DOI : 10.58993/ijh/2024.81.2.

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Indian J. Hortic. 81(2), June 2024: 135-141

Effect of pollen morphology on hybridization and seed setting in hibiscus

(Hibiscus rosa sinensis)
Praveen Naik K.T. 1, Jayoti Majumder1,*, Tanushree Koley1 and Kunal Adhikary2
1
Department of Floriculture and Landscaping, Bidhan Chandra Krishi Viswavidyalaya, Mohanpur,
Nadia, 741252, West Bengal, India

ABSTRACT
Pollen studies are crucial for successful hybridization in Hibiscus rosa sinensis. At BCKV, West Bengal,
an experiment on pollen morphometry and viability involving 10 hibiscus genotypes was conducted in 2021-
22. Versicolour Pinwheel showed the highest pollen viability (95.12%), followed by Cherry Glow (92.28%).
Three promising pollen parent genotypes, Brilliant, Versicolour Pinwheel, and Cherry Glow, were selected.
Hybridization (line × tester) was performed, revealing Cinnamon Girl’s had high compatibility with all three
pollen donors. Double Peach was compatible with Versicolour Pinwheel and Agni with Cherry Glow, resulting
in desirable seed set. Capsule set varied widely (0.0 - 45.67%) due to genotype compatibility, pollen viability,
pollen tube growth rates, and climatic conditions. This report aids breeders in selecting suitable pollen donor
parents for successful hybridization programmes.
Keywords: Acetocarmine, stigma, capsule, germplasm screening, seed set.

INTRODUCTION morphology in hibiscus, especially in the Indian

The genus Hibiscus, belonging to the family context, there is a focus on introducing new varieties
Malvaceae (Debut et al., 5), boasts numerous without a comprehensive understanding of their
species, around 105 of which are commonly found in pedigree. Therefore, a systematic breeding approach
is required to improve and unveil new varieties.
India (Janakiram and Patil, 9). Among these, Hibiscus
Improvement, domestication, and documentation
rosa sinensis stands out for year-round flowering and
efforts are essential to achieve these goals, which
high nutritional value. Packed with organic acids,
are hindered by insufficient information about pollen
β-carotene, vitamin C, proteins, and sugars, it holds
morphology and breeding techniques. This study aims
aesthetic and medicinal significance (Janakiram and
to fill this gap by investigating pollen morphology and
Patil, 9).
achieving successful crosses between compatible
Hibiscus flowers are typically bisexual and contain
parent varieties of Hibiscus rosa sinensis.
calyx, corolla, androecium, and gynoecium. These
vibrant flowers vary in size and colour, with single MATERIALS AND METHODS
and double varieties. The stigma, where pollen are
The study was conducted at Bidhan Chandra
collected, features five hairy spots on the pistil (Valdoz
Krishi Viswavidyalaya, West Bengal (co-ordinates:
et al., 19): Pollen travels through the style, connecting
22°56′42.88″N 88°32′0.86″E, altitude: 9.75 m), in
the stigma to the ovary. The male part comprises
2021-2022. It aimed to assess the pollen of ten
filament-like structures with anthers, each containing
Hibiscus rosa sinensis genotypes (Fig. 1). Pollen
250 to 500 pollen grains (Salamah et al., 15). Some
screening was done from June to December 2021,
pollen types have spines and mucilaginous substance
and hybridization was carried out from May to August
aiding attachment, while others lack spines (Debut et
2022, covering varied climatic conditions (highest
al., 5). Capsule-shaped fruits with five lobes house
temp: 36.3°C in July, lowest: 11.22°C in February).
the seeds (Valdoz et al., 19).
The region received 140.9 cm of annual rainfall.
The study of pollen development in hibiscus,
Pollens were collected from unopened flower
which is significantly influenced by plant and
buds of the ten hibiscus cultivars (enlisted below),
floral morphology, provides lucid insights into and preserved in Carnoy’s solution (4:1 ratio of
seed development. This process serves as the absolute ethanol and acetic acid). The varieties
initial stage for systematic breeding effort (Debut (Fig. 1) for the study are Brilliant, Versicolour Pin
et al., 5). Despite the limited research on pollen Wheels, Cherry Glow, Double Peach, Cinnamon
2
Department of Horticulture, ITM University, Gwalior, India Girl, Alipore Beauty, Agni, Celia, White, and Shaker
*Corresponding author email: jayotisarkar1@[Link] Bazaar Yellow. After 24 of fixation, buds were

