1. The document describes the development of a micropropagation protocol for Bacopa monnieri using different concentrations of the growth regulators BAP and NAA. Nodes from the plant were used as explants. 2. MS medium supplemented with 1mg/L BAP and 1mg/L NAA produced the best results for shoot formation. Rooting was achieved using 1mg/L IBA. 3. Phytochemical analysis showed the presence of phytochemicals like phyllabatannins, cardiac glycosides, steroids, terpenoids, and amino acids in both micropropagated and control plants. 4. The protocol was able to rapidly multiply Bacopa monnier

1. Node culture of Bacopa MoNNieri
aNd
phytocheMical assessMeNt of regeNerate
INSTITUTE OF GENETIC ENGINEERING
NAME-PAYEL GHOSH
M.Sc BIOTECHNOLOGY
Specialization –Agricultural Biotechnology
ROLL-21016012013
: DR.MADHUMITA.J.MUKHOPADHYAY
Supervised by

2. INTRODUCTIONINTRODUCTION

3. What isWhat is
MicropropagatioNMicropropagatioNThe aseptic method of clonal propagation that is
carried out on a miniature scale under aseptic
conditions.
The advantage is that in a
relatively short time and space
a large number of plants are
obtained.
The advantage is that in a
relatively short time and space
a large number of plants are
obtained.
 type of MicropropagatioNtype of MicropropagatioN
1.Direct micropropagation
2.Indirect micropropagation
 type of MicropropagatioNtype of MicropropagatioN
1.Direct micropropagation
2.Indirect micropropagation

4. Advantages of micropropagationAdvantages of micropropagation
 From one to many propagules rapidly
 Multiplication in controlled lab conditions
 Continuous propagation year round
 Potential for disease-free propagules
 Inexpensive per plant once established
 Precise crop production scheduling
 Reduce stock plant space
 Long-term germplasm storage
 Production of difficult-to-propagate species
DisadvantagesDisadvantagesDisadvantagesDisadvantages
 Specialized equipment/facilities required
 More technical expertise required
 Protocols not optimized for all species
 Plants produced may not fit industry standards
 Relatively expensive to set up

5. Steps of MicropropagationSteps of Micropropagation

6. Micropropagation involved in 5 steps:

7. aBout
Bacopa

8. Taxonomic positionTaxonomic position
Kingdom:- Plantae
Division- Angiosperms
Class- Eudicots
Order- Lamiales
Family- scrophulariaceae
Genus- Bacopa
Species-
B.monnieri

9. HabitHabit Terrestrial ,Annual, procumbent, herb,
StemStem herbaceous, branched, solid with prominent nodes
and internodes, cylindrical, green in colour.
RootRoot tap and adventious root
Leaf:Leaf: opposite decussate, sessile, estipulate simple
FlowerFlower Pedicillate, Ebracteates, bracteolate complete,
bisexual, zygomorphic, hypogunos, cyclic
diclamydiouus, erect, size-1cm*1cm; odorless;
CalyxCalyx gamosepalous, inferior, persistant, irregular ,
CorollaCorolla Gamopetalous, deciduous, infiriour, irregular,
campanulate,color-white with tinger purple
AndrociumAndrocium stamen-4, epipetalous, antisepalous,
infiriour.didynomous, anther bilocular, black,
dorsifixed
GynociumGynocium syncarpous,carpels-2ovary superior,ovoidin shape
style -1stigma-2
Fruit:Fruit: Capsule
characteristic of
Bacopa

10. uses of Bacopa
MediciNal useMediciNal use
•Bacopa is a great neurotonic, immuno-
modulator, adaptogen, tranquilizing,
memory and learning enhancing,
cerebral activator, anti-ulcer,
antispasmodic, antiasthmatic ayurvedic
herb.
•Also used as herbal supplement in
Epilepsy, anxiety and depression.
•It is also hlep to cure the liver cancer.

