HCI 1.0 mg/l, Pyridoxine HCI 1.0 mg/l, Nicotinic acid 1.0 mg/l, Glycine 4.0 mg/l. Myoinositol 100.0 mg/l, sucrose 30,000 mg/l, thidiazuron 1.5mg/l, hygromycin 5-20 mg/l, Cephotaxim 350mg/l & Augmentin300mg/I,. pH adjusted to 5.7 before adding agar, agar for gelling 8000 mg/l) After 2 months of culture, more callusing was observed instead of secondary embryos The above said medium was found to be less efficient in inducing secondary embryogenesis. The main advantages of the present invention are: 1. The development of efficient medium for regeneration of plants by secondary embryogenesis from somatic embryos of Coffea canephora after transformation usng Agrobacterium sp harboring a binary vector with a marker gene for the selection of transgenic secondary embryos . 2. Regeneration of plantlets from selected transgenic secondary embryos. 3. The improved culture medium for making transgenic plants from primary somatic embryos by organogenesis is highly efficient in contrast to the available literature. 4. According to this protocol a maximum of 7 months time is required to get tissue culture derived plantlets from primary somatic embryos. We claim: 1. An improved medium for regeneration of transgenic secondary embryos of Coffea canephora P.ex.Fr. which comprises media 1 comprising NH4NO3 0.08-0.09%, KNO3 0.08-0.095%, H3BO4 0.003-0.004%, KH2PO4 0.008-0.009% Kl 0.00004-0.000045 %, Na2MoO4.2H2O 0.0012-0.0013%, C0CI2 2H2O 0.00000121-0.00000129%, CaCl2.2H2O 0.021-029% , MgSO4.4H2O 0.0181-0.0189%, MnSO4.4H2O 0.001115- 0.00112%, ZnSO4.7 H2O 0.00041- 0.00049%, CUSO4.5H2O 0.00000121-0.00000129%, Na2EDTA.2H2O 0.001861-0.001869%, FeSO4. 7H2O 0.0013921-0.0013929%, vitamins ,Thiamine HCI 0.00004-0.00006%, Pyridoxine HCI 0.00004-0.00006%l, Nicotinic acid 0.00004-0.00006%, Glycine 0.00015-0.00025%, Myoinositol 0.005-0.015%, Sucrose 1.5-2.0% characterized in that supplemented with thidiazuron (TDZ) 0.0001-0.0002%, Hygromycin 0.0005-0.002%, Cephotaxime 0.025-0.035%, Augmentin 0.01-0.03%, and agar 0.6-0.8% at a pH level of 5.7. for 2-4 months in dark at 22-27°C for the formation of transgenic secondary embryos, and media II comprising NH4NO3 0.08-0.09 %, KNO3 0.08-0.095%, H3BO4 0.0003-0.0004%, KH2PO4 0.008-0.009%l, Kl 0.00004-0.000045 %, Na2MoO4.2H2O 0.0012-0.0013%, C0CI2 2H2O 0.00000121- 0.00000129%, CaCl2.2H2O 0.021-0.029%l, MgSO4.4H2O 0.0181- 0.0189%, MnSO4.4H20 0.001115-0.00112% ZnSO4. 7 H2O 0.00041-0.00049%l, CUSO4.5H2O 0.00000121-0.00000129%, Na2EDTA.2H2O 0.001861-0.001869%, FeSO4. 7H2O 0.0013921-0.0013929%, vitamins .Thiamine HCI 0.00004-0.0006%, Pyridoxine HCI 0.00004-0.00006 %, Nicotinic acid 0.00004-0.00006%, Glycine 0.00015-0.00025%, Myoinositol 0.005-0.015%, sucrose1-3%, thidiazuron(TDZ) 0.00001-0.0001%, Hygromycin 0.002-0.005%, Cephotaxime 0.025-0.035%. Augmentin 0.01-0.03% and agar 0.6-0.8% at a pH level of 5.7. for 3-4 months in dark at 22-27°C for the formation of plantlets from transgenic secondary embryos. 2. An improved culture medium as herein described with reference to examples accompanies specifications.

Documents:

397-del-2003-abstract.pdf

397-DEL-2003-Claims-(07-01-2009).pdf

397-del-2003-claims.pdf

397-del-2003-complete specification (granted).pdf

397-DEL-2003-Correspondence-Others-(06-01-2009).pdf

397-DEL-2003-Correspondence-Others-(07-01-2009).pdf

397-del-2003-correspondence-others.pdf

397-del-2003-correspondence-po.pdf

397-DEL-2003-Description (Complete)-(07-01-2009).pdf

397-del-2003-description (complete).pdf

397-del-2003-form-1.pdf

397-del-2003-form-18.pdf

397-DEL-2003-Form-2-(06-01-2009).pdf

397-DEL-2003-Form-2-(07-01-2009).pdf

397-del-2003-form-2.pdf

397-del-2003-form-3.pdf