Folia

Folia Hort. 35(1) (2023): 123–133

Horticulturae
Published by the Polish Society
DOI: 10.2478/fhort-2023-0009
for Horticultural Science since 1989

ORIGINAL ARTICLE Open access https://sciendo.com/journal/FHORT

Study on pollen viability and stigma receptivity

throughout the flowering period in the selected taxa
of the Gesneriaceae family
Françoise Jean Baptiste, Jong-Yi Fang*

Department of Tropical Agriculture and International Cooperation,

National Pingtung University of Science and Technology, Pingtung 91201, Taiwan

ABSTRACT
Plants in the Gesneriaceae family are appreciated for their decorative leaves and flowers, ease of cultivation, and shade
tolerance. Sexual hybridisation has long been carried out for producing novel hybrids. However, scientific knowledge is
lacking on the correct timing of pollination in this family. This study was conducted to elucidate the optimal timing for
pollination by screening pollen viability and stigma receptivity throughout the flowering period in eight gesneriad taxa.
Pollen viability was evaluated by the in vitro germination test and stigma receptivity was based on stigma morphology
and pistil length. The flowering duration varied from 10 days in Alsobia to 18 days in Streptocarpus. While the pollens of
Episcia, Kohleria, Saintpaulia, Sinningia, and Smithiantha had totally lost viability 2–5 days before the flowers withered,
a slightly contrasting situation was observed in the cases of Alsobia, Deinostigma, and Streptocarpus, where a small
portion of pollen grains remained viable towards the end of the flowering period. The highest pollen germination rate was
recorded from 1 day in Alsobia, Episcia, and Kohleria, to 9 days in Deinostigma. The reduction in pollen germination
was rapid in Alsobia, Saintpaulia, and Smithiantha, and moderate in the remaining taxa. The greatest pollen tube growth
occurred at 1–3 days after flower opening and decreased rapidly in all the taxa tested. The longest pistil of 2.03–3.50 cm was
observed at 3–8 days of anthesis depending on the plant tested. The findings in this study suggest that higher pollination
success may be achieved using pollen grains of newly opened flowers and stigmas of mature flowers in this family.

Keywords: gesneriad, pistil length, pollen germination, pollen tube length, pollination timing

INTRODUCTION
Gesneriaceae is an angiosperm family of the order importance and are frequently used for potted flowers
Lamiales with large-scale species richness. There or in landscaping. Breeding of gesneriads has long been
are roughly 160 genera and more than 3,300 species carried out to meet the market demand for novel hybrids,
in the family, including perennial herbs, shrubs, and and was mostly achieved via sexual hybridisation.
small trees, which are mostly distributed in tropical Pollen grains are sexual reproductive units in plants
and subtropical zones around the globe (Möller and and bearers of male genetic material (Halbritter et al.,
Clark, 2013; Weber et al., 2013). Many are of economic 2018). Understanding the variables that influence pollen

*Corresponding author.
e-mail: [email protected] (Jong-Yi Fang).

Open Access. © 2023 Baptiste and Fang, published by Sciendo. This work is licensed under the Creative Commons Attribution alone 3.0 License.
124 Pollen viability and stigma receptivity in the Gesneriaceae family

