1. name of the department/section: plant … of the department/section: plant biotechnology centre...

1. Name of the Department/Section: Plant Biotechnology Centre

Salient features: 1. Spacious and well planned building.

2. Well equipped laboratories

3. Trained and devoted teaching staff

4. Opportunity for independent work

2. About Department:

University has established an independent Plant Tissue Culture Laboratory in 1995. The

trained and devoted scientist having background of Biochemistry, Molecular Biology, Genetics

and plant breeding, Genetic Engineering, Tissue Culture and Plant Transformation techniques

were pooled from the various places in the jurisdiction of the university and placed under one

umbrella at central campus Dapoli in 2000. Consequently from the university receipts and the

Swaminathan Foundation Project and Mega seed project some of the major equipments required

for Biotechnology research were purchased and research work initiated. After realizing the

necessity and importance of biotechnology in Agriculture and research work done by the team

of the scientists with limited resources, Government of Maharashtra has kindly extended the

financial support for the construction of an additional building for Biotechnology along with all

well equipments required for the research and education in frontier areas of biotechnology from

the year 2007-08.

3. Academic Programmes:

Masters Programme

Name of the programme: M. Sc. (Ag. Biotechnolgy)

Semester

No.

Term

No.

Course

No.

Credits Title of the course offered by the department

I I MBB 501 2 + 1= 3 Principles of Biotechnology

MBB 502 3 + 0= 3 Fundamentals of Molecular Biology

MBB 503 3 + 0 = 3 Molecular Cell Biology

II II MBB 504 1 + 2 = 3 Plant Tissue culture and Genetic

Transformation

MBB 505 0 + 3 = 3 Techniques in molecular biology I

III I MBB 508 2 + 0 = 2 Genomics and Proteomics

MBB 555 2 +1 = 3 Introduction to Bioinformatics

IV II MBB 591 1 + 0 = 1 Master’s seminar

MBB 599 0 + 20=

20

Master’s research

Course Curricula syllabi:

MBB 501 PRINCIPLES OF BIOTECHNOLOGY 2+1

Theory

UNIT I

History, scope and importance; DNA structure, function and metabolism.

UNIT II

DNA modifying enzymes and vectors; Methods of recombinant DNA technology; Nucleic acid

hybridization; Gene libraries; PCR amplification; Plant and animal cell and tissue culture

techniques and their applications.

UNIT III

Molecular markers and their applications; DNA sequencing; Applications of gene cloning in

basic and applied research; Genetic engineering and transgenics; Genomics, transcriptomics and

proteomics.

UNIT IV

General application of biotechnology in Agriculture, Medicine, Animal husbandry,

Environmental remediation, Energy production and Forensics; Public perception of

biotechnology; Bio-safety and bioethics issues; Intellectual property rights in biotechnology.

Practical

i. Isolation of genomic and plasmid DNA

ii. Gel electrophoresis techniques

iii. Restriction enzyme digestion, ligation, transformation and screening of transformants

iv. PCR and molecular marker analysis

v. Plant tissue culture: media preparation, cell and explant culture, regeneration and

transformation.

MBB 502 FUNDAMENTALS OF MOLECULAR BIOLOGY 3+0

Theory

UNIT I

Historical developments of molecular biology; Nucleic acids as genetic material; Chemistry,

structure and properties of DNA and RNA.

UNIT II

Genome organization in prokaryotes and eukaryotes; Chromatin structure and function; DNA

replication; DNA polymerases, topoisomerases, DNA ligase, etc; Molecular basis of mutations;

DNA repair mechanisms.

UNIT III

Transcription process; RNA processing; Reverse transcriptase; RNA editing; Ribosomes

structure and function; Organization of ribosomal proteins and RNA genes; Genetic code;

Aminoacyl tRNA synthases.

UNIT IV

Translation and post-translational modifications; Operon concept; Attenuation of trp operon;

important features of gene regulation in eukaryotes.

MBB 503 MOLECULAR CELL BIOLOGY 3+0

Theory

UNIT I

General structure and constituents of cell; Similarities and distinction between plant and animal

cells; Cell wall, cell membrane, structure and composition of biomembranes, cell surface related

functions.

UNIT II

Structure and function of major organelles: Nucleus, Chloroplasts, Mitochondria, Ribosomes,

Lysosomes, Peroxisomes, Endoplasmic reticulum, Microbodies, Golgi apparatus, Vacuoles, etc.

UNIT III

Organellar genomes and their manipulation; Ribosomes in relation to cell growth and division;

Cyto-skeletal elements.

UNIT IV

Cell division and regulation of cell cycle; Membrane transport; Transport of water, ion and

biomolecules; Signal transduction mechanisms; Protein targeting.

