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WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES
Kalimuthu et al.
World Journal of Pharmacy and Pharmaceutical Sciences
SJIF Impact Factor 2.786
Volume 3, Issue 8, 785-793.
Research Article
ISSN 2278 – 4357
DIRECT AND INDIRECT MICROPROPAGATION OF TURNERA
ULMIFOLIA (L.) A MEDICINAL PLANT
*K. Kalimuthu R.Prabakaran and V. Preetha
Plant Tissue Culture Division, PG and Research Department of Botany,
Government Arts College (Autonomous), Coimbatore-641018, India.
Article Received on
20 May 2014,
Revised on 16 June
2014,
Accepted on 10 July 2014
ABSTRACT
The successful protocol for direct and indirect micropropagation of
Turnera ulmifolia was developed. In vitro direct regeneration,
multiplication and rooting of plantlets were observed from the nodal
culture on MS medium supplemented with BAP (2.22µM) and TDZ
*Correspondence for Author
Dr. K. Kalimuthu
Plant Tissue Culture Division,
(0.90 µM). The best callus proliferation and shoot formation from stem
explants were observed in MS medium containing BAP (8.88µM). The
PG and Research Department
higher percentage (87.34%) of root formation was observed in MS
of Botany, Government Arts
medium contain IBA (2.46 µM). The well rooted plants were
College (Autonomous),
acclimatized in shade house with mist chamber
Coimbatore-641018, India.
with 93.83 survival
percentage.
Key Words: Turnera ulmifolia, TDZ, IBA.
INTRODUCTION
In view of the tremendously growing world population, increasing anthropogenic activities,
rapidly eroding natural ecosystem, etc the natural habitat for a great number of herbs and
trees are dwindling. Many of them are facing extinction. Due to the unsustainable loophole in
different methods, researchers have introduced new biotechnological strategies that seem
would open up new vistas in the field of conservation1. To cope up with alarming situation,
the recent exciting developments in biotechnology have come as a boon. Tissue culture has
been successfully used for the commercial production of pathogen-free plants2, and to
conserve the germplasm of rare and endangered species3. The application of biotechnology
especially tissue culture provides an important tool to propagate the selected genotypes4.
Most of the plant raised through seeds are highly heterozygous and show great variations in
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growth, habit and yield and may have to be discarded because of poor quality of products for
their commercial release. Likewise, majority of the plants are not amenable to vegetative
propagation through cutting and grafting, thus limiting multiplication of desired cultivars.
Moreover many plants propagated by vegetative means contain systemic bacteria, fungi and
viruses which may affect the quality and appearance of selected items. In recent years, tissue
culture has emerged as a promising technique to obtain genetically pure elite populations
under in vitro conditions rather than have indifferent populations.
Turnera ulmifolia L belongs to the family Turneraceae. This family is one of at least 28
angiosperm family showing heterostyly5. The genus Turnera is one of the most important
genera of the family turneraceae comprising more than 100 species grouped in nine series6.
Which are distributed largely in tropical and subtropical region of Asia and Africa7. Turnera
ulmifolia L. is a polymorphic polyploid complex of perennial weeds commonly called
‘Yellow Alder’. This variety is a dense compact shrub that has dark green foliage and bright
yellow flowers. Turnera is adopted as ornamental plant, being used as foundation, border,
mass planting and ground cover8. Turnera is also used as a tea for the treatment of disease
related mainly to gastric dysfunction, research has produced data indicating that the plant
extract has a significant antiulcerogenic effect9. T. ulmifolia is popularly used to treat asthma,
rheumatism, indigestion and bronchitis10. It has antioxidant and anti-inflammatory
properties11, 12. To our knowledge there are few reports on tissue culture of T.ulmifolia15.
