Project 1: Establishment of callus and adventitious root cultures of Stevia

Rebaudiana Bertoni

Project 2: Establishment of callus, adventitious root cultures of Andrographis

Paniculata (Hempedu Bumi)

Name: Nurul Hamizah Binti HazaliSupervisor: Pn. Norazlina Bt Noordin

Objectives 1. To study the effects of different 2,4-Dichlorophenoxyacetic

Acid (2,4-D) concentrations on callus induction of Stevia Rebaudiana Bertoni and Andrographis Paniculata (Hempedu Bumi)

2. To study the effects of different Indole-3-Butyric Acid (IBA) concentrations on adventitious root induction of Stevia Rebaudiana Bertoni and Andrographis Paniculata (Hempedu Bumi)

Project 1: Establishment of callus and adventitious root culture of Stevia Rebaudiana Bertoni

• Discovered by Antonio Bertoni in 1887

• Have the natural sweetness to replace artificial sweetness

• 300x sweeter than sucrose• Known as honey leaf, sweet leaf

and sweet herb• No negative clinical report had been

reported

Materials and method

Establishment of callus and adventitious root cultures of Stevia Rebaudiana

Callus inductionExplant used: in- vitro leaf of

stevia Media: 2,4-D with 2, 4 and

6mg/l concentrationCondition: 24±2˚C with 16 hours

photoperiod

Adventitious root inductionExplant used: in- vitro leaf of

stevia Media: IBA with 1,3and 5mg/l

concentrationCondition: 24±2˚C with 16 hours

photoperiod

Result

2,4-D 2mg/l

2,4-D 4mg/l

2,4-D 6mg/l

control0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1 Callus induction of Stevia Rebaudiana after 3 weeks

Induction of Cal-lus

2,4-D concentrations

No o

f exp

lant

pro

ducin

g ca

llus o

ut o

f 16

exp

lant

s No formation of callus after

3weeks

IBA 1mg/l IBA 3mg/l IBA 5mg/l control0

1

2

3

4

5

6

Callus induction and adventitious root formation after 3 weeks

Induction of CallusFormation of Adventitious Root

IBA concentrations

No

of e

xpla

nt p

rodu

cing

callu

s an

d ad

ven-

titiou

s roo

t for

mati

on o

ut o

f 16

expl

ants

5/16 sample produce callus

31%

IBA 1mg/l IBA 3mg/l IBA 5mg/l Control 0

2

4

6

8

10

12

14

16

18

Callus induction and adventitious root formation after 2 months

Induction of Callus Adventitious Root For-mation

IBA concentrations

No

of e

xpla

nts p

rodu

cing

cal

lus a

nd a

dven

titiou

s roo

t fo

rmati

on o

ut o

f 16

expl

ants

5mg/l of IBA treatment pro-duce the highest callus induction 100%

Of 3mg/l IBA doesn’t change after 2months

3mg/l of IBA concentration produce highest adventitious root formation.

Pictures of callus and adventitious root formation after two months.

IBA 1mg/L

Direct adventitious

root

Adventitious root Direct

adventitious root

Indirect adventitious

root Callus

IBA 3mg/l

Callus Indirect adventitious

root

Indirect adventitious

root

Callus

Callus

• IBA 5mg/l

Callus Callus

2,4-D 2mg/l 2,4-D 4mg/l 2,4-D 6mg/l Control 0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

Callus induction after 2 months

Induction of Callus

2,4-D concentrations

No

of e

xpla

nt p

rodu

cing

callu

s ind

uctio

n ou

t of

16 e

xpla

nts

Highest callus in-duction on 4mg/L concentration

Callus

Subculturing for multiplication of adventitious root and callus of Stevia Rebaudiana

Adventitious root Clump of callus

Conclusion for project 1: Establishment of callus and adventitious root cultures of Stevia Rebaudiana Bertoni

• The formation of adventitious root was observed either from direct or

indirect induction. • All concentrations of IBA treatment showed production of callus and

adventitious roots. • However, 3mg/L of IBA showed highest formation of adventitious roots.• Highest callus induction occurs on the IBA treatment with 5mg/L

concentrations. • After almost two months, the of callus induction on 2,4-D treatment can

be observed.• For 2,4-D concentration, 4mg/L showed highest production of callus.

