FIELD OF THE INVENTION

The present invention is in relation to a method for increasing the yield of active chemical constituents in plants. More particularly, the present invention proposes a method for over expression of chemical constituents in plants using Agrobacterium mediated gene transfer and pathway engineering.

BACKGROUND AND PRIOR ART OF THE INVENTION

Metabolites purified from plants provide many pharmacologically active compounds including leading chemotherapy drugs. As it is generally true for metabolites, over all productivity is low, making commercial production expensive. Alternative production methods remain impractical, leaving the plants as the best source of these valuable compounds. Recently, significant progress in characterizing the biosynthetic pathways leading to various metabolites has been made, and a number of relevant genes have been cloned using Agrobacterium mediated transformation. Genetic engineering employing such genes provides a promising technology for improved productivity in plant cell cultures, plant tissue culture, or intact plants.

Many vital agronomical and pharmaceutical species are routinely transformed using Agrobacterium. In some developed countries, a high percentage of the acreage of such economically important crops is transgenic; and increasing number of these transgenic varieties are generated by Agrobacterium mediated, as opposed to particle bombardment mediated transformation in which predictable and stable expression of transgenes remains problematic. Basic Agrobacterium biology is utilized to develop Agrobacterium as a tool for plant genetic engineering. In this study the objective is to generate genetically improved/transgenic Dioscorea prazeri in which the range and scope of natural product is modified to provide commercially and an economically viable plant as it is medicinally very important as well as an endangered species.

Dioscorea prazeri is one of the best sources of Diosgenin that is a pharmaceutically important steroidal sapogenin, and is a precursor of sex hormones (progesterone), corticosteroids (corticosone) and contraceptives (Coursey D.G, 1967, On Wueme, 1978). It causes an inhibition of fibroblast like synoviocytes from human rheumatoid arthritis, with apoptosis induction associated with cylooxygenase-2 up-regulation (Liagre B et. al., 2004) and diosgenyl saponins induce apoptosis and mitotic arresting on human leukemia cell lines (Ming-Jie, 2004). Dioscin, a derivative compound from Diosgenin, has been reported to induce HeLa cells apoptosis through caspase-9 and caspase-3 (Cai J et. al, 2002). Diosgenin induces apoptosis in HeLa cells via activation of caspase pathway and inhibit HeLa cell growth (Huo Rui et. al, 2004). Diosgenin could be considered as an adjunctive hypocholesterolemic agent in future (Malinow M.R et. al, 1987). It has been reported to have various effects, such as hypocholesterolemic action in rat (Accatino L et. al., 1998), antioxidant activity in HIV patients with dementia (Turchan J et. al., 2003) and cyclooxygenase activity in osteosarcoma cells (Moalic S et. al, 2001). Wild yams are embraced with important therapeutic properties such as anti- conceptive, steroids (e.g. prednisone), as muscle relaxants, anesthetic agent and for abdominal surgery.

OBJECTIVES OF THE PRESENT INVENTION

The principle object of the present invention is to provide a method for Agrobacterium mediated gene transformation in plants.

Another objective of the present invention is to provide a method for Agrobacterium mediated gene transformation in Dioscorea prazeri and Nicotiana tobacum.

Yet another objective of the present invention is to introduce the gene 3-hydroxy-3- methyl glutaryl coenzyme A reductase (HMGR) from H. brasiliensis to Dioscorea prazeri via Agrobacterium mediated transformation. Still another objective of the present invention is to shorten the time required to achieve the levels of active chemical constituents in plants, particularly Dioscorea.

Still another objective of the present invention is to increase in the yield of precursor(s) in plants.

Still another objective of the present invention is to study the pathway engineering studies on Dioscorea prazeri with regarding to Diosgenin.

BRIEF DESCRIPTION OF ACCOMPANYING DRAWINGS

Figure 1: Gel picture of HMGR1 gene amplification with gene specific primers Lane 1-4: The amplification of HMGR1 gene. Lane 5: Amplification of genomic DNA as PCR positive control. Lane 6: Negative PCR control. Lane M-Molecular marker Figure 2: pGEM®-T Easy Vector Map and sequence reference points Figure 3: (a).Gel picture of colony PCR: Lanel-12: The amplified colonies with vector specific primers. Lane 3: The positive clone with 1.94 kb transcript along with few bases from the vector (arrow indicates). Lane 3: PCR positive control. Lane 12: Negative control. Lane M: Molecular marker (b).Gel picture of Colony PCR of genomic DNA clones: Lane 1-31: The amplication of colonies with vector specific primers. Lane 1-12: Positive clones showing 2.5 kb transcript. Lane N: Negative. Lane +: PCR Positive control. Lane M: Molecular marker.

Figure 4: Gel picture of Restriction digestion of plasmid DNA with Eco Rl enzyme: Lane P: Plasmid. Lane D: Digested DNA with Eco Rl. Lane M: Molecular marker

Figure 5: The complete sequenced analyzed from positive clone

Figure 6: pCAMBIA Vector 1305.1 where Gus Plus has been replaced by HMG Coenzyme A Reductase 1

Figure 7: Colony PCR 1-35 with positive control and per negative control with primers 1296 F, Bgl II F, Nos ER and 405 R. The positive clones have given two bands at lOOObp and 550 base pair (indicated by arrow)

Figure 8: (a) Plasmid DNA of 1 Clone 13 (lane 1), 14 (lane 2), 19 (lane 3), 1305.1 (lane 4) and standard (300 ng) (lane 5) (b) Restriction digestion of the Vector 1305.1 containing gene of interest with restriction enzymes Bgl //and Nhel for elution of the gene (lane 1) with 1 kb molecular marker(lane 2) (c) Positive clones 13, 14, 19, 1305.1 with Sac II and Bst XI restriction enzyme along with plasmid DNA

