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Footnote to the table: Values in bold in Table 3 indicate the best results for viable pollen stained with 2,3,5-triphenyltetrazolium chloride (TTC).
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Original	Article
Increasing pollination possibilities in Paspalum species: in vitro and in
vivo viability of cryopreserved pollen to address flowering asynchrony
Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other
rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and
applicable law.
Naiana Barbosa Dinato ✉
Email : naiana.dinato@gmail.com
Bianca Baccili Zanotto Vigna
Email : bianca.vigna@embrapa.br
Frederico de Pina Matta
Email : frederico.matta@embrapa.br
Alessandra Pereira Fávero
Email : alessandra.favero@embrapa.br
Affiliationids : Aff1, Correspondingaffiliationid : Aff1
Affiliationids : Aff2
Affiliationids : Aff2
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Aff1 Center for Biological and Health Sciences, Federal University of São Carlos, Washington Luís, Km 235, São Carlos, SP, 13565-905,
Brazil
Aff2 Embrapa Southeastern Livestock, Brazilian Agricultural Research Corporation, Rodovia Washington Luís, Km 234, CP 339, São
Carlos, SP, 13560-970, Brazil
Received: 21 June 2024 / Accepted: 23 October 2024
Abstract
Paspalum is a vital forage and turf grass in tropical and subtropical regions, AQ1 yet its breeding programs face challenges due to the
lack of natural flowering synchronization between some parent species. AQ2 Pollen cryopreservation offers a potential solution to this
issue. AQ3 This study aimed to adapt a cryopreservation protocol for the pollen of P. atratum, P. malacophyllum, AQ4 and P. regnellii,
and to evaluate the viability of cryopreserved pollen grains (CPG) for hybridization purposes. AQ5 Two dehydrating agents (LiCl and
silica gel) were tested for different durations (30, 60, and 120 min) alongside a non-dehydration treatment. The effectiveness of
cryopreservation was assessed over multiple time points (1, 10, 30, 90, 180, 270, and 365 days) with freshly harvested grains as
controls. Pollen viability was determined using 0.25% 2,3,5-triphenyltetrazolium chloride staining. Viability of CPG ranged from 40.67
to 80.67% across treatments. Optimal dehydration involved LiCl for 30 min and silica gel for 120 min, achieving an average viability
of 66% after 12 months, comparable to fresh pollen. In vivo germination tests confirmed successful pollen tube germination with the
combinations P. urvillei × P. malacophyllum; P. urvillei × P. regnellii and hybrid (P. plicatulum 4PT × P. guenoarum cv. Azulão) × P.
atratum, although pollen tubes did not reach the micropyle in some crosses. This study established effective pollen cryopreservation
protocols for P. atratum and P. malacophyllum, facilitating in vivo germination and enhancing the potential for hybridization in
Paspalum breeding programs, thereby addressing flowering asynchrony and broadening crossing opportunities within the genus.
Keywords
Germplasm
Cryopreservation
Grasses
Interspecific hybridization
Introduction
The genus Paspalum L. is prominent within the Poaceae family in the Americas, with around 350 species distributed across tropical and
temperate regions globally (Morrone et al. 2012 ). Over 200 of these species are found in Brazil (Valls et al. 2024 ), showing great forage
potential and adapting to various environments. The rich genetic diversity and remarkable adaptability drive research in genetic
improvement, expanding the use of Paspalum in pastures (Gouvea et al. 2020 ; Batista and Godoy 1993 ) and lawns (Souza et al. 2020 ;
Matta et al. 2023 ).
Apomixis, an asexual reproductive mechanism present in approximately 15% of angiosperm families, is prevalent in Paspalum, with most
accessions being tetraploid (Asker and Jerling 1992 ). Although some species present diploid sexual cytotypes and even tetraploid sexual
cytotypes (Ortiz et al. 2013 ), apomixis limits genetic recombination, hindering the introduction of new traits through conventional
crossbreeding.
The agronomic importance of the Paspalum species is well known, mainly as forage (Matta et al. 2023 ); nevertheless, some species with
important traits for the breeding programs (BP) present flowering asynchrony, which also poses a challenge to crossbreeding and the
introduction of new traits. Although flowering physiology is influenced by factors such as hormones, temperature, and photoperiod (Lee
and Lee 2010 ), manipulating these factors to synchronize flowering remains a challenge.
An effective strategy to overcome flowering asynchrony is the preservation of pollen grains at extremely low temperatures.
