Comparative Pollen Viability and Pollen Tube Growth of Two ...

PRIMARY RESEARCH PAPER| Philippine Journal of Systematic Biology

© Association of Systematic Biologists of the Philippines

Volume 11 Issue 2 - 2017

Comparative Pollen Viability and Pollen Tube Growth of Two

Endemic Philippine Etlingera (Zingiberaceae, Alpinioideae)

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

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. dalican had 88.56% pollen viability while E. philippinensis had only 40.69%. The rate of

pollen tube growth was faster in E. dalican (17.75 µm per day) than E. philippinensis (8.17

µm per day). The possible pollinators observed for the two species were butterflies of the

genus Catopsilia, ants and flies. Additional information on the inflorescence and flower

description of the two species are herein presented.

KEY WORDS :

Etlingera dalican

Etlingera philippinensis Philippine Native Gingers

Pollen germination

Pollen morphology

1Department of Biology, College of Arts and Sciences, Central

Mindanao University (CMU), University Town, Musuan, Bukidnon 2Center for Biodiversity Research and Extension in Mindanao

(CEBREM), Central Mindanao University (CMU), University Town,

Musuan, Bukidnon *Corresponding Author: [email protected] Date Submitted : 01 December 2016 Date Accepted : 18 May 2017

INTRODUCTION

Zingiberaceae, also called “gingers”, are herbaceous plants

that produce essential oils (Larsen et al., 1999; Larsen &

Larsen, 2006). These plants are widely used in traditional

medicine to relieve colds, inflammations, diarrhea, stomach

disorders, fever and cramps. They are also valuable as food

flavoring and spice agents, and are generously included in

postpartum diets (Larsen et al., 1999; Leong-Skornickova &

Gallick, 2010; Boonmee et al., 2011). According to Pelser et

al. (2011), the Philippine Zingiberaceae includes 14 genera

with 107 species.

Etlingera, a large genus of about 100 species which is

distributed in the Indo-Pacific region has 9 species in the

Philippines (Poulsen, 2006; Pelser et al., 2011) of which two

species are considered in this study. Etlingera dalican (Elmer)

A.D. Poulsen is a Philippine endemic species which was

renamed in 2003 of its former name Amomum dalican (Elmer)

Merr. and known for its local names “dalikan” in Bukidnon and

“tagbak” in the Visayas (Acma, 2010; Pelser et al., 2011). The

seeds of its ripe fruits are considered edible and some parts

of the plant are boiled to cure fever as reported by the local

people (Acma, 2010). Etlingera philippinensis (Ridl.) R.M.

Sm. is another Philippine endemic species also locally

known as “tagbak” in the Visayas which holds a new record

of reported occurrence in the eastern Mindanao corridor

(Acma, 2010).

Reconnaissance observations of these two species by the

authors indicate the rarity of fruits in E. philippinensis compared to E. dalican populations. Although many

physical, nutritional and biological factors in general could

influence fruit setting in plants, the aspect on pollen

viability, pollen tube growth and pollen morphology are

given specific attention because of their taxonomic and

phylogenetic values. Hence, this study presents empirical

information on pollen characteristics of two Etlingera species that could be scrutinized in future evaluation and

taxonomic characterization of these species.

MATERIALS AND METHODS

Collection of Samples. Fresh pollen samples were collected

from matured stamens of 10 plants in the same population

during anthesis (flowers fully open) between the months of

November 2015 and January 2016 from 5:00 AM to 6:00

AM. Pollen collection was in accordance with the natural

flower opening time for each species. Herbarium

specimens and spirit collections were deposited at the

Botany Section of Central Mindanao University Herbarium

(CMUH) with the accession number 00010859 for E.

dalican and 00010860 for E. philippinensis. All pollen

Noe P. Mendez1*, Heidi C. Porquis1,2, Evangeline B. Sinamban1, and Florfe M. Acma1,2

mailto:[email protected]


Philippine Journal of Systematic Biology | Mendez et al.: Comparative Pollen Viability of Two Endemic Philippine Etlingera

Volume 11 Issue 2 - 2017 | 2

samples used in the study were freshly collected from plants

growing in the living ginger collections at the Mt. Musuan

Zoological and Botanical Garden (MMZBG) of Central

Mindanao University (CMU) and Acma’s residence at the

Market Site of CMU, University Town, Musuan, Bukidnon.