MBB 504 PLANT TISSUE CULTURE AND GENETIC TRANSFORMATION 1+2

Theory

UNIT I

History of plant cell and tissue culture; Culture media; Various types of culture; callus,

suspension, nurse, root, meristem, etc.; In vitro differentiation: organogenesis and somatic

embryogenesis; Plant growth regulators: mode of action, effects on in vitro culture and

regeneration; Molecular basis of plant organ differentiation.

UNIT II

Micropropagation; Anther and microspore culture; Somaclonal variation; In vitro mutagenesis;

In vitro fertilization; In vitro germplasm conservation; Production of secondary metabolites;

Synthetic seeds.

UNIT III

Embryo rescue and wide hybridization; Protoplast culture and regeneration; Somatic

hybridization: protoplast fusion, cybrids, asymmetric hybrids, etc.

UNIT IV

Methods of plant transformation; Vectors for plant transformation; Genetic and molecular

analyses of transgenics; Target traits and transgenic crops; Biosafety issues, testing of

transgenics, regulatory procedures for commercial approval.

Practical

i. Laboratory set-up.

ii. Preparation of nutrient media; handling and sterilization of plant material; inoculation,

subculturing and plant regeneration.

iii. Anther and pollen culture.

iv. Embryo rescue.

v. Suspension cultures and production of secondary metabolites.

vi. Protoplast isolation, culture and fusion.

vii. Gene cloning and vector construction

viii. Gene transfer using different methods, reporter gene expression, selection of transformed

tissues/plants, molecular analysis.

MBB 505 TECHNIQUES IN MOLECULAR BIOLOGY-I 0+3

Practical

UNIT I

Good lab practices; Biochemical techniques: Preparation of buffers and reagents, Principle of

centrifugation, Chromatographic techniques (TLC, Gel Filtration Chromatography, Ion

exchange Chromatography, Affinity Chromatography).

UNIT II

Gel electrophoresis- agarose and PAGE (nucleic acids and proteins); Growth of bacterial culture

and preparation of growth curve; Isolation of plasmid DNA from bacteria; Growth of lambda

phage and isolation of phage DNA; Restriction digestion of plasmid and phage DNA; Isolation

of

high molecular weight DNA and analysis.

UNIT III

Gene cloning – Recombinant DNA construction, transformation and selection of transformants;

PCR and optimization of factors affecting PCR.

UNIT IV

Dot blot analysis; Southern hybridization; Northern hybridization; Western blotting and ELISA;

Radiation safety and non-radio isotopic procedure.

MBB 508 GENOMICS AND PROTEOMICS 2+0

Objective

To familiarize the students with recent tools used for genome analysis and their applications.

Theory

UNIT I

Structural genomics: Classical ways of genome analysis, large fragment genomic libraries;

Physical mapping of genomes; Genome sequencing, sequence assembly and annotation;

Comparative genomics, etc.

UNIT II

Functional genomics: DNA chips and their use in transcriptome analysis; Mutants and RNAi in

functional genomics; Metabolomics and ionomics for elucidating metabolic pathways, etc.

UNIT III

Proteomics - Protein structure, function and purification; Introduction to basic proteomics

technology; Bio-informatics in proteomics; Proteome analysis, etc.

UNIT IV

Applications of genomics and proteomics in agriculture, human health and industry.

MBB 555 INTRODUCTION TO BIOINFORMATICS 2+1

Theory

UNIT I

Introduction, biological databases – primary, secondary and structural, Protein and Gene

Information Resources – PIR, SWISSPROT, PDB, genebank, DDBJ. Specialized genomic

resources.

UNIT II

DNA sequence analysis, cDNA libraries and EST, EST analysis, pairwise alignment techniques,

database searching, multiple sequence alignment.

UNIT III

Secondary database searching, building search protocol, computer aided drug design – basic

principles, docking, QSAR.

UNIT IV

Analysis packages – commercial databases and packages, GPL software for Bioinformatics,

web-based analysis tools.

Practical

i. Usage of NCBI resources

ii. Retrival of sequence/structure from databases

iii. Visualization of structures

iv. Docking of ligand receptors

v. BLAST exercises.

4. Infrastructure:

a. Laboratories: Art-of-state laboratory facilities are created by the department. Independent

laboratories for Molecular biology, plant tissue culture, genetic engineering, media preparation

and sterilization are available. Each lab is spacious and 20 students can work at a time.

b. Name of the important instruments/facilities:

Sr.

No.

Name of the equipment Sr.

No.

