Journal of Medicinal Plants Studies 2014; 3(1): 04-09

ISSN 2320-3862
In vitro plant production through apical meristem
JMPS 2014; 3(1): 04-09 culture of Gentiana kurroo Royle
2014 JMPS
Received: 30-11-2014
Shiwani Kaushal, Arushdeep Sidana and Kamal Dev
Accepted: 08-12-2014
Abstract
Shiwani Kaushal Gentiana kurroo Royle is a critically endangered medicinal herb belonging to the family Gentianaceae.
Department of Biotechnology, The roots and rhizomes of the plant have been extensively used as a bitter tonic, antiperiodic,
Shoolini University of expectorant, antibilious, anthelmintic, anticancer, immunomodulatory, antinflammatory and analgesic. A
Biotechnology and Management method for rapid micropropagation of Gentiana kurroo through apical meristem has been developed.
Sciences Solan, Himachal
Apical meristem was excised to a length of 0.2-2 mm from the shoot tips. Among different treatments of
Pradesh, 173229, India.
growth regulators either alone or in combination, the growth of meristem was best observed on
Murashige and Skoog (MS) medium supplemented with 6- Benzyl amino purine (BAP) (1.0 mg/l) and
Arushdeep Sidana
Indole acetic acid (IAA) (0.5 mg/l). The maximum response for meristem proliferation was 83.3% with
Department of Biotechnology,
Shoolini University of
an average mean number of 8.1 0.2 leaves /explant. Three weeks old sprouted meristems were
Biotechnology and Management transferred to the MS medium supplemented with 0.5 mg/l each of Kinetin (KN) and BAP for shoot
Sciences Solan, Himachal elongation and proliferation resulting in 5- 6 shoots/ explant. In vitro regenerated shoots developed roots
Pradesh, 173229, India. in six weeks when transferred to half strength MS medium supplemented with 0.5 mg/l Indole butyric
acid (IBA) with a survival rate of 86%.
Kamal Dev
Department of Biotechnology, Keywords: Gentiana kurroo, apical meristem culture, MS medium, growth regulators.
Shoolini University of
Biotechnology and Management 1. Introduction
Sciences Solan, Himachal Gentianaceae is a family of flowering plants comprising approximately 300 species in the
Pradesh, 173229, India. world [1]. In India, the family is represented by 16 genera and approximately 145 species.
Gentiana kurroo Royle belonging to the family Gentianaceae is an important native Indian
species used for medicinal purposes. It is a rosetteforming small perennial herb also known as
Indian Gentian, Neelkanth, karu and chireta. It is mainly found in Kashmir and Himachal
Pradesh with adjoining hills of North-Western Himalayas at altitudes of 1500-3400 m. Plants
of this species also occur in Pathrala thatch, Karol Tibba Solan, and Mangarh area of District
Sirmour at an altitudinal range of 1700-2000 m at mean sea level [2]. In India, rhizomes and
roots of this plant are used as bitter tonic, antiperiodic, expectorant, antibilious, anthelmintic,
astringent, antipsychotic, anti-inflammatory, sedative, cholagogue, emmenagogue, febrifuge,
refrigerant, blood purifier and carminative. The roots of this plant are a source of iridoid
glycosides such as gentiopicrine, gentiamarin and the alkaloid gentianin [2]. Phytochemical
screening of the plant also showed that the roots of G. kurroo are rich in various active
ingredients like flavonoids, alkaloids and terpenoids, which are responsible for its effects as
analgesic, anticancer and immunomodulatory [3, 4]. Unfortunately, the pharmaceutical
industries are largely dependent on natural reserve, which lead to its extinction. Therefore, the
red data book of Indian plants has listed this species as endangered and its status as critical [2].
Previously reported studies established the shoot formation using nodal segments and shoot
tips [5], seedlings and leaves as explants [6], but plant regeneration through apical meristem has
not been reported till date. Recently, indirect and direct organogenesis has been carried out
using leaves, roots and petioles as explants, with a maximum response of 86.6% for shoot
regeneration from callus cultures [7]. Meristem-tip culture is an important technique for the
production of disease free plantlets and for rapid clonal multiplication. Although, many
species such as Gentiana scabra have been found to be infected with aphid-borne viruses[8],
Correspondence: but there is no report illustrating whether Gentiana kurroo is virus infected or not. In any case,
Kamal Dev meristem culture is the most effective method to raise virus free plants, not only for Gentiana
Department of Biotechnology, species; but for all the pharmaceutical important medicinal plants. The tissue culture technique
Shoolini University of
Biotechnology and Management
would be useful for conservation of rare and endangered plants for the production of
Sciences Solan, Himachal industrially important phytochemicals. Therefore, the present study was undertaken to develop
Pradesh, 173229, India. a protocol for plant regeneration through apical meristem.

