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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: [email protected] 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: [email protected] E-mail: [email protected]
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: [email protected]: 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: [email protected]
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