Name of the equipment

1 Horizontal gel electrophoresis 14 Refrigerators

2 Vertical gel electrophoresis 15 Particle gun

3 Thermal cyclers 16 Rotary incubator shaker

4 Laminar Flow cabinet 17 Oven

5 Gel documentation unit 18 Incubators

6 Hybridization chamber 19 Rotary shaker

7 High speed refrigerator centrifuge (Table top

model)

20 Culture racks

8 Weighing balance 21 Autoclaves

9 Refrigerated Micro centrifuge 22 Quartz double distillation unit

10 ELISA reader and washer 23 Ice flaking machine

11 UV visible spectrophotometer 24 Milli Q water purification system

12 Tube Rotator 25 Thermostatic water bath

13 -200C, -400C and -860C deep freeze 26 Green house type III

Teachings Aids

1. LCD 3. Over head projector

2. Computers

c. Activities:

1. Micropropogation technique of commercially important horticulture crop like banana Cv.

Grand naine and Safed velchi.

2. Micropropogation technique of endangered forest species.

3. Creation of variability in small millets through callus culture.

4. Marker Assisted Selection (MAS) in rice for developing resistance against biotic and abiotic

stress.

5. Development of transgenic in pulses for pod borer resistance.

6. Finger printing of released varieties university through molecular markers.

d. photographs:

5. Faculty

a. Academic staff: Assistant Professor and above with the details of the staff as given

below

Name of the Faculty Dr. Nitin Bhaskar Gokhale

Post Held In-Charge

Date of Birth 28/6/1960

Qualification M. Sc. (Biochemistry) Ph. D. (Agri.)

Area of Specialization Molecular Biology

Experience (Years) 31 years

Recent Photograph

Research Projects guided

Ph. D. 3

M. Sc. 9

Present area of research Biochemistry and Molecular Biology

Contact details

Land line No. 02358-282415(O)

Mobile 9422534721

Fax. 02358-282108

Email [email protected]

Name of the Faculty Dr. Santosh Vishnu Sawardekar

Post Held Associate Professor


Qualification M. Sc. (Agri.) Ph. D. (Genetics and Plant

Breeding)

Area of Specialization Genetic Engineering


Research Projects guided

M. Sc.

6

Present area of research Genetic Engineering and Molecular Biology

Contact details

Land line No. 02358-282415(O)

Mobile 9420376668

Fax. 02358-282108

Recent

Photograph

Email [email protected]

b. Research staff: The name of the research staff member like SRA and JRA.

Name of the Faculty Mrs. Sangita Sanjay Sawant

Post Held Junior Research Assistant


Qualification M. Sc. (Agri.) with NET (Genetic and Plant

Breeding)

Area of Specialization Genetic and Plant Breeding and Tissue culture


Present area of research Tissue culture

Recent Photograph

Contact details

Land line No. 02358-282415(O)

Mobile 9422379759

Fax. 02358-282108

Email -

6. Instructional Farm

Availing the facilities available with Dr. B. S. Konkan Krishi Vidyapeeth, Dapoli.

7. Research Activities and Achievements (including projects)

a. Variety/Implements released: -

b. Research Recommendations: -

c. Research Outcome/Findings:

1. Standardized the micro propagation technique in banana and medicinal plants.

2. Sex determination in Kokum (Garcinia indica) through molecular markers.

3. Sex determination in Nutmeg. (Myristica fragranns Houff) through molecular

markers.

4. Developed in vitro somaclones in Nagli (Eleusina coracana L.) and analysed

variation through RAPD.

5. Standardized in vitro Regeneration protocol in Pigeon pea.

6. Developed genetic transformation protocol in pigeon pea and dolichos bean.

7. Marker Assisted Selection in rice for biotic and abiotic stress.

8. Finger printing of released rice varieties of DBSKKV, Dapoli.

d. Completed Research Projects/ Programmes/ Schemes

1. Title: Standardization of tissue culture technique in cashew nut

UR No.: -

Objectives:

1. To optimise the culture media and culture conditions for the in vitro propagation of high

yielding hybrids of cashew (Vengurla-3, 4, 5, 6) via enhanced development of axillary

buds.

2. To optimise the culture media and culture conditions for the callus culture and somatic

embryo development of high yielding hybrids of cashew (Vengurla- 3, 4, 5, 6).

3. To study the influence of different with yielding cashew (Vengurla- 3, 4, 5, 6) on the in

vitro propagation and rapid multiplication.

4. To develop the technique for planting out for the maximum survival and establishment of

high yielding cashew hybrids (Vengurla- 3, 4, 5, 6).

5. To develop a protocol for large scale micropropagation, clonal multiplication and to

study the economic aspects of tissue culture technique for high yielding cashew hybrids.

Name of PI:- Dr. B. L. Lad

Co-PI:- Dr. M. B. Magdum

Sponsoring Agency: DBT, New Delhi.

