AcademicPres
Akinropo MS et al. (2020)
Notulae Scientia Biologicae 12(1):74-89
DOI:10.15835/nsb12110604
Research Article
Notulae Scientia Biologicae
In vitro callus and shoot regeneration in Enterolobium cyclocarpum
(Jacq.) Grised. - a fast timber yielding species
Michael S. AKINROPO*, Benjamin E. AYISIRE, Ejeoghene R. OGBIMI
Obafemi Awolowo University, Department of Botany, Ile-Ife, Osun State, Nigeria; mickoseun@yahoo.com (*corresponding author)
Abstract
This study was conducted to investigate the in vitro callus induction and rapid shoot regeneration
potential in Enterolobium cyclocarpum, a plant native to central Mexico but widely introduced into Africa. The
leaf, stem and nodal explants of E. cyclocarpum were cultured on full strength Murashige and Skoog (MS)
medium supplemented with different concentrations of Cytokinins - Benzyladenine (BA) and/or Kinetin and
Auxins - Naphthalene acetic acid (NAA) and/or 2,4-Dichlorophenoxylacetic acid (2,4-D) each alone and in
combination. The leaf explants did not respond to these treatments. The Nodal explants were best for
caulogenesis, while the explant responses were in the order- nodal > stem > cotyledon for callogenesis in MS
medium supplemented with BA and/or Kin combined with NAA and/or 2,4-D. The varied combinations
induced white compact callus. The highest callus production was observed on MS medium supplemented with
2.7 µM NAA + 2.2 µM BA and 5.4 µM NAA alone. Nodal and cotyledon explants developed callus and
multiple shoots on MS supplemented with a combination of cytokinin (BA and/or Kin.) and auxin (NAA
and/or 2,4-D). The maximum number of 3.98 ± 0.37 and 2.1±0.11 shoots/explants were recorded for nodal
and cotyledon explants on MS medium supplemented with a combination of 8.8 µM BA+2.7 µM NAA and
2.2µM BA+2.7 µM NAA respectively. On the basal medium, 10% of the excised shoots rooted successfully.
Thus, this in vitro method can be exploited for conservation and mass propagation of this fast timber yielding
tree and also utilized for embryogenesis studies.
Keywords: callus; caulogenesis; callogenesis; explants; multiple shoots
Introduction
The anthropogenic deforestation occurring throughout the world has always been increasing, with an
alarming rate in the last 3 decades in particular in West Africa primarily due to urbanization, unsustainable
logging, agricultural farming and collection of fuel wood (FOMERCU, 1999; Sayer et al., 2010). The gradual
increase in deforestation practices for economic and/or social reasons without any simultaneous replanting is
a global threat to the sustainability of the environment (FAO, 2005; Odediran et al., 2013). Africa has the
second highest rate of tropical deforestation in the world. The tropical forests in this region have declined at
an annual rate of 3.4 million hectares between 2000 and 2010 because of degradation and deforestation
processes (FAO, 2010; Eleanya, 2014).
Received: 14 Nov 2019. Received in revised form: 12 Feb 2020. Accepted: 28 Mar 2020. Published online: 31 Mar 2020.
Akinropo MS et al. (2020). Not Sci Biol 12(1):74-89.
75
Nigeria, alone lost a range between 3.5 to 3.7% of forest land per annum (Ladipo, 2010; UN-REDD,
2013;). Attendant consequences of deforestation are erosion, flooding, global warming, loss of habitat for
earth’s land animals and plants and not the least desertification (Farinola et al., 2013). In order to mitigate these
consequences, reforestation is urgently needed and this can be done by introducing fast-growing tree species
such as Enterolobium cyclocarpum.
Enterolobium is an exotic species introduced to Nigeria and other tropical regions (Ezenwa, 1999;
Rodríguez-Sahagún et al., 2007). It is native to tropical America, where it is among the most invaluable and
majestic tree tolerant of a wide range of rainfall level, temperature and soil condition. E. cyclocarpum is naturally
distributed from western and central Mexico to the northern part of South America (Neimbro-Rocas et al.,
2003). E. cyclocarpum usually called monkey ear or ear pod tree belongs to the family Fabaceae. The genus
Enterolobium contains only five species, all native to South and Central America. E. cyclocarpum is the only
widely distributed and is the national tree of Costa Rica and prized for its ornamental, timber and large feathery
shady relief from the intensity of the sun. It is a semi-deciduous, medium- to large-sized, and fast-growing tree
species reaching about 35-50 m in height, wide spread of about 35 m or more (Rodríguez-Sahagún et al., 2007).
The trunk is branched with many stems, with light gray bark, and with prominent dark brown vertical fissure.
The tree crown is broader and widely spreading especially those that reach canopy (Figures 1 and 2). The
bipinnate compound leaves are alternate, 15-40 cm in length, with 20-25 leaflets. The inflorescent consists of
fragrant, white flowers, with about 20 filamentous stamens, as its main features, and a single pistil bounded
together by a short green corolla and calyx. (Neimbro-Rocas et al., 2003; Harmon, 2008; Pier, 2008).
