International Research Journal of Biotechnology (ISSN: 2141-5153) Vol. 2(9) pp. 220-227, December, 2011
Available online http://www.interesjournals.org/IRJOB
Copyright © 2011 International Research Journals
Full Length Research Paper
In vitro plant propagation for rapid multiplication and
conservation of Fraxinus micrantha: A Himalayan tree
species of high medicinal value
Hemlata Bisht1*, Vinay Prakash2 and A. R. Nautiyal3
1
Department of Botany and Microbiology, School of Life Sciences, HNBG Central University, Srinagar (G), Uttarakhand,
India, 246 174
2
Department of Seed Science and Technology, School of Agriculture and Allied Sciences, HNBG Central University,
Srinagar (G), Uttarakhand, India, 246 174.
3
High Altitude Plant Physiology Research Centre, School of Agriculture and Allied Sciences HNBG Central University,
Srinagar (G), Uttarakhand, India, 246 174.
Accepted 03 November, 2011
Fraxinus micrantha, is a Himalayan temperate multipurpose tree species and extensively extracted for
its medicinal properties. To overcome the problem of low germination potential and unsuccessful
vegetative propagation in F. micrantha, in vitro plantlet proliferation through micropropagation was
investigated. For which epicotyle, hypocotyle and cotyledonary node part of in vitro raised seedlings
were used. Murashige and Skoog (MS) and Woody plant medium (WPM) with different combinations and
concentrations of cytokinins and auxins were tried for different phases of micropropagation. Epicotyl
part showed greater regeneration potential (80%) than hypocotyls (8%) and cotyledonary node (5%)
through callus induction but multiple shoot formed by cotyledonary node (80%) only. MS medium hardly
responded to the explants of F. micrantha but WP medium supplemented with cytokinins
(Benzyleaminopurine and kinetin) in association with auxins was found to be the most suitable medium
for callusing as well as shoot root induction. Combinations of Benzyleaminopurine (BAP), kinetin,
Indole- 3-butyric acid (IBA) and α-Naphthaleneacetic acid (NAA) have been most widely utilized to elicit
regeneration response and approximately 45-60% well developed rooted plantlets were obtained in the
medium with these combinations. However induction of auxins alone was non-responsive for different
phases of micropropagation in this species. Requirement of cytokinins in the optimum amount (2-4mg/l)
was found to be promising for callusing, shoot formation and rooting also. Although the frequency of
shoot formation was high in WPM supplemented with, IBA, NAA and BAP but embryogenic calli failed to
initiate the root and shoot if added in more than 5mg/l. The Observations revealed that the epicotyle part
of F. micrantha seedlings if used for micropropagation in WPM medium with combination of low
concentrations of cytokinin (BAP and kinetin) and auxins respond well and proved for rapid
multiplication in vitro.
Keywords: Fraxinus micrantha, Woody Plant Medium, auxins, cytokinins, epicotyle and embryogenic callus
INTRODUCTION
Fraxinus micrantha, Lingelsh (Vernacular name-Angu,
*Corresponding author E-mail: hvnautiyal@gmail.com
Abbreviations
MS; Murashige and Skoog, WPM; Woody Plant Medium, BAP;
Benzyleaminopurine, IBA; Indole- 3-butyric acid, 2,4-D; 2,4Dichlorophenoxy acetic acid, NAA; α-Naphthaleneacetic acid.
English name- Ash and family-Oleaceae) is a temperate
Himalayan multipurpose tree species of high medicinal
value and ethenobotanical importance (Iqbal, 2008,
Yadav et al., 1986). Fraxinus species have been used in
folklore medicine for their diuretic and purgative effects
due to presence of several glycosides including fraxin, a
coumarin glycoside, which is an active diuretic agent. The
leaves and the bark are also used for the treatment of
constipation, arthritis, rheumatic pain, cystitis and itching
Bisht et al. 221
scalp (Iqbal, 2008). Interestingly, the bark of the F.
micrantha used as bandage in fracture for humans as
well as animals. The leaves and bark extract used in
fever and blood dysentery also (Iqbal, 2008).
In addition to medicinal value, this plant is also well
known for its silvicultural importance. The wood of the
tree is white with a light reddish tinge with no heartwood,
(Van sambeek and Preece, 2007) Its shock resisting
ability is 40% superior and 25% harder than teak. So the
wood is used as fuel wood, extensively for making
agricultural implements, walking sticks, hockey sticks,
skies and other sports goods. In the hills, the wood is
highly prized for house building and furniture.
