Cytogenetic and pollen analysis in Passiflora triloba (Passifloraceae)

Análises citogenéticas e polínicas em Passiflora triloba (Passifloraceae)

Abstract

Passiflora species are prized and grown as ornamental plants due to their stunning blooms. P. triloba has a unique petal count within the genus and remarkable flowers. To obtain cytogenetic and pollen data for this wild species, the following were performed: (i) karyotype and CMA₃/DAPI banding; (ii) meiotic and post-meiotic analyses; (iii) pollen viability and in vitro pollen germination; and (iv) in vivo pollination. The species is diploid, with 2n = 2x = 18, and n = 9. The karyotype showed only metacentric chromosomes and CMA⁺ and DAPI^– blocks in all centromeres and in the terminal regions of two chromosome pairs, indicating two pairs of satellites. Meiotic analysis revealed a low percentage of irregularities such as early and lagging chromosomes, asynchrony, and transverse spindle fibers. There was a predominance of interstitial chiasmata with a recombination index of 18.88. In the post-meiotic analysis, few irregularities such as dyads and triads were found, and the meiotic index was 99.08%. Pollen viability was 80.54%, but non-viable pollen grains were also observed with contracted cytoplasm (0.38%), empty (9.9%), pulverized (8.44%), and micrograin (0.64%), while 31.88% of pollen grains emitted pollen tubes after in vitro germination testing. Pollen tube emission and fertilization occurred within 2 hours of in vivo pollination. The cytogenetic and pollen analyses in this study indicate that P. triloba has the chromosome number typical of the Passiflora subgenus, regular meiotic behavior, high pollen viability, and the ability to emit pollen tubes and achieve fertilization, supporting its potential use in interspecific hybridization.

Keywords:
Passifloraceae; passion fruit; meiosis and post-meiosis; CMA/DAPI; pollen viability; in vivo and in vitro germination

Resumo

– Espécies de Passiflora são apreciadas e cultivadas como plantas ornamentais devido às suas flores deslumbrantes. P. triloba tem uma contagem de pétalas única dentro do gênero e flores notáveis. Para obter dados citogenéticos e do pólen para esta espécie silvestre, foram realizados: (i) cariótipo e bandamento CMA3/DAPI; (ii) análises meióticas e pós-meióticas; (iii) viabilidade polínica e germinação de pólen in vitro; e (iv) polinização in vivo. A espécie é diploide, com 2n = 2x = 18 e n = 9. O cariótipo mostrou apenas cromossomos metacêntricos e blocos CMA+ e DAPI– em todos os centrômeros e nas regiões terminais de dois pares de cromossomos, indicando dois pares de satélites. A análise meiótica revelou uma baixa porcentagem de irregularidades, como cromossomos precoces e retardatários, assincronia e fibras transversais do fuso. Houve predominância de quiasmas intersticiais com índice de recombinação de 18,88. Na análise pós-meiótica, poucas irregularidades como díades e tríades foram encontradas, e o índice meiótico foi de 99,08%. A viabilidade do pólen foi de 80,54%, mas grãos de pólen não viáveis ​​também foram observados com citoplasma contraído (0,38%), vazio (9,9%), pulverizado (8,44%) e microgrão (0,64%), enquanto 31,88% dos grãos de pólen emitiram tubos polínicos após o teste de germinação in vitro. A emissão e fertilização do tubo polínico ocorreram dentro de 2 horas após a polinização in vivo. As análises citogenéticas e polínicas neste estudo indicam que P. triloba tem o número de cromossomos característico do subgênero Passiflora, comportamento meiótico regular, alta viabilidade polínica e capacidade de emitir tubos polínicos e atingir a fertilização, suportando seu uso potencial na hibridação interespecífica.

Palavras-chave:
Passifloraceae; passifloras; meiose e pós-meiose; CMA/DAPI; viabilidade polínica; germinação in vivo e in vitro

1. Introduction

Passiflora L. species have significant economic value, being cultivated for in natura consumption, processed products, herbal medicines, and ornamental purposes (Lopes et al., 2017; Ferraz and Lot, 2007; Abreu et al., 2009). The fruit of sour passionfruit trees is very used for food and widely commercialized, and genetic diversity studies are performed to increase quality and productivity (Morillo et al., 2023, 2024). However, passionflowers have also been used for ornamentation due to the beauty of their flowers (Abreu et al., 2009). Their flowers are hermaphroditic but require cross-pollination for fertilization, as they are typically allogamous and self-incompatible (Jesus et al., 2017). The species Passiflora triloba Ruiz & Pavón ex DC. has shown flowers up to 8 cm long, with a purplish-red color, contrasting with the violet and white striped corona; the number of sepals and petals is unique within the genus, having six of each (Ulmer and MacDougal, 2004).

