Title of Invention	"METHOD OF DIRECT REGENERATION AND SHIKONIN INDUCTION IN CALLUS OF ARNEBIA HISPIDISSIMA SP"
Abstract	The present method provides a rapid and efficient method for direct regeneration of whole plant of Aniebia hispidissitna. The invention also provides a method for high frequency direct plant regeneration from shoot tip or other suitable explants by supplementing the MS media with various plant growth regulators. The invention further provides a media composition for callus production and a method for regeneration of whole plant from callus cultures of Arnebia hispidissima. The invention also provides an in vitro method for induction of shikonin in callus cultures.

Title of Invention

"METHOD OF DIRECT REGENERATION AND SHIKONIN INDUCTION IN CALLUS OF ARNEBIA HISPIDISSIMA SP"

Abstract

The present method provides a rapid and efficient method for direct regeneration of whole plant of Aniebia hispidissitna. The invention also provides a method for high frequency direct plant regeneration from shoot tip or other suitable explants by supplementing the MS media with various plant growth regulators. The invention further provides a media composition for callus production and a method for regeneration of whole plant from callus cultures of Arnebia hispidissima. The invention also provides an in vitro method for induction of shikonin in callus cultures.

Full Text	Method of direct regeneration and shikonin induction in callus of Arnebia hispidissima FIELD OF INVENTION The present invention relates to a method of direct plant regeneration, and callus production and regeneration of plants from callus in Arnebia species. The invention also provides an in-vitro method for induction of Shikonin in callus cultures of Arnebia hispidissima. BACKGROUND OF THE INVENTION Arnebia (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 Arnebia as well as some other species of Boraginaceae family from the genera Lithospermum, Onosma, Echium provides the source of naphthoquinonous red pigment, Shikonin. 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, antifungal activities and exhibits anti-inflammatory and wound healing properties. The anti-allergic, antipyretic, antihydropic effects of Shikonin and its derivatives as well as antineoplastic activities are reported in the literature (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). Shikonin and its derivatives are contained in the outer surface of roots of Arnebia. Shikonin has an applicable pharmacological effect as an antibacterial agent, granulation tissue forming activity and anti-ulcer activity (Hayashi et al. 1969; Propageorgiou, 1980). Shikonin content depends upon the age of plant and other environmental factors. Middle aged plants with 6-8 flowering stems, show highest level of Shikonin (Pimenova and Tareeva, 1980). Wild plant species from the Boraginaceae family, accumulating Shikonin fail to provide sufficient raw material for commercial production, owing to which tissue culture technique becomes an excellent tool to overcome these short comings. Arnebia tissue culture was first reported by Rabinovich and Davydenkov (Davydenkov et al. 1991). However, no detailed studies have so far been reported on high frequency direct plant regeneration in this plant species and do not teach the present invention. This invention deals with high frequency direct plant regeneration and callus production from various explants of Arnebia species preferably Arnebia hispidissima. Further, methods are developed for the production of calli to induce production of Shikonin derivatives by varying the culture conditions such as light and level of auxins in the tissue-culture media. OBJECTS OF THE INVENTION An object of the present invention is to provide a rapid and efficient method for direct regeneration of Arnebia species preferably Arnebia hispidissima. Another object of the present invention is to provide a method for high frequency direct plant regeneration of Arnebia hispidissima from explants selected from shoot tips, leaf segments, nodal and inter-nodal segments by supplementing the media with various plant growth regulators. Another object of the invention is to provide a method for regeneration of true-to-type plantlets of Arnebia hispidissima. Another object of the invention is to provide an optimized media composition for direct regeneration of Arnebia hispidissima. Still another object of the invention is to provide method for production of callus cultures from various explants of Arnebia hispidissima. 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. SUMMARY OF THE INVENTION The present invention relates to a novel and efficient method of direct plant regeneration, callus production and regeneration of plants from callus in Arnebia species. The invention also provides an in vitro method for induction of shikonin in callus cultures of Arnebia hispiclissima. Another aspect of the present invention is to provide a method for high frequency direct plant regeneration from various explants wherein the explants are selected from a group consisting of shoot tips, nodal and internodal explants, 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 Murashige and Skoog media (MS) containing suitable 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, Indole-3-butyeric acid (IBA), A-naphthalene acetic acid (NAA), and indole-3-acetic acid (IAA). The invention further provides a media composition for callus production using various explants of Arnebia hispidissima. The invention further provides a method for regeneration of Arnebia 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 Arnebia 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 shoot initiation FIG. 2: Regenerated plantlets showing rooting FIG. 3: Green callus under light condition showing no pigmentation FIG. 4: Red pigmented callus showing enhanced shikonin production DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel and rapid method for high frequency direct plant regeneration, callus production and regeneration of plants from calli in Arnebia species. The invention also provides an in-vitro method for induction of shikonin in callus cultures of Arnebia hispidissima. An embodiment of the present invention relates to a method for direct regeneration of Arnebia plant, where the method comprising steps of culturing the explants obtained either from seedlings or whole plant on shoot induction medium to obtain multiple shoots; transferring the shoots on the root induction medium to obtain rooted plantlets and transferring the rooted plantlets in soil after hardening to obtain regenerated whole plant. A preferred embodiment of the present invention relates to providing a method for high frequency direct plant regeneration from various explants wherein the explants are selected from a group consisting of shoot tip, nodal and internodal explants, leaf segment and other suitable explants. Another embodiment of the invention relates to a method for direct regeneration of Arnebia plant, wherein the shoot induction medium is MS medium containing Benzyl Amino Purine (BAP) in the range of 0.25-1.0 mg/1, Kinetin (KN) in the range of 0.25-1.0 mg/1, Indole Acetic Acid (IAA) in the range of 0.1-1.0 mg/1, and Casein Hydrolysate (CH) 100 mg/1. Yet another embodiment of the invention relates to a method for direct regeneration of Arnebia plant wherein the root induction medium consisting of MS medium containing 0.5-2.0 mg/1 Indole Butyric Acid (IBA) or 0.5-2.0 mg/1 Napthalene Acetic Acid (NAA) or 1.0-3.0 mg/1 Indole Acetic Acid (IAA). Yet another embodiment of the present invention relates to providing a media composition and other conditions for callus induction in cultures of Arnebia species preferably Arnebia hispidissima, wherein the tissue culture medium is MS medium containing 0.2-2.0 mg/1 Kinetin (KN); 0.5-2.0 mg/1 2,4-Dichloro-phenoxyacetic acid (2,4-D); and 50-150 mg/1 Caesin Hydrolysate (CH). Yet another embodiment of the present invention relates to providing a media composition for induction of pigment shikonin in callus cultures of Arnebia hispidissima, wherein the tissue culture medium is MS medium with 0.2-2.0 mg/1 Kinetin (KN), 0.01-2.0 mg/1 Indole Acetic Acid (IAA), 0.2-2.0 mg/1 Benzyl Amino Purine (BAP), and Caesin Hydrolysate (CH) 50-150 mg/1. Direct Regeneration of plantlets from shoot tip culture of Arnebia hispidissima. For regeneration of Arnebia hispidissima, field grown plants were obtained from Guru Jambheshwar