Research Journal of Applied Biotechnology (RJAB) (2025)Vol.

11 (1):Pp 53-64

Evaluation of Pollen Vitality in Different Sugarcane Genotypes Utilizing

Various Techniques and Time Intervals
Wafaa E. Grad1, Shimaa H. Elhoffey1, Amr M. El-Sheikh1, Mohamed A. Ghonema1

1
Department of Breeding & Genetics, Sugar Crops Research Institute (SCRI), Agricultural Research Center (ARC),
Egypt.

DOI: 10.21608/rjab.2025.346852.1060
Received: 24 December 2024 Accepted: 11 January 2025

*Corresponding author1: Dr. Wafaa E. Grad

E-mail: [email protected]

ABSTRACT
This study was conducted to contrast two staining techniques for determining pollen viability in sugarcane.
At three diverse times (6:30, 8:00, and 9:30 a.m.), pollen from seven sugarcane genotypes was gathered,
and its vitality was assessed using Lactophenol blue and Iodine stains. For every genotype, using a drop
of the respective stain, three others were crushed on a glass slide. Pollen viability percentages were
determined using an optical microscope. Variance analysis was used to analyze the factors (staining
methods, genotypes, and times) and their interaction, and the means were compared. Lactophenol blue
staining proved more sensitive than Iodine staining in detecting the natural decline in pollen viability in
sugarcane. This study underscores the effectiveness of Lactophenol as a reliable medium for assessing
pollen viability, offering a more accurate representation of the physiological state of pollen grains at
various times (6:30, 8:00, and 9:30 a.m.) for different sugarcane genotypes. The genotypes exhibited
variations in pollen grain viability percentage during time intervals. Genotypes G2004/27 and EH78-26-
11 showed superior pollen grain vitality in both staining methods at early time, whereas genotype
G2003/47recorded the lowest values at the later time.
Key words: sugarcane, pollen, viability, stain, time.

1. INTRODUCTION grains determined by staining and direct

Hybridization processes are commonplace counting methods. High pollen viability in the
for sugarcane breeders (Saccharum spp.), these male parent is essential for successful crossing.
procedures usually include crossing of Prior research has demonstrated that flowering
extremely fertile sugarcane genotypes (as is a complex process comprising various
male) with male sterile kinds (as female) and developmental stages, each characterized by
then cultivating the fuzz, which are the actual distinct environmental and physiological
sugarcane seeds. The degree of anther requirements. Environmental conditions
dehiscence seen via a hand lens may reveal a encompass factors including diurnal
flowering variety's sexuality, but microscopic temperatures, day lengths, elevation, and the
examination of pollen grains provides a more requirements for temperature and moisture
precise way to identify flowering sugarcane (Coleman, 1963; Van Breemen et al., 1963;
types as male or female (Olaoye et al., 2014). Clements and Awada, 1967; Gosnell, 1973;
Research on the induction of flowering has Moore, 1987; Moore and Nuss, 1987 and
always been a primary focus of sugarcane Araldi et al., 2010) rainfall patterns and
breeding programs. Pollen viability is crucial distribution Olaoye (1996) sub-optimal
for hybridization or crossing, typically photoperiods (Nayamuth et al., 2003 and
expressed as the percentage of viable pollen Berding, 2005) increasing atmospheric CO2

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 Wafaa E. Grad et al.