Figure 9: Colony PCR of Agrobacterium tumefaciens strain with gene of interest with using primers Cam V F and Nhe I R 13+clones (lane 1-4) and 14+clones (lane 5-7) with positive control (lane 8) negative control (lane 9) with molecular marker (lane 10) Figure 10: (a) Control, D. Prazeri- Gus gene were not expressed (b) GUS Transient expression analysis - Dioscorea prazeri: Indigo blue spots show transient expression of GUS genes in different parts of the plant, (c) PCR based approach for the GUS gene transformation: Lane 1,2,3, plants were in selection, 4,5 were control plants and lane six was negative control, lane 8 was positive control of GUS gene plasmid and lane 8 is marker. The arrow indicate 2.0 Kb insert of GUS gene

Figure 11: (a) The growth phases of transgenic plants in standardized media of regeneration for D. prazeri A: Control plantlets (left) and Transgenic plantlets (right); B: Initiation of Rooting from transgenic plant; C: The proliferation of shooting and multiple shooting of transgenic plants; D: Adventatious shoot formation on transgenic plants; E: Initiation of tuber formation from transgenic plants, (b) Amplification of gene from transgenic plants by PCR with Bgl II forward and NOS end reverse primers (I row) and CaMV end forward and Nhe I Reverse (II Row) pic. Lane 1: Molecular marker, Lane 2,3 and 4:Control plants, 5 and 9: Non-transformed plants, 6, 7, 8, 10 showed the transcript of 1.8 Kb along with few base pair from the promoter region, Lane 12: positive control, Lane 12: negative control

Figure 12: Complete sequence analyzed from the transgenic plants along with the original sequence of the candidate gene

Figure 13: Chromatogram obtained on analysis of extract from transgenic and controlled plants: Standard: lmg/ml of diosgenin, Sample 1: control plant extract (5 months old plants), Sample 2: Transgenic plant extract (5 months old plants), Sample 3: Extract from young tubers of transgenic plants (5 months old plants), Sample 4: Extract from young tubers of control plants (5 months old plants).

STATEMENT OF THE PRESENT INVENTION

Accordingly, the present invention is in relation to a method for over expression of chemical constituents in plants, said method comprising step of introducing Agrobacterium having gene encoding 3-hydroxy-3-methylglutaryl-Coenzyme A reductase 1 into the plant; and a transformed plant over expressing chemical constituents.

DETAILED DESCRIPTION OF THE PRESENT INVENTION

The present invention is in relation to a method for over expression of chemical constituents in plants, said method comprising step of introducing Agrobacterium having gene encoding 3-hydroxy-3-methylglutaryl-Coenzyme A reductase 1 into the plant.

In another embodiment of the present invention, said plant is selected from a group comprising Dioscorea prazeri and Nicotiana tobacum.

In yet another embodiment of the present invention, said chemical constituents include diosgenin and sterols.

In another embodiment of the present invention, said gene is hmg gene isolated from Hevea brasiliensis.

The present invention is in relation to a transformed plant over expressing chemical constituents.

In another embodiment of the present invention, said transformed plants include Dioscorea prazeri and Nicotiana tobacum.

In yet another embodiment of the present invention, said transformed plant is introduced\ with Agrobacterium having gene encoding 3-hydroxy-3-methylglutaryl-Coenzyme A reductase 1.

In still another embodiment of the present invention, said gene is hmg gene isolated from Hevea brasiliensis.

In another embodiment of the present invention, said hmg gene is hmg 1.

Productivity of Diosgenin from Dioscorea is low, making commercial production expensive. Alternative production methods remain impractical, as plants are the best source of these valuable compounds. Availability of Dioscorea prazeri for commercial scale extraction is insufficient. Takes up to 3 years for the tubers to accumulate commercially viable levels of Diosgenin. Agrobacterium mediated transformation got application in many areas of research and provide commercially and economically viable plant. It will improve the range and scope for production of Diosgenin in endangered and indigenous medicinal plant D.prazeri by enhancing the production of the active component, which is medicinally very important.

The transient expression of GUS gene has done on Dioscorea prazeri for the standardisation of experimental conditions of the transformation studies and for finding out whether the Dioscorea prazeri is suitable system for the studies proposed.

Reporter genes are markers widely used for analysis of gene regulation and localization. Escherichia coli GUS (P-glucuronidase) gene is extensively used as a gene fusion marker for analysis of gene expression in transformed plants (Jefferson RA, 1987). E.coli P-glucuronidase enzyme hydrolyzes P-glucuronic acid conjugated through a β-O-glycosidic linkage to a glycone. The compound 5-bromo-4-chloro3-indolyl-pD-glucuronide (X-GlcA) is a GUS substrate, which upon hydrolysis produces an indigo blue precipitate. It is used in histochemical staining of plant tissues expressing the E.coli GUS enzyme (Jefferson RA, 1989)

The explant, Dioscorea prazeri nodal explants infected with Agrobacterium tumefaciens with GUS gene, pCambia 1301 GV 3101 and the control plants in the selection media were taken for the GUS transient expression analysis. The staining solution was added to the vials containing tissue so that the tissue gets fully covered in solution. The vials were covered to avoid light. The vials were degassed for five minutes in vacuum desiccator in dark. Covered the containers with lids and incubate at 37°C for 21 hours. The sections were rinsed in ethanol then mounted for microscopy. The chlorophyll was removed by destaining the explants with absolute alcohol for overnight for better observation.

The clear indigo blue stain was observed in nodal explants infected with Agrobacterium tumefaciens with GUS gene, pCambia 1301 GV 3101 with the substrate 5-bromo-4-chloro3-indolyl-PD-glucuronide by histochemical localization of B-glucurunidase activity. The control plants were colourless and not stained on hisochemical assay. The indigo blue stain developed with the infected plants shown the GUS transient expression in Dioscorea prazeri explants.