The flower samples were placed separately in zip–lock

cellophane bags to prevent drying and were transported to

the laboratory for further processing within 15-20 min from

collection and were maintained at room temperature of 20°C.

Five plants per species were morphologically measured and

described. Lengths of the live specimens were measured

using a meter stick. Three (3) inflorescences per species

were also collected and brought to the laboratory for

dissection and measurement of parts.

Pollen Morphology and Micromorphological Studies. Fresh

pollen samples for light microscopy examination were

removed from the anther sacs using forceps, mounted with

water in a glass slide, examined under microscope and

described as to: a. size b. shape c. color d. aperture and e.

ornamentation. Measurable features like the size of the pollen

were determined from scanning electron microscope (SEM)

photographs. Pollen color was visually classified based on

hydrated pollen which were examined under light microscope

(LM). Samples were not acetolyzed based on the reports of

Liang (1988) and Saensouk et al. (2009) that members of the

Zingiberaceae are mostly not resistant to acetolysis due to

their very thin exine and thick intine wall. For the other pollen

morphological features such as shape, aperture and

ornamentation, they were classified following the criteria of

Liang (1988), Liang (1990), Theilade et al. (1993), Saensouk

et al. (2009), and Chen & Xia (2011).

The pollen samples for SEM examination were sent to

Mindanao State University–Iligan Institute of Technology

(MSU-IIT), Iligan City. The possibility of dehydration of the

pollen samples was considered due to the 5-6 hrs travel

between CMU and MSU-IIT. The pollen samples were

mounted on the sample holder using carbon tape. Then the

mounted pollen were coated with platinum using the JEOL

JFC-1600 Autofine Coater to make the surface of samples

suitable for SEM characterization. The SEM images of the

samples were taken using the JEOL JSM-6510LA Analytical

SEM coupled with an energy dispersive x-ray spectrometer at

200x, 500x and 1000x magnifications.

Testing for Starch and Pollen Viability. A common method to

assess pollen viability is by staining and direct counting

(Barrett, 1985; Dudash, 1991; Willis, 1999). Parfitt and

Ganeshan (1989) have determined that the pollen stain tests

(acetocarmine, Alexander Stain’s, TTC, MTT, and NBT) are

not reliable or consistent and are not positively correlated with

in vitro germination tests (as cited by Bolat and Pirlak, 1999).

Hence, this study used IKI (iodine + potassium iodide)

solution for the stain test. Fresh pollen were mounted on

glass slides and tested with a drop of IKI solution to reveal

the presence of starch. Another set of pollen samples for the

determination of the pollen viability was prepared separately

with pollen added with a drop of IKI solution. These were

covered with a cover slip and subjected to light microscopy

using low power objective (100x) and counted for the

percentage viability. In getting the percentage viability, a

modified formula was used as shown below:

(Viable pollen /Total # of pollen) x 100 = Percentage Viability

Determination of Pollen Germination

Pollen germination assays. Pollen samples from

another set of 5 plants for each species from the same

population were collected for germination test. These

samples were processed right after the collection. Pollen

collection time was limited to 15-20 min to avoid exposure to

contaminants. The germination medium consists of 100 g

sucrose (C12H22O11), 500 mg calcium nitrate [Ca(NO3)

2_4H2O], 120 mg magnesium sulfate (MgSO4), 100 mg

potassium nitrate (KNO3) and 120 mg boric acid (H3BO3)

were dissolved in 1000 ml deionized water following Karni &

Aloni (2002). The pH of the germinating medium was 6.37 at

55°C. An estimated 10 ml of the medium was poured/

dispensed into three Petri plates for each species in the

treatment and allowed to cool for about 15 min to allow the

agar to solidify. The plates were covered and incubated and

maintained at a room temperature of 20°C. No staining of the

pollen was needed (Ozler et al., 2009) and each plate was

considered a replicate. The petri plates with the germinating

medium were exposed under the ultraviolet light for 15 min.

