In vitro Regeneration of Irvingia gabonensis by Somatic
Donfagsiteli Tchinda Nehemie
Omokolo Ndoumou Denis
A productive genotype of Irvingia gabonensis were
cultured in vitro for induction embryogenic calli, somatic embryogenesis
and regeneration of plantlets. Fragments of young leaves were used as
primary explants. Callogenesis was initiated by culture of explants during
30 days on Murashige and Skoog medium half strength (MS/2) supplemented
with 1-6 mg L-1 of 2,4-dichlorophenoxyacetic acid (2,4-D).
The highest percentage of explants forming calli is 85.1% at 3 mg L-1
of 2,4-D. Somatic embryos were obtained after a sub-culture of embryogenic
calli during 60 days on MS/2 supplemented with 1-3 mg L-1 of
BAP. The highest percentage of embryogenic calli which differentiates
somatic embryos is 63.8 ± 2.3% at 1 mg L-1 of 6-benzylaminopurine
(BAP). The highest number of somatic embryos per callus which is 43.6
is obtained with 2 mg L-1 of this phytohormone. When isolated
from calli and sub-cultured during 30 days on MS/2 supplemented with 2
mg L-1 of BAP, somatic embryos germinate with a highest percentage
of 83%. The sub-culture of germinated somatic embryos on the same Basal
Medium (BM) supplemented with 4 mg L-1 of BAP and 2 mg L-1
of Naphthalene Acetic Acid (NAA) during 80 days gives rise to the plantlets
with 82.7 ±4.8% of success. With this combination, each plantlet
has average length of 5.6 cm, bears 3.3 leaves and 7.2 roots with 1 or
2 pivoting roots. Plantlets acclimatized on a mixture sterilized soil/vermiculite
at equal volume survive at 93%. Results of this study constitute a new
way for a production of Irvingia gabonensis seedlings with pivoting
root and they permit to arrest the difficulties of natural and horticultural
Irvingia gabonensis from the family of Irvingiaceae
is known as bush mango and as non timber forest products (Vivien and Faure,
1996). It is a fruit tree native to moist lowland tropical forest in central
and West Africa. The uses of this tree are varied. The kernel of the nut
is a food additive. It is used to thicken and flavour soups. In Gabon,
the nut is used in the preparation of Dika bread. Oil can be extracted
from the nut. The quality of this oil is comparable to sheaf oil. The
mesocarp of fruit is appreciated as a fresh fruit. It can be also used
for the preparation of juice, jelly and jam. The sugar concentration of
this juice is comparable with pineapples and oranges (Akubor, 1996). This
juice contents higher concentration of ascorbic acid (67 mg/100 mL). The
wood of the tree is used in carpentry (Ngoye, 1998). Anti diarrhoeic and
anti ulcer properties has been reported in Irvingia gabonensis (Raji
et al., 2001). Despite the vast array of potentialities, this species
is not yet been integrated in the farming system and fruits are still
harvested mainly from wild trees. The International Centre for Research
in Agro Forestry (ICRAF) identifies Irvingia gabonensis as a priority
wild fruit tree species for domestication (Lapido et al., 1996).
Irvingia gabonensis is believed to be predominantly out crossing
and insect pollinated with seed dispersed by humans during migration and
through the digestive system of mammals (Lowe et al., 2000). These
indicate that Irvingia gabonensis is highly heterozygote. This
results in a great diversity of vegetative and organoleptic characters.
Therefore, the problem of multiplication of selected individuals exists.