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 Indian Journal of Horticulture, June 2024

Fig. 1. Selected hibiscus genotypes used for the pollen study.

plunged to 70% ethanol and refrigerated (Engin Table 1. Details of parental genotypes used in hybridization.
and Gokbayrak, 6). Anthers were counted with a
Cross Seed parent (♀) Pollen parent (♂)
magnifying lens. For pollen production estimation,
ten anthers were collected from fresh flowers of C1 Agni Brilliant
each variety pre-anther dehiscence and were stored C2 Cinnamon Girl Brilliant
in vials inside desiccators (Shaheen et al., 17). C3 Double Peach Brilliant
After anther dehiscence, 1.5 ml of 1.0% Teepol-
water mixture was added. Glycerine was added C4 Agni Versicolor Pinwheel
to even pollen dispersion. A small drop of this C5 Cinnamon Girl Versicolor Pinwheel
suspension was added to the counting chambers C6 Double Peach Versicolor Pinwheel
of a Spencer Bright line Haemocytometer. Pollen C7 Agni Cherry Glow
characteristics such as shape, area, diameter, and
spine length were recorded for all varieties. Pollen C8 Cinnamon Girl Cherry Glow
grains were observed under a Photo-emission C9 Double Peach Cherry Glow
electric microscope (40x) (Labomed®, USA) with
safranin stain (Shaheen et al., 17; Arora, 2). Pollen
pollination. Hand emasculation and bagging of
fertility was assessed using the acetocarmine test
(Xiong et al., 33) and the stigmatic surface-based mature buds in chosen seed parents were done
pollen germination method (Khanduri et al., 11). in the evening before pollination (Stetter et al.,
Viable grains, displaying normal morphology and 18). Pollination occurred in the next morning by
well-staining, were counted from ten microscopic transferring male-parent pollen to emasculated
fields. For in-vivo pollen germination on stigma, flower stigmas using a brush. Pollinated flowers were
emasculated buds were bagged overnight and then bagged and tagged with parent names and pollination
pollinated with fresh pollen of the same variety. dates (Anuragi, 1). After pollination, pollen tube
Styles were fixed, preserved, boiled, stained, and growth on the seed parent’s stigma was observed.
examined using a photoelectric microscope (40x) Flowers were collected after 30 min, and pistils were
after gentle squashing. isolated and preserved in acetic alcohol overnight.
For hybridization, three pollen parent varieties Styles were then separated, treated with lactophenol
were selected (Table 1). Balloon-staged buds were and stained with 1% acid fuchsin. Observations
collected, anthers detached, and placed on butter were made using a 40x magnification photoelectric
paper for dehiscence in partial shade. Pollen from microscope (Labomed ®, USA), with pollen tube
selected donor varieties was stored a day before images captured using Diwinter Calliper Pro software.