The pollen samples were inoculated aseptically to the petri

plates containing the germinating medium. Lids of petri

plates with pollen samples were sealed with a tape, labeled

accordingly and brought to the growth room with the same

maintained temperature.

A. Evaluation of the onset of pollen germination. After

24–h of incubation, the pollen were counted for pollen

germination. Pollen samples inoculated on the Petri

plates were mounted on glass slide and viewed under

the light microscope. Those found within the

microscopic field (100x magnifications) were considered

for scoring. A pollen was classified as germinated if at

least the beginning of a developing pollen tube could be

seen emerging from one of the pores or reach the

pollen diameter (Steiner & Gregorius, 1998; Tosun &

Koyuncu, 2007). Percentage pollen germination was

calculated following the formula of ISTA (1999) as

shown below:

(Pollen germinated / Total # of pollen) x 100 = %Germination

B. Pollen tube lengths. Pollen were considered



Volume 11 Issue 2 - 2017 | 3

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.

Bracts are oblong, tips notched and hairy, pink towards the

top and white below. Bracteoles are tubular, tips hairy, pink

towards upper portion while white at base. Calyx elongated,

fused, tubular, three tipped and pink. Corolla lobes are

oblanceolate, yellow in color, and corolla tube white in color;

stigma reddish color. Anther position is lower than stigma and

the pollen are located between corolla and anther sacs (Fig.

1). Populations of E. dalican are seen near streams and/or

forest habitat where there is no direct sunlight. The flowers of

the plant do not open at the same time.

E. philippinensis on the other hand, reaches a height of 2–2.5

m and the inflorescence (with 5-8 flowers) is cone-shaped, 9 x

2 cm. Bracts are elliptic-lanceolate, reddish but white at base;

bracteoles tubular. Calyx elongated, fused, tubular, three

tipped, red in color except the basal part which is white. The

corolla is oblong, red but base is white. Labellum is very

bright red or scarlet with ovate shape. All parts are lighter in

color at the bottom and darker towards the tip. Pollen samples

are also located in the anther sacs (Fig. 2). Rhizomes are

long; creeping along the soil, inflorescences either grow

distantly from the main rhizome or are partially buried in the

soil. This species is also found in shady places.

E. philippinensis have dominantly reddish color in its entire

inflorescences. Like in E. dalican, individual flowers of E.

philippinensis do not open at the same time.

In the report of Melati (2015) on Zingiber officinale Rosc.

temperature and relative humidity could influence the time of

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

distributed on the entire plant particularly on the leaves and

inflorescences (Fig. 4). The probability that they could be

possible pollinators is considered. Pollinators (butterflies

and ants) are known to frequently visit plant species due to

nectar secretion. The amount of reward offered to a flower

visitor also depends on the concentration of sugar in the

nectar. However, humidity, temperature and soil moisture

strongly influence high rate of nectar secretion (Brown, 1959;

Real & Rathcke, 1991; Aswani et al., 2013).

Pollen Morphology. The characters of pollen studied using

LM and SEM are summarized in Table 1. The results on the

morphological features of the pollen showed resemblances

as well as differences between the species in their features.

According to McGrath (1999), pollen of many plants can be

used to classify the genus and sometimes the species on the

basis of such characteristics like the size, shape, aperture,

Figure 1: Etlingera dalican Floral Dissection: inflorescence (inf),

sterile bract (sb), fertile bract (fb), flower (fl), bracteole (bt), calyx

(cx), corolla lobes (cl), epigynous gland (eg), style (st), stigma (s),

labellum (lm) and anther sacs (as).

Figure 2: Etlingera philippinensis Floral Dissection: inflorescence

(inf), flower (fl), fertile bract (fb), bracteole (bt), calyx (cx), corolla

lobes (cl), labellum (lm), stigma (s) and anther sacs (as).



Volume 11 Issue 2 - 2017 | 4

and ornamentation.

The present study revealed that E. dalican has bigger pollen

compared to E. philippinensis in which pollen size range for E.

dalican was 68 - 75 µm while for E. philippinensis was 60 - 65

µm. E. dalican pollen appeared greenish-yellow in color

having spheroidal shape (hydrated pollen) while E.

philippinensis pollen appeared greenish in color having

spheroidal shape (hydrated pollen). These findings on the

pollen size ranges and shapes agree with the reports of

Theilade et al. (1993), Chen & Xia (2011) and Saensouk et al.