Cryopreservation in liquid nitrogen (−196 °C) is considered the most promising method for long-term pollen storage, maintaining viability
for extended periods (Kartha 1985 ). From a theoretical standpoint, it is believed that the physiological metabolism of biomaterials is
extremely slow at low temperatures and nearly halted in liquid nitrogen, leading to a state of non-cellular division (Kartha 1985 ; Bajaj
1995 ). The viability of biomaterials would be preserved indefinitely as long as their structure remains intact, allowing them to resume
activity after thawing (Snyder and Clausen 1974 ; Medeiros and Cavallari 1992 ; Benson et al. 1998 ; Carvalho and Vidal 2003 ). The most
common method for pollen cryopreservation involves direct immersion in liquid nitrogen after prior dehydration. Lyophilization (Akihama
et al. 1980 ) and the use of desiccants such as silica gel (Ahlgren and Ahlgren 1978 ; Pereira et al. 2002 ; Winston and Bates 1960 ) or
saturated salt solutions (Ferreira et al. 2007 ) are frequently employed techniques for dehydrating pollen before freezing. Yateset al. (1991 )
indicate that a water content between 7 and 20% is ideal for long-term preservation, regardless of the temperature used.
Pollen viability, essential for the success of hybridizations, is assessed through direct (in vivo or in vitro germination) and indirect
(staining) methods (Dafni 1992 ; Shivanna and Rangaswamy 2012 ). Staining, a quick and cost-effective method, uses dyes such as acetic
carmine, aniline blue (Sharma and Sharma 1994 ; Stanley and Linskens 2012 ), and 2,3,5-triphenyltetrazolium chloride (TTC) (Shivanna
and Rangaswamy 2012 ; Stanley and Linskens 2012 ) to differentiate between viable and non-viable pollen grains. The in vivo germination
test involves pollinating receptive stigmas and subsequently counting the pollen tubes that penetrate the stigma (Wan et al. 2024 ). The
fluorescence technique, which uses fluorochromes to visualize pollen tubes, allows the analysis of characteristics such as growth rate and
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germination (Sun et al. 1991 ; Yi et al. 2006 ). This technique, based on secondary fluorescence (Kho and Baer 1968 ), is widely used in
breeding programs to assess pollen viability and identify incompatibilities between genotypes (Kearns and Inouye 1993 ).
Efficient pollination requires estimation of pollen quantity, quality, viability and germination capacity (Gómez et al. 2015 ) which are
extremely important to estimate the reproductive potential of the species (Gadže et al. 2011 ; Piccinini et al. 2012 ). Pollen viability is
considered high when more than 70% of the pollen grains stain, which is suitable for breeding programs (Ruggiero et al. 1996 ). Aiming to
enable crossbreeding between forage species of Paspalum with asynchronous flowering, this study had the following objectives: (1) adapt
the protocol for pollen cryopreservation of the late flowering forage species Paspalum atratum Sw. and Paspalum malacophyllum Trin. and
(2) identify the stainability and in vivo viability of cryopreserved pollen grains of these species and of Paspalum regnellii Mez as an
indicator of their success in interspecific hybridizations.
Methods
Plant	material
The selection of the species for this study was based on the forage potential of the male parents and the availability of sexual species with
forage potential and/or crossability with the male parents of interest. The following species are all forage grasses (Acuña et al. 2019 ) and
present different flowering times along the year in São Carlos, São Paulo State, Brazil (21°57′42″ S, 47°50′28″ W): Paspalum plicatulum
Michx. (Plicatula group) and Paspalum regnellii Mez (Virgata group) flower between January and March, while Paspalum atratum Sw.
(Plicatula group) and Paspalum malacophyllum Trin. (Malacophylla group) show late flowering, from April to June.
Paspalum urvillei Steud. (genome IIJJ, Burson 1979 ) was chosen as a female parent, as it is a sexual species and has affinity with P.
malacophyllum (genome MMMM) (Bolat and Pirlak 1999 ; Bennett and Bashaw 1966 ), with Paspalum dilatatum Poir. (Bennett and
Bashaw, 1966 ) and with Paspalum juergensii Hack. (Burson et al. 1973 ). The latter two species present genomic composition (IIJJX and
JJ) similar to the species from Virgata group (IIJ2J2), to which P. regnellii belongs (Cidade et al. 2013 ). Furthermore, species of the
Plicatula group (P. plicatulum, P. guenoarum and P. atratum) are known to hybridize among them (Novo et al. 2017 ), so a sexual hybrid
from this group was chosen as a female parent for the crosses with P. atratum.
