Title of Invention

METHOD FOR TRANSFORMATION OF ARNEBIA SPECIES

Abstract A method for enhanced production and recovery of Shikonin from Arnebia hispidissima through induction of hairy root, wherein the said method comprising the steps of Sterilizing the explants of Arnebia hispidissima, Inoculating the said sterilized explants in MS medium to form clusters of multiple shoots, obtaining explants of Arnebia hispidissima either from seedlings or whole plant characterized by, infecting the explants of step (c) with Agrobacterium rhizogenes, co-cultivating and transforming the explants of step (d) on co-cultivation medium with Agrobacterium rhizogenes for 3-5 days in complete darkness, selecting and growing transformed plant cells of step (e) on a root induction medium to obtain hairy roots supplemented with antibiotic, transferring the hairy roots obtained from the step (f) on Shikonin induction medium to induce Shikonin production in the hairy roots, wherein the said hairy root culture is free of NH4+ ions and are incubated in dark on rotary shaker at 23-25 C for 24 hrs for enhanced Shikonin induction, Recovering Shikonin of step (g) using conventional methods.
Full Text Field of invention:
The present invention relates to a method of Agrobacterium rhizogenes mediated genetic transformation in Amebia species. The present invention also provides co-cultivation for the production of plant secondary, metabolites, Shtkonin.
Background of the invention:
The hairy root disease is a pathological syndrome of dicotyledonous plants following wounding and infection with Agrobacterium rhizogenes. The rhizogenicity is conferred to plant cells by a fragment of ONA (Ri T-DNA), which is transferred from the large root inducing (Ri) plasmid, harbored by the bacterium, to the genome, where it is stably integrated and expressed. Integration of a DNA segment (T-DNA) of pRi into the host genome leads to active proliferation of adventitious hairy roots at or near the site of infection. The role genes in RiT-DNA induced changes in sensitivity in plant hormone or in the metabolism of plant hormones (Maurel et al 1994; Moritz and Schinulling 1998; Shen etal 19S8). Furthermore transformation of plant tissue by infection with Agrobacterium rhizogenes increases the production of certain metabolites (Ermayanti etal 1994; Mano etal 1986; Sim etal 1994). If the Agrobacterium rhizogenes containing modified Ri plasmid is used to infect plant cells, it is possible to transfer the target genes to plant tissues and to induce regeneration of transgenic plants. Therefore, Agrobacterium rhizogenes can be used in genetic manipulation of higher plants for improving plant resistance, quality and possible utilization. Many reports about hairy root transformation of various medicinal plants have been published including Lithospermum erythrorhizan. (Hazaki etal, 1998) However Agrobacterium rhizogenes mediated transformation of Amebia hispidissima and induction of Shikonin production in hairy root cullures of Amebia hispidissima by employing Agrnbacterium rhizogenes mediated genetic transformation has not been reported yet.
Amebia (family Boraginaceae) is a genus of hispid herbs, mostly confined to Asia with a few species occurring in drier part of North Africa.
Seven species are known to occur in India. These include A. benthamii. A. euchroma, A. guttata, A. hispidissima and A. nobilis.
The plant of genus Amebia as well as some other species of Boraginoceae from the genera Lithospermum, Onosma, and Echium etc. provide the source of naphthoquinonous , a red pigments, Shikonin(Fukui et al, 1984). Shikonin has been known since ancient times as a dye used for Silk* and Food Products. At the same time, Shikonin is recognized as a remedy, showing a wide range of medical applications. It possesses antibacterial
( Tanak a and odani 1972 ) antifungal activitues and exhibits anti-
inflammatory (Hayashi et al.1969), anti-tumor activities (Papageorgiou 1980) and wound healing properties. The anti-allergic, antipyretic, antihydropic effects of Shikonin and its derivatives as well as its antineoplastic activities are reported (Terada et al; 1990). Most of the species of Boraginaceae yield Ratanjot, which is used as a red dye and also as a medicine to treat a variety of ailments (Khatoon et al; 1994). Moreover its roots are important crude source of drug in Asian countries. Shikonin derivatives accumulate solely in the roots of the intact plant
Shikonin biosynthesis has been one of the most intensively
studied topics in the area of secondary metabolites of higher
plants ( M. Tabata 19 96), and many of the regulatory factors in the
biosynthetic pathway have been identified. The strongest inhibitor
for Shikonin biosynthesis is light {Tabata et al. 1974; Heide -et al.
1989). Several cDNAs of dark-expressed genes have been cloned from
Lithospermum erythrorhizon by sub tractive hybridization (Yazaki et ai.1998),
but neither their function in; vivo nor the molecular mechanism of light
inhibition is known because no stable transformation system isavailabie. The
major constraints with Lithospermum cell culture is that they are genetically
unstable and cultured cell tend to produce low yield of secondary metabolites
with passage of time. In the present invention provide a method for
transformation of Amebia hispidissima by Agrobacterium rhizogenes wild
type strains A4. The hairy roots continuously produce Shikonin and its
