Table Of ContentPRIMARY RESEARCH PAPER| Philippine Journal of Systematic Biology
Comparative Pollen Viability and Pollen Tube Growth of Two
Endemic Philippine Etlingera (Zingiberaceae, Alpinioideae)
Noe P. Mendez1*, Heidi C. Porquis1,2, Evangeline B. Sinamban1, and Florfe M. Acma1,2
ABSTRACT
The pollen viability and pollen tube growth of Etlingera dalican and Etlingera philippinensis
(Zingiberaceae) were examined from fresh samples under the light microscope (LM) and
scanning electron microscope (SEM). Pollen measurements were 68-75µm for E. dalican
and 60-65µm for E. philippinensis, having a spheroidal shape for hydrated pollen and an
irregular shape for dry pollen for both species. E. dalican pollen has greenish-yellow color
KEY WORDS : while that of E. philippinensis is greenish. Both species have inaperturate pollen but differ
in their ornamentation, which is gemmate in E. dalican and psilate in E. philippinensis. E.
Etlingera dalican dalican had 88.56% pollen viability while E. philippinensis had only 40.69%. The rate of
Etlingera philippinensis pollen tube growth was faster in E. dalican (17.75 µm per day) than E. philippinensis (8.17
Philippine Native Gingers µm per day). The possible pollinators observed for the two species were butterflies of the
Pollen germination genus Catopsilia, ants and flies. Additional information on the inflorescence and flower
Pollen morphology description of the two species are herein presented.
INTRODUCTION people (Acma, 2010). Etlingera philippinensis (Ridl.) R.M.
Sm. is another Philippine endemic species also locally
Zingiberaceae, also called “gingers”, are herbaceous plants known as “tagbak” in the Visayas which holds a new record
that produce essential oils (Larsen et al., 1999; Larsen & of reported occurrence in the eastern Mindanao corridor
Larsen, 2006). These plants are widely used in traditional (Acma, 2010).
medicine to relieve colds, inflammations, diarrhea, stomach
disorders, fever and cramps. They are also valuable as food Reconnaissance observations of these two species by the
flavoring and spice agents, and are generously included in authors indicate the rarity of fruits in E. philippinensis
postpartum diets (Larsen et al., 1999; Leong-Skornickova & compared to E. dalican populations. Although many
Gallick, 2010; Boonmee et al., 2011). According to Pelser et physical, nutritional and biological factors in general could
al. (2011), the Philippine Zingiberaceae includes 14 genera influence fruit setting in plants, the aspect on pollen
with 107 species. viability, pollen tube growth and pollen morphology are
given specific attention because of their taxonomic and
Etlingera, a large genus of about 100 species which is phylogenetic values. Hence, this study presents empirical
distributed in the Indo-Pacific region has 9 species in the information on pollen characteristics of two Etlingera
Philippines (Poulsen, 2006; Pelser et al., 2011) of which two species that could be scrutinized in future evaluation and
species are considered in this study. Etlingera dalican (Elmer) taxonomic characterization of these species.
A.D. Poulsen is a Philippine endemic species which was
renamed in 2003 of its former name Amomum dalican (Elmer) MATERIALS AND METHODS
Merr. and known for its local names “dalikan” in Bukidnon and
“tagbak” in the Visayas (Acma, 2010; Pelser et al., 2011). The Collection of Samples. Fresh pollen samples were collected
seeds of its ripe fruits are considered edible and some parts from matured stamens of 10 plants in the same population
of the plant are boiled to cure fever as reported by the local during anthesis (flowers fully open) between the months of
November 2015 and January 2016 from 5:00 AM to 6:00
1Department of Biology, College of Arts and Sciences, Central
AM. Pollen collection was in accordance with the natural
Mindanao University (CMU), University Town, Musuan, Bukidnon
2Center for Biodiversity Research and Extension in Mindanao flower opening time for each species. Herbarium
(CEBREM), Central Mindanao University (CMU), University Town, specimens and spirit collections were deposited at the
Musuan, Bukidnon Botany Section of Central Mindanao University Herbarium
*Corresponding Author: [email protected] (CMUH) with the accession number 00010859 for E.
