Title of Invention

"A PROCESS FOR TRANSFERRING FOREIGN DNA INTO CALLUS CELLS OF TAXUS SP."

Abstract

The present invention relates to a process for introducing GUS reporter gene and NPT II and HPT plant selection marker genes into callus cells of Taxus sp., said genes being harbored in a binary vector plasmid PTOK 233, said process comprising: (a) obtaining callus culture of Taxus sp. in a manner as herein described, characterised by (b) infecting said cultured callus cells with Agrobacterium tumefaciens (LBA4404) containing a binary vector plasmid pTOK233 harboring a GUS reporter gene and NPT II and HPT plant selection marker genes and present in T-region in Tiplasmid, and a Vir-region in an auxiliary plasmid, therein; (c) incubating said infected callus cells and subjecting said incubated cells to a second sub- culturing; wherein said T-region comprises said GUS gene and NPT-II and HPT flanked on both sides by border sequences as present in wild-type of Agrobacterium and wherein said T-region is transformed and stably integrated into the genome of said callus cells.

Full Text

FIELD OF THE INVENTION The present invention relates to a process for transferring foreign DNA into callus cells of Taxus sp'by Agrobacterium tumafadens mediated genetic transformation. BACKGROUND OF THE INVENTION Paclitaxel a compound obtained from Taxus sp. is widely used as an anticancer drug in treating breast, ovarian and lung cancers. The supply of the drug is currently by extracting from dried bark and needles of Taxus sp. (Wani et al., 1971; Miller et al., 1981). The extraction of paclitaxel from naturally growing Taxus trees is quite limited because a large number of trees need to be harvested to obtain a sufficient amount of paclitaxel. Hauser Chemical Research reported in 1992 that their production capacity of Paclitaxel was 130 kg/year, this required the collection of 1,600,000 Ib of yew bark in 1991 (Suffness and Wall, 1995). Several alternative sources of paclitaxel have been identified. They are semi-synthesis (Denis et al., 1988), total synthesis (Holton et al., 1994; Nicolaou et al., 1994); Fungus production (Stierle et al., 1993); production by cell and tissue cultures (Wickremsinhe and Arteca, 1991; Gibson et al., 1995; Chee, 1994). However, it is possible to improve paclitaxel production further through genetic manipulation. There were few attempts in this direction. Wild strains of Agrobacterium tumafadens and A., rhizogenes have been used to transform Taxus. Han et al (1994) used stem segments of mature Taxus brevifolia and T. baccatta as starting material and infected with A.tumafadens (strain Bo542). The highest frequency of gall formation (28.3%) was achieved with T.baccatta. Taxus hairy roots have been established using Taxus embryos as starting material (Plaut-Carcasson (1994). Luan et al. (1996) described transient GUS expression in zygotic embryos of T. brevifolia. However, Taxus callus cultures have not been used for transformation. Therefore, we have developed Agrobacterium mediated transformation of callus cultures of Taxus spp. using a super binary vector pTOK233 (Heie et al., 1994). This vector carries three chimeric genes, GUS, HPT and NPT-II. This binary plasmid carries three additional vir genes, virB, vir C, and vir G which help in efficient transformation (Hiei etal., 1994). A mega plasmid (Ti-plasmid), present in soil bacterium Agrobacterium tumafadens, is responsible for crown gall induction in gymnosperm plants (Han et al, 1994). A specific part of the Ti-plasmid, called T-region, is transferred by the bacterium to the plant cell upon infection is integrated into the core DNA (Chilton et al., 1977). The integrated DNA (T-region)is expressed (Willmitzer et al.1981) and this expression forms the molecular basis of the plant disease Crown Gall. Resulting tumour cells, in contradistinction to normal plant cells, can be grown on synthetic media without adding the plant hormones auxine and cytokinine. (U Braum, 1958). The tumour cells also contain specific compounds, called opines, which are not present in normal cells, the genetic information of which lies coded on the transferred T-DNA (Bomhoff et al., 1976); Schroder etal, 1981). Besides the T-region, which is present in A.tumefaciens, the Ti-plasmid contains a second region called Vir-region which is essential for the virulence qualities of the bacterium and helps in excision and transfer of T-region from Agrobacterium to plant cells(Omms et al.,1980; Garfmkel et al.,1980). This region was never seen in tumour cells and genetic analyses have shown that mutations in this area (leading to much weakened virulence or complete avirulence) are trans complementable by wild types of genes, which are present on clones or R Prime Plasmids (Hille et al. Plasmid 7, 197-118 (1982); Klee et al., J. Bacteriol. 