EFFECT OF MEDIUM PH AND SUCROSE CONCENTRATIONS ON
ADVENTITIOUS ROOTS INDUCTION OF LABISIA PUMILA
1
NOR’AISHAH HASAN, 2SOBRI BIN HUSSEIN, 3RUSLI IBRAHIM
1
Faculty of Applied Science, Universiti Teknologi MARA (UITM), Kuala Pilah, Negeri Sembilan,
Malaysia
2,3
Agrotechnology & Bioscience Division, Malaysian Nuclear Agency, Bangi, Kajang, Selangor, Malaysia
E-mail: [email protected]
Abstract- Adventitious roots are roots that arise from parts of the plant not originating from the embryonic root; such as roots
originating from stems or leaves. Adventitious roots are a potential source for the production of valuable plant secondary
metabolites on a commercial scale. A study was undertaken to investigate the effects of medium pH and sucrose concentrations
on adventitious roots induction from leaf explants of Labisia pumila. Full strength of Murashige and Skoog (MS) medium
supplemented with 5 mg/L of indole-3-butyric acid (IBA) were used in this study. Tukey’s analysis showed that there was a
significant difference between the parameter studied with the percentage of rooting, number of roots per explants and dry
weight of roots. pH 6.0 is the optimum pH value for the induction of L. pumila roots compared to pH 4.0, 5.0, 5.8 and 7.0. The
highest rooting efficiency was evidenced by the highest value recorded in rooting percentage (89.3 ± 9.5 %), (13.4 ± 0.99)
roots per explants and (0.0364 ± 0.005g) dry weight of roots. It showed that 5% of sucrose was the optimum concentration for
L. pumila as compared to 0, 1, 2, 3, 4, 6, and 7 % that produced lower rooting efficiency. The highest value was recorded at 5
% of sucrose with a rooting percentage (72 ± 13.2 %), (10 ± 4.45) roots per explants and (0.044 ± 0.014 g) dry weight of roots.
No adventitious roots formed in MS media without sucrose.
Index Terms- Adventitious root, carbon source, Labisia pumila, medium pH, sucrose
The presence of sucrose in the induction phase may
cause cells to be recruited for root inducing thus
improving the rooting response [7]. Studies had been
made by [8] using Chrysanthemum morifolium using
sucrose as a carbon sources. In addition, pH medium
also one of the important factors that affect the growth
and development of the plants. According to the ‘acid
growth hypothesis’, plants regulate cell expansion
through modifying the pH around the cell wall and
thereby its extensibility, which increase at low pH [9].
A large body of evidence supported this hypothesis in
shoot coleoptiles or expanding leaves [10]. However
results on root have been less conclusive. Lowering
the pH of the medium may either promote root
elongation or have little effect [11]. The ‘acid growth
hypothesis’ of roots, although well-known has been
subjected to only limit experimental testing with only
few studies focused on rooting [12]. Of these studies,
only examined elongation of root segments have been
reported by [13].Therefore, this study was conducted
to investigate the effects of these two factors; medium
pH and carbon source on the adventitious roots
induction from leaf explants of L. pumila. Results
obtained may be used as a recommendation to
establishment of root culture system of L. pumila by
adventitious roots induction in bioreactor for the
production of pharmaceutically and nutraceutically
important metabolites.
I. INTRODUCTION
Labisia pumila is an important medicinal plant widely
used for post partum illnesses and human dermal
fibroblast [1]. It is native plant of Southeast country
and locally known as "Kacip Fatimah". Its roots are
the main source of flavanoid and phenolic possessing
various pharmaceutical activities such as antioxidant,
anti-inflammatory [2], anti-microbial [3] and anti
cancer activity [4]. Multiple use of this plant species
have necessitated its large scale collection as raw
material to the pharmaceutical industry, leading to
over exploitation and disappearing in the wild. The
conventional propagation of this species is difficult
therefore in vitro propagation methods developed by
many researchers, to satisfy the increasing demand for
L. pumila [5]. In vitro root culture has become an
alternative method for the production of valuable
secondary metabolites on commercial scale. In in vitro
method, different cultivars of the same species differ
in terms of requirements for growth condition. Thus, it
is necessary to optimize culture condition to achieve
satisfactory micropropagation rates. Besides the plant
growth regulators, another factor limiting successful
plant micropropagation is carbon sources and medium
pH. Plant tissue needs carbohydrates as metabolic
“building block” and energy sources. The carbon
sources responsible for the development of shoots,
shoots elongation and number of leaves growth.
