Int.
J. Life. Sci. Scienti. Res., 4(3):
1736-1743, May 2018
Regeneration
of Plantlets from Rhizome Bud Explants of Lasia spinosa (Lour.) Thwaites- A Medicinal Plants of Assam
Puspita
Hore1* and Bhaben Tanti2
1Research Scholar, Department of Botany, Gauhati University, Assam, India
2Professor, Department of Botany, Gauhati University, Assam, India
*Address
for Correspondence: Puspita Hore, Research Scholar, Department of
Botany, Gauhati University, Assam, India
ABSTRACT-
Innumerable medicinal plants are commercially
propagated through tissue culture for large production of elite material.
Rhizome buds of Lasia spinosa (Lour.)
Thwaites could be induced on Murashige and Skoog (MS)
medium supplemented with different concentrations and combinations of kinetin (kin) and 6-benzylaminopurine (BAP) alone and in combination with Naphthaleneacetic acid (NAA). Lasia
is one of the traditionally important plants of Assam, which is employed
in the treatment of gastrointestinal diseases,
respiratory diseases and skin infections. This plant is also a rich source of
dietary fibers, reported containing polyphenols,
ascorbic acid, and hydrocyanic acid. The present study aimed to establish
producible protocol for in vitro regeneration
of Lasia spinosa using rhizome bud explants.
For shoot proliferation, among the various concentrations, 3.0 mgL-1
BAP showed the highest shoot regeneration frequency of 88.2±2.8%, the highest
number of shoot were recorded as 1.9±0.45 in L. spinosa, but the highest
shoot length (4.5±0.07cm) was observed at reduced concentration of BAP(1.0 mgL-1). Plantlets rooted on ½
strength MS medium augmented with 0.1-1.0 mgL-1 either NAA or IBA for L. spinosa for root formation. The highest percentage (79.5±2.6%),
maximum number of rootlets/ shootlet (4.0±0.46) and mean length of rootlets
(3.25±0.06cm) were observed in L.
spinosa. Our findings have paved a way for future investigation on another
mode of regeneration like haploid production, anther
culture etc and also for the commercial and rapid propagation of L. spinosa.
Key words: Lasia spinosa, Rhizome bud, Plantlets,
Kinetin, 6-benzylaminopurine
INTRODUCTION- Lasia spinosa
(Lour.) Thwaites (Araceae)
is an important plant in folklore medicine. Traditionally, the leaves of this herb
are commonly employed in the treatment of gastrointestinal diseases,
respiratory diseases and in skin infections. The plant parts of Lasia
spinosa has a number of medicinal uses like leaves and corms are used to
cure piles [1], tubers are used for the treatment of rheumatoid
arthritis, constipation, and to purify blood in Rajshahi
and Natore district of Bangladesh [2],
rhizome possesses antioxidant capacity [3,4], antimicrobial property
and cytotoxic activity [5,6]. In Northeast India, leaves and rhizomes
are commonly used in traditional medicine for treating joint-pain and skin
infections [7]. The genetic
diversity of medicinal plants in the world is getting endangered at an alarming
rate because of ruinous harvesting practices and over-harvesting for production
of medicines. As conventional propagation method through rhizome axillary buds is time consuming and provides a limited
number of propagules, it is necessary to promote
rapid production of L. spinosa through tissue culture techniques for
its commercial availability and conservation.
MATERIALS AND METHODS
Explant sterilization- Excised
micro-cutting of rhizome bud from the source plants were used as explants. The
explants were coarsely trimmed to a size of 3 cm and washed thoroughly under
running tap water for 10 min and then treated with liquid detergent (5% (v/v)
Tween-20) for 15 min. Later these explants were washed with double-distilled
water for 10 min. The explants were then sterilized with 0.1% (w/v) mercuric
chloride (HgCl2) for 5 min and washed several times with sterile H2O
to remove all traces of HgCl2. After a final wash, the explants were
spread on the pre-sterilized petri-dishes lined with
sterile blotting paper inside a laminar airflow chamber. They were then trimmed
finely to the appropriate size.
Inoculation and incubation- Excised micro-cuttings of rhizome bud (1-2cm) was dissected out
and all the inoculation operations were carried out under strict aseptic condition
inside a Laminar Air Flow chamber, which was made sterile by the incessant
exposure of germicidal UV rays for half an hour before use. All operations were
carried out using pre-sterilized instruments and glassware. Explants were then
aseptically introduced into culture vessels. The culture tubes
were then plugged tightly with non-absorbent cotton plugs and the culture
bottles and petri-plates were sealed tight with
sealing film. All cultures were
incubated under irradiance of 70 μmol m-2
s-1 for 16 hours photoperiod and temperature of 25±10oC
and with a relative humidity of 55 - 60%.