The plants used as male parents (pollen donors) from the species P. atratum, P. malacophyllum and P. regnellii were obtained from the
Paspalum Germplasm Bank (GB) from Embrapa Southeast Livestock (São Carlos, São Paulo State, Brazil), where the cryopreservation
tests have been performed. The plant material used was: two genotypes of P. malacophyllum BGP 6 (VSsSi 5095) and BGP 293 (V14606),
two from P. atratum BGP 98 (VSW 9880) and BGP 308 (V14525), and one from P. regnellii BGP 215 (Lr2) (Table 1 ). Seeds from these
accessions were germinated in seedling trays (5.3 × 33.7 × 67.2 cm) with 162 cells with 3.5 × 3.5 cm each and 31 cm volume, filled with
Carolina Soil substrate and later placed in seedling bags (10 × 16 cm) with 20 replications for each accession. After 3 months, the
seedlings were transplanted to pots (22.5 × 23 cm) with 8 L capacity, with Carolina Soil substrate for plants and conserved in a
greenhouse.
Table 1
Paspalum species studied for pollen AQ6 viability after cryopreservation and their use in intra- and inter-specific hybridizations used as male parents
Genotype Species Collector
code
Local Botanic informal
group
Reproductive
mode
Genome Ploidy References
BGP 6 Paspalum
malacophyllum VSsSi 5095 Itumbiara/GO-
BR Malacophyla Apomictic MMMM Autotetraploid Hojsgaard et al.
(2008 )
BGP 293 Paspalum
malacophyllum V 14606 Japorã/MS-BR Malacophyla Apomictic MMMM Autotetraploid Hojsgaard et al.
(2008 )
BGP 98 Paspalum atratum VSW 9880 Terenos/MS-BR Plicatula Apomictic – Segmental
Allopolyploid
Quarín et al.
(1997 )
BGP 308 Paspalum atratum V 14525 Terenos/MS-BR Plicatula Apomictic – Segmental
Allopolyploid
Quarín et al.
(1997 )
BGP 215 Paspalum regnellii Lr 2 Itirapina/SP-BR Virgata Sexual RRSS Autotetraploid Norrmann (1981 )
Codes of sample collectors: V = J. F. M. Valls; S = C. Simpson; Si = J. C. S. Silva; Ss = S. M. Sano; W = W. Werneck; Lr = L. A. R. Batista
As female parents, P. urvillei BGP 393 (Af34) and hybrid F1 #37 from the cross P. plicatulum 4PT × P. guenoarum cv. Azulão (Novo et al.
2017 ) were used (Table 2 ). Twenty-one seedlings from the hybrid F1 #37 were placed in seedling bags (10 × 16 cm) in the greenhouse.
The P. urvillei material was obtained from a collection on the embankment of Estrada Municipal Guilherme Scatena, close to the
Ecological Park in São Carlos, SP, and 20 seedlings were transplanted into seedling bags in the same way. After 6 months, all the female
plants were transplanted into 2-L pots (18 × 15 cm).
Table 2
Paspalum materials used as female parents in the hybridizations for cryopreserved pollen in vivo viability test
Genotype Species
Collector
code
Local
Botanic
informal
group
Reproductive
mode
Genome
formula
Ploidy References
Codes of sample collectors: Af = A. P. Fávero
Material provided by the Paspalum genetic breeding program of Universidad Nacional del Nordeste, Argentina
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Genotype Species Collector
code
Local
Botanic
informal
group
Reproductive
mode
Genome
formula
Ploidy References
Hybrid F1
#37
(Paspalum plicatulum 4PT) × 
(Paspalum guenoarum cv.
Azulão)
– Plicatula Sexual – Tetraploid Novo et al.
(2017 )
BGP 393 Paspalum urvillei Af 34
São
Carlos/SP—
BR
Dilatata Sexual IIJJ Autotetraploid Bennett and
Bashaw (1966 )
Codes of sample collectors: Af = A. P. Fávero
Material provided by the Paspalum genetic breeding program of Universidad Nacional del Nordeste, Argentina
Collection	of	pollen	grains
Inflorescences of male parents plants in pots were covered with paper bags (25 × 16 cm) on the day before anthers dehiscence and kept
that way until the following morning, according to Novo et al. (2017 ). Inflorescences of plants located in the field (P. regnellii) were
collected, placed in plastic cups, and covered with paper bags as described above.