levels (Rosenzweig et al., 1995) and pollution classification of inflorescence as either pollen
levels. Pollen longevity is greatly influenced by donor or receptor is complemented by the
environmental factors, particularly temperature importance of pollen viability for effective
and relative humidity. Depending on the cross-pollination.
species and conditions, viability can diminish Pollen quality is vital for breeders. The aim
within minutes to hours. Once shed, pollen is of this study is to evaluate pollen viability
highly susceptible to dry air and high among seven sugarcane (Saccharum spp.)
temperatures, which further shorten its viability genotypes. The viability was assessed using
and lifespan. Consequently, pollen has a two different stains, Iodine and Lactophenol
limited lifespan, referred to as the "viability Blue, at three distinct times: 6:30 a.m., 8:00
window." Notably, pollen from the Poaceae a.m., and 9:30 a.m.
family is especially vulnerable and has a
notably short lifespan (Barnabas and Kovacs, 2. MATERIALS AND METHODS
1997). This study was conducted during the March
However, in the genetic enhancement of 2022 growing season at the El-Sabahia Station
sugarcane, cross-breeding is limited by the lack in Alexandria, Sugar Crops Research Institute
of flowering synchrony between parent plants. (SCRI), Agricultural Research Center (ARC),
The primary methods to address this issue Egypt (31° 22' N, 29.94° E). Flowering
include staggered planting of accessions, induction occurred during the optimal cycle
employing darkrooms with controlled when daylight hours gradually decreased from
photoperiods, and the application of flowering 12:00 to 11:30 between late September and
inducers or inhibitors (Araldi et al., 2010). mid-October, aligning with favorable
Viable pollen grains are crucial for species temperature and humidity conditions, as shown
dispersal, adaptability, and the survival of in Figure (1).
subsequent plant generations. They are also 2.1. Source of genetic materials
vital for targeted plant breeding, leading to crop Pollen grains were collected from seven
improvement. Pollen viability encompasses synchronized genotypes (Table1 and Figure2)
various aspects of pollen performance, that flowered at the same age under natural
including stainability, germinability, and conditions in late November 2022.
fertilization ability (Dafni and Firmage, 2000).
Moreover, the advancement of dependable 2.2. Pollen grain characterization
techniques for assessing pollen's functional Inflorescences were collected from three
quality aids in tracking pollen vitality during stalks in each plot to evaluate pollen viability at
storage, genetic research, pollen-stigma three distinct times: 6:30 am, 8:00 am, and 9:30
interaction studies, crop development and am. This assessment utilized a pair of staining
breeding, as well as incompatibility and techniques, Iodine and Lactophenol blue. The
fertility investigations, as noted by (Melekber chosen times correspond with the typical
Sulusoglu and Aysun Cavusoglu, 2014). schedules of sugarcane breeding programs,
It is essential to understand the ability of the which commence operations around sunrise.
inflorescence to produce pollen for sugarcane The experiment was conducted in three
breeding programs. In a cross between two repetitions, each involving an inoculated slide.
genotypes, one genotype's inflorescence Pollen grains from each slide were examined
functions as the pollen donor (male), while the under an optical microscope (PB/OPTI1,
other acts as the pollen receptor (female) (Bull England) using 10X and 40X objectives. The
and Glasziou 1975; McIntyre and Jackson 2001 viability of pollen was assessed using Iodine
and Cheavegatti-Gianotto et al., 2011). The and Lacto-phenol (cotton blue) staining

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Research Journal of Applied Biotechnology (RJAB) (2025)Vol. 11 (1):Pp 53-64

methods, following the protocols of (Radford et pollen grains were further categorized into
al., 1974; Machado Jr, 1987 and Asghari, sulcate (Figure 3b) (with numerous pores
2000). For each genotype, three mature others visible under a light microscope) or colpate
had been crushed on a glass slide before (Figure 3c) (with two apertures evident after
staining with a few drops of each stain (Iodine viability testing). This classification aligns with
0.1 N and Lactophenol blue) and sealed with a the findings of Olaoye et al. (2014), who
cover slip to prevent displacement of the pollen categorized pollen morphology of genotypes as
grains. With Iodine staining, viable pollen either sulcate or colpate using Lactophenol
grains appeared brownish, while non-viable stain blue.
ones were yellowish. In the lacto-phenol The study demonstrated a highly significant
staining method, viable pollen turned blue, impact on all assessed factors (Genotype, Time,
whereas non-viable pollen remained staining technique) and their interactions, with
transparent. By using an optical microscope the exception of the interaction between Time
(PB/OPTI1, England) and the scanning and Staining method. (Table 2).
technique, the percentage of viable pollen for Figure (4a) illustrates marked disparities in
each staining procedure was determined until pollen grain viability across the studied
100 pollen grains per slide were evaluated. genotypes. The highest viability was noted in
Ph8013 and EH 78-26-11, succeeded by G.T.
2.3. Statistical analysis 54-9 and G2004/27, while G2006/41 and F161
The effects of staining procedures, exhibited reduced viability. On the other hand,
genotypes, timing, and their interactions were The genotype G2003/47 showed the lowest
assessed by analysis of variance and the F-test viability. A significant discrepancy was noted
(P<0.01). The total viable pollen percentage between the two staining methods; as
was converted to angle values in degrees ARC- illustrated in Figure (4b), the Lactophenol
Sin (Evwin et al., 1966). Mean comparisons staining method demonstrated superior values
were conducted using the LSD test at a 5% of pollen viability compared to the Iodine
significance level (Waller and Duncan, 1969) staining method. Additionally, the three
with Minitab® 21.4.2 software. distinct time points demonstrated notable
3. RESULTS AND DISCUSSION differences in pollen viability, with the earliest
The examination of pollen grains from time point (6:30 a.m.) displaying the highest
different genotypes, gathered at various times viability and the latest time point (9:30 a.m.)
and subjected to multiple staining techniques, showing the lowest percentage of viable pollen,
revealed that they could be distinguished as as referenced in Figure (4c).
viable or unviable based on their structure and The interaction between the two staining
features (Figure, 3). Viable pollen grains were methods and genotypes resulted in highly
large, perfectly round and darkly colored, significant values (Figure 5a). The Lactophenol
indicating fertility, whereas unviable pollen stain showed higher pollen viability than the
grains were transparent, hollow and lacked Iodine stain for genotypes Ph 8013, GT 54-9,
color indicating infertility. and G2003/47. Conversely, it showed lower
Pollen grains that stained dark brown were values compared to Iodine for F161 and
considered alive, in contrast to the yellowish G2004/27 genotypes, while it yielded identical
ones deemed unviable by the Iodine staining values for EH 78-26-11 and G2006/41
method (Figure 3a). Additionally, viable pollen genotypes. Compared to Iodine staining,
grains were round and absorbed the blue stain Lactophenol blue showed greater sensitivity in
(Lactophenol or cotton blue), while unviable identifying alterations in pollen viability that
ones remained transparent (Figure 3d). Viable naturally occur in sugarcane genotypes.