Among the types of carbon sources, sucrose was the
most effective treatments for shoots development
compared to fructose, glucose and maltose. According
to [6], the ability of sucrose to easy translocatable and
resistant to enzymatic degradation due to its
non-reducing nature is the best quality to be chosen.
II. METHODOLOGY
A. Plant Sample
In vitro plantlets of Labisia pumila that cultured on
basal Murashige and Skoog (MS) medium [14] were
used as plant materials in this study. Only young and
Proceedings of The IRES 14th International Conference, Paris, France, 7th November 2015, ISBN: 978-93-85832-27-7
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Effect Of Medium Ph And Sucrose Concentrations On Adventitious Roots Induction Of Labisia Pumila
fresh leaf explants were selected throughout the
experiment.
B. Culture medium
The leaf explants were cultured in full- strength MS
medium
supplemented
with
5 mg/L
of
indole-3-butyric acid (IBA) as described by [15] In
this study, MS basal medium without sucrose served
as control. A total of 5 explants were placed on each
culture medium. Each treatment, which consisted of 5
explants, was repeated three times. All root cultures
were incubated at 25 ± 1oC.
C. Effect of carbon sources and pH medium
Leaf explants were used to induce adventitious roots
from L. pumila. The leaf (0.5 cm x 0.5 cm) explants
were aseptically excised from intact in vitro plant. In
the study of effects of sucrose concentration on the
adventitious roots formation, the leaf explants were
cut transversally along the leaf mid-vein and cultured
with the abaxial surface in contact with the medium
containing 0, 1, 2, 3, 4, 5, 6, and 7% (w/v) of sucrose.
MS basal medium without sucrose served as control.
Meanwhile, for the determination of effects of
medium pH on adventitious roots induction, the leaf
explants were cultured on the MS medium containing
5% (w/v) sucrose and 5 mg/L of IBA. The pH of the
medium was adjusted to pH 4.0, 5.0, 5.8, 6.0 and 7.0
with 0.1M HCl and 0.1M NaCl prior to autoclaving.
All the cultures were incubated in a culture room and
allowed to grow at 25oC ± 1oC.
D. Data Collection
The initiation of root explants was recorded on the
basic of visual observations. Results were expressed as
mean ± standard deviation. Observation on explants,
the rooting percentage (%), dry weight (g), and
number of roots formed per explants and any
morphology changes were recorded continuously for 4
weeks. As for dry weight, the adventitious root
induced in each treatment were excised and collected
after 4 weeks of culture. Prior to blotting of the roots,
the weight of the filter paper was determined and
recorded. The roots were then collected on filter paper
and allowed air dry for 15 minutes. In order to measure
the dry weight, the roots collected were oven-dried at
mild temperature until a constant weight was obtained.
E. Statistical Analysis
All data collected from each treatment were analyzed
statistically using One Way Analysis of Variance
(One-way ANOVA) followed by the Tukey’s HSD
test with the mean comparison was made by at least
significant differences at the 5% probability level (p ˂
0.05). All the statistical analysis was conducted using
the statistical software of SPSS (version 12.0).
that affecting the adventitious roots formation. pH
value is expressed by the amount of hydrogen ion
concentration of the medium. Extremely low or high
pH was found to affect the nutrients and mineral
uptake of plant cell from its surrounding. It is known
that pH not only affect the utilization of medium
components such as macroelements, microelements
and growth regulators [16] but also on chemical
reactions, especially those catalyzed by enzymes [17].
In this study, pH 6.0 was indicated as the optimum pH
value for adventitious root induction from leaf
explants of L. pumila as it exhibited the highest
rooting percentage (89.3 ± 9.5 %), (13.4 ± 0.99) roots
per explants and (0.0364 ± 0.005) dry weight of roots.