When pollen is compatible with the stigma, pollen tubes grow towards the ovary (Souza et al., 2000; Oliveira and Samuelsson, 2022). When there is compatibility between the pollen and the stigma, the pollen tubes grow towards the ovary (Madureira et al., 2014), and the occurrence of a good fertilization percentage is directly influenced by pollen grain viability (Cabral et al., 2013), for stigma receptivity (Belo et al., 2015) and pollination time (Souza et al., 2004b). When incompatible, pollen is typically inhibited in the pistil, stopping at the stigma surface. A reorganization can be observed in response to incompatibility, causing a disruption in the protoplasm and resulting in a rounded shape of the pollen tube (Madureira et al., 2014).

Cytogenetic studies support taxonomic analyses and aid in understanding group evolution (Oliveira et al., 2019; Silva and Souza, 2020). According to evolutionary studies, Passiflora species exhibit varying chromosome numbers and can be grouped based on their basic chromosome number (x), with x = 6, x = 9, x = 10, and x = 12 (Melo and Guerra, 2003). Information on species fertility and chromosome behavior are important tools for selecting genotypes for interspecific crosses in breeding programs. Analyses conducted during meiosis, such as chromosome pairing, genetic recombination, levels of meiotic irregularities, post-meiosis, and gamete viability, complement each other and provide insights into the species' crossing potential (Souza et al., 2003; Kiihl et al., 2011).

Despite evolutionary stability, meiotic division can undergo changes resulting in cells with altered chromosome numbers, lack of homologous chromosome pairing, irregularities in spindle fiber orientation, formation of micronuclei, and chromosome bridges (Lavinscky et al., 2021). To ensure proper division, meiotic events are regulated by checkpoints that control progression from one phase to the next if everything is normal. However, meiotic mutants can bypass these checkpoints (Singh, 2003). During prophase I, chromosome pairing is one of the most crucial events in meiosis, as it reveals genetic compatibility through the recombination mechanism. Moreover, proper pairing ensures accurate chromosome segregation. Chromosome segregation is directly linked to the formation of balanced gametes (Zamariola et al., 2014), ensuring viable post-meiotic products, in other words, fertility is linked to the proper meiotic cycle (Souza et al., 2003).

Gamete viability may be reduced if chromosomes are not evenly distributed during meiosis, which can occur due to errors in chromosome pairing, bivalent maintenance, or spindle fiber formation (Souza et al., 2003), resulting in low productivity. The formation of monads, dyads, triads, polyads, micronuclei and aneuploid or polyploid cells are consequences of meiotic abnormalities (Souza et al., 2003; Lavinscky et al., 2021). Irregularities in cytokinesis can also affect post-meiotic products (Souza and Pereira, 2011).

The present study on P. triloba aimed to: (i) perform cytogenetic characterization through mitotic analysis, (ii) analyze the occurrence of CMA₃⁺/DAPI^- band distribution, (iii) study meiotic and post-meiotic behavior to observe the species' meiotic stability and possible irregularities during division, (iv) estimate pollen grain viability that is essential for species maintenance, (v) conduct in vitro germination to observe pollen tube emission, and (vi) observe if fertilization occurs through manual in vivo pollination by geitonogamy.

2. Materials and Methods

2.1. Plant material

The wild species Passiflora triloba (Figure 1) was used, maintained in the Passiflora Working Collection (CT-Passifloras/UESC), in a greenhouse at the State University of Santa Cruz, Ilhéus, Bahia (lat. 39°10' west longitude 14°39’S; 78m). Observations under the cultivation conditions of this study showed that P. triloba flowers open early in the morning, around 6:00 am, and remain open throughout the day, lasting an average of 12 hours; under ideal conditions, P. triloba blooms year-round.

Figure 1
Passiflora triloba flower. (a) Front view; (b, c) Side view.

