Vol. 11(9), pp. 362-368, September 2017
DOI: 10.5897/AJPS2017.1550
Article Number: F03266265858
ISSN 1996-0824
Copyright © 2017
Author(s) retain the copyright of this article
http://www.academicjournals.org/AJPS
African Journal of Plant Science
Full Length Research Paper
Assessment of the modes of pollen dispersal of
Vernonia amygdalina Del. and Vernonia calvoana Hook
Nguimkeng Gaintse Eric Dumas1, Zapfack Louis2, Ntsomboh-Ntsefong Godswill1,3, Temegne
Nono Carine1 and Youmbi Emmanuel1,4*
1
Laboratory of Biotechnology and Environment, Department of Plant Biology, Faculty of Science, University of
Yaounde I, P. O. Box 812 Yaounde, Cameroon.
2
Laboratory of Botany and Ecology, Department of Plant Biology, University of Yaounde I, Faculty of Science, P. O. Box
812 Yaounde, Cameroon.
3
Institute of Agricultural Research for Development (IRAD), CEREPAH La Dibamba, B. P. 243 Douala, Cameroon.
4
Laboratory of Tissue Culture, CARBAP, Njombe, Cameroon.
Received 24 March, 2017; Accepted 24 April, 2017
The study of pollen dispersal and mode of fertilization of Vernonia amygdalina and Vernonia calvoana
is a prerequisite to the understanding of genetic diversity and elaboration of improvement programs for
the Vernonia genus. In this study, precise experimental designs were made for morphological and
biological observations on Vernonia species capitulum in order to assess the distinctive effects of each
of the three possible pollen transportation agents (insects, wind and rain water) on pollination. Results
obtained show that the exclusive mode of pollen dispersal is entomophilous. Even though allogamy
and autogamy are observed as two possible modes of fertilization in Vernonia spp., some arguments
tend to favour allogamy. However, there is no clear cut position on the issue, hence the need for
confirmation by further experimentation in controlled pollination. This study paves the way for the
establishment of a genetic improvement program for this genus based on the results on pollen
dispersal.
Key words: Vernonia species, allogamy, autogamy, pollination agent.
INTRODUCTION
Vernonia amygdalina Del., commonly called Ndolé or
bitterleaf, and Vernonia calvoana Hook, known as sweet
Ndolé or sweet bitterleaf, are the two tropical species of
the Asteraceae family. V. amygdalina is a perennial shrub
(Farombi and Owoeye, 2011), indigenous to tropical
Africa occurring wild or cultivated all over sub-Saharan
Africa (Igile et al., 1995; Oboh and Masodje, 2009). The
leaves which generally have a bitter taste are eaten after
crushing and washing to remove the bitterness. V.
amygdalina is ubiquitous while V. calvoana has a more
circumscribed habitat (Biholong, 1986). V. amygdalina is
mainly found in secondary forests near villages or in
*Corresponding author. E-mail: youmbi_emmanuel@yahoo.fr.
Author(s) agree that this article remains permanently open access under the terms of the Creative Commons Attribution
License 4.0 International License
Nguimkeng et al.
fallows around forest zones and peri-forest savannahs in
Cameroon (Biholong, 1986). Vernonia species is adapted
to diverse climatic conditions. V. amygdalina is
widespread in Africa, particularly in Cameroon, Benin,
Togo, Senegal, and Côte d’Ivoire (Kalanda and Lisowski,
1995). V. calvoana is found in Camerron, Uganda,
Kenya, Tanzania, Nigeria, and in Ethiopia (Biholong,
1986). In Cameroon (Nkambé, Nkonsamba, Buéa,
Dschang, etc.), it grows near mountainous forests at
about 2,000 m altitude. It is an annual plant but
sometimes exhibits a perennial character depending on
the nature of the soil. Plants aged two to three years with
about 3 m of height can be found near streams (Fube
and Njonga, 1987). As compared to V. amygdalina, its life
span is shorter and its comestible leaves are less bitter.