MATERIALS AND METHODS
Turnera ulmifolia (L.) was collected from the Garden, Department of Botany, Government
Arts College, Coimbatore. Healthy, young and disease free portion of the branches were
selected and used as explants. Healthy explants like nodal explants axillary buds were
selected and washed thoroughly under running tap water for 15 min to wash off the microbes
present on the surface. The explants were cut (1-2 cm) separately and they were washed with
Tween 20 detergent solution for 10 min. After, they were thoroughly washed under running
tap water until the traces of Tween 20 was removed. Remaining steps of surface sterilization
was carried out under aseptic conditions in laminar air flow chamber. The shoots were then
subjected to 70% ethanol treatment for one min and again washed with sterilized double
distilled water at least three to four times. After washing with sterilized double distilled
water, surface sterilization was done with mercuric chloride (0.12%w/v HgCl2) solution for 3
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min and rinsed four to five times with sterilized double distilled water. Sterilized explants
were inoculated on medium for callus and shoot induction.
(i) Culture media and conditions for plant regeneration
The basal medium consisted of MS mineral salts and vitamins13, with 3% sucrose (HI Media,
India) solidified with 0.8% (w/v) agar was used for the culture. Medium without plant growth
regulators served as control. Callus from these primary cultures were transferred to MS
medium containing different concentration of BAP for callus proliferation, BAP and TDZ for
shoots and NAA, IAA and IBA for roots. The pH of the medium was adjusted to 5.8 by 1N
NaOH or 1N HCL after adding the growth regulators prior to autoclaving. The media were
steam sterilized in an autoclave under 15 psi and 121ºC for 20 min. All of the cultures were
incubated under 50µ mol-2S-1 provided by cool white fluorescent lamp for a photo period of
16 h at 25±2ºC and 70-80% relative humidity.
(i) Callus initiation and shoot multiplication
For callus induction, leaf disc, internode and nodal explants were cultured on MS medium
supplemented with growth regulator BAP, at different concentrations for rapid callus
induction and shoot initiation (Table1). Twenty explants were used for each culture. The
percent of explants responding for callus formation were recorded after 35 days. In the
subsequent sub cultures, the multiple shoots from the callus were carried out at the regular
interval of 15-20 days.
(ii) Shoot initiation and multiplication
For shoot induction, the nodal explants were cultured on MS medium supplemented with
plant growth regulators like BAP and TDZ in combinations for rapid shoot organogenesis
(Table 2). Twenty explants were used for each culture. The percent of explants responding
for shoot formation were recorded after 35 days. In the subsequent sub cultures, the multiple
shoots from the nodal explants were carried out at the regular interval of 15-20 days.
Rooting of in vitro multiple Shoots
Shoots with 5-6 cm height were separated and individual shoots were transferred for rooting
to MS medium containing different concentration of NAA, IAA and IBA. The cultures were
incubated under 16 h photoperiod for 15-20 days until the micro shoots developed the roots.
Then the rooting frequency was measured (Table 3).
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Acclimatization and transplantation of plantlets
The well-developed plantlets were removed from the culture bottles and washed with tap
water to remove trace of agar and dipped in fungicide for few minutes. Then the plantlets
were planted on to net pot contains different type of potting media (Table 4) and survivability
rate were determinate after 20 days of step-wise hardening processes. Hardened plants were
transferred to pot containing mixture of decomposed coir waste, garden soil and vermiculite
(1:1:1 ratio). The pots were watered at two days interval under shade house condition. After
60 days, the frequency of survival was calculated.