2.2. Preparation of metaphase chromosomes

To obtain rootlets, cuttings were taken from the middle part of the mother plant's branches and placed in 1.5 L polyethylene bags containing washed sand for rooting. The samples consisted of root tips, collected at approximately 1.0 cm and pretreated in 0.002 M 8-hydroxyquinoline antimitotic solution at room temperature (RT) for 1 h and at +10 °C for 21 h. The samples were washed twice for 5 min in distilled water on a shaker platform and fixed in Carnoy solution (3:1 ethanol: acetic acid; Johansen, 1940) for 3 h at room temperature, then stored at -20 °C until use. To obtain metaphases (Silva et al., 2018), the samples were washed twice in distilled water with agitation, placed on slides, and incubated in 50 µL of enzymatic solution containing 2% cellulase and 20% pectinase for cell wall and cytoplasm digestion in a humid chamber for 75 min at 37 °C. After incubation, the samples were transferred to Petri dishes, washed twice in distilled water for 5 min, and macerated on a slide containing 10 µL of 45% acetic acid, using needles to separate the cells. Then an 18×18 mm coverslip was placed over the material and using a syringe with a thick-tipped needle, the material was spread, and excess acetic acid was removed with filter paper. Subsequently, using filter paper, the coverslip was gently pressed with fingers to spread the chromosomes, and the slide was taken for freezing in liquid nitrogen. After about 8 min, the coverslips were removed, the slides were air-dried and stored at -20 °C until use.

2.3. Karyotype analysis

The staining for karyotype analysis involved immersing slides in 2% Giemsa solution for 15-20 min, followed by a quick rinse in distilled water, air drying at RT, and mounting with an 18×18 mm coverslip using Neomount medium. Metaphase spreads were photographed using an Olympus BX41 light microscope equipped with an Olympus DP25 5M digital camera and DP2-BSW software. Measurements were taken using Imagetool software version 3.0. Five metaphases (replications) were analyzed in a randomized experimental design for the following karyotypic characteristics: chromosome number, short arm length (SA), long arm length (LA) and satellite length, arm ratio (LA/SA), mean chromosome length (χ), relative chromosome length (%), haploid lot length (HLL), karyotype formula, and asymmetry index (TF%; Huziwara, 1962). The homologues were distinguished according to Guerra (1999). For the creation of the karyogram, Adobe® Photoshop SC5 software was used. The data were subjected to ANOVA (p < 0.05) using SISVAR software, version 5.0, and means were compared between chromosome pairs using Tukey test.

2.4. Double staining with CMA₃ and DAPI fluorochromes

The chromosome banding technique for locating GC- and AT-rich base-specific heterochromatin was performed using the fluorochromes CMA₃ (chromomycin A₃) and DAPI (4',6-diamidino-2-phenylindole), respectively. The slides used were removed from the freezer (-20 °C), air-dried at RT, aged for three days in a dark box, and stained with CMA₃/DAPI (Guerra and Sousa, 2002). The solutions were applied to the slides in dark conditions and in a humid chamber. The double staining procedure involved applying 20 μl of CMA₃ (0.25 mg/ml) to the slide with a coverslip for 1 h; after rinsing with distilled water and drying with cold air, 15 μl of DAPI (0.5 mg/ml) was applied for 30 min. After this time, the slides were washed with distilled water until the coverslip came off, dried with cold air, and mounted with 15 μl of glycerol/McIlvaine mounting medium (1:1 v/v) containing 2.5 mM MgCl₂ (Schweizer and Ambros, 1994). The preparations were covered with a 20×20 mm coverslip and aged for three days at RT. For DAPI visualization, a U-UTH filter (330-385 nm excitation/400 nm dichroic cut-off emission/>420 nm) was used. For CMA₃ visualization, a U-MWV filter (450-480 nm excitation/500 nm dichroic cut-off emission/>515 nm) was used. Photographic documentation was carried out using an Olympus CX41 epifluorescence microscope equipped with an Olympus 5M digital camera, utilizing the DP25 5-megapixel system and DP2-BSW software (Olympus). The overlays and karyogram preparation were performed using Adobe Photoshop CS5 software.

2.5. Meiotic and post-meiotic analyses

Flower buds at various developmental stages were randomly collected throughout the flowering period, fixed in Carnoy solution (3:1 ethanol; acetic acid; Johansen, 1940) with changes made at 30 and 180 min at RT, and stored at -20 °C for 24 h. The buds were then transferred to 70% ethanol and kept at +10 °C until analysis. For each slide, one anther from each bud was used. The samples were prepared using squash technique. The anthers were softened with two drops of 60% acetic acid on the slide for 7 min. For data recording, acetic acid was removed using filter paper and the material was stained with 2% acetic orcein. Fifty cells were observed in each meiotic phase. The frequency of interstitial and terminal chiasmata was analyzed to calculate the recombination index (RI = [Σ total number of chiasmata ÷ number of cells analyzed] + haploid number (n) of the species; Darlington, 1958). One chiasma was considered for rod-shaped bivalents and two chiasmata for ring-shaped bivalents (Senda et al., 2005). The percentage of meiotic irregularities observed in each phase was recorded. For post-meiosis analysis, four slides (replicates) from different flower buds, randomly collected, were used to record the quantity of post-meiotic products (monads, dyads, triads, tetrads, and polyads) and calculate the meiotic index (MI = [number of normal tetrads × 100] ÷ number of post-meiotic products counted) (Love, 1951).