To fight against this problem, vegetative propagation like grafting, cutting
and aerial layering are done (Shiembo et al., 1996). But these
classic methods are limited by the difficulty of rooting and field growth
performance after transplantation as in many woody plants (Hamzah, 1992;
Newton et al., 1992). Till date, very little research has been
carried on the tissue culture of Irvingia gabonensis compared to
the other wild plants such as Bauhina vahlii (Dhar and Upreti,
1999), Tilophora indica (Jayanthi and Mandala, 2001) mango (Litz
et al., 1998), Acacia tortilis (Sane et al., 2000)
and Plumbago species (Das and Rout, 2002). However, Omokolo et
al. (2004) showed that it is possible to propagate this species by
in vitro micro cutting. But, plantlets produced by this method
have a spread root system, which can not permit them to resist to the
hard climatic factors. However, the production of plantlets with pivoting
root system through somatic embryogenesis, capable to resist to hard climatic
factors has been reported in some species such as Theobroma cacao (Alemano
et al., 2001), Hevea brasiliensis (Blanc et al.,
2002), Tlophora indica (Jayanthi and Mandala, 2001). But in literature,
none research has been reported on somatic embryogenesis of Irvingia
gabonensis. However, this method can be used for the rapid and mass
production of Irvingia gabonensis plantlets capable to resist to
the hard conditions and therefore, contributes to the protection of plant
The aim of this study is to define in vitro the
conditions for regeneration and propagation of Irvingia gabonensis
through somatic embryogenesis. The effects of 2,4-D, NAA, BAP and different
combinations of BAP/NAA on different stages of somatic embryogenesis has
MATERIALS AND METHODS
Preparation of explants: Young leaves of Irvingia gabonensis were collected
on the apical zone of branches of productive genotype tree (8 years old)
found in the Obala region in centre province of Cameroon during the period
from July to December of the year 2006. These leaves were washed twice
with 1% solution of tween 20 for 10 min each. They were then disinfected
in 3% solution of sodium hypochlorite for 20 min and rinsed 5 times with
sterile distilled water. The leaves were then cut into pieces (1 cm wide),
transversally across the leaf lamina. Leaves fragments were placed on
the culture media with the adaxial surface in contact with the medium.
Media and conditions of calli induction: Calli were induced on Murashige and Skoog (MS) medium
(1962) modified as followed: half-strength macro salts and chelated iron
(MS/2) (Morel and Wetmore, 1951), vitamins 3% sucrose, 400 mg L-1
glutamine and 0.8% agar (Difco). This Basal Medium (BM) was supplemented
with 0 (control), 1, 2, 3, 4, 5 and 6 mg L-1 of 2,4-D and its
pH was adjusted to 5.8 by addition of NaOH 1 N or HCl 0.1 N. Media were
sterilized by autoclaving at 115°C for 30 min under a pressure of
1.6 kg cm-2. The control was the basal medium without 2,4-D.
Six leaves were cut into about 50 segments and placed in flasks for each
medium. The cultures were maintained at 26±1°C with 16 h photoperiod
under fluorescent light at 80 μmol m-2 sec-1.
The calli induction percentage was calculated in each medium after 30
days of culture. The growth of calli in different media was expressed
by measuring the average fresh weight.
Media of somatic embryogenesis: After 30 days of culture, calli were sub-cultured on
the basal medium supplemented with BAP in the range of 0 (control), 1,
2 and 3 mg L-1. Sub-cultures were placed under the same environmental
conditions as during calli induction. Embryos were induced after 60 days
of sub-culture. The percentage of calli which differentiate somatic embryos
and the average number of embryos per callus were evaluated.
Statistical analysis: For all experiment, the control was the basal medium
without phytohormon. Fifty explants were used for each calli induction
medium and 30 calli were used for each somatic embryogenic medium. All
experiences were repeated thrice. The data were analysed using the Duncan`s
multiple range tests. Significant differences between treatment means
were determined at p<0.05.