Productivity of Diosgenin from Dioscorea is low, making commercial production expensive. Alternative production methods remain impractical, as plants are the best source of these valuable compounds. Availability of Dioscorea prazeri for commercial scale extraction is insufficient. The tubers of Dioscorea take up to 3 years to accumulate commercially viable levels of Diosgenin. Agrobacterium mediated transformation got application in many areas of research and provide commercially and economically viable plant. It will improve the range and scope for production of metabolite, which is medicinally very important.

The literature survey of the related study suggests that 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) is an important control point for the synthesis of many plant isoprenoids. Plants regulate HMGR gene family members post-translationally by enzyme modification (Stermer A.B et. al., 1994). HMGR was considered to be regulatory enzyme in sterol biosynthesis (Benveniste P, 1986). The post squalene biosynthetic pathway was clearly characterized by critical rate limiting steps (Wentzinger F.L, 2002, Hartmann M.A., 2002). Co-expression of native and introduced genes reveals cryptic regulation of HMGR expression in Arabidopsis. In eukaryotes also isoprenoid biosynthesis was regulated by HMGR (Edward B. Re et. al., 1995). The strategy in this study was to introduce gene encoding 3-hydroxy-3-methylglutaryl-Coenzyme A Reductase 1 in to Dioscorea prazeri via Agrobacterium mediated transformation and to study the influence of this gene on production of steroidal sapogenin, diosgenin and for the enhancement of it.

Expression of the Hevea brasiliensis 3-hydroxy-3-methylglutaryl-Coenzyme A Reductase 1 (HMGR I) introduced into Nicotiana tabacum via Agrobacterium mediated transformation results in sterol overproduction. Transgenic plants were morphologically indistinguishable from control wild-type plants and displayed the same developmental pattern. In H. brasiliensis, HMGR I was encoded by hmg 1 gene. Transgenic lines showed increased levels of total sterol upto 6-fold, because of an increased expression level of hmg 1 mRNA with respect to control lines not expressing the hmg I transgene. A corresponding increased enzyme activity for HMGR was also observed. Northern blot analysis indicated that hmg I was more highly expressed in lacticifers than in leaves, suggesting strongly that one (hmg 1) of the three hmg genes in H. brasiliensis was specifically involved in rubber biosynthesis. H. brasiliensis hmgl gene was expressed in a plant unable to synthesize rubber (lactifer cells are absent in tobacco) and influence isoprenoid biosynthesis with in it (Schaller H et. al., 1995).

EXAMPLE 1:

The Genomic DNA, RNA and mRNA were isolated from Hevea brasiliensis for the pathway engineering studies. The amplification profile and the cycling conditions were standardised to obtain the gene. The RNA and DNA were reverse transcribed with Oligo(dT) and amplified with gene specific primers, Bglll forward and Nhel reverse to obtain the gene of interest 3-hydroxy-3-methyl-glutaryl-CoA reductase I, a 1.8 kb transcript. It was cloned to pGEM-T Easy cloning vector, transformed to competent cells to obtain the positive clones. The plasmid DNA was isolated and confirmed by restriction digestion. The plasmid was then sequence analysed.

The Hevea brasiliensis were obtained from Forest department, Government of Karnataka, Sulya, Puttur. The plants were being planted in the green house.

1.Isolation of Genomic DNA from Hevea brasiliensis

The Genomic DNA extracted from young leaves of Hevea brasiliensis by lithium chloride method for aromatic and medicinal plants (Anna Maria et. al., 2001). The high quality DNA obtained from this method was used for further experiments. Checked the quality and concentration of the DNA with a spectrometer and on 1% agarose gel.

2. Isolation of Total RNA from Hevea brasiliensis

Total RNA was isolated from young leaves of H brasiliensis (~100mg) using standard Trizol and Kit method (QIAGEN RNeasy Kit). The total RNA isolated from young leaves of Hevea brasiliensis was quantified on 1% of agarose gel by electrophoresis in an RNase free environment.

3.Isolation of mRNA from Hevea brasiliensis

Isolated the mRNa from lmg of Total RNA of H. brasiliensis using Oligotex mRNA Spin column protocol (QIAGEN). To ensure maximum yield, pipetted another l00µL hot buffer OEB heated at 70°C onto the column and re-suspending the resin, and centrifuged for 1 minute at maximum speed.

4. Reverse transcription and polymerase chain reaction (RT-PCR) for isolation of the gene of interest Superscript III first strand synthesis system for RT-PCR Kit from Invitrogen (Catalogue No. 18080-051) had used for the Reverse transcription of the candidate gene from Hevea brasiliensis because of increased specificity, higher yields of cDNA, and full length product. cDNA synthesis was performed in the first step using both mRNA and Total RNA of H. brasiliensis was primed with oligo(dT). Total RNA and mRNA (2ug) with the oligo(dT) primers and with dNTPs of a total reaction volume of l0µL were kept it at 65°C for 5 minutes in a thermocylcer (Eppendorf). The content was placed in ice immediately after this step for 1 minute. Added the cDNA synthesis mix to the reaction mixture and kept it at 50°C for 50 minutes for cDNA synthesis—. A terminating reaction was set at 85°C for 5 minutes in a thermocycler (Eppendorf). For removing the RNA added 1|JLL of RNase H and incubated for 20 minutes at 37°C. 10 uL of aliquot was removed from this mix for polymerase chain reaction. In the second step the PCR was performed using Advantage2PCR kit (catalogue No.K1910-Y, 639207 Ref.) of BD Biosciences/ Clonetech. The reverse transcribed product was amplified according to the standardized profile with gene specific primers with Tap polymersase enzyme (lU).The cycling parameters according to the fragment size of the gene of interest (1.8Kb) were initial denaturation of 95°C for 1 minute (1 cycle) template denaturation of 95°C for 30 seconds, extension of 68°C 3 minutes (35 cycles) and final extension of same temperature for 1 minute.

