effect of growth hormone on shoot bud multiplication of bacopa monnieri (brahmi-a medicinal plant)

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To Study the Effect of growth hormone on shoot bud multiplication of Bacopa

Monnieri (bramhi)-a medicinal plant

Presented by: Avishek Bhattacharjee Roll no-09210082012 6th semesterDepartment of BIOTECHNOLOGYInstitute of Genetic Engineering

Guided By: Dr. Madhumita J. Mukhopadhay

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MICROPROPAGATIONThe 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.





GLASSWARE WASHING AND STORAGE AREA

MEDIA PREPARATION AND STERILIZATION AREA

PRIMARY GROWTH ROOM

ASEPTIC TRANSFER AREA

DESIGNING PLANT MICROPROPAGATION LABORATORY





METHODS OF MICROPROPAGATION

Establishment

1. collection of explant(s)

2. Surface sterilization of explants

3. Placement of small part of plant tissue on growth medium.

4. Grows and differentiation of plant tissue.





Multiplication

1. Repeated cycle of establishment stage.

2. Producing a number of copies of required plant





Pre-transplant

• Transferring the plantlets to a growth medium containing Auxin or Cytokinin which stimulate root or shoot initiation respectively .

• Moving the plants to a location high in humidity, such as a green house with regular mist watering





Transfer from culture

• The plantlets are removed from the plant media and transferred to soil or (more commonly) potting compost for continued growth by conventional methods.





STEPS OF MICROPROPAGATION





Why need micropropagation of Bacopa monnieri ?

Rapid mass propagation to produce genetically uniform plants.

To develop a rapid and stable mass propagation protocol of this important

medicinal plant.

To obtain large number of virus free plant within a short time span

and a comparatively low cost.

Micropropagated plants may be tested in futures studies for it’s alcolied content (bacoposides)





ADVANTAGES OF MICROPROPAGATION Micropropagation produce disease-

free plants.

Micropropagation produces rooted plantlets ready for growth, rather

than seeds or cutting.

It can have an extraordinarily high fecundity rate, producing

thousands of propagules while conventional techniques might only

produce a fraction of this a number.

It is the only viable method of regenerating genetically modified cells or cells after protoplast

fusion.

It is useful in multiplying plants which produce seeds in uneconomical

amounts.





Bacopa monnieri L. Penn. commonly known as “Brahmi” is an important medicinal herb of the family Scrophulariaceae.It is the foremost brain tonic herb of the Indian System of Medicine and other traditional systems, use primarily as a nerve tonic, to treat insomnia and nervous tension.





Habitat and Botanical Description

Bacopa monnieri has originated in India. A genus of spreading herbs, commonly growing in damp and marshy places throughout India, ascending up to an attitude of 1,320 m.

Leaves:-are sessile, opposite decussate,succulent, obovate or oblanceolate in shape , short petiolate, 0.6-2.5 cm in size

Branches :-ascendingStem :-softAngular.

Flowers:- solitary axillary, blue or white in colour with purple veins, campanulate ,pentamerous , capsules ovoid Flowers and fruits appear in summer.





Kingdom:- PlantaeUnranked- Angiosperms Unranked- EudicotsOrder- LamialesFamily- scrophulariaceaeGenus- BacopaSpecies- B.monnieri





Why it is taken?♯ Uses as a leafy vegetable and/or as a medicinal plant.♯Bacopa also has important antioxidant vitamin E properties and acts as a metal chelator, removing excess damaging metals from the blood, thus limiting the propagation of free radicals. Bacopa may even be able to revitalize intelligence

Additionally, research on genetic improvement, propagation, field production, potential for processing and marketing.





MEDICINAL 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.





CHEMISTRY

Plant contains two saponins, bacoside A and B The carbohydrate mostly of bacoside A was shown to be arabinosyl glucose with the arabinose unit as the terminal sugar. Bacoside B was found to be dextrorotatory where as bacoside A was laevo rotatory. The haemolytic action of bacoside B is twice that of bacoside A.

Bacopa also contains D-mannitol, betulic acid, beta-sitosterol, octacosane, nicotine, and amino acids such as serine.





Objective of the work

Develop a rapid mass propagation protocol of Bacopa monnieri.Standardization of surface sterilization protocol

Standardization of suitable media for aseptic culture initiation, establishment and multiplication.Standardization of a suitable media for shooting and rooting in order to achieve quality plantlet.

To test the effect of growth hormone on shoot bud multiplication of Bacopa monnieri





Materials and Methods

Aseptic culture technique:Main requirement are surface sterilized explants of Bacopa monnieri (bramhi) for in vitro propagation.

