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Nutan Malpathak

on 1 November 2013

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Transcript of CAS2012

Dr. Nutan P. Malpathak
Teaching experience: 22 years
Joined as a lecturer in the Dept. of Botany,
University of Pune, in 1992.
Appointed as selected Reader in the Dept. of Botany, University of Pune on temporary basis 2000-2002.

Appointed as regular Reader through CAS from 16th March 2002
Course in charge for courses: Cell Biology,
Teaching other courses Plant Biotechnology and Bioinformatics,
Plant Biodiversity
1. In Vitro Cell Dev. Biol.-Plant 41:291-295. (2005)
2. Plant Cell Tissue Organ Culture 80:247-257  (2005)
3. J. Plant Biol. 31(2):95-99 (2005)
4. Current Science 87(10): 1442-1447(2004)
PCR of A4 and LBA 9402 (pBIN 19) mediated primary and secondary hairy root clones for rol A.
Patent granted entitled “Process of extraction of solasodine” ref no: 955/MUM/Y/310304 dt 15.9.03
Genetic Transformation in Solanum khasianum
Patent filed entitled” process for enhancing production of furanocoumarines using tailored cell cultures (2500/MUM/2008, dt 28/11/08)
New Biotechnology 25(1):85-91 (2008)

Bioorganic & Medicinal Chemistry 17:7052-7055 (2009)

In Vitro Cellular and Dev Biol. –Plant 46:108-116 (2010)

Appl Biochem Biotechnol 163:756–764 (2011)

Bioremediation, Biodiversity and Bioavailability 5(1): 1-9 (2011)

Journal of Botany (2012) (in press)

Transformation with LBA 4404 harboring pCAMBIA 2301
Establishment of lines: Cell Cultures & Multiple Shoot cultures, Histochemical localization
Exploring Ruta graveolens L cultures for enhanced production of furanocoumarins
Modification of non-phytopathogenic bacteria to mediate gene transfer to diverse plants

Non-Agrobacterium Rhizobiaceae members shown capable of gene transfer to plant cells

Exploring gene transfer capacity of diverse species of bacteria (TransBacterTM) in Ruta graveolens
Such Ex-Agrobcaterium mediated gene transfer has been reported so far only in few extensively worked out plant systems like Arabidopsis, tobacco, rice, soyabean, canolla, coton, corn and potato

It remains to be determined whether factors affecting transformation in Agrobacterium also affect transformation by TransBacterTM

(Broothaerts, W., Mitchell, H. J., Weir, B., Kaines, S., Smith, L. A., Yang, W., Mayer, J. E., Roa-Rodrı´guez, C. and Jefferson R. A. (2005) Gene transfer to plants by diverse species of bacteria, Nature. 433, 629-633)
GUS Plus screening
Factors that were optimized using Design of experiments (DOE)
After performing experiments according to DOE incubate shoots in X-GlcA (200ug/ml)
‘Screen’ blue-stained foci/ explants

GUS plus – Staphylococcus
codon usage optimized for plants, non-destructive, more sensitive
In vivo assay-GRP signal peptide directs GUSPlusTM to cell wall apoplastic space more efficiently than extensin signal peptide

Based on the observations, plant tissues could survive and continue to regenerate after incubation in a low concentration X-GlcA solution, and in the presence of the end product of GUS cleavage (indigo).

extrapolating beyond actual experimentation regions
Manually reduce runs
Design summary
pH, PEG, temperature, and interactive effect of pH and PEG (B, E,A, BE) have a significant positive influence the transformation efficiency.
Whereas ca depletion has a negative influence.