The positive reaction of the PCR reaction was the genomic DNA of H. brasiliensis with the gene specific primers (Figure 1). The PCR amplification was also done with the Eppendorff RT PCR kit for the confirmation of amplification at different PCR conditions with different cycling conditions. The standardised thermal cycling parameters for RT -PCR with Eppendorf kit using cMaster PCR enzyme mix for the gene of interest were initial denaturation of 95 °C for 3minutes (1 cycle) Template denaturation of 95°C for 30 seconds, Primer annealing of 55 °C 30 seconds and Extension of 72 °C for 2 minutes (40 cycles) and a final elongation for 7 minutes at 72 °C( 1 cycle).

The positive reaction of the PCR reaction was the genomic DNA of H, brasiliensis with the gene specific primers. The confirmation experiment of RT reaction product was done with primer for constitutively expressed gene, Actin with the thermal cycling parameters standardised for the target size 1.8Kb were initial denaturation of 95°C for 3minutes (1 cycle) Template denaturation of 95°C for 30 seconds, Primer annealing of 55 °C 50 seconds and Extension of 72 °C for 1.30 minutes (32cycles) and a final elongation for 7 minutes at 72 °C( 1 cycle).

5. The ligation of amplified product of the RT-PCR into PCR cloning vector (pGEM-T Easy Vector) andplasmid isolation The amplified product of gene of interest, 3-hydroxy-3-methyl-glutaryl-CoA reductase I that is a 1.8 kb transcript of RT-PCR had ligated to pGEM-T Easy cloning vector (Figure 2). Ligation mixture was incubated at 4°C overnight. 5.0ul of ligation mix was used for transformation with E.coli DH10B competent cells. The thawed competent cells along with the ligation buffer was transferred to the electroporation cuvette and given the pulse. LB broth was immediately transferred to the cells for recovery. The tubes were incubated at 37°C at 220 rpm for 1 hour. 200µL of transformation mixture was plated on LB media with antibiotic (lOOµL ampicillin), IPTG and X-gal for the selection. The plates were incubated at 37°C overnight. The positive clones were formed white colonies. Those were screened by PCR method.

6. Procedure for screening of colonies by PCR 25 colonies were randomly picked and patched on LB amp plate along with the positive and negative control. Also they were screened for positives by colony PCR using vector specific primers like T7 and SP6. Using the same toothpick, cells were suspended in l0µL of water taken in the PCR tubes. All the samples were incubated at 95 °C for 10 minutes to obtain the lysate. The PCR reaction was prepared and 15µL of it was added to the lysate and amplified with the cycling conditioned mentioned. The standardized thermal cycling parameters for colony PCR (Hb transcript) with vector specific primers T7 and SP6 were standardized as 94°C for 2 minutes (1 cycle) Template denaturation of 94°C for 30 seconds, Primer annealing of 50 °C 30 seconds and elongation of 72 °C for 2.0 minutes (35cycles) and a final elongation at 72°C for 7 minutes (1 cycle).

The product was stored at -20°C. The amplified PCR product was loaded on the gel for confirmation of positive colonies. After confirmation, the positive clones were inoculated into 5 ml LB amp broth from the patch. The colonies were showing the fragment of expected size was inoculated on LB medium with antibiotic Ampicillin and the genomic DNA clone (Figure 3) also made as a control for the experiments (2.4 kb transcript) The tubes were incubated at 37°C overnight at 225 rpm. The culture was taken for the glycerol stocks and for the plasmid DNA preparation. The plasmid DNA had taken for restriction digestion with EcoRl, Bglll and Nhel for confirming the transcript size.

7. Isolation of Plasmid DNA from the positive clone

The plasmid DNA was isolated from the overnight grown culture. The cells were harvested from the culture broth by centrifugation at 7500 rpm at room temperature for 2 minutes and the pellet was resuspended in 200 µl, of solution I (50mM glucose, 25mM Tris and l0mM EDTA). 300|iL of solution II (0.2N sodium hydroxide and 1% SDS) was added to the above mixture and mixed by inverting the tubes. 300uL of solution III (containing potassium acetate of 3M concentration of pH 4.8) was added, vortex for 10 minutes at 13,000 rpm at room temperature. RNase A (0.04^g final concentration) was added to the supernatant and incubated at 37°C for 20 minutes in water bath. 400uL of Chloroform wash was done twice and centrifuged at 13,000 rpm for 5 minutes. 0.7 Volume of Isopropanol was added to the aqueous layer and mixed by inverting the tube centrifuged at 13,000 rpm for 10 minutes. The pellet was washed and precipitated with 70% ethanol and centrifuged at 13,000 rpm for 5 minutes. The dried pellet was dissolved in HPLC grade water and quantified by electrophoresis on 1% agarose gel It was stored at -20°C. The plasmid DNA obtained was sequence analysed, Restriction mapped with different restriction enzymes for further pathway engineering studies (Figure 4).

Results and Discussion

The genomic DNA was isolated from Hevea brasiliensis by Lithium chloride method. The quality and quantity was checked on Nano drop and gel electrophoresis. This DNA was used to obtain genomic DNA clones, amplification and for sequence analysis. The RNA was isolated and used for the isolation of mRNA and to obtain the candidate gene. The mRNA was reverse transcribed with Oligo(dT) primers and amplified with gene specific primers. Many methods (hot start, multi step RT, and gradient conditions) were standardised to obtain the transcript of 1.8kb without any mutations. The temperature tried for amplification were 42, 50, 55 and 58°C and the cycles from 30 to 40 cycles. This was then optimized as annealing at 58°C with 40 cycles of amplification with the gene specific primers. The hot start PCR also gave improved results without any mutation of gene with Taq polymerase (Clonetech, Japan) and the normal PCR with Taq polymerase enzyme mix (Eppendorf). The amplified product was cloned to pGEM-T easy cloning vector and transformed to E.coli DH10B by electroporation and did the blue white screening. The Colonies were further screened by PCR with vector specific primers. The plasmid DNA was isolated from the overnight grown culture and sequence analysed and for used for further cloning, plant transformation and for the pathway engineering studies.

