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

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 : Ph.D. (Botany)-Thesis Submitted (09-02-2011) 2005-2007 : Master of Science (Botany with specialization in Plant Physiology) from Department of Botany, University of Rajasthan, Jaipur (71.33%)

2002-2005 : Bachelor of Science (Biology) from University Maharaja’s College,

University of Rajasthan, Jaipur (71%)

1999-2000 : Sr. Secondary (Biology) from Govt. Sr. Hr. Sec. School Lalsot, Dausa, Rajasthan Board of Secondary Education, Ajmer (68.62%)

1997-1998 : 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 ½ 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) 105:135-140 (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) 18:249-252 (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 : 9th August, 1984

Father’s Name : Rakesh Kumar Jain

Nationality : Indian

Sex : Male

Marital Status : Single

Language Skills : Hindi, English

References:

Prof. S. L. Kothari Dr. Shailesh Godika

Dean, Faculty of Science, Assistant Professor, Director, Centre for Converging Technologies Agriculture Research Station, Naugaon Professor of Botany, SK Agricultural University, Bikaner University of Rajasthan, Jaipur E-mail: shaileshgodika@gmail.com E-mail: slkothari@lycos.com

Rohit Jain

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

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

NO.3/1/3/Next-1 00/JRF/07 -MPD

Dated: 31st August, 2007

11328Rohit JainS/o SIl. Rakesh Kumar Jain,Sainara Shawan, Near Telephone ExchangeNew Colony, LalsotDai.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 15thJuly 2007, as Junior research Fellow in ICMR funded Research Projects/Scheme. Thisplacement is subject to fulfilling the conditions for appointment under the project/scheme.

You are required to contact along with your Sio-data with potential ResearchInvestigators located in various Medical colleges/Research Organization/National laboratoriesincluding ICMR Institutes who are in receipt of ICMR funding for their project for seekingplacement as JRF depending upon your area of specialization. This letter will enable you to findsuch placement in ICMR funded projects in these Institutions. Please note your stipend for JRFwould be paid from project grant if you secure placement in the project. The Council would notbe able to provide you stipend directly. Details of ICMR Institutes and advanced centers can beobtained from Website: icmr.nic.in

The emoluments/stipend for JRF at present is RS.8000/- plus HRA as appiicabie permonth. On finding placement in above-mentioned institutions you will be paid this amount fromthe 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 bepermitted to enroll yourself for pursuing Ph.D. from any university while working in the scheme (ifallowed by the university).

In case, you are found ineligible at any stage during your research work that may be dueto false certification or any other reason (including computer error), the award may be withdrawnby the Institution/ICMR.

Kindly acknowledge the receipt of the letter.

Agricultural Scientists Recruitment BoardProvisional Result of National Eligibility Test (NET) 2010 held on 19th September, 2010

Qualified for NET

Roll Number : 030304100200

Name : ROHIT JAIN

Father’s Name : RAKESH KUMAR JAIN

Category : UR

Physically Challenged : No

Discipline Name : BASIC PLANT SCIENCES

Exam. Centre Code : 03

Exam. Centre Name : BIKANER

The candidate whose Roll Number is given above has qualified the NET subject to fulfillment of eligibilityconditions laid down in the notification for NET-2010.

1. The Candidates who had not submitted the attested copy of their Master’s 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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Developed and Maintained by Vivek Dubey, ARIC, DIPA, ICARContent updation by Mitali Ghosh Roy, ARIC, DIPA, ICAR

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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*1Department of Botany, University of Rajasthan, Jaipur 302 004, India2Centre 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 andan important natural source of withanolides. Prolific multiplication of axillary buds occurred from the nodal segments takenfrom 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 segmentsand shoot tips of elongated microshoots also behaved the same way in cultures and formed multiple shoots through axillarybud multiplication. Addition of PG (0.5 mg l-1) in the regeneration medium significantly improved induction and elongationof shoot buds. Elongated shoots were placed on filter paper bridges soaked in MS medium with CC (10 mg l-1) and PG (0.5 mgl-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 4subcultures 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

autoclaving at 1.06 kg cm-2 (121°C) for 20 min was used in

all the experiments. The cultures were incubated at 25 ±

1°C under a 16-h photoperiod with 25µmol m-2s-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 waterand inoculated onto MS medium supplemented with BA orKn 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 withoptimal cytokinin concentration. Shoot buds induced inprimary cultures were sectored in clumps of 3-4 andcultured on fresh medium for further multiplication of shootbuds.