In the next morning, the anthers dehiscence was around 8:00 am (GMT-3) for P. malacophyllum, and P. regnellii, and 10:30 am (GMT-3)
for P. atratum. Inflorescences were slightly shaken to induce pollen release. Pollen samples were placed in glassPetri dishes (100 × 
15 mm) and taken to the seed laboratory for the long-term conservation tests.
Pollen	dehydration	and	cryopreservation
The pollen dehydration is a crucial step for cryopreservation (Santos 2000 ) and the period of time of conservation in LN tend to not
influence the quality of the pollen germination after cryopreservation (Linskens 1964 ). Two dehydrating agents and seven periods of
cryopreservation were chosen, as described in Fig. 1 . A non-dehydrated treatment was also evaluated for cryopreservation and a control
with fresh pollen grains was included for dehydration test.
Fig. 1
Experimental design for Paspalum atratum and Paspalum malacophyllum pollen grains dehydration and cryopreservation evaluations
The dehydration treatments silica gel (15 g) and saturated solution of lithium chloride (LiCl) were chosen as pollen dehydration agents
according to the best results obtained in Paspalum notatum pollen cryopreservation (Dinato et al. 2018 ). They were placed in an open
Petri dish (90 × 15 mm) into a clear plastic box (11 × 11 × 3.5 cm) and under a stainless-steel screen (11 × 11 cm) (Fig. 2 ). AQ7 A Petri
dish with the fresh pollen was placed on top of this screen and the clear plastic box was kept closed at different time intervals: 30, 60, and
120 min at 25 °C ± 2 (Dinato et al. 2018 ). Then pollen was transferred to transparent gelatin capsules purchased in pharmacies, capsules
size number zero and one, inside cryovials and immersed in LN. A treatment with non-dehydrated pollen was included in the
cryopreservation tests to verify the effect of moisture content on pollen. However, the moisture content was not measured due to the very
low mass of the collected pollen. For each treatment, three tubes with pollen grains were conserved inside the cryotank and a control was
evaluated, which consisted of the fresh pollen, recently harvested from inflorescences. Samples of the different treatments were stored in
LN for 1, 10, 30, 90, 180, 270, and 365 days, totaling 49 treatments (Table 3 ). AQ8
Fig. 2
Pollen grain dehydration procedure and subsequent preparation for cryopreservation. a Fresh pollen grains (yellow) with anthers (black)
collected in a Petri dish. b Top view of the clear plastic box with pollen grains placed in a Petri dish, on top of a mesh that stays above a
lithium chloride solution for pollen dehydration. c Lateral view of the same dehydration system, but using silica gel (blue). d Storage of
pollen grains in gelatin capsules. e Capsules placed in cryotubes for subsequent cryopreservation
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Table 3
Average percentage of viable pollen stained with 2,3,5-triphenyltetrazolium chloride (TTC) after dehydration with two dehydrating agents (LiCl and S = 
silica gel) during three different periods of dehydration (30, 60 and 90 min) and cryopreservation with seven different periods of conservation (1, 10, 30, 90
180, 270 and 365 days)
Cryopreservation period Dehydration period
BGP 6 BGP 293 BGP 98 BGP 308
LiCl Silica gel LiCl Silica gel LiCl Silica gel LiCl Silica gel
1 day
30 min 65.33 a 54.00 c 80.33 a 60.00 b 68.67 a 50.67 c 75.33 a 51.33 c
60 min 54.00 c 60.67 c 64.33 b 69.33 b 56.67 bc 58.67 c 59.33 b 60.67 b
120 min 49.00 c 69.00 a 54.00 c 78.67 a 48.00 c 68.33 ab 51.33 c 70.67 a
10 days
30 min 68.67 a 52.00 c 80.67 a 58.00 bcd 68.33 a 50.00 c 76.67 a 49.00 c