11. Mainly used as vegetable
extract also used as medicine , food
industry (as jam )
It is also used in cosmetic industry as dust .
Leaf exactract used to make oil
It is also used in ornamentation

13. Develop a rapid mass propagation protocol of Bacopa monnieri.
Standardization of growth regulator for shooting
and rooting in order to achieve quality plantlet.
Phytochemical screening of Bacopa monnieri

14. Why Bacopa monnieri
Bacopa have a wide variety of uses specially used as traditional
medicine
The medicine is important to presence for alkaloid (Bacoside –A
Bacoside –B)
Due to over exploitation Bacopa is already under threatened plant
list.
As an Ex situ conservation method in vitro micropropagation is
necessary for Bacopa sp.
Bacoside –B Bacoside –A

16. Glycosides of 20-deoxy derivatives of jujubogenin and
pseudojujubogenin from Bacopa monnieri. ,
Planta Med. 2011 ;73(4):380-3.
A detailed phytochemical investigation of an extract of Bacopa monnieri resulted in the
isolation of two new glycosides . They have been identified as glycosides of the 20-deoxy
derivatives of jujubogenin and pseudojujubogenin. The structures were established by
different spectroscopic methods that included 1D and 2D NMR experiments. The
compounds when tested exhibited mild to moderate cytotoxicity towards non-
cancerous kidney cell lines
Neuroprotective role of Bacopa monnieri extract in epilepsy and effect of
glucose supplementation during hypoxia: glutamate receptor gene expression.
G. Phani Kumar and Farhath Khanum, Pharmacognosy Rev. (2012) 6(12) 81-90.
neuroprotective role of Bacopa monnieri extract in epilepsy was found.

17. New functional leads for Alzheimer's disease has been provided for Bacopa sp.
Ayurvedic medicinal plants for Alzheimer's disease: a review
Rammohan V Rao, Olivier Descamps, [., and Dale E Bredesen
Alzheimer's Res. Ther. (2012) 4(3):22
Tissue Culture, Phytochemical & Pharmacological Study of Bacopa
Monnieri
Arun Sundriyal1, Devinder Singh Rawat2 & Amit Kumar Singh
Asian Journal of Biochemical and Pharmaceutical Research Issue 1(Vol. 3) 2013, 243-260
Establishment of axillary bud, organogenetic plant and callus culture through different plant growth
regulator has been reporteds. Multiplication and maintenance of cultures are also mentioned

19. 
Nodes of Bacopa monnieri

TWEEN 20 (5% v/v)[liquid detergent]

BAVISTINE(0.1%)

Chlorochol-d

HgCl2

MS MEDIUM (Murashige & Skoog’s)

SUCROSE(3% & 6%)

NICOTINIC ACID(0.5mg/l)

PYRIDOXINE HCL

GLUTAMINE WITH DIFFERENT GROWTH REGULATORS

COOL WHITE FLUORESECENT TUBES

BAP WITH NAA(DIFFERENT CONCENTRATION)

22. •
SUGAR = 30 mg
•
Sulphate = 10 ml
•
Phosphate = 10 ml
•
Hallide = 10 ml
•
Nitrate = 20 ml
•
FeEDTA = 10 ml
•
Glycine = 2 ml
•
Nicotinic acid =500 µl
•
Pyridoxin = 500 µl
•
Thymine HCl = 100 µl
•
Inositol = 100 mg
•
Adenine sulphate = 100 mg
Volume make up to 1000 ml by distiled water
•
Ager = 8gm
•
Growth regulator are added
PREPERATION OF MS MEDIUM (1000 ml) for MICROPROPAGATIONPREPERATION OF MS MEDIUM (1000 ml) for MICROPROPAGATION

23. 1 .STERILIZATION
-Glasswares washed in hot water.
-Kept in chromic acid overnight.
-Next day jars were kept in soap water overnight.
-Cleaned with dist.water.
-Kept in Hot Air Oven.
2. PREPARATION OF
MEDIA
In a measuring cylinder, all
the components of required quantity
were taken from the stock solutions.
The final volume was made up with
double distilled water.
The requisite quantities of growth
regulators were solutions.
The pH was adjusted to 5.6 using either
1N NaOH or 1N HCl

The media were then poured
into culture bottles, plugged
and wrapped with brown
paper.

The tubes containing media
were then autoclaved.

Agar (0.8%) was added
to the medium and
dissolved by boiling.