Date Submitted : 01 December 2016
dalican and 00010860 for E. philippinensis. All pollen
Date Accepted : 18 May 2017
© Association of Systematic Biologists of the Philippines Volume 11 Issue 2 - 2017
Philippine Journal of Systematic Biology | Mendez et al.: Comparative Pollen Viability of Two Endemic Philippine Etlingera
samples used in the study were freshly collected from plants Hence, this study used IKI (iodine + potassium iodide)
growing in the living ginger collections at the Mt. Musuan solution for the stain test. Fresh pollen were mounted on
Zoological and Botanical Garden (MMZBG) of Central glass slides and tested with a drop of IKI solution to reveal
the presence of starch. Another set of pollen samples for the
Mindanao University (CMU) and Acma’s residence at the
determination of the pollen viability was prepared separately
Market Site of CMU, University Town, Musuan, Bukidnon.
with pollen added with a drop of IKI solution. These were
The flower samples were placed separately in zip–lock
covered with a cover slip and subjected to light microscopy
cellophane bags to prevent drying and were transported to
using low power objective (100x) and counted for the
the laboratory for further processing within 15-20 min from
percentage viability. In getting the percentage viability, a
collection and were maintained at room temperature of 20°C.
modified formula was used as shown below:
Five plants per species were morphologically measured and (Viable pollen /Total # of pollen) x 100 = Percentage Viability
described. Lengths of the live specimens were measured
using a meter stick. Three (3) inflorescences per species Determination of Pollen Germination
were also collected and brought to the laboratory for Pollen germination assays. Pollen samples from
dissection and measurement of parts. another set of 5 plants for each species from the same
population were collected for germination test. These
Pollen Morphology and Micromorphological Studies. Fresh samples were processed right after the collection. Pollen
pollen samples for light microscopy examination were collection time was limited to 15-20 min to avoid exposure to
removed from the anther sacs using forceps, mounted with contaminants. The germination medium consists of 100 g
water in a glass slide, examined under microscope and sucrose (C H O ), 500 mg calcium nitrate [Ca(NO )
12 22 11 3
described as to: a. size b. shape c. color d. aperture and e. _4H O], 120 mg magnesium sulfate (MgSO ), 100 mg
2 2 4
ornamentation. Measurable features like the size of the pollen potassium nitrate (KNO ) and 120 mg boric acid (H BO )
3 3 3
were determined from scanning electron microscope (SEM) were dissolved in 1000 ml deionized water following Karni &
photographs. Pollen color was visually classified based on Aloni (2002). The pH of the germinating medium was 6.37 at
hydrated pollen which were examined under light microscope 55°C. An estimated 10 ml of the medium was poured/
(LM). Samples were not acetolyzed based on the reports of dispensed into three Petri plates for each species in the
Liang (1988) and Saensouk et al. (2009) that members of the treatment and allowed to cool for about 15 min to allow the
Zingiberaceae are mostly not resistant to acetolysis due to agar to solidify. The plates were covered and incubated and
their very thin exine and thick intine wall. For the other pollen maintained at a room temperature of 20°C. No staining of the
morphological features such as shape, aperture and pollen was needed (Ozler et al., 2009) and each plate was
ornamentation, they were classified following the criteria of considered a replicate. The petri plates with the germinating
Liang (1988), Liang (1990), Theilade et al. (1993), Saensouk medium were exposed under the ultraviolet light for 15 min.
et al. (2009), and Chen & Xia (2011). The pollen samples were inoculated aseptically to the petri
plates containing the germinating medium. Lids of petri
The pollen samples for SEM examination were sent to plates with pollen samples were sealed with a tape, labeled
Mindanao State University–Iligan Institute of Technology accordingly and brought to the growth room with the same
(MSU-IIT), Iligan City. The possibility of dehydration of the maintained temperature.