University, Hissar. Different types of explants such as shoot tips, leaf segments, nodal segments and internodal segments were used for direct regeneration of the plantlets. The surface sterilized explants were cultured on various shoot induction media for shoot regeneration. The shoot regeneration from various explants of Arnebia hispidissima using various shoot induction media combination is shown in Table 2. Various medium compositions were analyzed to identify the ideal media combination for shoot regeneration from various explants. Details are given in the Example 1 and 2. Regeneration of plants from callus of Arnebia Various explants namely leaf segments, internodal segments, nodal segments and shoot tips were cultured on tissue culture media. Various media compositions were used for the regeneration of Arnebia plants like MS basal medium with or without 2, 4-Dichloro-phenoxyacetic acid, 6-Benzyl Amino Purine, Kinetin, Indole-3-Acetic Acid. The surface sterilized explants were cultured on various callus induction media to obtain callus. Murashige and Skoog (MS) medium containing Kinetin (0.5 mg L"1) and 2, 4-D (1.0 mg L" ) resulted in the callus fomiation from various explants. Calli obtained with various explants appear green under white light and yellowish white in the dark conditions. Callus cultures do not produce any shikonin pigment even after several subcultures in the media combinations shown in table 4. The callus production from various explants of Arnebia hispidissima using various media combination is shown in Table 4. Various medium compositions were analyzed to identify the ideal media combination for callus induction from various explants. Details are given in the Example 3. Callus production and in vitro induction of Shikonin Various explants namely leaf segments, internodal segments, nodal segments and shoot tips were cultured on different tissue culture medium supplemented with various growth regulators alone or in combination. Surface sterilized explants were cultured on various callus induction media to obtain callus. Callus obtained on the medium was transferred to shikonin induction medium. Various medium compositions were analyzed to identify the ideal media combination for shikonin induction in the callus obtained from various explants. Medium composition for shikonin induction in callus obtained from various explants is given in Table 5. Details are given in Example 4. Pigmentation with accumulation of shikonin was observed only when callus cultures were transferred to culture medium containing 1.0 mgL' of IAA instead of 2,4- D and allowed to grow in the dark. Undifferentiated callus tissues of Arnebia hispidissima were capable of synthesizing shikonin derivative or pigment which are normally formed in the cork cells of the roots. Thus by transferring callus cultures to MS media containing IAA (1.0 mgL-1) and incubating in the dark for 4-5 weeks it was possible to obtain production of shikonin in the calli. Thus, it was observed that biosynthesis of shikonin pigment in callus cultures is inhibited by weak auxin and light during tissue culture. Pigment content increased linearly with time after a lag phase of about 4 weeks when callus tissues were grown on culture medium containing IAA in the dark. The callus cultures containing shikonin were transferred to MS media supplemented with 2,4-D and incubated in the dark for several weeks. It was found that pigment content decreased with time. The interesting observation is that pigment content decreased significantly when 2, 4-D was substituted for IAA even though the callus cultures were incubated in the dark. Several experiments were carried out for in vitro induction of shikonin pigment in callus cultures. The ideal media combination was MS supplemented with BAP (0.25 mg L" ) and IAA (1.0 mgL"1) for production of callus as well as induction of shikonin production. It was evident that shikonin was produced only if the callus cultures were incubated under dark conditions. Another observation was that pigment synthesis is repressed by irradiating the callus cultures in white light. For example, old callus cultures grown in MS media containing IAA and kinetin and under illumination produced no pigment during 4-6 weeks of culturing but pigments were produced under continuous dark period at 25 C. Comparative studies of cytokinin and auxins showed that kinetin is not necessary for shikonin production. It was seen that shikonin pigment was present in MS media combination with growth regulators kinetin, IAA and BAP, IAA but more percent in BAP, IAA combination (80.0 %). The following examples are for better understanding of the invention and should not be construed as to limit the scope of the invention. Examples Example: 1. Direct regeneration of plantlets from Shoot tip cultures a) Selection and preparation of explants: Field grown plants were obtained from Guru Jambheshwar University, Hisar. For shoot tip cultures, shoot tips (4-6 mm long) which consist of 10-15 developing leaves surrounding the meristem were selected. Bud scales and outer developing leaves were removed and the shoot tip was excised using a sharp, sterile scalpel blade. All the explants were surface sterilized with standard detergent followed by several rinses in tap water. These were then sterilized with 0.1% HgCb for 5-7 minutes and subsequently washed three times with double distilled water to remove traces of HgCk. Explants were placed with the abaxial side down on medium in glass jars with caps. b) Media preparation and culture conditions Tissue culture media was prepared by employing the standard MS media containing sucrose (3 %) and bacteriological agar (0.8%). (Murashige & Skoog, 1962). Growth regulators were incorporated into the media and the pH was adjusted to 5.8. The media was then sterilized in an autoclave at 15 psi at 121°C for 20 minutes. The growth chamber conditions maintained for in vitro cultures are 25±2°C, 50-60% relative humidity (RH) with 16 h photoperiod with fluorescent light (1000 µmol M-2 S-1 photosensitivity), followed by an 8-h dark period. Unless otherwise stated, all growth regulators added to MS medium are in mg/1. The various media combinations are given in Table 1-5. c) Initiation and proliferation of shoot regeneration Shoot tips were cultured on various media combinations such as MS medium alone or supplemented with one of the following plant growth regulators; 6-BenzyI amino purine (BAP; 0.25, 0.50, 1.0 mgL-1); Kinetin (KN; 0.25, 0.50, 1.0 mgL-1); Indole-3-aceticacid (IAA, 0.1, 0.25, 0.5, 1.0 mgL"1) and Casein hydrolysate (100 mgL" ') (Tablel). This study showed that MS media supplemented with BAP (0.25 mgL-1); Kinetin (0.50 mgL-1); IAA (0.1 mgL-1) and Casein hydrolysate (100 mgL-1) was an ideal media combination for shoot regeneration. This combination led to a high percentage of shoot regeneration from shoot tips (Table 2). It was observed that 1.5-5 cm long linear and lanceolate leaves were developed within 4-7 weeks in 93.3% of cultures resulting into a cluster formation at the end of 3-4 weeks of cultures (Figure 1 A). MS media containing only BAP hormone was ineffective in inducing shoot proliferation. Generally shoot tip explants are more responsive than nodal explants for shoot regeneration. On media supplemented with various BAP, Kinetin and IAA concentrations, 93.3% of shoot tip explants and 60% nodal explants developed more than 14 shoots (Figure 1 B). Tissue culture response, viability, intensity of shoot development and quality of shoots (as measured by the case of dividing shoot cultures, shoot elongation and manifestation of abnormalities such as fasciation, vitrification or etiolation) was recorded weekly for upto 8 weeks. The numbers of shoots regenerated from various explants were analyzed after 4 weeks. This experiment was repeated several times. Flowering was also induced in some of the cultures in vitro. The calyx of the in vitro induced flowers was 6-8 mm long divided almost to base, very hispid; and the corolla was yellow, densely pubescent outside at base and apex. Stamens were inserted at or in the throat and half exerted in the short style flowers; stigma small flattened on inside, rounded outside. The induced flowering appeared phenotypically same as in the field grown plants. d) Induction of rooting in regenerated shoots: Shoots longer than 15 mm length with four or more leaves were excised out and transferred to various media combinations such as, MS medium