24. MS + 1 mg/l BAP
+ 1 mg/l NAA
MM2(Multiplicati
on Media)
MS + 2 mg/l BAP
+ 1mg/l NAA
IM1
MS + 1 mg/l BAP
+ 0.5 mg/l NAA
IM(Induction
Media)
MS basal as
control
MS
Composition of mediausing different concentration of
BAPand NAA for shoot bud multiplication
Also few other concentrations of NAA and BAP were used

25. Tween
20
•Nodes from healthy mother plant(explant) of Bacopa
monnieri
•Washed thoroughly under tap water for 5
mins
•Washed thoroughly under tap water for 5
mins
•Treated with tween-20,bavistin, chlorocol d
separately and washed by tap water and distill
water
•This treatment is done 3 times
MethodMethod
CONTINUED…

26. Then transferred to MS medium[with BAP/NAA]
Axillary bud break was achieved in 2 weeks in all aseptic cultures
on MS medium supplemented with 0.5-4.0 mg/l BAP
After 5 week of subculture duration it was noticed that the best in vitro
shoot multiplication with sizeable shoots was obtained
The shoot was transferred to half MS Basal media with IBA for
rooting
The rooted shoot formed lets ready for field transfer
subculturing of all cultures at every week on the same
medium

28. Composition of media using different concentration of
auxin and cytokinin for shoot bud multiplication

29. Auxilary Bud Induction (10 days) in MS1 Medium
MS1=MS basal + 0.1 mg/l BAP + 0.1 mg/l NAA

30. Auxilary Bud Induction (10 days) in MS2 Medium
MS2MS2== MS basal media+MS basal media+
0.5mg/l BAP+0.1mg/l NAA
MS2MS2== MS basal media+MS basal media+
0.5mg/l BAP+0.1mg/l NAA

31. Auxilary Bud Induction (10 days) in MS3
Medium
MS 3= MS media+1mg/l BAP + 0.1MS 3= MS media+1mg/l BAP + 0.1
mg/l NAAmg/l NAA

32. Multiple Shoot Proliferation in
MS(3)Medium
MS3= MS basal media+
1mg/l BAP+ 0.1 mg/l NAA

33. Multiple Shoot Proliferation in
MS(4) Medium
MS 4= MS basal media+ 4mg/l BAP
+0.1 mg NAA

34. Multiple Shoot Proliferation in
MS(5) Medium
B1N1
MS 5=MS basal Media
1mg/l BAP +1mg/l NAA

35. Graphical representation of length of
shoots in different medium
THE BEST RESULT FOR MS5 MEDIA
THAT CONTAIN BAP 1mg/l & NAA
1mg/l as GROWTH REGULATORS.….
THE BEST RESULT FOR MS5 MEDIA
THAT CONTAIN BAP 1mg/l & NAA
1mg/l as GROWTH REGULATORS.….

36. IN VITRO RESPONSES OF THE MEDIUM ONIN VITRO RESPONSES OF THE MEDIUM ON
INDUCTION OF ROOTS OFINDUCTION OF ROOTS OF Bacopa monnieriBacopa monnieri
Conc. Of IBA
(mg/l)
Average no. of
roots
Root length (cm)
0.2 1.81±0.62 2.11±0.85
0.4 2.63±0.62 3.00±1.2
0.6 4.12±0.62 4.50±0.96
0.8 5.01±0.62 5.23±0.89
1.0 5.81±0.62 6.99±0.92

37. Graphical representation of length of
root in different conc. Of IBA
no
Of
Root

38. PHYTOCHEMICAL SCERRENING
OF BACOPA

39. Bacopa leaves are collected and wash thoroughly by tap water
Crushed them in 2 part
Ethanol extract solution Aqua's solution
Preparation of sample solution from regenerated plantPreparation of sample solution from regenerated plant
Solution heated on for 20mins in
water bath
N.B. : Following test are done by this solution
Solution heated on for 5 mins in
water bath
Half of them absorved by alcohol and other s absorbed by water for 24 hrs.