derivatives and are released into the culture medium with high efficiency.OBJECTS OF THE INVENTION
An object of the present invention is to provide a method for Agrobacterium rhizogenes mediated genetic transformation in Arnebia species preferably Arnebia hispidissima.
Another object of the present invention is to provide a method for co-cultivation for the production of plant secondary metabolites, Shikonin in transformed Arnebia species preferably Arnebia hispidissima.
Another object of the invention is to provide a method for regeneration of true to type plantlets of Arnebia hispidissima.
Still another object of the invention is to provide method for production of callus cultures from various explants of Arnebia hispidissima.
Another object of the invention is to provide an optimized media composition for callus production of Arnebia hispidissima.
Another object of the invention is to provide an optimized media composition and other conditions for transformation of Arnebia hispidissima from callus culture.
Another object of the present invention is to provide a method for plant regeneration of Arnebia hispidissima from explants selected from shoot tips, leaf segments, Nodal and internodal segments.
Another object of the present invention is to provide a method for plant regeneration of Arnebia hispidissima from callus culture by culturing callus on shoot induction media supplemented with various plant growth regulators.
Still another object of the invention is to provide an in vitro method for induction of Shikonin in callus cultures of Arnebia hispidissima.
Another object of the invention is to provide an optimized media composition and other conditions for induction of Shikonin in callus cultures of Arnebia hispidissima.
STATEMENT OF INVENTION
According to this invention there is provided a method for enhanced production and recovery of Shikonin from Arnebia hispidissima through induction of hairy root, wherein the said method comprising the steps of Sterilizing the explants of Arnebia hispidissima, Inoculating the said sterilized explants in MS medium to form clusters of multiple shoots, obtaining explants of Arnebia hispidissima either from seedlings or whole plant characterized by, infecting the explants of step (c) with Agrobacterium rhizogenes, co-cultivating and transforming the explants of step (d) on co-cultivation medium with Agrobacterium rhizogenes for 3-5 days in complete darkness, selecting and growing transformed plant cells of step (e) on a root induction medium to obtain hairy roots supplemented with antibiotic, transferring the hairy roots obtained from the step (f) on Shikonin induction medium to induce Shikonin production in the hairy roots, wherein the said hairy root culture is free of NH4+ ions and are incubated in dark on rotary shaker at 23-25 C for 24 hrs for enhanced Shikonin induction, Recovering Shikonin of step (g) using conventional methods.
SUMMARY OF THE INVENTION;
The present method provides a novel and efficient method for rapid and efficient method of direct regeneration of whole plant of Arnebia hispidissima.
Another aspect of the present invention is to provide a method for high frequency direct plant regeneration from various explants wherein the explains are selected from a group consisting of shoot tips, nodal and internodal explains, leaf segment and any other suitable explants.
Another aspect of the present invention is to provide a method for high frequency direct plant regeneration wherein the explants are cultured on* suitable Murashige and Skoog media (MS) containing plant growth regulators for shoot regeneration.
The invention provides a media composition for direct plant regeneration wherein the explants are cultured on MS medium alone or supplemented with one of the following plant growth regulators, namely, 6-Benzylaminopurine (BAP), Kinetin (KN), Indole-3-aceticacid (IAA).
The invention also provides a method wherein the multiplied shoot buds are further transferred to rooting media containing MS medium alone or MS media in combination with one of the growth regulators: lndole-3-butyeric acid (1BA), A-naphthalene acetic acid (NAA), indole-3-acetic acid (IAA).
The invention further provides a media composition for callus production using various explants of Amebia hispiclissima.
The invention further provides a method for regeneration of Amebia hispidissima plant from callus culture by culturing the explants on callus promoting medium at a temperature in the range of 25-27 °C and photoperiod of 12 hrs to obtain callus culture.
The invention further provides a method for regeneration of Amebia hispidissima plant by culturing the callus on shoot induction medium to obtain multiple shoots and root induction medium to obtain rooted plantlets. The invention further provides an in vitro method for the induction of the pigment Shikonin in callus cultures. The pigment production was observed when callus tissues were transferred to the Shikonin induction medium containing IAA and incubated in the dark. The pigment production in the callus was observed within two weeks of incubation of the callus in Shikonin induction medium.
BRIEF DESCRIPTION OF ACCOMPANYING DRAWINGS
FIG. 1: Explants showing hairy root initiation and callus induction from
Amebia hispidissima
FIG. 2: Explants showing hairy root induction from Amebia hispidissima
on the optimized medium with appearance of red spots.
