Seed germination of Southern Chihuahuan desert cacti: Effect of mucilage, light and phytohormones

Journal Pre-proof Seed germination of Southern Chihuahuan Desert cacti: Effect of mucilage, light and phytohormones ´ Ernesto Mascot-Gomez (Investigation) (Formal analysis) (Writing original draft), Joel Flores (Conceptualization) (Supervision), ´ Nguyen E. Lopez-Lozano (Resources) (Writing - review and ˜ editing), Laura Ya´ nez-Espinosa (Methodology) (Visualization)

PII:

S0367-2530(19)30532-8

DOI:

https://doi.org/10.1016/j.flora.2019.151528

Reference:

FLORA 151528

To appear in:

Flora

Received Date:

26 July 2019

Revised Date:

3 December 2019

Accepted Date:

5 December 2019

´ ´ ˜ Please cite this article as: Mascot-Gomez E, Flores J, Lopez-Lozano NE, Ya´ nez-Espinosa L, Seed germination of Southern Chihuahuan Desert cacti: Effect of mucilage, light and phytohormones, Flora (2019), doi: https://doi.org/10.1016/j.flora.2019.151528

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Seed germination of Southern Chihuahuan Desert cacti: Effect of mucilage, light and phytohormones Ernesto Mascot-Gómez1, Joel Flores*1, Nguyen E. López-Lozano1, and Laura YáñezEspinosa2 1

IPICYT/División de Ciencias Ambientales. Camino a la Presa San José No. 2055, Colonia

Lomas 4a. Sección, San Luis Potosí, S.L.P. 78216, México. Universidad Autónoma de San Luis Potosí, Instituto de Investigación de Zonas Desérticas,

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* Corresponding

author: Tel.: +52 444 834 2000x2036.

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Altaír No. 200, Colonia del Llano, C.P. 78377, San Luis Potosí, S.L.P., México.

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E-mail addresses: [email protected] (E. Mascot-Gómez), [email protected]

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(J. Flores), [email protected] (N.E. López-Lozano), [email protected] (L. Yáñez-Espinosa).

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Running head title:

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Mucilage, dormancy and germination in cactus seeds

Highlights

Seeds from five cactus species from the Chihuahuan Desert have mucilage.

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Mucilage resulted in higher germination percentage in three species.

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Mucilage resulted in faster germination in two species.

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For F. latispinus, mucilage removal promoted germination at high GA3

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

concentration.

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Abstract The aim of this study was to evaluate the effect of mucilage and its removal, as well as phytohormones [gibberellic acid (GA3) and indole-3-acetic acid (IAA)] in light and in darkness on germination of five cactus species (Coryphanta maiz-tablasensis, Echinocactus platyacanthus, Ferocactus latispinus, Ferocactus pilosus and Stenocereus queretaroensis)

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from the Chihuahuan Desert. Three of them, C. maiz-tablasensis, E. platyacanthus and F.

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pilosus, are at risk. The mucilage layer occurred in all species. The sterilization treatment removed the mucilage even from the micropyle. Mucilage resulted in higher germination

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percentage in E. platyacanthus (88.5% vs. 21.1% without mucilage), F. latispinus (88.5%

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vs. 48.2%) and S. queretaroensis (96.0% vs. 1.0%), as well as a lower germination time for E. platyacanthus (10.0 days vs. 19.5 days without mucilage), F. pilosus (14.1 days vs. 16.4

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days) and F. latispinus (7.8 days vs. 14.0 days). GA3 did affect germination percentage in E. platyacanthus (higher at 500 and 1000 mg l-1 than at 50 and 100 mg l-1), F. latispinus

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(higher at 1000 mg l-1 of GA3 than at the control and at the other concentrations) and F. pilosus (higher at 1000 mg l-1 of GA3 than at control and 50 mg l-1). The interaction of mucilage layer and GA3 was only significant for F. latispinus in that seeds with mucilage had higher germination at 0, 50, 100 and 250 mg l-1 of GA3 than seeds without mucilage,

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but at 500 and 1000 mg l-1 germination was high with and without mucilage. Most cactus seeds had no germination in darkness and auxins did not promote germination. Mucilage covers the micropyle and seeds without mucilage were internally more colored than seeds with mucilage in most species. We suggest that mucilage layer in the micropyle can function like a barrier regulating the passage of water to the inner seed.

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Keywords: Auxin, Cactaceae, Conservation biology, Gibberellin, Photoblasticism.

