“IN VITRO MICROPROPAGATION,

ACCLIMATIZATION AND ASSESMENT OF GENETIC

FIDELITY THROUGH RAPD ANALYSIS OF Eclipta
alba(L.) : AN IMPORTANT MEDICINAL PLANT

Submitted by
Priyanka Priyadarshinee
Guided by-Mrs. S.S. Sardar
09BOT/13
PG 4TH SEM.
COTENTS
INTRODUCTION
OBJECTIVES
MATERIAL S AND METHODS
RESULTS AND DISCUSSION
CONCLUSION
REFERENCE
INTRODUCTION
• Eclipta alba (L.) Hassk. (Asteraceae) is a small,
branched, annual herb with white flower heads, native
to tropical and subtropical regions of the world.
The plant is erect as prostate with prolific branching,
hairy stem. The leaves are opposite, sessile, lanceolate
& 2.5-7.5 cm long. The stem is circular and brown in
colour.
The plant is traditionally used in the treatment of liver
diseases, skin disorders, premature graying of hair, and
enhance the memory .
To fulfill the demand of pharmaceutical industries & to
avoid using contaminated raw materials, there is need
to establishment of an efficient invitro regeneration
system.
Plant tissue culture offers a method for large scale
multiplication of various medicinal herbs such as
Eclipta alba and also secondary metabolite production.
The present study was undertaken to study in vitro
propagation and acclimatization from nodal explant of
Eclipta alba using various plant growth regulators and
to check their genetic homogeneity through RAPD
analysis.
OBJECTIVES
The objectives of present work
1.Effect of basal media and cytokinins on shoot regeneration.
2.Effect of carbohydrates and nutrients on shooting.
3. Effect of cytokinin on shoot proliferation.
4. Effectof cytokinins and auxin on shoot proliferation and
multiplication.
5. Effect of cytokinin and auxin on callus proliferation.
6. Effect of auxins on rooting.
7. Field Acclimatization.
8. Genetic fidelity analysis through RAPD markers.
MATERIALS AND METHODS
 The plant Eclipta alba was collected and grown in the
garden.
Nodal segments were used as explants. Surface
sterilization of the explant was done by 0.1% mercuric
chloride solution.
The MS basal media was taken for tissue culture.
The above media was supplemented with different
concentrations and various combinations of plant
growth regulators (BAP,IAA,IBA,NAA,2,4-D) for
induction of multiple shoot and roots.
The pH of the media was adjusted to 5.6-5.62.
 The agar was added for solidification of media.
The media was sterilized at 121 ̊c and 1.06 kg/cm2
pressure for 15 minutes.
After sterilization of media the explants were
inoculated into the medium under aseptic conditions
and kept in the culture room at 26 ̊c for 16 th
photoperiod.
Explants with well developed shoots and roots were
subcultured at an interval of 2-3 weeks in culture.
When both in vitro grown plantlets and callus
regenerated plants having well developed shoots and
roots, they were transferred to pots containing sterile
soil, sand and vermicompost and kept in green house
for atleast 20 days.
Genomic DNA was isolated from both mother and
tissue cultured plant by C-TAB method.
Under optimized PCR condition RAPD analysis was
carried out.
RESULTS AND DISCUSSION
1.Effect of basal media and cytokinins on
shoot regeneration.
• Nodal explants of E. alba were cultured on three types of
media and supplemented with various concentrations of
BAP for shoot regeneration.
Among the three different media and growth regulators
tested, MS medium was found to support a better response
for shoot regeneration than B5 and SH media .
 MS medium was more effective than other media for other
medicinal plants as well as E.alba.
Among the three different media used, the explants in MS
medium appeared healthy and grew vigorously.
 

2.EFFECT OF CARBON SOURCES AND SHOOT REGENERATION

Nodal segments were cultured on MS medium

containing 3% (w/v) glucose, fructose, and sucrose,
respectively.
These media were supplemented with 1mg/l BAP and
0.8% (w/v) agar. Among the three carbon sources,
sucrose proved to be better for shoot regeneration
than fructose and glucose .
3. EFFECT OF PLANT GROWTH REGULATOR BAP ON
SHOOT INDUCTION OF E.alba.