FIG. 3: Hairy roots from Amebia hispidissima cultured on hormone free
MS medium showing reddish roots turning white.
FIG. 4: Shikonin production in hairy roots from Amebia hispidissima
cultured on the NH4+ ion free RC medium.
FIG. 5: Confirmation of presence of roIB genes in transformed hairy roots
of Amebia hispidissima
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides a method for enhanced production of shikonin in Amebia species. The present invention also provides a method for co-cultivation of various explants of Amebia species for hairy root culture with Agrobacterium rhizogenes.
In one embodiment, the invention provides a method for induction of shikonin production in hairy root cultures of Amebia hispidissima comprising:
a. obtaining explants either from seedlings or whole plant or in
vitro raised plantlets,
b. infecting the explants with Agrobacterium rhizogenes,
c. co-cultivating the explants on co-cultivation medium with
Agrobacterium rhizogenes for 3-5 days,
d. selecting transformed plant cells on a root induction medium
to obtain hairy roots,
et transferring the hairy roots obtained on shikonin induction medium to induce shikonin production in the hairy roots cultures,
f. recovering shikonin obtained from hairy root culture of step (e)
using conventional methods.
g. Confirming the presence of roIB genes in transformed hairy
roots of Amebia.
The present invention provides a method for induction of multiple shoots from various explants of Amebia plant. Further the method comprising of culturing the explants obtained either from seedlings or field grown plant on shoot induction medium to obtain multiple shoots.
Another embodiment of the present invention provides a method for co-cultivation of the explants obtained from the in vitro raised Amebia plantlets with Agrobacterium rhizogenes for the induction of hairy roots, wherein the explants are selected from a group consisting of shoot tip, nodal and internodal explants, leaf segment and other suitable explants.
Still another embodiment of the present invention provides a composition of the root induction medium wherein the medium is MS medium with or without 2 mg/1 IBA and 100 µ M acetosyringone.
Still another embodiment of the present invention provides a composition of the medium for selecting the transformed hairy roots wherein the medium is MS medium with or without 2 mg/1 IBA, 100 µ M acetosyringone and 250 mg/1 cefotaxime.
Still another embodiment of the present invention provides a method for induction of shikonin in the hairy root cultures of the Amebia plant.
Still another embodiment of the present invention provides a composition of the medium for shikonin induction wherein the medium is NH4+ free root culture liquid medium.
Present invention provides a evidence for the presence and integration of rolB genes in transformed hairy roots of Amebia.
Plants of Amebia hispidissima were employed for establishing hairy root cultures of Amebia hispidissima. Various explants namely leaf, shoot tip, nodal and internodal segments are employed for genetic transformation studies with Agrobacterium rhizogenes wild type A4 strain. The details of the explant preparation and multiple shoot production are given in Example 1.
The media composition for growing Agrobacterium rhizogenes is given in Example 2. Agrobacterium rhizogenes was grown in the media described in Example 2. The conditions for culturing Agrobacterium rhizogenes are given in Example 3. Other culture media known in the art may be used for culturing the Agrobacterium rhizogenes strains.
Leaf explants derived from in vitro grown plants of Amebia were infected with Agrobacterium rhizogenes wild type strain A4 (Source ATCC) and the details are given in Example 4. Further, the explants were co-cultivated in suitable medium for 3-7 days. This incubation in suitable medium leads to the production of hairy roots as described in Example 5 and 6.
The hairy roots when cultured on Murashige and Skoog (1962) semi solid medium do not produce any shikonin or its derivatives. However, the hairy roots when cultured on the NH4+ free semi-solid or liquid root culture (RC) medium (Thomas and Davey, 1982) produce a
large amount of shikonin, which is released into the medium. The details of induction of shikonin production are given in Example 7.
The shikonin pigment was extracted and quantified spectrophotometerically as described in Example 8.
The isolation of genomic DNA, agarose gel electrophoresis and Polymerase Chain Reaction (PCR) analysis of the Agrobacterium rhizogenes-mediated genetically transformed Hairy Root Cultures for confirmation of integration and presence of rolB genes in transformed hairy roots of Amebia hispidissima have been described in Example 9.
It was observed that shikonin production by hairy root cultures is much higher as compared to the normal roots. Hairy roots also display light dependent inhibition of shikonin biosynthesis similar to that of Amebia hispidissima cell cultures. The present invention demonstrates the effectiveness of transformation of Amebia hispidissima with Agrobacterium rhizogenes for hairy root culture for the production of plant secondary metabolite, shikonin. This is the first report of induction of shikonin production in hairy root cultures of an important medicinal plant Amebia hispidissima by employing Agrobacterium rhizogenes mediated genetic transformation.