The fruits are flattened, glossy, dark-brown indehiscent pod, shaped like an orbicular disk (Figures 3 and
4). The pod contains about 8-16 radially arrange seeds. The seed is hard-coated resembling small stone in
strength and durability, which avoids germination until a structural modification (scarification) allows the
hydration of the embryo. Enterolobium seeds are big, red-brown and marked with a conspicuous light brown
ring. Seed are rich in protein (about 35%) and amino acid (Rodríguez-Sahagún et al., 2007). They also contain
iron, calcium, Phosphorus and ascorbic acid (Harmon, 2009). Also, seeds and bark contain tannins (Barwick,
2004).
The wood of E. cyclocarpum is lightweight and is resistant to termite attack (Uphof, 1959; Standley and
Steyermark, 1976), which makes it feasible for house construction, furniture and shipbuilding (NiembroRocas, 2003; Rodríguez-Sahagún et al., 2007). It is also useful as firewood due to its high caloric content
(Rodríguez-Sahagún et al., 2007). It is widely grown as a shade tree to shelter plantation, livestock, even its
fruits and leaves are used as fodder directly from the tree or as a nutritional complement (Carranza-Montaño
et al., 2003; Mota et al., 2005). Syrup and gum obtained from the bark and truck respectively are used in the
treatment of colds and medicine for chest affections (Uphof, 1959; Burkil, 2004).
In Mexico, the boiled seeds are consumed in sauces, soups and as a coffee substitute, and several
medicinal properties have been attributed to them (Carranza-Montaño et al., 2003; Niembro-Rocas, 2003).
Moreover, several biotechnological applications such as its gum as a fungi culture substrate or for the
production of ice cream and yogurt have recently been proposed for this tree species (Rincón et al., 2005, 2006).
The conventional method of propagating E. cyclocarpum is mainly sexual i.e from seeds. However, the
regeneration rate of most leguminous trees in natural habitats is low due to seed dormancy (Nanda et al., 2004).
This limitation consequently affects the germination rate and the death of young seedlings under natural
conditions. The conventional method of propagation cannot meet the need for forest restoration programs,
hence micropropagation, is required.
To date, micropropagation offers a rapid means for reforestation, multiplying woody biomass and for
conserving elite and rare germplasm (Xie and Hong, 2001). There is limited information on in vitro techniques
of propagating E. cyclocarpum. Rodríguez-Sahagún et al. (2007) in Mexico has used nodal segments and apical
segment from axenic seedlings were cultured on MS basal medium supplemented naphthaleneacetic acid
Akinropo MS et al. (2020). Not Sci Biol 12(1):74-89.
76
(NAA) in combination with benzyl adenine (BA) or kinetin (KIN). They reported multiple shoot induction
using nodal segment collected from in vitro germinated seedling when cultured on MS medium was
supplemented with combination BA and NAA. However, E. cycocarpum as an established exotic species to
Nigeria, little or no studies had been done on it in vitro propagation.
Thus, the objective of this study was to evaluate the effects of Cytokinin-BA and Auxin- NAA on
multiple shoot induction using ex vitro explants of leaf, stem and node of E. cyclocarpum - a protocol needed
for the mass propagation as well as genetic improvement programs.
Figure 1. Habit of Enterolobium cyclocarpum tree
Figure 2. Ear pod fruits (a) and the hard-coated seeds (b)
Materials and Methods
Plant materials
The seeds of E. cyclocarpum were obtained from indehisced pods collected from an actively growing tree
in Reforestation Garden of Botany, Department of Botany, Obafemi Awolowo University, Ile - Ife. Seeds were
selected based on their non-dehised seed coat, scarified using emery paper. The scarified seed was surface
sterilized using 10% NaOCl for 15 minutes, and then kept under running tap water for 30 min. Then they
were soaked in distilled water for 15 mins to allow for imbibition. Finally, the seeds were planted in petri- dishes
for 2 weeks before they were transferred to top soil and were out in a randomized environment in which they
can access various environmental factors.
Explant source and sterilization
The leaf, stem and node ex vitro explants of E. cyclocarpum were obtained from 3-8 weeks old seedlings
and were kept under running water for 10-15 minutes before culturing.
Akinropo MS et al. (2020). Not Sci Biol 12(1):74-89.
77
Media and culture of explants
The explants were cultured on full strength MS (Murashige and Skoog, 1992) basal medium containing
3% sucrose per liter. The pH of the media was adjusted to 5.7±0.02 with 1 N NaOH or 1 N HCl solutions,
solidified with 0.8% agar (BDH) per liter prior to autoclaving at 121 °C and 103 kpa for 15 minutes. The basal
medium alone served as control and was also supplemented with cytokinin separately and/or with auxins.
The leaf, stem and Nodal explants were disinfected with 70% ethanol for 3-4 minutes and surface
sterilized by washing with 10% NaOCl containing two drops liquid detergent (tween-20) for 15 minutes after
which they were rinsed in at least three changes of sterile distilled water. The leaf explants were sectioned to
about 10 mm2 in size while the stem and the node were about 10 mm in length. The explants were cultured in
15 cm3 test tube containing 8 ml of the culture medium. The leaf explants were cultured with the adaxial surface
in contact with the medium while the nodes and stem cutting were slantingly explanted on the medium.
Culture conditions
The culture tubes were covered with non-adsorbent cotton wool and wrapped with aluminum foil, after
which they were incubated at a temperature of 25 ± 2 ºC and a photoperiod of 16 h light with cool white
fluorescent light (20 μmol m-2s-1).