The candidate gene, 3-hydroxy-3-methyl-glutaryl-CoA reductase I, was reverse transcribed and amplified by polymerase chain reaction from mRNA of Hevea brasiliensis. The gene was cloned into pGEMT-Easy Vector and sequence analysed for further studies. It was used for the Agrobacterium genetic transformation and pathway engineering studies.

EXAMPLE 2
Isolation of the gene from pGEM-T Easy vector

The pGEM T easy vector with the gene was digested with Bgl II and Nhe I enzymes. The digestion mixture was electrophoreised on agarose gel. The gel along the fragment of candidate gene was excised and transferred. The gel was weighed and dissolved in buffer QG completely. Isopropanol was added and incubated at room temperature for 10 minutes. The content was transferred to gel elution column (Eppendorf) and centrifuged for 1 minute at 13,000 rpm. The column was washed with buffer PE and centrifuged at 13,000 rpm for 1 minute, column was allowed to dry and eluted with sterile water and quantified by 1% agarose gel electrophoresis.

2. The cloning of gene topCAMBIA 1305.1 vector

The restriction mapping and the sequence analysis have done for the cloning experiments. The vector series is based on a new reporter gene GUS plus. The new gene has got improved enzymatic characteristics and is more efficiently secreted than E. coli when fused with signal peptide.

Plasmid DNA of 1305.1(25 ng) was electro-transformed to DH10B competent cells. The cells were plated on LB media (Kanamycin selection) after incubation (37° C for 1 hour) to obtain the positive clones. The plates were incubated at 37" C overnight at 225 rpm. The plasmid DNA was isolated by alkaline lysis method. Restriction mapping and sequence analysis were done.

The pCAMBIA vector 1305.1 was digested with Bgl II and Nhe I to introduce the candidate gene in the place of GUS. The pGEMT Easy vector was digested with the same enzymes to isolate the candidate gene. The gene along with the vector was ligated overnight at 4° C with T4 DNA ligase enzyme. It was electro-transformed to DH10B Electro competent cells. The transformed cells were plated to get the positive clones with pCAMBIA 1305.1 vector with the candidate gene HMG Co A red 1. The clone was PCR amplified with the primers 1296 forward, Bgl II forward, Nos end reverse and 405 reverse primers to find out the positive clone with right orientation.

The plasmid DNA of positive clones was isolated using alkaline lysis method. The overnight grown bacterial culture was centrifuged for 2 minutes for pelleting. The supernatant was discarded and re-suspended with Glucose Tris EDTA buffer, added fresh lysis buffer and mixed gently by inverting the tube 5 times, incubated the culture for 5 minutes and immediately added potassium acetate buffer, mixed gently, incubated on ice for 10 minutes. RNase was added, incubated at 37° C for 30 minutes 400 µL of chloroform was added; repeated the step with the supernatant. Equal volume of chloroform and isopropanol were added to the supernatant, incubated on ice for 20 minutes. The mixture was centrifuged for 10 minutes at 4 ° C. The pellet was washed with 70% ethanol and dried at 37 ° C for 10 - 15 minutes. Dissolve the pellet in TE buffer (pH 8.0). The plasmid DNA was quantified on 1% agarose gel and by Nanodrop spectrophotometer.

The pCAMBIA vector along with the gene of interest was confirmed positive by restriction mapping with the enzyme Bst Xl that has restriction sites at 10,789 base pair and at 1485 base pair in gene. Restriction with Bst XI and Sac II enzymes (4056 and 4349 base pair) also was done to find out the positive clones.

The DNA was sequence analysed to check the integrity of the clone. The reliable positive clones were taken for Agrobacterium mediated plant transformation studies. The sequence of positive clones were analysed with the primers Bgl II primer Forward, Nhe I primer Revrse, The complete gene sequence of clone was analysed with the primers 322 F, 865 F, 1296 F, 405 R and 311 R. (Figure 5)

3. Preparation of Agrobacterium Competent cells

The Agrobacterium strain was streaked on LB agar media from the glycerol stock from competent cell preparation. The competent cells were aliquoted (50 µL) to 0.5mL tubes and frozen immediately in liquid nitrogen/quick freezer. The aliquotes of competent cells were stored at -80° C. To one of the aliquots, 1 uL (10 ng) of plasmid DNA was electro-transformed and inoculated for 1 hr at 37° C, 10 uL of transformed cells with 90 µL of LB and 50 uL of transformed cells with 50 uL of LB were plated on to the LB agar medium and incubated for two days and checked the efficiency.

4. Electro-transformation {Agrobacterium strain GV)

The agrobacterium competent cells were thawed along with 400ng of DNA. The mixture was electro-transformed and cultured on LB media with antibiotics (kanamycin, gentamycin and rifampicin). The transformed cells were incubated for 3 days at 28° C. The single colonies were isolated and streaked on LB selection medium and then made a lysate for the PCR amplification. The lysate was denatured for 10 minutes at 95 ° C and amplified it with gene specific primers to confirm the clone.

Results and discussion

The restriction mapping of pGEM-T Easy vector has the candidate gene with the Enzyme Bgl II and Nhe I showed DNA fragment of 1.8 KB. The gene was gel eluted and cloned to pCAMBIA 1305.1 vector (Figure 6). The PCR amplification of colony lystate with 1296 forward and NOS end reverse primers resulted in amplicon of size 750 base pair (Figure 7). The clones with right orientation were given the fragment of size of lOOObp and 500 bp with all the four primers of Bgl II F, nos ER 1296 F and 405 R. The isolated colonies formed were amplified with the primers Bgl II primer forward and Nhe I primer reverse.