Plantlets of Amebia hispidissima were maintained in vitro as multiple shoot clusters. Explants from field grown plants or in vitro raised plantlets were cultured for three weeks at low light intensity on MS medium with acetosyringone (100 µM). The etiolated explants grown under low light intensity are infected with A. rhizogenes A4 strain (wild type) in petri plates for 15- 30 min and incubated in dark or in light of low intensity. Different strains of A. rhizogenes known in the art may be
used fof infection of Amebia explants. After infection the explants were dried on sterile blotting paper and transferred to suitable tissue culture medium for co-cultivation. Five days after co-cultivation on MS medium with acetosyringone, the explants became thick, curled and expanded 1-2 times. These explants were transferred onto MS medium supplemented with antibiotic 250 mg/1 cefotaxime. Other tissue culture medium was also used for co-cultivation of the explants after infection with A. rhizogenes. Within two weeks hairy roots begin to appear at the cut surfaces of the explant. Further, callusing of the hairy roots was observed on the MS medium containing antibiotics (Fig. 1). After elimination of Agrobacterium on cefotaxime supplemented media, the explants were transferred to hormone free MS solid medium, where the formation of hairy roots with red spots of shikonin pigment was observed (Fig 2). Further, the hairy roots were also obtained directly from the explants when transferred on suitable medium as given above. It was further observed that the reddish roots having shikonin pigment turned white after a second sub-culture on the same medium as shown in Fig. 3. It is clear that the new roots do not show the presence of shikonin pigment in the medium on incubation. Shikonin and its derivatives are known to possess tremendous medicinal values of antibacterial, antifungal, anti-allergic, anti-inflammatory, antihydropic, antineoplastic and wound healing properties as reported by Terada et al. (1990); Khatoon et al. (1994, 2003).
Therefore, it is clear that MS medium is not suitable for the production of shikonin in hairy root cultures. A number of different tissue culture media were screened for the induction of shikonin production in the hairy roots of Amebia. The ideal tissue culture media is the NH4+ ion free RC medium for the Shikonin production in roots. This medium showed rapid growth of roots and produced large amount of
Shikonin which was released into the medium as shown in Fig.4. It is clear from Fig.4 that the roots show the presence of enhanced Shikonin production. Another important observation that the Shikonin production is highest when incubated in the dark at a temperature of 22-27° C. The pigment, Shikonin, is present all over the roots as seen in Fig. 4. After 3 to 4 weeks of culture in the RC medium in dark conditions, Shikonin production by hairy roots increases as compared to the normal roots. However, it has been observed that addition of charcoal in the RC medium inhibits Shikonin production. The isolation of genomic DNA, agarose gel electrophoresis and Polymerase Chain Reaction (PCR) analysis of the Agrobacterium rhizogenes-mediated genetically transformed Hairy Root Cultures for confirmation of integration and presence of rolB genes in transformed hairy roots of Amebia hispidissima have been described in Example 9 and shown in Fig. 5.
The present invention demonstrates the effectiveness of co-cultivation of Amebia species explants for induction of hairy root cultures for enhanced in vitro production of commercially important secondary metabolite the Shikonin. The hairy roots were obtained either from calli or directly from explants. The leaf explant is the best explant (70.7%) for hairy root production as compared to other explants such as shoot tip (52%), nodal segment (38.7%) and internodal segment (9.3%).
EXAMPLES
It should be understood that the following examples described herein are for illustrative purposes only and that various modifications or changes in light will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and the scope of the appended claims.
Example 1
Preparation of plant material
The explants namely leaf, node, inter node and shoot tips were excised from field grown plants and surface sterilized with HgCl2 (0.1%) for 5-7 minutes and subsequently washed three times with double distilled water to remove traces of HgCl2 and inoculated on MS medium (Table 1) supplemented with BAP 0.25 mg L-1, Kinetin 0.5 mg L-1 IAA 0.1 mg L-1 and Casein Hydrolysate (CH) 100 mg L-1. Shoots obtained were multiplied on the same medium for 3 weeks at low light intensity. This leads to the formation of clusters of multiple shoots. The leaf explants were obtained from in vitro grown plants which serve as starting material for co-cultivation and genetic transformation of Amebia.
Example 2
Preparation of LB medium for Agrobacterium culture
All the components of LB (Luria Broth) media (Table 2) are dissolved in distilled water and final volume is made up to one liter. The pH of the medium is adjusted to 7 with NaOH solution and agar is added to the above medium. After melting the agar, measured quantity of medium is dispensed in each flask or test tube and autoclaved at 15 psi at 121°C for 15-20 minutes.
Example 3 Agrobacterium culture