alone or in combination with supplemented adjuvants with one of the following combinations of growth regulators: Indole-3-butyeric acid (IBA, 0.50, 1.0, 1.5, 2.0 mgL-1), A-naphthalene acetic acid (NAA, 0.50, 1.0, 2.0 mgL-1), indole-3-acetic acid (IAA,1.0, 2.0, 2.5, 3.0 mgL ) with or without Charcoal (0.1%) or half strength MS media alone or half strength MS media supplemented with one of the following combination of growth regulators : IBA (0.5, 1.0, 2.0 mgL-1), NAA (0.5, 1.0, 2.0 mgL-1), IAA (1.0, 2.0, 2.5, 3.0 mgL"1) The number of rooted shoots and quality of roots was recorded after three weeks of culture. Among the various auxins investigated, IBA (2.0 mgL" ) was most effective in initiating roots on shoots isolated from shoots induced in culture media (Figure 2). Shikonin production was observed in the roots. There was a higher amount of shikonin production in older roots as compared to younger ones. Hormones namely IAA and NAA were less effective for root induction. It was also observed that addition of charcoal (0.1%) in both MS medium and half strength MS medium with IBA (2.0 mgL-1) inhibited the Shikonin pigment production. The ideal media combination for root induction is MS media supplemented with 2.0 mgL"1 of IBA (Table-3). The Murashige and Skoog (MS) medium (Murashige & Skoog, 1962) supplemented with BAP (0.25mgL-1), Kinetin (0.25 mg L-1) IAA (0.1 mg L-1) and Caesin hydrolysate (CH,100 mg L" ) shows 53.3 and 40 percent shoot regeneration from shoot tip and nodal explants, respectively (Table 2). e) Acclimatization of regenerated plantlet and field trials: Rooted explants with four or more leaves and three to four roots were washed to remove the adhering sucrose/agar medium and then transferred to pots containing a mixture of sand: Vermiculite compost in the ratio 2:1 (v/v) and exposed to natural light and 70±5% Relative humidity in the green house. The light weight and porous texture of soil substitute prevents root damage, allows better root growth and hence development of a healthier plant. The whole process took 2-3 months from the start until a regenerated plant could be transferred to the green house. Example 2: Direct regeneration of Arnebia from Nodal segments Nodal segments were prepared by cutting the stem into 5-10 mm length, where each nodal segment has an axillary bud. Nodal segments were from third to sixth node from the tip of the branch. These explants served as the basic material for the present invention. The nodal segment explants were surface sterilized as described in Example 1. Tissue culture media was prepared by employing the standard MS media containing sucrose (3%) and bacteriological agar (0.8%). (Murashige and Skoog, 1962) as described in Example 1. Growth regulators were incorporated into the media and the pH was adjusted to 5.8. The details of media preparation and sterilization are as described in Example 1. The treated explants were cultured on MS medium alone or supplemented with one of the following growth regulators; 6-Benzylaminopurine (BAP; 0.25, 0.50, 1.0 mgL-1); Kinetin (KN; 0.25, 0.50, 1.0 mgL-1); Indole-3-aceticacid (IAA, 0.1, 0.25, 0.5, 1.0 mgL-1). MS media supplemented with BAP (0.25 mgL-1), Kinetin (0.50 mgL-1), IAA (0.1 mgL"1) and CH (100 mgL-1) showed 1.5-2.5 cm long leaves developed within 4-6 weeks So considerable shoot elongation was observed in the presence of BAP (0.25 mgL"1), Kinetin (0.5 mgL-1) and IAA (0.1 mgL-1) Shoot tip cultured on Murashige & Skoog medium supplemented with BAP (0.25 mgL-1), Kinetin (0.5 mg L-1), IAA (0.1 mg L-1) and Casein hydrolysate (100 mg L-1) produced 60 % shoot regeneration from nodal segments (as shown in Table-2). Thus the ideal combination of BAP, Kinetin and IAA was identified for effective and enhanced shoot regeneration from the various explants. Further, shoots were allowed to elongate in the same medium up to a length of 1.5 to 2 cm and were cultured on root induction medium for rooting. Different rooting media combinations were tested for root induction and these combinations are shown in Table 3. The Ideal media combination for root inductions was found to be MS medium supplemented with Indole-3-butyric acid (IBA 2.0 mg L-1). The in vitro rooted shoots resumed growth after a short period of acclimatization and resulted in plantlets which were transferred to the soil after hardening. These regenerated plantlets were successfully established in soil. In vitro flowering of tissue culture raised plantlets were observed when these plantlets were maintained for long periods in tissue culture media. For root induction shoots longer than 1.5 cm, with four or more leaves were excised out and transferred to MS medium alone or in combination with supplemented adjuvants with one of the following combinations of growth regulators: Indole-3-butyeric acid (IBA, 0.50, 1.0, 1.5, 2.0 mg L-1), naphthalene acetic acid (NAA, 0.50, 1.0, 2.0 mg L-1), indole-3-acetic acid (IAA, 1.0, 2.0, 2.5, 3.0 mg L-1) with and without charcoal (0.1%) or half strength MS alone or half strength MS supplemented with one of the following combination of growth regulators: IBA (0.5, 1. 0,2.0 mg L-1), NAA (0.5, 1.0, 2.0 mg L"'), 1AA (1.0, 2.0, 2.5, 3.0 mg L-1). Among the various auxins tested, IBA (2.0 mgL-1) was found to be most effective in initiating roots on shoots isolated from shoots induced in culture medium (Table-3). Rooted plantlets were then transferred to pots containing a mixture of sand: Vermiculite compost in the ratio 2:1 (w/w) and exposed to natural light and 70±5% relative humidity in the green house for acclimatization and hardening of the regenerated plantlets. Example 3 Regeneration of plants from callus of Arnebia Various explants namely leaf segments, internodal segments, nodal segments and shoot tips were cultured on different media combinations given below: 1) MS medium alone. 2) MS medium supplemented with 2, 4-Dichloro-phenoxyacetic acid (2, 4-D, at following concentrations namely, 0.25, 0.5, 1.0, 1.5, 2.0 mgL-1). 3) MS medium containing 2, 4-D (1.0 mgL"1) in combination with 6- Benzylaminopurine (BAP, at following concentrations'namely, 0.25, 0.50, 1.0, 1.5, 2.0 mgL-1). 4) MS medium containing 2, 4-D (1.0 mgL-1) in combination with Kinetin (KN, at following concentrations namely, 0.25, 0.50, 1.0, 1.5, 2.0 mgL-1). 5) MS medium containing Indole-3-aceticacid (IAA, at following concentrations namely, 0.25, 0.50, 1.0, 2.0 mgL-1) alone or in combination with BAP (at following concentrations namely, 0.25, 0.50, 1.0 mgL-1) and Kinetin (at following concentrations namely, 0.25, 0.50, 1.0 mgL-1). Murashige and Skoog (MS) medium containing Kinetin (0.5 mg L-1) and 2, 4-D (1.0 mg L" ) resulted in the callus formation from various explants. Calli obtained with various explants were green (Figure 3) under white light and yellowish white in the dark conditions. The calli obtained were sub-cultured on various shoot induction media. The ideal media combination is MS supplemented with BAP (0.25 mgL-1) Kinetin (0.5 mgL" ) IAA (0.1 mgL-1) and CH (100 mgL-1) and maintained in growth chamber with following conditions, namely temperature 25±2°C, relative humidity (RH) of 50-60% and 16h photoperiod with fluorescent light (2000 umolM"2S-2) followed by an 8h dark period. After incubation for 3-4 weeks, several green shoots were obtained from the callus cultures. The multiple shoots were transferred on the same medium for elongation. The shoots of length 1.5 cm were subsequently transferred to root induction medium for obtaining roots. After 3-4 weeks, well developed roots were formed at the base of the shoots. The ideal media combination for rooting was found as MS media supplemented with IBA 2.0 mgL-1. The in vitro rooted shoots were transferred to soil for hardening. The hardened regenerated plantlets were successfully established in soil. The regenerated plants were allowed to grow till maturity.. The seeds were harvested from these plants to screen for the germination rate and other traits. Example 4: Production of Callus and induction of Shikonin Explants were selected from Shoot tips, Leaf segments, Nodal segments and internodal segments. Nodal segments were prepared by cutting the stem into 5-10 mm length, where each node segment has an axillary bud. Nodal segments were from third to sixth node from the tip of the branch. The nodal segment explants were surface sterilized as described in Example 1. The surface sterilized explants were cultured on various media for callus induction. The