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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.