Statistical analysis
The data in terms of the number of induced shoot buds, shoot length, callus induction, morphology,
and colour of callus were recorded after 4-5 weeks in culture. Each treatment consisted of 10 replicates and all
experiments were repeated twice. The data were analyzed statistically using standard error (S.E) of the mean
and were separated using Duncan Multiple Range Test (DMRT). All statistical analyses were performed with
Statistical Package for Social Sciences (SPSS, version 11.5).
Results
There was no callus initiation with the cotyledon explants of E. cyclocarpum cultured in the MS medium
alone i.e. the control. Initiation of callus was observed within 2 weeks in the cotyledon explants on MS media
containing low concentrations of auxin alone or their various concentrations combined with the cytokinins.
The proliferation rate of callus induced from the cotyledon explants was generally slow. Shoot bud formation
became evident from the scanty callus induced by some auxin /cytokinin combinations after 3 weeks of culture
(Figure 3a).
A maximum mean number of 2.1 shoots per explant was observed with cotyledon explants grown on
MS medium containing 2.7 µM NAA + 2.2 µM BA which also induced the highest frequency (58.47±1.19%)
of callus formation (Tables 1 and 3). The concentration of 2.7 µM NAA + 4.7 µM kinetin in its case induced
highest frequency (51.57±1.88%) of off white callus (Table 5) and the highest mean number of 1.5
shoots/explant although this was not significantly (p ≤ 0.05) different from 1.4 shoots/explant induced from
cotyledon explants cultured on MS medium containing 2.7 µM NAA + 2.35 µM kinetin (Tables 2, 4 and 5).
Little extension growth was shown by the shoot buds when subcultured. The shoot buds that formed from the
cotyledon-derived callus on MS medium supported with 2.7 µM NAA + 4.7 µM BA developed further when
subcultured, 40% of the shoots produced thick roots in MS hormonal free medium forming complete plantlets.
Akinropo MS et al. (2020). Not Sci Biol 12(1):74-89.
78
Figure 3. Scanty (with shoot bud) and compact callus induced from cotyledon and stem explant of E.
cyclocarpum after three weeks of culture respectively on MS medium supplemented with 4.5 μM 2, 4-D +
9.4 μM Kinetin (a) and 5.4 µM NAA alone (b)
Table 1. The effects of various concentrations of NAA, 2,4-D alone or each separately combined with BA
S/N
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
MS medium + PGR
% response of
% response of
concentration (µM)
cotyledon explant
nodal explant
(mean ±S.E)
(mean ±S.E)
BA
NAA
2,4-D
0.00±0.00a
0.000±0.00a
2.25
34.59±3.01cd
48.47±1.099cde
4.5
34.22±3.62cde
43.40±1.47bc
9.0
0.00±0.00a
39.73±1.88b
cd
2.7
33.23±3.50
55.37±4.33e
ef
5.4
42.34±4.00
51.43±2.38de
a
10.8
0.00±0.00
48.13±1.40cd
a
2.2
0.00±0.00
66.67±2.14f
bc
2.2
2.25
29.76±0.68
74.50±2.05ghijk
2.2
4.5
40.5±2.22def
75.27±2.30ghijk
2.2
9.0
35.23±3.30cdef
68.67±1.54fgh
2.2
2.7
58.47±1.19g
81.57±1.92k
f
2.2
5.4
43.09±2.14
80.60±2.19jk
def
2.2
10.8
40.44±2.94
75.80±3.23hijk
a
4.4
0.00±0.00
71.90±1.96fghi
c
4.4
2.25
31.14±3.45
80.57±1.25jk
cd
4.4
4.5
34.24±3.56
76.10±2.81hijk
b
4.4
9.0
22.32±2.71
75.20±2.84ghijk
dcf
4.4
2.7
41.24±3.49
76.10±2.81hijk
def
4.4
5.4
21.49±6.32
79.83±1.91jk
b
4.4
10.8
23.54±2.10
73.23±1.8fghij
8.8
0.00±0.00a
70.43±2.77fghi
8.8
2.25
30.84±2.05c
69.73±2.52fghi
8.8
4.5
28.54±4.44c
68.90±2.59fghi
8.8
9.0
0.00±0.00a
67.50±2.04fg
bc
8.8
2.7
29.53±3.54
75.07±2.85ghijk
b
8.8
5.4
23.07±2.50
71.63±1.79fghi
b
8.8
10.8
22.34±2.15
76.67±3.11ijk
on callus formation from cotyledon, stem and nodal explants of E. cyclocarpum
% response of
stem explant
(mean ±S.E)
0.00 ± 0.00 a
43.80± 2.50defg
48.10 ±4.31eg
41.60±2.31cdef
44.37 ± 3.14 de
51.83 ±5.811efg
32.17 ± 5.97 b
0.00 ± 0.00 a
45.83 ± 4.54 de
33.63 ± 3.80 bc
25.97 ± 2.41 b
62.33i ± 3.49h
69.60 ± 1.54 ij
66.70± 3.30 hij
0.00 ± 0.00 a
35.77 ± 3.84 bcd
32.13 ± 2.40 b
0.00 ± 0.00 a
73.03 ± 3.58 jk
82.20 ± 2.63 k
66.17 ± 3.27 hij
0.00 ± 0.00 a
35.67 ± 2.94 bcd
42.77± 3.71 cde
0.00 ± 0.00 a
56.40±3.79 fgh
59.57± 5.088 ghi
51.93 ±6.058efg
Akinropo MS et al. (2020). Not Sci Biol 12(1):74-89.