The plasmid DNA of good quality and high concentration was obtained using alkaline lysis method (Figure 8). Restriction digestion of plasmid DNA of pCAMBIA vector 1305.1 with gene, with Bgl //enzyme showed linearised DNA fragment as it got unique site with the vector. Digestion of plasmid DNA with Nhe I showed the specific band size of 8405bp and 344 lbp. The restriction digestion with Bgl II and Nhe I enzymes showed the fragment of 1800 base pair. The pCAMBIA vector along with gene resulted in fragment of expected size. Restriction with Bst XI and Sac II enzymes (4056 and 4349 base pair) (Figure 8) also had shown the mapping in right orientation.

The complete gene sequence in clone was analysed with the primers 365 F, 822 F, 1296 F, 405 R and 311 R proved integrity of gene (Figure 5). The reliable positive clones were transformed to competent cells of agrobacterium strain GV for plant agrobacterium mediated transformation studies on D. prazeri. The lysate of agrobacterium colonies with the gene of interest were denatured for 10 minutes at 95 ° C and amplified it using primers Cam V F and Nhe I R 13+clones and 14+clones with negative control given the 1800 base pair fragments (Figure 9). The D. prazeri plants were transformed with different transformation conditions moved beyond antibiotic selection and are in growth stage. Further molecular, biochemical and morphological analysis has to be done with transgenic plants.

The clones showed accurate fragmentation through restriction mapping and sequence-analysed clones were taken for Agrobacterium mediated genetic transformation studies. Different conditions were experimented for finding the most favorable conditions for transformation experiments and obtaining healthy transgenic plants with higher regeneration efficiency. Further biochemical and molecular analysis has been carried out for the study with transgenic plants.

EXAMPLE 3

Agrobacterium mediated plant transformation experiments From the experimental study conducted for the plant selection marker, the effect of hygromycin on explants excised from Dioscorea showed that at 25 mgl"1 concentration of Hygromycin the explants turned pale and yellowish and on 5 days of Inoculation. The explants gradually turned brown from the base and showed necrosis at the laminar end 10 days after Inoculation. Slight growth of shoot buds was observed at this stage in few cases but in most of the newly formed Shoot buds were turning pale. After 15 days of inoculation in 25 mgl"1 hygromycin the explants turned completely brown and showed no growth. At 5 to 15 mgl"1 concentration of hygromycin the explants were growing normally in comparison with control explants .At 20 mgl"1 the growth was affected slightly but was not lethal. 30 to 50 mgl"1 of hygromycin was found to be phytotoxic and highly lethal to the explants in short span of time.

Based on the above experiment 25 mgl"1 hygromycin would be the recommended concentration for the positive selection of Dioscorea prazeri explants in genetic engineering studies.

The effect of the antibiotic (cefotaxime) to prevent the overgrowth of bacterium for the genetic transformation study of Dioscorea was studied. The explants excised from healthy and exponentially growing plants were observed. A positive influence of cefotaxime on frequency of shoot development, length of shoots, number of leaves and colour of leaves and shoots were observed in the explants. The effect varied depending on the concentration of cefotaxime. The concentration studied were 50 mgl"1, 100 mgl"1, 200 mgl"1, 250 mgl"1, 300 mgl"1' 350 mgl"1, 400 mgl"1, 500 mgl"1 and 1000 mgl"1 along with the control plants were used for the experimental studies. High concentration of cefotaxime showed an inhibitory effect on the frequency of shoot development and length of shoots; however at the lesser concentration did not affect the growth of the explants and it was observed to induce profuse shoot formation and growth. The results shown that the concentration should be used for the transformation studies to prevent overgrowth for Dioscoreaprazeri should be 300 mgl"1

1. GUS gene transient expression analysis in Dioscorea

The endangered plants were propagated from nodal explants using standardized protocol for finding out monocot plant is a suitable system for transformation studies with Agraobactrium tumefaciens.

The explants from D. prazeri were infected with Agrobacterium tumefaciens with GUS (beta glucuronidase) gene along with the control plants. The transient expression analysis was conducted. The tissue was fully immersed with staining solution. The vial were protected from light and degassed for 5 minutes in vacuum desiccators in dark. It was incubated at 37 for 21 hours. The section were rinsed in ethanol and mounted for microscopy. The chlorophyll was removed by de-staining the explants with absolute alcohol for overnight for better observation on histochemical assays. The plants crossed selction were amplified by PCR with GUS forward and GUS reverse primer with Taq polymerase enzyme. The experiments were repeated with more number of explants for stable permanent transformation.

2. Agrobaterium mediated transformation with gene of interest to enhance the transformation efficiency and to obtain transgenic plants oiD.prazeri

The explants were excised and sterilized. Different explants were used petiole, leaf lamina, internodal explants, axillary buds and tubers. The explants were pre-cultured for 2 to 7 days in dark in B.O.D. The Agrobacterium tumefaciens with the gene of interest were inoculated into Luria Bertani medium (LB) medium with kanmycin, gentamycin and rifampicin from glycerol stock and incubated in shaker at 28°C at 180 rpm. 1% inoculum from starter culture was inoculated to the fresh medium with antibiotics. The culture was grown to get the active growth phase and checked the absorbance at 600 nm. The different absorbance of cultures was experimented out from 0.2 to 0.8 to find out the favorable condition for the transformation and for getting the highest efficiency with D. prazeri. The culture was centrifuged at 4 °C for 20 minutes at 4000 rpm. The supernatant was discarded and the pellet was dissolved in MS media with hormones and acetosyringone to make the induction media. The absorbance of the induction media was verified. The explants were infected with freshly made induction media after wounding with sterile blade. The infection period with the induction media was experimented from 15 to 90 minutes. The infection period was standardized from the study of various infection periods and the regeneration efficiency of the transgenic plants. The explants were air dried for 10 - 25 minutes. The explants were transferred to co-cultivation media. The explants were incubated for different period of incubation at B.O.D f from 2 days to 7 days to enhance the efficiency of the monocot plant Dioscorea.