This result is align with [18] stated that a slightly
acidic pH (at the range 6.3 - 6.8) was the ideal pH for
most plant, as at this range there is a well balanced
availability of all nutrients. However, according to
[19] pH value in the range of 5.5-5.8 is recommended
for in vitro culture of the majority species. In contrast,
the most effective pH value is specific to the
individual plant species and even cultivars, and has to
be determined experimentally. Low pH medium (4.0,
5.0 and 5.8) present the lower rooting efficiency
compared to pH 6.0 (Fig. 1, 2 and 3) with (4.0 ± 0.9 %,
12.0 ± 5.3 % and 58.6 ± 8.0 % ) of rooting percentage,
(0.6 ± 0.3, 0.8 ± 0.5, 8.8 ± 1.23) number of roots per
explants and (0.002 ± 0.001g, 0.008 ± 0.003g and
0.0168 ± 0.0032g) of dry weight, respectively.
Theoretically, low pH condition (high H+), net H+
release by H+ ATPase activity is restricted, thus lead to
a shortage of energy supply to the H+ ATPase and
limiting dry matter production during plant growth.
Net H+ release by H+ ATPase activity is essential for
nutrient uptake, turgor generation, external
acidification for cell wall loosening and cytoplasmic
pH regulation [20]. It is unlikely that root growth was
totally inhibited because of lack of nutrient uptake.
Since the external medium was acidified by the pH
adjusted, H+ activity in low pH treatments should have
been sufficient for cell wall loosening. It was also
reported that root growth at high acidity condition was
primarily affected not in the meristematic tissue but in
the extension zone. This was in correlation to the
results obtained in this study whereby at low pH
condition, certain degree of roots was still induced but
further elongation was inhibited.
III. RESULTS AND DISCUSSIONS
This study reports the effect of medium pH and
sucrose concentration on adventitious root induction
of L. pumila. The results express as the percentage of
rooting, number of roots per explants formed and dry
weight of roots. The medium pH is a limiting factor
Proceedings of The IRES 14th International Conference, Paris, France, 7th November 2015, ISBN: 978-93-85832-27-7
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Effect Of Medium Ph And Sucrose Concentrations On Adventitious Roots Induction Of Labisia Pumila
In addition, acidic condition (pH 3.0 and 4.0)
contribute to the low availability of calcium (Ca) and
phosphorus (P) thus resulted to the low rooting
growth. This is because phosphorus will react with
Ferric (Fe) and Aluminium (Al) to produce insoluble
complexes that are not readily available for plant cell
uptake while Calcium wills insufficient supply for leaf
explants [21]. Ca and P is important mineral required
for plant cell in root growth and development.
According to [22], at lower pH value (pH 3.0 and 4.0),
Al, Fe and Manganase (Mn) may be soluble in
sufficient quantities to be toxic to the growth of some
plants. Aluminium toxicity can limit the plant growth
in acidic medium and cause detrimental effects to
plant root development. Thus as shown in this study,
some adventitious roots were found in acidic pH, the
amount of roots induced was relatively low.
Tukey’s analysis showed that there was a significantly
difference (p ≤ 0.05) between pH medium and
percentage of rooting, number of roots per explants
and dry weight of roots. Rooting efficiency of L.
pumila leaf explants increased to the increase of
medium pH from 4.0 to 6.0. It was demonstrate by the
percentage of rooting, number of roots per explants
and dry weight of roots (Fig. 1, 2 and 3). However,
percentage of rooting, number of roots per explants
and dry weight of roots was decreased from pH 6.0 to
7.0 with (89.3 ± 9.5 % to 75.3 ± 4.2 %), (13.4 ± 0.99 to
11.3 ± 4.2) and (0.00364 ± 0.0053 to 0.0250 ± 0.01g),
respectively. [23] reported that increasing culture
medium alkalinity to pH greater that 7 may reduce the
availability of P, Ca, K, Mg, Fe, Mn, Zn, Cu and Co.
As a result, the minerals will form insoluble complex
and become limited and harmful for plant cells as they
are important in root development and elongation.
In this study, control medium without addition of
sucrose (0% w/v) showed no adventitious roots
formation until 4 weeks of culture. This result is
accordance with Perez et al (2004), stated that most in
vitro cultures are not able to proliferate properly
without an exogenous supply of carbohydrates. The
MS medium containing 5% of sucrose showed the
optimum concentration which exhibits the highest
rooting percentage (72 ± 13.2 %), (10 ± 4.45) roots per
explants and (0.044 ± 0.014 g) dry weight of roots.