Histological studies of embryogenic calli and morphology
of somatic embryos: Embryogenic calli were fixed in AFA (mixing of 90 mL
of 70°C ethanol, 5 mL of formaldehyde and 5 mL of acetic acid). They
were then washed on a tap water current during 24 h and were dehydrated
by three successive baths in 90°C ethanol during 30 min each. After
three treatments of 30 min each in a mixture of 95°C ethanol and toluene
in the respective proportions: 0.75-0.25; 0.5-0.5 and 0.25-0.75, calli
were then included in liquefied paraffin during 32 h. Sections of 10 μm
thickness were then realised using rotary microtome (Shibuya Optical Co.,
Ltd). Paraffin was removed on these sections by soaking successively in
toluene during 1 h, 95°C alcohol during 1 h and 70°C alcohol during
10 min. They were then rinsed three times in distilled water during 30
min each and stained with haematoxylin of Regaud-Safranine O. Observations
were done using photonic microscope (Nikon 233729, Japan) equipped with
Development of somatic embryos: Somatic embryos were isolated from embryogenic calli
and sub-cultured on the basal medium without phytohormon during 7 days
for weaning. After this stage, somatic embryos were submitted to the second
sub-culture in BM supplemented with 2 mg L-1 of BAP during
30 days for germination.
Regeneration of plantlets: Germinated embryos were sub-cultured on the basal medium
supplemented with 1, 3, 4 or 5 mg L-1 of BAP and 2 mg L-1
of NAA (BAP/NAA ratio) during 80-90 days for regeneration of plantlets.
The average length of plantlets and the average number of leaves and roots
per plantlet as growth parameters were measured or counted for each BAP/NAA
ratio. For each ratio, 40 germinated embryos were used and the experience
was repeated thrice. The significant difference means were determined
using Duncan`s multiple range tests at p<0.05.
Acclimatization of plantlets: Plantlets were acclimatized in polyethylene bags containing
a sterile mixture soil/vermiculite at equal volume (v/v) under a temperature
of 26±1°C, 72-76% of relative humidity and 16 h per day of
light (80 μmol m-2 sec-1). During acclimatization,
the relative humidity was reduced progressively and plantlets were watered
firstly with sterile tap water during 30 days and secondary with tap water
during 60 days.
Induction of calli: Callogenesis was initiated from young tender
leaves fragments cultured during 30 days on BM supplemented with 2.4-D.
The percentage of callogenesis increases from 52.7±2.1% at 1 mg
L-1 and reaches the maximum value of 85.1±1.6% at 3
mg L-1 and then this percentage decreases and reaches the lowest
value of 13.4 at 6 mg L-1 (Table 1). No calli
were initiated on control medium. The fresh weight of calli also increases
from 0.33 g at 1 mg L-1 and reaches the maximum value of 0.78
g at 3 mg L-1, then decreases and reaches the lowest value
of 0.23 g at 6 mg L-1 (Table 1). Calli induced
in the presence of 1-5 mg L-1 of 2,4-D are yellowish, friable
and embryogenic (Fig. 1a), while those produced in the
presence of 6 mg L-1 of 2,4-D are brownish and non embryogenic.
The embryogenic calli are characterised by the presence of green piles
of embryogenic nodules (en) (Fig. 1a). The transversal
section of embryogenic calli shows that each embryogenic nodule is a pile
of cells in division (pcd) (Fig. 1b).
Induction of somatic embryos: Somatic embryos were initiated by sub-culture of embryogenic
calli on the basal
Effects of 2,4-D
at various concentrations on the induction of calli from leaves
fragments of Irvingia gabonensis after 30 days of culture
Each value represented
the mean±SE of three cultures each with 50 leaves fragments
per medium. Means followed by the same letter(s) within columns
are not significantly different at p<0.05 according to Duncan`s
multiple range test
Effects of BAP
at various concentrations on somatic embryos differentiation from
calli after 60 days of culture
Each value represented
the mean±SE of three sub-cultures each with 30 calli per
medium. Means followed by the same letter(s) within columns are
not significantly different at p<0.05 according to Duncan`s
multiple range test
medium supplemented with BAP during 60 days. The highest percentage of calli
which differentiates somatic embryos (63.8±2.3%) was obtained with
this phytohormon at 1 mg L-1
, while the average number of somatic
embryos per callus (43.6) was obtained at 2 mg L-1
After 60 days of sub-culture, when somatic embryos are isolated from calli,
they present different shapes but the predominant one is heart-shaped
). In fact, 77.1±4% of embryos present
this form. The heart-shaped somatic embryos are characterised by the presence
of two poles; apical pole (ap) and basal pole (bp) (Fig.1c
They are bipolar somatic embryos. When sub-cultured on BM supplemented
with 2 mg L-1
of BAP during 30 days, the bipolar somatic embryos
gives rise to germinated embryos. In fact, the section at the level of
apical pole shows the cellular organised zone (coz) as in meristematic
zone of plants (Fig. 1d
). The basal pole differentiates
the first root (fr) (Fig. 1e
). Then the somatic embryos
with cellular organised zone and first root are known as germinated embryos
). The percentage of bipolar somatic embryos
which gives rise to germinated embryos in these conditions is 83.7±3.8%.