Regeneration of
plantlets- Basal medium supplemented with different
concentrations of kinetin (1.0, 2.0, 3.0, 4.0
mgL-1) and Benzyl amino purine
(BAP) (1.0, 2.0, 3.0, 4.0 mgL-1) individually and in combinations
with Naphthalene acetic acid (NAA) (0.5, 1.0 mgL-1) were tested for
the induction of callus and regeneration of shoot and root from micro-cutting
of rhizomes bud explants [8]. Sub-culturing was done at 14 day intervals
onto fresh medium for 6 weeks to induce in vitro regeneration of shoot. Shoot
buds were further cultured for elongation in the same medium supplemented with
low concentration of cytokinin. The responses of each
explant with regard to the induction of shoots, the
length of shoot and the percentage of response were recorded after 6 weeks in
culture.
In vitro rooting- In vitro
regenerated shoots were rooted on half strength medium supplemented with
different concentrations of auxin [9] (NAA
and IBA) alone. The response of each explant with
regard to the number of roots induced and root lengths per shoot after 2 weeks
in culture were recorded.
Hardening and acclimatization- In vitro grown
plantlets were gently removed from culture tubes and washed with slightly warm
(37°C) sterile double distilled H2O to remove all traces of nutrient
medium [10]. After removing media, plants were dipped in 1% w/v
solution of Bavistine to prevent any fungal infection
to newly developed plants. After Bavistine treatment the
plantlets were carefully planted in plastic pots containing soilrite.
The plantlets were irrigated by sprinkling with 0.5x MS inorganic salts for
three to four times per day for seven days. Plantlets were acclimatized for two
weeks in an aseptic culture room under (16 hrs photoperiod at 28 ± 2°C; 8 h in
dark at 25 ± 2°C) conditions. Further, the plantlets were exposed gradually to
sunlight for acclimatization and were maintained in a garden.
Statistical analysis- Statistical data for the percentage of response
per explants with different concentrations and combinations of cytokinin and auxin with basal MS medium (shoot regeneration, shoot
lengths, number of roots and root lengths) were recorded. Thus obtained data
were analyzed statistically using SPSS 16.0 software (IBM Corporation SPSS,
North America). [11]
RESULTS- Regeneration
potential of micro-cutting of rhizome bud
was explored on MS medium supplemented with various plant growth regulators and
results are summarized in Table 1.
Micro-cutting of rhizome bud explant
remained green and fresh but failed to develop multiple shoots in growth
regulators free MS medium (control). All micro-cuttings of rhizome bud cultured
on MS medium supplemented with various concentrations of kinetin and BAP
individually and in combination with NAA have developed healthy shoots. Micro
cuttings of rhizome bud cultured on MS medium fortified with cytokinin alone induced multiple shoots at a lesser
frequency compared to the media supplemented with combination of cytokinin and auxin (Fig. 1). All
the concentrations of BAP and kinetin facilitated shoot bud differentiation but
BAP being more efficient than kinetin in terms of percent regeneration, number
of shoots and shoot length. Among the various concentrations of BAP and kinetin
tested, 3.0 mg/L BAP showed the highest shoot regeneration frequency of 88.2 ±
2.8%, the highest number of shoot were recorded as 1.9 ±0.45 in L. spinosa, but
the highest shoot length (4.5 ± 0.07cm) was observed at reduced concentration
of BAP (1.0 mg/L).
The synergistic influences
of auxin with cytokinin was
evident when combination of optimal concentration of each cytokinin
with different concentrations of NAA (0.5 and 1.0 mg/L) were tested (Table 1).
Addition of NAA markedly enhanced the percent regeneration and number of shoots
for the Lasia spinosa used for in vitro propagation. Among all
the cytokinin and auxin
combinations, the maximum percent regeneration was found as 90.6±2.8 and number
of shoots (3.6±0.55) per explants were obtained at 3.0 mg/L BAP + 1.0 mg/L NAA.
But the highest shoot length (4.21± 0.06cm) was recorded at the combination of
1.0 mg/L BAP + 0.5 mg/L NAA.
