Node culture of Bacopa Monnieri and

phytochemical assessment of regenerate

INSTITUTE OF GENETIC ENGINEERINGNAME-PAYEL GHOSH

M.Sc BIOTECHNOLOGYSpecialization –Agricultural Biotechnology

ROLL-21016012013: DR.MADHUMITA.J.MUKHOPADHYAY

Supervised by

INTRODUCTIONINTRODUCTION

What is MicropropagationWhat is Micropropagation

The 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

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

Steps of MicropropagationSteps of Micropropagation

Micropropagation involved in 5 steps:

About Bacopa

Taxonomic positionTaxonomic position

Kingdom:- PlantaeDivision- Angiosperms

Class- EudicotsOrder- Lamiales

Family- scrophulariaceaeGenus- Bacopa

Species- B.monnieri

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

USEs of bacopaUSEs 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.

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

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

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.

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

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.

New functional leads for Alzheimer's disease has been provided for Bacopa sp.

Ayurvedic medicinal plants for Alzheimer's disease: a reviewRammohan 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

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)

• 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 MICROPROPAGATION PREPERATION OF MS MEDIUM (1000 ml) for MICROPROPAGATION

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.

MS + 1 mg/l BAP + 1 mg/l NAA

MM2(Multiplication 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 media using different concentration of BAP and NAA for shoot bud

multiplication

Also few other concentrations of NAA and BAP were used

Tween20

•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…

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

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

Auxilary Bud Induction (10 days) in MS1 Medium

MS1=MS basal + 0.1 mg/l BAP + 0.1 mg/l NAA

Auxilary Bud Induction (10 days) in MS2 Medium

MS2MS2= = MS basal media+MS basal media+0.5mg/l BAP+0.1mg/l NAAMS2MS2= = MS basal media+MS basal media+0.5mg/l BAP+0.1mg/l NAA

Auxilary Bud Induction (10 days) in MS3 Medium

MS 3= MS media+1mg/l BAP + 0.1 MS 3= MS media+1mg/l BAP + 0.1 mg/l NAA mg/l NAA

Multiple Shoot Proliferation inMS(3)Medium

MS3= MS basal media+ 1mg/l BAP+ 0.1 mg/l NAA

Multiple Shoot Proliferation inMS(4) Medium

MS 4= MS basal media+ 4mg/l BAP+0.1 mg NAA

Multiple Shoot Proliferation inMS(5) Medium

B1N1

MS 5=MS basal Media1mg/l BAP +1mg/l NAA

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.….

IN VITRO RESPONSES OF THE MEDIUM ON IN VITRO RESPONSES OF THE MEDIUM ON INDUCTION OF ROOTS OF INDUCTION 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

Graphical representation of length of root in different conc. Of IBA

noOfRoot

PHYTOCHEMICAL SCERRENING OF BACOPA

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.

Test of Market sample Culture sample observation conclusion

Eth solution Aq solution Eth solution Aq solution

pholobatannins

Sample+1%HCl

Sample+1%HCl

Red ppt are found

pholobatanninsPresent

Flavonoid Sample+conc.H2SO4+5ml NH4OH

Sample+conc.H2SO4+5ml NH4OH

Yellow color appeared

Flavonoid Are present

Steroid 2 ml Acetic anhydride+0.5ml sample+2ml H2SO4

2 ml Acetic anhydride+0.5ml sample+2ml H2SO4

Violet blue green color appeared

Steroid are present

Terpinods Sample+2mlCH3Cl+conc.H2SO4

Sample+2ml CH3Cl+conc.H2SO4

Radish brown color

Terpinodes are present

Cardiac glycoside

2ml glacial acetic acid +ferric chloride solution+extract+conc H2SO4

2ml glacial acetic acid +ferric chloride solution+extract+conc H2SO4

Brown ring formation

Cardiac glycosideare present

Amino acid Sample +few drops ninhydrin+water bath heating

Sample +few drops ninhydrin+water bath heating

Violet color Amino acid are present

Test of pholobatannins:Sample solution +1%HCl= Red ppt are found

Pholobatannins test Flavonoid test

Test of flavonoidSample+conc.H2SO4+5ml NH4OH =Yellow color appeared

• Test of Steroid2 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

• 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

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

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 GNeuronutrient impact of Ayurvedic Rasayana therapy in brain aging. Biogerontology. 2008 Dec;9(6):369-74. doi: 10.1007/s10522-008-9185-z.

Kulhari A1, Sheorayan A1, Bajar S2, Sarkar S3, Chaudhury A1, Kalia RK1. Investigation of heavy metals in frequently utilized medicinal plants collected from environmentally diverse locations of north western India. 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 determination of bacopaside I and bacoside A by HPLC. 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 system in vitro.

Food Funct. 2014 Jan 22.

I AM MOST THANKFUL TOPROF.A.CHAKRAVARTHY

DR.SUDIPA CHAKRAVARTHYDR.MADHUMITA.J.MUKHOPADHYA

ANDDEBRAJ SIR

FOR HELPING ME WITH THIS PROJECT.