Requirements of Glasswares in lab :Sterile bottlesSterile test tubesConical flasksMeasuring cylindersBeakers

Requirements other than glasswares :70% ethyl alcoholTissue papersSterile scalpels & forcepsBurner





Lab Equipment

Continued…

Oven for dry sterilization: Although oven can be used for dry sterilization, of scalpels and glassware's such as Petri-dishes , test tubes & others.Autoclave :Autoclaves in different sizes are commercially available.Moist heat is applied in it in an atmosphere of steam.Moisture improves heat penetration ,making sterilization by moist heat more effective than dry heat.





Laminar airflow cabinet: These cabinets are commercially available in various sizes & shapes.where large dust particles are separated, & subsequently passes through a HEPA filter.

Water distillation Water distillation apparatus :-apparatus :-Distilled Distilled water is to prepare of water is to prepare of media.media.

Microwave oven:Microwave oven: Used for melting of Used for melting of solidified agar for use in solidified agar for use in media preparation & for media preparation & for heating water.heating water.





Monopan Balance:Monopan Balance: Used for weighing Used for weighing powdered substances for powdered substances for use in plant tissue use in plant tissue culture.culture.

pH meter:pH meter:Adjustment of pH Adjustment of pH generally at 5.8 by generally at 5.8 by (using 1(N) KOH or 1(N) (using 1(N) KOH or 1(N) HCl).HCl).





Sterilization of glassware





Media Composition

Murashige and Skoog (1962) Medium

Constituents Amounts (mg/l)

1. MacronutrientsNH4NO3 1650KNO3 1900CaCl2.2H2O 440MgSO4.7H20 370KH2PO4 1702. MicronutrientsMnSO4.4H20 16.90FeSO4.7H20 27.80ZnSO4.7H2O 8.60H3BO3 6.20KI 0. 83Na2MoO4.2H2O 0.25CoCl2.6H20 0.025CuSO4.7H2O 0.025Na2EDTA.2H2O 30.00





Media Composition

.Vitamins Myoinositol 100Glycine 2.0Nicotinic acid 0.5Pyridoxine HCL 0.5Thiamine HCL 0.1

4. Sucrose 3%(Merck)

5. Agar 0 .8%6. Growth Regulators- as required alpha-Naphthalene acetic acid(NAA)- 6-Benzyl amino purine(BAP)& Kinetin .





Collection of explantsBacopa planting material was collected from Garden. Rinsed in running water for 15 minutes

Surface sterilization explants of Bacopa monnieri (brahmi)

Continued…

1. Explants were washed with fresh tap water to clean dust & other loose surface contamination

2. Explants were shaken for 10 minutes with 2 drops of Tween 20(surfectant)





Rinsed in running water for 15 minutes

Shaken with 0.2 % Bavistin(antifungal agent) 2-3 mins

The explants were finally treated with 0.1% mercuric chloride for 3-4 mins followed by rinsing in sterile distilled water for 4-5 times.





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 distil water.

•The requisite quantities of growth regulators were added from the stock solutions.

•The pH was adjusted to 5.6 using either 1N NaOH or 1N HCl





Preparation of media•Agar agar(0.8%) was added to the medium and dissolved by boiling.

•The media(20ml)were then poured into culture tubes(25-150mm),plugged and wrapped with brown paper.

•The tubes containing media were then autoclaved.

•Slants were then prepared by keeping the tubes at 45degree C while cooling at room temperature.





Transfer of shoot in the culture tube

• Sterilized explants were transferred aseptically to sterilized glass plate under the laminar flow hood.

• Then a cut was given on both basal as well as the top portion of the explants.

The forceps were earlier rinsed in the 70% ethanol and were flamed and then kept for sometime to get cool. Then the lid from one test tube was removed and test tube's mouth was flamed to avoid any chance of contamination.





•Each nodal explants was then placed in an erect position in the test tube containing medium with the help of long forceps.

•The forceps were then again rinsed with 70% alcohol to avoid any chance of cross contamination.

•These test tube were finally kept in the growth room with temperature conditions 25± 2 °C, with a photoperiod of 16 hours daylight and 8 hrs night break under the (cool white fluorescent tube light 40 W GE).





IN-VITRO ROOTING OF SHOOTLET•In the laminar flow, under sterile conditions the cotton plug was removed from the test tube in sterile condition (laminar flow hood) and with the help of sterile forceps the multiplied shoots were removed from the medium and placed on the sterile glass plate.•With the help of sterile scalpel elongated shoots up to 1-2 cm in length were cut and placed into the rooting medium. The test tube were then plug and placed in the growth room under the same condition. The time required for in vitro rooting of shoots may vary from 10 – 25 days.