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Various parameters such as capsule development anthers per flower may have contributed to lower
time, cross-specific capsule set, maturation duration, counts per anther, as observed in previous studies
seeds per capsule, seed set percentage, and 10-seed (Salamah et al., 15). Regarding pollen area (Fig. 3),
weight were recorded for analysis. Versicolour Pinwheel had the maximum (2.03 mm²),
The data collected from this study underwent with Alipore Beauty closely behind (1.98 mm²), while
statistical analysis using Randomized Block Design Agni exhibited the minimum (1.12 mm²) (Table 2).
with three replicates, each consisting of three plants. These findings differ from pollen size data in other
The statistical analysis of the observed traits was studies for H. rosa sinensis (Naggar, 12), suggesting
performed utilizing both MS Excel and the OPSTAT study-dependent variation likely due to different
software. The study included correlation analyses and hibiscus genotypes being analyzed. Table 2 reveals
multiple linear regression (MLR) analyses to assess significant pollen diameter variation in the hibiscus
the connection between hibiscus varieties and pollen genotypes (Fig. 3). Versicolour Pinwheel had the
morphological traits. largest diameter (0.51 mm), along with Alipore Beauty
(0.51 mm), and White Satin the smallest (0.30 mm).
RESULTS AND DISCUSSION
Earlier reports on H. rosa sinensis also highlighted
Pollination is crucial for genetic transmission in diameter differences (Naggar, 12), typically ranging
regenerative plants. This study aims to link pollen from 124 to 165 μm.
morphology with hybridization compatibility. Pollen Versicolour Pinwheel had the most pollen spines
grain morphology of Hibiscus showed significant (61.50), followed by White Satin (57.33), while Celia
diversity in size, diameter, and spine presence had the fewest (5.0). The longest spines were in
(Table 2, Fig. 2). Anther count varied among
Versicolour Pinwheel (0.06 mm), similar with Brilliant
hibiscus genotypes (Table 2), ranging from 82.67
(0.06 mm), with Celia and Agni having the shortest
to 123.67, likely influenced by genetic differences
(0.02 mm each). Taxonomically significant variations
and climate (Bell, 3; Arora, 2). Pollen shape across
in Malvaceae pollen grain spines have been noted
hibiscus genotypes generally followed a spherical,
34-colporate, and polyforate structure per Erdtmans’ (Naggar, 12).
classification (Erdtmans, 7). All 10 varieties exhibited Table 2 indicates that the Versicolour Pinwheel
spined pollen (Fig. 2), consistent with findings by had the highest Cherry Glow (92.28%), which was
Debut et al. (5). significantly higher than the others. Conversely,
Pollen grain count per anther varied from 62.5 to Agni displayed the lowest viability (62.85%). This
236.00 among the varieties (Table 2, Fig. 3). Cherry aligns with findings by Khanduri et al. (11). Table
Glow had the highest count (236.00), followed closely 2 and Fig. 3 illustrate pollen growth on stigmatic
by Shaker Bazaar Yellow, while Agni showed the surfaces among selected varieties. Versicolour Pin
lowest count (62.5). Larger pollen size and more Wheel had the highest growth (9.24 mm), followed

Table 2. Pollen morphology of 10 Hibiscus rosa sinensis genotypes.

Genotypes Anther Pollens/ Pollen Pollen Pollen Spine/ Spine Pollen growth Pollen
flower anther shape area dia. pollen length on stigma viability
(mm²) (mm) (mm) (mm) (%)
Brilliant 84.67 114.00 Spherical 1.43 0.42 54.00 0.06 6.34 85.01(67.21)
Versicolour Pinwheel 108.67 175.00 Spherical 2.03 0.51 61.50 0.06 9.24 95.12(77.21)
Cherry Glow 104.33 236.00 Spherical 1.56 0.41 50.67 0.03 9.04 92.28(73.76)
Double Peach 109.33 174.00 Spherical 1.76 0.49 14.33 0.04 8.69 76.19(60.73)
Cinnamon Girl 113.33 200.00 Spherical 1.41 0.43 52.00 0.05 6.73 71.22(57.54)
Alipore Beauty 113.67 167.00 Spherical 1.98 0.51 45.67 0.06 6.62 78.28(62.17)
Agni 82.67 62.50 Spherical 1.12 0.37 26.00 0.02 5.17 62.85(52.83)
Celia 123.67 134.00 Spherical 1.65 0.42 5.00 0.02 5.09 81.17(64.23)
White Satin 84.67 145.00 Spherical 1.47 0.30 57.33 0.05 5.47 81.42(64.45+)
Shaker Bazaar Yellow 101.33 211.00 Spherical 1.39 0.46 51.00 0.04 7.72 90.63(72.15-)
CD0.05 11.531 44.795 0.436 0.072 10.821 0.014 1.518 9.139
SE m± 3.881 15.077 0.147 0.024 3.642 0.005 0.511 3.076