Ndolé leaves are comestibles and exhibit antimicrobial,
antihypertensive, antidiabetic, and laxative properties
(Grubben and Denton, 2004; Dibong et al., 2011; Audu et
al., 2012). In fact, all parts of Vernonia spp. are
pharmacologically useful in phytomedicine (Oboh and
Masodje, 2009; Farombi and Owoeye, 2011); roots and
leaves are used to treat fever, hiccups, kidney disease,
and stomach ache (Ngatu et al., 2014) in addition to their
reported antihelmintic, antitumorigenic, and antimalarial
properties (Hamowia and Saffaf, 1994; Abosi and
Raserika, 2003; Izevbigie et al., 2004; Oboh and Masodje,
2009). Akah and Okafor (1992), Nwanjo (2005), and
Atangwho et al. (2010) also reported hypoglycaemic and
hypolipidaemic effects of Ndolé leaf extracts in
experimental animals. Moreover, Oboh and Masodje
(2009) and Udochukwu et al. (2015) revealed the
moisture, protein, ash and mineral content and
antimicrobial effects of bitter leaf leaves. Recently, a
comprehensive review done on the medicinal potentials
of plants of the genus Vernonia (Toyang and Verpoorte,
2013) also highlighted most of the aforementioned useful
properties of bitter leaf.
These are probably the main reasons why V.
amygdalina is much consumed all over sub-Saharan
Africa and as exotic food in some parts of Europe and
America. In sub-Saharan Africa especially in Central and
West Africa, Ndolé constitutes a much appreciated
delicacy in homes and modern restaurants. Also, the
number of African restaurants is on a rapid increase in
France and other European countries (Le Roux, 1996;
Defrance, 1997). Meanwhile, the demand for exotic food
products like Ndolé is on the rise in Europe (Volatier,
1997; Gillet, 1997) where the market for such foods
currently represents over 10 billion of CFA Francs
(Bidima and Voufo, 2007). For instance, the sales of
Vernonia (Ndolé) leaves as vegetable is strongly
developed in France and Belgium where there are big
Cameroonian communities (Bidima and Voufo, 2007;
Tabuna, 2000). In France, for example, about 37% of
consumers buy exotic products (Gillet, 1997; Volatier,
1997). The sales of Ndolé are extending to other
European countries like the United Kingdom, Germany
363
and Switzerland which constitute a potential important
market outlet (Tabuna, 2000).
Due to the importance of Ndolé, it is important for plant
breeders to master the production and improvement
procedures. In addition to having important medicinal
properties, comestible Vernonia can play a role in food
safety. More and more, it is being cultivated by gardeners
in subsistence agriculture in rural areas and marshy
zones especially in urban areas. However, this is done
without any well-conceived genetic improvement program
in place. In fact, the realization of such a program is
feasible through a better understanding of the
reproductive biology of the plant. In this regard, Judd et
al. (1999) believe that Asteraceae in general are
allogamous and that their pollen dispersal is
anemophilous,
entomophilous,
and
hydrophilous.
Concerning the mode of fertilization, the flower of
Vernonia is considered to exhibits protandry like other
Asteraceae in general (Perry and Hirons, 1967; Tyler and
Arthur, 2006; Guerrina et al., 2016). This situation
naturally imposes allogamy on this plant. However, it is
observed that the flowers of the same capitulum do not
open at the same time, giving the possibility of selffertilization between flowers of the same capitulum and
hence autogamy. With regards to the floral morphology, it
seems not appropriate to conclude on the different
modes of pollen dispersal given that water which is
obviously subject to gravity will not easily transport pollen
located under the stigma to fertilize it. These
observations led us to put in place a protocol to
investigate the modes of pollen dispersal of the two
Vernonia spp. in this study.
MATERIALS AND METHODS
Plant material and study site
Plant materials used for this study were two species of Vernonia: V.
amygdalina and V. calvoana. The study was conducted in Mfou in
the Centre Region of Cameroon, from November 2013 to February
in 2014. Mfou is the head quarter of Mefou-and-Afamba Division,
located at 4°27’00’’ latitude north and 11°38’00’’ longitude east. The
Mfou zone is characterized by an equatorial Guinean climate with
four seasons: a long rainy season from mid-August to midNovember, a long dry season from mid-November to mid-March, a
short rainy season from mid-March to mid-June, and a short dry
season from mid-June to mid-August. Average daily temperature
varies from 23 to 24°C, and annual rainfall is 1,600 to 1,800 mm.