Composition of media using different concentration of auxin

and cytokinin for shoot bud multiplicationMedium Code Medium details Bud breakage

ResponseCluster formation

MS MS basal as control + +MS1 MS + 0.1 mg/l BAP +

0.1 mg/l NAA+ ++

MS2 MS + 0.5 mg/l BAP + 0.1 mg/l NAA

++ +++


++ ++


+ +


+++++ ++++

MS6 MS + 1.0 mg/l BAP + 1.0 mg/l Kn

++

MS7 MS + 0.5 mg/l BAP + 0.5 mg/l Kn

++ ++





Graphical representation of length of shoots in different medium

0

5

10

15

20

25

M S M S1 M S2 M S3 M S4 M S5 M S6 M S7

10d20d30d40d





RESULT CONTINUED………

•Multiplication of shoot cultures was carried out by culturing nodal segments/clusters excised from garden raised plants.

•Maximum numbers of plants were obtained in the medium containing NAA(1 mg/l) and BAP (1mg/l) in shoot tip and nodal culturesof Bacopa.





Auxilary Bud Induction (25 days) in MS(1)+BAP(1) Medium





Multiple Shoot Proliferation inMS5 Medium





Multiple Shoot Proliferation in

MS(2)Medium





Multiple Shoot Proliferation in MS 2

and MS 5 Medium





RESULTCONTINUED………

The regenerated shoots were transferred to ½ concentration of the modified MS basal medium containing variable concentrations of IBA





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

OF OF Bacopa monnieriBacopa monnieriConc.(mg/l) Average no.

of rootsRoot length (cm)

0.2 1.81±0.62 2.11±0.850.4 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





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

monnieri

1 2 3 4 5

IBA

Average No. of roots

Root length (cm )

01234567

IBAAverage No. of rootsRoot length (cm )





Multiple Shoot Proliferation in MS5+IBA(1)Medium





TRANSPLANTATIONExplants used for carrying out hardening

experiment using soil mixture were grown in MS basal medium as reported before. After

sufficient rooting, Bacopa plantlets were transferred to polybags having soil mixture in

for hardening.

Agar was removed from the rooted plantlets and then plantlets were washed with tap water.

The polybags were sprinkled with water. The roots of each plant were into the soil mixture. dipped in 0.1% Bavistin solution to avoid future fungal attack and then roots of explants were inserted carefully





Then plants were transferred to the polypacks in a shaded place.

Finally they were transferred to the open area

for 9-10 days before transferring them to the

field.





Herbs are being used since ancient time to maintain health, to treat disease and regain the healthy state of mind and body. They have been used in traditional forms of Indian medicine.

Observations and conclusions in the present work

•.

Conclusions:

The Medium MS 5 Supplemented with 1.0 mg/l BAP + 1.0 mg/l NAA gave best result for Bud breakage, multiplication and maximum shoot length. When shootlets were implanted in ½ strength MS mediasupplemented with 1 mg/l IBA maximum number of roots were initiated with about 7 cm length in 4-5 weeks.





Agenda K and D’Souza L (1999) In vitro propagation of Ayurvedic plants. In :

Khan I A and Khanum A (eds) Role of Biotechnology in Medicinal and Aromatic plants Vol 2: 207-237.Ukaaz Publication Hyderabad· Raman L U, Verma P C, Singh Digvijay, Gupta M M and Banerjee S (2002) Bacoside production by suspension cultures of Bacopa monnieri (L.) Pennell.Biotechnology Letters 24 (17) : 1427-1429 *· Rani G, Virk G S and Nagpal A (2003) Callus induction and plantlet regeneration in Withania somnifera (L.) Dunal. In Vitro Cellular and Development Biology – Plant 39 (5): 468-474Micropropagation of Bacopa monneiri L. Penn.- an important medicinal plant-A DISSERTATION ByMs. NEETU SHARMADepartment of Biotechnology and Environmental Sciences, Thapar Institute of Engineering and Technology Patiala , India.

REFERENCES





ACKNOWLEDGEMENT

I AM MOST THANKFUL TOPROF.A.CHAKRAVARTHYDR.SUDIPA CHAKRAVARTHY , DR.M.J.MUKHOPADHYAY AND Mr. D. MODAK FOR HELPING ME WITH THIS PROJECT.




effect of growth hormone on shoot bud multiplication of bacopa monnieri (brahmi-a medicinal plant)

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