Identifying key factors influencing transformation efficiency
Production of Flavonoids by Psoralea corylifolia cultures
Time course of accumulation of daidzein (A) and genistein (B) in cultures of P. corylifolia.
J Nat Med (2010) 64:346–353
Food Chemistry 118 (2010) 128–132
Biotech and Bioprocess Engi 14: 612-618
Biotech. Bioprocess Eng. (2009)14:288-294
Rec. of Natural Products (2009) 3(1):38-45
Bioresource technology (2009)100:1833-39
Pharmacocognosy Reviews (2008)2(3):43-53
Jr of Biodiv and Eco. Sciences (JBES)(2012) 1(4): 1-9
Daidzein and genistein obtained from extract of cultures of P. Corylifolia
Bacterial strain: A4, LBA 9402
Explant used: Cotyledon, hypocotyl, shoot.
Culture conditions: Incubation at 25 oC with 16/8 hr of light and dark photoperiod
Agrobacterium rhizogenes mediated transformation of Psoralea corylifolia.
Study of genetic diversity in Psoralea corylifolia populations from Maharashtra
Location of populations of P. corylifolia from different regions of Maharashtra.
The binary matrix data was analyzed using NTSys software (Rohlf, 1994) and the statistical analysis was performed using SPSS software.
The RAPD profile yielded a total of 225 polymorphic loci with 99.56% polymorphism at species level.
RAPD analysis
Variance among populations V (A): 5.72669123200 (23.53%)

Variance within populations V (B): 18.61136306400 (76.47%)
Cluster analysis among 9 populations of Psoralea corylifolia based on Nei’s genetic distances.
3 different groups are formed
1st group comprises of localities from
Pune Shrigonda and Nashik.
2nd group comprises of Kopargaon and
3rd group comprises of Nagar and Baramati.
One way ANOVA for the nine populations of P. corylifolia.
In each column, means with the same superscript letter were not significantly different, using the LSD tests (P< 0.05), and values represent mean ± SD.
Hierarchical cluster analysis based on the biochemical data in populations of P. corylifolia.
Study of biochemical diversity in Psoralea corylifolia populations
Column chromatography
Preparation of extracts:
300g of pulverized seed of Psoralea corylifolia were extracted repeatedly in 95% ethanol.
The ethanolic extract was concentrated using rota vapour. Part of the extract was coated on silica gel and was used for column chromatography
Isolation of compounds by column chromatography
Compounds identified from seed fraction of Psoralea corylifolia
The compound was isolated from stem fraction using 6:4 and 5:5 hexane: ethyl acetate gradient. The single blue fluorescent band was purified by preparative TLC and the identification of the compound was done by TLC by comparing the sample with standard. Further confirmation was carried out by LC MS and IR studies. LC MS analysis showed a single peak at 14.136 and mass spectrum peak of this compound exhibited molecular ion peak at 185. The IR analysis also showed overlapping peaks of the sample along with the standard and thus the compound was deduced as Psoralen. The molecular weight of this compound is 186.20 as per literature survey.
Compounds identified from stem fractions
Effect of methanolic and ethanolic extracts on inhibition of growth of cancer cell lines in vivo
The cell lines HEK293, S180,HT29 and A431 were purchased from NCCS (Pune) and were cultured in RPMI 1640 medium supplemented with 2% fetal bovine serum.
The cell lines were incubated overnight in a CO 2 incubator at 5% humidity and were then plated in a 96-well micro titer plate. 10μl of the extract (10-100μg) was added to each well and the cells were incubated for 24hrs.
The viability of the cells was assayed by performing MTT assay.
Anticancer assay:
SDS PAGE analysis showed dianthin 30 to be present in all the varieties
Leaves of Dianthus Caryophyllus L. contain dianthin ,dianthin 30 of molecular weight 29,500 dalton and dianthin 32 of molecular weight 31700 dalton.
Lane 1:Protein Molecular Weight Marker
Lane 2-5:Gaudina,Yellow Firato, Bizet, Flanel.
Detection of Dianthin by SDS Page
Polymerase chain reaction based detection of RIPs
Specific PCR amplifications of either pre-dianthin 30 or
dianthin 30.
Biosynthetic potential of different cultivars for Dianthin production
Different carnation cultivars - Gaudina, Yellow Firato, Bizet and Flanel were obtained from KFBioplants Pvt. Ltd.
Rooted plantlets were established in pots in the greenhouse of the Department.
Values are a mean of three replicates
17 multiple shoots per explants were obtained.
Repeated subculture did not affect regeneration potential.
A high frequency regeneration protocol was established.
Establishment of in vitro cultures.
“Biotechnology for a better future” Edts: .D’Souza et al (2004) , SAC Publications, Mangalore, India Pages 219-230
Values are a mean of three replicates
Table1: Effect of growth regulators on multiple shoots initiation.
PCR analysis conforming dianthin from in vitro cultures
Fig 6 : Dianthin amplification in in-vitro cultures
Dianthin was observed in 0.25 M and 0.3M Nacl gradient.
Both callus cultures and shoot cultures showed the presence of dianthin
M:Protein molecular weight marker, C: Callus cultures, S:Shoot cultures
Dianthin isolation was carried out by gel filtration (Sephadex g 75) using protein extract of in-vitro cultures was used (5 g of shoots, callus extracted in 50 mM phosphate buffer pH 7)