(2015). On the other hand, the outline of dry pollen in the two

species as seen in polar view are irregular specifically its

infoldings, and their pollen are inaperturate. The main

difference for the two species is in its ornamentation in which

E. dalican is gemmate while E. philippinensis is psilate (Figs.

5-8).

In this observation, it is clear that E. dalican have bigger

pollen compared to E. philippinensis. The inaperturate pollen

of the two species supports the findings of Furness & Rudall

(1999) regarding widespread occurrence of inaperturate

pollen among monocotyledonae. These data closely

resembled the inaperturate pollen in Curcuma kwangsiensis

and Boesenbergia longiflora of Chen and Xia (2011) which

range from ovoid to spherical and varied from 51.9 ± 7.2µm

in C. kwangsiensis and 109.4 ± 12.5µm in B. longiflora.

Furthermore, it was also reported that B. albomaculata and

B. longiflora pollen are spherical, 83.0 ± 7.8µm in diameter

and appeared nonaperturate. It was likewise observed that

the most inner layer was developed only in certain regions

and appeared to have excrescences similar to B. tiliiflora

(Mangaly and Nayar, 1990) with a circular area around the

distal pole where the intine is thinner.

Theilade et al. (1993) studied the Zingiber pollen and

reported that the species of said genus possess a spherical

shape (with a cerebroid or reticulate sculpturing) or

ellipsoidal pollen with a diameter ranging 55-85 µm. Pollen

were also reported that no structures indicating the presence

Figure 3 (left)

Observed insect

visitor of E.

dalican. Figure 4 (right)

Observed insect

visitors of

E. philippinensis.

Table 1. Pollen Morphology and Micromorphological Features of E. dalican and E. philippinensis under LM and SEM.

Species Pollen

size

Pollen

shape (hydrated

pollen)

Pollen

shape (dry pollen)

and infolding

Pollen color Pollen

aperture

Pollen

ornamentation

E. dalican 68–75 µm Spheroidal Irregular Greenish–yellow Inaperturate Gemmate

E. philippinensis 60–65 µm Spheroidal Irregular Greenish Inaperturate Psilate



Volume 11 Issue 2 - 2017 | 5

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

agents of reproduction and their features can often be used

to identify a particular taxon (Uno et al., 2001).

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

that pollen remain viable longer at low relative humidity

levels (Stanley & Linskens, 1974). However, recent studies

Figure 5. Pollen of E. dalican showing spheroidal shape (hydrated

pollen) under LM (100x).

Figure 6. Pollen of E. dalican showing its sizes (68-75µm); irregular

shape (dry pollen); inaperturate and gemmate ornamentation under

SEM (1,000x).

Figure 7. Pollen of E. philippinensis showing spheroidal shape

(hydrated pollen) under LM (100x).

Figure 8. Pollen of E. philippinensis showing its sizes (60-

65µm); irregular shape (dry pollen); inaperturate and psilate

ornamentation under SEM (1,000x).



Volume 11 Issue 2 - 2017 | 6

pointed out that pollen of different species need a high level of

relative humidity to germinate as well (Corbet & Plumridge,

1985; Digonnet–Kerhoas & Gay, 1990; Mulugeta et al., 1994;

Loupassaki & Vasilakakis, 1995). The link between pollen

viability and fruit and seed set has been earlier evidenced

(Stone et al., 1995). As to whether this connectivity is applied

to the two Etlingera species studied still requires further

investigation.

Pollen Germination and Pollen Tube Growth. Culture medium

containing a gelling agent (agar or gelatin) has been used

successfully in many species (Jayaprakash & Sarla, 2001;

Kakani et al., 2002; Kozai et al., 2008). In this study, agar was

used instead of gelatin due to the report of Stanley & Liskens

(1974) that there are several advantages of using agar

germination tests such as the ease of taking carbohydrate,

creating stable relative humidity and providing aerobic

conditions. The germination medium used for this study

followed the formulation of Karni & Aloni (2002). This is in

accordance with the suggested reports of Khan and Perveen

(2006) and Imani et al. (2011) that culture medium aside from

having carbohydrates (particularly sucrose) should also

contain germination–stimulating substances such as boric

acid, calcium nitrate, potassium nitrate and magnesium

sulfate wherein all aforesaid substances are the major

components.