79
PGR: Plant growth regulators;
Dichlorophenoxylacetic acid
BA:Benzyl
Adenine;
NAA:Naphthaleneacetic
Acid;
2,
4-D:2,
4-
Table 2. The effects of various concentrations of NAA, 2, 4-D alone or each separately combined with
kinetin on callus/shoot induction from cotyledon, stem and nodal explants of E. cyclocarpum
S/N
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
MS medium + PGR concentration
(µM)
KIN
NAA
2,4-D
2.25
4.50
9.00
2.70
5.40
10.80
2.35
2.35
2.25
2.35
4.50
2.35
9.00
2.35
2.70
2.35
5.40
2.35
10.80
4.70
4.70
2.25
4.70
4.50
4.70
9.00
4.70
2.70
4.70
5.40
4.70
10.80
9.40
9.40
2.25
9.40
4.50
9.40
9.00
9.40
2.70
9.40
5.40
9.40
10.80
-
% response of
cotyledon explant
(mean ±S.E)
0.00±0.00a
34.59±3.01cd
34.22±3.62cde
0.00±0.00a
48.40±1.24f
41.03±2.48de
0.00±0.00a
0.00±0.00a
44.50±4.75ef
41.23±1.37de
0.00±0.00a
49.43±2.048f
48.67±4.50f
24.90±2.25b
0.00±0.00a
35.70±2.76cd
35.47±2.71cd
0.00±0.00a
51.57±1.88f
47.50±4.62ef
0.00±0.00a
0.00±0.00a
29.63±2.14bc
33.73±2.64c
0.00±0.00a
35.03±3.43cd
31.73±2.21c
0.00±0.00a
% response of
nodal explant
(mean ±S.E)
0.00 ± 0.00a
48.47±1.10cd
43.40 ±1.47bc
39.73±1.88 b
55.37±43de
51.43 ±2.38d
48.13 ±1.40cd
70.90±0.86 ghijk
73.73 ± 1.45 jkl
79.2±2.77 l
67.20± 3.24 fghij
79.87 ± 2.43l
78.13 ±1.24 jkl
71.00 ±3.27ghijk
72.23±2.01 hijkl
74.87 ±2.71jkl
78.20 ±3.93 jkl
64.60± 3.18 fgh
73.43l ±1.35ijk
74.60 ±2.81 kl
69.70 ±2.98 ghij
62.77 ±2.98 fg
68.40±2.11 ghij
68.03±1.87 ghij
59.80 ±2.06 ef
68.83± 2.39 ghij
65.40± 2.84 fghi
64.57± 2.86 fgh
% response of
stem explant
(mean ±S.E)
0.00 ± 0.00a
43.80± 2.50defg
48.10 ±4.31eg
41.60±2.31cdef
45.8 ±3.06efg
50.37±6.07 gh
45.67±2.83 efg
0.00 ± 0.00a
41.47±1.91 cdef
50.27±1.21 gh
35.57±2.80 bcd
42.83±2.70 cdefg
43.97± 2.53 defg
47.73 ±3.36 fg
0.00±0.00a
61.73 ± 1.84 ij
75.53 ± 2.88 l
71.67 ± 2.02 kl
62.90 ±2.80 ij
67.567 ±2.39 jk
56.10 ± 4.80 hi
0.00±0.00a
40.93± 0.88 cdef
41.30± 2.45 cdef
29.10 ± 0.81 b
39.70± 5.11 cdef
34.73 ± 2.96 bc
38.83 ± 1.71 cde
PGR: Plant growth regulators; KIN:Kinetin; NAA:Naphthaleneacetic Acid; 2, 4-D:2, 4-Dichlorophenoxylacetic acid
Table 3. The effects of various concentration of NAA, 2, 4-D alone or each separately combined with BA
on caulogenesis from stem, cotyledon and nodal explants of E. cyclocarpum
MS medium + PGR
Degree of
No. of shoot
Degree of
No. of shoot
Degree of
Concentration (µM)
S/N
cotyledon callus per cotyledon nodal callus
per nodal
stem callus
formation
explants
formation
explants
formation
BA NAA
2,4-D
1.
No callus
No callus
No callus
2.
2.25
+
+
+
3.
4.5
+
+
++
4.
9.0
No callus
+
++
5.
2.7
+
+
+
6.
5.4
+
+
+
7.
10.8
No callus
+
+
8.
2.2
No callus
++S
1.50±0.40abc
No callus
a
bcd
++S
1.70±0.30
9.
2.2
2.25
+S
++
0.80±0.12
Akinropo MS et al. (2020). Not Sci Biol 12(1):74-89.
80
a
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
2.2
2.2
2.2
2.2
2.2
4.4
4.4
4.4
4.4
4.4
4.4
4.4
8.8
2.7
5.4
10.8
2.7
5.4
10.8
-
4.5
9.0
2.25
4.5
9.0
-
+S
+
+S
+
+
No callus
+S
+
+
+S
+
+
No callus
1.40±0.20
23.