Duration: 3 years

Total Outlay: 7 lakhs

Summary of Achievements: Soaking of Nodal segments of cashew seedlings and grafts

(V4) in the mixture of 500 ppm cetrimide + 100 ppm Bactrinol + 100 ppm citric acid solution

with 2.3 drops of teepol for 60 minutes followed by HgCl2 (0.1%) solution treatment for 5

minutes was best for asceptic culture establishment in vitro the medium . The medium MS (½

N) + 10 ppm BA + 5 ppm NAA + 5 ppm GA3 + 0.05 % charcoal and gelled with 0.9% agar agar

was suitable for axillary bud growth of cashew in vitro. The suitable size of explants for in vitro

culture of cashew was 20 mm. The medium MS (½ N) + 10 ppm BA + 5 ppm NAA + 5 ppm

GA3 + 0.05 % charcoal gelled with 0.9% agar agar was suitable for obtaining 2.3 shoots by

using cotyledonary sprouts of cashew seedlings in vitro. Single root was developed in vitro by

using asceptically grown cashew explants (of seedlings) using lioyd and McCown’s medium

supplemented with 2.5 ppm BA + 5 ppm NAA + 5 ppm GA3 + 0.05 % charcoal with 0.9 % agar

agar. Initial one week incubation in dark followed by light could induce roots within 2.3 weeks.

2. Title: Strengthening of tissue culture laboratory

UR No.: -

Objectives:-

1. Propagation by tissue culture in horticultural crops.

2. Varietal improvement through tissue culture in field crops.

Name of PI:- Dr. B. L. Dhonukshe

Co-PI:- Dr. B. L. Lad

Sponsoring Agency: Govt. of Maharashtra

Duration: 3 years


Summary of Achievements: On research front, the project helped to a great extent in

pursuing tissue culture, clonal multiplication, somaclonal variation and somatic embryogenesis

studies in the different crops. As many as ten crops viz. Cashew, Mango, Banana, Kokum,

Jackfruit, Orchids, Bamboo, Ragi, Cowpea and Field bean were studies. The significant

achievements include standardization of protocols for Jackfruit, Banana, Ragi and Orchids.

Preliminary success was achieved in somatic embryogenesis in mango and shoot development in

Kokum. The tissue cultured banana plantlets have reached the stage of farm trials. Five students

completed their M. Sc. Degree on tissue culture based research. The Plant Biotechnology Unit

now stands poised to deliver impact making results of research being pursued.

Title: Establishment of tissue culture in public places


Title: Embryo rescue techniques in mango


Title: Standardization of tissue culture techniques in kokum

UR No.: - F16(10)/98 Hort-I, dated 2/9/99

Objectives:

1. To asses the totipotency potential of elite strains of Kokum.

2. To evaluate the regenerative response using different explants of various types, sizes and

ages.

3. To establish the aseptic cultures of above explants for proliferation.

4. To standardize the media conditions and growth regulator combinations for in vitro

organogenesis of proliferated cultures.

5. Commercial scaling of micropropagation technique for mass multiplication of Kokum.

Name of PI: Dr. B. L. Dhonukshe

Sponsoring Agency: ICAR, New Delhi

Duration: 3 years


Summary of Achievements: The scheme has really helped in boosting our efforts on

standardization for tissue culture technique for Kokum. In the first year of the scheme, we have

succeeded in standardizing surface sterilizing treatments, explants and media for shoot

regeneration. We have succeeded in regenerating shoots in as many as 17 elite genotypes of

Kokum maintained by Dr. B. S. Konkan Krishi Vidyapeeth, Dapoli and now, our efforts are on

to induce rooting.

Title: Plant health Diagnosis


Title: Seed production in agril crops and fisheries

Total Outlay: 36.90 lakhs

Title: Swaminathan committee’s Recommendation project

UR No.: -

Objectives:

1. Strengthening of research and education work on genetic engineering and molecular

biology.

2. Utilization developed technique for crop improvement.

Name of PI:- Dr. N. B. Gokhale

Co-PI:- Dr. S. V. Sawardekar

Sponsoring Agency: State Government of Maharashtra

Duration: 5 years


Summary of Achievements: During the 5 years of project infrastructure facilities

required for development research and education on Genetic Engineering and molecular biology

has been developed. Genetic transformation protocol has been standardized in pulses like tur and

dolichos bean. Through molecular biology technique finger printing of released rice varieties has

been done. Similarly, through marker assisted selection screening has been done for blast, BLB

and BPH through SSR markers.

e. Ongoing Research Projects/Programmes/Schemes:

Title: Use of irradiation technique for creation of variability in finger millet and assessment of

mutants through molecular marker

UR No.: No. 2011/35/11/BRNS/1452/20 Sep. 2011

Objectives: 1. Irradiation of promising genotypes of finger millet.