E. dalican and E. philippinensis as observed in this study

have similar percentage germination of 0% after 1 day of

incubation. However, for pollen tube growth, the observation

after 2 days of incubation indicates that the first pollen tube

length in E. dalican measures 3.6–4.4 µm on the (2nd day

observation) and 72.3–79.4 µm in the 14th day observation.

On the other hand, pollen tube length in E. philippinensis on

the 2nd day measures 0.8–2.4 µm and 33.6–38.3 µm in the

14th day observation. As shown in Table 2, average increase

in pollen tube length of E. dalican is higher compared to that

of E. philippinensis.

Germination has already been used to indicate viability of

pollen (Schoenike & Stewart, 1963) and a linear relationship

is seen between pollen viability and germination capability in

many fruit species (Stanley & Linskens, 1974). Moreover,

Einhardt et al. (2006) concluded that if pollen seems non–

viable, and if vigorous pollen tubes are present, it is still good

enough to ensure at least a moderate efficient fruit set,

despite the low germination rate. Pollen germination and

tube growth thus emerge as highly dynamic and coordinated

processes, integrating many different signals from the local

environment to regulate growth and development (Rodriguez

-Enriquez et al., 2013). The present data on pollen viability

and pollen tube growth in E. dalican and E. philippinensis

collected in Central Mindanao University appear to provide

additional support to these earlier observations.

CONCLUSIONS AND RECOMMENDATIONS

The distinguishing features of pollen in E. dalican and E.

philippinensis are presented. The pollen of E. dalican are

distinguished from that of E. philippinensis by their sizes and

exine ornamentation in which E. dalican measured 68-75 μm

and has gemmate ornamentation while E. philippinensis

measured 60-65 μm and has psilate ornamentation.

Furthermore, E. dalican pollen viability and pollen tube

growth is higher than that of E. philippinensis.

In view of our results, we recommend further studies on

pollen of other ginger species to be examined/characterized

using SEM in order to delineate species. Implications on the

different stages of germination should also be noted and

described. Moreover, these species should be germinated/

propagated using other germination media and recording the

tube growth as well. More research using molecular and

population genetic studies are needed to investigate the

relationships of the genus Etlingera. Further studies to

establish relationship between pollen viability and

germination with fruit setting in the two species must be

done.

ACKNOWLEDGEMENT

The authors would like to extend their genuine appreciation

and recognition to the following, who one way or another

have contributed to the success of the study: (1) Central

Mindanao University (CMU); (2) CMU Natural Science

Table 2. Two (2) weeks observation for Pollen Tube Growth of E. dalican and E. philippinensis under the light microscope (400x).

Species 2nd day 4th day 6th day 8th day 10th day 12th day 14th day

Average

microns (per

day)

E. dalican 3.6–4.4 µm 7.8–9.2 µm 13.6–

21.6 µm

28.6–33.3

µm

43.4–49.6

µm

63.4–

66.9 µm

72.3–

79.4 µm 17.75 µm

E. philippinensis 0.8–2.4 µm 2.4–4.4 µm 4.8–6

µm

13.6–15.3

µm

22.9–24.1

µm

28.8–

31.4 µm

33.6–

38.3 µm 8.17 µm



Volume 11 Issue 2 - 2017 | 7

Research Center (NSRC)–Plant Tissue and Spore Culture

Laboratory (PTSCL) headed by Dr. Chris Rey C. Lituañas; (3)

CMU NSRC–Tuklas Lunas Development Center (TLDC)/

Molecular Biology and Biotechnology Laboratory (MBBL)

headed by Dr. Reggie Y. Dela Cruz; (4) Center for

Biodiversity Research and Extension in Mindanao (CEBREM)

headed by Dr. Victor B. Amoroso; (5) Hon. Felix G. Mendez

and Mrs. Sarah P. Mendez for the financial support; (6) Asst.