The in vitro-raised microshoots (2–3 cm in length)were harvested for rooting. Two step rooting procedurewas followed. Step one involved the pulse treatment ofindividual 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 ½ or ¼ MS, agar-gelled semisolid medium

*Corresponding author. E-mail: kothari-sl@uniraj.ernet.inAbbreviations: BA - 6-benzylaminopurine, CC - choline chloride,IAA - indole-3-acetic acid, IBA - indole-3-butyric acid, Kn - kinetin,NAA - α-naphthaleneacetic acid, PAA - phenylacetic acid, PG -phloroglucinol, RAPD - random amplified polymorphic DNA

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 (-

196°C) and stored at -20°C 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 94°C for 5 min followed by 35 cycles of 94°C for 30 sec,

50°C for 45 seconds and 72°C for 1 min, and a final

extension at 72°C 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 Fischer’s 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

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 1. Shoot bud formation from nodal segments of W. coagulanscultured on MS medium supplemented with BA and Kn

BAP Kn Percent response Mean No. of(mg l-1) (mg l-1) (%) Buds/Explant ± S.E.

0.5 0 57 3.8a ± 0.4

1 0 68 6.4b ± 0.4

2 0 74 7.0c ± 0.4

3 0 79 9.0d ± 0.4

5 0 83 11.2e ± 0.5

0 0.5 43 2.4f ± 0.1

0 1 55 3.0c ± 0.4

0 2 55 3.7d ± 0.2

0 3 66 5.0x ± 0.3

0 5 73 3.2g ± 0.30.5 0.5 83 18.6 h ± 0.5

S.E. – Standard errorMeans in a column followed by different letters are significantlydifferent from each other

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

PG CC Nodal segments Shoot-tips

(mg l-1) (mg l-1) Mean No. of Mean length of Mean No. of Mean length ofBuds/Explant ± S.E. Shoots (cm) ± S.E. Buds/Explant ± S. E. Shoots (cm) ± S. E.

0 0 20.9a ± 0.3 0.5a ± 0.1 22.3a ± 0.4 0.3a ± 0.10.5 0 23.4b ± 0.2 4.3b ± 0.2 24.6b ± 0.3 4.7b ± 0.21 0 21.1c ± 0.3 4.1b ± 0.1 23.3c ± 0.1 4.4bc ± 0.12 0 19.2d ± 0.3 3.5c ± 0.2 20.4d ± 0.3 4.2c ± 0.23 0 18.3e ± 0.2 3.1d ± 0.1 19.3e ± 0.3 3.9d ± 0.25 0 15.5f ± 0.5 3.0d ± 0.1 17.5f ± 0.2 3.6d ± 0.10 0.5 21.0c ± 0.4 3.8e ± 0.2 23.9g ± 0.3 4.6be ± 0.20 1 17.7g ± 0.5 3.3f ± 0.1 22.4a ± 0.3 4.3e ± 0.10 2 14.3h ± 0.3 2.8d ± 0.2 21.5h ± 0.5 3.7d ± 0.20 3 13.5i ± 0.2 2.4g ± 0.1 19.1i ± 0.6 3.4d ± 0.00 5 13.1j ± 0.3 2.2g ± 0.1 14.5j ± 0.3 2.9e ± 0.1

S.E. – Standard errorMeans 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 withBA (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 (0.5mg 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 MSmedium 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. coagulansshowing 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 eachleaf, a distinct meristematic zone of small densely stainedcells was present over a differentiated zone. A ring ofmultiple shoot primordia could be observed arising directlyfrom base of cultured shoot tip (Fig. 1c). In the culturednodes, at a later stage of development, vertical andsideways expansion of the meristematic zone occurred(Fig. 1d).

The maximum frequency of root formation (80%),highest number (11.5±0.7) of roots and root length(7.9±0.3cm) were seen after pulse treatment of shoots inMS medium containing 10 mg l-1 CC and 0.5 mg l-1 PGfollowed by their transfer to ½ strength MS medium withIBA (0.25 mg l-1), PAA (0.5 mg l-1) and CC (2 mg l-1) after 7days (Fig. 1e). Two-step procedure for rooting has beenused to advantage in several woody species (14). Theincorporation of CC at different concentrations enhancedthe response of rooting of shoots significantly. CC and PGhave enhanced rooting in Bambusa tulda (15). Thesecompounds are reported to enhance rooting by acting asauxin protectors to increase the free endogenous IAA levelsduring the inductive phase of rooting (16).

