In vitro Regeneration of Aconitum balfourii Stapf: A Rare Medicinal Herb from Himalayan Alpine Through Root Explants
Aconitum balfourii Stapf is a rare medicinal herb belonging to genus Aconitum and family Ranunculaceae. Excessive illegal collection from wild due to pharmacological significance along with quiescent nature of seeds and lengthy cultivation cycle are collectively responsible for its endangered status. Therefore, conservation applying tissue culture is a vital need and has been developed nowadays. In the present study, in vitro regeneration of A. balfourii from root explants has been attempted. Root explants were cultured on MS-Medium supplemented with different combinations of BA and NAA and callus induction took place at 13.4 μM NAA and 5.55 μM BA. Such induced calluses were transferred to shooting media. The best shooting were observed in 8.88 μM BA and 0.54 μM NAA. These shoots were subculture on 7.99 μM BAP. Furthermore the result showed that increasing the concentration of BA caused a reducing length of shoots; also presence of low concentration of NAA has been necessary for shoot induction. Some factors such as shoot length, no. of shoots and induction frequencies of shooting and rooting were also studied. The well developed micro shoots were cultured on rooting media for complete regeneration of plant. Rooting of micro shoots occurred in media containing 1.43 μM IAA and 1.23 μM IBA.
Received: August 04, 2011;
Accepted: November 26, 2011;
Published: January 12, 2012
The genus Aconitum belongs to the family Ranunculaceae is a diverse
genus with nearly 300 species worldwide (Kadota, 1987)
and has been found rich source of diterpenoid alkaloids. In, India, the genus
is represented by about 26 species mainly distributed in subalpine and alpine
zones of Himalayas from Kashmir to Uttarakhand and extending to the hills of
Assam (Chaudhary and Rao, 1998). A. balfuorii
Stapf. is an significant and highly valued herb of this genus. A. balfourii
Stapf. is prevalent in different zones of Garhwal and Kumaon regions of Uttarakhand
i.e., Valley of flowers, Kedarnath, Tungnath, Madhyamaheshwar and Panwalikantha
on shady slopes at altitudes between 2800-4200 m. (Nautiyal
et al., 2002; Chopra et al., 1984; Samant
et al., 1998). It is an erect, glabrous shrub that becomes more than
1.5 m in height. Root tubers are 7-12 cm long and extraordinary heavy (Gaur,
1999) Tubers contain a crystalline toxic alkaloid called pseudoaconitine
(0.4-0.5%) and aconitine, balfourine, bikhaconitine in small amount (Khetwal,
2007). The alkaloid isolated from the roots of this herb, has exhibited
anti-inflammatory, vermifuge, anti-rheumatic, analgesic and cardio tonic activities
and used in all types of pains and inflammations (Ameri,
1998). Hence, it has been frequently used in Ayurvedic, Homeopathy and Unani
system of medicine since long time so the commercial demand is very high. To
accomplish the demand excessive illegal collection and sale of Aconitum
have been continuously carried out. Due to over exploitation and destruction
of natural habitats along with poor seed germination and seedling establishment
under natural conditions, A. balfourii is identified as an endangered
(Nautiyal et al., 2002) in Garhwal Himalaya and
vulnerable in entire Uttarakhand by CMAP (2003). The
importance of this plant, recalcitrant nature of its seeds and its endangered
status has necessitated an urgent need to work on its proper regeneration. Therefore,
in the present study an attempt has been made to establish in vitro regeneration
protocol of A. balfourii Stapf, through root explants. Although, diverse
sources of explants have been reported for regeneration, but root explants have
some advantages over other explants in terms of higher regeneration potential,
easy manipulation and higher susceptibility of Agrobacterium rhizogene transformation
(Morton and Browse, 1991). This is first time report
of regeneration protocol of A. balfourii Stapf. from root explants.
MATERIALS AND METHODS
Plant material: Actively growing mature plants and tubers of Aconitum
balfourii Stapf. were collected from Tungnath region (3300 m). These plants
and tubers were kept for establishment under controlled environment containment
facility at College of Basic Sciences and Humanities, GBPUA&T, Pantnagar.
The explants for in vitro regeneration were obtained from green house
(25°C, 65.0% RH).
Callus induction: Callus induction was attempted with different explants
viz., leaves, shoot tips and roots. All explants were excised in 0.5 cm length
and pre-cleaned with double glass distilled water containing 1% v/v Tween-20
for 5 min. The explants were surface sterilized with 0.1% aqueous mercuric chloride
solution for 1 min, rinsed thoroughly 4-5 times in sterile distilled water and
inoculated aseptically on Murashige and Skoog (1962) basal
medium containing 3% sucrose and 0.7% agar. The medium (MS) was supplemented
with growth regulators in various combination of BA (0-6.66 μM) and NAA
(0-13.42 μM) for callus induction. The pH of the medium was adjusted to
5.8±0.1 with 0.1 N NaOH or 0.1 N HCl prior to autoclaving for 20 min
at 120°C and 1.05 Kg cm-2. The cultures were maintained at 25±2°C
under 14 h photoperiod (55 mmol m-2 sec-1) from cool,
white fluorescent tube lights. All explants were cultured in vessels each containing
50 mL of medium. To avoid blackening, due to release of phenolics, the medium
was supplemented with 0.05% PVP. After eight weeks of culture, calli proliferated
from the excised leaf explants, shoot explants and root explants as well and
these were subculture in the fresh medium at a 2-weeks interval for continuous
growth. Four explants were used per flask and four flasks were used per treatment.