Prof. Majvell Kay G. Odarve-Vequizo of MSU-IIT for

processing the samples for SEM; (7) Ruth C. Alincastre for

helping the authors obtain the SEM results. Sincere gratitude

is also extended to the Association of Systematic Biologists of

the Philippines (ASBP) for the opportunity to publish and

considering this paper as a finalist to the Young Systematic

Biologists Forum (YSBF) during the 2016 ASBP on May 19-

20, 2016 at the University of Santo Tomas (UST), España,

Manila.

LITERATURE CITED

Acma, F.M.,2010. Biosystematics of the genus Amomum

Roxb. (Family Zingiberaceae) in the Philippines. Doctor

of Philosophy (Botany) Dissertation. University of the

Philippines, Los Baños.

Aswani, K., M. Sabu & K.P. Smisha,2013. Reproductive

Biology of Etlingera elatior (Jack.) R.M.Sm. ornamental

torch ginger. Taxonomy division, Department of Botany,

University of Calicut, Kerala - 673 635. India, 3(4): 2231–

4490.

Barrett, S.C.H.,1985. Floral trimorphism and monomorphism

in continental and island populations of Eichhornia

paniculata. Biological Journal of the Linnean Society, 25:

41–60.

Bolat, I. & L. Pirlak,1999. An Investigation on Pollen Viability,

Germination and Tube Growth in Some Stone Fruits.

Turkish Journal of Agriculture and Forestry, 23: 383-388.

Boonmee, A., C. Srisomsad, D. Chokchaichamnamkit, A.

Karnchanatat & P. Sangvanich,2011. A proteomic

analysis of Curcuma comosa Roxb. Rhizome BioMed

Centra: the open access publisher. Proteome Sci;

Accessed: http:www.ncbi.nlm.nih.gov/sites. Accessed on:

November 19, 2014.

Brown, H.D.,1959. Effects of respiratory inhibitors upon nectar

secretion in Antirrhinum. Bulletin of the Torrey Botanical

Club, 86: 290-295.

Chen, J. & N.-H. Xia,2011. Pollen morphology of Chinese

Curcuma L. and Boesenbergia Kuntz (Zingiberaceae):

Taxonomic implications. Flora, 206: 458–467.

Chen, Z.Y.,1989. Evolutionary patterns in cytology and pollen

structure of Asian Zingiberaceae. In: Holm-Nielsen L.B.,

I.C. Nielsen & H. Balslev (eds) Tropical Forests: Botanical

Dynamics, Speciation and Diversity, Academic Press.

Pp.185–91.

Corbet, S.A. & J.R. Plumridge,1985. Hydrodynamics and the

germination of soil-seed rape pollen. Journal of

Agricultural Science, 104: 445-451.

Darjanto & S. Satifah, 1990. Basic Science of Floral Biology

and Artificial Fertilization. Gramedia Press, Jakarta. 156

pp.

Digonnet-Kerhoas, C. & C. Gay,1990. Qualite´ du pollen:

definition et estimation. Bulletin. Societe Botanique

Francaise, 137: 97-100.

Dudash, M.R.,1991. Plant size effects on female and male

function in hermaphroditic Sabatica angularis

(Gentianaceae). Ecology, 72: 1004–1012.

Einhardt, P.M., E.R. Correa & M.C.B. Raseira,2006.

Comparação entre métodos para testar a viabilidade de

pólen de pessegueiro. Revista Brasileira de Fruticultura,

28: 5-7.

Firmage, D.H. & A. Dafni,2001. Field tests for pollen viability:

a comparative approach. Acta Horticulturae, 561:87–94.

Frankel, R. & E. Galun,1977. Pollination Mechanisms,

Reproduction and Plant Breeding.– Springer, New York.

Furness, C.A. & P.J. Rudall,1999. Inaperturate pollen in

monocotyledons. International Journal of Plant Sciences,

160: 395–414.

Imani, A., K. Bargezar, S. Piripireivatlou & H.S.

Masomi,2011. Storage of apple pollen and in vitro

germination. African Journal of Agricultural Research,

Victoria Island, 6: 624–629.

ISTA (International Seed Testing Association),1999.

International rules for seed testing. Seed Science and

Technology, 21: Pp. 288.

Jayaprakash, P. & N. Sarla,2001. Development of an

improved medium for germination of Cajanus cajan (L.)