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2. Material and Methods acclimatized and transferred to the greenhouse for hardening.
2.1 Plant material and culture conditions Hardening was continued for 3 weeks, or until they were
Cultures of G. kurroo were obtained from Dr. Y.S. Parmar successfully acclimatized. The survival rate was recorded after
University of Horticulture and Forestry, Nauni, Solan and was 45 days of plantation in the earthen pots.
maintained in MS medium supplemented with 0.5 mg/l, each
of KN and BAP, under controlled temperature (25 C), 2.3 Data analysis
humidity (70-75%) and light (10 h dark and 14 h light cycle) The data for the percentage of shoot response and average
conditions in a growth chamber. Apical meristems were number of leaves/explant was determined after 6 weeks of
excised aseptically under the dissection microscope with the subculture. Different developmental stages of excised apical
help of hypodermic needles and scalpel. The apical meristem meristem of Gentiana kurroo were observed under the light
consisting of the apical dome with one or two leaf primordia microscope. Twelve replicates were tested in each treatment
ranging from 0.1- 2 mm were gently removed from parental and each experiment was repeated thrice. Means and standard
tissues and cultured on MS medium supplemented with errors were calculated for each experiment. The significance of
different concentrations of growth regulators such as BAP the results was calculated using Graph pad prism 5.02
(0.5-1.0 mg/l), KN (0.5-1.0 mg/l), IAA (0.5-1.0 mg/l), NAA software. The overall variation in a set of data was analysed by
(0.5-1.0 mg/l) and GA3 (0.5-1.0 mg/l) as shown in the Table1. one way analysis of variance (ANOVA). A value of P <0.05
The pH of the medium was adjusted to 5.8 0.1, prior to was considered significant.
addition of 0.8 % (w/v) agar and was sterilized at 121 C for
15 min. 3. Results
3.1 Dissection and Microscopic analysis of apical meristem
2.2 In vitro shoot multiplication, root induction and Meristems were excised aseptically under the dissection
acclimatization microscope with the help of hypodermic needles and scalpel.
The proliferated meristem was multiplied and rooted on MS The apical meristem sections without leaf primordia were
medium supplemented with different concentrations of KN, excised to a range of 0.1- 2 mm in diameter and observed
BAP, IAA, IBA and NAA, either alone or in combination for under a light microscope. Apical meristem appears as a shiny
shoot elongation and root induction. After about 6 weeks in dome under the light microscope (Fig 1a). With the increasing
the rooting medium, the plantlets were transferred to the number of days in the culture medium containing essential
earthen pots containing a pre-autoclaved mixture of farm yard growth regulators, the excised meristem dome develops
manure and clay loam (1: 1). The potted plants were covered bipolar axis during reorganization (Fig b-f). Multiple leaves
with plastic sheet or glass beaker to maintain the humidity. become evident after 10 days of culture and fully developed
Fully developed shoots with healthy roots were then leaf whorl on the 14th day (Fig g- i).

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Fig 1: Developmental stages of apical meristem of Gentiana kurroo. (a) Light micrographs of excised meristem showing apical dome; (b) Leaf
primordia regenerating from the apical dome; (c) Basal part of the apical meristem; (d-f) Leaflets regenerating from the apical meristem; (g-i)
Formation of fully developed leaf whorls.