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 Indian Journal of Horticulture, June 2024

Fig. 2. Pollen micro-morphology in 10 varieties of hibiscus

Fig. 3. Observations during hybridization and post-hybridization in Hibiscus.

by Double Peach (9.05 mm), while White Satin Cherry Glow, Versicolour Pinwheel, and Brilliant in
had the lowest (5.09 mm). Differences in pollen separate clusters. Positive correlations are denoted
germination and tube growth on stigma among as red, and negative correlations are denoted as
hibiscus varieties have been discussed by Anuragi blue. Versicolour Pinwheel shows strong positive
(1). Patil et al. (13) observed high pollen germination correlations with various pollen traits, as does
rates (80–90%) in Abelmoschus spp. shortly after Brilliant. Shaker Bazaar Yellow, conversely, exhibits
anther opening, suggesting barriers rather than negative correlations with pollen traits. Similar
pollen vigour may cause germination failure. As in the analyses were performed by Kabre et al. (10) for
methodology, a correlation study assessed variable hibiscus diversity analysis using heatmaps.
relationships via a correlation matrix (Fig. 4). The Hybridization blends traits from different
heatmap simplifies these correlations visually, aiding germplasms to create desired offspring, which
in identifying key variables and avoiding duplication. may become novel varieties or foundational lines
The dendrogram highlights three clusters, with for future breeding. In hibiscus, flowers are known

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growth variation to flower morphology in Hibiscus

trionum, while Arora (2) attributed differences to
pollen competitiveness in varied environments. Patil
et al. (13) found normal pollen tube growth in both
directions of the Abelmoschus esculentus × A. caillei
cross, implying genetic enhancement potential.
In the crosses, C5 (Versicolour Pinwheel ×
Cinnamon Girl) had the highest capsule formation at
46.2% (Table 3, Fig. 3). However, some crosses like
Agni × Brilliant (C1), Double Peach × Brilliant (C3),
Agni × Versicolour Pinwheel (C4), and Double Peach
× Cherry Glow (C9) didn’t form capsules (Table 4).
Lack of seed development might be due to issues
like endosperm degeneration (Shaheen et al., 17)
or early style and stigma abscission (Fakir et al., 8).
Capsule formation varied from 4.67 to 6.67
days after pollination. Certain crosses like Agni ×
Brilliant (C1), Double Peach × Brilliant (C3), Agni
× Versicolour Pinwheel (C4), and Double Peach ×
Fig. 4. Heatmap depicting correlations between expression Cherry Glow (C9) didn’t result in capsule formation.
patterns of ten selected hibiscus genotypes and The longest time was in Cinnamon Girl × Cherry
pollen morphology. Glow (C8) at 6.67 days, followed by Double Peach
× Versicolour Pinwheel (C6) at 6.33 days. Capsule
for their hypoglycemic (Janakiram and Patil, 9) maturation duration aligned with findings by Fakir et
and hypotensive effects (Bell, 3). Hibiscus rosa- al. (8). C7 (Agni × Cherry Glow) displayed the highest
sinensis exhibits rich vegetative and floral traits, 10-seed weight at 2.25 mg. At the same time, some
enhancing genetic diversity through self-crossing crosses produced no seeds Agni × Brilliant (C1),
and crossbreeding with other varieties (Kabre et al., Double Peach × Brilliant (C3), Agni × Versicolour
10). Pollen tube growth on the stigmatic surface of Pinwheel (C4), and Double Peach × Cherry Glow
seed parents in one-line crosses showed significant (C9). Physiological maturity, marked by maximum
variation (Table 3, Fig. 3). Notably, Cherry Glow dry weight in seeds, is highlighted by Raifa et al. (14)
pollen on Cinnamon Girl (C8) led to rapid tube in hibiscus.
growth (6.78 mm), while the slowest growth was This study offers insights into pollen viability,
in Double Peach with Cherry Glow and Brilliant aiding hibiscus breeding. Versicolour Pinwheel,
pollens (0.68 mm and 0.67 mm, respectively) in C9 Brilliant, and Cherry Glow were found to be potential
and C3. In Hibiscus, Chachalis et al. (4) linked tube pollen donors (male parent candidates). The study