To document the different stages of meiosis and post-meiosis, the slides were stained with DAPI, according to Lavinscky et al. (2021). The anther was softened with two drops of 60% acetic acid for 7 min, after which the squash technique was performed. After placing the coverslip, the slide was carefully exposed to liquid nitrogen vapor, first by positioning it near the opening of the nitrogen tank ±10 min. Following this initial cooling, it was then lowered approximately 10 cm into the vapor ±5 minutes, remaining in the vapor throughout. After this period, the coverslips were quickly removed using a scalpel blade and the slides were air-dried at RT. 15 µl of DAPI (0.5 mg/ml) was added and the sample was covered with a 22×22 mm coverslip. For DAPI visualization and photographic documentation the same methodology mentioned in the previous item (double staining) was used. Plates were produced using the software Adobe® Photoshop CS5.

2.6. Pollen viability - stainability

For pollen grain (PG) viability analysis, Alexander stain (Alexander, 1969) was used, consisting of anhydrous ethanol (10 ml), 1% malachite green in 95% ethanol (1 ml), distilled water (50 ml), glycerol (25 ml), phenol (5 g), chloral hydrate (5 mg), 1% acid fuchsin in water (5 ml), 1% orange G in water (0.5 ml), and glacial acetic acid (to reach a final volume of 100 ml). This solution stains the cytoplasm red and the PG wall green/blue, with stained PG with intact cytoplasm considered viable. Five fully open flowers were used, totaling five replications, in a randomized experimental design. One anther per flower was analyzed. Morphological aspects and stainability were observed, followed by counting 1000 PGs per anther, totaling 5000 PGs. Non-viable PGs were classified according to Lavinscky et al. (2021) and Souza et al. (2004a): empty (absence of cytoplasm), shrunk (contracted cytoplasm), pulverized (cytoplasm with granular appearance and low stainability), and micrograin (cytoplasm present and up to 50% of the size of viable PG). The slides were examined under an Olympus BX41 light microscope equipped with a 5M Olympus DP25 digital camera and DP2-BSW software. The data were subjected to ANOVA (p < 0.05) using SISVAR software, version 5.0.

2.7. In vitro germination

For in vitro germination of PG, the autoclaved culture medium proposed by Bruckner et al. (2000) was used, composed of 0.10g/L boric acid (H₃BO₃); 50 g/L sucrose; 0.3 g/L calcium nitrate tetrahydrate (Ca(NO₃)₂.4H₂O); 0.2 g/L magnesium sulphate heptahydrate (MgSO₄.7H₂O) and 0.1 g/L potassium nitrate (KNO₃). For this analysis, five replications were used in a randomized experimental design. The PGs were transferred to a slide containing a drop of culture medium. The material was covered with a coverslip and incubated in a humid chamber at 37 °C for 24 h. Counting was performed using a light microscope, considering germinated PGs as those whose pollen tube emission exceeded their own diameter. A total of 1000 PGs were counted per repetition, amounting to 5000 PGs analyzed. The data were subjected to ANOVA (p < 0.05) using SISVAR software, version 5.0.

2.8. In vivo pollination

Flowers were bagged one day before anthesis. Manual pollination was performed using different flowers (geitonogamy - pollen is transferred from one flower to another on the same plant), with a total of five plants analyzed. Two hours after pollination, the flowers were collected and fixed in 70% FAA (5% formalin, 5% acetic acid, 90% ethanol) for 1 h, then placed in 5M sodium hydroxide (NaOH) for 24 h. After this period, the stigma and ovary were sectioned and stained with 0.1 N aniline blue. Observations were made to determine if pollen tube emission occurred and if it reached the ovule. For negative control, a previously protected and unpollinated flower was collected for visualization and comparison following the same methodology. The record was made using an Olympus CX41 epifluorescence microscope equipped with an Olympus 5M digital camera using the DP25 5 megapixels system and DP2-BSW software. A U-UTH filter (330-385 nm excitation/400 nm dichroic cut-off emission/>420 nm) was used.