The soil of this site is ferralitic, acid and is strongly desaturated with
ruddle aspect, presenting a concrete horizon that protrudes on hill
tops. Nine (9) plants of V. amygdalina were chosen at random from
the fallow, while nine plants of V. calvoana were planted at 5 m
interspacing, after clearing a surface area of 50 m 2. The study site
was a two-year old fallow dominated by Chromolaena odorata (L.).
The experimental designs (Figures 1, 2, and 3) were put in place
once the first floral bud appeared. It was considered that the agent
being tested (wind, rain water or insects) influenced pollination if at
the end of the process the achene obtained germinated. Seeds
were put in envelopes, labeled, and conserved in the same non
humid conditions. Germination test was done on the seeds
obtained from field experiments in the laboratory of Biotechnology
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Afr. J. Plant Sci.
and Environment of the University of Yaounde I. The seeds were
germinated in Petri dishes on cotton soaked with tap water.
Experimental design to limit insects and wind
Figure 1. Design to study the role of rain water in dispersal of
Vernonia spp. pollen.
This design was established to determine the role of rain water in
pollen dispersal. For the construction of the fence (Figure 1), four
poles, each 3 m long, and higher than the Vernonia plant were
placed at the four corners around the plant. The four walls were
covered with plastic transparent paper while the roof was left open
to rainfall. Three distinct fences around three different plants were
constructed for each of the two Vernonia spp. A webbed cone was
constructed using iron wires covered with impregnated mosquito
net. An adhesive tape was used to hold the pointed end of the cone
on the branch of Vernonia under study (Figure 1).
Three different branches of the same plant were isolated with the
webbed cones. For each species of Vernonia, nine branches
belonging to three plants (three branches per plant) were isolated to
prevent insect visits. The aim of this design was to avoid the action
of wind and insects on the pollination process while maintaining the
action of rain water which could fall through the open roof.
Experimental design to limit insects and rain water
This design was devised to analyze the role of wind as pollen
dispersal agent. Eight wooden poles were used to construct a shed
with open walls (Figure 2) around the plant. The roof was covered
with a transparent plastic paper to avoid rainfall on the plant. For
each species of Vernonia, three sheds were constructed over three
different plants. Three branches per plant with flowers were
protected with webbed cones against insect pollination. The goal
here was to exclude the action of rain water and insects in the
process of pollination while preserving only the action of wind.
Experimental design to determine the role of insects as pollen
dispersal agents
Figure 2. Experimental design to study the effect of wind
on dispersal of Vernonia spp. pollen.
This third design helped to avoid the action of rain water in pollen
dispersal by using a transparent plastic tissue over the plant,
supported by four wooden poles. Another barrier was created on
the four walls around the plant to screen it from wind flow. A 2 m
space was allowed between the plastic roof and the upper edge of
the walls to permit insect visitation of the plant (Figure 3). Six plants
were used in this experiment; three per species. On each plant,
three flowered branches were isolated using the webbed cones
(Figure 3). All these plants were exposed to wind and rain during
the field experimentation period. At the end of the process, pollen
grains from insect-isolated and non-isolated branches were
harvested from the plants. The non-isolated grains constituted the
control treatment of the entire experiment.
Data analysis
Figure 3. Experimental design to put into evidence the
role of insects on dispersal of Vernonia spp. pollen.
Data obtained on the germination percentage of pollen grains of the
two Vernonia spp. studied was treated by analysis of variance
(ANOVA) with the Minitab version 16 software (Ryan et al., 2004).
Comparison and classification of means was done using StudentNewman-Keuls test at the threshold of 5%. The ANOVA was
performed with data after ensuring conformity of these data with
ANOVA assumptions (normality and homogeneity of variance).
Non-compliant data to the ANOVA assumptions were analyzed by
the Kruskal-Wallis test and averages were compared by the MannWhitney U test at the threshold of 5% using IBM SPSS software.
Germination (%)
Figure 4. Germination percentages of two Vernonia species in
the presence of rain water and the absence of wind and insects.
T: Absence of wind and insects, presence of water; T0:
presence of wind, insects, and water; VA :Vernonia amygdalina,
VC: Vernonia calvoana; Values followed by the same letter are
not significantly different at the threshold of 5% with the StudentNewman-Keuls test.