(Anonymous, 1969)
Natural regeneration in F. micrantha is very low and
even in the controlled conditions the sowing of intact
samaras do not yield good results as the pericarp of the
seed acts as the barrier for seed germination (Preece et
al., 1995). Moreover, the genus Fraxinus also attracted
the attention of the scientists for the dormancy of its
seeds and also the clonal propagation by cuttings too
(Bedell, 1998; Van Sambeek et al., 2002; Aftab et al.,
2005,). In the present study it is of interest to devise in
vitro propagation techniques for its regeneration and
rapid multiplication. Many studies on in-vitro regeneration
of Fraxinus species viz. F. angustifolia (Tonon et al.,
2001), F. pennsylvanica (Aftab et al., 2005; Du and Pijut,
2008), F. excelsior (Meier-Dinkel, 2007; Hammatt, 1996),
F. americana and F. pennsylvanica (Palla and Pijut,
2010; Van Sambeek and Preece, 2007) have been
undertaken earlier but no such reports are available on F.
micrantha. Present study deals with the development of a
standardized technique for callus induction, somatic
embryogenesis and plantlet proliferation in F. micrantha.
The protocol could enable its effective use not only in
obtaining quick planting material through rapid
multiplication but also in evolving superior genotype by
in-vitro breeding methods.
MATERIALS AND METHODS
Ex-plant source and surface sterilization
Ex-plant i.e. mature leaves, leaf buds petioles and nodal
explants from mature tree of F. micrantha were collected
in the month of July 2009 from its natural habitat. During
initial phase of experimentation these ex-plants were
inoculated in culture medium but they did not show early
and good response to micropropagation. So the
hypocotyle, epicotyle and cotyledonary node of 25 days
old in vitro raised seedlings were inoculated to nutrient
media under aseptic conditions for in vitro propagation
and the satisfactory results were obtained. To obtain the
seedlings, seeds of F. micrantha were collected from the
trees growing naturally at 2000 m in Western Himalayan
region of India and stored in proper storage conditions in
laboratory. As per requirement seeds were excised and
washed repeatedly in tap water. Excised seeds were
treated with 0.15% mercuric chloride for 5 minutes and
thoroughly washed with sterilized water several times.
These seeds were then kept for germination in WPM
basal media. After 25 days seedlings were obtained and
hypocotyle, epicotyle and cotyledonary node were
individually inoculated in culture vessels (conical flasks,
100 ml) filled with different culture mediums (25 ml each)
after proper surface sterilization. For surface sterilization,
seedlings were treated with mild detergent tween-20 (1%)
and quick dip in 70% alcohol followed by 0.1% mercuric
chloride for 2 minutes. then properly washed with
sterilized water several times. Explants were surface
sterilized in laminar flow cabinet only. No other
sterilization method proved successful in establishing
contamination free cultures.
Culture media preparation
After surface sterilization, the seedlings were dissected
aseptically and implanted on the MS (Murashige and
Skoog, 1962) and WP (woody plant) culture medium
(Lloyd and McCown, 1980) with 0.4% agar and different
combinations of growth regulators. Initially, MS medium
with different concentrations and combinations of
cytokinins (BAP, kinetin) and auxins (IAA, NAA, and 2,4D) were tried for callus proliferation as well as shoot root
induction. But the discouraging results (2-4% explants
responded) were found, so not descried here in detail.
Woody plant medium coded as WPMA1-A3 (WPM with
BAP, IBA and NAA), WPMB1-B3 (WPM with Kinetin, BAP
and IBA), WPMC1-C3 (WPM with BAP and IBA), WPMD1-D3
(WPM with Kinetin and BAP), WPME1-E3 (WPM with IBA
and NAA), WPMF1-F3 (WPM with BAP and NAA), WPMG
(WPM without any growth hormones) and WPMH1-H3
(WPM with BAP and 2,4-D) with different concentrations
of above mentioned growth hormones (describe in detail
in table 2) were used. Basal WP medium supplemented
with growth hormones and 3% sucrose were made
semisolid with 0.4% Agar. The pH was adjusted to 5.8
before autoclaving. Twenty vessels for each combination
were used for ex-plant inoculation. Basal media was
routinely autoclaved for 20-30 minutes at 121°C
temperature and 1.2 kg/cm2 pressure depending on size
of culture vessels.