1. Introduction

The Cactaceae is an endemic family to the Western hemisphere, except for Rhipsalis baccifera, which also occurs in Africa and Asia (Ortega-Baes et al., 2010). It comprises 124

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genera and 1,427 species (Hunt, 2006). The populations of these species are drastically

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affected due to overcollection and severe perturbation of their habitats (Goettsch et al., 2015). People collect cacti for food, fodder, medicinal and ornamental plants and as a

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source of wood (Ortega-Baes et al., 2010; Goettsch et al., 2015). Thus, 140 Mexican cactus

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species are threatened and 32 species are Critically Endangered following IUCN Red List (Goettsch et al., 2015).

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Seed germination studies help conservation of this natural resource; the possibility of obtaining valuable plants through propagation could decrease the demand for wild-

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collected material (Flores et al., 2008). The knowledge of factors affecting germination is relevant in the propagation of wild species for conservation purposes (Khurana and Singh, 2001) and to understand germination ecology (Baskin and Baskin, 2014). The germination of seeds is one of most risky stages in the reproduction of cacti, many seeds do not

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germinate because they are killed by animals or pathogens, or because environmental conditions are – permanent or temporarily – unsuitable for plant species (Bregman and Graven, 1997).

Seed germination is inhibited when seeds are dormant; seed dormancy is a temporary failure or block of a viable seed to complete germination under physical conditions that normally favor this (Kucera et al., 2005). Seed dormancy is an adaptation to increase the 3

germination probabilities in a safe site and safe time and have success in the establishment of a seedling (Baskin and Baskin, 2014). Seed germination is a key aspect in plant biology and breaking seed dormancy is necessary for many cactus species to germinate (RojasAréchiga and Vázquez-Yanes, 2000). Seeds from several cactus species have physiological dormancy (Rojas-Aréchiga and Vázquez-Yanes, 2000). Some species need a period of after-ripening to break dormancy

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and the embryos have low growth potential, like Opuntia tomentosa (Orozco-Segovia et al.,

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2007), O. streptacantha, O. leucotricha and O. robusta (Delgado-Sánchez et al., 2011, 2013), Turbinicarpus lophophoroides and T. pseudopectinatus (Flores et al., 2008). Other

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species like O. compressa and O. macrorhiza need a stratification period to obtain high

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germination percentages (Baskin and Baskin, 1977).

Hormones such as gibberellic acid (GA3) play a key role in physiological dormancy

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release and in the promotion of germination in several plant species (Baskin and Baskin, 2004; Kucera et al., 2005). GA3 is involved in breaking seed dormancy in several cactus

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species (McDonough, 1964; Brencher et al., 1978; Zimmer and Buttner, 1982; Deno, 1994; Sánchez-Venegas, 1997). GA3 may act as a partial alternative for light requirement in Carnegiea gigantea and Lemaireocereus thurberi (Stenocereus thurberi) (McDonough, 1964), Gymnocalycium oursellianum (G. monvillei), Hamatocactus setispinus (Thelocactus Heliabravoa

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setispinus),

chende

(Polaskia

chende),

Pachycereus

hollianus

(Lemaireocereus hollianus), Rebutia marsoneri and R. minuscula (Brencher et al., 1978), Gymnocalycium mihanovichii, Eulychnia longispina (E. breviflora) and E. castanea (Zimmer and Buttner, 1982). Auxins are growth regulators and can also promote seed germination in some species (Kucera et al., 2005). Although auxin by itself is not a necessary hormone for seed 4

germination, according to the analyses regarding the expression of auxin related genes, auxin is present in the seed radicle tip during and after seed germination (Miransari and Smith, 2014). There are only studies of auxins in two cactus species, Ferocactus histrix and F. latispinus and no promoting effect was found (Amador-Alférez et al., 2013). However, Bacillus isolates from rhizosphere of cacti shorten germination time in Mammillaria magnimamma, these bacteria produce indole-3-acetic acid, an auxin (Chávez-Ambriz et al.,

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2016).

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Light has a stimulating effect on the germination of many cacti (Flores et al., 2006, 2011, 2016; Meiado et al., 2016). In some cactus species the light requirement is

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conditioned to seed washing prior to sowing, e.g. Melocactus curvispinus sp. caesius seeds

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when sown just after harvest did not germinate under light or dark conditions, but washing before sowing stimulated germination giving a 100% germination under light conditions

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(Arias and Lemus, 1984). Perhaps phenolics present in the seed testa inhibit germination (Debeaujon et al., 2000). However, seeds from other cactus species, e.g. Echinopsis

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thionanta and Gymnocalycium gibbosum, have a secretory layer (mucilage) that absorbs water and distributes it over the seed surface; a protein-enriched layer that appears to be hydrophilic surrounds the ripe seed and improves germination under relatively dry conditions (Bregman and Graven, 1997).