The plantlets were successfully hardened inside theculture room under diffused light on MS medium for 2weeks, followed by their establishment in pots containing(1:1) soil and manure in greenhouse. About 75% of themicropropagated plants survived after transfer to soil andorganic manure (1:1). All the established plants wereapparently uniform and did not show any detectablevariation.

Clonal fidelity of the regenerated shoots was checkedthrough RAPD. Of 12 random primers, 8 generated distinct,reproducible products. A total of 580 amplification productswere detected. The primers OPA-5 and OPA-19 (Fig. 1f)gave highly reproducible banding pattern. Fingerprintingprofiles of regenerants were monomorphic and there wasno variation amongst mother and tissue culture raisedplants. There are number of reports demonstrating thesuitability of enhanced axillary branching for raising trueto type plants (17). Similar results have been obtained inpresent 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 Agarwal R, Diwanay S, Patki P & Patwardhan B, JEthnopharmacol, 67 (1999) 27.

2 Bhandari MM, Flora of the Indian desert, MPS Repros,Jodhpur, India (1995) pp 246.

3 Hemalatha S, Wahi AK, Singh PN &Chansouria JPN, JEthnopharmacol, 93 (2004) 261.

4 Maurya R, Jayendra A, Singh AB & Srivastava AK, BioorgMed Chemis Lett, 18 (2008) 6534.

5 Atta-ur-Rahman, Yousaf M, Gul W, Qureshi S,Choudhary MI, Voelter W, Hoff A, Jens F & Naz A,Heterocycles, 48 (1998) 1801.

6 Mesaik MA, Zaheer-ul-Haq, Murad S, Ismail Z, AbdullahNR, Gill HK, Atta-ur-Rahman, Yousaf M Siddiqui RA,Ahmad A & Choudhary MI, Mol Immun, 43 (2006)1855.

7 Murashige T & Skoog T, Physiol Plant, 15 (1962) 473.

8 Johansen DA, Plant microtechnique, Mc Graw-Hill BookCompany, Inc., New York, USA (1940).

9 Doyle IJ & Doyle JL, Focus, 12 (1990) 13.

10 Gomez KA & Gomez AA, Statistical procedures foragricultural research, John Wiley and Sons, New York (1984)

11 Sarkar D & Naik PS, Plant Cell Tiss Org Cult, 60 (2000) 139.

12 Ibanez MR & Amo-Marc JB, Plant Growth Reg, 26 (1998)49.

13 Rastogi S, Rizvi SMH, Singh RP & Dwivedi UN, Biol Plant,52 (2008) 743.

14 Husain MK & Anis M, In Biotechnology for a better future(L D’Souza, M Anuradha, S Nivas, S Hegde, K Rajendra,Editors). SAC, Mangalore (2004) p 294.

15 Mishra Y, Patel PK, Yadev S, Shirin F & Ansari SA, SciHort, 115 (2008) 315.

16 Faivre-Rampant O, Kevers C & Gaspar T, Plant Sci, 153(2004)73.

17 Rani V & Raina SN, In Vitro Cell Dev Biol-Plant, 36 (2000)319.

1 23

Plant Cell, Tissue and OrganCulture (PCTOC)Journal of Plant Biotechnology ISSN 0167-6857Volume 105Number 1 Plant Cell Tiss Organ Cult(2011) 105:135-140

DOI 10.1007/s11240-010-9840-3

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

1 23

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RESEARCH NOTE

Adventitious shoot regeneration and in vitro biosynthesisof 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 devel-

oped on Murashige and Skoog (MS) medium supplemented

with 2.3 lM kinetin (Kn) and lower levels of 6–benzylade-

nine (BA) (13.3 lM) while multiple adventitious shoot bud

differentiation occurred on medium fortified 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 trans-

ferred to a greenhouse for hardening and further growth.