Culture conditions
All the cultures were maintained at 25±2°C temperature
and 55±5% relative humidity under cool white fluorescent
tube, incandescent lamps and diffused light (150-200 lux
ca.) having photosynthetically active radiation using 16
hours light and 8 hours dark cycle. Regular observations
were made for callus induction, somatic embryo
222 Int. Res. J. Biotechnol.
Table 1. Responses of different explants of F. micrantha during micropropagation
Explant
Cotyledonary
node
Epicotyle
Hypocotyle
Plantlet production without somatic
embryogenesis (%)
Multiple Rooting
Plantlets
shooting
Production
80
80
75
25
20
18
17
10
08
formation and shoot root proliferation. At 15 days interval
cultures were transferred to specified medium for shoot
and root initiation and elongation till plantlet proliferation.
Plantlet production through somatic
embryogenesis (%)
Embryo - Root /Shoot
Plantlets
genesis
Induction
Production
15
10
05
90
15
85
08
80
08
medium on different phases of in vitro propagation in F.
micrantha described below.
Plantlet production through direct multiple shooting
Acclimatization and transplantation
In vitro obtained plantlets were removed from glass
vessels and transplanted in 5 cm square plastic pots
containing vermiculite supplied with half strength Hogland
nutrient solution once in every 10 days. Pots were kept in
mist chamber at 28±2°C where 90 sec misting at 10
minute interval was given to attain relative humidity 8595% for ex vitro acclimatization. After 3 weeks,
acclimatized plants were finally moved to polybags
containing FYM :soil:sand::1:2:1 mixture and transplanted
to field as per requirement.
Statistical Analysis
Analysis of variance (ANOVA) was carried out to analyze
the significant variation in callusing, shooting and rooting
under different combination and concentration of growth
regulators in combination with woody plant medium.
RESULTS
Initially, when the explants from mature trees were used
for micropropagation disappointing results were obtained.
Moreover MS medium supplemented with different
concentrations and combinations of cytokinins and auxins
were also tried but satisfactory results were not found in
any of the combinations. MS medium hardly responded
to micropropagation in F. micrantha and tissues dried
after few days of inoculation. Later on, epicotyle,
hypocotyle and cotyledonary node from in vitro raised
seedlings were used as explant and inoculated in WP
medium for in vitro propagation, the medium responded
positively and satisfying results were obtained.
Micropropagation of F. micrantha was considerably
influenced by different combinations and concentration of
growth hormones as well as the ex-plant type used for in
vitro propagation. The effect of different concentrations of
auxins and cytokinins supplemented with WP basal
Cotyledonary node of in vitro raised seedlings resulted
into multiple shooting in 80% culture vessels if inoculated
in WPMA1 medium supplemented with IBA and NAA (both
in 3-7mg/l conc.). Direct multiple shooting in 80%
cotyledonary
node
(maximum
8
shoot/ex-plant
irrespective of the medium) was noticed within one month
of inoculation (Table 1). Shoots having 3-4 cm length
were dissected from ex-plant and subcultured in the
medium having IBA in different concentration and
combinations. Root induction in such shoots were noticed
in the medium in which IBA was present. 75% shoots
resulted into plantlets in WPM A, WPM B and WPMc.
Epicotyl and hypocotyl were not responded well for the
production of multiple shoots directly and plantlet
formation (Table 1) if compared with cotyledonary node.
Formation of embryogenic callus
Development of pale green and compact callus was
noticed after 10 days of inoculation (Figure 1 A). The
presence of auxins (IBA, 2,4-D and NAA) together with
cytokinin (BAP and Kinetin) has been reported to be
indispensable for the induction of callusFast growing,
healthy and proliferating callus was obtained in 75-80%
culture vessels within 10-15 days in WPMA, WPMB,
WPMC, WPMD and WPMF culture mediums, in all these
mediums BAP (3-7 mg/l) was present (Table2) . In all the
treatments, callus induction was more than 70% except
WPME, where BAP was lacking. Callus initiation from all
the explants was significantly influenced by the hormonal
composition of the medium (Table 3).
