Effect of explants type on rapid multiplication of Bacopa monnieri in in vitro cultures and analysis of homogeneity of the

population obtained by RAPD analysis.

Submitted By:BrajBala Mishra

M.Sc IV SEM Biotechnology

Guidance of : Dr.Tejovathi Gudipathi

BOSTON COLLEGE FOR PROFESSIONAL STUDIES

INTRODUCTION

Bacopa monnieri L. is an important plant of Madhya Pradesh. It has been placed second in a priority list of the most important Indian medicinal plants evaluated on the basis of medicinal importance, commercial value and potential for further research and development (Anonymous, 1997).

Bcopa monnieri (l.) wettst. (family sacrophulariaceae) is a creeping , glabrous, s Flowers of beahmi are white or pale –bluish in colour ,axillary , solitary arranged on long slender pedicles ucculent herb , rooting at nodes . Bacopa shows antidiabetic, antioxidant and hepatoprotective activity

GROWTH REGULATORS

Growth regulators are the compounds produced naturally in plants and active in controlling growth and other function of plant cells and tissues. Cytokinines are a class of plant growth substances that promote cell division, or cytokinesis, in plant root and shoot. They are involved primarily in cell growth and differentiation, but also affect apical dominance, axillary bud growth and leaf senescence. F. Skoog discovered their effects using coconut milk in the 1940s at the university of wiskonsin- Madison.

Auxins are another class of naturally occurring or synthetic organic (carbon-containing) plant growth substances (often called phytohormones or plant hormones) that increase, in low concentrations, the rate of cell elongation in stems, among other influences. Auxins play an essential role in coordination of many growth and behavioral processes in the plant life cycle.

INOCULATION OF EXPLANT

For the present project work on effect of growth regulators on plant regeneration and the molecular analysis of regenerated plants , mature leaf explants of Bacopa material plants collected from shivpuri was used . Three explants – Leaves, nodes and internodes were used from the vigorously growing plants for the present tissue culture and molecular studies.In the present studies three growth regulators- Kinetin (Kn), 6-benzylaminopurine (BAP) and 2,4-dichlorophenoxy acetic acid) 2,4-D were used for callus initiation and plant regeneration studies. Each growth regulator stock of 200ppm strength was prepared separately. Growth regulator was first dissolved in small amount of IN HCl/ IN NaOH solution and made up to 100ml.All the solutions were preserved at 5oC and brought to room temperature before preparing the medium

In the present study, leaf explants from shivpuri accession , maintained at College green House has been used. The leaf explants were inoculated on MS medium with growth regulators. 2,4-D, BAP and Kn were supplemented at 1.0 to 4.0 mg/l concentration into MS medium. The results obtained after 4 weeks were collected and presented in the Table -

MS + Kn No. of explant No. shoot % plantlet regeneration

LEAF

1.0 30 12.01 55.1

2.0 30 12.21 50.1

3.O 30 29.2 45.2

4.O 30 30.2 40.2

NODE

1.O2.0

3030

3.412.4

100100

INTERNODE

1.0 30 12.22 30

2.0 30 14.02 50

3.0

4.0

30

30

17.0

13.7

64

54

DNA Isolation of Baccopa

In vitro plant regeneration for the micropropagation of important plant biodiversity is a excellent alternative for rapid multiplication and conservation of the germplasm. However, testing of the homogeneity of the in vitro propagated plants is also essential for the maintenance of germplasm. Molecular markers are important tools in assessing the variability, if any, in the in vitro produced clones. In the present study, we have used RAPD- Randomly apmplified polymorphic DNA method and three randomly selected regenerated plants R1 ,R2andR3), Young leaves without necrotic areas and lesions were selected, for the molecular studies

POLYMERASE CHAIN REACTION

Polymerase Chain Reaction primarily consists of three basic steps:

Denaturation of the double stranded template DNA.Annealing of primers to denatured strands of template.Extension of primers to form new products resulting in doubling of amplicon in each cycle.Polymerizing enzyme used is a heat resistant Taq DNA polymerase obtained from Thermus aquaticus (survives in hot spring waters).Polymerase chain reaction is achieved by using an automatic thermal cycler. Sufficient amplification of a target DNA is obtained in about 25-45 cycles. The DNA amount doubles in each cycle resulting in an exponential accumulation of the PCR product.

Table gives the solutions used and their volume in the reaction mixture.

S.NO STOCKS REAGENT VOLUME/REACTION

1. 2X PCR Master mix 10ul

2. - Primers 2.0ul

3. Nuclease free Water 7.0ul

4. 40- 50 mg /ul Genomic DNA 1.0ul

- Total 20.0ul

All the L1, N2, and 1N3 plant DNA was amplified using four operon primers. The reaction mixture was prepared as given above table for each DNA sample and with each of the four primers. PCR program was fixed as given in the Table , standardized by Pratima et. al (unpublished

PCR PROGRAM

S.NO

1 SETP 1 Initial denaturation

940 c 5 min

2 STEP 2 Denaturation 940 c 1min

3 STEP 3 Annealing 370 c 1min

4 STEP 4 Extension 72o c 2min

5 STEP 5 Final Extension

72o c 10min

6 STEP 6 Hold 40 c 30min

Amplification cycles (step2-4) – 25 cycles.The samples were placed in the Thermocycler machine and the program for a set for 25 cycles and the PCR was performed

AGAROSE GEL ELECTROPHORESIS

Agarose is linear polymer of alternate residues of D & L –galactose extracted from the sea weed . Agarose gel is a matrix .It acts as molecular sieve through which DNA fragment move on application of electric current . Higher concentration of agarose gives a gel with smaller pore size , hence DNA with smaller size can easily move through the small size pores , while lower concentration of agarose helps in the movement of large size DNA fragment as the spaces between the cross linked molecules is more

In the present project, Random amplified polymorphic DNA analysis was performed to check the genetic fidelity of in vitro regenerated plants. Plants obtained from different media containing various PGRs were used for DNA isolation for RAPD analysis.3decamer random oligonucleotide primers (OP5, CAGCCCAGAG; OP6, ACCTCAGCTC; OP7, TGCCGGCTTG ) were used for the PCR reactions. Figure 5,7, and 8 show RAPD amplification patterns obtained with primers OP5, OP6 and OP7, respectively. The number of bands produced by each primer ranged from 1-7.

P05 primer CAGCCCAGAG

P07 primer TGCCGGCTTG

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