various media combinations are given in Table 1. Tissue culture media was prepared by employing the standard MS media containing sucrose (3%) and bacteriological agar (0.8%) as described in Example 1 with various Growth regulators combination. Callus induction from various explants of Arnebia hispidissima using various media combination is shown in Table 4. MS media alone or in combination with kinetin (0.2-2.0 mgL"1), 2,4-D (0.5-2.0 mgL-1) and CH (50-150 mgL-1). The ideal media for callus induction was observed to be MS medium supplemented with kinetin (0.5 mg L-1), 2,4-D (1.0 mg L-1) and CH (100 mgL-1) under photoperiod of 12 hrs at 25 ± 2°C showed 86.7 ± 0.09 percent callus induction from nodal explants (Table 4). After producing callus on MS media in combinations with Kinetin (0.2-2.0 mgL-1), 2,4-D (0.5-2.0 mgL-1) and CH (50-150 mgL-1) under 6-12 hrs photoperiods at 25 ± 2C, the callus was sub cultured after 4 weeks on media containing MS medium, kinetin (0.2-2.0 mgL-1), IAA (0.01-2.0 mgL-1) and CH (50-150 mgL-1). Callus start producing shikonin pigment under photoperiod of 6-12 hrs per day at 25 ± 2°C but more pigmented callus was observed under dark conditions of 12-24 hrs at 25 ± 2°C (Figure 4). It was observed that shikonin derivative content increased when kinetin was substituted with BAP either in the same or less concentration. The ideal conditions for induction of shikonin pigment was observed on media containing MS medium, BAP 0.25 mgL-1, IAA 1.0 mgL-1 and CH 100 mgL-1 under dark condition for 12-24 hrs at 25 ±2°C Advantages: 1. Rapid and efficient method of regeneration of plants from explants throughout the year. 2. Unlike the conventional methods of plant propagation, direct plant regeneration & micro propagation of even temperate species can be carried out throughout the year devoid of callus phase. 3. Tissue cultured plants are generally free from fungal and bacterial diseases and true to-type planting material can be produced. 4. In-vitro conditions optimized for induction of shikonin production by varying hormone and photo periodic conditions. References: Mizukami H, Konoshima M and Tabata M (1997): Effect of nutritional factors on Shikonin derivative formation in Lithospermum callus culture. Phytochemistry 16: 1183-1186. Yazaki K, Tanaka S, Matsuoka H, Sato F (1998): Stable transformation of Lithospermum erythrorhizone by Agrobacterium rhizogenes and Shikonin production of transformants. Plant Cell Report, 18:214-219. Fujita Y (1988): Shikonin production by plant (Lithospermum erythrorhizone) cell culture. In Bajaj YPS (cell) Biotechnology in agriculture and Forestry, Vol. 4. Medicinal and aromatic plants I. Springer, Berlin Heidelberg, New York. Pp. 225-226. Zakhlenjeek OV and Kunakh VA: In vitro culture and the production of Shikonin and other secondary metabolites by plant (Arnebia euchroma). In Bajaj, YPS, Biotechnology in agriculture and Forestry, Vol. 41. Medicinal and aromatic plants (X). Springer, Berlin Heidelberg, New York. Yu HJ, Oh SK, Oh MH, Choi DVV, Kvvon YH, Kim SG: Plant regeneration from callus cultures of Lithospermum erythrorhizone Plant Cell Report. Berlin, W.Ger: Springer International Feb. 1997. Vol. 16(5): 261-266. Srinivasan V, Ryu DDY: Improvement of Shikonin productivity in Lithospermum erythrorhizone cell culture by alternating carbon and nitrogen feeding strategy. Biotechnol-Bioeng., New York: Wiley, 1962 Sept. 20, 1993 V 42 (7): pp. 793-799. Kim DJ, Chang HN: Effect of growth hormone modification on Shikonin production from Lithospermum erythrorhizone cell culture with in situ extraction. Biotechnology letters. 1990, 12(4):289-294. Laszlo M: Production of secondary metabolites by plant cell and tissue cultures Napjaink-Biotechnologiaja. 1990, 26: 34-39. Sokha V, Nikolaeva L, Panu F (1996). The Shikonin products in plant tissue culture: selection of cell lines with high productivity. Wang WJ, Bai JY, Liu DP, Xue LM, Zhu XY 1995. The anti-inflammatory activity of Shikonin and its inhibitory effect on Leukotriene B4 biosynthesis: Acta Pharmaceutica-Sinica. Tabata M, Mizukami H, Hiraoka N. and Konoshima M (1974) Phytochemistry 13,297. Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassay in tobacco tissue cultures. Physiol plant 15: 473-497. (Table Removed) (a)* Dissolved 100 mg (CuS045H20) in 100 ml distilled water (b) Dissolved 1 g (Na2MoO4.2H20) in 100 ml distilled water (c) Dissolved 100 mg (CoCI,.2H20) in 100 ml distilled water NOTE: — 1. Add one ml of cone. HCI in stock solution No.l to avoid precipitation. 2. Heat slightly stock solution No. 4 to avoid precipitate. **:— Myo inositol and sucrose are added in solid form while preparing the final volume. pH 5.8 (Table Removed) (2 to 5) I / We claim: 1. A method for direct regeneration of Arnebia plant, said method comprising steps of: a. obtaining explants either from seedlings or whole plant, b. culturing the explants of step (a) on shoot induction medium to obtain multiple shoots, c. culturing the shoots of step (b) on root induction medium to obtain rooted plantlets, and d. transferring the plantlets of step (c) in soil after hardening to obtain regenerated whole plant. 2. A method for direct regeneration of Arnebia plant as claimed in claim 1, wherein in step (a) the explants are selected from a group consisting of shoot tips, leaf segments, nodal and internodal segments. 3. A method for direct regeneration of Arnebia plant as claimed in claim 1, wherein the shoot induction medium of step (b) is MS medium comprising Benzyl Amino Purine (BAP) in the range of 0.25-1.0 mg/1, Kinetin (KN) in the range of 0.25-1.0 mg/1, Indole Acetic Acid (IAA) in the range of 0.1-1.0 mg/1, and Casein Hydrolysate (CH) 100 mg/1. 4. A method for direct regeneration of Arnebia plant as claimed in claim 3, wherein the shoot induction medium is MS medium comprising 0.25 mg/1 of BAP, 0.5 mg/1 of Kinetin, 0.1 mg/1 of IAA, and 100 mg/1 of CH. 5. A method for direct regeneration of Arnebia plant as claimed in claim 1, wherein the root induction medium of step (c) is selected from a group consisting of MS medium containing Indole Butyric Acid (IBA) in the range of 0.5-2.0 mg/1; MS medium containing Napthalene Acetic Acid (NAA) in the range of 0.5-2.0 mg/1 and MS medium containing Indole Acetic Acid (IAA) in the range 1.0-3.0 mg/1, preferably MS medium containing 2.0 mg/1 Indole Butyric Acid (IBA). 6. A method for induction of shikonin in Arnebia cultures, wherein the said method comprising steps of: a. obtaining explants from either seedlings or whole plant, b. culturing the explants on tissue culture medium to obtain callus, c. transferring the callus obtained from step (b) on shikonin induction, and d. medium and incubating in dark for 3-5 weeks at temperature in the range of 24 to 27 °C to obtain shikonin in the cultures, 7. A method for induction of shikonin in Arnebia cultures as claimed in claim 6, wherein in step (a) the explants are selected from a group consisting of shoot tips, leaf segments, nodal and internodal segments. 8. A method for induction of shikonin in Arnebia cultures as claimed in claim 6, wherein the tissue culture medium of step (b) is MS medium comprising Kinetin (KN) in the range of 0.2-2.0 mg/1; 2, 4-Dichloro-phenoxyacetic acid (2,4-D) in the range of 0.5-2.0 mg/1; and Caesin Hydrolysate (CH) in the range of 50-150 mg/1. 9. A method for induction of shikonin in Arnebia cultures as claimed in claim 8, wherein the tissue culture medium MS medium comprising 0.5mg/l Kinetin (KN); 1.0 mg/1 2,4-Dichloro-phenoxyacetic acid (2, 4-D); and 100 mg/1 Caesin Hydrolysate (CH). 10. A method for induction of shikonin in Arnebia cultures as claimed in claim 6, wherein the shikonin induction medium of step (c) is selected from a group consisting of MS medium with Kinetin (KN) in the range of 0.2-2.0 mg/1, Indole Acetic Acid (IAA) in the range of 0.01-2.0 mg/1 and Caesin Hydrolysate (CH) in the range of 50-150 mg/1; MS medium with Indole Acetic Acid (IAA) in the range of 0.01-2.0 mg/1 and Caesin Hydrolysate (CH) in the range of 50-150 mg/1; MS medium with Benzyl Amino Purine (BAP) in the range of 0.2-2.0 mg/1, Indole Acetic Acid (IAA) in the range of 0.01-2.0 mg/1 and Caesin Hydrolysate (CH) in the range of 50-150 mg/1. 11. A method for induction of shikonin in Aniebia cultures as claimed in claim 10, wherein the Shikonin induction medium is MS medium comprising 0.25 mg/1 Benzyl Amino Purine (BAP), 1.0 mg/1 Indole Acetic Acid (IAA), and 100 mg/1 Caesin Hydrolysate (CH).