Somatic embryogenesis
Somatic embyogenesis is highly efficient method for
rapid and mass scale
production
of plant
propagules and offers a potential for storage and
germplasm conservation of desired species also.
Bisht et al. 223
A
Figure 1 A. Embryonic callus formation during In Vitro propagation in Fraxinus micrantha
from epicotyle part
Table 2. Effect of different combinations and concentrations of growth hormones supplemented in
WPM during different phases of micropropagation in F. micrantha
Medium
Code
BAP
WPMA1
WPMA2
WPMA3
WPMB1
WPMB2
WPMB3
WPMC1
WPMC2
WPMC3
WPMD1
WPMD2
WPMD3
WPME1
WPME2
WPME3
WPMF1*
WPMF2*
WPMF3*
WPMG**
WPMH1
WPMH2
WPMH3
3.0
5.0
7.0
4.0
5.0
6.0
2.0
3.0
2.0
2.0
3.0
4.0
0
0
0
3.0
2.0
4.0
0
0
4.0
2.0
A1-A3
Hormones Used (in mg/l)
Kinetin IBA (mg/l) 2,4-D NAA (mg/l)
0
0
0
2.0
2.0
2.0
0
0
0
4.0
3.0
2.0
0
0
0
0
0
0
0
0
0
0
3.0
5.0
7.0
4.0
5.0
6.0
2.0
2.0
3.0
0
0
0
5.0
4.0
6.0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
5.0
4.0
2.0
B1-B3
3.0
5.0
7.0
0
0
0
0
0
0
0
0
0
5.0
6.0
4.0
3.0
4.0
2.0
0
0
0
0
Responses (%)
CE S R
80 72 50
85 74 60
60 15 10
86 40 45
88 40 43
65 30 30
85 00 55
83 05 52
80 00 55
80 60 45
70 65 35
75 63 48
05 00 55
05 05 55
00 00 55
85 04 55
83 00 50
80 05 53
60 00 50
70 00 20
80 10 10
65 00 10
C1-C3
WPM
(WPM with BAP, IBA and NAA), WPM
(WPM with Kinetin, BAP and IBA), WPM
D1-D3
E1-E3
(WPM with BAP and IBA), WPM
(WPM with Kinetin and BAP), WPM
(WPM with IBA and
F1-F3
G
H1NAA), WPM
(WPM with BAP and NAA), WPM (WPM without any growth hormones) and WPM
H3
(WPM with BAP and 2,4-D), CE-Callusing and embryogenesis, S-Shoot induction, R-Root
induction
*Micronutrients and vitamins of MS medium were used, **WPM Basal medium without hormone
224 Int. Res. J. Biotechnol.
Table 3. ANOVA summary Table
Callusing
(C)
Emergence
of
Shoot
(SE)
Rooting
(R)
Variance
Source
Between
Groups
Within
Gorups
Total
Between
Groups
Within
Gorups
Total
Between
Groups
Within
Gorups
Total
SS
df
MS
F
P-value
F-Crit
48015.65
23
2087.637
995.4296
1.14153E56
1.756759
100.6667
48
2.097222
48116.32
52396.99
71
23
2278.13
p>0.001
3814.543
highly significant
1.1968E1.756759
70
28.66667
48
0.597222
52425.65
16920.99
71
23
735.695
p>0.001
696.9743
highly significant
5.74494E1.756759
53
50.66667
48
1.055556
16971.65
71
p>0.001
highly significant
B
Figure 1 B. Germinating somatic embryos and proliferating shoots during In Vitro
propagation in Fraxinus micrantha from epicotyle part
desired species also. In the present study proliferating
callus were transferred in all the WP medium described
under material and methods for somatic embryogenesis
and organogenesis (Table 2). Green colored somatic
A
B
embryos were appeared in (Figure 1B) WPM , WPM
D
F
WPM and WPM mediums, in all these treatments
different concentration and combination of BAP was
present. Somatic embryos were then separated from
callus and subculture to fresh medium specified for shoot
and root induction. Constant supply of BAP appeared
essential for all phases. The role of BAP was statistically
correlated with the micropropagation in F. micrantha
(Table 3) as the medium without BAP or kinetin (WPME)
almost failed to induce callusing, embryogenesis and
shoot/root initiation during entire tissue culture process
(Table 2).
Shoot/Root induction and plantlet proliferation
Organogenesis leading adventitious shoot production
from somatic embryos was frequently occurred by the
epicotyle if cultured in WPMA and WPMB mediums.