Project two: Establishment of Callus, Adventitious Root cultures of Andrographis Paniculata (Hempedu Bumi)

• Also called as “King of Bitter”• It is annual, branched, and herbaceous plant• Leaves and roots are use for medicinal purposes• Treatment for diabetes, high blood pressure, asthma,

chest pain, snake bites and venom sting.

Materials and method

Establishment of Callus, Adventitious Root and shoot cultures of Andrographis Paniculata (Hempedu Bumi)

Callus InductionExplant used: leaves

of A. Paniculata Media: 2,4-D with 2,

4 and 6mg/l concentration

Condition: 24±2˚C with 16 hours photoperiod

Adventitious root induction

Explant used: leaves of A. Paniculata

Media: IBA with 1, 3 and 5mg/l

concentrationCondition: 24±2˚C with 16 hours photoperiod

Shoot cultures Explant used: nodal

and shoot segment of A.Paniculata

Media: 1 miligram/L meta-Topolin mediaCondition: 24±2˚C

with 16 hours photoperiod

Different elements used in different protocol of surface sterilization

Cycle Elements used

1 10% of clorox (10mins) and 20% of clorox (10minutes)

2 10% of clorox (10mins), clorox treatment repeated 5x

3 10% of clorox (5mins), 20% of clorox (repeated 5x and 1min for each immersion)

Clean cultures

Pictures of surface sterilization

Percentage of clean culture No of cycle Total no of sample No of clean culture Percentage of clean

culture

1 72 28 38%

2 60 19 31%

3 108 64 59%

Result of callus induction on 2,4-D treatment

2,4-D 2mg/L 2,4-D 4mg/L 2,4-D 6mg/L Control 0

1

2

3

4

5

6

7

Callus Induction of Andrographis Paniculata after 2 weeks

Callus Induction

2,4-D concentration

no o

f exp

lant

pro

duci

ng ca

llus o

ut o

f 16

exp

lant

s

6/16 explants pro-ducing callus at 4mg/l 2,4-D con-centration 38%

Formation of callus after 2weeks

Formation of callus from mid-rib of the leaf after

2weeks in dark condition Leaves of Andrographis Paniculata right after sub

culturing

Callus induction of Andrographis Paniculata after 3weeks

2,4-D 2mg/L 2,4-D 4mg/L 2,4-D 6mg/L Control 0

2

4

6

8

10

12

Callus Induction of Andrographis Paniculata after 3 weeks

Callus Induction

2,4-D concentration

no o

f exp

lant

pro

duci

ng ca

l-lu

s out

of 1

6 ex

plan

ts

10 out of 16 explants producing callus at 4mg/l of 2,4-D concentration63%.

At 2,4-D 2mg/l after 3weeks

At 2,4-D 4mg/l after 3weeks

Andrographis Paniculata on IBA treatment

The explant of Andrographis Paniculata on IBA treatment : brown or dormant (maintain)

The formation of adventitious root is yet to be observed

Recommendation for future research: 1. Using IAA media treatment to induce adventitious roots2. Using NAA media treatment to induce adventitious roots

3. Using in combination of IAA and NAA media treatmentt to induce roots

Shoot cultures of Andrographis Paniculata

Nodal segment of

Andrographis Paniculata

shoot segment of Andrographis

Paniculata

Conclusion for project 2:

• The explant on the IBA treatment with all of respective concentrations either turn brown or dormant( maintain).

• The formation of adventitious root on IBA treatment is yet to be observed.

• All of 2,4-D treatment showed formation of callus, however best or optimum concentration is 4mg/L.

Others activities

• Subculturing of Eurycoma Longifolia Jack (Tongkat Ali)

• Sub culturing of Josepine (pineapple) • Sub culturing and prepare media for rose. Ferric

media.• Learned on how to do Biotis• Prepare 30 L of BAP media for bioreactor uses• Subcuturing of Stevia Rebaudiana into lmg m-T

media treatment ( shoot parts)

Acknowledgement