TABLE-1
Treatment Medium(mg/l) Shoot length No. of leaves Average no. of Percentage of
No. (cm) (M±SE) per explant shoots per response
(M±SE) explant
(M±SE)
T1 MS+ 0.5 BAP 8.1±0.28 4.2±0.36 2.6±0.33 70 %

T2 MS+1.0 BAP‫٭‬ 5.8±0.27 8.9±0.59 16.2±0.42 96%

T3 MS+1.5 BAP 7.5±0.43 7.3±0.54 10.1±0.36 65 %

T4 MS+2.0 BAP 6.8±0.36 5.8±0.57 8.6±0.56 55%

T5 MS+2.5 BAP 6.1±0.25 4.6±0.41 6.3±0.43 40%

T6 MS+3.0 BAP 5.4±0.30 4.1±0.44 5.4±0.48 25%

Replicates-15
Cultural periods: 2weeks
M±SE: Mean±Standard error
4.EFFECT OF PLANT GROWTH REGULATORS BAP AND IAA ON
SHOOT MULTIPLICATION OF E.alba.
TABLE-2
Treatment Medium(mg/l) No. of shoots Shooting length
No. per explant (M±SE)
(M±SE)
T1 MS+1.0 BAP+0.25 IAA‫٭‬ 8±0.58 6.5±0.56

T2 MS+1.0 BAP+0.5 IAA 3.7±0.67 3.9±0.49

T3 MS+1.0 BAP+1.0 IAA 1.6±0.52 3.1±0.27

T4 MS+2.0 BAP+0.25 IAA 4±0.51 2.4±0.33

T5 MS+2.0 BAP+0.5 IAA 6±0.26 2.9±0.45

T6 MS+2.0 BAP+1.0 IAA 3±0.44 1.7±0.30

Replicates-15
Cultural periods: 2weeks
M±SE: Mean±Standard error
9

5
No of shoots per
explant
4
Shoot length
3

0
T1 T2 T3 T4 T5 T6
5. EFFECT OF PLANT GROWTH REGULATORS BAP IN
COMBINATION WITH 2,4-D ON CALLUS INDUCTION OF E.alba.
TABLE-3
Treatment Medium(mg/l) Callus induction Response Remarks
No. frequency(%)
T1 MS+0.5 BAP+0.5 2,4-D 20 C+
MS+0.5 BAP+1.0 2,4-D‫٭‬ 90 C+++ Callus was green,
T2 fragile, healthy and fast
growing.
T3 MS+0.5 BAP+1.5 2,4-D 60 C++
T4 MS+0.5 BAP+2.0 2,4-D 55 C++

T5 MS+0.5 BAP+2.5 2,4-D 40 C+

T6 MS+0.5 BAP+3.0 2,4-D 15 C+

T7 MS+1.0 BAP+0.5 2,4 D 18 C+

T8 MS+1.0 BAP+1.0 2,4 D 50 C++
T9 MS+1.0 BAP+1.5 2,4 D 37 C+
T10 MS+1.0 BAP+2.0 2,4 D 22 C+

T11 MS+1.0 BAP+2.5 2,4 D 07 C+

T12 MS+1.0 BAP+3.0 2,4 D 0 C+

Replicates-15
Cultural periods: 2weeks
M±SE: Mean±Standard error
100

90

80

70

60

50 Callus induction
frequency
40

30

20

10

0
T1 T2 T3 T4 T5 T6 T7 T8 T9 T10 T11 T12
6. EFFECT OF PLANT GROWTH REGULATORS BAP AND IAA ON
MULTIPLE SHOOT REGENERATION FROM CALLUS OF E.alba.