RESULTS AND DISCUSSION
Table 1: Effect of MS medium and different concentrations of BAP on callus inductions
and shoot formation of Turnera ulmifolia
S. No
MS medium
BAP µM/l
% of stem
producing
callus
% of callus
forming
shoots
1
2.22
39 ±2.09
18±2.0
2
4.44
63±3.25
21±2.31
3
6.66
75±4.02
78±3.54
4
8.88
90±3.01
91±2.58
84±4.70
81±5.87
5
11.10
6
13.32
52±2.60
37±1.72
7
Basal medium
-
-
Nature of
the callus
Days taken for
callus
induction
Green
friable
Green
friable
Green
friable
Friable
and dark
green
Friable and
dark green
Friable and
dark green
-
15
14
10
8
8
9
-
Table 2: Effect of BAP and TDZ on initiation and multiple shoot induction from nodal
explants of Turnera ulmifolia cultured on MS medium
S.NO
BAP µM
TDZ µM
Response %
Shoot
No/explant
1
2
3
4
5
6
2.22
4.44
6.66
8.88
11.10
13.32
0.90
0.90
0.90
0.90
0.90
0.90
86.50
83.33
75.33
56.66
40.66
11.83
2.33±1.36
2.00±0.89
1.83±0.75
1.66±0.42
1.55±0.54
1.33±0.33
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Shoot
No/explants
subculture
4.33±1.03
3.66±0.81
2.83±0.75
2.33±0.51
2.16±0.75
1.66±0.33
Shoot length
cm
4.83±0.75
4.33±1.03
3.33±0.81
2.50±0.54
1.50±0.54
0.66±0.51
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1
2
3
4
5
6
2.22
4.44
6.66
8.88
11.10
13.32
1.8
1.8
1.8
1.8
1.8
1.8
72.16
70.50
55.66
41.33
29.16
18.33
2.00±0.63
1.66±0.51
1.50±0.54
1.50±0.54
1.33±0.51
1.16±0.40
3.33±0.51
3.00±0.63
3.16±0.30
2.66±1.03
2.33±0.33
2.00±0.44
3.00±0.63
2.33±0.51
2.66±0.81
2.00±0.81
1.50±0.54
0.91±0.04
1
2
3
4
5
6
2.22
4.44
6.66
8.88
11.10
13.32
2.7
2.7
2.7
2.7
2.7
2.7
57.16
45.16
32.50
30.83
21.50
11.16
1.83±0.75
1.66±0.81
1.66±0.49
1.33±0.33
1.33±0.51
1.00±0.57
2.00±0.57
2.00±0.63
1.50±0.54
1.33±0.51
1.00±0.63
0.85±0.71
2.83±0.75
2.50±0.54
1.83±0.75
1.50±0.54
1.51±0.03
0.83±0.75
Basal
medium
-
-
-
-
-
-
Table 3: Effect of auxins on root formation of Turnera ulmifolia in MS medium
S.NO
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
MS
medium
+NAA
µM
5.35
10.74
16.05
21.40
26.75
Basal
medium
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MS
medium
+IAA
µM
2.86
5.72
8.58
11.44
14.30
-
MS
medium
+IBA
µM
2.46
4.92
7.38
9.84
12.30
-
-
Response%
70.33
63.83
51.66
24.16
50.16
46.53
34.82
23.62
21.43
13.16
87.34
84.83
44.66
24.16
50.16
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Mean
number of
roots/shoot
Mean root
length (cm)
4.16±0.47
3.26±0.04
2.50±0.42
2.00±0.36
1.16±0.13
3.66±0.51
2.33±0.61
2.00±0.63
1.16±0.75
0.80±0.83
6.00±.63
5.5±0.54
4.33±0.51
2.83±0.75
1.50±0.83
3.85±0.69
3.66±0.51
2.85±0.69
3.00±0.63
1.16±0.75
2.85±0. 75
2.00±0.63
2.00±0.63
1.00±0.89
0.85±0.69
5.66±0. 81
4.83±0.75
3.26±0.04
2.42±053
1.53±0.02
-
-
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Table 4: Evaluation of different planting substrates for acclimatization of in vitro
Plantlets of Turnera ulmifolia
No. of
No. of plants
Survival (%)
Planting substrates
plants
S.NO
transferred
survived
1
Garden soil
50
28
55.83
2
3
4
Vermiculite
Decomposed coir waste
Hardening media(decomposed coir waste:
garden soil: vermiculite)
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50
50
50
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30
40
60.83
80.50
47
93.83
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Kalimuthu et al.