From the 25% glycerol stocks v/v of Agrobacterium rhizogenes strain A4, culture is streaked on to LB plate by using the inoculation loop and incubated at 28°C for 12-24 hrs. Single colonies of Agrobacterium rhizogenes are further streaked for three successive days on solid LB medium (Table 2) for 24 hr and grown at 28°C. The single colony from the actively growing culture is inoculated in 30 ml of liquid LB medium at 28°C at 200 rpm in incubator shaker. Bacterial culture is
allowed'to grow to an absorbance of 0.8-1.0 at OD600. From the actively growing cultures, fresh inoculation is made in LB and allowed to grow under similar conditions mentioned above till the OD600 of 0.30 is obtained.
Example 4
Infection of explants with Agrobacterium rhizogenes
The Agrobacterium cells are pelleted at 3000 rpm for 10 minutes. The supernatant is discarded leaving only the pellet which is immediately re-suspended in 30 ml liquid MS (without vitamins, sucrose and hormones) with 100 µM acetosyringone (Sigma chemicals Co; USA). In the above suspension, the MS media without vitamin and sucrose are poured in the sterilized plate and in-vitro produced explants are placed in the plates. The plate is sealed with Parafilm and stirred at 50 rpm for 15-20 minutes to ensure that the cut surfaces have contact with the bacteria. Explants are blotted dry on sterilized filter paper to get rid off the excess bacteria adhering to the tissue.
Example 5 Co-cultivation
The infected explants are co-cultivated in Petri dishes containing semi-solid MS medium supplemented with or without IBA (2.0 mg L-1) and 100 µM acetosyringone. Petri plates are sealed with Parafilm. These cultures are incubated for 5 days in complete dark at 25 ± 2°C.
Example 6
Induction of hairy roots
Five days after the co-cultivation, the explants are transferred onto same medium as discussed in Example 5. Additionally, it is supplemented with cefotaxime 250 mg/1. After elimination of Agrobacterium on cefotaxime supplemented media, the explants were
transferred to hormone free MS solid medium, where the formation of hairy roots at the cut surfaces of the explant with red spots of shikonin induction in the older parts of the tissue was observed (Fig 1,2). These were separated using scalpels, blades and sub-cultured on semi-solid hormone free MS medium for 5 days.
Example 7
Induction of shikonin in hairy root culture
The reddish roots turned white after a second sub-culture on the medium described in Example 6 (Fig. 3). Hairy roots are further sub-cultured in NH4+ free RC root culture liquid medium (RC) (Table 3). The cultures are kept on a rotary shaker at 100 rpm, at 25°C in the dark for 24 hr. Hairy roots cultured on the NH4+ free RC medium showed fast growth and produced large amount of shikonin which was released into the medium (Fig. 4). After 3 to 4 weeks of culture in the RC medium, shikonin production by hairy roots increases as compared to the normal roots.
Example 8
Extraction of Shikonin
The protocols for extraction of Shikonin content and its derivatives from hairy roots are optimized using the method given below. Hairy roots are homogenized with chloroform (CHCl3) and left in the dark for 12-24 hr. After drying over MgSO4, the extract is evaporated to dryness and 2.5% KOH solution is added. Blue color is expected to develop after 10 min. The shikonin content is assayed by employing UV Spectrophotometer and recording the absorbance at 622 nm (Fukui et al. 1988; Yazaki et al. 1998).
Example 9
Isolation of Genomic DNA, agarose gel electrophoresis and Polymerase Chain Reaction (PCR) analysis of the Agrobacterium rhizogenes-mediated genetically transformed Hairy Root Cultures of Amebia hispidissima.
Genomic DNA of young leaves, callus, normal roots and transformed Hairy Roots of Amebia hispidissima were isolated using CTAB method of Saghai Maroof et al. (1984) with some modifications. Approximately 3-5 g of plant material was grinded to a fine powder using liquid nitrogen in a sterilized pestle and mortar. Powder was transferred immediately into 50 ml polypropylene tube (Tarsons Products Pvt. Limited; Kolkata, India) taking care that thawing does not take place. Added 15 ml of preheated (65°C) CTAB buffer to the powder mixture. The samples were thoroughly mixed by gently inverting the tubes several times. The tubes were then incubated at 65°C for 90 min in a water bath (Pharmacia Biotech, USA) with regular mixing of samples. After incubation, samples were cooled to room temperature and 15 ml of chloroform: isoamyl alcohol (24:1) mixture was added to each tube. Contents in the tubes were mixed gently for 10-15 min. Tubes were then centrifuge (Remi Instruments, Mumbai, India) at 10,000 rpm for 10 min at 25°C and the upper aqueous layer was transferred into fresh sterilized 50 ml tube using micropipette. Equal volume of 15 ml isoamyl alcohol was added to the tube and mixed gently for 10-15 min, centrifuged at 10,000 rpm for 10 min. The aqueous phase was carefully transferred to fresh tubes.
Equal volume of ice cold isopropanol was subsequently added, mixed and tubes kept at room temperature for 15 min. The DNA was spooled out with micropipette, washed in 70% ethanol and allowed to air dry. It was dissolved in 500 µl of 1XTE buffer. For removal of RNA