TABLE-4
Medium(mg/l) Total no. of Explant showing Percentage
Treatment explants multiple shoot of response
No. taken regeneration(M±SE)

T1 MS+1 BAP+0.25 IAA 15 3.1±0.45 20.7

T2 MS+2 BAP+0.25 IAA 15 7.5 ±0.59 50

T3 MS+3 BAP+0.25 IAA‫٭‬ 15 12.2±0.74 81.3

T4 MS+1 BAP+0.5 IAA 15 4±0.51 26.7

T5 MS+2 BAP+0.5 IAA 15 5.3±0.42 35.3

T6 MS+3 BAP+0.5 IAA 15 8.7±0.72 58

Replicates-15
Cultural periods: 2weeks
M±SE: Mean±Standard error
90

80

70

60

50
Column1
Explant showing multiple
40
shoot regeneration

30

20

10

0
T1 T2 T3 T4 T5 T6
-7. EFFECT OF PLANT GROWTH REGULATORS OF IAA,IBA,NAA ON ROOTING OF IN VITRO
SHOOTS OF E.alba

TABLE-5
Treatment No. Medium(mg/l) Rooting No. of roots per Root length
. Response (%) explant (M±SE) (cm) (M±SE)
T1 MS+0.5 IBA 80 19.8±0.82 2±0.51
T2 MS+1.0 IBA‫٭‬ 95 30.6±0.87 4.1±0.71
T3 MS+2.0 IBA 73 8.2±0.41 3.9±0.75
T4 MS+0.5 IAA 75 5.6±0.56 3±0.28
T5 MS+1.0 IAA 62 8.8±0.69 3.8±0.32
T6 MS+2.0 IAA 50 8.1±0.82 3.2±0.28
T7 MS+0.5 NAA 58 8.5±0.38 1.2±0.19
T8 MS+1.0 NAA 59 10.6±0.49 2±0.28
T9 MS+2.0 NAA 40 13.2±0.67 1.4±0.21

Replicates-15
Cultural periods: 2weeks
M±SE: Mean±Standard error
100

90

80

70

60

50

40 Column2
30

20

10

0
IBA IAA NAA
35

30

25

20

15

Column2
10

0
IBA IAA NAA
 4

3.5

2.5

Root length
1.5

0.5

0
IBA IAA NAA
8. HARDENING OF ROOTED PLANTLETS
TABLE-6
Serial Hardening medium pH Porosity Water Survival
No. holding
capacity

1. Soil 6.36 Highest 10% 6%

2. Soil:Sand(1:1) 5.15 Least 19% 2%

3. Soil:Vermicompost 5.64 In combination with soil 15% 94%

(1:1) enhances the soil
porosity
100%

90%

80%

70%

60%

50%
Water holding capacity
40% Column1

30%

20%

10%

0%
SOIL SOIL:SAND SOIL:V.C
A-Callus induction
B-Callus multiplication and differentiation
(A )Adventitious shoot emerging from the edge of the explant. (B) Induction of multiple shoot
from peripheral region of explant. (C) Induction of multiple shoot from basal region of
explant. (D) Regeneration of multiple shoots into a clump. (E) Shoot multiplication
(F) Rooting in regenerated shoot on growth regulator free MS medium.
(G) Well-hardened plants of E. alba.
RAPD banding pattern of micropropagated and
field-grown mother plants of Eclipta alba
Primer Sequence Total bands Range of
amplicons(bp)
OPA7 GAAACGGGTG -
OPA9 GGGTAACGCC
OPC2 GTGAGGCGTC
OPC20 ACTTCGCCAC
OPD8 GTGTGCCCCA
OPD18 GAGAGCCAAC
OPN4 GACCGACCCA
OPN11 TCGCCGCAAA
OPAF5 CCCGATCAGA
OPAF15 CACGAACCTC
CONCLUSION
 The In vitro micropropagation of E.alba through
nodal explant shows both direct and indirect
regeneration, through callus.
Both the regenerations shows multiplication of
shoots.
All the shoots were rooted well in IBA(1.0).
All the in vitro propagile shows 94% of hardening in
soil:vermicompost (1:1).
All the micropropagated plantlets after hardening
shows the genetic fidelity with the mother.
So, there is no genetic variability.
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