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 Wafaa E. Grad et al.

However, the Iodine staining method pollen (King 1960 and Rodriguez-Riano and
demonstrated higher stability and simplicity for Dafni 2000). Conversely, the Lactophenol blue
classifying inflorescence at all evaluated times staining method binds to the cytoplasm of live
(6:30, 8:00, and 9:30 a.m.) likened to pollen, which does not guarantee germination
Lactophenol. Figure (5b) demonstrated a (Nepi and Franchi 2000). Nonetheless, this
highly significant interaction between the three stain indicated a decrease in pollen viability
times and seven genotypes, with the earliest over time (h), likely due to changes in the
time showing the highest viability values and cytoplasm associated with loss of viability.
the latest time showing the lowest for various This decline was particularly evident,
genotypes. especially in GT 54-9 and G 2004/27 genotypes
A negligible difference was observed that exhibited initially high viability,
between the two staining methods for pollen suggesting that even robust pollen can be
grains collected at each of the three times susceptible to environmental stressors over
(Figure 5c). time. The Lactophenol blue staining method
Table 3 and Figure 6 demonstrate that the not only allowed for the visualization of live
performance varied among different genotypes, pollen but also facilitated a deeper
with G2004/27 and EH 78-26-11 showing the understanding of the physiological changes
highest pollen viability across the staining occurring within the pollen grains (Figure7).
techniques at early time (6:30 a.m.), whereas Kılıç et al. (2024) utilized five chemical
G2003/47 showed the lowest viability. When staining methods, Iodine-potassium iodide,
comparing staining methods at each evaluation 2,3,5-triphenyl tetrazolium chloride,
time, differences in pollen viability were noted Lactophenol cotton blue, safranin, and
between genotypes at 6:30, 8:00, and 9:30 a.m., acetocarmine to evaluate pollen viability. They
for both Iodine and Lactophenol blue staining observed that different chemical techniques
methods. Notably, at 8:00 a.m. and 9:30 a.m., had different efficacy depending on species and
there was a marked decrease in pollen viability variation, which causes significant differences
for each genotype when assessed with either in outcomes even for the same species or
staining method, as indicated in (Table 3 and variety. While other techniques could stain
Figure 6). pollen that has already diminished in viability,
Except for the G2003/47 genotype, which others might stain pollen before maturity.
showed no variance in pollen viability at the This study suggests that the decline in pollen
later times due of its very low pollen viability viability was primarily due to reduced air
at 6:30 a.m., the Lactophenol blue staining moisture throughout the day, rather than
method was the only one to reveal variations in temperature, which ranged from 22°C at 6:30
pollen viability over time among the evaluated am to 24°C at 9:30 am in November. Low
genotypes. By tracking the decrease in pollen humidity likely caused desiccation, negatively
viability over time, the Lactophenol blue affecting pollen hydration and reducing
staining technique proved more sensitive. successful germination, which is essential for
The sensitivity of the Iodine staining method fertilization in flowering plants. Notably, the
differed from that of the Lactophenol blue stable temperature during this period indicates
method. The Iodine staining consistently that moisture content, rather than heat, is
indicated similar pollen viability across time crucial for pollen health. These findings
points, likely due to differences in the staining highlight the importance of monitoring
mechanisms. Iodine staining relies primarily on environmental factors, especially humidity, to
starch, which is predominantly found in viable gain insight into plant reproductive success.
pollen grains but can also be present in aborted Sufficient moisture is essential for initiation