contamination, 2 ul of RNase A (10 mg ml-1) was added to each tube. Samples were mixed gently and then incubated at 37°C for 1 h. DNA was again extracted by adding equal volume of chloroform: isoamyl alcohol (24:1) mixture. Samples were mixed well and then centrifuged at 10,000 rpm for 10 min. Supernatant was transferred to new eppendorf tubes (Tarsons Products Pvt. Limited; Kolkata, India) and 1/10 volume of 3 M sodium acetate and 2 volumes of chilled absolute alcohol were added. The eppendorf tubes were centrifuged at 10,000 rpm for 10 min to pellet down the DNA. The Pellet was washed with 70% alcohol, air dried and finally dissolved in appropriate volume 1XTE buffer. Samples were stored at -20°C until further use.
PCR amplified DNA fragments were separated by submerged horizontal agarose gel electrophoresis (1% w/v). Gel casting tray (Bio Rad Res. Lab. USA) was washed, air dried and its ends were sealed with tape. Agarose was melted by boiling in 1X TBE buffer, cooled to 60-65°C and 2-3 drops of ethidium bromide (5 µgml-1) were added. Gel solution was then poured into gel casting tray with an appropriate comb inserted. Gel was allowed to set for 30 min. After gelling of agarose, sealing tapes were removed from both the ends and the tray was placed in electrophoresis chamber.1X TBE buffer was added to submerge the gel and comb was removed gently. Samples were prepared by adding 2 µl of 6 X loading dye and centrifuged briefly in a micro centrifuge for proper mixing. DNA samples were than loaded in the wells and electrophoresis was carried out at constant voltage (3v/cm of gel) till the dye migrated to the other end of gel. A 100 bp ladder was employed as the molecular weight marker on gels. PCR amplified products were visualized under UV light and photograph was taken using gel documentation system (Gene Genius, Syngene, UK). Total genomic DNA was extracted from normal roots, transformed primary roots, transformed secondary roots, callus tissue and leaves for PCR analysis. Lanes 1-2 non transformed callus
tissues, Lanes 3-5 transformed hairy root cultures, non transformed normal roots lanes 6-7 and non transformed leaves lane 8. Sharp and distinct bands were obtained as shown in lanes 1-8 in gel photograph A (Fig 5).
Plasmid DNA was isolated from wild type Agrobacterium rhizogenes strain A4 for PCR analysis. 1% Agarose gel electrophoresis revealed a sharp and distinct band of > 20 kb lanes 2 & 3 when both □φpxl74 DNA/Hae III digest and 1 kb DNA marker lanes 1 8B 6 were included as reference in gel photograph B (Fig 5).
The forward (FrolB) primers ATG GAT CCC AAA TTG CTA TTC CTT CCA CAG (Tm values 61.4) and reverse (RrolB) primers TTA GGC TTC TTT CTT CAG GTT TAC TGC AGC (Tm values 60.4) for roIB gene (25 nm DNA Oligo, standard desalted, 30 bases each) were synthesized from Integrated DNA Technologies, Inc. CA, USA.
PCR amplification was performed in a Thermocycler (PTC-100™, MJ Research Inc., USA). The rolB gene in the T-DNA of pRiA4 plasmid upon transfer to the plant genome was detected in the plant and bacterial genome by PCR amplification using the procedure of Hamill et al. (1991) with some modifications. PCR amplification was carried out in a total volume of 30 µl, 1 µl samples of Amebia hispidissima genomic DNA, 20 pmol of each of Fro© and Rro© primers, 200 µM of each of dATP, dCTP, dGTP, dTTP and 0.5U Taq DNA polymerase obtained from Genei, Banglore, India in a standard PCR incubation buffer (10 raM Tris HC1 pH 9.3, 15 mM KC1, 1.5 mM MgCl2). The thermocycler was programmed to the following temperature profile: 30 cycles of denaturation at 94°C for 1 min, annealing at 55°C, extension at 72°C for 1 min and final extension at 72°C for 3 min, followed by a soak at 4°C. Sterile distilled water instead of genomic DNA served as a negative control. The amplification reactions were preformed twice.
The presence of roIB genes in transformed hairy roots of Amebia hispidissima was confirmed by PCR analysis using forward and reverse primers of roIB genes as shown in gel photograph C (Fig 5). Total genomic DNA extracted from transformed roots formed on MS medium without plant growth regulators yield fragment of ~0.8 kb which coincide with the fragment expected after amplification of the genes for T-DNA of Ri plasmid of Agrobacterium rhizogenes. Agarose gel electrophoresis revealed distinct bands of 0.8 kb from genomic DNA of transformed Hairy Root Cultures (lanes 2, 4, 5 and 8) and plasmid DNA of Agrobacterium rhizogenes (lane 1). Thus, it has been confirmed at molecular level that the gene for T-DNA of Ri plasmid had been expressing into the genome of the transformed Hairy Root Culture. No amplification was observed in negative controls (Lane 10) and no amplification in the genomic DNA of non transformed normal roots, callus tissue and leaves (lanes 3 and 6, 7 and 9).
ADVANTAGES
Shikonin is known to have tremendous applications for cosmetic, dyeing, food, medicinal, and pharmaceutical industry. The present invention demonstrates the effectiveness of transformation of Amebia hispidissima with Agrobacterium rhizogenes for induction of hairy root culture for the production of plant secondary metabolite, Shikonin. This is the first report of induction of Shikonin production in hairy root cultures of an important medicinal plant Amebia hispidissima by employing Agrobacterium rhizogenes mediated genetic transformation. It is envisaged that simple and reproducible method developed here through induction of hairy root cultures in Amebia hispidissima will be very useful for meeting the ever increasing demand of Shikonin for pharmaceuticals, medicinal and food industries.