Mill sp. pollen in vitro. Journal of Experimental Botany, 52:

851–855.

Kakani, V.G., P.V.V. Prasad, P.Q. Craufurd & T.R.

Wheeler,2002. Response of in vitro pollen germination

and pollen tube growth of groundnut (Arachis hypogaea

L.) genotypes to temperature. Plant, Cell & Environment,

25: 1651-1661.

Karni, L. & B. Aloni,2002. Fructokinase and hexokinase from

pollen grains of bell pepper (Capsicum annuum L.):

Possible role in pollen germination under conditions of

high temperature and CO2 enrichment. Annals of Botany,

90: 607-612.

Kelly, J.K., A. Rasch & S. Kalisz,2002. A method to estimate

pollen viability from pollen size variation. American

Journal of Botany, 89: 1021–1023.

Khan, S.A. & A. Perveen,2006. Germination capacity of

stored pollen of Abelmoschus esculentus L. (Malvaceae)

and their maintenance. Pakistan Journal of Botany, 38:

233-236.

Kozai, N., I. Kataoka, O. Chusri, Y. Yonemoto & T.

Ogata,2008. Improvement of artificial medium for passion

fruit pollen germination. Acta Horticulturae, 787: 155–158.



Volume 11 Issue 2 - 2017 | 8

Larsen, K., H. Ibrahim, S.H. Khaw & L.G. Saw,1999. Gingers

of Peninsular Malaysia and Singapore. Natural History

Publications (Borneo). Kota Kinabalu. Pp. 1-30.

Larsen, K. & S.S. Larsen,2006. Gingers of Thailand. Queen

Sirikit Botanical Garden, The Botanic Garden

Organization, Ministry of Natural Resources and

Environment. Pp. 22–127.

Liang, Y.H.,1988. Pollen morphology of the family

Zingiberaceae in China-pollen types and their significance

in the taxonomy. Acta Phytotaxonomica Sinica, 26: 265–

286.

Liang, Y.H.,1990. A study on the pollen wall of the Chinese

Zingiberaceae. Acta Botanica Austro Sinica, 6: 56–65.

Lincoln, R.J., G.A. Boxshall & P.F. Clark,1982. A Dictionary of

Ecology, Evolution and Systematics. Cambridge University

Press. New York.

Loupassaki, M. & M. Vasilakakis,1995. The effect of

temperature and relative humidity on the in vitro

germination of the pollen of avocado. Proceeding of the 3rd

World Avocado Congress: Pp. 42–45. Accessed on http://

www.avocadosource.com/WAC3/WAC3_TOC.htm.

Luza, J.G., V.S. Polito & S.A. Weinbaum,1987. Staminate

bloom date and temperature responses of pollen

germination and tube growth in two walnut (Juglans regia

L.) species. American Journal of Botany, 74: 1898–1903.

McGrath, K.A.,1999. World of Biology. Gale Group. Detroit.

Mangaly, J.K. & J. Nayar,1990. Palynology of South Indian

Zingibearaceae. Botanical Journal of the Linnanaean

Society, 103: 351–366.

Melati, E. R. Palupi & N. Bermawi,2015. Floral biology of

ginger (Zingiber officinale Rosc.). International Journal of

Current Research in Biosciences and Plant Biology, 2(4): 1

-10.

Mulugeta, D., B.D. Maxwell, P.K. Fay & W.E. Dyer,1994.

Kochia (Kochia scoparia) pollen dispersion, viability and

germination. Weed Science, 42: 548-552.

Ozler, H., Z. Kaya & S. Pehlivan,2009. Pollen morphology of

some Centaurea L., Psephellus Cass. and Cyanus Miller

taxa. Acta Biologica Cracoviensia Series Botanica 51/2: 53

–66.

Pacini, E.,1990. Harmomegathic characters of Pteridophyta

spores and Spermatophyta pollen. In: M. Heese and F.

Ehrendorfer (eds.) Morphology, Development and

Systematic Relevance of Pollens and Spores. Plant

Systematics and Evolution. Supplement, 5. Pp. 53–69.

Paoletti, E.,1992. Effects of acidity and detergent on in vitro

pollen germination and tube growth in forest tree species.