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and growth, as well as for timing emergence, temperatures affect sensitive reproductive traits
anthesis, and regulating seed set. Flower like pollen viability and to anticipate potential
opening and anthesis are influenced by relative reductions. Additionally, the impact of
humidity, since both processes often transpire temperature increases on pollen
few hours before to daylight, when the plant is thermotolerance should be evaluated along
well hydrated and relative humidity is high. with other environmental factors, such as
Anthesis occurs when the relative humidity humidity.
decreases near sunrise (de Calvino, 1925; To improve crossbreeding, it's crucial to
Mclntosh, 1930 and Dutt et al., 1938). consider pollen viability, as neglecting it can
Sugarcane pollen viability is short-lived, result in misclassifying inflorescences as male
significantly affected by humidity and or female, leading to increased self-fertilization
temperature, with a half-life of just 12 minutes and the production of unviable seeds, which is
after dispersion (Sartoris 1942; Scarpari and undesirable in sugarcane breeding programs.
Beauclair, 2008). Higher humidity levels The categorization of sugarcane inflorescences
increase pollen longevity, whereas in as male or female is affected by regional
conditions of rapid desiccation, pollen has a climate, which may vary owing to climate
half-life of merely 20 to 30 minutes (Moore, change. Olaoye et al. (2010) indicated that
1976). Low humidities at anthesis leads to poor flowering behavior and the sexuality of
seed set (Nuss, 1979). In temperate South sugarcane inflorescences whether male or
Africa, pollen fertility has been shown to be female alter in response to climatic changes.
limited at temperatures below 21°C (Berding, Finally, the study found that the
1981). Due to the short lifespan of sugarcane Lactophenol staining intensity decreased over
pollen grains, common in many Poaceae family time, indicating a gradual loss of cytoplasmic
plants (Hanna and Towill, 1995), it is integrity, which is crucial for pollen
recommended to conduct pollen tests at 5:00 functionality. These results underscore the
a.m. Currently, the plants are turgid, and the importance of timely pollen collection and
anthers are more fully exposed, resulting in application, particularly in breeding programs
greater pollen viability (Moore, 1987). After where pollen viability is essential for successful
this period, survival becomes difficult due to fertilization. The findings of the current study
water loss and the maintenance of dehydration highlight Lactophenol efficacy as a reliable
in natural conditions (Nepi and Pacini, 1993 medium for assessing pollen viability,
and Lisci et al., 1994). It is to begin breeding providing a more accurate representation of the
programs early, before advisable sunrise, as the pollen grains' physiological state. In contrast,
high number of inflorescences to evaluate the conventional Iodine staining method can
makes it challenging to limit the pollen yield inconsistent results, as it depends on the
viability test to a few hours. In Alexandria, presence of starch, which does not necessarily
Egypt, favorable temperature and humidity reflect pollen's overall viability. Consequently,
conditions have been conducive to flower these findings suggest that future research on
growth, especially during the optimal cycle pollen viability should include Lactophenol
when daylight hours gradually reduced from staining to enhance the reliability of results and
12:00 to 11:30 between late September and to obtain a better understanding of the
mid-October (Ghonema, 2017). Iovane et al., reproductive capabilities of the plant species
(2022) found that pollen viability significantly being investigated. Moreover, to ensure the
declines when exposed to high humidity and vitality and longevity of pollen grains across all
temperatures. Moreover, in light of climate genotypes, it is advisable to initiate the
change, it is essential to assess how rising hybridization process early under favorable