Random amplification of polymorphic DNA (RAPD)

showed monomorphic bands in all the plants thereby con-

firming clonality of the regenerants. Thin layer chromatog-

raphy (TLC) showed the presence of withanolides in the

regenerated plants. Quantification through reverse-phase

HPLC revealed increased concentration of withanolides in

the regenerated plants compared to the field-grown mother

plant. Accumulation of withaferin A and withanolide A

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 6–benzyladenine

CC Choline chloride

DAD Diode array detector

IAA Indole–3–acetic acid

IBA Indole–3–butyric acid

Kn Kinetin

MS Murashige and Skoog

NAA a–naphthaleneacetic acid

PAA Phenylacetic acid

PG Phloroglucinol

RAPD Random amplification 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-inflamma-

tory, antitumor, hepatoprotective, antihyperglycemic, car-

diovascular, immunosuppressive, free radical scavenging

and central nervous system depressant activities of the

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

123

Plant Cell Tiss Organ Cult (2011) 105:135–140

DOI 10.1007/s11240-010-9840-3

plant have also been demonstrated (Maurya and Akanksha

2010). Pharmacological investigations have elucidated

association of these activities with the specific steroidal

lactones known as withanolides present in Withania (Atta-

ur-Rahman et al. 1998). Withaferin A, withanolide A and

withanone are the major withanolides present in W. som-

nifera and W. coagulans. Overexploitation and the repro-

ductive 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 field 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 regen-

erated plants for the first time.

Materials and methods

Plant material and establishment of in vitro cultures

from leaf explants

Leaf explants (0.8–2 cm) were collected from the field-

grown plants spotted in Ajmer (Rajasthan) in 2007. The

species was identified 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 4–5 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 combi-

nations of different plant growth regulators (Sigma, India)

including 6–benzyladenine (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-3-

butyric acid (IBA; 0.9, 1.4 and 2.4 lM), phenylacetic acid

(PAA; 1.4, 2.2 and 3.6 lM) and a–naphthaleneacetic 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 121�C temperature for 20 min. Leaf explants

with or without petiolar parts were placed abaxially on the

medium. Cultures were maintained at 26 ± 1�C under 16/

8 h photoperiod with 25 lmol m-2 s-1 photosynthetic

photon flux density provided by white fluorescent tubes

(40 W; Philips, India). Twenty replicates were maintained

for each treatment. The numbers of responding explants

and shoot buds developed per explant were recorded and

shoot buds were subcultured on first 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 appro-

priate 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 flask 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 -20�C until used for DNA extraction by CTAB

method (Doyle and Doyle 1990). The PCR amplification

conditions were: an initial denaturation at 94�C for 4 min

followed by 40 cycles of 94�C for 45 s, 37�C for 45 s and

72�C for 2 min, and a final extension at 72�C 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 profiling was done through TLC

while quantification 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 ace-

tate: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 110�C. HPLC anal-

ysis 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; Agi-

lent) using water (A) and methanol (B), each containing

0.1% acetic acid, as solvent and online UV-Diode Array

136 Plant Cell Tiss Organ Cult (2011) 105:135–140

123

Detector (UV-DAD) at 227 nm. The solvent gradient was set

as A:B, 60:40–25:75, 0–45 min; 10:90, 45–60 min at a flow

rate of 0.6 ml min-1. Sample volume of 10 ll was injected

and the column temperature maintained at 27�C during the

run. Authentic withanolides including withaferin A, witha-

none 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 Fischer’s

least significant 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

(2.2–13.3 lM). Kn alone (Murch et al. 2004) or in com-

bination 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 (13.3–22.2

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 (2.2–22.2 lM) with NAA (1.0–

2.6 lM). Kn in combination with auxins initiated forma-

tion of pale and non–morphogenic callus.

The use of 2.3 lM Kn in combination with BA

(2.2–13.3 lM) promoted the initiation and development of

shoot buds along with callus (Fig. 1a). Clusters of adventi-

tious 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 regen-

eration 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 elon-

gation of the shoot buds did not occur (Fig. 1c). A combi-

nation 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 2–3 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). Elon-

gated 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 significantly. 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

1–10 and OPT 1–10) 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

Table 1 Shoot bud formation from leaf explants of W. coagulanscultured on MS medium supplemented with BA and Kn

BA (lM) Kn (lM) % response Shoot buds

(Mean ± SE)