Among all the media tried WPMA1, WPMA2, WPMD1,
WPMD2and WPMD3 favored the shoot initiation from
somatic embryos. Maximum (74%) organogenic callus
resulted into shoot emergence (Table 2 and Figure 1B) in
A1
A2
WPM and WPM medium with 3-5 mg/l BAP, IBA, and
NAA. But more than 5 mg/l concentrations (WPMA3)
reduced shoot initiation percentage and only in 15%
embryos shoot induction was noticed. Supply of 3 mg/l
cytokinins (3mg/l BAP and kinetin in WPMD2) extended
shoot induction upto 65% (Table 2).
The auxin supply alone did not favour callusing as well
Bisht et al. 225
C
D
Figure 1. Different phases of in vitro propagation in Fraxinus micrantha through
epicotyle part (C: Root induction in subcultured proliferating shoots D: One month old
plantlet ready to be transferred for hardening)
as percent shoot induction at any of the concentrations
tested (WPME1, WPME2 and WPME3) but increased in the
shoot length appeared. For which, the continuous
subculturing at the interval of 7 days is advisable to avoid
the desiccation of callus or emerging shoot. Multiple
shoots raised through embryogenic calli of epicotyle were
A
B
differentiated after 20 days in WPM and WPM medium.
Further increase in shoot length (up to 4 cm) was
achieved within 30 days of explant inoculation on auxin
and cytokinin rich medium (Table 2 and Figure 1C).
Emerging shoots were gently separated and
A
H
transferred to all the culture medium (WPM -WPM )
described in table 2 for root initiation and its development
(Figure 1C, 1D). The rooting of elongated shoots was
easily achieved on WPMA, WPMB, WPMD and WPMF
medium. This showed that the presence of IBA, NAA and
BAP (3-5 mg/l) markedly improved the rooting
percentage (50%) of developed shoots on time to time
serial transfer on same culture medium. Root induction
(35-40%) in in developed shoots of F. micrantha was
found easy even in auxin free WPMD medium.
Interestingly, rooting in somatic embryos before and after
shoot emergence was noticed in WPMG basal media
without growth regulators (Table 2).
Sometimes inoculation of hypocotyle/epicotyle in media
H1
containing auxin (WPM ) directly produced roots first in
15-20% explants without callusing or shooting. These
rooted explants if transfer to WPMA, WPMB, and WPMD
shoots were developed successfully and resulted into
seedlings. Explants inoculated in culture media placed in
dark or light respond similarly in terms of different phases
of tissue culture and significant results were not found. It
was interesting to observe that growth hormone free WP
medium supplemented with micronutrients of MS medium
(WPMG) failed to induce callus but, if already produced
callus (produced in other mediums) were transferred to
this medium, somatic embryos appeared in it. This WPMG
medium also promoted root development
in somatic
embryos as well as developing shoots but had no
promotive effect on shoot multiplication and elongation.
The presence of IBA, NAA and BAP (5 mg/l each) in
WPM seems effective in callusing, embryogenesis as
well as root/shoot emergence and elongation/growth,
A2
therefore (WPM ) was considered as the best culture
226 Int. Res. J. Biotechnol.
medium for in vitro multiplication of F. micrantha (table 2).
In this medium rapid production of plantlets were also
noticed within 30-32 days.
On the basis of ANOVA, variation in callusing, shoot
emergence and rooting were found highly significant
(p>0.001) with respect to the WP basal medium
supplemented
with
different
concentration
and
combination of growth regulators used for different
phases of in vitro multiplication (Table 3).
Plantlets Hardening and Transplantation
In vitro raised plantlets were transplanted to pots filled
with sterilized vermiculite and kept under high humidity
for hardening in a mist chamber. Micropropagated
plantlets were transfer to pot after 2 weeks of root
initiation when the developing roots acquired 6cm length.
During hardening hardly 5-6% seedlings were desiccated
and all the other seedlings acclimatized and hardened
well. After 2 weeks the seedlings were transferred to
nursery and transplanted as per requirement wherever
needed with an aim to conserve and restore the species
in its natural habitat.