2. Assessment of genetic variability of mutants through molecular markers.

3. To develop blast resistant mutant in finger millet.

Name of PI: Dr. N. B. Gokhale

Co-PI: Dr. S. V. Sawardekar

Sponsoring Agency: Government of India, Department of Atomic Energy (DAE), Board of

Research in Nuclear Sciences (BRNS)

Duration: 3 years

Total Outlay: Rs. 1766350/-

8. Repository of abstracts of the theses:

Name of the candidate: S. N. SABALE

Degree for which the thesis/project report submitted: M. Sc. ( Ag. Biotechnology )

Year of submission: 2012

Name of the Guide/ Co guide: Dr. N. B. GOKHALE

Abstract:

Elimination of bacterial contamination by using antibiotics in micropropagtion of

banana (musa spp.) Cv. Grand naine.

Abstract

The investigation entitled, “Elimination of bacterial contamination by use of antibiotics

in micropropagtion of banana (Musa spp.) Cv. Grand naine” was undertaken in Completely

Randomized Design with 3 replications. This experiment was carried out to eliminate the

bacterial growth by using the antibiotics in culture media during the micropropagation of banana

Cv. Grand naine. The four antibiotics namely, Augmentin, Amoxicillin, Rifampicin, Cefotaxime

were tested for its efficiency to control the bacterial contamination occurring during

micropropagation.

The media combination MS + 6.5 mgl-1

BAP + 1000 ppm Amoxicillin shown maximum

elimination of bacterial contamination at 15 DAI. While, the media combination MS + 5 mgl-1

BAP + 2 mgl-1

IAA + 1000 ppm Amoxicillin shown maximum elimination of bacterial

contamination at 30 DAI. Highest percent of explants establishment was on MS + 6.5 mgl-1

BAP

+ 1000 ppm Amoxicillin and highest percent of shoot initiation was on MS + 5 mgl-1 BAP + 2

mgl-1 IAA + 1000 ppm Amoxicillin. The minimum number of days to shooting was recorded

24.75 days on the media combination MS + 5 mgl-1

BAP + 2 mgl-1

IAA + 1000 ppm

Cefotaxime and the maximum number of shoots was recorded 6.24 shoots per explant in the

media combination MS + 5 mgl-1

BAP + 2 mgl-1

IAA + 750 ppm Amoxicillin at 30 DAI.

The media combination supplemented with 1000 ppm Amoxicillin was effective in

elimination of maximum bacterial contamination without any phytotoxic effect on explants at 30

DAI, saving maximum cultures during establishment and proliferation ultimately increasing the

plantlets during micropropagation.

Name of the candidate: K.A. Lipne

Degree for which the thesis/project report submitted: M. Sc. ( Ag. Biotechnology)


Name of the Guide/ Co guide: Dr. S.G. Bhave

Abstract:

Genetic transformation of dolichos bean (lablab purpureus (l.) Sweet) for pod borer

resistance

ABSTRACT

The present investigation was aimed to standardize regeneration and Agrobacterium-

mediated genetic transformation protocols in popular dolichos bean variety Konkan bhushan.

Different explants viz, mature embryo axes with single cotyledon, mature embryo axes and

shoot tip were cultured on MS basal medium supplemented with different levels of BAP (2 to 6

mg/l), NAA (0.5 mg/l) and Kinetin (2 to 6 mg/l) for multiple shoot induction. Frequency of

multiple shoot induction was significantly more in MEASC explants than in MEA and shoot tip.

BAP at 2 mg/l and NAA at 0.5 mg/l induced shooting in most explants and recorded highest

number of multiple shoots. Addition of 0.5 mg/l kinetin and 0.5 mg/l BAP in MS medium

resulted in higher frequency of shoot elongation. The elongated shoots were rooted on MS

medium supplemented with 0.1 mg/l NAA.

MEASC explants were co-cultivated with Agrobacterium strain EHA 105 carrying

pBinBt3 construct having cryIIAa gene. The result of the present study indicate that highest

transformation efficiency (0.952) could be achieved by 15 min colonization and 48 hour

cocultivation followed by washing of explants in 400 mg/l cefotaxime. The genetic engineering

approaches have potential of greatly enhancing the productivity of dolichos bean by increasing

the resistance against pod borer. To be of value the transgenic plants must efficiently express

transgene. However, the present study showed that Agrobaterium-mediated transformation is a

possible approach to develop transgenic plant in dolichos bean.

Name of the candidate: V. D. Padwale

Degree for which the thesis/project report submitted: M. Sc. (Ag. Biotechnology)


Name of the Guide/ Co guide: Dr. N. B. Gokhale

Abstract:

Assessment of genetic variation in Mango (Mangifera indica L.) Cv. Alphonso by using

RAPD markers

ABSTRACT

Assessment of genetic variation in Mango (Mangifera indica L.) Cv. Alphonso is

fundamental for the conservation of genetic resources and utilization in breeding programme.

The objective of this study was to assess the genetic variation in mango (Cv. Alphonso) at

various locations in Konkan region.