viability is critical in genetic breeding programmes MATERIALS AND METHODS

(Dane et al., 2004). Pollen viability encompasses several
features of pollen performance, including germination, Plant material
pollen tube elongation, and fertilisation abilities (Dafni Eight selective taxa of the Gesneriaceae family were
and Firmage, 2000). Staining, in vitro germination, and used in this study, which included two species namely
seed set as well as semi-in situ germination on the excised Deinostigma eberhardtii (Pellegr.) D.J. Middleton &
stigma are common methods for determining pollen H.J. Atkins and Streptocarpus saxorum Engl., and six
viability. Although staining techniques can distinguish horticultural hybrids such as Alsobia ‘Cygnet’, Episcia
between abortive and non-aborted fresh pollen, they ‘Thad’s Yellow Bird’, Kohleria ‘Brimstone’, Saintpaulia
frequently fail to distinguish between different levels of ‘Lyon’s Party Parasol’, Sinningia ‘HCY’s Peach
pollen viability (Ge et al., 2011). In the last two methods, Fragrance’, and Smithiantha ‘An’s Hanabi’ (Figure 1).
contrasting dye is used to detect pollen tube growth Each species or hybrid was propagated to increase the
towards or on stigmas, and the results are thought to number of individuals for the study. Plants of Alsobia
provide the most precise seed set estimates (Dionne ‘Cygnet’, Deinostigma eberhardtii, Episcia ‘Thad’s
and Spicer, 1958). Nevertheless, incompatibilities, post- Yellow Bird’, Saintpaulia ‘Lyon’s Party Parasol’, and
fertilisation obstacles, and restricted measurability may Streptocarpus saxorum were propagated by stem
limit the accuracy of these assays (Dafni and Firmage, or leaf cuttings to yield ca. 10 individuals, whereas
2000). In vitro pollen germination test has been adopted plants of Kohleria ‘Brimstone’, Sinningia ‘HCY’s
to evaluate pollen viability in many plants by tracking Peach Fragrance’, and Smithiantha ‘An’s Hanabi’
the percentage of pollen germination and the length of were propagated by rhizomes or tubers to produce
the pollen tube over time (Shivanna and Mohan Ram, three to six individuals. All the plants were grown in
1993). Pollen grains that fail to germinate frequently have a 2:1:1 substrate combination of peat moss, perlite, and
weak pollen tube growth and are therefore ineffective vermiculite inside 5″ pots. Each month, the plants were
during sexual fertilisation (Lin et al., 2017). Different fertilised with Hi-Control fertiliser (14N-11P-13K). The
medium formulations are required for different plant greenhouse was maintained at 26 ± 4 °C and ≈75–80%
species to germinate their pollen grains (Baloch et al., relative humidity without supplemental lighting. Water
2001). Among the medium supplements, sucrose, boric was applied directly to the substrate twice a week
acid, and calcium nitrate are among the most commonly without wetting the leaves of the plants.
used in various studies (Tushabe and Rosbakh, 2021).
Stigma is a glandular organ whose key functions
Evaluation of pollen viability
are to receive, recognise, and provide the pollen grains A preliminary trial was conducted on the in vitro pollen
with germination substrate (Hiscock and Allen, 2008). germination of Smithiantha ‘An’s Hanabi’ to evaluate the
At various phases of flower development, stigma requirement of sucrose, boric acid (H3BO3), and calcium
receptivity determines the rate of pollination success nitrate [Ca(NO3)2 ∙ 4H2O] for pollen germination. A
(Galen et al., 1987). Stigma receptivity can be determined total of 12 media containing 10%, 20%, or 30% sucrose,
using morphology, seed set ability, and cytochemical 50 mg ∙ L –1 or 100 mg ∙ L –1 boric acid, and 200 mg ∙ L –1 or
testing for the existence of enzyme activity (Dumas et 400 mg ∙ L –1 calcium nitrate were tested, together with
al., 1984). Stigma characterisation has been extensively the water agar (WG) medium devoid of sucrose, boric
illustrated, both anatomically and biochemically (Heslop- acid, and calcium nitrate as the control. Pollen grains
Harrison and Shivanna, 1977). According to the pattern were collected from three flowers of mixed ages and of
of secretion, two types of stigmas are recognised in different flowering individuals. The pollen grains were
histology: wet and dry stigmas (Heslop-Harrison and suspended in distilled water and 10 drops of 1 μL each
Shivanna, 1977). The wet stigma’s secretion is exposed of pollen suspension were inoculated on the surface of
to the environment, but the dry stigma contains a 10 mL pollen germination medium contained in a 6-cm
proteinaceous pellicle overlying a discontinuous cuticle Petri dish. The Petri dishes were incubated at 25 ± 2 °C
that keeps secretions within the stigma (Lersten, 2004). in the dark for 24 h. Pollen germination and pollen tube
Research on pollination ecology and pollen growth were assessed under a light microscope with 10x
morphology has been conducted in some gesneriads magnification. The pollen grains were considered as
over the last 70 years, but mostly focussed on native germinated if the length of the pollen tube was equal to
and endangered species (Erdtman, 1952; Melhelm and or greater than the grain diameter (Kakani et al., 2005).
Mauro, 1973; Fritze and Williams, 1988; Luegmayr, The numbers of germinated and non-germinated pollen
1993; Gao et al., 2006; Lazarevic et al., 2013). Aside grains were counted in 10 fields (one field per drop) in
from the cited articles, pollen studies in commercially each Petri dish. The percentage of pollen germination
important gesneriads are scarce. Therefore, this study was evaluated using Eq. (1) (Kakani et al., 2005).
aimed to provide scientific information on pollen Pollen tube length was analysed with ImageJ software,
viability and stigma receptivity throughout anthesis in version 1.52v (National Institutes of Health, Bethesda,
eight popular gesneriad species in order to determine MD, USA) and Java 1.8.0_112. The mean pollen tube
the best pollination timing for this family. length (in centimetres) was estimated as the average of
Baptiste and Fang 125