3. Results

3.1. Karyotype analysis and CMA₃/DAPI banding

The species P. triloba exhibited the somatic chromosome number of 2n = 18 (Figure 2), with all metacentric chromosomes, resulting in a karyotype formula of 18M. Chromosome length ranged from 4.17 µm to 2.91 µm (Table 1). The HLL was 31.39 µm. The χ was 3.48 µm and the TF% was 44.08%. The analysis of variance for chromosome length showed a significant difference (p < 0.05) (Table 2). According to Tukey test, chromosome pairs 4 to 7 showed no statistically significant difference (Table 1).

Figure 2
Metaphase chromosomes in Passiflora triloba (2n = 18). (a) Mitotic metaphase; (b) Karyogram with conventional staining; (c) DAPI banding karyogram; (d) CMA₃ banding karyogram; (e) CMA₃/DAPI overlay karyogram. The arrows indicate CMA₃⁺ and DAPI^- blocks in terminal regions and arrowheads point to CMA₃⁺ blocks and all centromeres; (f) Ideogram showing CMA₃⁺ and DAPI^- blocks in terminal regions of pairs 5 and 7 and in centromeres of all chromosomes. Bar = 10µm.

Table 1
Average lengths of chromosome pairs in Passiflora triloba.

Chromosome	SA (µm)	LA (µm)	TCL (µm)	SD	LA/SA (µm)
1	1.85	2.32	4.17 a	0.35	1.25
2	1.64	2.24	3.88 ab	0.35	1.36
3	1.60	2.09	3.69 abc	0.41	1.31
4	1.62	1.98	3.60 abcd	0.40	1.22
5	1.53	1.96	3.50 abcd	0.32	1.28
6	1.46	1.91	3.37 bcd	0.31	1.31
7	1.48	1.71	3.19 bcd	0.25	1.15
8	1.36	1.73	3.08 cd	0.26	1.27
9	1.30	1.60	2.91 d	0.34	1.23

• SA = short arm; LA = long arm; TCL = total chromosome length; SD = standard deviation; LA/SA = arm ratio (long arm/short arm). Values with the same letter indicate statistically equal means by Tukey’s test at 5% significance level.

Table 2
Meiotic behavior irregularities in Passiflora triloba.

Early or lagging chromosomes						Asynchrony			Spindle irregularities
MI	AI	TI	MII	AII	TII	MII	AII	TII	MII	AII	TII
8%	6%	10%	-	-	-	2%	8%	2%	14%	22%	8%

• M = metaphase; A = anaphase; T = telophase; I = meiosis I; II = meiosis II.

CMA₃/DAPI banding revealed CMA₃⁺ and DAPI^– bands in the terminal region of the long arm of chromosome pairs 5 and 7, with the presence of two pairs of satellited chromosomes. All chromosomes showed CMA₃⁺ and DAPI^– markings in the centromeric regions (Figure 2). No DAPI⁺ bands were observed.

3.2. Meiotic and post meiotic analysis

The species P. triloba exhibited the gametic chromosome number of n = 9 (Figure 3). Irregularities were observed in meiosis I and II (Table 2; Figure 3). During meiosis I, early or lagging chromosomes were observed in metaphase, anaphase, and telophase. In meiosis II, asynchrony was observed in the cell division process, with cells showing one group of chromosomes in pre-metaphase and another in metaphase, or one group of chromosomes in anaphase and another in telophase. Irregularities in the spindle fibers, transverse type, were also observed during metaphase, anaphase, and telophase of meiosis II, where chromosome groups aligned irregularly on the metaphase plate, forming a “T” shape. During diakinesis, two types of chromosomal association were observed: interstitial rod-shaped and terminal ring-shaped (Figure 3), with a predominance of interstitial chiasmata. The RI was 18.88 (Table 3). The analyzed post-meiotic products showed a low percentage of irregularities, with only dyads (0.06%) and triads (0.87%) found (Figure 3), which did not affect the MI, which was 99.08%.

Figure 3
Meiotic and post-meiotic behavior in Passiflora triloba (2n = 18). (a) Diakinesis with 9 bivalents (arrowhead shows ring association: terminal; arrow indicates rod association: interstitial); (b) Regular metaphase I; (c) Metaphase I with early chromosome (arrow); (d) Regular anaphase I; (e) Early anaphase I with lagging chromosomes (arrow); (f, g) Late anaphase I with lagging chromosome (arrow); (h) Regular telophase I; (i) Telophase I with lagging chromosome (arrow); (j) Regular metaphase II; (k) Metaphase II with asynchrony (arrow shows pre-metaphase); (l) Regular anaphase II; (m) Anaphase II with tripolar spindle irregularity (arrow); (n) Regular telophase II; (o) Normal tetrad; (p) Triad. Bar = 20 µm.

