Journal of Cell and Molecular Research (2010) 2 (2), 81-85
Conservation of Colutea gifana, a rare and potential
ornamental species, using in vitro method
Mahnaz Kiani1*, Homa Zarghami1, Ali Tehranifar2 and Farshid Memariani3
1
Ornamental Plant Department, Research Center for Plant Sciences, Ferdowsi University of Mashhad, Mashhad, Iran
2
Department of Horticulture, Faculty of Agriculture, Ferdowsi University of Mashhad, Mashhad, Iran
3
Botany Department, Research Center for Plant Sciences, Ferdowsi University of Mashhad, Iran
Received 10 November 2010
Accepted 18 December 2010
Abstract
In vitro methods provide a variety of tools to supplement traditional methods for collection, propagation and
preservation of endangered plant species. In this study, an efficient protocol was developed for in vitro
propagation of Colutea gifana, a rare and endangered plant species with limited reproductive capacity that
grows in a narrow area of Iran. Single node explants were used for a series of experiments to select the
appropriate disinfection method and growth regulators for establishment, proliferation and rooting stages.
Explants showed the highest establishment percent after treatment with 2% Sodium hypochlorite (NaOCl) for
15 min, cultured in MS medium containing 2.2 µM 6-benzylaminopurine (BA) and 1 µM indole-3-butyric acid
(IBA). In proliferation stage, 8.8 µM of BA was more effective cytokinin than Kinetin (Kin) and Thidiazuron
(TDZ) for growth induction of axillary shoots. In vitro rooting of proliferated shoots was induced in halfstrength MS medium in all concentrations of both tested auxins i.e. IBA and α-naphthalene acetic acid (NAA).
Eighty percent of the plantlets were successfully acclimatized to ex vitro conditions, showed normal
development. These plants were used to replenish declining populations in the collection sites and conserve C.
gifana from extinction.
Keywords: conservation, extinction, Fabaceae, germplasm, micropropagation
∗
Introduction
In vitro propagation of endangered plants offer
considerable benefits for the rapid multiplication of
species that are at risk, have limited reproductive
capacity and exist in threatened habitats (Fay,
1992). These methods are essential components of
plant genetic resources management and become
increasingly important for conservation of rare and
endangered plant species (Sudha et al., 1998;
Benson et al., 2000; Iankova et al.,2001; Bhatia et
al., 2002;). This is especially important since in
vitro propagation allows the establishment of
cultures from the minimum amount of starting
material coupled with the possibility of further
multiplication (Benson, 2000). In combination with
another in vitro culture technique, referred to as
slow growth, it is possible to establish long term
collections of germplasm with minimal resources
(Watt et al., 2000). Similarly, these techniques
facilitate the application of genetic manipulation
∗Corresponding author E-mail:
mhkiani@um.ac.ir
procedures (Knapp et al., 2001; Ueno et al., 1996).
Colutea gifana Parsa is a local endemic plant in
N Khorasan province, NE Iran. This species is a
dwarf shrub with yellowish-green branches,
glabrous paired leaves and inflorescences that
average 1.5-2.5 cm long with 4-5 yellow flowers
(Rechinger, 1984) that has a high potential to be
used as a new ornamental plant. Due to the limited
extent and occurrence of natural populations, this
species is considered vulnerable (Jalili and Jamzad,
1999) and facing extinction in the medium-term
future, therefore needs urgent in situ and ex situ
conservation. Furthermore, as low seed set makes
problems in seed propagation of this species, in
vitro multiplication might be used as an alternative
method for reintroduction of this species into the
natural environment (Wochock, 1981) and reducing
the risk of extinction (Nadeem et al., 2000;Chandra
et al., 2006). However, the members of Fabaceae
family are generally recalcitrant under in vitro
conditions (Trigiano et al, 1992; Jha et al, 2004).
This study was conducted to develop a protocol for
in vitro propagation of this endemic and rare plant,
to help with its future in situ and ex situ
conservation.