Figure 1. The eight gesneriad taxa used in this study. (A) Alsobia ‘Cygnet’. (B) Deinostigma eberhardtii. (C) Episcia
‘Thad’s Yellow Bird’. (D) Kohleria ‘Brimstone’. (E) Saintpaulia ‘Lyon’s Party Parasol’. (F) Sinningia ‘HCY’s Peach
Fragrance’. (G) Smithiantha ‘An’s Hanabi’. (H) Streptocarpus saxorum. Bars = 1 cm.

30 randomly selected pollen tubes from each of the 10 day to eliminate variation caused by non-experimental
fields and calculated using Eq. (2). treatment conditions. Graphs were made using Microsoft
Excel software (Microsoft Corporation, Redmond,
Pollen germination (%) Washington, USA). Data means were subjected to one-
Germinated pollen grains per field way ANOVA for analysis and compared with Duncan’s
= × 100  (1)
multiple range test using Statistical Package for Social
Total pollen grains per field
Sciences (SPSS) for Windows, version 26 (International
Business Machines Corporation, Armonk, New York,
Pollen tube length (cm) USA).
Total length of 30 selected pollen tubes  (2)
=
30 RESULTS
Subsequently, the in vitro pollen germination test was
Evaluation of pollen viability
narrowed down to four germination media containing
10% sucrose, 50 mg ∙ L –1 or 100 mg ∙ L –1 boric acid, and In the preliminary trial on pollen germination of
200 mg ∙ L –1 or 400 mg ∙ L –1 calcium nitrate, together Smithiantha ‘An’s Hanabi’, it was found that the highest
with the control (CK) medium devoid of boric acid and pollen germination rates were recorded on A1–A4
calcium nitrate on eight gesneriad taxa. media containing 10% sucrose (54%–92%), followed
Pollen viability was evaluated by determining pollen by B1–B4 media containing 20% sucrose (10%–13%)
germination and pollen tube length of eight gesneriad and WG medium (15%) (Figure 2). None of the pollen
taxa at each day of the flowering period. Pollen grains grains germinated on C1–C4 media were enriched with
were inoculated on the optimised in vitro germination 30% sucrose. The pollen tube lengths were also greater
medium. The pollen germination percentages and pollen on A1–A4 media with 10% sucrose (1.47–1.64 cm)
tube lengths were evaluated as described previously. compared to B1–B4 media with 20% sucrose (0.59–
0.62 cm) and WG medium (0.86 cm). Based on this,
Evaluation of stigma receptivity the in vitro pollen germination test was performed only
The morphology of the stigma was monitored daily on media containing 10% sucrose in the subsequent
and the day at which the stigma opened was recorded experiment involving eight gesneriad taxa.
for the plant analysed. Evaluation of pistil length was When testing the eight gesneriad taxa, higher
conducted each day, from 7:00 a.m. to 10:00 a.m. Three germination percentages were observed in A1 and A2
flowers of each plant were recorded for the pistil length media than in A3 and A4 media for D. eberhardtii,
by using a ruler. The length of the pistil was measured Episcia ‘Thad’s Yellow Bird’, Kohleria ‘Brimstone’,
from the top of stigma to the base of the ovary. Saintpaulia ‘Lyon’s Party Parasol’, Sinningia ‘HCY’s
Peach Fragrance’, and Smithiantha ‘An’s Hanabi’
Experimental design and data analysis (Figure 3). In Alsobia ‘Cygnet’, the media A1, A2, and
All experiments were arranged in a completely A3 produced higher pollen germination percentages
randomised design with three replicates per treatment compared to the medium A4. The pollen germination
and/or taxon. All trials were conducted on the same percentages were equivalent across all four media
126 Pollen viability and stigma receptivity in the Gesneriaceae family