3.2 Meristem establishment and shoot multiplication and tested either alone or in combination for shoot elongation
The apical meristem was cultured on MS medium and multiplication (Fig. 3). Among different combinations, the
supplemented with different concentrations and combinations MS media with 0.5 mg/l each of BAP and KN was the best
of growth regulators. No morphogenic response was observed combination for shoot elongation and proliferation with an
when nodal explants were cultured on MS medium devoid of average of 5.0 0.2 shoots/ explants (Fig. 2d-f & Fig. 3). BAP
plant growth regulators. About 83.3% of the apical meristem alone was significantly more efficient (4-5 shoots/explant)
sprouted within 10-12 days of culture in MS medium than KN alone (1-2 shoots/ explant) for promoting shoot
supplemented with 0.5 mg/l IAA and 0.8 mg/l BAP (Table 1; multiplication, which resulted in two fold increase in the
Fig 2b &c). Also, IAA (0.5 mg/l) in combination with KN number of shoots/ explant (Fig. 3). All other concentrations,
(0.8 mg/l) showed excellent growth with a success rate of except 0.5 mg/l each of KN and BAP showed a marginal
66.6% with an average number of 5.60.2 leaves/ explant after increase in the number of shoots/ explant (5.0 0.2) as
20 days of inoculation (Table 1). After three weeks, the compared to higher and lower concentrations. When KN and
established primary meristem culture was aseptically BAP were used in combination at higher concentration (1.0
subcultured on semisolid MS medium supplemented with mg/l), the shoot number and shoot length was reduced (3.9
different concentrations of KN and BAP. BAP and KN (0.25, 0.2 shoots/explant) with callusing at the basal ends (Fig. 3).
0.5, 0.75 and 1.0 mg/l) were used at equimolar concentrations

Table 1: Effect of growth regulators on development stages of apical meristem of G. kurroo.

*Average
Growth regulators mean number
Percentage (%)of shoot response
(mg/l) of leaves/explant
Control (basal) 0.0 0.0 0.0
0.25 + 0.25 0.0 0.0 0.0
NAA + KN 0.25 + 0.75 1.08 0.2 8.3
0.30+ 1.0 4.1 0.2 41.6
0.25 + 0.25 0.0 0.0 0.0
NAA + BAP 0.25 + 0.75 3.9 0.18 50.0
0.3+ 0.8 5.10 0.12 58.3
0.25 + 0.25 0.0 0.0 0.0
BAP + KN 0.25 + 0.50 3.7 0.18 8.3
0.5+ 0.5 5.9 0.2 50.0
0.8 + 0.8 6.2 0.21 58.3
0.25+ 0.5 0.0 0.0 0.0
IAA + BAP 0.25+ 0.75 4.12 0.17 50.0
0.5 + 0.8 8.16 0.2 83.3
0.25 + 0.5 2.3 0.2 8.3
IAA + KN 0.25 + 0.75 3.7 0.23 25
0.5+ 0.8 5.16 0.2 66.6
0.1 + 0.25 0.0 0.0 0.0
GA + BAP 0.25 + 0.25 1.08 0.2 16.6
0.25+ 0.5 3.67 0.14 41.6
0.1 + 0.25 0.0 0.0 0.0
GA + NAA 0.1 + 0.3 1.08 0.2 16.6
0.25 + 0.5 3.3 0.13 33.3

* Each value represents mean SE of 12 replicates per treatment.

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Fig 2: Propagation of Gentiana kurroo through apical meristem. (a) Asceptically excised apical meristem (~ 2 mm) as an explant
inoculated on MS medium supplemented with 0.5 mg/l IAA and 0.8 mg/l BAP; (b) & (c) Differentiated meristem with leaf
primordia after 10 and 20 days of culture respectively; (d) & (e) Shoot induction from in vitro established meristem on MS
semisolid medium with 0.5 mg/l KN and 0.5 mg/l BAP after 30 and 40 days respectively; (f) Shoot proliferation on MS medium
supplemented with 0.5 mg/l KN and 0.5 mg/l BAP after 60 days of inoculation (g) Root induction from in vitro established
meristem derived shoot on half strength MS semisolid medium with 0.5 mg/l IBA after 45days of culture in the rooting medium;
(h) Plantlet with healthy roots kept in a mixture of sterilized FYM and Clay loam (1:1) under glass beaker to maintain the
humidity; (i) Meristem derived hardened plant of Gentiana kurroo in pot after 60 days of hardening.