82
Materials and Methods
Plant material and preparation of explants
Young shoots (20 cm long) were collected from
six Colutea gifana shrubs in N Khorasan province
in May 2009. Collected shoots were washed with
running tap water for 1 h and surface sterilized with
70% ethanol (30s) and different concentrations and
time intervals of sodium hypochlorite (NaOCl) and
mercuric chloride (HgCl2) (Table 1). After rinsing
three times with distilled water, the terminal buds
and leaves were removed. Single node explants
with 2 cm long were cultured in Murashige and
Skoog basal medium (Sigma-Aldrich, USA; pH
adjusted to 5.8) with 2.2 µM BA and 1 µMIBA.
Cefotaxime at 100 mg/l concentration was also
used in some disinfection treatments to evaluate the
effect of this treatment for further contamination
control (Table 1). Cultures were maintained at
24±1ºC and illuminated by white fluorescent tubes
(40 µmol m-2 s-1) 16 h per day. Fifteen replicates
were used for each treatment. Number of surviving
explants and explants with developing axillary
shoots were recorded after four weeks of culture.
For the proliferation stage, developed axillary
shoots were cultured on MS medium supplemented
with BA (2.2, 4.4, 8.8 or 17.7 µM), Kinetin (2.2,
4.4, 8.8 or 17.7 µM) or TDZ (0.5, 1.1, 2.2 or 4.5
µM) and IBA (1 µM). After transferring to the
proliferation medium, number of shoots per explant
and shoot length were recorded after four weeks. At
rooting
stage,
well-developed
shoots,
approximately 4 to 6 cm in length, were cultured on
half-strength MS medium supplemented with IBA
or NAA (1.3, 2.7, 5.4or 10.8 µM).
After 5 weeks, rooted shoots were gently washed
with distilled water and transferred to plastic cups
with drainage holes containing a 1:1 mixture of
perlite and fine sand for the first month of
acclimatization stage. For the initial seven days,
plantlets were covered with a transparent cup and
irrigated with a half-strength MS solution (sucrose
free) every other day. After seven days, plantlets
were exposed to the atmosphere and irrigated with
both distilled water and a half-strength MS
solution. Surviving plantlets were transferred to
Conservation of Colutea gifana, a rare and potential…
larger containers with a 1:1 mixture of perlite and
peat before moving plants to outdoor and collection
sites.
Data analysis
Collected data were subjected to analysis of
variance (ANOVA) using SAS (SAS Institute, Inc.,
Cary, NC) software. Mean values were separated
according to Duncan’s multiple range test at
probability of 0.05 level.
Results
Explants showed the highest establishment rate
(less contamination and higher growth) after a 2%
NaOCl disinfection treatment for 15 min.
Cefotaxim didn’t show any positive effect on
contamination control (Table 1). Although antibiotic
treatments are the most widely used method to
eliminate bacterial contaminations, they are not
always effective against different bacterial strains
and several cases of their toxicity for plants have
been reported (Cassels 1997; Falkiner 1997).
Bud breakage and growth of shoots were noticed
in the pre-existing axillary meristems of nodal
explants in the presence of all tested cytokinins
(BA, Kinetin, TDZ) in MS medium.BA was more
effective cytokinin considering shoot numbers and
at concentration of 8.8 µM resulted in the highest
multiplication rate with average values of 5.3
shoots per explants (table 2). The superiority of BA
over the other cytokinins has been also reported in
micropropagation of some species from Fabaceae
family (Rout, 2005; Prakesh et al, 2006). As BA
concentration increased, shoot growth became
distorted, showed deformed leaves, pale color, and
hyperhydricity, which may be related to the excess
of cytokinin (Arora et al., 2009). An inhibitory
effect of high concenteration of BA has also been
reported in Pterocarpus santalinus L. from
Fabaceae (Prakash et al, 2006). TDZ was less
effective for shoot proliferation in this experiment,
although it has been reported that induces high rates
of axillary shoot proliferation in some Fabaceae
species, such as Swainsona Formosa (Jusaitis
1997), Glycine max (Kaneda et al, 1997) and
Swainsona salsula (Yang et al, 2001).
83
Journal of Cell and Molecular Research
Table 1.Effect of different disinfection treatments on infection percent and growth of C. gifana explants.