pollen samples was considered due to the 5-6 hrs travel A. Evaluation of the onset of pollen germination. After
between CMU and MSU-IIT. The pollen samples were 24–h of incubation, the pollen were counted for pollen
mounted on the sample holder using carbon tape. Then the germination. Pollen samples inoculated on the Petri
mounted pollen were coated with platinum using the JEOL plates were mounted on glass slide and viewed under
JFC-1600 Autofine Coater to make the surface of samples the light microscope. Those found within the
microscopic field (100x magnifications) were considered
suitable for SEM characterization. The SEM images of the
for scoring. A pollen was classified as germinated if at
samples were taken using the JEOL JSM-6510LA Analytical
least the beginning of a developing pollen tube could be
SEM coupled with an energy dispersive x-ray spectrometer at
seen emerging from one of the pores or reach the
200x, 500x and 1000x magnifications.
pollen diameter (Steiner & Gregorius, 1998; Tosun &
Koyuncu, 2007). Percentage pollen germination was
Testing for Starch and Pollen Viability. A common method to
calculated following the formula of ISTA (1999) as
assess pollen viability is by staining and direct counting
shown below:
(Barrett, 1985; Dudash, 1991; Willis, 1999). Parfitt and
Ganeshan (1989) have determined that the pollen stain tests
(Pollen germinated / Total # of pollen) x 100 = %Germination
(acetocarmine, Alexander Stain’s, TTC, MTT, and NBT) are
not reliable or consistent and are not positively correlated with
B. Pollen tube lengths. Pollen were considered
in vitro germination tests (as cited by Bolat and Pirlak, 1999).
© Association of Systematic Biologists of the Philippines Volume 11 Issue 2 - 2017 | 2
Philippine Journal of Systematic Biology | Mendez et al.: Comparative Pollen Viability of Two Endemic Philippine Etlingera
germinated when its tube length equaled the grain
diameter (Luza et al., 1987). The pollen tube lengths
were measured within 14 days at 2 days interval after
initial plating. Observations were done with an ocular
micrometer fitted to the ocular/eyepiece of the
microscope. The determination of the percentage pollen
tube lengths depended upon the pollen tubes that
emerged outside the pollen wall.
RESULTS AND DISCUSSION
Plant Description. E. dalican plants reach a height of 2.5–3 m
and the inflorescence (with 10-18 flowers) is obconic,
measuring 8 x 4 cm with a truncated top when flowers open.
Figure 2: Etlingera philippinensis Floral Dissection: inflorescence
Bracts are oblong, tips notched and hairy, pink towards the
(inf), flower (fl), fertile bract (fb), bracteole (bt), calyx (cx), corolla
top and white below. Bracteoles are tubular, tips hairy, pink
lobes (cl), labellum (lm), stigma (s) and anther sacs (as).
towards upper portion while white at base. Calyx elongated,
fused, tubular, three tipped and pink. Corolla lobes are E. philippinensis have dominantly reddish color in its entire
oblanceolate, yellow in color, and corolla tube white in color;
inflorescences. Like in E. dalican, individual flowers of E.
stigma reddish color. Anther position is lower than stigma and
philippinensis do not open at the same time.
the pollen are located between corolla and anther sacs (Fig.
1). Populations of E. dalican are seen near streams and/or
In the report of Melati (2015) on Zingiber officinale Rosc.
forest habitat where there is no direct sunlight. The flowers of
temperature and relative humidity could influence the time of
the plant do not open at the same time.
anthesis and increase in temperature and relative humidity
accelerates the opening of flowers. However, as to whether
the same factors are behind the anthesis in these two
Etlingera species needs further investigation. Likewise,
Darjanto & Satifah (1990) also stated that changes in these
factors change plants’ response to flowering.
Throughout the study, it was observed that the two Etlingera
species were often frequented by two species of butterflies
belonging to genus Catopsilia. This observations support the
earlier report of Larsen et al. (1999) and Larsen & Larsen
(2006) that Etlingera species are pollinated by butterflies. E.
dalican was further observed hosting black-colored ants in its
inflorescences (Fig. 3). E. philippinensis, on the other hand,
harbored fruit flies and red-colored ants. These insects were
Figure 1: Etlingera dalican Floral Dissection: inflorescence (inf),
distributed on the entire plant particularly on the leaves and
sterile bract (sb), fertile bract (fb), flower (fl), bracteole (bt), calyx
inflorescences (Fig. 4). The probability that they could be
(cx), corolla lobes (cl), epigynous gland (eg), style (st), stigma (s),
possible pollinators is considered. Pollinators (butterflies
labellum (lm) and anther sacs (as).