Figure 2. Pollen germination percentages and pollen tube lengths of Smithiantha ‘An’s Hanabi’ in different germination
media. Medium specification (WG: 0C-0B-0C; A1: 10S-50B-200C; A2: 10S-50B-400C; A3: 10S-100B-200C; A4:
10S-100B-400C; B1: 20S-50B-200C; B2: 20S-50B-400C; B3: 20S-100B-200C; B4: 20S-100B-400C; C1: 30S-50B-
200C; C2: 30S-50B-400C; C3: 30S-100B-200C; C4: 30S-100B-400C; S: sucrose; B: boric acid; C: calcium nitrate).
Means with identical lowercase letters are not significantly different (Duncan test, *p < 0.05). WG, water agar.

A B

C D

E F

G H

Figure 3. Pollen germination percentages of eight gesneriad taxa in different germination media. (A) Alsobia ‘Cygnet’,
(B) Deinostigma eberhardtii, (C) Episcia ‘Thad’s Yellow Bird’, (D) Kohleria ‘Brimstone’, (E) Saintpaulia ‘Lyon’s
Party Parasol’, (F) Sinningia ‘HCY’s Peach Fragrance’, (G) Streptocarpus saxorum, and (H) Smithiantha ‘An’s
Hanabi’. Medium specification (CK: 10S-0B-0C; A1: 10S-50B-200C; A2: 10S-50B-400C; A3: 10S-100B-200C;
A4: 10S-100B-400C; S: sucrose; B: boric acid; C: calcium nitrate). Means with identical lowercase letters are not
significantly different (Duncan test, *p < 0.05).

in S. saxorum. Regarding pollen tube length, longer media. In Alsobia ‘Cygnet’ and Kohleria ‘Brimstone’,
pollen tubes were found in the A1 medium than in the media A1, A2, and A3 exhibited greater pollen
the A2, A3, and A4 media for Saintpaulia ‘Lyon’s tube lengths than the A4 medium. There was no
Party Parasol’, Sinningia ‘HCY’s Peach Fragrance’, significant difference in pollen tube lengths among
and Smithiantha ‘An’s Hanabi’ (Figure 4). In the four media in Episcia ‘Thad’s Yellow Bird’. In all
D. eberhardtii and S. saxorum, the A1 and A2 media the plants tested, the medium CK yielded the lowest
produced longer pollen tubes than the A3 and A4 pollen germination percentages and pollen tube
Baptiste and Fang 127

A B

C D

E F

G H

Figure 4. Pollen tube lengths of eight gesneriad taxa in different germination media. (A) Alsobia ‘Cygnet’,
(B) Deinostigma eberhardtii, (C) Episcia ‘Thad’s Yellow Bird’, (D) Kohleria ‘Brimstone’, (E) Saintpaulia ‘Lyon’s
Party Parasol’, (F) Sinningia ‘HCY’s Peach Fragrance’, (G) Streptocarpus saxorum, and (H) Smithiantha ‘An’s
Hanabi’. Medium specification (CK: 10S-0B-0C; A1: 10S-50B-200C; A2: 10S-50B-400C; A3: 10S-100B-200C;
A4: 10S-100B-400C; S: sucrose; B: boric acid; C: calcium nitrate). Means with a common lowercase letter are not
significantly different (Duncan test, *p < 0.05).