365
Germination (%)
Germination (%)
Nguimkeng et al.
Figure 6. Percentage germination of pollen from the two
species of Vernonia sp. in the presence of insects and in
the absence of water and wind. T1: Absence of water and
wind, presence of insects; T0: presence of wind, insects
and water; VA: Vernonia amygdalina; VC: Vernonia
calvoana; Values followed by the same letter are not
significantly different at the threshold of 5% with the
Student-Newman-Keuls test.
grains of V. amygdalina and V. calvoana resulting from
plants grown in the absence of wind and insects. Results
show that the pollen germination percentage of control
(wind + water + insects) is 11.67% for V. amygdalina and
25.67% for V. calvoana, as against 0% for the treatment
(rain water only) on the two species. No germination was
observed in the treatment (Figure 4) indicating that water
is probably not the agent of Vernonia pollen dispersal.
Pollen dispersal by wind
Figure 5. Percentage germination of two Vernonia species in
the presence of wind and in the absence of water and insects.
T1: Absence of water and insects, presence of wind; T0:
presence of wind, insects, and water; VA: Vernonia amygdalina,
VC: Vernonia calvoana; Values followed by the same letter are
not significantly different at the threshold of 5% with the StudentNewman-Keuls test.
Results of germination (%) of pollen grains of Vernonia
amygdalina and V. calvoana in the absence of water and
insects are shown in Figure 5. It can be observed (Figure
5) that percentage germination of pollen of the control
(wind + rain water + insects) is 14.67% for V. amygdalina
and 26.67% for V. calvoana. The percentage germination
of pollen from the treatment (wind alone) is 0% for both
species (Figure 5). The fact that no germination was
observed in the treatment (wind) indicates that wind is not
the pollen dispersal agent in Vernonia spp.
Pollen dispersal by insects
RESULTS
Mode of pollen dispersal
Pollen dispersal by rain water
Figure 4 presents the germination percentages of pollen
The germination percentages of pollen from V.
amygdalina and V. calvoana in the absence of water and
wind are illustrated in Figure 6. The germination
percentages of pollen from the control (wind + water +
insects) for V. amygdalina (11.00%) and V. calvoana
(28.33%) are statistically identical to those of the
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Afr. J. Plant Sci.
treatment (insects only): V. amygdalina (13.33%) and V.
calvoana (28.22%). This observation shows that insects
are responsible for the dispersal of pollen grains of
Vernonia spp.
Possible mode of fertilization of Vernonia spp.
Based on morphological analysis of Vernonia spp,
flowers and pollen morphology (Nguimkeng et al., 2015),
and on experimentation done on the mode of pollen
dispersal of the two Vernonia spp. studied, it can be
hypothesized as is currently admitted that fertilization in
Vernonia spp. is done by allogamy. Mindful of the fact
that the flowers of the same capitulum do not open at the
same time in this genus, a second hypothesis could be
that fertilization in Vernonia spp. is done by autogamy
between florets of the same capitulum. However, it is
necessary to verify these hypotheses by further thorough
experimentation. In this study, observations made on the
capitulum of Vernonia spp. show that the florets of the
same capitulum are not of the same age and are found to
be at different developmental stages. Central florets are
younger than those on the peripheries. The expulsion of
pollen grains out of the anthers by plunger
pollination mechanism is a common procedure in
Asteraceae. By this mechanism, pollen grains are
released out of the anther lobes by the style which
progressively scrapes pollen grains as it grows, to put
them at the disposal of pollinators.
During the vertical elongation of the style, it
progressively opens horizontally along a median axis to
form two symmetrical ligulate parts folded outward. At
maturity, the upper surface bears the stigma while the
lower surface is covered by pollen grains. Analysis of
pollen morphology and mode of pollen dispersal helped
to hypothesize on the possible allogamous mode of
fertilization in V. amygdalina and V. calvoana. However,
there is need to experimentally test the mode of
fertilization in order to confirm this hypothesis. This could
be done by examining seed germination upon controlled
self-pollination and controlled cross-pollination of
Vernonia spp. flowers.