60 min 54.33 c 62.33 bc 65.00 b 69.00 b 57.33 bc 60.67 bc 59.67 b 61.67 b
120 min 49.67 c 71.33 a 55.33 bc 79.00 a 48.33 c 68.33 ab 49.33 d 69.67 a
30 days
30 min 66.00 a 55.00 c 80.67 a 58.00 bcd 68.00 a 52.33 c 77.00 a 49.67 c
60 min 54.33 c 63.33 bc 64.00 b 68.33 b 58.00 b 62.33 bc 60.33 b 60.33 b
120 min 48.67 c 68.33 a 55.67 bc 78.33 a 48.33 c 69.00 a 49.33 d 70.33 a
90 days
30 min 64.67 a 53.67 c 80.33 a 58.67 bc 68.33 a 53.33 bc 75.67 a 52.00 c
60 min 54.00 c 65.00 b 64.33 b 68.33 b 57.00 bc 64.67 b 59.33 b 59.33 b
120 min 49.33 c 69.33 a 56.00 b 78.33 a 48.67 c 70.33 a 51.00 c 67.33 a
180 days
30 min 66.00 a 53.67 c 70.00 a 51.00 de 79.00 a 57.00 b 74.33 a 53.67 c
60 min 54.67 c 63.00 bc 57.67 bc 62.00 c 66.33 b 65.33 b 60.00 b 61.00 b
120 min 50.00 c 69.00 a 47.67 d 70.67 a 58.00 bc 77.00 a 51.33 c 69.33 a
270 days
30 min 65.67 a 53.00 c 69.67 a 49.00 e 69.33 a 49.67 c 62.00 ab 42.00 d
60 min 55.00 c 63.00 bc 58.00 bc 61.67 c 56.67 bc 58.67 c 53.33 c 49.33 c
120 min 49.00 c 70.00 a 48.00 d 70.33 a 48.33 c 68.67 a 43.67 d 60.00 b
365 days
30 min 65.67 a 52.67 c 69.33 a 51.33 cde 69.00 a 51.00 c 61.33 b 40.67 d
60 min 54.67 c 63.33 bc 55.67 bc 62.00 c 56.33 bc 60.67 bc 54.33 c 48.67 c
120 min 48.00 c 68.00 a 47.67 d 71.00 a 48.33 c 69.00 a 42.33 d 61.00 b
0 day C 70.67 a 77.67 a 73.67 a 69.00 a
1 day ND 33.67 f 44.00 d 35.00 f 35.67 f
10 days ND 32.67 f 42.00 e 35.00 f 33.67 f
30 days ND 32.33 f 42.67 e 34.67 f 33.00 f
90 days ND 31.67 fg 41.33 e 34.33 f 32.33 f
180 days ND 30.67 fg 40.00 e 36.00 ef 32.67 f
270 days ND 30.00 g 33.00 fg 32.67 f 29.67 g
365 days ND 30.00 g 31.67 fg 31.67 fg 29.33 g
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Cryopreservation period Dehydration period
BGP 6 BGP 293 BGP 98 BGP 308
LiCl Silica gel LiCl Silica gel LiCl Silica gel LiCl Silica gel
C—fresh pollen, ND—non-dehydrated pollen
p > 0.05; cv: 1.922873
The slow thawing consisted of thawing pollen for 30 min in the freezer (−20 °C), 30 min in the refrigerator (+4 °C), and 30 min at room
temperature (+25 °C) (Dinato et al. 2018 ). Then the percentage of stained pollen was evaluated using 2,3,5-triphenyltetrazolium chloride
solution, according to the protocol explained in the next item.
TTC	viability	test
The viability test using TTC indirectly indicates the germination potential of the cryopreserved pollen grains. The test was carried out
immediately after the pollen was collected from the anthers (fresh pollen as control) and after each cryopreservation treatment for
accessions BGP 6, 293, 308, and 98, including the dehydrated and non-dehydrated treatments. The results for P. regnellii cryopreserved
pollen are not shown. One sample was collected from each replication and the pollen was stained with TTC solution (0.25%). A drop of
this solution was placed on a microscope slide and the pollen sample was placed subsequently. The slide was placed in a clear plastic box
with a filter paper moistened with distilled water, which was kept in an oven at 27 °C for 2 h. Pollen stainability (%) was estimated by
counting randomly under an optical microscope the number of stained pollen grains in relation to the total pollen grains. In each slide, 200
grains were evaluated. Three replications were made for each treatment. Pollen was considered viable when the grains were well
developed and well-stained, while grains poorly developed or partially stained were considered non-viable. The experimental design was
completely randomized. Data were subjected to analysis of variance and the means were compared using the Tukey test (p ≤ 0.05), using
the Statistical Analysis System software (2004).