Table 3. Observations recorded after hybridization in Hibiscus genotypes.

Crosses (♀ × ♂) Pollen tube growth Capsule set Days to Wt. of

on stigma (mm) (%) capsule set 10-seeds (mg)
C1 (Agni × Brilliant) 1.67 0.00 (0) 0.00 0.00
C2 (Cinnamon Girl × Brilliant) 4.00 38.67 (38.35+) 4.67 1.33
C3 (Double Peach × Brilliant) 0.67 0.00 (0) 0.00 0.00
C4 (Agni × Versicolour Pinwheel) 2.00 0.00 (0) 0.00 0.00
C5 (Cinnamon Girl × Versicolor Pinwheel) 5.33 46.20 (42.82) 5.33 2.07
C6 (Double Peach × Versicolour Pinwheel) 4.83 34.33 (35.85) 6.33 1.00
C7 (Agni × Cherry Glow) 3.50 29.33 (32.77) 5.33 2.25
C8 (Cinnamon Girl × Cherry Glow) 6.78 36.67 (37.23) 6.67 1.68
C9 (Double Peach × Cherry Glow) 0.67 0.00 (0) 0.00 0.00
CD0.05 1.34 3.405 1.562 0.613
[Link] ± 0.447 1.136 0.521 0.204

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 Indian Journal of Horticulture, June 2024

guides breeders and cultivators toward successful calyx protein content in Hibiscus sabdariffa L.
hibiscus choices, contributing to breeding strategies var. sabdariffa. J. Agrofor. Environ. 6: 1-4.
and future efforts in the field.
9. Janakiram, T. and Patil, M.S. 2017. Breeding in
AUTHORS’ CONTRIBUTION Hibiscus: A review. Indian J. Agric. Sci. 87: 159-
Conceptualization (JM), Methodology (PKTN, 66.
JM), Investigation (PKTN), Data curation and Formal 10. Kabre, N.V., Sawadogo, B., Kiebre, M., Kiebre,
analysis (PKTN, JM), Writing original draft (PKTN),
Z., Nanema, R.K. and Bationo-Kando, P. 2019.
Resources, Software, Validation (TK and KA),
Estimates of phenotypic diversity and genetic
Writing, review and editing (PKTN, TK and KA).
parameters of Hibiscus cannabinus L. grown in
DECLARATION Burkina Faso. Int. J. Biol. Chem. Sci. 13: 1903-
The authors declare that they do not have any 17.
conflict of interest. 11. Khanduri, P., Chaudhary, A., Uniyal, P.L.
ACKNOWLEDGEMENT and Tandon, R. 2014. Reproductive biology
of Willisia arekaliana (Podostemaceae), a
The authors are grateful to the Hon’ble Vice freshwater endemic species of India. Aquat.
Chancellor of BCKV, West Bengal, for providing a farm Bot. 119: 57-65.
and laboratory facilities.
12. Naggar, S.M. 2004. Pollen morphology of Egyptian
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Received : March, 2024; Revised : May, 2024;

Accepted : June, 2024

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