RAPD profile for all Mango plants of various location (Cv. Alphonso) were generated

with 38 random decamer primers. Out of 38 primers screened 10 primers gave scorable DNA

fragments and each of the 10 random primers revealed polymorphism. These primers generated

201 DNA fragments in the average range of 348.3 bp to 812.2 bp, of which 140 were

polymorphic. The average level of polymorphism generated by the primers was high (67.37%).

The primers OPF-20, OPM-12, and OPU-08 produced distinct RAPD patterns (100%

polymorphism) for all the Mango plants. The average discrimination power among the 10

primers was 52.5 per cent.

The overall range of the similarity among all Mango samples was found to be very wide,

ranging from 0.086 to 0.571 which indicates there was high variability among the Alphonso

cultivars under study. The cluster analysis based on RAPD data showed that Mango samples

formed two main groups. The 900 Alphonso cultivars occupied a unique position and was most

diverse from rest of the Alphonso cultivars. The study indicated that RAPD markers are suitable

for the assessment of genetic diversity among Mango samples and identification of diverse

sources in crop germplasm collection.

Name of the candidate: D. M. Patil

Degree for which the thesis/project report submitted: M. Sc. (Ag. Biotechnology)


Name of the Guide/ Co guide: Dr. S. V. Sawardekar

Abstract:

Genetic diversity analysis in cowpea (vigna unguiculata (l.) Walp.) By using RAPD

markers

ABSTRACT

Assessment of genetic variability within Vigna unguiculata (L.) Walp. is fundamental for

the conservation of genetic resources and its utilization in hybridization programme. The

objective of this study was to estimate the genetic diversity among 30 genotypes of cowpea

through molecular characterization by using RAPD markers.

RAPD profiles for all 30 genotypes were generated with 20 random decamer primers.

Out of 20 primers screened 17 primers gave scorable DNA fragments and each of the 17 random

primers revealed polymorphism. The primers generated 1238 DNA fragments in the average

range of 381.94 bp to 1131.71 bp, of which 908 were polymorphic. The level of polymorphism

generated by the primers was high (71.20%). The primers OPA-04, OPA-05, OPC-02, OPC-05

and OPC-08 produced distinct RAPD patterns (100% polymorphism) for all the 30 genotypes.

These primers can be used for identification of the genotypes studied. The average

discrimination power among the 17 primers was 62 per cent.

The overall range of the similarity among 30 genotypes was found to be very wide,

ranging from 0.321 to 0.800 which indicates there was high variability among the cowpea

cultivars under study. The cluster analysis based on RAPD data showed that genotypes formed

two main groups. The genotype PCP-97223 occupied a unique position and was most diverse

from rest of the genotypes. The study indicated that RAPD markers are suitable for the

assessment of genetic diversity among group of genotypes and identification of diverse sources

in crop germplasm collection. This study could identify the diverse genotype like DCP-11 and

Pusa Phalguni for their use in hybridization programme of cowpea.

Name of the candidate: P. C. Ingale

Degree for which the thesis/project report submitted: M. Sc. ( Ag. Biotechnology )


Name of the Guide/ Co guide: Dr. N. B. Gokhale

Abstract

Sex determination in nutmeg (myristica fragarns houtt.) By using RAPD markers

ABSTRACT

Determination of sex in nutmeg is of utmost importance from the commercial

agricultural point of view, since the sexuality cannot be distinguished prior to flower initiation.

The use of bio-molecular techniques in sex determination of nutmeg presents a potential

theoretical significance and economic value. For this analysis quality DNA is a prerequisite.

Since this crop is having lot of phenolic compounds, optimization of DNA isolation protocol and

use of RAPD markers to determine sex of nutmeg has been used in the present investigation.

The DNA was extracted from young leaves of nutmeg from 5 male and 5 female plants.

The standardization of buffer constituents for DNA isolation was carried out. In Rapid method

five components of the extraction buffer were standardized and in CTAB method three

concentrations of CTAB powder were used. The results obtained using 0.900g glucose, 0.100g

PVP,0.040g Sodium bisulphate, 0.050g Sodium Lauryl Sulphate, 500µl Sarcosine in Rapid

method and 2% CTAB powder in CTAB method of DNA isolation are encouraging.

The most suitable combination of 100 mM Tris–HCl pH 8.0, 50 mM EDTA, 1 M NaCl

as standardized buffer constituents showed clear and specific banding pattern. Modifications in

extraction procedure indicated that the decrease in sample size, use of PVP, 0.3%

mercaptoethanol, twice the volume isopropanol and washing with 70% ethanol produced better

and clear bands when subjected to PCR.

Modifications carried out in PCR analysis showed that the most favorable conditions of

dNTPs 1µM (1µl) concentration showed clear bands, Taq polymerase 0.5 units (1µl) proved

better than others in proper band formation and annealing temperature 370C for 45 sec yielded

good results.

