Vitro Clonal Propagation And Phytochemical Studies Biology Essay

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Plumbago zeylanica is an important medicinal plant and has been used in ayurveda from ancient times. In the present study we have observed the effect of variable concentration of auxin and cytokinin on the regeneration potential. P. zeylanica has shown the better micropropagation result when nodal bud was used as explants. The budding part respond best in the medium C having IAA and BAP concentrations 4 mg/l & 5mg/l respectively in MS media, resulting in the development of shoot regeneration. After three weeks shoot regeneration increases without further development in the roots. At the same time it has been observed that explants has shown regeneration potential in media D and G, (BAP-3.5mg/l, NAA-2.5 mg/l) but the growth rate was slower. Phytochemical analysis of crude extract of this plant indicates the presence of photochemical i.e. alkaloids, glycosides and phenolic compound etc.

Introduction

Plumbago zeylanica (known vernacularly as Chitraka, Chitramulamu, Tellachitramulamu, Agnichela, Agnimaala or by its trade or popular names of "Lead wort-white flowered" and "Ceylon Lead wort") of the Plumbaginaceae, is distributed as a weed in throughout the tropical and subtropical countries of the world.  This plant is a spreading or subscandent, herbaceous, suffrutescent plant, 1 - 2 m in length. The root contains medicinally important compound known as 'Plumbagin'. Plumbagin is present in all the variety of plumbago to a maximum of about 0.91%. This active chemical is vesicant, has properties of vitamin K, and is antibiotic on several human pathogens (Burkill, 1985). The plant is continuously collected from the wild and over exploited from ancient times. In view of this, there is an need for alternative means of clonal propagation with a view to preventing the plant from extinction.Biotechnological tools such as In-Vitro micropropagation can play important role in the conservation of endangered medicinal plants as well as for rapid multiplication and quality improvement of valuable medicinally important plant species. In-vitro regeneration of Plumbago zeylanica, plants via callus cultures (Rout et al., 1999) and through direct organogenic differentiation have been worked out in recent years (Selvakumar et al.,2001; Verma et al., 2002). Clonal multiplication of Plumbago rosea (Kumar and Bhavanandan,1988) and Plumbago indica (Chetia and Handique,2000) had also been reported earlier. In-vitro production of "plumbagin"- the desired secondary metabolite of P.zeylanica, from A.rhizogenes mediated hairy root cultures had also been published currently (Verma et al., 2002).

Methodology

The experiment was performed in the tissue culture laboratory of IMS Engineering college, Ghaziabad in the year 2008-09 Fresh and healthy plant materials of P. zeylanica collected from the local nursery and nodal bud and leaf were used as explants for the present study. The standard MS media were used during entire study with variable concentration of Auxin and Cytokinin The pH of each medium was adjusted to 5.8 prior to the addition of 8gm/litere of agar (Quligens). Media and instruments were sterilized by autoclaving for 15 - 30 min at 121°C (1 atm). Aseptic transfers were performed in a laminar flow hood (S.M Scientific). The explants were washed thoroughly with liquid detergents and followed by under running tap water for 30 minutes and final sterilization was carried out with 0.1% HgCl2 and rinsed properly with sterile distilled autoclaved water for 4-5 times . After the surface sterilization of explants it was aseptically transferred into the pre- sterilized media boottles (Conical flask). Cultures were maintained 240C in the culture rooms and exposed to 8-10 hours light. Contaminated cultures were treated with 0.1% HgCl2 and sub cultured in fresh media.

The crude extract of Plumbago zelanica was extracted through soxlet apparatus taking leaf and flower parts as plant material using ethanol and hexane as solvent. Solvent is evaporated using vacuum rotatory evaporator. Crude extract was analyzed through chemical reaction for the presence of different photochemical

Result & Analysis

The entire experiment were performed in 3 cycles which gives the following results in variable concentration of auxin and cytokinin

Cycle I

Observations

IBA-4

BAP-5

IBA-4

BAP-4

IAA-4

BAP-4

IAA-4

BAP-5

SURVIVAL

3

2

3

4

GROWTH

2

1

1

1

CONTAMINATION

0

2

2

0

DEAD

1

1

0

1

CYCLE II:

Observations

IBA-3mg/ml

NAA-2.5mg/ml

IBA-2.5mg/ml

NAA-2mg/ml

IBA-3.5mg/ml

NAA-3.0mg/ml

GROWTH

2

3

5

CONTAMINATION

4

1

2

DEAD

0

0

0

Conformation analysis of different phytochemical present in the Plumbago (leaves) plant material

TESTS

CATEGORY

RESULT

Alkaloid

Dragandroff's reagent

positive

Carbohydrates

Molish test

positive

Glycosides

Balijet test

negative

Phenolic

Lead acetate

positive

Flavanoids

Ammonia test

Negative

Protein & amino Acid

Xanthoprotein

Positive

Conformation analysis of different phytochemicals present in the Plumbago (flower) plant material

TESTS

CATEGORY

RESULT

Alkaloid

Dragandroff's

reagent

Positive

Carbohydrates

Molish test

Positive

Glycosides

Balijet test

Negative

Phenolic

Lead acetate

positive

Ferric chloride

negative

Flavanoids

Ammonia test

negative

Protein & amino acid

Xanthoprotein

positive

Saponins

Foam

negative

P. zeylanica has shown the better micropropagation result when nodal bud was used as explants. The budding part respond best in the medium C having IAA and BAP concentrations 4 mg/l & 5mg/l respectively in MS media, resulting in the development of shoot regeneration. After three weeks shoot regeneration increases without further development in the roots. At the same time it has been observed that explants has shown regeneration potential in media D and G,(BAP-3.5mg/l, NAA-2.5 mg/l ) but the growth rate was slower. Phytochemical analysis of crude extract of this plant indicates the presence of photochemical i.e. alkaloids, glycosides and phenolic compound etc.

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