lengths. The medium A1 showing the highest pollen (94%). High percentages of pollen germination were
germination percentages (from 87% in D. eberhardtii maintained for approximately 3 days following anthesis
to 95% in Episcia ‘Thad’s Yellow Bird’ and in Saintpaulia ‘Lyon’s Party Parasol’ (88%–90%),
Smithiantha ‘An’s Hanabi’) and pollen tube lengths S. saxorum (92%–93%), and Smithiantha ‘An’s Hanabi’
(from 2.60 cm in Kohleria ‘Brimstone’ to 4.96 cm in (93%–95%). In Sinningia ‘HCY’s Peach Fragrance’,
Alsobia ‘Cygnet’) was selected for screening pollen maximum pollen germination rates were recorded for 4
viability throughout the flowering period. consecutive days (88%–92%). Deinostigma eberhardtii
The percentages of pollen germination at each day was the only plant that had high pollen germination
during the flowering period of eight gesneriad taxa are percentages (92%–89%) lasting for 9 days of the
shown in Figure 5. The flowering duration varied from flowering period. The reduction of the germination
10 days in Alsobia ‘Cygnet’ to 18 days in S. saxorum. potential of pollen grains was more rapid in Alsobia
In general, the pollen germination percentages were ‘Cygnet’, Saintpaulia ‘Lyon’s Party Parasol’, and
the highest at anthesis, and then decreased as the flower Smithiantha ‘An’s Hanabi’, and was more moderate in
aged. While in Episcia ‘Thad’s Yellow Bird’, Kohleria the remaining taxa.
‘Brimstone’, Saintpaulia ‘Lyon’s Party Parasol’, The pollen tube lengths of eight gesneriad taxa
Sinningia ‘HCY’s Peach Fragrance’, and Smithiantha during each day of the flowering period are shown in
‘An’s Hanabi’ the pollen grains had completely lost Figure 6. It can be seen that the pollen tube length was
viability 2–5 days before the flowers withered, a slightly greatly affected by the time of pollen collection. The
contrasting situation was observed in the case of Alsobia longest pollen tubes were found at the first day of flower
‘Cygnet’, D. eberhardtii, and S. saxorum, where a small opening in D. eberhardtii (3.48 cm), Episcia ‘Thad’s
portion of pollen grains remained viable by the end of Yellow Bird’ (4.20 cm), Saintpaulia ‘Lyon’s Party
the flowering period. Parasol’ (3.35 cm), Sinningia ‘HCY’s Peach Fragrance’
The speed at which the germination rates of pollen (4.37 cm), S. saxorum (3.88 cm), and Smithiantha ‘An’s
grains decreased varied from plant to plant. High pollen Hanabi’ (4.61 cm). In Kohleria ‘Brimstone’, the longest
germination percentages were recorded for only 1 day of pollen tubes were recorded for 2 days after flower
the flowering period in Alsobia ‘Cygnet’ (89%), Episcia opening (4.04–4.14 cm). The only plant with high pollen
‘Thad’s Yellow Bird’ (97%), and Kohleria ‘Brimstone’ tube lengths that lasted for 3 days after flower opening
128 Pollen viability and stigma receptivity in the Gesneriaceae family

Figure 5. Daily pollen germination percentages throughout the entire flowering period of eight gesneriad taxa.
(A) Alsobia ‘Cygnet’, (B) Deinostigma eberhardtii, (C) Episcia ‘Thad’s Yellow Bird’, (D) Kohleria ‘Brimstone’,
(E) Saintpaulia ‘Lyon’s Party Parasol’, (F) Sinningia ‘HCY’s Peach Fragrance’, (G) Streptocarpus saxorum, and
(H) Smithiantha ‘An’s Hanabi’. Means with a common lowercase letter are not significantly different (Duncan
test, *p < 0.05).