.png)
Fig. 1: Different
stages of in-vitro regeneration
of Lasia spinosa from
micro-cutting of rhizome bud explant A= Plant in wild
condition, B= Inoculation of micro cutting of rhizome bud in MS medium, C=
Initial days after inoculation in MS medium, D= Direct organogenesis from explant in 3:1 mgL-1 of BAP and NAA in MS
medium, E-F= multiple shoot regeneration in 3:1 mgL-1 of BAP and
NAA, G= Shoot elongation in 1 mgL-1 BAP, H-I= Stages of rooting in
0.5 mg/L IBA in ½ MS medium
Table 1: Effect of cytokinin and auxin individually
and in combinations for organogenesis from micro cuttings of rhizome bud of L. spinosa (6 weeks)
|
Concentration of plant growth regulators
(mgL-1)
|
Response of micro cutting of
rhizome bud (%)
|
Number of shoots/explant
(Mean ± SD)
|
Shoot length/explant
(cm)
(Mean ± SD)
|
|
Kinetin
|
BAP
|
NAA
|
|
Control (PGR free)
|
0
|
0
|
0
|
|
1
|
|
|
0
|
0
|
0
|
|
2
|
|
|
69.6±2.8
|
1.6±0.42
|
3.60±0.06
|
|
3
|
|
|
72.6±2.8
|
1.2±0.46
|
3.01±0.02
|
|
4
|
|
|
67.2±2.8
|
1.0±0.43
|
2.5±0.02
|
|
1
|
|
0.5
|
0
|
0
|
0
|
|
2
|
|
1
|
72.0
|
1.8±0.44
|
3.06±0.12
|
|
3
|
|
1
|
82.3±2.8
|
2.1±0.32
|
2.77±0.12
|
|
4
|
|
0.5
|
70.6±2.8
|
1.2±0.24
|
2.04±0.05
|
|
|
1
|
|
83.6
|
1.8±0.34
|
4.5±0.07
|
|
|
2
|
|
76.3±2.8
|
1.2
± 0.56
|
3.84±0.05
|
|
|
3
|
|
88.2±2.8
|
1.9±0.45
|
3.28±0.06
|
|
|
4
|
|
78.0
|
1.4±0.42
|
2.94±0.06
|
|
|
1
|
0.5
|
82.6±2.8
|
2.1±0.42
|
4.21±0.06
|
|
|
2
|
1
|
84.6±2.8
|
2.8±0.56
|
3.58±0.05
|
|
|
3
|
1
|
90.6±2.8
|
3.6±0.55
|
3.42±0.10
|
|
|
4
|
0.5
|
77.6±2.8
|
2.6±0.42
|
2.52±0.05
|
Root induction- The
in vitro raised shootlets were sub-cultured on ½ strength MS medium
augmented with 0.1 -1.0 mgL-1 either NAA or IBA for L. spinosa for root
formation. At 14th day, the in vitro raised shootlets were
produced in vitro rootlets without any callus proliferation. Medium
containing 0.5 mgL-1 of IBA was proved to be the most effective for
rooting of micro-shoots than that containing any other concentrations of NAA in
case of both the plants evaluated (Table
2). Here, NAA did not significantly improve the parameters evaluated. Highest
percentage (79.5±2.6%), maximum number of rootlets/ shootlet (4.0±0.46) and
mean length of rootlets (3.25±0.06cm) were observed in L. spinosa. There are several reports regarding the effectiveness
of IBA in rooting of in vitro induced shoots of medicinal plants. [12,13]
Table 2: Effect of auxins for root
induction of Lasia spinosa
|
Auxin concentration (mgL-1)
|
Response (%)
|
Numbers of roots/shoot
|
Root length/culture
|
|
NAA
|
IBA
|
|
0.1
|
|
37.6±2.6
|
1.7±0.54
|
2.66±0.12
|
|
0.5
|
|
54.2±
2.6
|
2.2±0.54
|
2.22±0.12
|
|
0.8
|
|
48.4±
2.6
|
2.8±0.56
|
2.69±0.14
|
|
1
|
|
44.2±
2.6
|
2.7±0.46
|
2.46±0.15
|
|
|
0.1
|
66.3±
2.6
|
2.6±0.45
|
2.76±0.05
|
|
|
0.5
|
79.5±
2.6
|
4.0±0.46
|
3.25±0.06
|
|
|
0.8
|
63.2±
2.6
|
1.3±0.56
|
3.15±0.07
|
|
|
1
|
60.4±
2.6
|
3.1±0.42
|
3.00±0.07
|
Acclimatization and hardening- The rooted plantlet was successfully hardened off inside the
growth room in sterile soilrite for 2 weeks and
eventually established in natural soil. There was no detectable
variation among the potted plants with respect to morphological and growth
characteristics (Fig. 2).
After 15 days, in
vitro raised plantlets were hardened in polycups
with soilrite, irrigated with 0.5× MS liquid medium.
The plants were kept in a culture room for 14 days. Approximately, 75% of
plants were successfully established in polycups.
After 15 days the polycups hardened plants, these
were transferred to pots placed in and kept in poly house. Seventy percentages
of plantlets were well established for Lasia
spinosa in the poly house condition. After one month, regenerated plants
were successfully transferred to the field.
The protocol
optimized here for in-vitro propagation
of Lasia spinosa using micro-cuttings
of rhizomes bud through direct organogenesis was found to be efficient,
reproducible and provide a rapid technique for mass propagation and
multiplication of this potential medicinal plant and also could further be used
in its improvement programme.