Table 3
Average number and location of chiasmata observed per cell in diakinesis and recombination index in Passiflora triloba.

Terminal		Interstitial		TC	RI
Mean	(Min-Max ± SD)	Mean	(Min-Max ± SD)	Mean
1.8	(0-10 ± 2.43)	8.08	(4-9 ± 1.20)	9.88	18.88

• Min = minimum; Max = maximum; SD = standard deviation; TC = total chiasmata; RI = recombination index.

3.3. Pollen and pollination studies

A high rate of pollen viability was observed, as 80.54% of the pollen grains showed intact cytoplasm and wall and were considered viable (Figure 4). Four types of non-viable PGs were observed: shrunk (8.44%), empty (9.9%), micrograin (0.64%), and contracted (0.48%). The analysis of variance between viable PGs and different types of non-viable PGs showed a significant difference (Table 4). In culture medium, 31.88% of PGs produced pollen tubes (Figure 4). In vivo pollination allowed observation of pollen tube emission and fertilization after controlled manual pollination (Figure 5), with the plant being self-compatible due to fruit formation. The negative control conducted with previously protected flowers allowed observation of the color difference between fertilized and unfertilized ovules. Pollen tube growth was observed from the stigmatic surface to the ovule entry.

Figure 4
Pollen grains (PG) in Passiflora triloba. (a) Viable PG (arrowhead) and non-viable powdery PG (arrow); (b) Non-viable micro-grain type PG (arrow); (c) Empty non-viable PG; (d) Non-viable PG with contracted cytoplasm (arrow). e In vitro germination showing pollen grain (PG) with pollen tube emission (arrow) and PG without emission (arrowhead). Bar = 20 µm (a-d), 50 µm (e).

Table 4
Summary of variance analysis for chromosome length and pollen grain types in Passiflora triloba.

Chromosome length
SV	DF	MS
Treatments	17	0.740441*
Error	72	0.115869
CV (%)		9.76
Average		3.4870000
Type of pollen grains
SV	DF	MS
Treatments	4	582071.900000*
Error	20	1424.520000
CV (%)		18.87
Average		200.000000

• ^*
  p ≤ 0.05.
• SV = source of variation; DF = degree of freedom; CV = coefficient of variation; MS = mean square.

Figure 5
In vivo pollination in Passiflora triloba showing pollen tube growth stained with aniline blue. (a) Pollen grains (arrowhead) deposited on the stigma surface emitting pollen tubes (arrow) towards the ovary; (b) Pollen grains emitting pollen tubes (arrow); (c) Fertilized ovule showing the pollen tube (arrow) moving towards the nucleus; (d) Negative control showing unfertilized ovule; (e) Path taken by the pollen tube (arrow) from the stigma surface towards the ovule. Bar = 100µm.

4. Discussion

The phylogeny of the genus Passiflora recognizes the basic chromosome number of x = 6, but diversification during the Miocene led to a change from n = 6 to n = 9 through ascending dysploidy and genome expansion via retrotransposon proliferation (Sader et al., 2019). P. triloba, like most species in the subgenus Passiflora L., has a chromosome number of 2n = 18 and n = 9 (Oliveira et al., 2021; Souza et al., 2020; Silva et al., 2018; Viana and Souza, 2012). The mean chromosome length of P. triloba (3.48µm) is very close to that of P. edulis Sims (3.28µm) (Viana and Souza, 2012) and P. gardneri Mast. (3.31 μm) (Silva et al., 2018), and high comparable to P. capsularis L. (2.66 μm) and P. rubra L. (2.77 μm) (Amorim et al., 2014). Chromosome length is directly correlated with genome size, and within the Passiflora genus, species with 2n = 18 show up to a 10-fold variation (Souza et al., 2004a; Yotoko et al., 2011).

Studies on other species in the genus reveal that at least one chromosome pair is submetacentric, a feature not observed in P. triloba, where all chromosomes are metacentric. The asymmetry index (TF) of 44.08% found in P. triloba indicates a symmetric karyotype according to Huziwara (1962), demonstrating that it is a less derived species than P. gardneri and P. gibertii N.E. Brown, for instance, which have an asymmetry index of 43.48% and 43.39%, respectively (Silva et al., 2018). Karyotypic variation results from chromosomal rearrangements and adaptive processes (Sader et al., 2019; Yotoko et al., 2011). The greater chromosomal symmetry suggests that P. triloba has undergone few chromosomal changes throughout its evolution compared to other Passiflora species. However, in the cultivated species P. edulis, different chromosome lengths and asymmetry indices were observed in various cultivars, suggesting that hybridization processes in genetic improvement strategies have a significant influence on the karyotypic polymorphism of this species (Marroquín et al., 2023).