150, 327-331)). Seven genetic loci have been determined in this Vir (Virulence) region, called Vir A, B, C, D, E, F and G (Klee et al., J. Bacteriol. 150, 327-331 (1982); Hille et al (1982). The Vir region and the T-region can be physically separated from one another on two compatible plasmids without any negative effect on the capacity of Agrobacterium tumefaciens to incorporate the T-region or artificial T-region into the plant cell (Hoekema et al.,1983)). A binary vector system based on this invention can be used for the genetic manipulation of plant cells. (Described in Dutch patent specification no. 83 00698 and European patent application no. 84 202396, and in Dutch patent application 84 01048). In Dutch patent application 84 01048 then reported that monocotyledonous species, which was thought not to be susceptible to Agrobacterium, was indeed able to respond to Agrobacterium. After wounding the plants and infecting with Agrobacterium it appeared from the experiments that (1) cells of monocotyledonous plants were positively transformed by A. tumefaciens; (2) plant material isolated from non-wounded or wounded but not infected monocotyledonous plants contained neither octopine nor nopaline. Opines were not discovered in plant material isolated from plants infected with avirulent strains while plant cells obtained from wound sites infected with virulent A. tumefaciens strains appeared to nearly always contain opines. Consequently, it was concluded that the Ti plasmid is suitable as a vector for monocotyledonous plant cells. Summary of the invention The T-region of the Ti (tumor inducing) plasmid of Agrobacterium tumefaciens is transferred in nature to cells of higher plants upon infection, where from DNA from T-region is introduced into the genome and is expressed. Transfer of the T-region or any aritificial T-region also occurs when this region is present in the Agrobacterium on a separate plasmid, next to a plasmid which contains the essential virulence genes on a Vir-region. (This is the so-called binary vector system described in Dutch Patent Application No. 83 00698, and also mentioned in Dutch patent application no. 8401048 for genetic manipulation of monocots). The surprising, novel invention now described is based on an extension of the binary vector system. In the invention artificial T-region is present in binary vector pTOK 233 harbouring GUS reporter gene and NPT-II and HPT plant selection marker genes are introduced into the genome of Taxus callus cells with a Vir-region present on an auxiliary plasmid. The T-region introduced into the callus cells in this way appears to be transferred to callus cells efficiently, where it is integrated into plant genomes and is expressed. With this new invention new procedures can be developed for genetic manipulation of gymnosperms plant cells. The invention creates new possibilities for the incorporation of foreign DNA carrying beneficial genes that encode therapeutic proteins etc. into the genome of gymnosperms plant cells or other kind of plant cells. The result presented here show that the presence of the components of the binary system on plasmids is an essential condition. The T-region can be integrated into the genome of Taxus callus cells without any negative effect of the transfer of the T-region or artificial T-region to plant cells followed by integration into plant genomes. This invention throws a vivid light on the transfer of T-region into the callus cells of a conifer which enables the genetic manipulation of said cells for biotechnological applications. The present invention relates to a process for introducing GUS reporter gene and NPT II and HPT plant selection marker genes into callus cells of Taxus sp., said genes being harbored in a binary vector plasmid PTOK 233, said process comprising: (a) obtaining callus culture of Taxus sp. in a manner as herein described; characterised by (b) infecting said cultured callus cells with Agrobacterium tumefaciens (LBA4404) containing a binary vector plasmid pTOK233 harboring a GUS reporter gene and NPT II and HPT plant selection marker genes and present in T-region in Tiplasmid, and a Vir-region in an auxiliary plasmid, therein; (c) incubating said infected callus cells and subjecting said incubated cells to a second sub-culturing; wherein said T-region