We Claim:
1. A method for enhanced production and recovery of Shikonin from
Arnebia hispidissima through induction of hairy root, wherein the said
method comprising the steps of:
a. Sterilizing the explants of Arnebia hispidissima,
b. Inoculating the said sterilized explants in MS medium to form
clusters of multiple shoots,
c. obtaining explants of Arnebia hispidissima either from
seedlings or whole plant characterized by,
d. infecting the explants of step (c) with Agrobacterium
rhizogenes,
e. co-cultivating and transforming the explants of step (d) on co-
cultivation medium with Agrobacterium rhizogenes for 3-5 days
in complete darkness,
f. selecting and growing transformed plant cells of step (e) on a
root induction medium to obtain hairy roots supplemented
with antibiotic,
g. transferring the hairy roots obtained from the step (f) on
Shikonin induction medium to induce Shikonin production in
the hairy roots, wherein the said hairy root culture is free of
NH4+ ions and are incubated in dark on rotary shaker at 23-25
C for 24 hrs for enhanced Shikonin induction,
h. Recovering Shikonin of step (g) using conventional methods.
2. A method as claimed in claim 1, wherein the said explants were
surface sterilized in HgCl2 for 5-7 minutes and subsequently washed
with distilled water to remove traces of HgCl2.
3. A method of claim 1, wherein the Agrobacterium rhizogenes strain is selected from a group consisting of A4, K84, R1601, 1855, 8196 and 15834 and the LB medium is prepared by dissolving all the
components of Luria Broth in distilled water, pH adjusted to 7.0 with NaOH solution and agar was added after melting it and autoclaved at 15 psi at 121°C for 15 to 20 minutes.
4. A method of claim 1, wherein the co-cultivation medium is done in semi solid MS medium supplemented with or without 2.0 mgL-1 IBA (Indole Butyric Acid) and 100 uM acetosyringone.
5. The method as claimed in claim 1, wherein said antibiotic used is 250 mgL1 Cefotaxime for removing Agrobacterium.
6. The method as claimed in claim 1, wherein said MS medium comprising Kinetin in the range of 0.2-2.0 mgl-1 , 2,4-dichlorophenoxyacetic acid (2,4-D) in the range of 0.5-2.0 mgl-1 and Casein Hydrolysate (CH) in the range of 50-150 mgH , MS medium comprising BAP in the range of 0.5-2.0 mgl-1 , 2,4-D in the range of 0.5-2.0 mgl1 and CH in the range of 50-150 mgl-1 , MS medium containing Kinetin in the range of 0.2-2.0 mgl-1 , IAA in the range of 0.1-2.0 mgl"1 and CH in the range of 50-150 mgl-1 and MS medium containing BAP in the range of 0.1-2.0 mgl1, IAA in the range of 0.01-2.0 mgl-1 and CH in the range of 50-150 mgl1.
7. The method as claimed in claim 1, wherein in step (b) the explants selected from a group consisting of shoot tips, nodal segments, internodal segments, leaf segment and shoot tips were pre-cultured for three weeks at low light intensity on MS medium supplemented with 100 uM acetosyringone at a temperature of 22 to 27°C.
8. The method as claimed in claim 4, wherein co-cultivation is done in MS medium comprising Kinetin in the range of 0.2-2.0 mgl-1, IAA in the range of 0.01-2.0 mgl-1 and Casein Hydrolysate (CH) in the range of 50-150 mgl-1 and MS medium containing BAP in the range of 0.1-2.0 mgl-1, IAA in the range of 0.01-2.0 mgl"1 and CH in the range of 50-150 mgl-1.