Plantlets regeneration and acclimatization: Plantlets were regenerated
by sub-culture of germinated somatic embryos on the basal medium supplemented
with different ratios of BAP/NAA during 80 days (Table 3).
The highest percentage of germinated embryos which develops into
and regeneration of plantlets of Irvingia gabonensis from leave
explants. (a) Embryogenic callus with embryogenic node (ne) obtained
from leave fragments cultured on MS/2 supplemented with 3 mg L?1
of 2.4-D during 30 days. (b) Section of embryogenic node showing
the piles of cells in division (pcd). (c) Heart-shaped somatic
embryos with apical pole (ap) and basal pole (bp) obtained from
embryogenic calli sub-cultured on MS/2 supplemented with 1 mg
L?1 BAP during 60 days. (d) Section of apical pole of heart-shaped
somatic embryos showing the cellular organised zone (coz). (e)
Germinated somatic embryos showing the first root (fr) obtained
after maintained heart-shaped somatic embryos during 30 days on
MS/2 supplemented with 2 mg L?1 BAP. (f) Apex (ax) obtained by
the development of the apical pole of germinated somatic embryos
on MS/2 supplemented with 4 mg L?1 of BAP and 2 mg L?1 of NAA
during 21 days. (g) Plantlets with elongated pivoting roots (epr)
obtained on MS/2 supplemented with 4 mg L?1 of BAP and 2 mg L?1
of NAA during 80 days and (h) 90 days olds acclimatized plantlet
on sterilized mixture soil vermiculite (v/v)
Effect of different
ratio of BAP/NAA on the regeneration and growth of plantlets of
Irvingia gabonensis from germinated somatic embryos cultured
during 80 days
Each value represented
the mean±SE of three sub-cultures each with 40 germinated
somatic embryos per medium. Means followed by the same letter(s)
within columns are not significantly different at p<0.05 according
to Duncan`s multiple range test
plantlets (82.7±4.8 %) was obtained with 4/2 mg L-1
while the lowest percentage (22.9±1.7%) was obtained with 5/2 mg
L-1 ratio (Table 3). The apical pole of germinated
embryos develops after 21 days of sub-culture and gives rise to the apex
(ax) with the average length of 200±20 mm (Fig. 1f).
After 80 days of sub-culture, the apex had developed and gave rise to
leafing stem; while on the basal pole the first root elongates and gives
rise to pivoting root with average length of 3.5±0.6 cm (Fig.
1g). Then, this elongated pivoting root ramifies and gives rise to
root system. Well developed plantlets were obtained with 4/2 mg L-1
BAP/NAA. In fact, with this ratio, each plantlet has the average growth
parameter: 5.6 cm of length, 3.3 leaves and 7.2 roots (Table
Under the conditions of acclimatization, regenerated plantlets survive
at 93%. They developed during 90 days and gave rise to vigorous dark green
plants (Fig. 1h). Each plant at this stage has an average
length of 32.4±2.7 cm, presents one or two ramifications and bears
an average number of 14.7±2.6 leaves (Fig. 1h).