Figure 6. Daily pollen tube lengths throughout the flowering period of eight gesneriad taxa. (A) Alsobia ‘Cygnet’, (B)
Deinostigma eberhardtii, (C) Episcia ‘Thad’s Yellow Bird’, (D) Kohleria ‘Brimstone’, (E) Saintpaulia ‘Lyon’s Party
Parasol’, (F) Sinningia ‘HCY’s Peach Fragrance’, (G) Streptocarpus saxorum, and (H) Smithiantha ‘An’s Hanabi’.
Means with a common lowercase letter are not significantly different (Duncan test, *p < 0.05).
Baptiste and Fang 129

was Alsobia ‘Cygnet’ (3.52–3.75 cm). In all genera, the differently enlarged and showed a small opening. At
pollen tube lengths decreased rapidly throughout the the completion of 8–10 days after anthesis, the stigma
flowering period. cavity was clearly visible to the naked eye for the
various species tested (Figure 7). At 11–13 days, the
Evaluation of stigma receptivity papillae were collapsed at stigmatic tissue in an older
Morphological observations of eight gesneriad taxa stigma, and the cells missed turgidity. After 14 days,
revealed wet stigmas in S. saxorum and Saintpaulia the stigmatoid tissue was completely degenerated.
‘Lyon’s Party Parasol’, and dry stigmas in the For the wet stigma, secretion was already observable
remaining taxa. The dry stigmas had a hydrated at Day 1 of flowering. The secretion was produced
cuticular layer or pellicle at maturity but no free- by the stigmatoid tissue located under the receptive
flowing secretion. The dry stigma is clear white and papillae surface. The deficiency of papillae turgidity
slightly larger than the style at anthesis. The stigmatic is preceded by the beginning of exudate production.
surface was closed with unicellular papilla during the The stigmas and papillae were turgid by the fourth day
first days of flowering. At Day 4, the stigma lobe was after flowering (Figure 7).

Figure 7. Stigma morphology of eight gesneriad taxa. Dry stigmas of (A) Alsobia ‘Cygnet’, (B) Deinostigma
eberhardtii, (C) Episcia ‘Thad’s Yellow Bird’, (D) Kohleria ‘Brimstone’, (E) Sinningia ‘Mark Twain’, and (F)
Smithiantha ‘An’s Hanabi’ at Day 8–10 of flowering. Wet stigmas of (G) Streptocarpus saxorum and (H) Saintpaulia
‘Lyon’s Party Parasol’ at Day 1 of flowering. Bars = 1 cm.

Figure 8. Daily pistil lengths throughout the flowering period of eight gesneriad taxa. (A) Alsobia ‘Cygnet’,
(B) Deinostigma eberhardtii, (C) Episcia ‘Thad’s Yellow Bird’, (D) Kohleria ‘Brimstone’, (E) Saintpaulia ‘Lyon’s
Party Parasol’, (F) Sinningia ‘Mark Twain’, (G) Streptocarpus saxorum, and (H) Smithiantha ‘An’s Hanabi’. Means
with a common lowercase letter are not significantly different (Duncan test, *p < 0.05).
130 Pollen viability and stigma receptivity in the Gesneriaceae family