and ants) are known to frequently visit plant species due to
E. philippinensis on the other hand, reaches a height of 2–2.5 nectar secretion. The amount of reward offered to a flower
m and the inflorescence (with 5-8 flowers) is cone-shaped, 9 x visitor also depends on the concentration of sugar in the
2 cm. Bracts are elliptic-lanceolate, reddish but white at base; nectar. However, humidity, temperature and soil moisture
bracteoles tubular. Calyx elongated, fused, tubular, three strongly influence high rate of nectar secretion (Brown, 1959;
tipped, red in color except the basal part which is white. The Real & Rathcke, 1991; Aswani et al., 2013).
corolla is oblong, red but base is white. Labellum is very
bright red or scarlet with ovate shape. All parts are lighter in Pollen Morphology. The characters of pollen studied using
LM and SEM are summarized in Table 1. The results on the
color at the bottom and darker towards the tip. Pollen samples
morphological features of the pollen showed resemblances
are also located in the anther sacs (Fig. 2). Rhizomes are
as well as differences between the species in their features.
long; creeping along the soil, inflorescences either grow
According to McGrath (1999), pollen of many plants can be
distantly from the main rhizome or are partially buried in the
used to classify the genus and sometimes the species on the
soil. This species is also found in shady places.
basis of such characteristics like the size, shape, aperture,
© Association of Systematic Biologists of the Philippines Volume 11 Issue 2 - 2017 | 3
Philippine Journal of Systematic Biology | Mendez et al.: Comparative Pollen Viability of Two Endemic Philippine Etlingera
Figure 3 (left)
Observed insect
visitor of E.
dalican.
Figure 4 (right)
Observed insect
visitors of
E. philippinensis.
and ornamentation.
of the two species supports the findings of Furness & Rudall
(1999) regarding widespread occurrence of inaperturate
The present study revealed that E. dalican has bigger pollen
pollen among monocotyledonae. These data closely
compared to E. philippinensis in which pollen size range for E.
resembled the inaperturate pollen in Curcuma kwangsiensis
dalican was 68 - 75 µm while for E. philippinensis was 60 - 65
and Boesenbergia longiflora of Chen and Xia (2011) which
µm. E. dalican pollen appeared greenish-yellow in color
range from ovoid to spherical and varied from 51.9 ± 7.2µm
having spheroidal shape (hydrated pollen) while E.
in C. kwangsiensis and 109.4 ± 12.5µm in B. longiflora.
philippinensis pollen appeared greenish in color having
Furthermore, it was also reported that B. albomaculata and
spheroidal shape (hydrated pollen). These findings on the
B. longiflora pollen are spherical, 83.0 ± 7.8µm in diameter
pollen size ranges and shapes agree with the reports of
and appeared nonaperturate. It was likewise observed that
Theilade et al. (1993), Chen & Xia (2011) and Saensouk et al.
the most inner layer was developed only in certain regions
(2015). On the other hand, the outline of dry pollen in the two
and appeared to have excrescences similar to B. tiliiflora
species as seen in polar view are irregular specifically its
(Mangaly and Nayar, 1990) with a circular area around the
infoldings, and their pollen are inaperturate. The main
distal pole where the intine is thinner.
difference for the two species is in its ornamentation in which
E. dalican is gemmate while E. philippinensis is psilate (Figs.
Theilade et al. (1993) studied the Zingiber pollen and
5-8).
reported that the species of said genus possess a spherical
shape (with a cerebroid or reticulate sculpturing) or
In this observation, it is clear that E. dalican have bigger
ellipsoidal pollen with a diameter ranging 55-85 µm. Pollen
pollen compared to E. philippinensis. The inaperturate pollen
were also reported that no structures indicating the presence
Table 1. Pollen Morphology and Micromorphological Features of E. dalican and E. philippinensis under LM and SEM.
