This document summarizes an experiment that analyzed the effect of different explant types and growth regulators on the rapid multiplication of Bacopa monnieri in in vitro cultures. Leaf, node, and internode explants were cultured on media with different concentrations and combinations of kinetin, BAP, and 2,4-D. The highest rates of shoot regeneration were observed in nodes cultured with kinetin. DNA was then isolated from regenerated plants and analyzed using RAPD to check for genetic homogeneity between clones. PCR was performed using random primers and products were visualized on agarose gels. The gels showed 1-7 bands for each primer, indicating genetic similarity between clones.

1. 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

2. 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

3. 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.

4. 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

5. 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.O
30
3.41
100
2.0
30
2.4
100

6. 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

10. 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

11. 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.

12. Table gives the solutions used and their volume
in the reaction mixture.
S.NO STOCKS REAGENT VOLUME/REACTI
ON
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

13. 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

14. 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

15. 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.

16. P05 primer CAGCCCAGAG

17. P07 primer TGCCGGCTTG

18. THANK YOU