Full Text

Method of direct regeneration and shikonin induction in callus of
Arnebia hispidissima
FIELD OF INVENTION
The present invention relates to a method of direct plant regeneration, and callus production and regeneration of plants from callus in Arnebia species. The invention also provides an in-vitro method for induction of Shikonin in callus cultures of Arnebia hispidissima.
BACKGROUND OF THE INVENTION
Arnebia (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 Arnebia as well as some other species of Boraginaceae family from the genera Lithospermum, Onosma, Echium provides the source of naphthoquinonous red pigment, Shikonin. 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, antifungal activities and exhibits anti-inflammatory and wound healing properties. The anti-allergic, antipyretic, antihydropic effects of Shikonin and its derivatives as well as antineoplastic activities are reported in the literature (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). Shikonin and its derivatives are contained in the outer surface of roots of Arnebia. Shikonin has an applicable pharmacological effect as an antibacterial agent, granulation tissue forming activity and anti-ulcer activity (Hayashi et al. 1969; Propageorgiou, 1980). Shikonin content depends upon the age of plant and other environmental factors. Middle aged plants with 6-8 flowering stems, show highest level of Shikonin (Pimenova and Tareeva, 1980). Wild plant species from the Boraginaceae family, accumulating Shikonin fail to provide sufficient raw material for commercial production, owing to which tissue culture technique becomes an excellent tool to overcome these short comings. Arnebia tissue culture was first reported by Rabinovich and Davydenkov

(Davydenkov et al. 1991). However, no detailed studies have so far been reported on high frequency direct plant regeneration in this plant species and do not teach the present invention.
This invention deals with high frequency direct plant regeneration and callus production from various explants of Arnebia species preferably Arnebia hispidissima.
Further, methods are developed for the production of calli to induce production of Shikonin derivatives by varying the culture conditions such as light and level of auxins in the tissue-culture media.
OBJECTS OF THE INVENTION
An object of the present invention is to provide a rapid and efficient method for direct regeneration of Arnebia species preferably Arnebia hispidissima.
Another object of the present invention is to provide a method for high frequency direct plant regeneration of Arnebia hispidissima from explants selected from shoot tips, leaf segments, nodal and inter-nodal segments by supplementing the media with various plant growth regulators.
Another object of the invention is to provide a method for regeneration of true-to-type plantlets of Arnebia hispidissima.
Another object of the invention is to provide an optimized media composition for direct regeneration of Arnebia hispidissima.
Still another object of the invention is to provide method for production of callus cultures from various explants of Arnebia hispidissima.
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.
SUMMARY OF THE INVENTION
The present invention relates to a novel and efficient method of direct plant regeneration, callus production and regeneration of plants from callus in Arnebia

species. The invention also provides an in vitro method for induction of shikonin in callus cultures of Arnebia hispiclissima.
Another aspect of the present invention is to provide a method for high frequency direct plant regeneration from various explants wherein the explants are selected from a group consisting of shoot tips, nodal and internodal explants, 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 Murashige and Skoog media (MS) containing suitable 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, Indole-3-butyeric acid (IBA), A-naphthalene acetic acid (NAA), and indole-3-acetic acid (IAA).
The invention further provides a media composition for callus production using various explants of Arnebia hispidissima.
The invention further provides a method for regeneration of Arnebia 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 Arnebia 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 shoot initiation
FIG. 2: Regenerated plantlets showing rooting
FIG. 3: Green callus under light condition showing no pigmentation
FIG. 4: Red pigmented callus showing enhanced shikonin production
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to a novel and rapid method for high frequency direct plant regeneration, callus production and regeneration of plants from calli in Arnebia species. The invention also provides an in-vitro method for induction of shikonin in callus cultures of Arnebia hispidissima.
An embodiment of the present invention relates to a method for direct regeneration of Arnebia plant, where the method comprising steps of culturing the explants obtained either from seedlings or whole plant on shoot induction medium to obtain multiple shoots; transferring the shoots on the root induction medium to obtain rooted plantlets and transferring the rooted plantlets in soil after hardening to obtain regenerated whole plant.
A preferred embodiment of the present invention relates to providing a method for high frequency direct plant regeneration from various explants wherein the explants are selected from a group consisting of shoot tip, nodal and internodal explants, leaf segment and other suitable explants.
Another embodiment of the invention relates to a method for direct regeneration of Arnebia plant, wherein the shoot induction medium is MS medium containing Benzyl Amino Purine (BAP) in the range of 0.25-1.0 mg/1, Kinetin (KN) in the range of 0.25-1.0 mg/1, Indole Acetic Acid (IAA) in the range of 0.1-1.0 mg/1, and Casein Hydrolysate (CH) 100 mg/1.
Yet another embodiment of the invention relates to a method for direct regeneration of Arnebia plant wherein the root induction medium consisting of MS medium containing 0.5-2.0 mg/1 Indole Butyric Acid (IBA) or 0.5-2.0 mg/1 Napthalene Acetic Acid (NAA) or 1.0-3.0 mg/1 Indole Acetic Acid (IAA).
Yet another embodiment of the present invention relates to providing a media composition and other conditions for callus induction in cultures of Arnebia species

preferably Arnebia hispidissima, wherein the tissue culture medium is MS medium containing 0.2-2.0 mg/1 Kinetin (KN); 0.5-2.0 mg/1 2,4-Dichloro-phenoxyacetic acid (2,4-D); and 50-150 mg/1 Caesin Hydrolysate (CH).
Yet another embodiment of the present invention relates to providing a media composition for induction of pigment shikonin in callus cultures of Arnebia hispidissima, wherein the tissue culture medium is MS medium with 0.2-2.0 mg/1 Kinetin (KN), 0.01-2.0 mg/1 Indole Acetic Acid (IAA), 0.2-2.0 mg/1 Benzyl Amino Purine (BAP), and Caesin Hydrolysate (CH) 50-150 mg/1.
Direct Regeneration of plantlets from shoot tip culture of Arnebia hispidissima.
For regeneration of Arnebia hispidissima, field grown plants were obtained from Guru Jambheshwar University, Hissar. Different types of explants such as shoot tips, leaf segments, nodal segments and internodal segments were used for direct regeneration of the plantlets.
The surface sterilized explants were cultured on various shoot induction media for shoot regeneration. The shoot regeneration from various explants of Arnebia hispidissima using various shoot induction media combination is shown in Table 2. Various medium compositions were analyzed to identify the ideal media combination for shoot regeneration from various explants. Details are given in the Example 1 and 2.
Regeneration of plants from callus of Arnebia
Various explants namely leaf segments, internodal segments, nodal segments and shoot tips were cultured on tissue culture media. Various media compositions were used for the regeneration of Arnebia plants like MS basal medium with or without 2, 4-Dichloro-phenoxyacetic acid, 6-Benzyl Amino Purine, Kinetin, Indole-3-Acetic Acid. The surface sterilized explants were cultured on various callus induction media to obtain callus.
Murashige and Skoog (MS) medium containing Kinetin (0.5 mg L"1) and 2, 4-D (1.0 mg L" ) resulted in the callus fomiation from various explants. Calli obtained with various explants appear green under white light and yellowish white in the dark conditions. Callus cultures do not produce any shikonin pigment even after several subcultures in the media combinations shown in table 4.
The callus production from various explants of Arnebia hispidissima using various media combination is shown in Table 4. Various medium compositions were

analyzed to identify the ideal media combination for callus induction from various explants. Details are given in the Example 3.
Callus production and in vitro induction of Shikonin
Various explants namely leaf segments, internodal segments, nodal segments and shoot tips were cultured on different tissue culture medium supplemented with various growth regulators alone or in combination. Surface sterilized explants were cultured on various callus induction media to obtain callus. Callus obtained on the medium was transferred to shikonin induction medium. Various medium compositions were analyzed to identify the ideal media combination for shikonin induction in the callus obtained from various explants. Medium composition for shikonin induction in callus obtained from various explants is given in Table 5. Details are given in Example 4.
Pigmentation with accumulation of shikonin was observed only when callus
cultures were transferred to culture medium containing 1.0 mgL' of IAA instead of 2,4-
D and allowed to grow in the dark. Undifferentiated callus tissues of Arnebia
hispidissima were capable of synthesizing shikonin derivative or pigment which are
normally formed in the cork cells of the roots. Thus by transferring callus cultures to
MS media containing IAA (1.0 mgL-1) and incubating in the dark for 4-5 weeks it was
possible to obtain production of shikonin
in the calli. Thus, it was observed that biosynthesis of shikonin pigment in callus cultures is inhibited by weak auxin and light during tissue culture. Pigment content increased linearly with time after a lag phase of about 4 weeks when callus tissues were grown on culture medium containing IAA in the dark. The callus cultures containing shikonin were transferred to MS media supplemented with 2,4-D and incubated in the dark for several weeks. It was found that pigment content decreased with time. The interesting observation is that pigment content decreased significantly when 2, 4-D was substituted for IAA even though the callus cultures were incubated in the dark.
Several experiments were carried out for in vitro induction of shikonin pigment in callus cultures. The ideal media combination was MS supplemented with BAP (0.25 mg L" ) and IAA (1.0 mgL"1) for production of callus as well as induction of shikonin production. It was evident that shikonin was produced only if the callus cultures were incubated under dark conditions.