Tree Physiology, 10: 357–366.

Parfitt, D.E. & S. Ganeshan,1989. Comparison of procedures

for estimating viability of Prunus pollen. HortScience, 24

(2): 354-356.

Pelser, P.B., J.F. Barcelona & D.L. Nickrent (eds.), 2011. Co’s

Digital Flora of the Philippines. Accessed: http://

www.philippineplants.org. Accessed on: November 20,

2014.

Poulsen, A.D.,2006. Etlingera of Borneo. Natural History

Publications. Kota Kinabalu, Sabah. http://

www.nhpborneo.com/book. Accessed on: November 20,

2014.

Real, L. A. & B.J. Rathcke,1991. Individual variation in nectar

production and its effect on fitness in Kalmia latifolia.

Ecology, 72: 149-155.

Rodriguez-Enriquez, M.J., S. Mehdi, H.G. Dickinson & R.T.

Grant-Downton,2013. A novel method for efficient in vitro

germination and tube growth of Arabidopsis thaliana

pollen. New Phytologist, 97: 668–679.

Saensouk, P., P. ChantaranothaI & P. Theerakulpisut,2009.

Pollen morphology of the genus Cornukaempferia

(Zingiberaceae) in Thailand. Journal of Systematics and

Evolution, 47: 139–143.

Saensouk, P., P. Theerakulpisut, A. Thammathaworn, S.

Saensouk, C. Maknoi and P. Kohkaew,2015. Pollen

morphology of the genus Curcuma (Zingiberaceae) in

Northeastern Thailand. ScienceAsia, 41: 87–92.

Schoenike, R.E. & D.H. Stewart,1963. Fifth year results of

vacuum drying storage and additives on the viability of

some conifer pollen. Forest Science, 9: 96–97.

Simpson, M.,2006. Plant Systematics. Elsivier Academic

Press. New York.

Leong-Skornickova, J. & D. Gallick,2010. The Ginger

Garden. Singapore: National Parks Board Singapore

Botanic Garden. Pp. 1775.

Stanley, R.G. & H.F. Linskens,1974. Pollen Biology,

Biochemistry Management. Springer–Verlag, Berlin.

Steiner, W. & H-R. Gregorius,1998. In vitro pollen

germination experiments in Alnus and their relevance for

mating system analysis. Abteilung fiir F o r s t g e n e t i k

und Forstpflanzenzuchtung, Universitat Gottingen,

Biisgenweg 2, D-37077 Gottingen, Germany. 5(1): 47-

60.

Stone, J.L., J.D. Thomson & S.J. Dent-Acosta,1995.

Assessment of pollen viability in hand p o l l i n a t i o n

experiments: a review. American Journal of Botany,

82:1186-1197.

Theilade, I. & J. Theilade,1996. Ontogeny of pollen grains in

Zingiber spectabile (Zingiberaceae). Grana, 35: 162-

170.

Theilade, I., M.L. Maersk-Moller, J. Theilade & K.

Larsen,1993. Pollen morphology and structure of Zingiber

(Zingiberaceae). Grana, 32: 33s-342.

Tosun, F. & F. Koyuncu,2007. Investigations of suitable

pollinator for 0900 Ziraat sweet cherry c v : p o l l e n

performance tests, germination tests, germination

procedures, in vitro and in vivo pollinations pollinations.

Horticultural Science, 34 (2): 47–53.

Uno, G., K. Sporne & P. Endres,2001. Some observations

http://www.avocadosource.com/WAC3/WAC3_TOC.htm

http://www.avocadosource.com/WAC3/WAC3_TOC.htm

http://www.philippineplants.org

http://www.philippineplants.org

http://www.nhpborneo.com/book

http://www.nhpborneo.com/book



Volume 11 Issue 2 - 2017 | 9

on the evolution of pollen types in dicotyledons. New

Phytologist, 71(1): 181-185.

Willis, J.H.,1999. The role of genes of large effect on

inbreeding depression in Mimulus guttatus. Evolution, 53:

1678–1691.

Comparative Pollen Viability and Pollen Tube Growth of Two ...

Documents

Transcript of Comparative Pollen Viability and Pollen Tube Growth of Two ...