Callus of root explants was used for further shoot induction and multiplication.
Shoot proliferation: After callus was obtained from different explants, we have used root callus for further standardization of regeneration protocol. For Shoot bud induction and shoot differentiation, MS medium composed of BA (0-10.65 μM-1) and combination with 0.54 μM NAA was employed. These shoot buds were subculture onto maturation medium supplemented with 7.99 μM BA, 0.05% PVP and 3% sucrose for three weeks. Every possible care has been taken to prevent any further contamination.
Rooting of microshoots: For root induction, excised micro shoots (1-2 cm length) were transferred to MS basal medium supplemented with different concentrations of IAA (0-4.52 μM and IBA (0-5.71 μM) individually or in combinations and 2% (w/v) sucrose. One excised shoot was placed in each culture vessel having 50 mL of the culture media.
Data analysis: All the experiments were performed in replicates. Four replicates in callus induction and shoot proliferation and ten replicates in root induction for each combination of hormone treatments were analyzed, respectively. Mean values of various treatments were subjected to one way Analysis of Variance (ANOVA).
RESULTS AND DISCUSSION
Effect of various concentrations of BA and NAA on callus induction:
All the three explants viz., roots, stems and leaves used for callusing were
respond at different concentration of growth hormones supplemented to MS-Medium
along with 3% sucrose and 0.7% agar (Fig. 1a-c).
The optimal callus from root explants were obtained at 13.42 μM NAA and
5.55 μM BA. In case of stem and leaf explants the best callusing was observed
at 13.44 μM NAA and 4.44 BA and 16.11 μM NAA and 4.44 BA, respectively
(Table 1). The callus induction from leaf explants was previously
reported in different concentration of BA and NAA in A. balfourii (Pandey
et al., 2004; Bist et al., 2011).
Callus induction percentage was highest in leaf explants i.e., 90-91% followed
by root and stem 80-85%. After obtaining callus from different explants sources
the root callus was used for further regeneration, therefore sub culturing of
root callus was performed to every 2 weeks of culture for minimizing the phenolics
and increase the growth and mass of callus.
|| Effect of different combinations of NAA and BA on callus
induction from root, leaf and stem explants
|SEM: 1.649, SEM: 1.451, SEM: 1.672. Cd at 5% = 4.740. Cd at
5% = 4.170. Cd at 5% = 4.807 in root, leaf and stem, respectively. Values
are Mean±SE of four replicates and repeated three times. The data
were recorded after eight weeks of culture
||Callus induction from different explants, (a) Root callus,
(b) Shoot callus and (c) Leaf callus
Effect of different level of BA and NAA on shoot induction and proliferation:
In Table 2 effect of different concentration of BA and interaction
with NAA showed that the induction of shooting required a low level of NAA with
BA. Different shooting stages of shooting are shown in Fig. 2(a-d).
The maximum shoot induction 93.7% (14.50 shoots/flask) was obtained at 8.88
μM BA and 0.54 μM NAA. This finding is supported by earlier reported
data on different species of the genus Aconitum (Giri
et al., 1993; Watad et al., 1995).
Furthermore, it was also observed that as we increased the concentration of
BA from 0 to 10.65 μM, the shoot length was decreased and it also adversely
affects the average no. of shoots. It might be due to that BA at high concentration
causes decrease in apical dominance. This result is in line with what reported
by earlier studies on this plant (Jabeen et al.,
2006; Giri et al., 1993; Bist
et al., 2011). For further sub culturing of micro shoots the medium
supplemented with 7.99 μM BA alone was found to be optimum because shoot
length was maximum (3.20 cm) at this concentration. Cytokinin as a plant growth
regulator causes shoot proliferation by stimulating cell division (Ranjan
et al., 2003).
Effect of different auxin concentrations on rooting: As saw in Table
3 two different auxins IAA and IBA individually and with various combinations
were used to see the effect on rooting of excised shoots. The best rooting 72%
was observed at 1.43 IAA and 1.23 IBA. However, the rooting was observed at
every combination but with different level of response. Rooting of microshoots
and complete regenerated plant was depicted in Fig. 3 and
||Different stages of shoot induction and multiplication (a)
Shoot initiation from callus, (b) Shoot differentiation (initial stage),
(c) Shoot differentiation (later stage) and (d) Shoot multiplication
|| Effect of BA and NAA on shoot induction
|SEM: 1.451. Cd at 5% = 4.218. Values are Mean±SE of
four replicates. Data was taken after 8 weeks of culture
|| Rooting of microshoots
|| Complete regenerated plantlet
|| Rooting response on different combinations of IAA and IBA
|SEM: 1.889. Cd at 5% = 5.540. Data was taken 3 weeks of culture
and ±SE represents mean of 10 cultures per treatment
Considering Table 3, it showed that as we increase the concentration
of individual auxin decrease the rooting response. But the combination of both
auxins synergistically increases the rooting response. Our investigation is
consistence with earlier reports on combined response of IAA and IBA improve
the rooting in other plants (Samantary and Maiti, 2010).
Since A. balfourii Stapf. is a valued herb of the genus Aconitum due to presence of pharmacologically important alkaloids, in the present investigation an in vitro regeneration protocol was optimized through root explants. Although, in vitro regeneration protocols have been already reported but it is first time report of regeneration by using root explants. Based on the obtained result in this study, the protocol is helpful to regenerate whole plant after infection of A. rhizogene that is one of the most frequent approaches to enhance the production of important secondary metabolites from several medicinal plants.
We acknowledge National Medicinal Plant Board (NMPB), New Delhi for funding the project.
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