Treatment
3 min 0.1% HgCl2
6 min 0.1% HgCl2
3 min 0.1% HgCl2 + Cefotaxim
6 min 0.1% HgCl2 + Cefotaxim
20 min 1% NaOCl
30 min 1% NaOCl
30 min 1% NaOCl + Cefotaxim
10 min 2% NaOCl
15 min 2% NaOCl
15 min 2% NaOCl + Cefotaxim
Infection (%)
0
7.2
11.7
33.3
0
5
27.2
0
0
0
Growth (%)
37.5
14.2
30
16.6
40
5
0
26.3
70
36.8
Table 2. Effect of different cytokinins on axillary shoot proliferation.
Cytokinin ( µM)
Sprouting (%)
Shoot number
Shoot length (cm)
Mean number
of nodal segment
92
89
100
100
2.3 b
4.6 a
5.3 a
3.8 a
0.5 ab
0.8 a
0.8 a
0.5 ab
1.6 d
2.0 cd
2.9 bc
1.7 cd
100
100
93
100
1.0 b
1.3 b
1.0 b
2.4 b
0.6 ab
0.3 b
0.8 a
0.8 a
2.1 cd
2.4 bcd
2.8 bcd
2.8 bcd
93
100
93
86
1.8 b
1.2 b
2.1 b
1.2 b
0.7 ab
0.9 a
0.5 ab
0.8 a
2.3 bcd
2.2 bcd
2.6 bcd
4.0 a
BA
2.2
4.4
8.8
17.7
Kinetin
2.2
4.4
8.8
17.7
TDZ
0.5
1.1
2.2
4.5
*Values within each column followed by the same letters are not significantly different by the Duncan Multiple Rang test at 0.05%
probability level.
Rooting and Acclimation
Root initiation was observed within 10–15 d in
all treatments expect in auxin free medium (data
not shown). Micro-cuttings cultured on half
strength basal MS medium with IBA or NAA
produced normal roots without forming callus. The
highest frequency of root initiation (average of
55%) was observed in half strength MS medium
supplemented with IBA in comparison to NAA
after five weeks (table 3, figure1D). No significant
differences were found among treatments when
comparing the number of roots and root length
(table 3). Rooted plantlets were successfully
acclimatized to growth chamber conditions and
survival percentage was 80%. Acclimatized
plantlets were healthy and well developed,
phenotypically similar to the parental stock and
showed active growth (figure 1F). These plants
were successfully established in the collection sites
(N Khorasan province) and continued to grow.
84
Conservation of Colutea gifana, a rare and potential…
Figure 1. Complete micropropagation cycle of Colutea gifana: (A, B) Shoot initiation from nodal segments; (C)
Multiple shoot regeneration; (D) Root development; (E, F) Acclimatization stages.
Table 3. Effect of different auxin treatments on rooting percent and root growth on 1/2 MS medium.
Auxin (µM)
Rooting
(%)
Number
of root
Mean
root length/shoot (cm)
60
50
50
60
2.2 a
2 a
2.9 a
2.5 a
2.1 a
2.7 a
2.5 a
1.7 a
40
40
40
50
2.5 a
1.8 a
2.4 a
2.7 a
1.6 a
1.6 a
1.1 a
1.1 a
IBA
1.3
2.7
5.4
10.8
NAA
1.3
2.7
5.4
10.8
*Values within each column followed by the same letters are not significantly different by the Duncan Multiple Rang test at 0.05%
probability level.
Discussion
a
protocol
for
micropropagation of C. gifana, which is the
first report for this species. It is important to note
This
paper
presents
that the morphology of in vitro plantlets showed a
true-to-type growth habit, both in vitro and when
transferred to ex vitro growth conditions. There was
no evidence of callus formation at proliferation
stage and shoot multiplication occurred from pre-
existing axillary shoot primordia that eliminated the
risk of genetic instability. Our future, long-term
research will concentrate on the ex vitro transfer
stage for the possible introduction of this species as
a new ornamental plant and assessing the value of
the protocol for micropropagation of the other
Colutea species. To date, ex vitro transfer has been
based on the use of commercially available
substrates, however more consideration must be
given to the environment-specific needs of this
species. In the wild, C. gifana proliferates in
85
Journal of Cell and Molecular Research
serpentine soil, which is derived from ultramafic
bedrock. It may therefore, be appropriate to
simulate the environmental requirements of
C.gifana during acclimatization stage. The
developed protocol would be useful for large-scale
multiplication of C. gifana, ex situ and in situ
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