In Passiflora, the location and number of GC repeats vary, serving as a source of karyotypic polymorphism even within species (Marroquín et al., 2023). Chromosome banding using base-specific fluorochromes, such as chromomycin A₃ (CMA) which has affinity for GC bases and 4′-6-diamidino-2-phenylindole (DAPI) with affinity for AT bases, has been used in plants to allow distinct differential staining of different types of heterochromatin (Barros-Silva and Guerra, 2023). The presence of CMA₃⁺ and DAPI^- bands has been observed in Passiflora species with varying numbers and locations, ranging from one to three marked chromosome pairs, as well as heteromorphisms, with five marked chromosomes (Viana and Souza, 2012; Silva et al., 2018). P. triloba exhibited bands in the centromeric region on all chromosomes, revealing a constitutive pattern with CMA₃⁺ and DAPI^- centromeric bands, confirming the predominance of GC bases in its composition. In Pinus densiflora Siebold & Zucc., CMA⁺ centromeric bands corresponded to 18S and 26S rDNA sequences (Hizume et al., 2001), and in Crotalaria juncea L. the chromosomes showed CMA⁺ bands in the centromeric regions and colocalized with 35S sites (Hou et al., 2004). In P. triloba, two pairs of chromosomes showed CMA₃⁺/DAPI^- on long arms, and the presence of these bands in satellite regions is associated with nucleolus organizing regions (NOR), as observed in P. caerulea and P. cincinnata where rDNA 18-5, 8-26S coincided with the CMA₃⁺/DAPI^- bands (Chiapero et al., 2013; Melo et al., 2014).

Most Passiflora species have shown stable chromosome pairing and segregation, although anomalies occur at low frequencies (Souza and Pereira, 2011). During meiosis, crucial events occur such as the pairing of homologous chromosomes, which enables genetic recombination (crossing over), the formation of chiasmata, and the segregation of homologous chromosomes and sister chromatids. These processes are fundamental for genetic diversity, adaptation, and evolution of living organisms (Risso-Pascotto et al., 2003). Although meiosis is an evolutionary stable event controlled by multiple genes throughout its stages, mutations in these meiosis-related genes can affect gamete fertility (Santos et al., 2021; Lavinscky et al., 2021).

In P. triloba, chromosome behavior during meiosis was regular, with lagging chromosomes observed in meiosis I, but absent in meiosis II. This can be attributed to meiotic checkpoints, which monitor the division process and, when necessary, correct issues or allow time so that the cell can correct any problems (Sumner, 2003). The effectiveness of checkpoints was also observed in P. amethystina Mikan (Souza and Pereira, 2011), P. gardneri and P. gibertii (Lavinscky et al., 2021).

The anomalies observed in P. triloba, such as asynchrony in metaphase II and anaphase II (characterized by two chromosome sets in different phases within the same cell) or malformation of spindle fibers (when during the second meiotic phase, the occurrence of convergent or transverse spindle fiber organization leads to “V” and “T” shaped chromosomal configurations, respectively) can result in abnormal post-meiotic products, such as monads, dyads and polyads, or diploidized cells (2n) (Souza and Pereira, 2011). The post-meiotic products observed in P. triloba were mostly regular, with tetrad formation at the end of meiosis II and few occurrences of triads and dyads. The meiotic index refers to the percentage of cells that at the end of meiosis II have generated tetrads (instead of dyads, triads, polyads), reflecting the plant's ability to produce normal male gametes and generate stable progeny in terms of chromosome segregation and perpetuation of genetic characteristics. In P. triloba, the meiotic index was almost one hundred percent, indicating meiotic stability (Love, 1951), and the species is considered cytologically stable.

Chiasmata play a crucial role in maintaining bivalents at the metaphase plate and must be properly terminalized for homologous chromosomes to migrate accurately to the cell poles (Junqueira Filho et al., 2003). The frequency of chiasmata can be used to estimate the degree of genetic recombination in a population (Stebbins, 1971). A high frequency of chiasmata indicates the formation of diverse gametes through crossing over (Pagliarini, 2000). The analysis of interstitial, terminal, and total chiasma counts in diakinesis-stage cells of P. triloba revealed a predominance of interstitial chiasmata. The prevalence of this type of chiasma has already been observed in species with n = 9, such as P. amethystina, P. mucronata Lam., P. gibertii, P. malcophylla Mast. and P. cincinnata Mast. (Souza and Pereira, 2011). The average number of terminal chiasmata in P. triloba was 1.8, which is lower than the minimum value previously reported for other Passiflora species, such as P. mucronata with 2.65 (Souza and Pereira, 2011). Terminal chiasmata may be associated with the phenomenon of terminalization, being more visible in diplotene than in diakinesis (Lopes et al., 2007), and also have the physical function of maintaining the union of bivalents for correct segregation of homologues (Zarchi et al., 1972).