8.8
-
2.25
+
1.60±0.23
24.
25.
8.8
8.8
-
4.5
9.0
+
No callus
26.
8.8
2.7
-
+
27.
28.
8.8
8.8
5.4
10.8
-
++
+
1.40±0.17
2.1±0.11
d
1.30±0.18
bc
ab
bcd
1.90±0.17
cd
++
++
+++S
++S
+++
++S
++S
++S
++
++S
++S
++
++S
2.80±0.40efgh
2.50±0.50defg
2.00±0.30def
3.00±0.50fgh
2.70±0.40efgh
2.10±0.30def
3.00±0.60fgh
++
+
++
++
++
No callus
++
+
No callus
++
+++
++
No callus
++S
3.10±0.50fgh
+
1.80±0.50cde
2.70±0.20efgh
efgh
+S
+S
2.60±0.40
1.90±0.30cde
+
No callus
++S
3.98±0.50fghi
+
++S
+++S
2.70±0.50efgh
1.80±0.40cde
+
+
+ Slight callus formation; ++Moderate callus formation; +++ Massive callus formation
PGR: Plant growth regulators; BA: Benzyl adenine; NAA: Naphthaleneacetic Acid; 2, 4-D: 2, 4Dichlorophenoxylacetic acid; **S: Shoot initiation
S/N
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
Table 4. The effects of various concentrations of NAA, 2, 4-D alone or each separately combined with
kinetin on caulogenesis from stem, cotyledon and nodal explants of E. cyclocarpum
MS medium + PGR
Degree of
No. of shoot
Degree of
No. of shoot
Degree of
Concentration (µM)
cotyledon callus per cotyledon
nodal callus
per nodal
stem callus
formation
explants
formation
explants
formation
KIN NAA 2,4-D
No callus
No callus
No callus
2.25
+
++
+
4.50
+
++
++
9.00
No callus
+
++
2.70
+
++
+
5.40
+
++
+
10.80
No callus
+
++
2.35
No callus
++S
1.40±0.40abc
No callus
2.35
2.25
+
++S
1.30±0.30ab
+
2.35
4.50
++
++
+
2.35
9.00
No callus
+
b
2.35
2.70
+S
++
+S
2.30±0.11defgh
1.4±0.05
2.35
2.35
4.70
4.70
4.70
4.70
4.70
4.70
4.70
5.40
10.80
2.70
5.40
10.80
2.25
4.50
9.00
-
+S
+
No callus
++S
+
No callus
+S
+
No callus
a
0.8±0.11
0.17±0.11
ab
b
1.5±0.12
+S
+++
+S
++S
++S
+
++S
++S
+
2.40±0.50efgh
2.10±0.17cdfg
2.30±0.11defg
2.00±0.18bcdef
3.50±0.28ij
3.0±0.23hi
++
++
No callus
++
+++
++
++
+++
+
Akinropo MS et al. (2020). Not Sci Biol 12(1):74-89.
81
22.
23.
24.
25.
26.
27.
28.
9.40
9.40
9.40
9.40
9.40
9.40
9.40
2.70
5.40
10.80
2.25
4.50
9.00
-
No callus
+
+S
No callus
+
+
No callus
1.20±0.23
ab
+S
+S
+S
+S
+S
+S
+S
3.50±0.40ij
3.70±0.29j
2.50±0.40fgh
2.20±0.30defg
3.30±0.50ghi
3.60±0.50ij
0.28±0.35gh
No callus
+
+
+
+
+
+
+ Slight callus formation; ++ Moderate callus formation; +++ Massive callus formation
PGR: Plant growth regulators; KIN: Kinetin; NAA: Naphthaleneacetic Acid; 2, 4-D: 2, 4-Dichlorophenoxylacetic
acid; **S: Shoot initiation
Table 5. The effects of various concentrations of NAA, 2, 4-D alone or each separately combined with BA
on morphology of callus from stem, cotyledon and nodal explants of E. cyclocarpum
S/N
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
MS medium + PGR
concentration (µM)
BA
NAA
2,4-D
2.2
2.2
2.2
2.2
2.2
2.2
2.2
4.4
4.4
4.4
4.4
4.4
4.4
4.4
8.8
8.8
8.8
8.8
8.8
8.8
8.8
2.7
5.4
10.8
2.7
5.4
10.8
2.7
5.4
10.8
2.7
5.4
10.8
2.25
4.5
9.0
2.25
4.5
9.0
2.25
4.5
9.0
2.25
4.5
9.0
-
Morphology of
cotyledon callus
Morphology of
nodal callus
Morphology of
stem callus
Light brown and hard
Light brown and hard
Light brown and hard
Light brown and hard
Brown and granular
Light brown and hard
Brown and hard
Brown and hard
light and loose yellow
light and loose yellow
Light brown and hard
Light brown and hard
Light brown and granular
Light and friable yellow
Light yellow and friable
White and friable
White and friable
White and friable
White and friable
White and friable
White and friable
White and friable
White and friable
White and friable
White and friable
White and friable
White and friable
White and friable
White and friable
White and friable
White and friable
White and friable
White and friable
White and friable
White and friable
White and friable
White and friable
White and friable
White and friable
White and friable
White and friable
White and friable
Light brown and rather hard
Light brown and rather hard
Light brown and rather hard
Creamy/ semi hard
Creamy/ semi hard
Creamy/ semi hard
Light brown and semi-hard
Hard
Light yellow and rath. hard
Light yellow and hard
Light yellow and hard
Light brown and semi hard
Light brown and semi hard
Light brown and semi hard
Light brown and Friable
Light brown and Friable
Light brown and Friable
Light brown and semi hard
Light brown and semi hard
Light brown and Friable
Light brown and Friable
Light brown and Friable
Light brown and semi hard
Light brown and semi hard
Light brown and semi hard
Light brown and semi hard
PGR: Plant growth regulators; BA: Benzyl Adenine; NAA: Naphthaleneacetic Acid; 2, 4-D: 2, 4Dichlorophenoxylacetic acid
The stem explants of E. cyclocarpum explanted on MS medium alone grew in size, expanded
longitudinally and laterally at first but later the cut edges began to turn brown and the explants died ultimately.