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3.3 In vitro rooting and Hardening

Elongated shoots were excised individually and rooted in the
half strength MS medium supplemented with different types of
auxins (IAA, IBA and NAA). IBA was clearly the best of
three auxins tested, and each plantlet developed 24-25
roots/explant with 88.8% root response followed by IAA
(69.4%), NAA (60.8%) and 30% on MS half basal salts (Fig.
4a). Well developed rooted plantlets were hardened in a
mixtures containing sand and soil (1:1); soil and farm yard
manure (FYM) (1:1); sand, soil and farm yard manure (1:1:1);
clay loam and FYM (1:1) and coco peat. The survival rate
after six weeks of acclimatization was highest in clay loam and
FYM (86.0 %), followed by coco peat (80.5%), sand, soil and
FYM (44.4 %), soil and FYM (38.8 %) (Fig. 4b). No
morphological aberrations were observed after 2 months of
hardening.

Fig 3: Effect of different concentrations of growth regulators on

number of shoots/ explant of G. kurroo. Number of shoots/ explant
was determined based on the data as shown in Fig 2f. Data represents
an average and standard error of experiments performed in triplicate;
***p < 0.05.

Fig 4: Effect of auxins on rooting of Gentiana kurroo.(a) In half strength MS medium after six weeks; (b) Percentage survival (%)
of in vitro plantlets in different potting mixture after 2 months. Results represents the average and standard error of experiments
performed in triplicate; ***p < 0.05.

4. Discussions has been reported dealing with plant regeneration through

Apical meristem culture has recently become an important apical meristem. Hence, this study was carried out to describe
technique for virus elimination in plant tissue culture the morphogenetic events through microscope during the
technology. Shoot development directly from the apical development of plantlet through meristem culture. Shoot
meristem avoids callus formation, ensuring that genetic elongation and subsequent proliferation was effectively
instability and somaclonal variation are minimized [9, 10]. In the achieved on MS medium supplemented with a combination of
present study, apical meristem was excised and cultured on KN and BAP. Similar observations have been reported in
MS medium, which successfully developed into a new Dendrocalamus strictus and Arundinaria callosa [11, 12]. Shoot
plantlet. Previously reported studies have explained the use of proliferation was declined when the KN and BAP
shoot tips and nodal segments as explants for in vitro concentration was increased beyond 1.0 mg/l in the medium.
propagation of Gentiana kurroo [5]. Recently, indirect and Similar results were also reported in Stevia rebaudiana [13].
direct organogenesis has been carried out using leaves, roots The lower percentage of average number of leaves/ explant
and petioles as explants with maximum response (86.6%) of was observed on medium containing BAP and NAA due to the
shoot regeneration from callus cultures [7]. Despite these profuse callusing at the basal part of differentiated shoot buds.
studies for in vitro propagation of Gentiana kurroo, nothing Similar observations were also reported in Jatropha curcas [14].
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Journal of Medicinal Plants Studies

For the root induction, elongated shoots were individually 5. Sharma N, Chandel KPS, Paul A. In vitro propagation of
excised and cultured on half strength MS basal salts Gentiana kurroo an indigenous threatened plant of
supplemented with 0.5 mg/l IBA resulting in 91.6% root medicinal importance. Plant Cell Tissue Organ Culture
response with an average of 24.50.2 roots/ explant. IBA has 1993; 34:307309.
successfully been used in root induction for other species like 6. Fiuk A, Rybczynski JJ. Genotype and plant growth
Arbutus unedo, Hydrastis canadensis and Aegle marmelos [15, regulator dependent response of somatic embryogenesis
16, 17]
. In vitro rooted shoots were successfully transferred to from Gentiana spp. leaf explants. In vitro Cellular &
the plastic pots containing sterilized potting mixture of clay Developmental Biology 2008; 44:90-99
loam and farmyard manure (1:1), with a survival percentage of 7. Sharma A, Kaur R, Sharma N. In vitro morphogenic
86% after two months of acclimatization without any response of different explants of Gentiana kurroo Royle
somaclonal variations. Similar report on acclimatization of G. from Western Himalayas-an endangered medicinal plant.
kurroo has been achieved on coco peat with 80% survival rate Physiology and Molecular Biology of Plants 2014;
[7].
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culture which may be exploited in situations where the donor and Forestry Research Center, Japan, 2005.
plant is infected with viral, bacterial or fungal pathogens. The 9. Murashige T, Skoog F. A revised medium for rapid
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The authors are grateful to Prof. D. R. Sharma for the 5(1-2): 45-50.
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The authors declare that there is no conflict of interests J. An efficient protocol for clonal micropropagation of
regarding the publication of this paper. Mentha piperita (Pipperment). Asian Journal of Plant
Sciences 2012; 2(4):518523.
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