Another observation was that pigment synthesis is repressed by irradiating the callus cultures in white light. For example, old callus cultures grown in MS media containing IAA and kinetin and under illumination produced no pigment during 4-6 weeks of culturing but pigments were produced under continuous dark period at 25 C. Comparative studies of cytokinin and auxins showed that kinetin is not necessary for shikonin production. It was seen that shikonin pigment was present in MS media combination with growth regulators kinetin, IAA and BAP, IAA but more percent in BAP, IAA combination (80.0 %).
The following examples are for better understanding of the invention and should not be construed as to limit the scope of the invention.
Examples
Example: 1. Direct regeneration of plantlets from Shoot tip cultures
a) Selection and preparation of explants:
Field grown plants were obtained from Guru Jambheshwar University, Hisar. For shoot tip cultures, shoot tips (4-6 mm long) which consist of 10-15 developing leaves surrounding the meristem were selected. Bud scales and outer developing leaves were removed and the shoot tip was excised using a sharp, sterile scalpel blade. All the explants were surface sterilized with standard detergent followed by several rinses in tap water. These were then sterilized with 0.1% HgCb for 5-7 minutes and subsequently washed three times with double distilled water to remove traces of HgCk. Explants were placed with the abaxial side down on medium in glass jars with caps.
b) Media preparation and culture conditions
Tissue culture media was prepared by employing the standard MS media containing sucrose (3 %) and bacteriological agar (0.8%). (Murashige & Skoog, 1962). Growth regulators were incorporated into the media and the pH was adjusted to 5.8. The media was then sterilized in an autoclave at 15 psi at 121°C for 20 minutes. The growth chamber conditions maintained for in vitro cultures are 25±2°C, 50-60% relative humidity (RH) with 16 h photoperiod with fluorescent light (1000 µmol M-2 S-1 photosensitivity), followed by an 8-h dark period. Unless otherwise stated, all growth regulators added to MS medium are in mg/1. The various media combinations are given in Table 1-5.

c) Initiation and proliferation of shoot regeneration
Shoot tips were cultured on various media combinations such as MS medium alone or supplemented with one of the following plant growth regulators; 6-BenzyI amino purine (BAP; 0.25, 0.50, 1.0 mgL-1); Kinetin (KN; 0.25, 0.50, 1.0 mgL-1); Indole-3-aceticacid (IAA, 0.1, 0.25, 0.5, 1.0 mgL"1) and Casein hydrolysate (100 mgL" ') (Tablel). This study showed that MS media supplemented with BAP (0.25 mgL-1); Kinetin (0.50 mgL-1); IAA (0.1 mgL-1) and Casein hydrolysate (100 mgL-1) was an ideal media combination for shoot regeneration. This combination led to a high percentage of shoot regeneration from shoot tips (Table 2). It was observed that 1.5-5 cm long linear and lanceolate leaves were developed within 4-7 weeks in 93.3% of cultures resulting into a cluster formation at the end of 3-4 weeks of cultures (Figure 1 A).
MS media containing only BAP hormone was ineffective in inducing shoot proliferation. Generally shoot tip explants are more responsive than nodal explants for shoot regeneration. On media supplemented with various BAP, Kinetin and IAA concentrations, 93.3% of shoot tip explants and 60% nodal explants developed more than 14 shoots (Figure 1 B). Tissue culture response, viability, intensity of shoot development and quality of shoots (as measured by the case of dividing shoot cultures, shoot elongation and manifestation of abnormalities such as fasciation, vitrification or etiolation) was recorded weekly for upto 8 weeks. The numbers of shoots regenerated from various explants were analyzed after 4 weeks. This experiment was repeated several times. Flowering was also induced in some of the cultures in vitro. The calyx of the in vitro induced flowers was 6-8 mm long divided almost to base, very hispid; and the corolla was yellow, densely pubescent outside at base and apex. Stamens were inserted at or in the throat and half exerted in the short style flowers; stigma small flattened on inside, rounded outside. The induced flowering appeared phenotypically same as in the field grown plants.
d) Induction of rooting in regenerated shoots:
Shoots longer than 15 mm length with four or more leaves were excised out and transferred to various media combinations such as, MS medium alone or in combination with supplemented adjuvants with one of the following combinations of growth regulators: Indole-3-butyeric acid (IBA, 0.50, 1.0, 1.5, 2.0 mgL-1), A-naphthalene acetic acid (NAA, 0.50, 1.0, 2.0 mgL-1), indole-3-acetic acid (IAA,1.0, 2.0,

2.5, 3.0 mgL ) with or without Charcoal (0.1%) or half strength MS media alone or half strength MS media supplemented with one of the following combination of growth regulators : IBA (0.5, 1.0, 2.0 mgL-1), NAA (0.5, 1.0, 2.0 mgL-1), IAA (1.0, 2.0, 2.5, 3.0 mgL"1)
The number of rooted shoots and quality of roots was recorded after three weeks of culture. Among the various auxins investigated, IBA (2.0 mgL" ) was most effective in initiating roots on shoots isolated from shoots induced in culture media (Figure 2). Shikonin production was observed in the roots. There was a higher amount of shikonin production in older roots as compared to younger ones.
Hormones namely IAA and NAA were less effective for root induction. It was also observed that addition of charcoal (0.1%) in both MS medium and half strength MS medium with IBA (2.0 mgL-1) inhibited the Shikonin pigment production. The ideal media combination for root induction is MS media supplemented with 2.0 mgL"1 of IBA (Table-3).
The Murashige and Skoog (MS) medium (Murashige & Skoog, 1962) supplemented with BAP (0.25mgL-1), Kinetin (0.25 mg L-1) IAA (0.1 mg L-1) and Caesin hydrolysate (CH,100 mg L" ) shows 53.3 and 40 percent shoot regeneration from shoot tip and nodal explants, respectively (Table 2).
e) Acclimatization of regenerated plantlet and field trials:
Rooted explants with four or more leaves and three to four roots were washed to remove the adhering sucrose/agar medium and then transferred to pots containing a mixture of sand: Vermiculite compost in the ratio 2:1 (v/v) and exposed to natural light and 70±5% Relative humidity in the green house. The light weight and porous texture of soil substitute prevents root damage, allows better root growth and hence development of a healthier plant. The whole process took 2-3 months from the start until a regenerated plant could be transferred to the green house.
Example 2: Direct regeneration of Arnebia from Nodal segments
Nodal segments were prepared by cutting the stem into 5-10 mm length, where each nodal segment has an axillary bud. Nodal segments were from third to sixth node from the tip of the branch. These explants served as the basic material for the present invention.