2.2 2.3 80 4.6 ± 0.5 e

4.4 2.3 86 7.7 ± 0.6 d

8.9 2.3 73 9.3 ± 0.6 c

13.3 2.3 93 12.1 ± 0.2 b

22.2 2.3 80 17.6 ± 0.5 a

SE Standard error

Means in a column followed by different letters are significantly

different from each other at P = 0.05

Plant Cell Tiss Organ Cult (2011) 105:135–140 137

123

reproducible banding pattern (Fig. 2). DNA fingerprinting

profiles 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

the amount of respective withanolide over a range of

50–1,000 ng ll-1. The response was linear over the tested

concentration range. The identification of withanolides was

confirmed on the basis of retention time and absorption

spectra on UV-DAD (32.46 min, 215 nm; 38.38 min,

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 first 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

Fig. 2 Agarose gel electrophoresis of RAPD fragments showing

banding pattern amplified by OPF–3 primer. M Molecular marker,

C control

Fig. 3 TLC profile of W. coagulans. Lanes 1 standard withaferin A, 2standard withanolide A, 3 standard withanone, 4 sample extracted

from in vitro shoots, 5 samples extracted from field leaves, 6 samples

extracted from callus, 7 samples extracted from field roots

138 Plant Cell Tiss Organ Cult (2011) 105:135–140

123

230 nm; and 40.90 min, 230 nm for withaferin A (Fig. 4a),

withanolide A (Fig. 4b) and withanone (Fig. 4c), respec-

tively). The accumulation of all the three withanolides was

higher in regenerated plants than in the samples taken from

field-grown plants (Fig. 4d, e). A shift towards organ dif-

ferentiation 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 witha-

none content increased up to ten-fold in the regenerated

plantlets as compared to field-grown plants (Table 2).

Withanolide A accumulates in small amounts in shoots

(Fig. 4e) and more in roots (Fig. 4f) in field-grown plants,

but in the present study the amount of withanolide A was as

good in regenerated shoots as in the roots of field 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 estab-

lish cultures might have contributed to withanolide pro-

duction. The positive correlation between withanolide

synthesis and morphological differentiation suggests that

synthesis is regulated in a tissue-specific way and organ-

ogenesis 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

Fig. 4 DAD–HPLC chromatogram of standards. a Withaferin A, b withanolide A, c withanone. Samples from d in vitro developed shoots,

e field leaves, and f field roots (insets are UV-DAD spectra of the specified withanolide)

Table 2 Withanolide content in different tissues of W. coagulans

Sample Withanolide Content (mg gfw-1) Mean ± SE

Withaferin A Withanolide A Withanone

Field leaves 0.084 ± 0.004 0.059 ± 0.014 0.031 ± 0.001

In vitro leaves 0.192 ± 0.005 0.123 ± 0.009 0.282 ± 0.006

Field roots Nil 0.113 ± 0.009 Nil

Plant Cell Tiss Organ Cult (2011) 105:135–140 139

123

confirm the potential of this plant to biosynthesize the

active principle (withanolides) under in vitro culture con-

ditions. 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 Scientific

and Industrial Research (CSIR) in the form of R&D project: CSIR–

38(1178) EMR–II/2007 is gratefully acknowledged. Rohit Jain,

Arunima Sinha and Devendra Jain thank CSIR for the award of Senior

Research Fellowships.

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GenBank: GU727686.1

Gordonia lacunae strain CCC12 16S ribosomal RNAgene, partial sequence

LOCUS GU727686 1491 bp DNA linear BCT 20-MAR-2010DEFINITION Gordonia lacunae strain CCC12 16S ribosomal RNA gene, partial sequence.ACCESSION GU727686VERSION GU727686.1 GI:291061266KEYWORDS .SOURCE Gordonia lacunae ORGANISM Gordonia lacunae Bacteria; Actinobacteria; Actinobacteridae; Actinomycetales; Corynebacterineae; Gordoniaceae; Gordonia.REFERENCE 1 (bases 1 to 1491) AUTHORS Jain,D., Jain,R., Agarwal,V., Sharma,P., Srivastava,G., Kachhwaha,S. and Kothari,S.L. TITLE Direct Submission JOURNAL Submitted (22-JAN-2010) Department of Botany, University of Rajasthan, JLN Marg, Jaipur, Rajasthan 302004, IndiaFEATURES 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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Gordonia lacunae strainCCC12 16S ribosomal RNA

GU727686 (1)

centre for convergingtec... (8)

University ofrajasthan (11902589)

Gordonia (730)

22-03-2010 Nucleotide - Gordonia lacunae strain …

www.ncbi.nlm.nih.gov/…/291061266 1/2