Protein extract was eluted by using a linear gradient of Nacl (0.05 to 0.3 M) for partial purification of Dianthin. Fractions were further analyzed on SDS PAGE for confirmation.
Extraction and partial purification Dianthin from in-vitro cultures.
Total RNA was isolated from Nicotiana benthemina infected cucumber green mottle mosaic virus and treated with the crude extracts of callus and shoot cultures and RT-PCR was done.
Amplification was observed only in control and not in the treated indicating the RNase activity of the extracts
Lane 1:10kb DNA marker 2: CGMMV RNA
3:RNA treated by callus 4:RNA treated by in-vitro shoots
Rnase Activity
Cucumber Green Mottle Mosaic Virus (CGMM) RNA was obtained by RT-PCR of total RNA from Nicotiana benthemina infected with Cucumber Green Mottle Mosaic Virus with CGMM coat protein AV 77 & BM 85.
Protein extract from the callus and shoots cultures were incubated with total RNA for 30min at 4C and then used for cDNA synthesis.
E: In vivo apical meristem, F: Trichomes, G:In vivo root tip, H:In vivo midvein

Histochemical localization of CPT
A: In vitro apical meristem, B:Std CPT, C: Callus, D: in vitro stem
HPLC column used was RP18. Mobile phase used for the separation of camptothecin was acetonitrile: water (40:60) .Flow rate 1.0ml/min with sample size 20 ul. UV detection at 254 nm.
Micropropagation of Chonemorpha
HPLC profiles
In virto studies and secondary metabolite production in Chonemorpha fragrance (Moon) Alston.
Hairy root initiation and elongation
Separation of CPT by HPTLC
Examination duties performed
Citation index
Ph.D. completed: 5
Ph.D. Working: 8
M. Phil completed: 2
M. Phil working : 1
1. "Effect of polyamines and Gibberellins on flowering in vivo & in vitro in garlic” Funded by DST, New Delhi. (1998)
2. "Transformation and alkaloid production in Solanum khasianum using Agrobacterium rhizogenes induced hairy root cultures. Funded by: CSIR, New Delhi. (2000)
3.”Exploitation of transformed hairy root cultures of Solanum khasianum Clarke as a source for enhanced solasodine production” UGC Minor project, (2003)
4.“Exploring Ruta graveolense cultures for production of furanocoumarines” Funded by UGC, New Delhi. (2006)
5. “ Production of flavonoids by Psoralea cell cultures and somatic embryos” PU-BARC collaboration (2003)
6.“Isolation, purification and biological activity of furanocoumarins from Ruta graveolens” Hermes Chemical Company Pvt. Ltd, Hyderabad (2007-2008)
7.“Genetic transformation and metabolic engineering of Chonemorpha fragrans” UGC New Delhi, (2008-2011)
8.“Exploring Dianthus caryophyllous cultures for ribosome inactivating proteins” BCUD,UOP, (2009-2011)
Research Projects completed:
1. Developed protocol for hairy root cultures in Solanum khasianum, Psoralea corylifolia and for transformed shoots in Ruta

2. Development of micropropagation protocols for Solanum surratense, Solanum khasianum, Solanum indicum, Ruta graveolense, Psoralea corylifola, Dianthus caryophyllus, Chonemorpha fragrans

3. Developed scaling up protocol for
a.solasodine production using hairy root cultures of Solanum khasianum
b.furanocoumarin production in cell and shoot cultures of Ruta graveolens

Patent granted entitled “Process of extraction of solasodine” ref no: 955/MUM/Y/310304 , dt 15.9. 2003

Patent filed entitled” process for enhancing production of furanocoumarines using tailored cell cultures (2500/MUM/2008, dt 28/11/08)
Technology development:
Break through

Ø Isolation and Characterization of Furanocoumarins from in vitro cultures of Ruta graveolens.
Hermes Chemical Company Pvt. Ltd., Secunderabad, Andhra Pradesh, India.