comprises said GUS gene and NPT-II and HPT flanked on both sides by border sequences as present in wild-type of Agrobacterium and wherein said T-region is transformed and stably integrated into the genome of said callus cells. Detailed Description of the invention The present invention deals with a method for Agrobacerium-mediated genetic transformation of callus cultures of Taxus sp. which include T-cuspidata var.Capitata; T.cuspaidata var. Greenwave; T.csupidata var. Nigra; T.media var. Hatfieldii; T.media var. Tautonii; T.baccata var. fastigata Aurea; T.baccata var. Fastigata Robusta; T.baccata var.Dovastoniana Aureovariegata, with efficient transformation frequency which comprises the following steps It was observed that explants collected during monsoon period i.e. June - September is prone to severe fungal contamination. Our surface sterilization procedure procedure decreases the risk of contamination and facilitates the contamination free culture process. The callus cultures of Taxus sp. were raised on Murashige and Skoog's (1962) MS-medium (Table-1) supplemented with 2,4-D 1.0 mg/1 2,4-D and have been maintained on the same callus medium by periodical subcultures (once in a month). The freshly sub-cultured callus culture were utilized for transformation via Agrobacterium tumefaciens Transfer and the integration of a DNA sequences coding for β-glucuronidase (GUS) hygromycin phosphotransferase (HPT) and nemoycin phosphotransferase (NPT-II) into Taxus genome. Wherein said DNA sequences coding for bacterial GUS expresses in the transformed callus lines which was visualized (blue colour) by histochemical assay.(Jefferson et.al. 1987) Expression of said gene under the control to appropriate regulatory elements (i.e. cauliflower mosaic virus 35S promoter (US Patent No.5359142). The Agrobacterium tumefaciens method is performed over other gene transfer methods like particle gun because of the easy performance, less number of the transgene copy insertion and rearrangements in the plant genome. The said genes were introduced into Taxus genome by means of the Ti-plasmid of A.tumefaciens (Patent No. EP 0116718). The T-DNA containing all the three genes were transmitted to callus cells upon infection by A. tumefaciens, and are stably integrated into the Taxus genome. Under appropriate conditions known in the art, the callus cells can be transformed. The Agrobacterium strain LBA 4404 employed in the art of transformation (Patent No. US 4650759) Ti-plasmid contain two regions required for obtaining the transformed cells. One of them named transfer DNA (T-DNA), induces tumor formatting and the other region is responsible for the excision and transfer of the T-DNA into plant genome. Ti-plasmid contains three additional virulence genes, Vir B, Vir C and Vir G(Fig.l) which are responsible for the supervirulence of the vector (Hiei et.al. 1994). The T-DNA can be modified such a way that the foreign genes can be cloned in it without changing its ability of transfer into plant genome by removal of unwanted tumor-causing genes (disarmed Ti-plasmid vector). The plasmid construct used in the present invention comprises three chimeric genes GUS & HPT (US Patent No.4960704) and NPT-II (US Patent No. 5034322). The selection of transformed callus cells is enabled by the use of a selectable marker gene which is also transferred (Fig.2). This expression of marker gene confers a phenotypic trait that enables for selection The factor such as bacterial O.D., i.e. 0.4 O.D co-culture period , i.e. 10 min and 2 days respectively, the concentration of selection agent, i.e. 50 mg/1 hygromycin for selecting the transformed callus cells and the selection period, and the addition of 100 uM acetosyringone and 3% sorbitol were found to be important and critical for successful transformation of Taxus callus cultures. In the present invention an efficient Agrobacterium -mediated genetic transformation was developed for callus cells of Taxus sp. using a supervirulence binary vector pTOK 213 that harbours three chimeric genes GUS, HPT and NPT-H. Several putative transformed callus lines were developed and the integration of transgenes was demonstrated, based on both PCR (Fig.3) and Southern hybridization analysis (Fig.4-A & 4-B). These transformed callus lines also exhibited high GUS activity. The novelty of this method is the high frequency of transformation and demonstration of critical role of certain factors like bacterial O.D., infection time and use of acetosyringone and sorbitol for successful transformation. The present invention will now be described in greater detail with reference to the accompanying drawings wherein: Fig. 1 shows linear maps of pTOK233. In the Figure, the legends have the following meaning: RB- right border; LB - left border; NPT II - neomycin phosphotransferase; GUS -P-glucuronidase; HPT - hygromycin phosphotransferase; NOS- nopaline synthase promoter; 35 S- 35 S promoter; TNOS -3' signal of nopaline synthase; T35S - 3' signal of 35S RNA; B - BamHl; E- EcoRl; H - Hind III; S.