Documents:

2483-DEL-2004-Abstract-(15-09-2010).pdf

2483-del-2004-assignment.pdf

2483-DEL-2004-Claims-(15-09-2010).pdf

2483-DEL-2004-Claims-(30-04-2010).pdf

2483-del-2004-claims.pdf

2483-DEL-2004-Correspondence-Others (27-11-2009).pdf

2483-DEL-2004-Correspondence-Others-(15-09-2010).pdf

2483-DEL-2004-Correspondence-Others-(30-04-2010).pdf

2483-del-2004-correspondence-others.pdf

2483-DEL-2004-Description (Complete)-(15-09-2010).pdf

2483-DEL-2004-Description (Complete)-(30-04-2010).pdf

2483-del-2004-description (complete).pdf

2483-del-2004-description (provisional).pdf

2483-DEL-2004-Drawings-(30-04-2010).pdf

2483-del-2004-drawings.pdf

2483-DEL-2004-Form-1-(27-11-2009).pdf

2483-del-2004-form-1.pdf

2483-del-2004-form-13.pdf

2483-del-2004-form-18.pdf

2483-DEL-2004-Form-2-(30-04-2010).pdf

2483-del-2004-form-2.pdf

2483-del-2004-form-26.pdf

2483-DEL-2004-Form-3-(15-09-2010).pdf

2483-DEL-2004-Form-3-(30-04-2010).pdf

2483-DEL-2004-Form-5-(30-04-2010).pdf

2483-del-2004-form-5.pdf

2483-del-2004-form-6.pdf

2483-DEL-2004-GPA-(27-11-2009).pdf

2483-DEL-2004-Petition-137-(15-09-2010).pdf


Patent Number 244496
Indian Patent Application Number 2483/DEL/2004
PG Journal Number 50/2010
Publication Date 10-Dec-2010
Grant Date 08-Dec-2010
Date of Filing 14-Dec-2004
Name of Patentee DEPARTMENT OF BIO & NANO TECHNOLOGY
Applicant Address GURU JAMBHESHWAR UNIVERSITY OF SCIENCE & TECHNOLOGY, HISAR-125001, HARYANA, INDIA.
Inventors:
# Inventor's Name Inventor's Address
1 ASHOK CHAUDHURY GURU JAMBESHWAR UNIVERSITY, HISAR-125001, HARYANA, INDIA
2 MINAKSHI PAL GURU JAMBESHWAR UNIVERSITY, HISAR-125001, HARYANA, INDIA
PCT International Classification Number C12N 15/00
PCT International Application Number N/A
PCT International Filing date
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 NA