The second hypothesis which stipulates that autogamy
is equally possible in Vernonia spp. was also considered
probable (Figure 7). It is therefore possible that
fertilization in Vernonia spp. is done both by allogamy
and autogamy.
DISCUSSION
The results obtained from this work have put into
evidence the existence of a single mode of pollen
transport in natural milieu for the two species studied
(Figure 6). In fact, 11.00% germination of pollen grains
was obtained for V. amygdalina and 28.33% germination
for V. calvoana when the flowers were exposed to
pollination agents in natural milieu. These results
corroborate those of Judd et al. (1999) and Grubben and
Denton (2004) who observed that pollination of Vernonia
is entomophilous. However, our results are contrary to
those of the same authors who declared that pollination
in Asteraceae is equally hydrophilous and anemophilous.
The results of our experimentation clearly demonstrated
that in the case of Vernonia, pollination is exclusively
entomophilous (by insects). Nguimkeng et al. (2015)
believe that pollen morphology of Vernonia adapts this
genus to this mode of pollen transfer since it is echinulate
and covered by a substance which sticks it easily to the
pollinator. The presence of this same gelatinous sticky
substance does not facilitate an anemophilous mode of
pollen transport since there is need of more force to
unstuck the pollen from the style given their tiny sizes.
Rain water does not also constitute a medium of pollen
transfer in Vernonia spp. This is because pollen formed
at the end of floret maturation process is located under
the stigma. The law of gravity imposes that this male
gametophyte located under the stigma be carried by rain
water downwards and not up over to the stigma located
on the part above it.
The observations by Nguimkeng et al. (2015) suggest
that fertilization in Vernonia spp. is possibly allogamous
(Figure 7). Perry and Hirons (1967) and Judd et al.
(1999) suggested this mode of fertilization in Asteraceae
in general, on the basis of observations done in natural
milieu. Vernonia flowers exhibit the phenomenon of
protandry which is naturally proper to allogamy since,
pollen from a flower attains maturity when the gynoecium
is not yet matured. In such conditions, if the life span of
this male organ is short as is the case with this species,
the pollen is destined to obviously pollinate the
gynoecium of another matured flower.
Based on observations in this study (Figure 7), it could
be considered that allogamy is a mode of fertilization in
Vernonia spp. even though autogamy is not to be
excluded as a mode of fertilization as long as evidence of
incompatibility has not been established. Even though the
flowers exhibit protandry, those of the same capitulum do
not mature at the same time. The central florets are
younger than those at the periphery. It is therefore
probable that pollen of central florets fertilize the
peripheral florets whose gynoecia have attained maturity.
In this case, we assume autogamy since the florets of the
same capitulum result from the same genetic material.
Another possible consideration is the fertilization of a
floret belonging to a different capitulum of the same plant
as illustrated in Figure 7. Further detailed studies of
Vernonia spp. on the flower lifespan, exact stage and
time (hours of the day) of pollen release, stigma
receptivity, proper pollen storage and germination
conditions (Monteiro et al., 2015) are recommended.
These considerations are valuable for eventual
establishment of an efficient regeneration system through
Nguimkeng et al.
367
Figure 7. Possible model of pollination system in Vernonia amydalina and Vernonia calvoana. 1:
Matured peripheral floret; 2: young central floret; 3: receptacle of capitulum; 4: peduncle of capitulum;
5: receptacle of floret; 6: aigrette; 7: petal; 8: stigma.
classical breeding or micropropagation (Min et al., 2015)
for Vernonia spp. cultivation.
Conclusion
Results obtained showed that the exclusive mode of
pollen dispersal of V. amygdalina and V. calvoana was
entomophilous. Based on observations and pending
thorough experimentation, fertilization in the two species
can either be allogamous or autogamous with allogamy
more preferential. It was found that V. calvoana pollen
germinates better than that of V. amygdalina. Information
obtained on the mode of pollen dispersal and fertilization
in Vernonia spp. could be exploited for genetic
improvement of this important plant. It could also help to
understand the genetic diversity within the genus and the
preponderant role of insects in the preservation of
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Afr. J. Plant Sci.
biodiversity.
CONFLICT OF INTERESTS
The authors have not declared any conflict of interests.
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