The length of the pistils in all the species tested that boric acid at 50 mg ∙ L–1 or 100 mg ∙ L–1 enhanced
increased progressively from Day 1 until it reached pollen germination and pollen tube growth compared
the maximum at Day 3 for Sinningia ‘HCY’s Peach to media devoid of boric acid. The concentrations of
Fragrance’, Day 4 for Alsobia ‘Cygnet’ and D. eberhardtii, boric acid used in this study were coherent with those
Day 5 for Episcia ‘Thad’s Yellow Bird’, Day 6 for used by Wang et al. (2003) and Acar et al. (2010), where
S. saxorum, Day 7 for Smithiantha ‘An’s Hanabi’, and 100 mg ∙ L–1 boric acid enabled germination of pollen
Day 8 for Kohleria ‘Brimstone’ and Saintpaulia ‘Lyon’s grains and growth of pollen tubes in Pistacia, while
Party Parasol’ (Figure 8). Thereafter, the pistil length higher concentrations (2,000 mg ∙ L–1) hindered pollen
remained constant till the end of the flowering period. germination and growth of pollen tubes. Calcium plays
a role in cation balance and is required for the formation
of pollen tubes (Jayaprakash, 2018). Extracellular
DISCUSSION calcium has been shown to be essential for pollen tip
Sexual hybridisation can enrich the genetic diversity of development (Steinhorst and Kudla, 2013). Dickinson
flowering plants by creating new hybrids with combined (1967) showed that calcium regulates the permeability
parental characteristics. The timing of pollination is vital of pollen tube membranes. The absence of calcium in
for successful hybridisation due to possible disparity the medium increases membrane permeability, leading
between male and female reproductive maturity. Pollen to loss of internal metabolites (Dabgar and Jain,
viability and stigma receptivity are critical factors 2001). This could explain why the pollens germinated
affecting the timing of pollination since they occur only poorly in CK medium in this study. Our results did not
for a limited time and at a specific period during flower reveal any difference in pollen germination and pollen
growth (Heslop-Harrison, 2000). tube growth between 200 mg ∙ L–1 and 400 mg ∙ L–1
The in vitro pollen germination test has been calcium nitrate. These concentrations are comparable
considered an appropriate approach to evaluate to those used by Steinhorst and Kudla (2013), who
pollen viability (Sulusoglu and Cavusoglu, 2014; stated that the dicotyledonous plants required between
Gandadikusumah et al., 2017; Damayanti et al., 300 mg ∙ L–1 and 400 mg ∙ L–1 calcium nitrate for pollen
2021). However, the in vitro conditions for pollen germination and pollen tube growth. Also, Tuinstra and
germination vary widely among species, especially in Wedel (2000) showed that 300 mg ∙ L–1 calcium nitrate
terms of the medium requirement (Ahmad et al., 2012; was adequate for pollen germination as well as pollen
Mudi and Mondal, 2014). Medium components such tube development in sorghum. In this study, the medium
as boric acid, calcium nitrate, potassium nitrate, and A1, which contained a combination of 10% sucrose,
magnesium sulphate have all been shown to have a 50 mg ∙ L–1 boric acid, and 200 mg ∙ L–1 calcium nitrate,
great influence on pollen germination and pollen tube proved to be the best for pollen germination and pollen
growth (Sahar and Spiegel-Roy, 1984). In this study, tube growth in all taxa tested; accordingly, the same was
the in vitro pollen germination of different gesneriad selected for the evaluation of pollen viability throughout
species was conducted on media containing various the flowering period.
amounts of sucrose, boric acid, and calcium nitrate. Understanding when pollen grains possess high
Supplementation with an adequate amount of sucrose viability during the flowering period is essential prior
to the medium is crucial for pollen germination and to any cross-pollination attempt. When hybridisation
pollen tube development in gesneriads. In this study, works are undertaken, it is important that the pollen
higher germination percentages and growth of pollen grains are collected at an appropriate stage of maturation
tubes were obtained with 10% sucrose compared to to ensure pollination success. In this study, the pollen
20% sucrose, whereas no germination was found with grains were screened for viability at each day of the
30% sucrose. This result is in agreement with that of flowering period, which lasted from 10 days to 18 days
Fragallah et al. (2018), who found that both germination depending on the plant tested. The results indicated that
and pollen tube growth of Chinese fir was best in media the pollen grains were fully mature at the time of flower
containing less than 15% sucrose. According to Lin opening and showed maximum germination and pollen
et al. (2017), both pollen germination and pollen tube tube growth at anthesis. Thereafter, the pollen grains
elongation are hindered by high sugar concentrations. gradually decreased in viability with advancing flower
This could be due to the imbalanced osmotic pressure age, although the decline rate varied between examined
in the pollen grain, which inhibited pollen germination plants. According to Ferri et al. (2008), the quick decline
when pollen was immersed in media with high sucrose in pollen viability could be related to the lack of gut water
concentrations. Boron is well known for its function permeability, leading to a decrease in pollen hydration.
in pollen germination and pollen tube development by This pattern was also observed in Olea europaea
reducing pollen bursting (Patel and Mankad, 2014). ‘Picual’ (Aguilera and Valenzuela, 2013). In this study,
Pollen germination is generally hampered by a lack of the best time to collect pollen grains depended on the
boric acid, which has been demonstrated in a variety rate of pollen viability degradation, which was largely
of plant species (Ahmad et al., 2012; Liu et al., 2013; species-dependent. Pollen grains of Alsobia ‘Cygnet’,
Kavand et al., 2014). The results of this study showed Episcia ‘Thad’s Yellow Bird’, and Kohleria ‘Brismtone’
Baptiste and Fang 131