-Sad;X- Xbal; Vir -B, Vir-G and Vir-C are present on the backbone of plasmid i.e., outside the T-DNA region. Fig. 2 A shows the expression of GUS in callus transformed with pTOK233, while Fig. 2B shows absence of GUS expression in untransformed control calli. Arrows indicate the GUS stained blue spots. Fig. 3 shows the PCR analysis using hpt gene as primer for transformed Taxus callus lines of pTOK233. Fig 4-A shows southern hybridization of products of PCR using hpt gene as probe for transformed callus with pTOK233. Lane-1 DNA from untransformed control. Lane 2-7 DNA from transformed callus lines. Fig. 4-B shows southern hybridization of products of PCR using npt gene as probe for transformed callus with pTOK233. Lane-1 DNA from untransformed control. Lane 4-5 DNA from transformed callus lines The transformation can be performed as described in the following examples which are merely illustrative and not restrictive. In general, preparation of plasmid DNA, restriction enzyme digestion, agarose gel electrophoresis of DNA, Southern blot DNA hybridization, PCR, were carried out using standard methods (Sambrook et al, 1989). a) Method of surface sterilization: Young and actively growing branches of size 20-30 cm are collected from wild mature old plants or from managed nurseries. 2-3 inches explants which includes - green stem / rachis , needles, immature buds etc. was cut and washed thoroughly under running tap water for 30 min. and then treated with 0.1% detergent ("Savlon"- Cholrohexidine) for 10-12 min. The best sterilization method consisted as under: Explant was washed with sterile water and treated with 10% Clorox- Na Hyo Chorite peroxide for 15 min. followed by treatment with 0.5% (w/v) "Bavistin" (active compound: Carbendazim, broad spectrum fungicide) for 60 min. Then under laminar flow hood, these explants are further treated with 0.1% mercuric chloride for 7 min. and washed thrice with sterile distilled water prior to inoculation in nutrient medium. b) Induction and maintenance of callus culture Explants of size 1-2 inches were surface sterilized as described, are placed horizontally on nutrient medium ( M. S. medium) containing various hormones listed in Table No.l. We found that MS (Murashige & Skoog medium, 1962) salts, sucrose 3% (w/v), /wyo-inositol 200 mg/1, growth regulator 2,4-D 1.0 mg/1, gelling agent (agar) 0.8% (w/v), charcoal at 0.1% before adjusting pH of medium to 5.8 was best suitable medium for initiation and maintenance of callus cultures. The explants after surface sterilization placed horizontally on medium and cultures were incubated intially for a week in dark and then at 25+2°C in light (2000 lux) at 16 h photoperiod. Culturing was continued for one month. After 1 month explant gave rise small nodular to creamy callus at the cut end region. After 1 month they were subcultured to fresh medium with the same composition. One explant per one test tube containing 15-20 ml culture agar medium can be cultured. The cultures are incubated at temperature 20-25°C under 16 h photoperiod with light intensity of 2000-3000 lux. The culture may be continued till the usage. c) Transformation vector The binary plasmids pTOK233 (harbouring hygromycin phosphotransferase (HPT), neomycin phospho-transferase (NPTII) and β-glucoronidase (GUS) chimeric genes and three additional virulence genes (vir B, vir C and vir G)} (Fig. 1) (Hiei et al., 1994) were kindly provided by Prof. T. Komari (Japan Tobacco Inc. Shizuoka, Japan) d) Callus Transformation The A. tumefaciens strain LBA4404 harbouring the plasmid, pTOK233 was inoculated into YEM (yeast extract 4 g/1, manitol 10 g/1, NaCl 1 g/1, MgSO4 . 