DISCUSSION
Conservation of high value medicinal plants of Himalaya
through
rapid
and mass
multiplication
using
agrotechnological approaches (Prakash et al., 2011;
Kumar et al., 2011, Nautiyal et al., 2009) and
biotechnological means (Kondamudi et al., 2010, Jha et
al., 2011)) is always of interest of people working in this
sector. The present study deals with the in vitro
propagation of F. micrantha, a multipurpose tree species
of Himalaya needs a refined and rapid method of
multiplication. Overall observations suggest that WPMA
and WPMB medium supplemented with BAP, Kinetin, IBA
and NAA (in low concentrations of 2-5mg/l) supports the
callus induction, somatic embryogenesis as well as
root/shoot development and growth in all the seedling
segments. However, higher concentrations (>5mg/l) of
growth hormones proved less effective. Low
concentrations (1-5mg) of cytokinins and auxins
combinations in WPM or MS medium respond well for
shoot and root development in vitro culture of Saccharum
officinarum (Baksha et al., 2002), Artimisia vulgaris
(Kumar and Ranjitha Kumari, 2010) and Capsicum
annum (Ostroshy et al., 2011).
The presence of IBA, NAA and BAP (5 mg/l each) in
(WPMA2) seems effective in all the phases of in vitro
multiplication so considered as the one best culture
medium for F. micrantha (table 2). It shows the
synergistic effect of auxins with cytokinins for promoting
callus, shoot/root emergence and plantlet development
as compared to alone. Serial supply of BAP in nutrient
medium seems essential for all regeneration phases
through tissue culture in F. micrantha similar to F.
angustifolia (Tonon et al., 2001). But BAP failed to induce
adventitious structure and TDZ works well in F.
Americana (Hammatt, 1996) and F. excelsior (Bates et al.
1992) in contrast to F. micrantha and F. aungustifolia
where BAP respond well. Recent studies with Pongamia
pinnata (Srivastava and Kant, 2010) also showed similar
results in relation to direct multiple shooting and rooting in
in vitro raised seedlings as in F. micrantha here.
Similarly, in the present study the epicotyle segments
of F. micrantha callused in presence of 2, 4-D in WP
medium (WPMH1-H3) after 20 days. The callusing was
increased when kinetin was incorporated in the medium
like WPMB (Table 2). It is well reported that WPM
supplemented with auxins and cytokinines favours the
establishment of micropropagation from an explant in a
vast majority of tree species including Fraxinus viz.
Fraxinus angustifolia (Tonon et al., 2001), F. excelsior
(Meier-Dinkel, 2007), F. americana (Palla and Pijut
2010), Quercus rubra (Vengadesan and Pijut, 2009),
Pongamia pinnata (Srivastava and Kant, 2010) and
Memecylon edule (Elavazhagan and Arunachalam,
2010). Van Sambeek and Preece, 2007 and Du and Pijut
2008 reported in vitro propagation in Fraxinus using MS
and SKW medium supplemented with growth hormones
rather than WP medium.
Rooting in somatic embryos was noticed in WPM basal
media without growth regulators but rooting in emerged
shoots was developed in the medium in which IBA was
present in F. micrantha (Table 2). WPM without plant
growth regulators and containing BAP and IBA 4mg/l and
0.15mg/l) works well in root/shoot development during
micropropagation of some other Fraxinus species (PerezParron et al., 1994) also. Root induction in F. micrantha
(35-40%) was found easy in developed shoots even on
D
auxin free WPM medium, similar to F. angustifolia and
F. excelsior (Silviera and Cottignies, 1993).
CONCLUSION
Morphogenetic processes like somatic embryogenesis,
organogenesis and rooting are often described as
complex phenomena characterized by different phases
each with specific nutritional requirement but in case of F.
micrantha, WP medium supplemented with cytokinins
(BAP and kinetin) in combination with auxins (IBA, NAA
and 2,4-D) conclusively proved effective for callusing,
organogenesis, as well as differentiation rather than
alone. It is also concluded that the epicotyle part in
comparison to cotyledonary leaf and hypocotyle
segments showed higher potential to regenerate.
Cytokinins (BAP and Kinetin) along with auxins (IBA,
NAA and 2,4-D) combinations proved more satisfactory
for plantlet proliferation. In vitro propagation in F.
micrantha for regeneration and rapid multiplication seems
Bisht et al. 227
viable approach to conserve this valuable medicinal tree
species of Himalaya to restore the Himalayan ecosystem.
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