were best collected on Day 1 of flower opening, whereas different gesneriad taxa, suggesting that such variation
pollen grains of D. eberhardtii could be collected at any may have taxonomic value. Our result is in agreement
time between Days 1 and 9 during flowering. The other with those of Guo and Wang (2014), who reported that
gesneriad taxa showed intermediate duration of pollen the pistils of Oreocharis pumila reached maximum
viability where pollen grains could be collected between length at Day 3 of anthesis (ca. 18 mm) and lasted till
Days 1 and 3. The same results were observed in Day 6, a period during which 100% stigma receptivity
Aeschynanthus tricolor whereby the highest germination was recorded. It can be suggested that the pistil length
percentage was discovered on the day of anthesis (96%), can be used as an indicator of stigma receptivity and
whereas pollen collected 5 days following anthesis had the flowers have the greatest reproductive potential
the lowest germination (5.6%) (Gandadikusumah et al., towards the mid and end of the flowering period in
2017). It was suggested by Shivanna and Rangaswamy generiads.
(1992) that genotypic differences among plants are one
of the variables affecting pollen viability. In this study, CONCLUSIONS
only few taxa had viable pollen grains at the end of
The in vitro pollen germination of gesneriads performed
the flowering period, and most of the taxa lost pollen
the best on a medium supplemented with 10% sucrose,
viability completely a few days before the end of the
50 mg ∙ L –1 boric acid, and 200 mg ∙ L –1 calcium nitrate.
flowering period.
Screening of pollen viability throughout the flowering
In plants, stigma types have been classified as either
period revealed that pollen grains showed the highest
dry or wet, with the former having a proteinaceous
germination percentages and pollen tube lengths at
extracellular pellicle layer overlying the stigma surface
the first day of flower opening in all the gesneriad taxa
and generally lacking any free-flowing secretion, whereas
tested. Old stigma seemed to be more receptive than
the latter is characterised by a fluid secretion at the
young stigma and pollen grains should be transferred to
receptive state (Heslop-Harrison and Shivanna, 1977).
3–8 days-old stigmas depending on the plant considered.
In gesneriads, the stigmas can be of wet or dry type, and
The findings in this study suggest that higher success
1–2 lobed, with usually unicellular papillae (Watson and
rates of pollination may be obtained using pollen grains
Dallwitz, 1991). In this study, the gynoecium of Alsobia
collected from newly opened flowers and stigmas of
‘Cygnet’, Episcia ‘Thad’s Yellow Bird’, D. eberhardtii,
mature flowers in this family.
Smithinatha ‘An’s Hanabi’, Sinningia ‘Mark Twain’,
and Kohleria ‘Brimstone’ were revealed as having dry
stigmas, whereas S. saxorum and Saintpaulia ‘Lyon’s FUNDING
Party Parasol’ presented wet stigmas. Wet stigmas No funding for this study.
have resulted in the development of exudates high in
proteins, carbohydrates, free amino acids, and lipids, AUTHOR CONTRIBUTIONS
which created an ideal environment for hydration,
germination, and early tube formation of the pollen. J.Y.F. designed and supervised the experiments,
In Sanango racemosum, the stigmatic cells secrete a performed the critical revision of the figures and
viscous substance underneath the cuticle, and the pollen manuscript text. F.J.B. performed the experiments,
tubes penetrate the cuticle and grow towards the base of figures plotting, statistical analysis of data and drafted
the papilla in the space thus generated (Maldonado and the manuscript.
Otegui, 1997).
At certain stages of the floral life cycle, stigma CONFLICT OF INTEREST
receptivity may have a considerable impact on the The authors declare no conflict of interest.
percentage of pollination success (Galen et al., 1987).
As a result, testing the timing and duration of stigma
receptivity should be included in breeding trials or
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