The nodal segment explants were surface sterilized as described in Example 1. Tissue culture media was prepared by employing the standard MS media containing sucrose (3%) and bacteriological agar (0.8%). (Murashige and Skoog, 1962) as described in Example 1. Growth regulators were incorporated into the media and the pH was adjusted to 5.8. The details of media preparation and sterilization are as described in Example 1. The treated explants were cultured on MS medium alone or supplemented with one of the following growth regulators; 6-Benzylaminopurine (BAP; 0.25, 0.50, 1.0 mgL-1); Kinetin (KN; 0.25, 0.50, 1.0 mgL-1); Indole-3-aceticacid (IAA, 0.1, 0.25, 0.5, 1.0 mgL-1). MS media supplemented with BAP (0.25 mgL-1), Kinetin (0.50 mgL-1), IAA (0.1 mgL"1) and CH (100 mgL-1) showed 1.5-2.5 cm long leaves developed within 4-6 weeks So considerable shoot elongation was observed in the presence of BAP (0.25 mgL"1), Kinetin (0.5 mgL-1) and IAA (0.1 mgL-1)
Shoot tip cultured on Murashige & Skoog medium supplemented with BAP (0.25 mgL-1), Kinetin (0.5 mg L-1), IAA (0.1 mg L-1) and Casein hydrolysate (100 mg L-1) produced 60 % shoot regeneration from nodal segments (as shown in Table-2). Thus the ideal combination of BAP, Kinetin and IAA was identified for effective and enhanced shoot regeneration from the various explants.
Further, shoots were allowed to elongate in the same medium up to a length of 1.5 to 2 cm and were cultured on root induction medium for rooting. Different rooting media combinations were tested for root induction and these combinations are shown in Table 3. The Ideal media combination for root inductions was found to be MS medium supplemented with Indole-3-butyric acid (IBA 2.0 mg L-1). The in vitro rooted shoots resumed growth after a short period of acclimatization and resulted in plantlets which were transferred to the soil after hardening. These regenerated plantlets were successfully established in soil. In vitro flowering of tissue culture raised plantlets were observed when these plantlets were maintained for long periods in tissue culture media.
For root induction shoots longer than 1.5 cm, with four or more leaves were excised out and transferred to MS medium alone or in combination with supplemented adjuvants with one of the following combinations of growth regulators: Indole-3-butyeric acid (IBA, 0.50, 1.0, 1.5, 2.0 mg L-1), naphthalene acetic acid (NAA, 0.50, 1.0, 2.0 mg L-1), indole-3-acetic acid (IAA, 1.0, 2.0, 2.5, 3.0 mg L-1) with and without charcoal (0.1%) or half strength MS alone or half strength MS supplemented with one of the following combination of growth regulators: IBA (0.5, 1. 0,2.0 mg L-1), NAA

(0.5, 1.0, 2.0 mg L"'), 1AA (1.0, 2.0, 2.5, 3.0 mg L-1). Among the various auxins tested, IBA (2.0 mgL-1) was found to be most effective in initiating roots on shoots isolated from shoots induced in culture medium (Table-3).
Rooted plantlets were then transferred to pots containing a mixture of sand: Vermiculite compost in the ratio 2:1 (w/w) and exposed to natural light and 70±5% relative humidity in the green house for acclimatization and hardening of the regenerated plantlets.
Example 3
Regeneration of plants from callus of Arnebia
Various explants namely leaf segments, internodal segments, nodal segments and shoot tips were cultured on different media combinations given below:
1) MS medium alone.
2) MS medium supplemented with 2, 4-Dichloro-phenoxyacetic acid (2, 4-D, at
following concentrations namely, 0.25, 0.5, 1.0, 1.5, 2.0 mgL-1).
3) MS medium containing 2, 4-D (1.0 mgL"1) in combination with 6-
Benzylaminopurine (BAP, at following concentrations'namely, 0.25, 0.50, 1.0, 1.5, 2.0
mgL-1).
4) MS medium containing 2, 4-D (1.0 mgL-1) in combination with Kinetin (KN, at following concentrations namely, 0.25, 0.50, 1.0, 1.5, 2.0 mgL-1).
5) MS medium containing Indole-3-aceticacid (IAA, at following concentrations namely, 0.25, 0.50, 1.0, 2.0 mgL-1) alone or in combination with BAP (at following concentrations namely, 0.25, 0.50, 1.0 mgL-1) and Kinetin (at following concentrations namely, 0.25, 0.50, 1.0 mgL-1).
Murashige and Skoog (MS) medium containing Kinetin (0.5 mg L-1) and 2, 4-D (1.0 mg L" ) resulted in the callus formation from various explants. Calli obtained with various explants were green (Figure 3) under white light and yellowish white in the dark conditions. The calli obtained were sub-cultured on various shoot induction media. The ideal media combination is MS supplemented with BAP (0.25 mgL-1) Kinetin (0.5 mgL" ) IAA (0.1 mgL-1) and CH (100 mgL-1) and maintained in growth chamber with following conditions, namely temperature 25±2°C, relative humidity (RH) of 50-60% and 16h photoperiod with fluorescent light (2000 umolM"2S-2) followed by an 8h dark

period. After incubation for 3-4 weeks, several green shoots were obtained from the callus cultures.
The multiple shoots were transferred on the same medium for elongation. The shoots of length 1.5 cm were subsequently transferred to root induction medium for obtaining roots. After 3-4 weeks, well developed roots were formed at the base of the shoots. The ideal media combination for rooting was found as MS media supplemented with IBA 2.0 mgL-1. The in vitro rooted shoots were transferred to soil for hardening. The hardened regenerated plantlets were successfully established in soil. The regenerated plants were allowed to grow till maturity.. The seeds were harvested from these plants to screen for the germination rate and other traits.
Example 4: Production of Callus and induction of Shikonin
Explants were selected from Shoot tips, Leaf segments, Nodal segments and internodal segments. Nodal segments were prepared by cutting the stem into 5-10 mm length, where each node segment has an axillary bud. Nodal segments were from third to sixth node from the tip of the branch. The nodal segment explants were surface sterilized as described in Example 1. The surface sterilized explants were cultured on various media for callus induction. The various media combinations are given in Table 1. Tissue culture media was prepared by employing the standard MS media containing sucrose (3%) and bacteriological agar (0.8%) as described in Example 1 with various Growth regulators combination.
Callus induction from various explants of Arnebia hispidissima using various media combination is shown in Table 4. MS media alone or in combination with kinetin (0.2-2.0 mgL"1), 2,4-D (0.5-2.0 mgL-1) and CH (50-150 mgL-1). The ideal media for callus induction was observed to be MS medium supplemented with kinetin (0.5 mg L-1), 2,4-D (1.0 mg L-1) and CH (100 mgL-1) under photoperiod of 12 hrs at 25 ± 2°C showed 86.7 ± 0.09 percent callus induction from nodal explants (Table 4).
After producing callus on MS media in combinations with Kinetin (0.2-2.0 mgL-1), 2,4-D (0.5-2.0 mgL-1) and CH (50-150 mgL-1) under 6-12 hrs photoperiods at 25 ± 2C, the callus was sub cultured after 4 weeks on media containing MS medium, kinetin (0.2-2.0 mgL-1), IAA (0.01-2.0 mgL-1) and CH (50-150 mgL-1). Callus start producing shikonin pigment under photoperiod of 6-12 hrs per day at 25 ± 2°C but more pigmented callus was observed under dark conditions of 12-24 hrs at 25 ± 2°C

(Figure 4). It was observed that shikonin derivative content increased when kinetin was substituted with BAP either in the same or less concentration. The ideal conditions for induction of shikonin pigment was observed on media containing MS medium, BAP 0.25 mgL-1, IAA 1.0 mgL-1 and CH 100 mgL-1 under dark condition for 12-24 hrs at 25
±2°C
Advantages:
1. Rapid and efficient method of regeneration of plants from explants throughout the year.
2. Unlike the conventional methods of plant propagation, direct plant regeneration & micro propagation of even temperate species can be carried out throughout the year devoid of callus phase.
3. Tissue cultured plants are generally free from fungal and bacterial diseases and true to-type planting material can be produced.
4. In-vitro conditions optimized for induction of shikonin production by varying hormone and photo periodic conditions.
References:
Mizukami H, Konoshima M and Tabata M (1997): Effect of nutritional factors on Shikonin derivative formation in Lithospermum callus culture. Phytochemistry 16: 1183-1186.
Yazaki K, Tanaka S, Matsuoka H, Sato F (1998): Stable transformation of Lithospermum erythrorhizone by Agrobacterium rhizogenes and Shikonin production of transformants. Plant Cell Report, 18:214-219.
Fujita Y (1988): Shikonin production by plant (Lithospermum erythrorhizone) cell
culture. In Bajaj YPS (cell) Biotechnology in agriculture and Forestry, Vol. 4.
Medicinal and aromatic plants I. Springer, Berlin Heidelberg, New York. Pp. 225-226.
Zakhlenjeek OV and Kunakh VA: In vitro culture and the production of Shikonin and other secondary metabolites by plant (Arnebia euchroma). In Bajaj, YPS, Biotechnology in agriculture and Forestry, Vol. 41. Medicinal and aromatic plants (X). Springer, Berlin Heidelberg, New York.