The stem explants explanted on MS medium supplemented with either BA or kinetin alone did not show
callogenesis. With MS medium containing the individual auxin, callus initiation appeared after 3 weeks
Akinropo MS et al. (2020). Not Sci Biol 12(1):74-89.
82
followed by a very slow proliferation of creamy/ off white hard callus (Figure 3b). The NAA/ BA or 2,4-D/
BA combinations brought about callus initiation in the stem explants within 2 weeks to produce off white
friable or semi-hard (Tables 5 and 6). The largest callus size was produced in stem explants cultured on MS
medium containing 5.4 µM NAA + 4.4 µM BA (Figure 4a), and 82.2 ± 2.63% response was obtained (Table
1). The combination of NAA/ kinetin and 2,4-D/ kinetin induced callus from the stem explants of E.
cyclocarpum within 2 weeks, the calli were generally light brown and semi-hard. Massive callus induction was
observed with stem explants of E. cyclocarpum cultured on MS medium supported with 4.5 µM 2,4-D + 4.7
µM kinetin with the highest frequency of 75.53 ± 2.88% (Table 2).
Nodal explants of E. cyclocarpum cultured on MS medium alone enlarged in size without either
production of callus or axillary bud release. When the explants were grown on MS medium to which auxins
alone were added, the only callus formed and with auxin/cytokinin combinations, both callus and shoot buds
were observed within 2 weeks of culture. Calli formed at the basal cut ends of the explants in all the cases were
cream-coloured or white and friable.
The highest frequency of callus formation (81.57 ± 1.92) was induced from nodal explants cultured on
MS medium supplemented with 2.7 µM NAA + 2.2 µM BA (Table 1) and the maximum shoot/explants
induced (4.0) was obtained from nodal explant cultured on MS medium supplemented with 2.7 µM NAA +
8.8 µM BA (Table 3). Massive callus formed from nodal explants with the highest frequency (79.2 ± 2.77%)
when cultured on MS medium supplemented with 4.5 µM 2,4-D + 2.35 µM kinetin (Table 2). Callus and
shoot buds induced by 4.5 µM 2,4-D + 2.35 µM Kinetin, while callus and short buds induced from nodal
explants cultured on MS medium supported with 2.7 µM NAA + 2.2 µM BA after 4 weeks (Figure 4b and 5).
On the subculture of shoot buds so induced from nodal explant-derived callus, they showed extended growth
without root formation.
On the transfer of separated shoot buds to MS medium without hormones, less than 10% of them rooted
after 6 weeks of culture. In MS medium containing 4.9 µM IBA, short and thick roots formed in over 80% of
the shoots within 4 weeks of culture. Figure 6 shows the acclimatized plantlets induced from nodal explant of
E. cyclocarpum with thick root in a rooting medium of 4.9 µM IBA.
Figure 4. Callus and basal callus with shoot induced from stem and nodal explant of E. cyclocarpum
after four weeks of culture respectively on MS Supplemented 5.4 µM NAA + 4.4 µM BA for the stem(a)
and 4.5 µM 2,4-D+ 2.35 µM Kinetin for the node(b)
Akinropo MS et al. (2020). Not Sci Biol 12(1):74-89.
83
Figure 5. Basal callus with shoot buds obtained from nodal explant of E. cyclocarpum after four weeks of
culture on ms supplemented with 2.7 µM NAA+ 2.2 µM BA
Figure 6: Acclimatized plantlets of E. cyclocarpum with thick roots
Table 6. The effects of various concentrations of NAA, 2, 4-D alone or each separately combined with
kinetin on morphology of callus from stem, cotyledon and nodal explants of E. cyclocarpum
MS medium + PGR
Morphology of
Morphology of
Morphology of
concentration (µM)
S/N
cotyledon callus
nodal callus
stem callus
KIN NAA 2,4-D
1.
Light brown and hard
2.
2.25
White and friable
3.
-
-
4.50
Light brown and hard
White and friable
Light brown and rather hard
4.
-
-
9.00
-
White and friable
5.
-
2.70
-
Light brown and
rather hard
White and friable
Creamy/semi hard
6.
-
5.40
-
Light brown and
rather hard
White and friable
Creamy/semi hard
7.