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Table (1): Geographical origins of tested cane materials

No Genotypes Origin
1 G.T. 54-9 Giza, Taiwan
2 Ph 8013 Philippines
3 G2006/41 Egypt, Giza
4 F161 Taiwan
5 G2003/47 Egypt, Giza
6 G2004/27 Egypt, Giza
7 EH 78-26-11 Egypt, Hawamdeia

Table (2): Variance analysis of pollen viability in seven sugarcane genotypes at three times (6:30,
8:00 and 9:30 a.m.) using two staining methods (Iodine and Lactophenol blue)
Source of variation Degree of Freedom Mean Square F. Value
Hours 2 2572.66 167.85***
Genotypes 6 702.42 45.83***
Staining method 1 616.06 40.19***
Hours * genotypes 12 121.06 7.90***
Hours * staining method 2 6.07 0.40 ns
Genotype * staining method 6 465.11 30.35***
Hours * genotype * staining method 12 68.12 4.44***
Error 126 15.33
C. V. (%) 25.43

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Table (3): Mean and t-test results for pollen viability of seven sugarcane genotypes at three times
(6:30, 8:00 and 9:30 a.m.) using two staining methods (Iodine and Lactophenol blue)
Pollen viability Pollen viability
Time Genotype
Iodine (%) Lactophenol (%)
GT54-9 43.2875 d e f 58.4725 a
Ph8013 38.4000 f g h i 58.2175 a
G2004/27 52.1800 b 51.7075 b
6.30 EH78-26-11 51.5975 b 50.6275 b c
G2006/41 44.5875 d e 39.5100 e f g h
F161 41.6300 d e f g 38.1625 f g h i
G2003/47 29.8975 k l m n 36.9825 g h i
GT54-9 33.9125 i j k l 55.1475 ab
Ph8013 37.4825 g h i 53.2550 ab
G2004/27 50.4100 b c 35.3775 h i j
8.00 EH78-26-11 45.6500 c d 45.5250 c d
G2006/41 39.9600 e f g h 34.9450 h i j k
F161 41.7475 d e f g 38.3675 f g h i
G2003/47 28.9775 l m n 39.1875 e f g h i
GT54-9 27.4750 m n 30.3350 j k l mn
Ph8013 37.6925 g h i 51.2825 b
G2004/27 31.1800 j k l m 25.0375 n o
9.30 EH78-26-11 37.1100 g h i 40.8575 d e f g
G2006/41 27.9475 m n 34.5800 h i j k
F161 30.0550 j k l mn 27.4275 m n
G2003/47 21.4000 o 28.0025 m n

80
70
60
50
Average of
40 Temperture
30 °C

20 Average of
Humidity
10
0

Figure (1): Average of temperature (0C) and Humidity for the evaluation periods.

61
 Wafaa E. Grad et al.

Figure (2): Saccharum spp hyprid inflorescences and pollen harvesting (a), panicle
collected (b), panicle with production and exposition of anthers and
average portion (c) and harvesting of pollen with brushes (d).

Figure (3): Pollen morphology in sugarcane genotypes: (a) Iodine-stained pollen

grains at 40X and 100X, (b) sulcate viable pollen grains stained with lacto-
phenol, (c) colpate viable pollen grains, and (d) both viable and unviable
pollen grains.

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Research Journal of Applied Biotechnology (RJAB) (2025)Vol. 11 (1):Pp 53-64

a b c

Figure (4): Mean effects for pollen viability within genotypes (a), staining methods
(b) and different times (c).

b c

Figure (5): Mean effects for pollen viability of interaction between genotypes & stain
method (a), genotypes & Times (b) and stain method & times (c).

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 Wafaa E. Grad et al.

60
50
40
30
20
10
0

6.30 am I 6.30 am L 8.00 am I 8.00 am L 9.30 am I 9.30 am L

Figure (6): Comparisons between pollen viability of seven sugarcane genotypes at

three different times (6.30 a.m., 8.00 a.m. and 9.30 a.m.) obtained by two
staining methods (Iodine and Lactophenol blue).

a b c

d e f

Figure (7): Different absorption of Lactophenol blue during three times intervals for
two genotypes G 2004/27 (a: 6.30, b: 8.00, c: 9.30 a.m.) and GT 54 – 9
(d: 6.30, e: 8.00, f: 9.30 a.m.).

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