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Md Abul Barkat Dr. Mohd.MujeebM.Pharm. 3 rd sem.(2010-11) D/o Pharmacognosy and PhytochemistryD/o Pharmacognosy and Phytochemistry Faculty of PharmacyFaculty of Pharmacy Jamia HamdardJamia Hamdard

Presented byUnder the Supervision of


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Contents

I ntroduction

Aims and objectivePlan of work:Targets achievedTargets to be achieved

Rationale of the work References

Catharanthus roseus var.alba


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I ntroduction

The plant tissue culture is refers to techniqueof growing plant cells, tissues, organs, seeds or

other plant parts in a sterile environment on anutrient medium.

In the 1950s and 60s there was a great deal of research, but it was only after the developmentof a reliable artificial medium (Murashige &Skoog, 1962) that plant tissue culture reallytook off commercially


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T issue culture offers numerous significant benefits over traditional propagation methods

Propagation can be much more rapid than bytraditional means.It may be possible in vitro to multiply plantsthat are very difficult to propagate by cuttings

or other traditional methods.Large numbers of genetically identical clonesmay be produced.Seeds can be germinated with no risk of damping off/predation.


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U nder certain conditions, plant material can be stored in vitrofor considerable periods of time with little or no maintenance.Tissue culture techniques are used for virus eradication,genetic manipulation, somatic hybridization and other

procedures that benefit propagation, plant improvement, and basic research.

Tissue culture is an essential part of many genetictransformation protocols.


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A ims and Objectives

Selection of suitable culture conditions to initiate, developmentand maintenance of callus culture of the proposed plant.Development of the biomass contains higher concentration of secondary metabolites.Phytochemical analysis of the developed biomass.Optimization of the culture parameters for the enhancement of the secondary metabolites in cultures.


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Development and evaluation of formulation of the proposed plant.Phytochemical screening to confirm the presence or absence of phytopharmaceuticals.Pharmacological evaluation of callus culture with specificreferences to antioxidant and anti-diabetic activity.


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A nalytical work

Phytochemical screening to confirm the presence or absenceof the phytopharmaceuticals.Quantification of total phenolic and flavonoid contents.HPTLC fingerprinting profile of natural plant parts and their cultures.Quantification of secondary metabolite by the use of HPTLC/HPLC technique .

P harmacological work Anti-oxidant activityAnti-diabetic activity


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P rocurement and identification of plant materialThe plant material was collected from the herbal garden of Jamia Hamdard,and authenticated by Taxonomist, Department of Botany, faculty of Science,Jamia Hamdard, New Delhi-62

Surface sterilization of explant

S. No Sterilizing agent Conc. (%) Contact time (min.) Observation

1 Sodium hypochlorite

sol.

3 10 +

2 Sodium hypochlorite

sol.

4 8 +++

Observation : 4% Sodium hypochlorite sol.treatment for 7-10 min. showed healthy growthwithout contamination.

T argets A chieved


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Preparation of Culture mediaMurashige & Skoogs (MS) medium was selected as the optimal culturemedium for initiation and development of static culture from the leaf andseeds of Catharanthus roseus var.alba which was supplemented with thevarious combinations of phytohormones for the induction of calluscultures.

Abb reviations used further 2,4-D = 2,4-Dichlorophenoxyacetic acid

NAA= Naphthalen acetic acidIBA= Indolebutyric acidIAA= Indoleacetic acid

6-BA= Benzyladenine andK= Kinetin


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MS + HormonalCombination

(ppm)

Callus initiation Time (days) Observation Figure

MS + 6-BA(1ppm)

+++ 40 Initiation of callusfrom leaf

MS +NAA(1ppm)

++ 45 Initiation of callusfrom leaf

() = No initiatin, (+) = initiation, (++) = Good initiation and (+++) = better initiation


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T argets to be A chieved

Tissue culture studiesGrowth kinetic studiesDevelopment and maintenance of leaf and seed culture.Optimization of culture parameters for the enhancement of ajmalicinein callus culture.Development and evaluation of formulation of the proposed plant.

Analytical work Phytochemical screening to confirm the presence/absence of

phytopharmaceuticals.Quantification of total phenolic and flavonoid contents.HPTLC fingerprinting profile of natural plant parts and their cultures.Quantification of ajmalicine in natural plant parts and its cultures.

P harmacological work I n vitro antioxidant activity.Anti-diabetic activity.


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R ationale of the work

The natural source of ajmalicine is very few plant specieslike Vinca and Rauwolfia which contain low concentration.Due to the high Global demand of this compound, newmethods are needed to enhance its production .One of the strategies to enhance metabolite productionusing suspension cultured cells in which we use likeelicitors, precursor, adsorbent and immobilization or

biotransformation technique.


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I n the present investigation, an attempt will be made tooptimise the culture conditions by L.Almagro, A.J.LopezPerez and M.A.Pedreno response to using plant cell culture

based on the use of cyclodextrine for the enhancement of ajmalicine content in leaf callus of Catharanthus roseus .

W e will also develop novel methods to increase yields of ajmalicine in the callus of the white vinca . No such type of systemic tissue culture profile of this plant has beenestablished by any research worker and agencies.


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R eferences

Barcelo, P., Rasco-Gaunt, S., Thorpe, C. and Lazzeri, P. (2001).Transformation and gene expression. In Adv ances in botanical research , volume 34: Biotechnology of cereals (ed. P. R. Shewry, P.A. Lazzeri and K. J. Edwards), pp. 59126. Academic Press, London.

Dahleen, L. S. (1999). Donor-plant environment effects onregeneration from barley embryo-derived callus. Crop Science , 39 ,6825.

Dahleen, L. S. and Bregitzer, P. (2002). An improved media systemfor high regeneration rates from barley immature embryo-derivedcallus cultures of commercial cultivars. Crop Science , 42 , 9348.


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Dodds, J. H. and Roberts, L. W . (1995). Ex periments in plant tissueculture . Cambridge U niversity Press, Cambridge.Gamborg, O. L. (2002). Plant tissue culture. Biotechnology.Milestones. I n v itro Cellular an d D evelopmental BiologyPlant ,38 , 8492.Ramage, C. M. and W illiams, R. R. (2002). Mineral nutrition and

plant morphogenesis. I n v itro Cellular an d D evelopmental BiologyPlant , 38 , 11624.Torbert, K. A., Rines, H. W ., Kaeppler, H. F., Menon, G. K. and Somers, D.A. (1998). Genetically engineering elite oat cultivars. Crop Science , 38 ,16857.


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W alden, R. and W ingender, R. (1995). Gene-transfer and plant-regeneration techniques. T ren d s in Biotechnology , 13 , 32431.

Torbert, K. A., Rines, H. W ., Kaeppler, H. F., Menon, G. K. andSomers, D. A. (1998). Genetically engineering elite oat cultivars.Crop Science , 38 , 16857.

W alden, R. and W ingender, R. (1995). Gene-transfer and plant-regeneration techniques. T ren d s in Biotechnology , 13 , 32431.


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