The explants were transferred to standardized regeneration media supplemented with Benzyl Amino Purine (BAP) and Naphthalene acetic acid (NAA) with sucrose along with plant selection marker, hygromycin. The concentration of hygromycin was decided according to the standardized lethal concentration for normal plants. The plants that crossed primary selection (15mgr') were transferred to secondary selection media (20mgr') and then to tertiary selection (25mgl1). The plants incubated and treated at different transformation conditions and achieved the most favorable conditions for obtaining healthy transformed plants. The one end of the gene was flanked by strong promoter, CaMV 35S for expressing the tansgene in Dioscorea.

3. Analysis of the transgenic plants

3a. Molecular analysis

Molecular approach was done by genomic DNA extraction by lithium chloride method for aromatic and medicinal plants (Anna Maria et. al., 2001). The leaves were isolated from young plantlets of D. prazeri, which were crossed all the three stages of selection. The DNA was amplified with gene specific primers along with the primer designed for promoter regions (CaMV End Forward and NOS End reverse) with the standardized PCR conditions. The amplified gene was gel eluted and the product was purified. The purified product was sequence anlaysed with CaMv forward primer Bgl II forward,311 reverse,365 forward, 1296 forward ,1314 forward, Nhe I reverse and NOS end reverse to obtain the complete sequence for analysis.

3b. Morphological analysis

The 5 month old plantlets were examined for morphological variation if any with the control plantlets by observing the lamina, Internodes, petioles and primary stem formed. All the experiments were conducted with many replicates and confirmed the experiments conditions for genetic transformation studies and obtaining transgenic plants of Dioscorea.

3c. Biochemical analysis

(i) Extraction of active component, steroidal sapogenin (Diosgenin) with Petroleum Ether

The standard curve was plotted with the Standard Diosgenin procured from Sigma, USA (-98% pure). As a part of the study the control plantlets micropropagated and the tubers obtained from it was compared with the wild plants to find out the variation before the transgenic plant analysis.

The plantlets were dried at 45°C for two days, which were hydrolysed at 95°C for 3/4 hours in 20 ml of 2N hydrochloric acid. The pH on post hydrolysis (1.0) was neutralized (7.0) with 2N sodium hydroxide. The extract was centrifuged and the residue was dried at 78°C for 2 hours in hot air oven. The sample of 1.5 g of dry was subjected to soxhlet extraction with petroleum ether at 60 °C for 1 hour and then re-distilled at 75°C to 80°C for 1 hour in Biosox Unit (Techno Reach). On post- distillation the sample volume was around 5ml, which was then rotoevaporated in water bath at 50°C for 20 minutes with pressure of 300PSi and cooled. The plant extract was dissolved in 5ml of petroleum ether to Isopropanol in ratio 12:1. The extract dissolved was sonicated for 15 minutes. Dried samples were stored at -80°C. The extract was analysed on HPLC. The tuber extract of Dioscorea allata was used as a negative control. The samples injected were shown apparent peak on standardisation with the mobile phase. The peak of the samples was compared with diosgenin peak obtained. The yield of the extract was calculated.

The transgenic plants and the control plants were examined with the same procedure for examining the enhancement of the active component.

(ii) Extraction of active component, steroidal sapogenin (Diosgenin) with HPLC grade Methanol

The plantlets were dried at 45°C for two days, which were hydrolysed at 95°C for 3/4 hours in 20 ml of 2N hydrochloric acid. The pH on post hydrolysis (1.0) was neutralized (7.0) with 2N sodium hydroxide. The extract was centrifuged and the residue was dried at 78°C for 2 hours in hot air oven. The plantlets dried were subjected to soxhlet extraction with Methanol at 80 °C for 2 hours (Biosox Unit Techno Reach). On post- distillation the sample was completely dried by roto-evaporation apparatus in water bath at 50°C with pressure of 300PSi and cooled. The plant extract was dissolved in 1ml of HPLC grade methanol. The yield of the extract was calculated. The extract was analysed on HPLC with HPLC grade Methanol as a mobile phase. The samples injected were shown apparent peak on standardisation with the mobile phase. The peak of the samples was compared with diosgenin peak obtained.

Results and discussion

The GUS transformation for transient expression analysis showed that the Dioscorea prazeri, a monocot plant could be a suitable system for Agrobacterium mediated plant transformation. The indigo blue stain developed showed the Gus transient expression in different plant parts of D. prazeri explants, which were infected. The control explants that were not infected were not showing any indigo blue spots by histochemical assays (Figure 10). The transformed plants showed respective band of GUS gene in the PCR based approach in relation with positive control (Figure 10). The conditions were standardized for Agrobacterium-mediated transformation in D. prazeri.

The nodal explants and the shoot tips of Dioscorea prazeri were regenerated faster and showed the efficiency on transformation experiments. The explants were pre-cultured in Biological Oxygen Demand incubator at 26 °C for 4 days observed healthier resulted in more frequency of regeneration on infection and faster growth in with selection media. The OD600 0.5 to 0.8 were showing good transformation efficiency but the active phase at which OD600 0.75 showed the best results with more healthy transgenic plants. The plants infected with the agrobacterium at rest of the phases were clearly shown less efficiency on developing transgenic plants. The explants infected with the induction medium with Agrobacterium tumefaciens holding the gene of interest, for 40 minutes in dark at 25 to 28°C enhanced the transformation efficiency. The air- drying under sterile condition for 15 minutes enhanced the transformation efficiency; along with standardized concentration of cefotaxim overgrowth of the bacterium was completely prevented. The co-cultivation of the explants on Murashigue and Skoog (MS) medium supplemented with Benzyl Amino Purine (BAP) and Naphthalene acetic acid (NAA) required for the plant growth along with sucrose enhanced the survival rate of explants on transformation.