This result was aligning with the study of [24] using
Echinacea angusitifolia and [25] using Panax ginseng.
However, the result was contrast to the study by [26]
using Fagus species and [27] using Brassica napus.
Thus, it can be concluded that the optimal sucrose
concentration for induction of adventitious roots was
varied with different species of plants [28]
Fig. 1: Effect of the addition of medium pH on the adventitious
roots induction from in vitro explants of Labisia pumila. (A) pH
4 (B) pH 5 (C) pH 5.8 (D) pH 6 (E) pH 7
Fig. 2: Percentage of rooting from leaf explants of L. pumila
cultured on MS medium supplemented with different pH. Bars
indicate the standard deviation. Different letters within each
column indicate significant at p < 0.05 in Tukey’s test
Fig. 3: Number of roots per explants induced from leaf
explants of L. pumila cultured on MS medium supplemented
with different pH. Bars indicate the standard deviation.
Different letters within each column indicate significant at p <
0.05 in Tukey’s test
Fig. 4: Dry weight of adventitious roots induced from leaf
explants of L. pumila cultured on MS medium supplemented
with different pH. Bars indicate the standard deviation.
Different letters within each column indicate significant at p <
0.05 in Tukey’s test
Proceedings of The IRES 14th International Conference, Paris, France, 7th November 2015, ISBN: 978-93-85832-27-7
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Effect Of Medium Ph And Sucrose Concentrations On Adventitious Roots Induction Of Labisia Pumila
Values represent means ± standard deviations for three
replications. Values followed by same letter are
significantly different at the p ≤ 0.05 level, according
to the Tukey test.
A further increase in sucrose concentration from 5% to
7% resulted in lower rooting efficiency from leaf
explants of L. pumila. In term of percentage of rooting,
it showed that it decreased from 72 ±13.2%, 50.6 ±
2.0%. A similar pattern also showed in number of
roots formed per explants (10 ± 4.45 to 1.6 ± 1.2) and
dry weight of roots (0.012 ± 0.014g to 0.018 ±
0.001g), respectively. This may be due to excessive
amount of sucrose dissolved in the culture and give
rise to higher osmotic potential in culture medium than
in leaf explants. As a result plant cell will become less
sensitive and encountered a water deficiency problem
as water is important to dissolved plant hormones
especially exogenous auxins. Studied has been
reported in Orthosiphon stamineus in lower rooting
efficiency due to water deficiency [29].
CONCLUSION
pH medium and sucrose concentration play an
important factor affected the adventitious root
induction in L. pumila. As root have been recognize as
the major contributor for the production of secondary
metabolites, further studies should be conducted on
other factors such as concentration of plant growth
regulators and type of explants before a root culture
system are cultivated in bioreactor for the production
of pharmaceutically and nutraceutically important
metabolites.
Fig. 5: Effect of the addition of sucrose at various
concentrations on the adventitious roots induction from in vitro
explants of Labisia pumila. (A) 0 % (B) 1% (C) 2% (D) 3 %
(E) 4 % (F) 5 % (G) 6 % (C) 7 %
Tukey’s analysis showed that there was a
significantly difference (p ≤ 0.05) between sucrose
concentration with percentage of rooting, number of
roots per explants and dry weight of roots. Figure 2
showed there was an increasing trend in rooting
efficiency from 1% to 5 % of sucrose. It showed with
(8 ± 1.4% to 72 ± 13.2%) percentage of rooting, (0.4 ±
0.09 to 10 ± 4.45) number of roots per explants formed
and (0.0048 ± 0.003 to 0.044 ± 0.014g) dry weight of
roots. Low concentration of sucrose contributed to the
lower rooting efficiency. This may be due to
inadequate supply of carbohydrate for leaf explants to
generate sufficient energy which needed in rooting
process. As a result, less adventitious root was induced
from these concentrations.
ACKNOWLEDGEMENT
Author thank to Universiti Teknologi MARA,
Malaysian Nuclear Agency and Universiti Putra
Malaysia for providing greenhouse and funding during
the research activities. Research was fully funded by
RAGS (600-RMI/RAGS 5/3 (189/2013).
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