Pollen Pollen
Pollen Pollen Pollen
Species shape (hydrated shape (dry pollen) Pollen color
size aperture ornamentation
pollen) and infolding
E. dalican 68–75 µm Spheroidal Irregular Greenish–yellow Inaperturate Gemmate
E. philippinensis 60–65 µm Spheroidal Irregular Greenish Inaperturate Psilate
© Association of Systematic Biologists of the Philippines Volume 11 Issue 2 - 2017 | 4
Philippine Journal of Systematic Biology | Mendez et al.: Comparative Pollen Viability of Two Endemic Philippine Etlingera
Figure 5. Pollen of E. dalican showing spheroidal shape (hydrated Figure 8. Pollen of E. philippinensis showing its sizes (60-
pollen) under LM (100x). 65µm); irregular shape (dry pollen); inaperturate and psilate
ornamentation under SEM (1,000x).
of apertures were noted. Saensouk et al. (2015) on the other
hand reported that the majority of the genus Curcuma has
large sized pollen in the range of 50.5–86.9 µm. Chen (1989)
further reported that the exine sculpturing of the pollen of
Curcuma are psilate which is similar to E. dalican and E.
philippinensis.
It appears that the ranges of our pollen size fall between the
pollen sizes of related genera viz., Boesenbergia,
Cornukaempferia, Curcuma and Zingiber (e.g. Liang, 1988;
Liang, 1990; Mangaly and Nayar, 1990; Theilade et al.,
1993; Theilade and Theildae, 1996; Saensouk et al., 2009,
Chen and Xia, 2011; Saensouk et al., 2015). These reports
on pollen morphology are significant, since pollen function as
Figure 6. Pollen of E. dalican showing its sizes (68-75µm); irregular agents of reproduction and their features can often be used
shape (dry pollen); inaperturate and gemmate ornamentation under to identify a particular taxon (Uno et al., 2001).
SEM (1,000x).
Pollen Viability. Previous studies on pollen samples indicate
that pollen of E. dalican turn dark brown in the iodine test,
while E. philippinensis pollen turn yellowish as indications of
the presence of starch following Simpson (2006). Firmage &
Dafni (2001) also reported that if the pollen has starch it
means that they are viable. Using the test, the current study
shows that the percentage viability of E. dalican pollen
(88.56%) is higher than that of E. philippinensis (40.69%).
There are a number of nongenetic causes of pollen inviability
including pollen age and physical factors such as
temperature and humidity (Kelly et al., 2002). Viability also
mainly depends on relative atmospheric humidity at
shedding and during pollen transport (Frankel & Galun,
1977; Pacini, 1990; Paoletti, 1992). It has been considered
Figure 7. Pollen of E. philippinensis showing spheroidal shape
that pollen remain viable longer at low relative humidity
(hydrated pollen) under LM (100x).
levels (Stanley & Linskens, 1974). However, recent studies
© Association of Systematic Biologists of the Philippines Volume 11 Issue 2 - 2017 | 5
Philippine Journal of Systematic Biology | Mendez et al.: Comparative Pollen Viability of Two Endemic Philippine Etlingera
Table 2. Two (2) weeks observation for Pollen Tube Growth of E. dalican and E. philippinensis under the light microscope (400x).
Average
Species 2nd day 4th day 6th day 8th day 10th day 12th day 14th day microns (per
day)
13.6– 28.6–33.3 43.4–49.6 63.4– 72.3–
E. dalican 3.6–4.4 µm 7.8–9.2 µm 17.75 µm
21.6 µm µm µm 66.9 µm 79.4 µm
4.8–6 13.6–15.3 22.9–24.1 28.8– 33.6–
E. philippinensis 0.8–2.4 µm 2.4–4.4 µm 8.17 µm
µm µm µm 31.4 µm 38.3 µm
pointed out that pollen of different species need a high level of many fruit species (Stanley & Linskens, 1974). Moreover,
relative humidity to germinate as well (Corbet & Plumridge, Einhardt et al. (2006) concluded that if pollen seems non–
1985; Digonnet–Kerhoas & Gay, 1990; Mulugeta et al., 1994; viable, and if vigorous pollen tubes are present, it is still good
Loupassaki & Vasilakakis, 1995). The link between pollen enough to ensure at least a moderate efficient fruit set,
viability and fruit and seed set has been earlier evidenced despite the low germination rate. Pollen germination and
(Stone et al., 1995). As to whether this connectivity is applied tube growth thus emerge as highly dynamic and coordinated
to the two Etlingera species studied still requires further processes, integrating many different signals from the local
investigation. environment to regulate growth and development (Rodriguez
-Enriquez et al., 2013). The present data on pollen viability
Pollen Germination and Pollen Tube Growth. Culture medium and pollen tube growth in E. dalican and E. philippinensis
containing a gelling agent (agar or gelatin) has been used collected in Central Mindanao University appear to provide
successfully in many species (Jayaprakash & Sarla, 2001; additional support to these earlier observations.