The nutmeg DNA showed a poor amplification with RAPD primers studied. Of the total

60 decamer primers used in the investigation, only 14 primers showed amplification. Primers

OPA-14, OPA-15, OPQ-16 and OPQ-04 showed polymorphism which can be used to

differentiate male and female plants. A large number of RAPD primers failed to amplify.

However, the information generated by few polymorphic primers was able to differentiate in

between male and female plants. Such information will help the farmers to identify the sex of

the saplings at an early stage and avoid their economic loss. Thus widening the probability ratio

of male to female plants 1:9 as compared to present ration of 1:1 increasing more females,

thereby increasing the yield of nutmeg almost two folds is possible.

9. Extension Activities

a. The training programmes organized : Nil

b. Seminar/Symposia/Conference/workshop Organized : Nil

c. Farmer Melawa Organized : Nil

d. Radio/ TV Talks delivered by the staff members of the Department/Section:

Sr.

No.

Name of the

person

Topic Where Date of

delivered

1. Dr. N. B. Gokhale Jaiwatantradnyanadware

Poshan Muley Vrudhi

All India Radio,

Ratnagiri

2006

2. Jaiwatantradnyanadware

Jywik, Ajywik Wanachi

Nirmitte

All India Radio,

Ratnagiri

2006


Kadhani Pachayat

tantranancha Vikas.

All India Radio,

Ratnagiri

2007


kadhani paschhat

tantradnyanachi vikas warta.

All India Radio,

Ratnagiri

2009

5. Jaiwatantradnyan ani

krishividyappethach yogdan

Bhetwarta

All India Radio,

Ratnagiri

2010

6. B.T. Tantradnyan- samaj-

Gairsamaj

All India Radio,

Ratnagiri

2011

7. Haritagruhatil Bhagipala

Lagavad

All India Radio,

Ratnagiri

2011

8. Dr. S. V.

Sawardekar

Kharif Bhendichi lagwad All India Radio,

Ratnagiri

1997

9. Khar Jaminit Bhatachi

Lagwad

All India Radio,

Mumbai

2002

10. Khar Jaminit Bhagipala pike All India Radio,

Mumbai

2002

11. Paryavaran Sanrkshansathi

Jaivatantradnyan

All India Radio,

Ratnagiri

2008

12. Bhajipala Utpadanat Bt

Tantradnyan

All India Radio,

Ratnagiri

2009

13. Tissue culture Keli bagechi All India Radio, 2010

niga Ratnagiri

14. Jaivatantradwara falbag

vikas

All India Radio,

Ratnagiri

2011

15. Jaivatantradnyantil pragati

ani samsya

All India Radio,

Ratnagiri

2012

16. Mrs. S. S. Sawant Deliver the Radio talk on

Bhatachi Navin Jat

Phondaghat

All India Radio,

Ratnagiri

1998

17. Delived the Radio talk on

‘Jaivatantradnyan ani Krushi

Vikas’

All India Radio,

Ratnagiri

2006


‘Uti savardhanatun Aushadhi

Vanaspati Vikas’

All India Radio,

Ratnagiri

2008


‘Jaivatantradnyanatil Pragati

Ani Samasya’

All India Radio,

Ratnagiri

2010

e. Farmer-Scientist Forum:- Nil

f. Other Extension Activities:- Nil

g. Publications:

Journal Research Papers

1. Shirsat N.D.; H.R. Nadkarni; S.G. Bhave; N.B. Gokhale and S.S. Sawant (2008). Clonal

propagation of Ginger var. Varada through tissue culture. J. Maharashtra agric. Univ.

33(2):178-180.

2. Bhuwad A.M.; A.D. Rangawala; H.R. Nadkarni; S.G. Bhave and S.S. Sawant (2007). In

vitro study in orchid (Dendorbium moschatum) J. Ann. of Plant Physiol. 21 (2) :240-243.

3. Kharat S.G.; S.G. Bhave; H.R. Nadkarni; S.S. Sawant and V.W. Bendale (2008).

Micropropagation of Lesser yam (Dioscorea esculenta (Lour) Burk) Journal of Root

crop 34(1):65-69.

4. Patil S.M., S.V. Sawardekar, S.G. Bhave, S.S. Sawant, N.D. Jambhale and N.B. Gokhale

(2009). Development of somaclones and their genetic diversity analysis through RAPD

in finger millet (Eleusine coracana L. Gaertn.). Indian J. Genet, 69(2): 132-139.

5. Warang S.S., B.L. Dhonushe, H.R. Nadkarni, S.S. Sawant and N.B. Gokhale (2010).

Genotypic variability in tissue cultured plantlets of banana under greenhouse condition J.

Maharashtra agric. Univ., 35(2): 315-316.