The transgenic plants with the gene of interest on amplification with the promoter specific and gene specific primers resulted with the transcript of size 1.8kb (Figure 11). The sequence analysed from the transgenic plants showed that the insert introduced was without any mutation and it is matching with the original sequence of the candidate gene (Figure 12). The Morphological characters of the control plantlets and the transgenic didn't show any morphological abnormality. The primary stem growth observed with plantlets those were 5 months old was higher in number and healthier in comparison with the control plantlets. (Table 1) Table 1: Morphological Analysis of transgenic and control plantlets

The percentage of the frequency of obtaining transgenic plants examined was higher than ever recorded with this experiment as 10% with agrobacterium mediated transformation. The regeneration frequency obtained gone higher up to 16% without plant selection marker but the tedious post was to examine all the plants regenerated for transgenic plants. The results obtained on an average from various set of tranforamtion experiments from many replicates. The control explants showed 98% to 100% frequency of regeneration. The frequency of regeneration of transformants was observed as 8% to 10% with selection markers (Table 2). However, without selection marker, the frequency of regeneration of transformants was observed as 12% to 16%. (Table 3) Table 2:

Diosgenin assay was carried out by HPLC analysis to determine the secondary metabolite content, Diosgenin in the regenerants and the mother plant using the standard Diosgenin (Sigma) and no significant difference observed between these plants in terms of secondary metabolite product pattern with the in vitro grown and the mother plant on micropropagation. The sonication further increased the extraction of steroidal sapogenins than the usual procedure. The percentage of Diosgenin obtained from tubers was found to be high as 2.2 -2.4%. The negative control plantlet used for the experiment, D. allata did not reveal the presence of steroidal sapogenin. The results obtained were based on the peak from chromatogram with Diosgenin (standard) and from the extract containing Diosgenin from the dried material of the regenerated plants and the mother plant of D. prazeri, for the determination of secondary metabolite content. The biochemical analysis for the content of Diosgenin levels with pulverised tubers of control mother plantlets and the wild plants showed no significant variation, showed the Diosgenin content was the same in the regenerated plantlets as of mother plant. The methanolic extraction was given the clear peak even with the snmall amount of material. So the methanolic extraction was used for the chromatographic anlysis of transgenic plants.

The comparative analysis showed that the plants at a younger stage itself shown steroidal sapogenin, Diosgenin and there was a prominent enhancement with the transgenic plant in comparison with the control plants which were not transformed. The transgenic plantlets showed an enhancement of 3.7 to 4 fold in comparison with the control plants and the diosgenin level increases with the age of the plant (Figure 13). The healthy transgenic plants obtained with higher regeneration efficiency by introducing the gene of interest, 3-hydroxy-3-methyl glutaryl coenzyme A reductase (HMGR) from H. brasiliensis to Dioscorea prazeri via Agrobacterium mediated transformation were shown an expression of gene with strong promoter CaMV 35 S and enhanced the steroidal sapogenin content. At a very young stage of 5 months old plantlets showed a 3.7 to 4 old increase in the steroidal sapogenin, Diosgenin.

WE CLAIM:
1. A method for over expression of chemical constituents in plants, comprising steps of

(a) excising the explants

(b) transient expression of GUS gene in monocot plants,

(c) introduction of HMG-CoA reductase gene using Agrobacterium mediated plant transformation

(d) selection of transgenics using plant selection marker

(e) analyzing the transgenic plants for over expression of active components, and

(f) extraction of active components from transformed plants.

2. The method as claimed in claim 1, wherein said explant from the vegetative parts such as but not limited to, petiole, leaf lamina, internodal explants, axillary buds and tubers.

3. The method as claimed in claim 1, wherein said plant is selected from a group comprising Dioscorea sp., more specifically Dioscorea prazeri.

4. The method as claimed in claim 1, wherein said gene is 3-hydroxy-3-methylglutaryl-Coenzyme A reductase 1 isolated from Hevea brasiliensis.

5. The method as claimed in claim 1, wherein said chemical constituents include Diosgenin and sterols.

6. The method as claimed in claim 1, wherein the plant generated is transformed plant over expressing chemical constituents.

7. The transformed plants as claimed in claim 6, wherein said transformed plants include Dioscorea prazeri.

8. The transformed plant as claimed in claim 6, wherein said gene is 3-hydroxy-3-methylglutaryl-Coenzyme A reductase 1 isolated from Hevea brasiliensis.

9. The transformed plant as claimed in claim 6, wherein said enhanced chemical constituents include Diosgenin and sterols.

10. The transformed plant as claimed in claim 6, wherein said enhanced chemical constituents showed an enhancement of 3.7 to 4 fold.

11. The transformed plant as claimed in claim 6, wherein said enhanced chemical constituents showed an enhancement of 3.7 to 4 fold within 5 months of growth.

12. The transformed plant as claimed in claim 6, wherein the nodal explants and the shoot tips of Dioscorea prazeri were regenerated within 5 months with 8 to 16% regeneration frequency.

13. The transformed plant as claimed in claim 6, wherein the transformation efficiency was enhanced infecting explants with the induction medium having phenolic compound (Acetosyringone) of 80µM to 120µM, more specifically lOOµM with Agrobacterium tumefaciens holding the gene of interest.

14. The transformed plant as claimed in claim 6, wherein the transformation efficiency was enhanced infecting explants with the induction medium for 15 to 90 minutes, more specifically, 40 minutes in dark at 25 °C to 28°C followed by air drying for 10 to 25 minutes, more specifically, 15 minutes.