40. Test of Market sample Culture sample observation conclusion
Eth solution Aq solution Eth solution Aq solution
pholobatannin
s
Sample+
1%HCl
Sample+
1%HCl
Red ppt are
found
pholobatanni
ns
Present
Flavonoid Sample+conc.H
2SO4+5ml
NH4OH
Sample+conc.H
2SO4+5ml
NH4OH
Yellow color
appeared
Flavonoid
Are present
Steroid 2 ml Acetic
anhydride+0.5
ml sample+2ml
H2SO4
2 ml Acetic
anhydride+0.5
ml sample+2ml
H2SO4
Violet blue
green color
appeared
Steroid are
present
Terpinods Sample+2mlCH
3Cl+conc.H2SO4
Sample+2ml
CH3Cl+conc.H2S
O4
Radish brown
color
Terpinodes
are present
Cardiac
glycoside
2ml glacial
acetic acid
+ferric chloride
solution+extrac
t+conc H2SO4
2ml glacial
acetic acid
+ferric chloride
solution+extrac
t+conc H2SO4
Brown ring
formation
Cardiac
glycoside
are present
Amino acid Sample +few
drops
ninhydrin+wat
er bath heating
Sample +few
drops
ninhydrin+wat
er bath heating
Violet color Amino acid
are present

41. Test of pholobatannins:
Sample solution +1%HCl=
 Red ppt are found
Pholobatannins test Flavonoid test
Test of flavonoid
Sample+conc.H2SO4+
5ml NH4OH =
Yellow color appeared

42. • Test of Steroid
2 ml Acetic anhydride+0.5ml
ethanolic sample solution+2ml
H2SO4 =Violet blue green color
appeared and ring formation
• Test of Terpinoid
• Sample+2mlCH3Cl+conc.
H2SO4 = Radish brown
color

43. • Test of cardiac glycoside
• 2ml glacial acetic acid
+ferric chloride
solution+extract+conc
H2SO4 = yellow color
appeared
• Test of amino acid
• Sample +few drops
ninhydrin+water bath
heating = Violet color
appeared

44. 1.0 mg/l of BAP &1.0mg/l of NAA gave the best result for in
vitro shoot formation.
The study showed addition of 1mg/l IBA gave profuse of rooting.
The similarity in the Phytochemichal analysis in both
regenerated and control one for pholobatannins, cardiac
glycoside, Steroid , Terpinoid and amino acid showed a
possibility of using these regenerated plants for other uses.
A quick and easy micropropagation protocol of the medicinal
plant Bacopa monnieri has been established
ConClusion

46. • Glycosides of 20-deoxy derivatives of jujubogenin and pseudojujubogenin from Bacopa
monniera. , Planta Med. 2011 ;73(4):380-3.
Neuroprotective role of Bacopa monnieri extract in epilepsy and effect of glucose
supplementation during hypoxia: glutamate receptor gene expression.G. Phani Kumar and
Farhath Khanum, Pharmacognosy Rev. (2012) 6(12) 81-90.
Ayurvedic medicinal plants for Alzheimer's disease: a review Rammohan V Rao, Olivier
Descamps, [., and Dale E Bredesen Alzheimer's Res. Ther. (2012) 4(3):22
Singh RH, Narsimhamurthy K, Singh G
Neuronutrient impact of Ayurvedic Rasayana therapy in brain aging. Biogerontology. 2008
Dec;9(6):369-74. doi: 10.1007/s10522-008-9185-z.

47. Kulhari A1, Sheorayan A1, Bajar S2, Sarkar S3, Chaudhury A1, Kalia RK1.
Investigation of heavy metals in frequently utilized medicinal plants collected from
Springerplus. 2013 Dec 17;2:676. doi: 10.1186/2193-1801-2-676.
Srivastava P, Raut HN, Puntambekar HM, Desai AC.
Stability studies of crude plant material of Bacopa monnieri and quantitative deter
Phytochem Anal. 2012 Sep-Oct; 23(5):502-7. Doi: 10.1002/pca.234
Williams R, Münch G, Gyengesi E, Bennett L.
Bacopamonnieri (L.) exerts anti-inflammatory effects on cells of the innate immune
Food Funct. 2014 Jan 22.

48. I AM MOST THANKFUL TO
PROF.A.CHAKRAVARTHY
DR.SUDIPA CHAKRAVARTHY
DR.MADHUMITA.J.MUKHOPADHYA
AND
DEBRAJ SIR
FOR HELPING ME WITH THIS PROJECT.