Pollen	viability	by	in	vivo	germination
The following crosses were performed to evaluate the in vivo germination as indicative of the cryopreserved pollen germination viability:
P. urvillei (BGP 393) × P. malacophyllum (BGP 6; BGP 293); P. urvillei (BGP 393) × P. regnellii (BGP 215); hybrid (P. plicatulum 4PT × 
P. guenoarum cv. Azulão—plant F #37) × P. atratum (BGP 98; BGP 308). Female plants were placed in a humid chamber inside the
greenhouse in the afternoon before the anthesis. In the following morning, due to the high air relative humidity, dehiscence of the anthers
was delayed for a short period, whichprovided enough time to remove the anthers with tweezers before they opened. After emasculation,
the plant was removed from the humid chamber to allow the stigmas to dry. The stigmas of emasculated florets were examined under a
magnifying glass to check for contamination and the contaminated stigmas were extracted with a tweezer. The emasculated florets with
clean stigmas were pollinated with the cryopreserved pollen (Dinato et al. 2018 ). We used the pollen which was dehydrated with LiCl for
30 min or with silica gel for 120 min. Spikelets were collected at different time intervals for the different crossings to evaluate the
germination percentage over time, according to Burson and Young (1983 ). Then the structures were fixed in FAA (solution of 1
formaldehyde: 1 glacial acetic acid: 8 ethyl alcohol) for 30 min and stored in 70% ethanol (Burson and Young 1983 ). Subsequently, the
pistils were extracted and placed in a 1 N NaOH solution for 30 min and then placed in a 0.1% aniline blue solution for 30 min, adapted
protocol from Kho and Baer (1968 ). The pistils were examined under a Zeiss fluorescence microscope, using a T400lp light filter. The
percentage of germinated pollen grains was determined by randomly counting samples, following the protocol of Burson and Young
(1983 ).
To determine whether pollen germination rate was related to the dehydration treatment and to the times after hybridization with
cryopreserved pollen, we fitted a general linear model to the data. In these models, the germination state—germinated (when the pollen
tube was twice as long as the pollen grain) or not germinated—of each pollen grain was considered as a response and treatment variable
(Silica or LiCl), period as well as their interaction as explanatory variables. In these cases, we assume a binomial error distribution family
(link = logit). The significance of the models was assessed by comparing them with a null model in the Chi-square test. General trends in
germination rates were assessed by visual inspection of interaction plots and data were compared using analysis of variance (ANOVA).
These analyses were carried out using the R software (2020).
Results	and	discussion
There were differences in the stained pollen percentage after the different dehydration treatments (Fig. 3 ; Table 3 ) and among genotypes.
The best treatments were dehydration with LiCl for 30 min and with silica gel for 120 min. The treatments did not differ statistically from
the control (fresh pollen) and pollen viability that did not undergo dehydration presented low viability, of around 30% (Table 3 ). These
results were similar to that obtained for P. notatum (Dinato et al. 2018 ), so the dehydration protocols of LiCl for 30 min and silica gel for
120 min are adequate for species of the genus Paspalum, as low variability is observed among the pollen morphology of the genus
(Radaeski and Bauermann 2018 ; Dinato et al. 2024 ).
Fig. 3
TTC pollen viability from the accession BGP 98 (Paspalum atratum). a Fresh pollen without dehydration nor cryopreservation (control), b
pollen after dehydration with lithium chloride for 30 min and stored in liquid nitrogen for 180 days, c pollen without dehydration and stored in
liquid nitrogen for 12 months. Viable pollen (blue arrow) and non-viable pollen grains (red arrows) are indicated
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Pollen can be classified as tolerant or sensitive to dehydration and, in this context, binucleated pollen are classified as tolerant and
trinucleated one as sensitive (Hughes et al. 1991 ). Many authors describe that binucleated pollen have greater viability compared to
trinucleated pollen (Frankel and Galun 2012 ; Stanley and Linskens 2012 ). Therefore, it is necessary to have an adequate methodology for
drying trinucleated pollen, as the nuclear components of male cells can be damaged, reducing the viability. Grass pollen is trinuclear,
making it difficult to store male gametes (Gill 2014 ; Gómez et al. 2015 ).
Resistant pollen can be dehydrated to low water contents from 10 to 12%, using the same seeds methods, then placed directly in the liquid
nitrogen and thawed at room temperature (Hong et al. 1996 ). When the sensitive pollen can be partially desiccated, the water content levels
achieved should be higher for those that generate desiccation damage and lower for those where the water can freeze (Towill 2002 ). The
dehydrating agents used had relative humidity of 12% and 10%, respectively, at 25 °C (Winston and Bates 1960 ; Young 1967 ).
The reduction in pollen viability in the treatment without dehydration (Table 2 ) may occur because when the pollen is subjected to low
temperatures, moisture decreasing is necessary to prevent pollen burst caused by the intracellular water freezing. Barnabás and Rajki
(1976 ) corroborate this result and emphasize that pollen needs an adequate humidity to be stored. Similar to other dehydrating agents, LiCl
and silica gel are reported in the literature as efficient for dehydrating and adjusting pollen humidity for storage at low temperatures
(Connor and Towill 1993 ; Towill 2002 , 2010 ).