The average total number of chiasmata per cell observed in P. triloba was 9.88, which falls within the range of results previously obtained in studies of other species in the genus, varying from 8.70 in P. alata Curtis to 14.72 in P. malacophylla (Souza and Pereira, 2011). The maximum recombination index of a species depends on two factors: the number of chromosomes in the species and the position where crossing over occurs in the homologous pair (Pereira et al., 2017). There are regions known as hotspots where crossing over occurs more frequently. On the other hand, there are regions on chromosomes with very low occurrence, known as recombination cold spots (Wijnker and Dejong, 2008). The observed recombination index of 18.88 in P. triloba fell within the average range of indices previously observed in Passiflora species with n = 9, which varied from 17.6 in P. alata to 23.9 in P. malacophylla (Souza and Pereira, 2011).

In allogamous species, pollen grains carry genetic information because of heterozygosity, with a high probability of different allele combinations (Souza et al., 2002). Therefore, a higher quantity of viable pollen grains also provides a greater possibility of different allelic combinations and segregation of desirable traits. In P. triloba, the pollen viability rate was 80.54%, considered high, but higher percentages of pollen viability were observed in Passiflora species, above 90%, such as in P. miniata Mast., P. quadrangularis L., and P. alata, with averages of 90.80%, 91.3%, and 95.5%, respectively (Bispo et al., 2015; Valeriano et al., 2011; Souza et al., 2002, 2004b).

In vitro germination tests have been used to assess pollen viability by simulating stigma conditions and stimulating pollen tube growth (Almeida et al., 2011). The percentage of in vitro germinated pollen grains in P. triloba was 31.88%, a rate considered low, but demonstrating the ability of pollen grains from this species to produce pollen tubes. A very low index was also reported in P. suberosa L., as only 21.08% of pollen grains produced pollen tubes in vitro (Cruz et al., 2008), while in P. edulis f. flavicarpa O. Deg., used as a control for P. suberosa as it had already been tested (Bruckner et al., 2000), germination was nearly 100% (Cruz et al., 2008). Low in vitro germination rates may occur due to the need for methodological adaptation. On the other hand, in vitro germination tends to overestimate in vivo germination as it disregards the influence of environmental factors, genetic barriers, and stigma receptivity (Barnabás et al., 1988). So, this is a complementary test to others.

In vivo pollination can be performed to infer fertilization capacity and fruit set (Souza et al., 2024) and to estimate the fertilization capacity of the ovule of a given species through self-pollination or cross-pollination (Pound et al., 2003). Self-incompatibility in Passiflora and most angiosperms is an important reproductive mechanism that prevents self-pollination and promotes genetic diversity within species (Zhang et al., 2009). On the other hand, self-incompatibility becomes a barrier to genetic improvement programs (Bandeira et al., 2011). Despite the self-incompatibility of the genus, geitonogamous pollination in P. triloba showed pollen tube emission and fertilized ovules only two hours after controlled manual pollination, as well as fruit formation. Other studies have shown that fertilization has not occurred one hour after pollination, not considering the possibility of fertilization after two hours, since the next observation was only carried out 24 hours after pollination (Rêgo et al., 2000; Soares et al., 2020). P. triloba is a fertile, self-compatible species, as observed in P. rubra and P. capsularis (Amorim et al., 2011) and in P. edulis mutants (Souza et al., 2010). The occurrence of self-fertilization can lead to a decrease in heterozygote frequency (Allard, 1999), but it can be considered an additional tool in the strategy of fixing desirable traits.

Acknowledgements

Thanks to UESC, CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico) and FAPESB (Fundação de Amparo à Pesquisa do Estado da Bahia) for the financial support for research; CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior) for the scholarship granted to the first author.

Data Availability Statement

The datasets generated during and/or analyzed during the current study are publicly in this article.

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Edited by

• Editor:
  Takako Matsumura Tundisi

Publication Dates

• Publication in this collection
  26 Sept 2025
• Date of issue
  2025

History

• Received
  06 Dec 2024
• Accepted
  21 Mar 2025