-
10.80
-
Light brown and hard
White and friable
Creamy/semi hard
8.
2.35
-
-
-
Creamy and friable
-
Light brown and rather hard
Akinropo MS et al. (2020). Not Sci Biol 12(1):74-89.
84
9.
2.35
-
2.25
Light brown and hard
Creamy and friable
Light brown and semi hard
10.
2.35
-
4.50
Light brown and hard
Creamy and friable
Light brown and semi hard
11.
2.35
-
9.00
Creamy and friable
Light brown and semi hard
12.
2.35
2.70
-
Light brown and hard
Creamy and friable
Light brown and semi hard
13.
2.35
5.40
-
Light yellow and hard
Creamy and friable
Light brown and semi hard
14.
2.35
10.80
-
Light yellow and hard
Creamy and friable
Light brown and semi hard
15.
4.70
-
-
-
Creamy and friable
-
16.
4.70
-
2.25
Deep brown and hard
Creamy and friable
Light brown and semi hard
17.
4.70
-
4.50
Light yellow and hard
Creamy and friable
Light brown and semi hard
18.
4.70
-
9.00
-
Creamy and friable
Light brown and semi hard
-
Light brown and hard
Creamy and friable
Light brown and semi hard
19.
4.70
2.70
20.
4.70
5.40
-
Light yellow and hard
Creamy and friable
Light brown and semi hard
21.
4.70
10.80
-
-
Creamy and friable
Light brown and semi hard
22.
9.40
-
-
-
Creamy and friable
-
23.
9.40
-
2.25
Deep brown and hard
Creamy and friable
Light brown and semi hard
4.50
9.00
-
Light yellow and hard
Creamy and friable
Creamy and friable
Creamy and friable
Creamy and friable
Creamy and friable
Light brown and semi hard
Light brown and semi hard
Light brown and semi hard
Light brown and semi hard
Light brown and semi hard
24.
25.
26.
27.
28.
9.40
9.40
9.40
9.40
9.40
2.70
5.40
10.80
Light brown and hard
Light brown and hard
-
PGR: Plant growth regulators; KIN: Kinetin; NAA: Naphthaleneacetic Acid; 2, 4-D: 2, 4-Dichlorophenoxylacetic
acid
Discussion
The use of in vitro approaches to propagation, conservation and genetic improvement of forest trees has
been of increased interest since the last three decades (Chalupa, 1981; Tomar and Gupta, 1988; Xia and Hong,
2001; Chalupa, 2002; Anis et al., 2005; Faisal, 2007; Renukdas et al., 2010 and Sajeevan et al., 2013). Two
methods of plant regeneration widely used in rapid and large scale micropropagation or plant transformation
studies are organogenesis or embryogenesis. The success of these techniques requires good callus quality and
quantity (Lin et al., 2010).
The present studies showed that the stem, cotyledon, and nodal explants, but not leaf explants of E.
cyclocarpum species were callogenic. The initiation and proliferation of callus were promoted by using NAA,
2,4-D alone or separately combined with Kinetin or BA to different extents in different explants. It was
generally observed that the percentage of explants response in terms of callus induction was greater with auxin
combination with BA than with Kinetin in the explants studied. The auxins, NAA and 2,4-D are commonly
used with BA for callus induction in plant systems (Dhar and Joshi, 2005; Abbasin et al., 2010; Isikalam et al.,
2010; Ntui et al., 2012). Other research groups also induced callus, a consequence of wound reaction (Khal,
1983), in vitro without the use of plant growth regulators of several plant species (Handro and Floh, 2001;
Martin, 2002).
Cotyledon explants of E. cyclocarpum were found to produce callus together with shoot buds when
cultured on MS medium containing NAA combined with BA or Kinetin. The frequency of shoot induction
appeared favoured by high cytokinin relative to auxin. A combination of 2.2 µM BA + 2.7 µM NAA induced
the best shoot production from the cotyledon explants of E. cyclocarpum (2.1 shoots/explant). The influencing
Akinropo MS et al. (2020). Not Sci Biol 12(1):74-89.
85
effects of auxin and cytokinin combination on organogenic differentiation have been well documented in
several plant systems. Rahman et al. (2010) found that adventitious shoots were induced from leaf-derived
callus of Lagerstroemia speciosa grown on MS medium that was supplemented with 5 µM BA, 3 µM NAA, 10%
coconut milk and 568 µM ascorbic acid. Similarly, cotyledon derived calli induced by 0.5 µM NAA + 0.5 µM
BA produced high-efficiency shoot regeneration (4.2 shoots/explant) when subcultured on MS medium
supplemented with 5 µM BA (Agrawal and Sadar, 2007). Salvi et al. (2001) who used various Neem explants
including cotyledon, produced multiple shoots on MS medium containing 8.88 µM BA and 0.57 µM NAA. By
addition of 3 mg/L Hymexazol to a modified MS medium containing 2.22 µM BA and 0.05 µM NAA, Yin et
al. (2001) increased adventitious shoot development from cotyledon explants of Albizia julibrissin.