The results presented in this study corroborate other authors who regard cryopreservation as an efficient alternative for pollen conservation.
This method allows the biological material exposition to negative temperatures, which drastically reduces the plant material activities, such
as division and metabolic reactions, allowing storage for indefinite periods (Engelmann 2004 ; Bennett and Bashaw 1966 ; Sartor et al.
2013 ; Chen et al. 2011 ). The success of pollen conservation depends on several factors, including optimal temperature, pollen moisture,
and environmental storage humidity. The storage period itself does not impact the success of pollen conservation (Linskens 1964 ).
In vivo germination assays showed pollen tube germination in the stigma in all crosses (Fig. 4 ). All crosses but BGP 393 × BGP 215
showed differences in pollen germination across the different time after pollination (TAP). The crosses BGP 393 × BGP 215 and F1 #37 × 
BGP 98 presented differences between pollen desiccation treatments and the cross BGP 393 × BGP 6 showed interaction between treatment
and TAP (Figs. 5 , 6 ).
Fig. 4
Germination of the pollen tube (light blue) in the stigma of the female parent P. urvillei (BGP 393) after 5 h of pollination with cryopreserved
pollen from the male parents a P. regnellii (BGP 215) and b P. malacophyllum (BGP 293). Optical microscope images obtained with
fluorescence T400lp light filter × 40
Fig. 5
Pollen germination rate along the different times after hybridization with cryopreserved pollen in the crosses using Paspalum urvillei (BGP
393) as a female parent with two different dehydration treatments: LiCl (light blue) and silica gel (dark blue). a Cross between P. urvillei (BGP
393) × P. malacophyllum (BGP 293); b cross between P. urvillei (BGP 393) × P. malacophyllum (BGP 6); c cross between P. urvillei (BGP
393) × P. regnellii (BGP 215)
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Fig. 6
Pollen germination rate along the different times after hybridization with cryopreserved pollen in the crosses using the hybrid P. plicatulum
4PT × P. guenoarum cv. Azulão-plant F #37 as a female parent with two different dehydration treatments: LiCl (light blue) and silica gel (dark
blue). a Cross with P. atratum (BGP 308), b cross with P. atratum (BGP 98)
According to Figs. 4 and 5 , all crosses combinations reached the germination peak until 8 h after pollination, except for P. plicatulum
4PT × P. guenoarum cv. Azulão—plant F #37 crossed with P. atratum (BGP 308), which reached the germinationpeak after 18 h when
silica gel was used as desiccant. The variation observed in the germination peaks refers to the number of pollen initially deposited in the
stigma, which can influence the number of germinated pollen. Crossings using two different genotypes of P. malacophyllum as male parent
showed that the pollen tube reached the micropyle. This does not occur in the cross between P. urvillei (BGP 393) × P. regnellii (BGP 215).
In the crosses carried out between the hybrid P. plicatulum 4PT × P. guenoarum cv. Azulão-plant F #37 × P. atratum (BGP 98) although
there was a germinated pollen rate until 53%, the pollen tube did not reach the micropyle.
Pollen germination rates in the analyzed crosses varied depending on the time after pollination (TAP). This relationship between TAP and
germination rate has been previously described by Burson (1987 ) and Burson and Young (1983 ), who suggest that the amount of pollen
used and pollen tube growth can influence this process. In addition, studies such as that of Burson and Young (1983 ) show that pollen
germination rates in interspecific grass crosses can vary significantly over time, with little influence from environmental factors.
There are no studies on in vivo germination of cryopreserved pollen in grasses, but studies with fresh pollen show both the arrival of the
pollen tube in the micropyle and also the non-reaching of the micropyle, which would be expected mainly in interspecific crosses. In
Panicum species, pollen tubes development in interspecific hybridization ranged from 77 to 88% and, in some cases, the tube grew in the
style. No pollen tube penetrated the micropyle and there was also a loss of the pollen tube orientation. Many tubes grew beyond the
micropyle and it was the first time that this phenomenon was observed in Panicum or other grass species (Burson and Young 1983 ).
However, Heslop-Harrison (1982 ) mentioned that the loss of tube orientation with random growth and failure of the tubes to locate the
micropyle may be one of the causes of the pollen tube rejection, causing cross-incompatibility in grasses. Paspalum were similar to
Panicum for high germination rate in self-pollination and lower rates in interspecific crosses. Differently from Panicum, the pollen tube
reached the micropyle, indicating no cross-incompatibility between the Paspalum species (Burson 1987 ). Apparently, there is self-
incompatibility between the style tissue and the pollen tubes.