7H2O 2 g/1 and K2HPO4 5 g/1, pH 7.0) medium having 100 mg/1 kanamycin and grown at 28°C and 250 rpm for 48 h to obtain a growing culture of the bacterium which was then subcultured into the same medium and grown overnight to obtain an O.D of 0.2 - 0.4. The bacterial cells were pelleted at 3000 rpm at room temperature and resuspended in liquid MS medium supplemented with 3% sorbitol, along with 100 μM acetosyringone (Sigma Aldrich, USA). The calli subcultured for about one week were infected with the bacteria for 10 min and then placed on co-cultivation medium (MS+1.0 mg/1 2, 4-D +3% sorbitol + 100 μM acetosyringone) and incubated in the dark for 2-3 days. The co-cultured calli were then transferred to the selection medium (MS+1.0 mg/1 2, 4-D + 50 mg/1 hygromycin+500 mg/1 cefotaxime) and incubated in the light (16 h photoperiod) at 26 ± 1°C for 15 days. The growing callus pieces were subjected to second selection on the above selection medium for another 15 days. The growing callus pieces were then transferred to callus medium (MS + 1.0 2, 4-D mg/1 + 250 mg/1 cefotaxime) and incubated in the light for one month before being subcultured on the same callus medium. Such callus lines selected on the selection medium were utilized for GUS assay and molecular analyses. e) GUS assay GUS assay was performed as per the protocol developed by Jefferson (1987) for calli selected on the selection medium. f) Polymerase chain reaction The genomic DNA was isolated from the transformed and non-transformed control callus tissue using CTAB protocol (Doyle & Doyle 1990). PCR was performed using 100 ng of DNA and primers of hpt gene obtained from (Genmed Synthesis, Inc., USA). The primers (Forward Primers: 5' CGC ATG AAA AAG CCT GAA CTC ACC GCG 3' and Reverse Primers : 5' GCA GGC TCC CGT TTC CTT ATC GAT 3')were designed to amplify a 1 kb fragment in the hpt. The PCR amplification was carried out in 25 μl reaction mixture using PCR master kit (GIBCO-BRL, USA). The samples were denatured initially at 94 °C for 5 minutes, followed by 40 cycles of 1 min denaturation at 94°C, 1 min of primer annealing at 55 °C and 2 min of synthesis at 72°C, with a final extension step of 72°C for 10 min. The PCR products were analysed on a 1% agarose gel. The PCR products were confirmed by Southern blot DNA hybridization (Sambrook et al. 1989). using hpt gene as probe. g) Southern blot analysis The genomic DNA was restricted with Bam HI and Eco RI for pTOK233 transformed Taxus callus lines and probed with nptll gene for the integration of the transgene Southern blots were prepared according to the protocol given by Sambrook et al. (1989). The 1 kb hpt gene fragment was isolated from the plasmid pCAMBIA 1380 (harbouring HPT gene in the T-DNA region and kindly provided by R. A. Jefferson) and used as probe which was prepared by nick translation according to the suppliers guidelines (Boehringer Mannheim, Germany) and hybridized to the membrane (Hybond-N, Nylon 0.45 μM membrane, Amersham) (Sambrook et al. 1989). Autoradiographs were obtained by exposing the membrane to an X-ray film (XK-5 Kodak film). Table -1 (Table Removed) Media For growing Bacteria YEMmedium Yeast extract Mannitol NaCl K2HPO4 MgSO4 . 7H2O This is a minimal media used for Agrobacterium cultures. pH(6.8 - 7.0)is adjusted using 0.1 NHC1 Table-2 (Table Removed) Supplements like, 3% sucrose and myo-inositol- 200 mg/1 were added. Medium pH was 5.8 REFERNCES Patents • US Patent Documents No's.5792924 ; 5932782 ; 5359142; 4650759 • European Patent No. EP 0116718 • WIPO patent No. 94/20606; 83 00698; 84 02396 ; 84 01048 Journals • Bomhoff et al.,(1976) Mol. Gen. Genet. 145, 177:181 • Braum U, (1958) Proc. Natl. Acad. Sci 44, 344:349 • Chee PP (1994) In vitro culture of zygotic embryos of Taxus spp. Hortscience 29: 695- 697 • Chilton et al.,(1977) Cell 11, 263-271 • Denis JN, Greene AE, Guenard D, Gueritte-Voegelein F, Mangatal L and Potier P (1988). A highly efficient, practical approach to natural Paclitaxel. J. Am. Chem. Soc. 110: 5917- 5919 • Doyle JJ and Doyle JL (1990) Isolation of plant DNA from fresh tissue. Focus 12: 13-15 • Garfmkel et al., (1980) J. Bacteriol. 144, 732-743 • Gibson DM, Ketchum REB, Hirasuna TJ and Shuler ML (1995). Potential of plant cell culture of taxane production. In: Suffness M (ed) Paclitaxel Science and Applications (pp 71-95). CRC press. Boca Raton, NY • Han KH, Fleming P, Walker K, Loper M, Chilton WS, Mocek U, Gordon MP and Floss HG(1994).Genetic transformation of mature Taxus : an approach to genetically control the in vitro production of the anticancer drug, Paclitaxel. Plant Science 95: 187-196 • Hiei Y, Ohta S, Komari T and Kumashiro T (1994) Efficient transformation of rice (Oryza saliva L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA. Plant J. 6: 271-282 • Hille et al. (1982) Plasmid 7, 197-118 • Hoekema et al., (1983) Nature (London), 303, 179-180 • Holton RA, Kim HB, Somoza C, Liang F, Biediger RJ, Boatman PD, Shindo MS, Mith CC and Kim SC (1994). 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