The results of this study show that calli could be initiated
successfully from young leaves explants of Irvingia gabonensis cultured
on MS/2 supplemented with 2,4-D. During the past decade, 2,4-D has been
used to induce calli in some ligneous plants such as Bauhinia vahlii
(Dhar and Upreti, 1999), Citrus grandis (Huang et al.,
2002), Ricinodendron heudelotii (Fotso et al., 2007). But,
the frequency of callogenesis depends not only of the concentration of
2,4-D; also of the type and age of explants. In this study, the maximum
percentage of callogenesis (85.1±1.7%) was obtained with 3 mg L-1
of 2,4-D. These results support those obtained by Litz et al. (1998)
with Mangifera indica and Huang et al. (2002) with Citrus
grandis. Those authors showed that 2,4-D was more effective on callus
induction of woody plant explants than naphthalene acetic acid (NAA).
Calli induced were embryogenic. In contrast, Hatanaka et al. (1991)
and Gill et al. (1995) obtained non embryogenic calli with 2,4-D
on Coffea canephora and mandarine explants, respectively. In Irvingia
gabonensis, induced calli regenerate somatic embryos after being transferred
to differentiation medium supplemented with BAP with the highest percentage
of 63.8±2.3 at 1 mg L-1. In most of the reports on in
vitro propagation of woody plants by somatic embryogenesis, BAP seems
to be one of the best cytokinin used to induce somatic embryos on calli
(Omokolo et al., 1997; Jayanthi and Mandala, 2001; Imani et
al., 2001; Mohammed et al., 2002). Meanwhile, reports of Zhijian
et al. (1998) on Theobroma cacao and Von Arnold et al.
(1996) on Picea abies show that the differentiation and development
of somatic embryos depend not only of the concentration of BAP in the
medium, but also of the others factors such as photoperiods, temperature
and relative humidity. In Irvingia gabonensis, obtained results
seem to depend to the sum of these factors in addition with 2,4-D which
promoted embryogenic calli. In fact, 82.7±4.8% of germinated somatic
embryos give rise to plantlets. These results contrast with those obtained
by El Maataoui et al. (1990) in Quercus suber and Luo and
Koop (1997) in Arabidopsis thaliana. They showed that, only 10-20%
of mature somatic embryos obtained in both species give rise to plantlets.
In general many reports show that, the success of somatic embryogenesis
in higher woody plants depends particularly to the ecotype and genotype
of the species studied (Kielly and Bowley, 1992; West and Harada, 1993;
Imani et al., 2001). So, the highest percentage of regeneration
of plantlets obtained in this study can be also explained by the genotype
of the species studied. This regeneration is obtained in this study when
BAP is combined whit NAA in the same media. This result permits to approve
the existence of complementary and synergic action between auxins and
cytokinins as has been reported on somatic embryogenesis in many ligneous
species such as Coffea canephora (Hatanaka et al., 1991),
Mangifera indica (Litz et al., 1998), Theobroma cacao
(Alemano et al., 1997). Acclimatization of plantlets during
90 days in a sterile mixture soil/vermiculite gives 93% of success. This
result is similar to those obtained by Omokolo et al. (2004) with
plantlets of the same species regenerated by in vitro micro cutting
and acclimatized under the same conditions. But, it is higher than the
rate of acclimatized plantlets of Dacryodes edulis (51%) obtained
by Youmbi and Benbadis (2001) under the same conditions.
In conclusion, with the maxima conditions described at
different stage in this study (from callogenesis to acclimatization),
it is possible to regenerate through somatic embryogenesis 28±1
survival plantlets of Irvingia gabonensis from a single fragment
of leave. So, the present study constitutes a new way for a rapid production
of Irvingia gabonensis seedlings with pivoting root system which
could be used for the species propagation and domestication. But it is
important to use explants from reproductive genotype to pass round the
highly heterozygote characteristic of species after regeneration. Further
research should be required to determine appropriated stock plant management
The authors thank Drs. Mbouobda Hermann Desire and Minyaka
Emile for technical assistance.
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