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PLATE – 1 Turnera ulmifolia - Multiple shoots formation from callus and nodal
explants
A. Callus initiation B. Multiple shots from Callus . C, D. &E. Multiple shoots form node
F. Acclimatization
In this experiment, the first two expanded leaves (1-2 cm) at the apex of vigorous shoots were
used as explants. Callus induction was observed in stem explants inoculated in MS media
containing different concentrations of BAP. Depending upon the concentration of hormones
the callus get induced. The highest percentage of callus induction was observed in the
hormone BAP at 8.88 µM/l concentration (90±3.01) followed by 11.10 µM/l (84±4.70) (Plate
1 A and B). Similar results were observed on T. subulata, callus were induced from shoot tip
cultures on MS medium using NAA and BAP with 1.5% sucrose in diffused light as primary
cultures14.
The morphogenic response of nodal explants to BAP and TDZ are summarized in table 2.
Placing explants in a medium without growth regulators (control) induced no shoots.
However, the multiplication rate, shoot number was higher in culture supplemented with
growth regulators. The percentage of response varied with varying concentrations of growth
regulators used. All the concentrations of BAP and TDZ facilitated shoot bud differentiation.
Swelling of dormant axillary bud took place within 8 days of inoculation and then
differentiation into multiple shoots occurred after 35-40 days (Plate 1 C, D & E). Of the
various levels of BAP (2.22 to 13.32 µM) tested along with TDZ (0.90, 2.27 µM) the highest
response percentage (86.50 %), highest number of shoots per explants in initiation
(2.33±1.36), with an average number of shoots per subculture (4.33±1.03) with a mean length
of 4.83±0.75 cm was recorded in MS medium fortified with BAP (2.22 µM) and TDZ (0.90
µM) (Table 3). This is followed by MS+BAP (4.44 µM) and TDZ (0.90 µM) with response
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percentage (83.33%), number of shoots per explants in initiation (2.00±0.89) and an average
number of shoots per subculture (3.66.0±0.81) with a mean length of 4.33±0.75. However,
further increase in the concentration of BAP and TDZ decreased the percentage of response,
number of shoots in initiation, number of shoots per subculture and shoot length. Similar
results were observed on the medium for shoot multiplication in T.ulmifolia nodal explants
MS medium amended with BAP (2.0 mg/l) and IAA (0.1 mgl−1) for shoot induction,
Subsequently, elongation of regenerated shoots could be possible on liquid MS medium
supplemented with 0.5 mgl−1 BAP and Kin each along with 0.1 mgl−1 IAA15. Where as in the
present study in shoot initiation multiplication and shoot elongation occurred on the same MS
medium with BAP (2.22 µM) + TDZ (0.90 µM). For root induction in vitro shoots were
transferred to MS medium supplemented with NAA, IAA and IBA. Root formation from the
basal cut end of the shoots was observed seven days after transfer to the rooting medium
without callus formation. The highest percentage (87.34%) of rooting was achieved in MS
medium containing IBA (2.46µM). The rooting frequency, number of roots per shoot and
length of root were recorded after 20 days of culture with an average mean number of roots
per subculture (6.00±0.63) with a mean length of 5.66±0. 81cm was recorded. These results
are in consonant with root induction in T.ulmifolia half-strength of MS medium
supplemented with 2.0 mgl−1 (IBA). The well-developed healthy in vitro rooted plantlets
were washed thoroughly and planted in different planting substrate (Table 4). Among the four
different substrate used, hardening media composed of decomposed coir waste, garden soil
and vermiculite in the ratio of 1:1:1 in the best medium with the survival percentage of 93.83.
The simple, reproduceable protocol raised in the present investigation could be used for mass
multiplication of the plantlets of the T.ulmifolia.
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