In vivo pollen viability, pollen-pistil compatibility, pollen vigor, and the morphology of pollen tube development can be observed through
aniline blue staining (Wang et al. 2012 ). Fluorescence microscopy with aniline blue results in this work show that in crosses between P.
urvillei (BGP 393) × P. malacophyllum (BGP 6 and BGP 293), the pollen tube reached the micropyle after 2 h and 15 min of pollination for
crossing BGP 393 × BGP 6 and after 6 h for crossing BGP 393 × BGP 293, revealing that pollen can germinate and pollen tubes grow
through the style in all cross combinations. However, many abnormal pollen tubes have also been observed in crossings, such as non-
germinated pollen, as well as pollen tubes that stopped growing or did not grow directly into the ovary.
Germination rates (Fig. 6 ) were significant for both crosses with P. plicatulum 4PT × P. guenoarum cv. Azulão; however, most slides
showed pollen without germination as expected, indicating a possible incompatibility between parents. This shows that pre-zygotic barriers
are likely to occur, and pre-zygotic barriers cause a low rate or failure of egg fertilization (Bhat and Sarla 2004 ; Dickinson et al. 2012 ).
Pre-zygotic barriers due to differences between the style lengths of progenitors are also observed in other genera (Baldwin and Husband
2011 ). Ideally the crosses performed in the present work should also have been evaluated with fresh pollen, but it is not possible due to the
flowering asynchrony between species. Although this study did not primarily aim to identify pre-zygotic barriers, the scientific literature
corroborates the importance of these mechanisms in maintaining biodiversity. A complete understanding of the causes leading to
reproductive isolation between species remains a challenge for the scientific community (Christie et al. 2022 ). Therefore, future studies
that delve deeper into the mechanisms that prevent or hinder hybridization between the species analyzed in this study could provide
valuable insights into the processes of speciation and the evolution of species, significantly contributing to the advancement of knowledge
in the field.
In all crosses, it was observed that pollen was adhered to the stigma and mechanisms were induced for the development of the pollen tube,
since many pollen had a pollen tube with a considerable size germinating in the stigma; however, a small amount of pollen reached the
ovary and the micropyle. This was probably due to partial incompatibility between the parents, since the number of pollen grains observed
was smaller than expected.
Thus, in vitro germination and pollen tube elongation have been used as a powerful tool for genetic, physiological, biochemical, and
cytological studies in many plant species belonging to different families (Heslop-Harrison 2013 ); however, we cannot correlate in vitro
pollen germination with in vivo germination. In this context, our results show that cryopreserved pollen presents high staining rates and
germinate in vivo, circumventing the flowering asynchrony between genotypes of Paspalum in the genetic breeding program.
1
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Species with pollen grains morphologically similar to Paspalum are likely to respond similarly to the dehydration and cryopreservation
protocols tested in this study. This expectation is supported by previous research on other grasses (Poaceae), such as those conducted by
Lansac et al. (1994 ), Inagaki and Mujeeb-Kazi (1997 ), Gill (2014 ), and Rajasekharan and Rohini (2023 ), which have successfully
cryopreserved pollen.
Conclusion
Pollen cryopreservation is suitable for Paspalum malacophyllum and Paspalum atratum when the pollen is dehydrated with LiCl for 30 min
or silica gel for 120 min, as viability remained the same as fresh pollen. The pollen remained viable after cryopreservation and visualized in
vivo, as there was an effective germination of the pollen tube in the stigma in all evaluated crosses. In crosses where P. urvillei was used as
female parent and P. malacophyllum as male parent, the pollen tube reached the micropyle, which did not occur in crossings of the same
female parent with P. regnellii. The pollen tube did not reach the micropyle in the crossings that used P. plicatulum 4PT × P. guenoarum cv.
Azulão-plant F #37 as female parent. The results of this research can be used for genetic breeding programs for asynchronous species,
allowing new crossing possibilities in the program, and for the conservation of cryopreserved material. AQ9
Acknowledgements
Professor Dr. Vinícius Lourenço Garcia de Brito, from Federal University of Uberlândia, MG, Brazil, is acknowledged for helping with R
analyses.
Funding
No funding was received for conducting this study.
Declarations
Conflict	of	interest
On behalf of all authors, the corresponding author declares that there is no conflict of interest.
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