Dr. G.V. Ravishankar, CFTRI, Mysore for use of bioreactors. Trained the research student Asha Jacob for using different types of bioreactors

Dr. D. P. Fulzele, Nuclear Agricultural Division, BARC for Psoralea cell cultures and somatic embryos used in bioreactors for production of Isoflavonoids

Genes for metabolic engineering of phenylpropanoid pathway in Ruta
Dr. F. Bourgaud, INRA, France.
Metabolomics analysis in psoralea Dr. P J Young, Kyung Hee University, S.Korea
1. “Modular Training Course in Bioinformatics for Life Science Teachers”,Organised by:Bioinformatics Centre, University of Pune,Funded by :Department of Information Technology,Ministry of Information and Communications Technology (MCIT),Government of India, September 12--October 5, 2011

2.Participated in the 2nd International Training Program organized by science and technology park, University of Pune in collaboration with Michigan State University, USA and United Nations Asian and Pacific center for transfer of technology, New Delhi in “Intellectual Property Management and Technology Transfer” Science and Technology Park, University of Pune, 19th-25th January,2003
3. Participated in the training program on “Patent Search”, organized by C-DAC and TIFAC, C-DAC, University of Pune, 25th-26th July,2003
4. Participated in International training course on “Plant Cell Culture and Biotechnology” from 30th August’92 – 12th Sept.’92 held in shanghai, China, Sponsored by UNESCO. Travel assistance received from DST, New Delhi.
5. Training in the field of Genetic Transformation. Worked at Institute of Molecular Genetics, Shanghai, China from May – July’94 Recipient of UNESCO-BAC short term fellowship 
Workshops attended
1.Refresher course on "Quantitative methods of analysis in Life sciences" held in the Dept. of Botany, Univ. of Pune, sponsored by UGC and ASC, Univ. of Pune, 5th June-2nd July, 1996.
2.Refresher course on "Information Technology" held at Dept. of Space sciences, Univ. of Pune, sponsored by UGC and ASC, Univ. of Pune, 4th -31st August, 1999.
3.Refresher course on “Plant Biotechnology” held at CPMB, Osmania University, Hyderabad, 1st-20th March, 2004.
Refresher courses completed:
1.Co-ordinator for Refresher course in Quantitative methods in Life Sciences" 5th June-2nd July, 1997, held in Dept. of Botany, Univ. of Pune, Pune
2.Co-ordiated National seminar on "Recent Advances in Cell, Tissue and Embryo Culture", March, 1-3, 1997, at Department of Botany, Univ. of Pune, Pune
3.Co-ordinated orientation course for course BO 201a: Cell Biology, for teachers of postgraduate centers, 12-15th Dec.1996
4.Job training for partial fulfillment of 2nd year (Biotech) B.Sc. student from Ahamednagar College & Modern college, Ganeshkhind, Pune
5.Contributed as resource person for different refresher courses in Botany and Zoology Departments
6.Contributory teacher for Dept. of Zoology and Biotechnology.
Other Activities:
Study of Rnase Activity
Antiviral Activity

Cucumber Green Mottle Mosaic Virus was used for sap inoculation on 2 month old tobacco plants.
For evaluation of antiviral activity plants inoculated with virus were treated with protein extracts of in vivo shoots, in-vitro shoots and callus.
Infection symptoms, like curling of the leaf tips, mosaic appearance on the leaf were seen less when plants were treated with in-vivo plant extracts.
Indian Drugs (2006) 42(12):1001-1003
Phcog. Res. (2010) 2(5): 298-301
Intnl J of Pharma Sci and Res (IJPSR) (2011) 2(10): 2690-2693
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