Kakani et al., 2002; Kozai et al., 2008). In this study, agar was
used instead of gelatin due to the report of Stanley & Liskens CONCLUSIONS AND RECOMMENDATIONS
(1974) that there are several advantages of using agar
germination tests such as the ease of taking carbohydrate, The distinguishing features of pollen in E. dalican and E.
creating stable relative humidity and providing aerobic philippinensis are presented. The pollen of E. dalican are
conditions. The germination medium used for this study distinguished from that of E. philippinensis by their sizes and
followed the formulation of Karni & Aloni (2002). This is in exine ornamentation in which E. dalican measured 68-75 μm
accordance with the suggested reports of Khan and Perveen and has gemmate ornamentation while E. philippinensis
(2006) and Imani et al. (2011) that culture medium aside from measured 60-65 μm and has psilate ornamentation.
having carbohydrates (particularly sucrose) should also Furthermore, E. dalican pollen viability and pollen tube
contain germination–stimulating substances such as boric growth is higher than that of E. philippinensis.
acid, calcium nitrate, potassium nitrate and magnesium
sulfate wherein all aforesaid substances are the major In view of our results, we recommend further studies on
components. pollen of other ginger species to be examined/characterized
using SEM in order to delineate species. Implications on the
E. dalican and E. philippinensis as observed in this study different stages of germination should also be noted and
have similar percentage germination of 0% after 1 day of described. Moreover, these species should be germinated/
incubation. However, for pollen tube growth, the observation propagated using other germination media and recording the
after 2 days of incubation indicates that the first pollen tube tube growth as well. More research using molecular and
length in E. dalican measures 3.6–4.4 µm on the (2nd day population genetic studies are needed to investigate the
observation) and 72.3–79.4 µm in the 14th day observation. relationships of the genus Etlingera. Further studies to
On the other hand, pollen tube length in E. philippinensis on establish relationship between pollen viability and
the 2nd day measures 0.8–2.4 µm and 33.6–38.3 µm in the germination with fruit setting in the two species must be
14th day observation. As shown in Table 2, average increase done.
in pollen tube length of E. dalican is higher compared to that
of E. philippinensis. ACKNOWLEDGEMENT
Germination has already been used to indicate viability of The authors would like to extend their genuine appreciation
pollen (Schoenike & Stewart, 1963) and a linear relationship and recognition to the following, who one way or another
is seen between pollen viability and germination capability in have contributed to the success of the study: (1) Central
Mindanao University (CMU); (2) CMU Natural Science
© Association of Systematic Biologists of the Philippines Volume 11 Issue 2 - 2017 | 6
Philippine Journal of Systematic Biology | Mendez et al.: Comparative Pollen Viability of Two Endemic Philippine Etlingera
Research Center (NSRC)–Plant Tissue and Spore Culture germination of soil-seed rape pollen. Journal of
Laboratory (PTSCL) headed by Dr. Chris Rey C. Lituañas; (3) Agricultural Science, 104: 445-451.
CMU NSRC–Tuklas Lunas Development Center (TLDC)/ Darjanto & S. Satifah, 1990. Basic Science of Floral Biology
Molecular Biology and Biotechnology Laboratory (MBBL) and Artificial Fertilization. Gramedia Press, Jakarta. 156
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