The leaf explants of E. cyclocarpum formed no callus when cultured on either MS medium alone or
supplemented with various concentrations of either BA or NAA alone and in their combinations. The leaf
explants, however, were found to have lost chlorophyll turning light green the end. This is contrary to wound
reaction inducing mitosis in the cells from the cut surfaces and culminating in callus formation (Pérez-Francés
et al., 1995). The loss of response of the explants may suggest that the endogenous level of auxins/cytokinin or
the exogenously applied growth regulators were insufficient to induce callus. It is also known that different
tissues can respond in different ways during in vitro culture process (Jimenez, 2001; Banerjee et al., 2011), and
the requirements for plant growth regulators appear to be tissue-specific (Venkatachalam et al., 1999; Banerjee
et al., 2011). Vidoz et al. (2012) also reported that the leaf explants of Lotononis bainesii did not respond on
MS media to the absence of plant growth regulators. Conversely, leaf explants have been reported by several
authors as the best for callus induction and shoot initiation. Jiancan et al. (2011) obtained 95% callus induction
from leaf explants of Zizyphus jujuba in MS supplemented with 0.5 mg/L NAA.
In the absence of growth regulators in the culture medium, E. cyclocarpum stem explants neither formed
callus nor shoot buds. The stem explants inoculated either on MS medium alone or supplemented with BA
alone did not show callogenesis or caulogenesis but they first increased in size and gradually turned brown later.
Cultures of stem explants on MS medium supplemented with the different concentrations of auxin and
combined with BA started callus initiation after 2 weeks and proceeded with high proliferation.
Nodal explants exhibited the greatest tendencies to form callus and shoots under the influence of plant
growth regulators compared to stem, hypocotyl, leaf and cotyledon explants of the plant species investigated.
An average of 3.98 shoot / explant was generated from the nodal explant-induced callus of E. cyclocarpum by
2.7 µM NAA combined with 8.8 µM BA.
The nodal explants of several tree seedlings have been known to produce both callus and shoot buds in
vitro under the influence of some plant growth regulators. Rahman et al. (1993) reported that NAA or 2,4-D
separately combined with BA induced both callus and shoots within 3 weeks of culture of nodal explants of
Caesalpinea Pulcherima. Sugla et al. (2007) induced multiple shoots in vitro from nodal explants through
forced axillary branching in Pongamia pinnata using 7.5 µM BA while more recently, Sajeevan et al. (2012)
reported inducing multiple shoots from nodal explants of Morus alba L. variety VI cultured on MS medium
supplemented with 1.0 mg/L BAP, 0.1 mg/L TDZ and 0.25 mg/L NAA. Maximum shoot induction was
obtained from the nodal cutting of E. cyclocarpum seedlings cultured on MS medium supplemented with a
combination of 10.7 µM NAA and 2.2 µM BA (Rodriquez-Sahagun et al., 2007). Multiple shoots were also
obtained from nodal explants of 18-day-old in vitro seedlings of Pterocarpus marsupium Roxb. culture on MS
medium containing 4 µM BA, 0.5 µM NAA and 20 µM adenine sulphate (Husain et al., 2008). Shoot
proliferation from nodal explants of Melaleuca alternifolia (Tee Tee) cultured in liquid or on agar-based MS
medium containing 1.11 µM BA or 0.55 µM BA had also been reported in the literature (Yohana et al., 2010).
Root induction was also achieved in the rooting medium (MS + 4.9 µM IBA). Stout short single roots
were induced from the cotyledon and nodal explant-derived shoots of E. cyclocarpum. It appears that different
species have a different critical concentration of hormones below which root initiation will occur and above
Akinropo MS et al. (2020). Not Sci Biol 12(1):74-89.
86
which there will be inhibition. Furthermore, organogenic differentiation of callus leading to production of
shoots and roots are dependent on several factors aside from hormonal factors and these include level of salt
(e.g. PO43-), quality of light, and temperature, in addition to physiological state, size of explant, and orientation
of the medium (Razan, 2003). Chevre (1985) opined that rooting is often more difficult with the ligneous
plants than with the herbaceous plants. More recently, similar observations were reported by Lin et al. (2010)
stating that in general, establishing an efficient tissue culture technique was difficult in woody plants compared
to herbaceous plants. This in part is related to the phase change from juvenility to maturation that most woody
plants undergo (Trevor et al., 1990). The development of basal callus is one of the main physiological disorders
that affect rooting competence of micro shoots, a situation which is more severe in woody species (Bairu and
Kane, 2011). These authors explained that basal callus constitutes a sink trapping essential growth constituent
and consequently affecting many physiological processes of the shoot.
Conclusions
The optimal concentration of NAA/BA that can induce stem callus in a potential source for plant
regeneration (either by organogenesis or embryogenesis) and production of secondary metabolites were
determined for E. cyclocarpum. Also, the optimal concentration of NAA/BA for shoot induction and plantlets
formation of the plant species from cotyledon and nodal explants were also determined. Thus, it can be
concluded by means of in vitro culture of the cotyledon and nodal explants of E. cyclocarpum, true clone
plantlets can be produced. This plantlet can be employing for reforestation and/or forest restoration programs.
Acknowledgements
This research received no specific grant from any funding agency in the public, commercial, or private
sectors. The authors are grateful to Emanuel Uwadone for his assistance in statistical analysis and all the
anonymous reviewers.
Conflict of Interests
The authors declare that there are no conflicts of interest related to this article.
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