6. Warang S.S., H.R. Nadkarni, S.S. Sawant and N.B. Gokhale (2010). In vitro

multiplication of elite cultivar of banana through shoot tip culture. J. Maharashtra agric.

Univ., 35(1): 69-71.

7. Belanekar S.B., H.R. Nadkarni, S.S. Sawant and N.B. Gokhale (2010). Micropropagation

in gladiolus (Gladiolus grandiflorus L.) var. white friendiship. J. Maharashtra agric.

Univ. 35(1):66-68.

8. Bhave S.G., S.M. Patil, S.V. Sawardekar, S.S. Sawant and N.B. Gokhale (2010).

Variability studies for weight of callus in finger millet (Eleusine coracana L.) J.

Maharashtra agric. Univ. 35(1):72-76.

9. Bhave, S.G., S.S. Dapke, S.S. Sawant, N.B. Gokhale and S.N. Joshi (2010). In vitro

propagation of malkangni (Celastrus paniculatus Wild) a rare endangered medicinal

species. J. Maharashtra agric. Univ. 35(1):49-52.

10. Bhave S.G., S.S. Dapake, S.S. Sawant, N.B. Gokhale and S.N. Joshi (2010). Rapid in

vitro multiplication and restoration for malkangi (Celastrus paniculatus willd.) through

direct organogenesis. Timber forest products Vol. 17(4): 427-431.

11. Sawardekar S.V., N.B. Gokhale, M.S. Mote, S.N. Joshi and S.S. Sawant (2011). Sex

detection of kokum (Garcinia indica Choisy) by RAPD markers. Indian J. Gent., 71(1):

82-83.

12. Sawardekar S.V., S.N. Sarode, S.S. Sawant, S.G. Bhave and N. B. Gokhale (2011).

Efficient in vitro regeneration of pigeonpea (Cajanus cajan (L.) millsp.) through somatic

embryogenesis for genetic transformation. J. Arid Legumes 8(1): 58-64.

13. Sarode S.N., S.V. Sawardekar, S.S. Sawant, S.G. Bhave and N. B. Gokhale (2012).

Assessment of nature and weight of callus in diverse genotypes of pigeonpea [Cajanus

cajan (L.) millsp.] J. Agric. Res. Technol. 36(1):161-163.

14. Gokhale N. B., S.S. Sawant, S.V. Sawardekar and S.N. Joshi (2011). Genetic

engineering for salt tolerance. J. Indian Soc. Coastal Agric. Res. 29(1):78-81.

15. Sawardekar S. V. And I. S. Katageri (2011). Regeneration from hypocotyls derived

callus of chickpea (Cicer arietinum L.). J. Agric. Res. Technol., 36(2):212-217.

16. Sawardekar S. V., Mhatre N. K., Sawant S. S., Bhave S.G., Gokhale N. B., Narangalkar

A. L., Katageri I. S. And Anandkumar P. (2012). Agrobactrium mediated genetic

transformation of pigeonpea (Cajanus cajan L.) Nod borer resistance: Optimization of

protocol. Indian J. Genet. 72(3):380-383.

17. Mhatre N. K., Sawardekar S. V., Bhave S.G., Gokhale N. B., Sawant S. S. And Devmore

J.P. (2012). In vitro regeneration technique in pigeonpea (Cajanus cajan L. Milli sp.) Cv.

Konkan Tur-1, through direct organogenesis. Int. Journal of Biotechnology and

Biosciences 2(2): 163-166.

18. Sawardekar S. V., V. K. Jagdale, Bhave S. G., Gokhale N. B., Sawardekar S. V., Lipne

K. A. (2013). Genotypic difference for callus induction and plantlet regeneration in

cowpea [Vigna ungniculata (L.) WALP]. International Journal of Applied Biosciences

1(1): 1-8.

19. Patil D. M., Sawardekar S. V., Gokhale N. B., Bhave S. G., Sawant S. S., Sawantdesai S.

A., Lipne K. A., Sable S. N. And Joshi S. N. (2013). Genetic diversity analysis in

cowpea [Vigna ungniculata (L.) WALP] by using RAPD markers. International Journal

of Innovative Biotechnology and Biochemistry 1(1): 15-23.

10. Details of other activities (for e.g. seed production, production of other commodities

etc.) -Nil.

11. Contact Information

Name of the Head: Dr. N. B. Gokhale

Name of the Department:- Plant Biotechnology Centre

Postal Address:- College of Agriculture, Dapoli, Dr. B. S. Konkan Krishi Vidyapeeth,

Dapoli Dist. Ratnagiri – 415 712

Landline Number:- (02358)-282415

Mobile Number:- 9422534721

Fax:- (02358)-282108

Email:- [email protected]

12. News and Events:- -Nil.

1. name of the department/section: plant … of the department/section: plant biotechnology centre...

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