Fig.
2 (A, B): Acclimatization
in-vitro grown plantlets of Lasia spinosa in Poly-house condition
DISCUSSION- In
this study, an in-vitro propagation protocol has been developed for L.
spinosa using micro-cutting of rhizome bud. The
plants showed direct organogenesis from the micro-cutting of rhizome bud which
was found to be more suitable for regeneration when cultured on MS medium using
various concentrations of BAP (1.0-4.0) and kinetin (1.0-4.0) separately or in
combination with low concentration (0.5 and 1.0 mgL-1) of auxin (NAA). Intact rhizome buds of native ginger in
Borneo, Etlingera coccinea were
cultured on Murashige and Skoog medium supplemented with 0.1to 2.0µM Thidiazuron (TZD), 6-BAP and kinetin. Regenerated shoots developed
well in MS medium supplemented with 5.0µM BAP by promoting an average of
5.5±0.25 proliferated shoots per explants, 3.0±0.061 cm shoot length, and
6.5±0.35 leaves per explants. It was observed
that BAP in combination with NAA was more effective for shoot induction [8,14]. The best conditions for
propagating Homalomena pineodora
was found to be on MS medium supplemented with 3% sucrose and 0.5 mgL-1
BA (6- benzyladenine) under 24 hrs of cool
fluorescent light which produced an average of 3.8 shoot
per explants [15]. Treatments of BAP and NAA, 3.0 mgL-1
BAP + 1.0 mgL-1 NAA was found to be suitable and showed better
response in L. spinosa. In this concentration, 90.6% explants induced to
develop shoots. The number of shoot as well as length of shoot per explant was recorded as 3.6±0.55 and 3.42±0.10 cm
respectively. BAP is considered one of the most suitable cytokinin
for the multiplication of axillary buds reported by
many authors [16,19-21]. In the present investigation, combination
of BAP with NAA was found more suitable than BAP and kinetin alone. But,
highest shoot length was observed in low concentration of BAP i.e. 4.21±0.06
cm. In vitro formed shoots were excised and rooted on a separate root
inducing half strength basal MS medium. Regenerated shoots thus formed were
carefully excised and then rooted on basal MS medium. Rooting on proliferated
shoots of Anthurium andreanum
were successfully obtained on addition of PGRs viz., IBA (0.0, 0.5,
1.0 and 2.0 mg/L), NAA (0.0, 0.05, 0.1 and 0.25 mgL-1) and KIN (0.0
and 0.2 mgL-1) [17,22]. The in vitro raised
shootlets were subcultured on ½ strength MS medium
augmented with 0.1 -1.0 mgL-1 either NAA or IBA for L. spinosa for
root formation. At 14th day, the in vitro raised shootlets were produced
in vitro rootlets without any callus proliferation. The researcher
performed rooting of plantlets of Aglaonema
on MS medium containing 3 mgL-1 indole-3-butyric acid (IBA)
[10,23]. Rooting is usually induced by auxin,
and IBA is more effective for rooting compared with other auxins as reported
for Anthuriums [9,24-26].
Medium containing 0.5 mgL-1 of IBA was proved to be the most
effective for rooting of microshoots than that
containing any other concentrations of NAA in case of
both the plants evaluated. Here, NAA did not significantly improve the
parameters evaluated. The highest percentage (79.5±2.6%), maximum number of
rootlets/ shootlet (4.0±0.46) and mean length of rootlets (3.25±0.06cm) were
observed in L. spinosa. The rooted plantlet were successfully hardened
off, approximately 75% of plants were successfully established. After 15 days,
hardened plants were transferred to pots, where seventy percentages of
plantlets was found to be well established.
CONCLUSIONS- Numerous elite
material can easily be generate from callus as it has high potency. As well the
genetic diversity of medicinal plants in the world is getting endangered at an
alarming rate because of ruinous harvesting practices and over-harvesting for
production of medicines. There is a
strong need for proactive understanding in the conservation, cultivation and
sustainable usage of important medicinal plant species for future use. Hence
tissue culture may lay to overcome many of the difficulties in the present day
world.
Our
findings have paved a way for future investigation on another mode of
regeneration like haploid production, anther culture,
protoplast culture etc and also for the commercial and rapid propagation of L. spinosa. Above all regeneration of plants
through micropropagation is an alluring alternative for mass multiplication of
outstanding cultivars at faster rates than conventional methods.
ACKNOWLEDGEMENTS- The
financial assistance received as BSR-Junior Research Fellow under UGC-SAP
(DRS-I), Govt. of India is highly acknowledged.
CONTRIBUTION OF AUTHORS- Research concept and design was framed by
BT and practical implementation, data collection, and analysis was carried out
by PH. Final review of work was carried out by PH and BT.
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