Yu HJ, Oh SK, Oh MH, Choi DVV, Kvvon YH, Kim SG: Plant regeneration from callus cultures of Lithospermum erythrorhizone Plant Cell Report. Berlin, W.Ger: Springer International Feb. 1997. Vol. 16(5): 261-266.
Srinivasan V, Ryu DDY: Improvement of Shikonin productivity in Lithospermum erythrorhizone cell culture by alternating carbon and nitrogen feeding strategy. Biotechnol-Bioeng., New York: Wiley, 1962 Sept. 20, 1993 V 42 (7): pp. 793-799.
Kim DJ, Chang HN: Effect of growth hormone modification on Shikonin production from Lithospermum erythrorhizone cell culture with in situ extraction. Biotechnology letters. 1990, 12(4):289-294.
Laszlo M: Production of secondary metabolites by plant cell and tissue cultures Napjaink-Biotechnologiaja. 1990, 26: 34-39.
Sokha V, Nikolaeva L, Panu F (1996). The Shikonin products in plant tissue culture: selection of cell lines with high productivity.
Wang WJ, Bai JY, Liu DP, Xue LM, Zhu XY 1995. The anti-inflammatory activity of Shikonin and its inhibitory effect on Leukotriene B4 biosynthesis: Acta Pharmaceutica-Sinica.
Tabata M, Mizukami H, Hiraoka N. and Konoshima M (1974) Phytochemistry 13,297.
Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassay in tobacco tissue cultures. Physiol plant 15: 473-497.

(Table Removed)
(a)* Dissolved 100 mg (CuS045H20) in 100 ml distilled water
(b) Dissolved 1 g (Na2MoO4.2H20) in 100 ml distilled water
(c) Dissolved 100 mg (CoCI,.2H20) in 100 ml distilled water
NOTE: — 1. Add one ml of cone. HCI in stock solution No.l to avoid precipitation.
2. Heat slightly stock solution No. 4 to avoid precipitate. **:— Myo inositol and sucrose are added in solid form while preparing the final volume.
pH 5.8
(Table Removed) (2 to 5)

I / We claim:
1. A method for direct regeneration of Arnebia plant, said method comprising
steps of:
a. obtaining explants either from seedlings or whole plant,
b. culturing the explants of step (a) on shoot induction medium to obtain
multiple shoots,
c. culturing the shoots of step (b) on root induction medium to obtain rooted
plantlets, and
d. transferring the plantlets of step (c) in soil after hardening to obtain
regenerated whole plant.
2. A method for direct regeneration of Arnebia plant as claimed in claim 1, wherein in step (a) the explants are selected from a group consisting of shoot tips, leaf segments, nodal and internodal segments.
3. A method for direct regeneration of Arnebia plant as claimed in claim 1, wherein the shoot induction medium of step (b) is MS medium comprising Benzyl Amino Purine (BAP) in the range of 0.25-1.0 mg/1, Kinetin (KN) in the range of 0.25-1.0 mg/1, Indole Acetic Acid (IAA) in the range of 0.1-1.0 mg/1, and Casein Hydrolysate (CH) 100 mg/1.
4. A method for direct regeneration of Arnebia plant as claimed in claim 3, wherein the shoot induction medium is MS medium comprising 0.25 mg/1 of BAP, 0.5 mg/1 of Kinetin, 0.1 mg/1 of IAA, and 100 mg/1 of CH.
5. A method for direct regeneration of Arnebia plant as claimed in claim 1, wherein the root induction medium of step (c) is selected from a group consisting of MS medium containing Indole Butyric Acid (IBA) in the range of 0.5-2.0 mg/1; MS medium containing Napthalene Acetic Acid (NAA) in the range of 0.5-2.0 mg/1 and MS medium containing Indole Acetic Acid (IAA) in the range 1.0-3.0 mg/1, preferably MS medium containing 2.0 mg/1 Indole Butyric Acid (IBA).

6. A method for induction of shikonin in Arnebia cultures, wherein the said
method comprising steps of:
a. obtaining explants from either seedlings or whole plant,
b. culturing the explants on tissue culture medium to obtain callus,
c. transferring the callus obtained from step (b) on shikonin induction, and
d. medium and incubating in dark for 3-5 weeks at temperature in the range of
24 to 27 °C to obtain shikonin in the cultures,
7. A method for induction of shikonin in Arnebia cultures as claimed in claim 6, wherein in step (a) the explants are selected from a group consisting of shoot tips, leaf segments, nodal and internodal segments.
8. A method for induction of shikonin in Arnebia cultures as claimed in claim 6, wherein the tissue culture medium of step (b) is MS medium comprising Kinetin (KN) in the range of 0.2-2.0 mg/1; 2, 4-Dichloro-phenoxyacetic acid (2,4-D) in the range of 0.5-2.0 mg/1; and Caesin Hydrolysate (CH) in the range of 50-150 mg/1.
9. A method for induction of shikonin in Arnebia cultures as claimed in claim 8, wherein the tissue culture medium MS medium comprising 0.5mg/l Kinetin (KN); 1.0 mg/1 2,4-Dichloro-phenoxyacetic acid (2, 4-D); and 100 mg/1 Caesin Hydrolysate (CH).
10. A method for induction of shikonin in Arnebia cultures as claimed in claim 6, wherein the shikonin induction medium of step (c) is selected from a group consisting of MS medium with Kinetin (KN) in the range of 0.2-2.0 mg/1, Indole Acetic Acid (IAA) in the range of 0.01-2.0 mg/1 and Caesin Hydrolysate (CH) in the range of 50-150 mg/1; MS medium with Indole Acetic Acid (IAA) in the range of 0.01-2.0 mg/1 and Caesin Hydrolysate (CH) in the range of 50-150 mg/1; MS medium with Benzyl Amino Purine (BAP) in the range of 0.2-2.0 mg/1, Indole Acetic Acid (IAA) in the range of 0.01-2.0 mg/1 and Caesin Hydrolysate (CH) in the range of 50-150 mg/1.

11. A method for induction of shikonin in Aniebia cultures as claimed in claim 10, wherein the Shikonin induction medium is MS medium comprising 0.25 mg/1 Benzyl Amino Purine (BAP), 1.0 mg/1 Indole Acetic Acid (IAA), and 100 mg/1 Caesin Hydrolysate (CH).

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Patent Number

279667

Indian Patent Application Number

2066/DEL/2004

PG Journal Number

05/2017

Publication Date

03-Feb-2017

Grant Date

27-Jan-2017

Date of Filing

21-Oct-2004

Name of Patentee

GURU JAMBHESHWAR UNIVERSITY

Applicant Address

GURU JAMBHESHWAR UNIVERSITY OF SCIENCE & TECHNOLOGY, HISAR-125001, HARYANA, INDIA.

Inventors:

#	Inventor's Name	Inventor's Address
1	MANAKSHI PAL	DEPARTMENT OF BIO & NANO TECHNOLOGY, GURU JAMBHESHWAR UNIVERSITY OF SCIENCE & TECHNOLOGY, HISAR-125101, HARYANA, INDIA.
2	ASHOK CHAUDHURY	DEPARTMENT OF BIO & NANO TECHNOLOGY, GURU JAMBHESHWAR UNIVERSITY OF SCIENCE & TECHNOLOGY, HISAR-125101, HARYANA, INDIA.

PCT International Classification Number

A61K

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA