To,

Project Coordinator DBT-UR-IPLS Centre for Converging Technologies University of Rajasthan, Jaipur Sub: Application for Assistant Professor in the DBT-UR-IPLS at Centre for Converging Technologies University of Rajasthan, Jaipur Respected Sir, I am writing to apply for the position of Assistant Professor which was advertised in The Hindu, Dated 09-04-2011. Please find the following documents enclosed with my CV. I do hope that you will get the application in proper manner. Curriculum Vitae Proof of Date of birth (Secondary School Certificate) Senior School Certificate B.Sc. Marksheet M.Sc. Marksheet M.Sc. Provisional Degree Ph.D. Thesis Submission Certificate NET certificate ICMR-JRF certificate GATE 2007 Certificate ARS NET 2010 Certificate RPSC SET 2010 Certificate Madurai Kamraj University Training Course Certificate NBPGR, New Delhi Training Course certificate Certificates of Seminars/Conferences attended/organized Publications in peer reviewed Journals Yours Sincerely

Rohit Jain SRF-CSIR Department of Botany University of Rajasthan, Jaipur

CURRICULUM VITAE Rohit Jain Lab No. 7, Dept. of Botany University of Rajasthan Jaipur (Rajasthan), India-302 004 Email: jainrohit17@gmail.com Mobile: +91 9414040903

Objective: To secure a challenging position in the Biological Science by contributing in Research and Development through hard work, dedication and continuously updating my knowledge by assimilating the latest trends in science with a positive attitude. Current Position: Working as a Senior Research Fellow (CSIR) under the supervision of Prof. S. L. Kothari, Dean Faculty of Science, Department of Botany, University of Rajasthan, Jaipur.

Academic Details: 2007-2011 2005-2007 2002-2005 1999-2000 1997-1998 : : : : : Ph.D. (Botany)-Thesis Submitted (09-02-2011) Master of Science (Botany with specialization in Plant Physiology) from Department of Botany, University of Rajasthan, Jaipur (71.33%) Bachelor of Science (Biology) from University Maharajas College, University of Rajasthan, Jaipur (71%) Sr. Secondary (Biology) from Govt. Sr. Hr. Sec. School Lalsot, Dausa, Rajasthan Board of Secondary Education, Ajmer (68.62%) Secondary from Jyoti Secondary School, Lalsot, Dausa, Rajasthan Board of Secondary Education, Ajmer (84.55%)

Awards and Honors: Qualified National Eligibility Test (NET) for Lectureship (June 2007) Qualified ICMR JRF Test for Project Fellowship (2007) Qualified Graduate Aptitude Test for Engineering (GATE) 2007 97 Percentile Qualified RPSC State Eligibility Test (SET) 2010 Qualified ARS NET 2010

Training: Summer training on Recombinant DNA Technology (13th - 27th June 2009) at UGC Networking Resources Centre in Biological Sciences, Madurai Kamraj University, Madurai.

International workshop on Cryopreservation and in vitro techniques for conservation of plant genetic resources (15th 27th November 2010) at National Bureau for Plant Genetic Resources (NBPGR) IARI, PUSA, New Delhi.

Work Experience Senior Research Fellow in a major research scheme of CSIR entitled Collection, characterization and conservation of Withania coagulans (Stocks) Dunal chemotypes/biotypes and their metabolomic comparison with Withania somnifera (L.) Dunal counterparts (3 Years) Achievements: Successfully developed an efficient micropropagation protocol for ex situ conservation of endangered medicinal plant species Withania coagulans from shoot tip, node and leaf explants Enhancement of withanolides (bioactive compounds of Withania) in in vitro regenerated plants in Withania coagulans Developed a protocol for the synthesis and characterization of silver nanoparticles using spore crystal mixture of Bacillus thuringiensis Isolated and characterised a carotenoid producing novel Actinomycetes strain Gordonia lacunae (NCBI Gene Bank Accession GU727686)

Publications in Peer reviewed Journals (SCI Listed): 1. Jain R, Sinha A, Jain D, Kachhwaha S, Kothari SL Adevntitious shoot regeneration and in vitro biosynthesis of steroidal lactones in W. Coagulans (Stocks) Dunal. Plant Cell Tissue and Organ Culture (2011) 105:135140 (IF: 1.27) 2. Jain D, Kachhwaha S, Jain R, Srivastava G, Kothari SL Novel microbial route to synthesize silver nanoparticles using spore crystal mixture of Bacillus thuringiensis. Indian Journal of Experimental Biology (2010) 48: 1152 1156 (IF: 0.55) 3. Sinha A, Jain R, Kachhwaha S, Kothari SL Optimization of the level of micronutrient copper in the culture medium improves shoot bud regeneration in Indian Ginseng [Withania somnifera (L.) Dunal]. National Academy Science Letters (2010) 33: 11-16 (IF: 0.17) 4. Jain R, Sinha A, Kachhwaha S, Kothari SL Micropropagation of Withania coagulans (Stocks) Dunal. : A critically endangered medicinal herb. Journal of Plant Biochemistry and Biotechnology (2009) 18:249252 (IF: 0.41) 5. Jain D, Jain R, Agarwal V, Sharma P, Srivastava G, Kachhwaha S, Kothari SL. Gordonia lacunae strain CCC12 16S ribosomal RNA gene, partial sequence. (2010) NCBI Gene Bank Accession GU727686.

6. Singh-Adhayach P, Arora A, Darji BL, Jain R Pollen grain fertility as a biomonitoring parameter for air pollution. Our Earth (2010) 7: 17-18 7. Jain D, Rathod KS, Jain R, Singh H, Gupta V, Tanwar S, Kachhwaha S, Kothari SL Phytofabrication of iron oxide nanoparticles using Calotropis gigantea L. (Communicated) Digest Journal of Nanomaterials and Biostructures Abstracts/Poster Presented: 1. Jain D, Jain R, Kachhwaha S, Kothari SL, (2010) Molecular Characterization and PCR based detection of cry genes in native Bacillus thuringiensis strains isolated from the desert soils of Rajasthan in Indo-US international workshop on Plant genomics in crop improvement with special reference to biotic and abiotic stress at CCS Haryana Agriculture University, Hisar. 2. Jain D, Jain R, Kachhwaha S, Kothari SL (2009) Effect of Ramp rate on RAPD analysis: An important step in optimization and reproducibility of RAPD in National Workshop at Jaipur National University, Jaipur.

Experimental Expertise Molecular Biology : Plasmid isolation, Bacterial transformation, Restriction Enzyme digestion and ligation, Isolation of Genomic DNA, Isolation of RNA, cDNA preparation, SDS-PAGE, Elution of proteins, Construction and Screening of library, Southern and Western Blot analysis, Genetic Transformation by Biolistic Gene Gun (PDS 1000), Expression and purification of recombinant proteins PCR techniques: Designing of PCR primers, Cloning of PCR products, Gradient PCR and RT-PCR Biochemical Analysis: Extraction of Primary and Secondary metabolites, Thin Layer Chromatography, Column Chromatography, Liquid-liquid Partition Chromatography, Reverse Phase-HPLC, UV-Visible and Nano Drop Spectrophotometry Bioinformatics: ClustalW, Sequence analysis using BLAST and EMBL tools, BIOEDIT, NTSYS, FASTPCR, NETPRIMER Tissue culture techniques: Tissue culture of Withania Spp., Agrobacterium mediated transformation in Withania Nanotechnology: Nanomaterial synthesis through biological route, Characterization of nanomaterials using XRD, SEM-TEM

Computer Proficiency Expert in Computers in Windows/Linux environment, MS-Excel and Adobe Photoshop, on line and off line data retrieval, well versed with using bio-informatics tools and software Symposium & Seminar Attended /Organized National seminar on Water Auditing at University of Rajasthan, Jaipur National symposium on Cancer: Diagnosis, Awareness & Treatment at Dept. of Zoology Uni. of Raj. Jaipur. Organized a 3 days National workshop on Bioinformatics & Biotechnology at Centre for Converging Technologies, University of Rajasthan, Jaipur. National seminar on Biotechnology in Sustainable Agriculture and Environmental Management at Dept. of Botany, University of Rajasthan, Jaipur. Indo-US international workshop on Plant genomics in crop improvement with special reference to biotic and abiotic stress at CCS Haryana Agriculture University, Hisar. Personal Details Date of Birth Fathers Name Nationality Sex Marital Status Language Skills References: Prof. S. L. Kothari Dean, Faculty of Science, Director, Centre for Converging Technologies Professor of Botany, University of Rajasthan, Jaipur E-mail: slkothari@lycos.com Dr. Shailesh Godika Assistant Professor, Agriculture Research Station, Naugaon SK Agricultural University, Bikaner E-mail: shaileshgodika@gmail.com : : : : : : 9th August, 1984 Rakesh Kumar Jain Indian Male Single Hindi, English

Rohit Jain

Dr. K. K. SINGH DDG (Sr. Gr.) & Chief, Division of Manpower Development

INDIAN COUNCIL OF MEDICAL RESEARCH Ansari Nagar, New Delhi - 110029, India Phone: (Off.) 26589753; (Res.) 26266317 Gram: Scientific, Fax: 26588662 E.Mail: sinQhkeshari~vahoo.com

NO.3/1/3/Next-1 00/JRF/07 -MPD Dated: 31st August, 2007 11328 Rohit Jain S/o SIl. Rakesh Kumar Jain, Sainara Shawan, Near Telephone Exchange New Colony, Lalsot Dai.isa, :'dja:ilhan<J03503

Subject: ICMR JRF Examination held on 15thJuly 2007.

Dear Sir/Madam, I am pleased to inform you that, you have qualified ICMR JRF Examination held on 15th
July 2007, as Junior research Fellow

in ICMR funded Research Projects/Scheme.

This

placement is subject to fulfilling the conditions for appointment under the project/scheme. You are required to contact along with your Sio-data with potential Research Investigators located in various Medical colleges/Research Organization/National laboratories including ICMR Institutes who are in receipt of ICMR funding for their project for seeking placement as JRF depending upon your area of specialization. This letter will enable you to find such placement in ICMR funded projects in these Institutions. Please note your stipend for JRF would be paid from project grant if you secure placement in the project. The Council would not be able to provide you stipend directly. Details of ICMR Institutes and advanced centers can be obtained from Website: icmr.nic.in The emoluments/stipend for JRF at present is RS.8000/- plus HRA as appiicabie per month. On finding placement in above-mentioned institutions you will be paid this amount from the project fund for the duration of the project. You are advised to submit certificate of passing qualifying examination (postgraduate) and other documents by 3~thNovember 2007 to the undersigned. The offer is valid for the period of 2 years, (Please see ANNEXURE-I). You would be permitted to enroll yourself for pursuing Ph.D. from any university while working in the scheme (if allowed by the university). In case, you are found ineligible at any stage during your research work that may be due to false certification or any other reason (including computer error), the award may be withdrawn by the Institution/ICMR. Kindly acknowledge the receipt of the letter.

12-04-2011

Provisional Result of National Eligibilit

Agricultural Scientists Recruitment Board Provisional Result of National Eligibility Test (NET) 2010 held on 19th September, 2010
Qualified for NET
Roll Number Name Fathers Name Category Physically Challenged Discipline Name Exam. Centre Code Exam. Centre Name : 030304100200 : ROHIT JAIN : RAKESH KUMAR JAIN : UR : No : BASIC PLANT SCIENCES : 03 : BIKANER

The candidate whose Roll Number is given above has qualified the NET subject to fulfillment of eligibility conditions laid down in the notification for NET-2010. 1. The Candidates who had not submitted the attested copy of their Masters Degree Certificate or Provisional Degree Certificate (completed on or before 18th September, 2010 i.e. cut-off date) are requested to submit the same to the undersigned immediately otherwise NET certificate will not be issued and candidature for the examination will be cancelled as per the rules of the Notification. No further correspondence will be made in this regard. 2. No scrutiny of eligibility of the candidates has been done at this stage. The Board takes up the eligibility conditions with reference to documents/testimonials/certificates etc. only after the declaration of provisional result. 3. NET Certificate to the qualified and eligible candidate will be issued from 1st January, 2011 onwards. Therefore, no request for issue of NET certificate before January, 2011 will be entertained under any circumstances. 4. The qualified candidates must intimate this office about any change in their correspondence address. 5. The Board shall not be responsible for any technical error or typographical error of any kind for this examination. The decision of the Board in all matters of dispute shall be final. Note: Agricultural Scientists Recruitment Board is not responsible for any inadvertent error that may have crept in the details being published on int These Details are for immediate information to the candidates only.

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J. Plant Biochemistry & Biotechnology Vol. 18(2), 249-252, July 2009

Short Communication

Micropropagation of Withania coagulans (Stocks) Dunal: A Critically Endangered Medicinal Herb

Rohit Jain1, Arunima Sinha1, Sumita Kachhwaha1, 2 and S L Kothari1, 2*
1 2

Department of Botany, University of Rajasthan, Jaipur 302 004, India Centre for Converging Technologies (CCT), University of Rajasthan, Jaipur 302 004, India

An efficient micropropagation protocol has been developed for Withania coagulans, a highly endangered medicinal herb and an important natural source of withanolides. Prolific multiplication of axillary buds occurred from the nodal segments taken from adult plant, and cultured on MS medium enriched with BA (0.5 mg l-1), Kn (0.5 mg l-1) and PG (0.5 mg l-1). Nodal segments and shoot tips of elongated microshoots also behaved the same way in cultures and formed multiple shoots through axillary bud multiplication. Addition of PG (0.5 mg l-1) in the regeneration medium significantly improved induction and elongation of shoot buds. Elongated shoots were placed on filter paper bridges soaked in MS medium with CC (10 mg l-1) and PG (0.5 mg l-1) for the initial 7 days pulse treatment and thereafter, they were transferred to rooting medium containing IBA (0.25 mg l-1) + PAA (0.5 mg l-1) + CC (2 mg l-1). This protocol has the capacity of producing 1000 plants from one nodal segment after 4 subcultures of 2 weeks each. Key words: Withania coagulans, micropropagation, phloroglucinol, choline chloride.

Withania species (Solanaceae) are the natural source of withanolides (steroidal lactones) which have potential antitumor, antimicrobial and immunomodulatory properties (1). Fruits of W. coagulans are also used for milk coagulation (2). The extract of the plant exhibits free radical scavenging (3) and hypolipidemic activity (4). Coagulin-H (1), isolated from W. coagulans (5) has been identified as immunosuppressive drug (6).
The natural propagation of W. coagulans occurs through seeds but chances of seed setting get limited due to unisexual nature of flowers. Overexploitation and the reproductive failure have rendered the species highly vulnerable to complete extinction. To date, there have not been any reports of ex situ conservation of this plant through tissue culture. We now report an efficient and reproducible protocol for micropropagation of W. coagulans. Only two plants of Withania coagulans were spotted in the wild in Ajmer district and the explants were taken from one of these plants. MS (7) basal medium supplemented with 3% sucrose, pH adjusted to 5.8 before
*Corresponding author. E-mail: kothari-sl@uniraj.ernet.in Abbreviations: BA - 6-benzylaminopurine, CC - choline chloride, IAA - indole-3-acetic acid, IBA - indole-3-butyric acid, Kn - kinetin, NAA - -naphthaleneacetic acid, PAA - phenylacetic acid, PG phloroglucinol, RAPD - random amplified polymorphic DNA

autoclaving at 1.06 kg cm-2 (121C) for 20 min was used in all the experiments. The cultures were incubated at 25 1 C under a 16-h photoperiod with 25mol m -2 s -1 photosynthetic photon flux density (PPFD) provided by cool white fluorescent tubes (40 W; Philips, India). Nodal segments from field grown plant were thoroughly washed in 5% (v/v) Teepol, surface sterilized with 70% (v/v) ethanol for 30s, followed by an aqueous solution of 0.1% (w/v) freshly prepared HgCl2 solution for 3 min. Finally, the explants were thoroughly washed with sterile distilled water and inoculated onto MS medium supplemented with BA or Kn at 0.5, 1, 2, 3 and 5 mg l-1 either alone or in combination. Various concentrations (0.5, 1, 2, 5 and 10 mg l-1) of PG (Sigma, USA) and CC (Sigma, USA) were also tested with optimal cytokinin concentration. Shoot buds induced in primary cultures were sectored in clumps of 3-4 and cultured on fresh medium for further multiplication of shoot buds. The in vitro-raised microshoots (23 cm in length) were harvested for rooting. Two step rooting procedure was followed. Step one involved the pulse treatment of individual shoots with PG or CC (0.5, 1, 2, 5 and 10 mg l-1) either alone or in combination with IBA and PAA at 10, 50 and 100 mg l-1 for 7 days on MS liquid medium using a filter paper bridge. In step two, the pre treated microshoots were transferred onto or MS, agar-gelled semisolid medium

250 J Plant Biochem Biotech

with 3% sucrose supplemented with IBA /IAA/ NAA/ PAA (0.25 1 mg l-1) either alone or in combination. Cultures were evaluated after 4 weeks. Histological preparations were made as described (8). Plantlets were then removed from the vessels, washed gently with water and transferred to pots containing 1:1 mixture of garden soil and organic manure. DNA was extracted from the leaves of 19 randomly selected regenerated plants and from the leaves of mother plant (WM). The sample was powdered in liquid nitrogen (196C) and stored at -20C until use for DNA extraction by CTAB method (9). Twelve RAPD primers were taken to assess the clonal fidelity of the regenerated shoots. The PCR amplification conditions were, an initial denaturation at 94C for 5 min followed by 35 cycles of 94C for 30 sec, 50 C for 45 seconds and 72C for 1 min, and a final extension at 72C for 5 min. The data on shoot formation and rooting were collected after 4 weeks. Each treatment consisted of twenty replicates. Three explants were cultured per conical flask and single explant was cultured per test tube. All experiments were repeated twice. The data was analyzed statistically using one way analysis of variance (ANOVA) by Fischers least significant difference (P = 0.05; 10). The explants inoculated on MS medium responded differently on BA and Kn (Table 1). BA gave better response than Kn in terms of induction of shoot buds. BA (0.5 mg l-1) in combination with Kn (0.5 mg l-1) proved best for induction of multiple shoots. An average of 19 shoots (1cm) could be

Table 1. Shoot bud formation from nodal segments of W. coagulans cultured on MS medium supplemented with BA and Kn BAP (mg l-1) 0.5 1 2 3 5 0 0 0 0 0 0.5 Kn (mg l-1) 0 0 0 0 0 0.5 1 2 3 5 0.5 Percent response (%) 57 68 74 79 83 43 55 55 66 73 83 Mean No. of Buds/Explant S.E. 3.8a 0.4 6.4b 0.4 7.0c 0.4 9.0d 0.4 11.2e 0.5 2.4f 0.1 3.0c 0.4 3.7d 0.2 5.0x 0.3 3.2g 0.3 18.6 h 0.5

S.E. Standard error Means in a column followed by different letters are significantly different from each other

obtained after 3 weeks (Table 1; Fig. 1a). Proliferating shoot cultures were established by subculturing the shoots on MS medium with BAP (0.5 mg l-1) + Kn (0.5 mg l-1) in clumps of 3-4 buds. Nodal segments and shoot tips were also used from regenerated shoots after 4 weeks of shoot bud initiation. Each explant formed up to 21 shoot buds but these were too short (0.3-0.5 cm), and not suitable for micropropagation. PG is a phenolic compound that stimulates shoot and root growth in shoot cultures (11). The addition of PG (0.5 mg l-1) along with BA (0.5 mg l-1) and Kn (0.5 mg l-1) in MS medium improved the establishment of nodal explant cultures (Table 2, Fig. 1b). The use of PG during

Table 2. Shoot bud formation from nodal segments and shoot-tips of W. coagulans (excised from in vitro raised shoots) cultured on MS medium supplemented with BA (0.5 mg l -1) + Kn (0.5 mg l -1) and different concentrations of PG or CC PG (mg l ) 0 0.5 1 2 3 5 0 0 0 0 0
-1

CC (mg l ) 0 0 0 0 0 0 0.5 1 2 3 5
-1

Nodal segments Mean No. of Buds/Explant S.E. 20.9a 23.4b 21.1c 19.2d 18.3e 15.5f 21.0c 17.7g 14.3h 13.5i 13.1j 0.3 0.2 0.3 0.3 0.2 0.5 0.4 0.5 0.3 0.2 0.3 Mean length of Shoots (cm) S.E. 0.5a 4.3b 4.1b 3.5c 3.1d 3.0d 3.8e 3.3f 2.8d 2.4g 2.2g 0.1 0.2 0.1 0.2 0.1 0.1 0.2 0.1 0.2 0.1 0.1

Shoot-tips Mean No. of Buds/Explant S. E. 22.3a 24.6b 23.3c 20.4d 19.3e 17.5f 23.9g 22.4a 21.5h 19.1i 14.5j 0.4 0.3 0.1 0.3 0.3 0.2 0.3 0.3 0.5 0.6 0.3 Mean length of Shoots (cm) S. E. 0.3a 4.7b 4.4bc 4.2c 3.9d 3.6d 4.6be 4.3e 3.7d 3.4d 2.9e 0.1 0.2 0.1 0.2 0.2 0.1 0.2 0.1 0.2 0.0 0.1

S.E. Standard error Means in a column followed by different letters are significantly different at P = 0.05 from each other

Short Communication

251

Fig. 1. In vitro regeneration of W. coagulans. (a) Induction of shoot buds from nodal explants of W. coagulans cultured on MS medium with BA (0.5 mg l-1) + Kn (0.5 mg l-1), (b) Proliferation and elongation of shoot buds on MS medium with BA (0.5 mg l-1) + Kn (0.5 mg l-1) + PG (0.5 mg l-1), (c-d) Histological details of the shoot bud formation from the shoot tip (c) and nodal segments (d), (e) Rooting on half strength MS medium with IBA (0.25 mg l-1) + PAA (0.5 mg l-1) + CC (2 mg l-1), and (f) Agarose gel electrophoresis of RAPD fragments of W. coagulans showing banding patterns of 20 plants amplified by the primer OPA-19.

252 J Plant Biochem Biotech

multiplication has improved shoot multiplication in several species (12). Rastogi et al (13) have also advocated incorporation of PG in the medium for better growth of cultures. The ability of shoot multiplication was maintained up to 12 subcultures, at 2-wk interval, on MS medium supplemented with BA (0.5 mg l-1) and Kn (0.5 mg l-1). Histological studies revealed that in the axil of each leaf, a distinct meristematic zone of small densely stained cells was present over a differentiated zone. A ring of multiple shoot primordia could be observed arising directly from base of cultured shoot tip (Fig. 1c). In the cultured nodes, at a later stage of development, vertical and sideways expansion of the meristematic zone occurred (Fig. 1d). The maximum frequency of root formation (80%), highest number (11.50.7) of roots and root length (7.90.3cm) were seen after pulse treatment of shoots in MS medium containing 10 mg l-1 CC and 0.5 mg l-1 PG followed by their transfer to strength MS medium with IBA (0.25 mg l-1), PAA (0.5 mg l-1) and CC (2 mg l-1) after 7 days (Fig. 1e). Two-step procedure for rooting has been used to advantage in several woody species (14). The incorporation of CC at different concentrations enhanced the response of rooting of shoots significantly. CC and PG have enhanced rooting in Bambusa tulda (15). These compounds are reported to enhance rooting by acting as auxin protectors to increase the free endogenous IAA levels during the inductive phase of rooting (16). The plantlets were successfully hardened inside the culture room under diffused light on MS medium for 2 weeks, followed by their establishment in pots containing (1:1) soil and manure in greenhouse. About 75% of the micropropagated plants survived after transfer to soil and organic manure (1:1). All the established plants were apparently uniform and did not show any detectable variation. Clonal fidelity of the regenerated shoots was checked through RAPD. Of 12 random primers, 8 generated distinct, reproducible products. A total of 580 amplification products were detected. The primers OPA-5 and OPA-19 (Fig. 1f) gave highly reproducible banding pattern. Fingerprinting profiles of regenerants were monomorphic and there was no variation amongst mother and tissue culture raised plants. There are number of reports demonstrating the suitability of enhanced axillary branching for raising true to type plants (17). Similar results have been obtained in present investigation.

The protocol offers a potential system for a large-scale propagation and conservation of this medicinal plant and would facilitate its improvement programme using genetic transformation and metabolic engineering techniques.

Acknowledgements
We thank Council of Scientific and Industrial Research (CSIR), New Delhi for the financial support in the form of a R&D project: CSIR-38(1178)/EMR-II/07. Rohit Jain and Arunima Sinha also thank CSIR for the award of Senior Research Fellowships.
Received 20 January, 2009; accepted 8 July, 2009. Online published 18 July, 2009.

References
1 2 3 4 5 Agarwal R, Diwanay S, Patki P & Patwardhan B, J Ethnopharmacol, 67 (1999) 27. Bhandari MM, Flora of the Indian desert , MPS Repros, Jodhpur, India (1995) pp 246. Hemalatha S, Wahi AK, Singh PN &Chansouria JPN, J Ethnopharmacol, 93 (2004) 261. Maurya R, Jayendra A, Singh AB & Srivastava AK, Bioorg Med Chemis Lett, 18 (2008) 6534. Atta-ur-Rahman, Yousaf M, Gul W, Qureshi S, Choudhary MI, Voelter W, Hoff A, Jens F & Naz A, Heterocycles, 48 (1998) 1801. Mesaik MA, Zaheer-ul-Haq, Murad S, Ismail Z, Abdullah NR, Gill HK, Atta-ur-Rahman, Yousaf M Siddiqui RA, Ahmad A & Choudhary MI, Mol Immun, 43 (2006)1855. Murashige T & Skoog T, Physiol Plant, 15 (1962) 473. Johansen DA, Plant microtechnique, Mc Graw-Hill Book Company, Inc., New York, USA (1940). Doyle IJ & Doyle JL, Focus, 12 (1990) 13. Gomez KA & Gomez AA, Statistical procedures for agricultural research, John Wiley and Sons, New York (1984) Sarkar D & Naik PS, Plant Cell Tiss Org Cult, 60 (2000) 139. Ibanez MR & Amo-Marc JB, Plant Growth Reg, 26 (1998) 49. Rastogi S, Rizvi SMH, Singh RP & Dwivedi UN, Biol Plant, 52 (2008) 743. Husain MK & Anis M, In Biotechnology for a better future (L DSouza, M Anuradha, S Nivas, S Hegde, K Rajendra, Editors). SAC, Mangalore (2004) p 294. Mishra Y, Patel PK, Yadev S, Shirin F & Ansari SA, Sci Hort, 115 (2008) 315. Faivre-Rampant O, Kevers C & Gaspar T, Plant Sci, 153 (2004)73. Rani V & Raina SN, In Vitro Cell Dev Biol-Plant, 36 (2000) 319.

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Adventitious shoot regeneration and in vitro biosynthesis of steroidal lactones in Withania coagulans (Stocks) Dunal

Plant Cell, Tissue and Organ Culture (PCTOC) Journal of Plant Biotechnology ISSN 0167-6857 Volume 105 Number 1 Plant Cell Tiss Organ Cult (2011) 105:135-140 DOI 10.1007/s11240-010-9840-3

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Your article is protected by copyright and all rights are held exclusively by Springer Science+Business Media B.V.. This e-offprint is for personal use only and shall not be selfarchived in electronic repositories. If you wish to self-archive your work, please use the accepted authors version for posting to your own website or your institutions repository. You may further deposit the accepted authors version on a funders repository at a funders request, provided it is not made publicly available until 12 months after publication.

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Plant Cell Tiss Organ Cult (2011) 105:135140 DOI 10.1007/s11240-010-9840-3

RESEARCH NOTE

Adventitious shoot regeneration and in vitro biosynthesis of steroidal lactones in Withania coagulans (Stocks) Dunal
Rohit Jain Arunima Sinha Devendra Jain Sumita Kachhwaha S. L. Kothari

Received: 5 June 2010 / Accepted: 30 August 2010 / Published online: 19 September 2010 Springer Science+Business Media B.V. 2010

Abstract A micropropagation system through leaf explant culture has been developed for Withania coagulans. Shoot bud proliferation occurred through both adventitious and de novo routes depending on the hormonal regime of the culture medium. Green compact nodular organogenic callus developed on Murashige and Skoog (MS) medium supplemented with 2.3 lM kinetin (Kn) and lower levels of 6benzyladenine (BA) (13.3 lM) while multiple adventitious shoot bud differentiation occurred on medium fortied with 2.3 lM kinetin (Kn) and higher levels of BA (22.2 lM). Shoot buds were transferred to proliferation medium containing 2.2 lM BA, 2.3 lM Kn, and 3.9 lM phloroglucinol (PG) for further growth and development of shoot system. Elongated shoots were rooted using a two-step procedure involving pulse treatment of 7 days in a medium containing 71.6 lM choline chloride (CC) and 3.9 lM PG and then transferred to rooting medium containing MS, 1.2 lM IBA, 3.6 lM PAA, and 14.3 lM CC for 3 weeks. Well-rooted plants were transferred to a greenhouse for hardening and further growth. Random amplication of polymorphic DNA (RAPD) showed monomorphic bands in all the plants thereby conrming clonality of the regenerants. Thin layer chromatography (TLC) showed the presence of withanolides in the regenerated plants. Quantication through reverse-phase HPLC revealed increased concentration of withanolides in the regenerated plants compared to the eld-grown mother plant. Accumulation of withaferin A and withanolide A
R. Jain A. Sinha D. Jain S. Kachhwaha S. L. Kothari Department of Botany, University of Rajasthan, Jaipur 302004, India S. Kachhwaha S. L. Kothari (&) Centre for Converging Technologies (CCT), University of Rajasthan, Jaipur 302004, India e-mail: slkothari@lycos.com

increased up to twofold and that of withanone up to tenfold. Direct regeneration via leaf explants will be useful for Agrobacterium-mediated genetic transformation, and will facilitate pathway manipulation using metabolic engineering for bioactive withanolides. Keywords Micropropagation HPLC TLC RAPD Withania coagulans Withanolides Abbreviations BA 6benzyladenine CC Choline chloride DAD Diode array detector IAA Indole3acetic acid IBA Indole3butyric acid Kn Kinetin MS Murashige and Skoog NAA anaphthaleneacetic acid PAA Phenylacetic acid PG Phloroglucinol RAPD Random amplication of polymorphic DNA TLC Thin layer chromatography

Introduction Withania coagulans (fam. Solanaceae) is commercially important for its ability to coagulate milk, in the treatment of ulcers, rheumatism, dropsy, consumption and sensile debility (Bhandari 1995). Antimicrobial, anti-inammatory, antitumor, hepatoprotective, antihyperglycemic, cardiovascular, immunosuppressive, free radical scavenging and central nervous system depressant activities of the

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plant have also been demonstrated (Maurya and Akanksha 2010). Pharmacological investigations have elucidated association of these activities with the specic steroidal lactones known as withanolides present in Withania (Attaur-Rahman et al. 1998). Withaferin A, withanolide A and withanone are the major withanolides present in W. somnifera and W. coagulans. Overexploitation and the reproductive failures forced the species W. coagulans towards the verge of extinction (Jain et al. 2009b). The in vitro shoot cultures could provide an alternative to eld plant harvesting for the production of therapeutically valuable compounds (Sangwan et al. 2007). There are no reports of in vitro plant regeneration in W. coagulans except our earlier report using nodal and shoot tip explant cultures (Jain et al. 2009b). Here, we report regeneration from leaf explants and production of withanolides from the regenerated plants for the rst time.

and shoot buds developed per explant were recorded and shoot buds were subcultured on rst stage proliferation medium (MS, 2.2 lM BA, and 2.3 lM Kn) containing 3.9 lM phloroglucinol (PG) to further enhance growth and development of shoot buds. Regenerated shoots of appropriate length ([3 cm) were subjected to a two-step rooting procedure involving pulse treatment of 7 days on MS, 71.6 lM choline chloride (CC) and 3.9 lM PG and then transferred to rooting medium containing MS, 1.2 lM IBA, 3.6 lM PAA, and 14.3 lM CC prior to hardening as described previously (Jain et al. 2009b). The data on shoot bud formation and rooting were collected after 4 weeks. Three explants per ask and single explant per test tube was cultured. All experiments were repeated twice. RAPD analysis DNA was extracted from the leaves of 17 randomly selected regenerated plants and from the leaves of mother plant (WM). The leaf samples were powdered in liquid nitrogen and stored at -20C until used for DNA extraction by CTAB method (Doyle and Doyle 1990). The PCR amplication conditions were: an initial denaturation at 94C for 4 min followed by 40 cycles of 94C for 45 s, 37C for 45 s and 72C for 2 min, and a nal extension at 72C for 10 min. The amplicons were separated through 1.2% agarose (Himedia, India) gel electrophoresis and photographed using Gel Documentation System (Bio-Rad, Germany). Extraction of withanolides All the analytical and HPLC grade solvents, reagents and precoated silica gel TLC plates were purchased from Merck. Isolation of withanolides from various tissues was performed using the method described by Sangwan et al. (2007). Qualitative and quantitative analysis of withanolides Qualitative withanolide proling was done through TLC while quantication was carried out through HPLC as described by Sangwan et al. (2007). For TLC, 10 ll sample was loaded on precoated silica gel G-60 plates, performed in a solvent system consisting of chloroform:ethyl acetate:methanol:toluene (74:4:8:30, v/v), and development was done with anisaldehyde reagent (250 ll anisaldehyde in a mixture of 20 ml acetone, 80 ml water and 10 ml 60% perchloric acid) followed by heating at 110C. HPLC analysis was performed on Agilent (Germany) model 1200 and separation was achieved by a reverse-phase column (Eclipse XDB c-18, 4.5 mm 9 150 mm, particle size 1.8 lm; Agilent) using water (A) and methanol (B), each containing 0.1% acetic acid, as solvent and online UV-Diode Array

Materials and methods Plant material and establishment of in vitro cultures from leaf explants Leaf explants (0.82 cm) were collected from the eldgrown plants spotted in Ajmer (Rajasthan) in 2007. The species was identied by the Herbarium, Dept. of Botany, University of Rajasthan, Jaipur. Explants were thoroughly washed under running tap water for 15 min followed by treatment with 20% Extran (liquid detergent; Merck, India) for 5 min. Eventually, the explants were aseptically surface sterilized with 0.1% (w/v) HgCl2 (Merck, India) solution for 3 min. Explants were rinsed 45 times with sterile distilled water and cultured on full- and half-strength MS (Murashige and Skoog 1962) medium supplemented with 3% sucrose (Merck, India) and 0.9% agar (bacteriological grade; Merck, India). Various concentrations and combinations of different plant growth regulators (Sigma, India) including 6benzyladenine (BA; 2.2, 4.4, 8.8, 13.2 and 22.2 lM), kinetin (Kn; 2.3, 4.6, 9.2, 13.9 and 23.2 lM), indole-3-acetic acid (IAA; 1.1, 1.7 and 2.8 lM), indole-3butyric acid (IBA; 0.9, 1.4 and 2.4 lM), phenylacetic acid (PAA; 1.4, 2.2 and 3.6 lM) and anaphthaleneacetic acid (NAA; 1.0, 1.6 and 2.6 lM) were added in the medium to optimize growth and differentiation. The pH of the medium was adjusted to 5.8 followed by sterilization at 1.2 kg/cm2 pressure and 121C temperature for 20 min. Leaf explants with or without petiolar parts were placed abaxially on the medium. Cultures were maintained at 26 1C under 16/ 8 h photoperiod with 25 lmol m-2 s-1 photosynthetic photon ux density provided by white uorescent tubes (40 W; Philips, India). Twenty replicates were maintained for each treatment. The numbers of responding explants

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137 Table 1 Shoot bud formation from leaf explants of W. coagulans cultured on MS medium supplemented with BA and Kn BA (lM) 2.2 4.4 8.9 13.3 22.2 SE Standard error Means in a column followed by different letters are signicantly different from each other at P = 0.05 Kn (lM) 2.3 2.3 2.3 2.3 2.3 % response 80 86 73 93 80 Shoot buds (Mean SE) 4.6 0.5 e 7.7 0.6 d 9.3 0.6 c 12.1 0.2 b 17.6 0.5 a

Detector (UV-DAD) at 227 nm. The solvent gradient was set as A:B, 60:4025:75, 045 min; 10:90, 4560 min at a ow rate of 0.6 ml min-1. Sample volume of 10 ll was injected and the column temperature maintained at 27C during the run. Authentic withanolides including withaferin A, withanone and withanolide A (Chromadex, CA, USA) were used as markers to ascertain their discrete resolution from each other under these conditions for both TLC and HPLC. Computation of withanolide concentration in the samples was done through a calibration curve of concentration versus detector response (peak area) using different concentrations of standard solutions of withaferin A, withanolide A and withanone in methanol. The data was analyzed statistically using one-way analysis of variance (ANOVA) by Fischers least signicant difference (P = 0.05) (Gomez and Gomez 1984). HPLC data was analyzed with the Chemstation LC 3D software (Agilent).

Results and discussion Leaf explants cultured in the absence of growth regulators senesced without producing callus or adventitious buds, whereas they responded with enlargement and swelling at the cut petiolar end followed by callus formation on MS medium supplemented with Kn (2.3 lM) or BA (2.213.3 lM). Kn alone (Murch et al. 2004) or in combination with auxins (Kachhwaha and Kothari 1996; Reddy et al. 2004) and BA alone (Kulkarni et al. 2000; Sharma et al. 2003; Tilkat et al. 2009) or in combination with auxins (Koroch et al. 2002; Jain et al. 2009a; Kothari et al. 2010; Sinha et al. 2010) have most frequently been reported to induce in vitro plant regeneration in a wide range of monocotyledonous and dicotyledonous plants. Therefore, we also examined the effect of IAA, NAA or PAA in combination with BA or Kn on organogenesis. The combination of BA or Kn with auxins was not conducive to organogenesis. Brown, compact, nodular callus was observed on medium supplemented with BA (13.322.2 lM) and IAA (1.1 lM) or IBA (0.9 lM) or PAA (1.4 lM), but it could not induce any shoot buds. The amount of callus increased with increasing concentration of auxins. Rhizogenesis was observed all along the lamina cultured on medium with BA (2.222.2 lM) with NAA (1.0 2.6 lM). Kn in combination with auxins initiated formation of pale and nonmorphogenic callus. The use of 2.3 lM Kn in combination with BA (2.213.3 lM) promoted the initiation and development of shoot buds along with callus (Fig. 1a). Clusters of adventitious shoots (17.6 0.5) regenerated mostly from petiolar base of leaf explants or at leaf midrib region on medium supplemented with 22.2 lM BA and 2.3 lM Kn (Table 1, Fig. 1b). This clearly demonstrated that the combination of

BA and Kn was the most important factor for shoot regeneration from leaf explants of W. coagulans. Combination of BA with Kn for inducing shoot bud differentiation from the explants has also been reported in several other plants (Dayal et al. 2003; Baskaran and Jayabalan 2005; Sreedhar et al. 2008). Presence of petiolar part along with lamina was essential for morphogenesis as no response was observed when lamina without petiolar part was cultured. Previous reports have shown the same impact including petioles for enhancing shoot regeneration in several other plant species such as Paulownia tomentosa (Corredoira et al. 2008), Prunus persica (Gentile et al. 2002; Zhou et al. 2010), and P. serotina (Liu and Pijut 2008). Shoot buds induced on explants in the primary cultures were transferred to the proliferation medium containing 2.2 lM BA and 2.3 lM Kn for further differentiation of new shoot buds, but the elongation of the shoot buds did not occur (Fig. 1c). A combination of 2.2 lM BA, 2.3 lM Kn and 3.9 lM PG was required in the proliferation medium for the elongation of shoot buds up to 23 cm, a length which was required for rooting (Fig. 1d). PG has similarly been used by other workers (Sarkar and Naik 2000; Feeney et al. 2007). Elongated shoots ([3 cm) were transferred to MS medium containing 1.2 lM IBA, 3.6 lM PAA, and 14.3 lM CC after 7 days of pulse treatment with 71.6 lM CC and 3.9 lM PG for rooting. The incorporation of CC and PG enhanced rooting signicantly. These compounds have been reported to act as auxin protectors and increase the endogenous IAA levels during the inductive phase of rooting (Faivre-Rampant et al. 2004). Use of CC and PG in enhancing rooting has also been reported in Dendrocalamus hamiltonii (Sood et al. 2002) and Bambusa tulda (Mishra et al. 2008). The rooted plantlets (Fig. 1e) were successfully transferred to the greenhouse for hardening. The regenerated plants were subjected to RAPD analysis to check their clonality. Twenty random primers (OPF 110 and OPT 110) were used, of which 15 produced distinct and reproducible bands. A total of 1,197 amplicons were obtained and primer OPF-3 generated a highly

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Fig. 2 Agarose gel electrophoresis of RAPD fragments showing banding pattern amplied by OPF3 primer. M Molecular marker, C control

Fig. 1 Shoot bud induction from leaf explants of W. coagulans. a Indirect induction on MS, 13.3 lM BA and 2.3 lM Kn. b Direct induction from petiolar end on MS, 22.2 lM BA and 2.3 lM Kn. c Shoot buds developed on the rst stage proliferation medium. d Proliferation and elongation of shoots on MS, 2.2 lM BA, 2.3 lM Kn and 3.9 lM PG. e Rooting on MS, 1.2 lM IBA, 3.6 lM PAA and 14.3 lM CC

reproducible banding pattern (Fig. 2). DNA ngerprinting proles of regenerants revealed that there was no variation amongst mother and tissue culture-raised plants. There are many reports demonstrating the suitability of enhanced axillary branching for raising true-to-type plants (Rani and Raina 2000). Analysis of withanolide content in in vitro shoot cultures of W. somnifera has been reported by several workers (Ray and Jha 2001; Sangwan et al. 2004, 2007), but there are no such reports for W. coagulans. The study used an analytical reverse phase HPLC system providing symmetrical and high resolution peaks of three important withanolides in the plant. TLC of different extracts revealed that withaferin A, withanolide A and withanone were biosynthesized in regenerated plants of W. coagulans (Fig. 3). Withanolide content was analyzed by HPLC, and standard samples of withaferin A, withanolide A and withanone were used to construct a calibrated graph by plotting peak areas versus

Fig. 3 TLC prole of W. coagulans. Lanes 1 standard withaferin A, 2 standard withanolide A, 3 standard withanone, 4 sample extracted from in vitro shoots, 5 samples extracted from eld leaves, 6 samples extracted from callus, 7 samples extracted from eld roots

the amount of respective withanolide over a range of 501,000 ng ll-1. The response was linear over the tested concentration range. The identication of withanolides was conrmed on the basis of retention time and absorption spectra on UV-DAD (32.46 min, 215 nm; 38.38 min,

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139

Fig. 4 DADHPLC chromatogram of standards. a Withaferin A, b withanolide A, c withanone. Samples from d in vitro developed shoots, e eld leaves, and f eld roots (insets are UV-DAD spectra of the specied withanolide)

230 nm; and 40.90 min, 230 nm for withaferin A (Fig. 4a), withanolide A (Fig. 4b) and withanone (Fig. 4c), respectively). The accumulation of all the three withanolides was higher in regenerated plants than in the samples taken from eld-grown plants (Fig. 4d, e). A shift towards organ differentiation resulted in improved potential of the cultures to synthesize withanolides. The quantities of withaferin A and withanolide A increased up to two-fold while the withanone content increased up to ten-fold in the regenerated plantlets as compared to eld-grown plants (Table 2). Withanolide A accumulates in small amounts in shoots (Fig. 4e) and more in roots (Fig. 4f) in eld-grown plants, but in the present study the amount of withanolide A was as good in regenerated shoots as in the roots of eld plants (Table 2, Fig. 4d). Several factors, e.g., the difference in chemotype utilized as source for initiation of multiple shoot buds, and culture conditions such as basal media composition and growth regulator types utilized to establish cultures might have contributed to withanolide production. The positive correlation between withanolide synthesis and morphological differentiation suggests that

Table 2 Withanolide content in different tissues of W. coagulans Sample Withanolide Content (mg gfw-1) Mean SE Withaferin A Field leaves In vitro leaves Field roots 0.084 0.004 0.192 0.005 Nil Withanolide A 0.059 0.014 0.123 0.009 0.113 0.009 Withanone 0.031 0.001 0.282 0.006 Nil

synthesis is regulated in a tissue-specic way and organogenesis is the key regulatory factor which stimulates production of withanolides in vitro. The detection of higher content in differentiated cultures also points out that the enzymes responsible for biogenesis of withanolides in vitro might be optimally active in the culture conditions as has been shown earlier in W. somnifera (Sharada et al. 2007). Taken as a whole, our results demonstrate that leaves of W. coagulans have a great organogenic potential for shoot bud formation; however, the response is highly sensitive and directly related to the combinations of exogenous growth regulators in the culture medium. The results also

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Plant Cell Tiss Organ Cult (2011) 105:135140 Kulkarni AA, Thengane SR, Krishnamurthy KV (2000) Direct shoot regeneration from node, internode, hypocotyls and embryo explants of Withania somnifera. Plant Cell Tissue Org Cult 62:203209 Liu X, Pijut PM (2008) Plant regeneration from in vitro leaves of mature black cherry (Prunus serotina). Plant Cell Tissue Org Cult 94:113123 Maurya R, Akanksha J (2010) Chemistry and pharmacology of Withania coagulans: an ayurvedic remedy. J Pharma Pharmacol 62:153160 Mishra Y, Patel PK, Yadev S, Shirin F, Ansari SA (2008) A micropropagation system for cloning of Bambusa tulda roxb. Sci Hort 115:315318 Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473497 Murch SJ, Wierenga EJ, El-Demerdash MA, Saxena PK (2004) In vitro propagation of the Egyptian medicinal plant, Echinops spinosissimus turra. Plant Cell Tissue Org Cult 74:8186 Rani V, Raina SN (2000) Genetic delity of organized meristem derived micropropagated plants: a critical reappraisal. In Vitro Cell Dev Biol Plant 36:319330 Ray S, Jha S (2001) Production of withaferin A in in vitro shoot cultures of Withania somnifera. Planta Med 67:432436 Reddy PS, Rodrigues R, Rajasekharan R (2004) Shoot organogenesis and mass propagation of Coleus forskohlii from leaf derived callus. Plant Cell Tissue Org Cult 66:183188 Sangwan RS, Chaurasiya ND, Misra LN, Lal P, Uniyal GC, Sharma R, Sangwan NS, Suri KA, Qazi GN, Tuli R (2004) Phytochemical variability in commericial herbal products and preparations of Withania somnifera (ashwagandha). Curr Sci 86:446461 Sangwan RS, Chaurasiya ND, Lal P, Misra L, Uniyal GC, Tuli R, Sangwan NS (2007) Withanolide A biogeneration in in vitro shoot cultures of ashwagandha (Withania somnifera Dunal), a main medicinal plant in ayurveda. Chem Pharma Bull 55:13711375 Sarkar D, Naik PS (2000) Phloroglucinol enhances growth and rate of axillary shoot proliferation in potato shoot tip cultures in vitro. Plant Cell Tissue Org Cult 60:139149 Sharada M, Ahuja A, Suri KA, Vij SP, Khajuria RK, Verma V, Kumar A (2007) Withanolide production by in vitro cultures of Withania somnifera and its association with differentiation. Biol Plant 51:161164 Sharma PK, Tyagi P, Sharma KC, Kothari SL (2003) Clonal micropropagation of Crataeva adansonii (DC.) Prodr: a multipurpose tree. In Vitro Cell Dev BiolPlant 39:156160 Sinha A, Jain R, Kachhwaha S, Kothari SL (2010) Optimization of the level of micronutrient copper in the culture medium improves shoot bud regeneration in Indian ginseng [Withania somnifera (L.) Dunal]. Natl Acad Sci Lett 33:1116 Sood A, Ahuja PS, Sharma M, Sharma OP, Godbole S (2002) In vitro protocols and eld performance of elites of an important bamboo Dendrocalamus hamiltonii nees et arn. ex munro. Plant Cell Tissue Org Cult 71:5563 Sreedhar RV, Venkatachalam L, Thimmaraju R, Bhagyalaxmi N, Narayanan MS, Ravishankar GA (2008) Direct organogenesis from leaf explants of Stevia rebaudiana and cultivation in bioreactor. Biol Plant 52:355360 Tilkat E, Onay A, Yldrm H, Ayaz E (2009) Direct plant regeneration from mature leaf explants of pistachio, Pistacia vera L. Sci Hort 121:361365 Zhou H, Li M, Zhao M, Fan X, Guo A (2010) Plant regeneration from in vitro leaves of the peach rootstock Nemaguard (Prunus persica 9 P. davidiana). Plant Cell Tissue Org Cult 101:7987

conrm the potential of this plant to biosynthesize the active principle (withanolides) under in vitro culture conditions. In vitro regeneration of adventitious shoots is an essential component for most of the genetic transformation protocols. The system described here will be useful in this respect and for conservation of elite germplasm of this important medicinal plant species.
Acknowledgments Financial support from Council of Scientic and Industrial Research (CSIR) in the form of R&D project: CSIR 38(1178) EMRII/2007 is gratefully acknowledged. Rohit Jain, Arunima Sinha and Devendra Jain thank CSIR for the award of Senior Research Fellowships.

References
Atta-ur-Rahman A, Choudhary MI, Qureshi S, Gul W, Yousaf M (1998) Two new ergostanetype steroidal lactones from Withania coagulans. J Nat Prod 61:812814 Baskaran P, Jayabalan N (2005) An efcient micropropagation system for Eclipta alba available medicinal herb. In Vitro Cell Dev Biol Plant 41:532539 Bhandari MM (1995) Flora of the Indian desert. MPS Repros, Jodhpur Corredoira E, Ballester A, Vieitez AM (2008) Thidiazuron-induced high-frequency plant regeneration from leaf explants of Paulownia tomentosa mature trees. Plant Cell Tissue Org Cult 95:197208 Dayal S, Lavanya M, Devi P, Sharma KK (2003) An efcient protocol for shoot regeneration and genetic transformation of pigeonpea [Cajanus cajan (L.) Millsp.] using leaf explant. Plant Cell Rep 21:10721079 Doyle JJ, Doyle JL (1990) Isolation of plant DNA from fresh tissue. Focus 12:1315 Faivre-Rampant O, Kevers C, Gaspar T (2004) IAAoxidase activity and auxin protectors in nonrooting, rac, mutant shoots of tobacco in vitro. Plant Sci 153:7380 Feeney M, Bhagwat B, Mitchel JS, Lane WD (2007) Shoot regeneration from organogenic callus of sweet cherry (Prunus avium L.). Plant Cell Tissue Org Cult 90:201214 Gentile A, Monticelli S, Damiano C (2002) Adventitious shoot regeneration in peach (Prunus persica). Plant Cell Rep 20:10111016 Gomez KA, Gomez AA (1984) Statistical procedures for agricultural research. Wiley, New York, p 680 Jain P, Kachhwaha S, Kothari SL (2009a) Improved micropropagation protocol and enhancement in biomass and chlorophyll content in Stevia rebaudiana (Bert.) Bertoni by using high copper levels in the culture medium. Sci Hort 119:315319 Jain R, Sinha A, Kachhwaha S, Kothari SL (2009b) Micropropagation of Withania coagulans (Stocks) Dunal: a critically endangered medicinal herb. J Plant Biochem Biotechnol 18:249252 Kachhwaha S, Kothari SL (1996) Plant regeneration from immature embryo explants of Hordeum spontaneum and Hordeum vulgare. Cer Res Comm 24:2732 Koroch A, Juliani HR, Kapteyn J, Simon JE (2002) In vitro regeneration of Echinacea purpurea from leaf explants. Plant Cell Tissue Org Cult 69:7983 Kothari SL, Joshi A, Kachhwaha S, Ochoa-Alejo N (2010) Chilli peepersa review on tissue culture and transgenesis. Biotechnol Adv 28:3548

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22-03-2010

Nucleotide - Gordonia lacunae strain

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GenBank: GU727686.1

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Gordonia lacunae strain CCC12 16S ribosomal RNA gene, partial sequence
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LOCUS GU727686 1491 bp DNA linear BCT Run BLAST 20-MAR-2010 Pick Primers DEFINITION Gordonia lacunae strain CCC12 16S ribosomal RNA gene, partial sequence. Recent activity ACCESSION GU727686 Turn Off Clear VERSION GU727686.1 GI:291061266 KEYWORDS . Gordonia lacunae strain SOURCE Gordonia lacunae CCC12 16S ribosomal RNA ORGANISM Gordonia lacunae Bacteria; Actinobacteria; Actinobacteridae; GU727686 (1) Actinomycetales; Corynebacterineae; Gordoniaceae; Gordonia. centre for converging REFERENCE 1 (bases 1 to 1491) tec... (8) AUTHORS Jain,D., Jain,R., Agarwal,V., Sharma,P., Srivastava,G., Kachhwaha,S. and Kothari,S.L. University of TITLE Direct Submission rajasthan (11902589) JOURNAL Submitted (22-JAN-2010) Department of Botany, University Gordonia (730) of Rajasthan, JLN Marg, Jaipur, Rajasthan 302004, India See more... FEATURES Location/Qualifiers source 1..1491 /organism="Gordonia lacunae" /mol_type="genomic DNA" /strain="CCC12" /db_xref="taxon:417102" /country="India" rRNA <1..>1491 /product="16S ribosomal RNA" ORIGIN 1 gggcaaacgc tggcggcgtg cttaacacat gcaagtcgaa cggaaaggcc cagcttgctg 61 ggtactcgag tggcgaacgg gtgagtaaca cgtgggtgat ctgccctgca ctctgggata 121 agcctgggaa actgggtcta ataccggata tgaccaactg tcgcatggtg gttggtggaa 181 agcttttgcg gtgtgggatg ggcccgcggc ctatcagctt gttggtgggg taatggccta 241 ccaaggcgac gacgggtagc cgacctgaga gggtgatcgg ccacactggg actgagacac 301 ggcccagact cctacgggag gcagcagtgg ggaatattgc acaatgggcg caagcctgat 361 gcagcgacgc cgcgtgaggg atgacggcct tcgggttgta aacctctttc accagggacg 421 aagcgtgagt gacggtacct ggagaagaag caccggccaa ctacgtgcca gcagccgcgg 481 taatacgtag ggtgcgagcg ttgtccggaa ttactgggcg taaagagctc gtaggcggtt 541 tgtcgcgtcg tctgtgaaat tctgcaactc aattgcaggc gtgcaggcga tacgggcaga 601 cttgagtact acaggggaga ctggaattcc tggtgtagcg gtgaaatgcg cagatatcag 661 gaggaacacc ggtggcgaag gcgggtctct gggtagtaac tgacgctgag gagcgaaagc 721 gtgggtagcg aacaggatta gataccctgg tagtccacgc cgtaaacggt gggtactagg 781 tgtgggttcc ttttcacggg atccgtgccg tagctaacgc attaagtacc ccgcctgggg 841 agtacggccg caaggctaaa actcaaagga attgacgggg gcccgcacaa gcggcggagc 901 atgtggatta attcgatgca acgcgaagaa ccttacctgg gtttgacata caccagacgc 961 ggctagagat agtcgttccc ttgtggttgg tgtacaggtg gtgcatggct gtcgtcagct 1021 cgtgtcgtga gatgttgggt taagtcccgc aacgagcgca acccttgtcc tgtattgcca 1081 gcgggttatg ccggggactt gcaggagact gccggggtca actcggagga aggtggggat 1141 gacgtcaagt catcatgccc cttatgtcca gggcttcaca catgctacaa tggctggtac 1201 agagggctgc gataccgtga ggtggagcga atcccttaaa gccagtctca gttcggattg 1261 gggtctgcaa ctcgacccca tgaagtcgga gtcgctagta atcgcagatc agcaacgctg 1321 cggtgaatac gttcccgggc cttgtacaca ccgcccgtca cgtcatgaaa gtcggtaaca 1381 cccgaagccg gtggcctaac cccttgtggg agggagctgt cgaaggtggg atcggcgatt 1441 gggacgaagt cgtaacaagg tgccgtaccg gaagcacatt tatattttgg g //

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