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We hypothesized that for some cactus species from Southern Chihuahuan Desert: i)

seeds with mucilage have higher germination percentage and lower mean germination time than seeds without mucilage, and ii) cactus seeds are positively photoblastic but phytohormones like gibberellic acid (GA3) and indole-3-acetic acid (IAA) promote germination in the dark.

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2. Materials and methods

2.1 Studied species

Species studied were Coryphantha maiz-tablasensis Fritz Schwarz ex Backeb, Echinocactus platyacanthus (Link & Otto), Ferocactus latispinus (Haw.) Britton & Rose,

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Ferocactus pilosus (Galeotti) Werderm. and Stenocereus queretaroensis (F.A.C. Weber)

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Buxb. These species were chosen because of their ornamental use (all species) and edible fruits (S. queretaroensis) and stems (E. platyacanthus, F. latispinus and F. pilosus). Two

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species, E. platyacanthus and F. pilosus, are specially protected and C. maiz-tablasensis is

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threatened in the framework of the environmental laws and regulations of Mexico (Semarnat, 2010). Three of these species, E. platyacanthus, F. latispinus and S.

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queretaroensis, are widely distributed in the Southern Chihuahuan Desert and in semiarid regions of South and Central Mexico and two species, C. maiz-tablasensis and F. pilosus,

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have a narrow distribution in the Southern Chihuahuan Desert (Guzmán et al., 2003). Three species, E. platyacanthus, F. latispinus and S. queretaroensis, are positively photoblastic (Flores et al., 2011) and two species, E. platyacanthus and F. pilosus, have seed hydration memory (Contreras-Quiroz et al., 2016). Germination of C. maiz-tablasensis

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seeds has not been evaluated.

The number of seeds produced by a single fruit of cactus can be enormous, sometimes

more than 1,000 seeds per fruit (Pilosocereus chrysacanthus), or just a few (1 to 5 seeds per fruit in Epithelantha sp. and Pereskia aculeata) (Rojas-Aréchiga and Vázquez-Yánes, 2000). Seed number of our species is less than 100 seeds per fruit for Coryphanta spp. (Loza-Cornejo et al., 2012), 171 seeds per fruit for E. platyacanthus (Jiménez-Sierra et al., 6

2007) and 922 seeds per fruit for S. queretaroensis (Ibarra-Cerdeña et al., 2005). The number of seeds per fruit of F. latispinus and F. pilosus is unknown. Cactus seed mass ranges from 0.046 to 16 mg (Flores et al., 2011). Seeds of all our studied species are small, e.g. E. platyacanthus (2.62 mg), F. latispinus (0.26 mg) and S. queretaroensis (2.27 mg; Flores et al., 2011) and F. pilosus (1.20 mg; Contreras-Quiroz et

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al., 2016). Seed mass of C. maiz-tablasensis is unknown.

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2.2 Seed collection

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We collected seeds from ripe fruits of these five species in the Southern Chihuahuan

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Desert, Mexico. Seeds were extracted from fruits inside of a laminar flow chamber and cleaned, and then they were dried at room temperature (21 – 23 ºC) and stored in paper

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2.3 Imbibition test

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bags, also at room temperature for one month until the onset of the experiments.

Three groups of 15 seeds per species were weighed dry. Seeds were placed in Petri dishes with 25 ml of distilled water and weighed each 10 minutes the first two hours, then

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each hour until constant weight, which was considered the maximum imbibition. We removed water from the Petri dishes before each weighing; more water was added after that. Due to lack of seeds, C. maiz-tablasensis was not used in this test. The data were expressed as imbibition percentage (IP = (seed imbibition weight – seed dry weight) * 100), following Lima and Meiado (2017).

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2.4 Seed germination experiment

An experiment was performed to evaluate the effect of mucilage (with and without it), light condition (light and darkness), and phytohormones (six concentrations of gibberellic acid (GA3) and indole-3-acetic acid (IAA)) on germination percentage and mean germination time, MGT (Ranal et al., 2009). A factorial experimental design was used for

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all experiments. There were three replicate dishes per treatment, with 10 seeds in each

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replicate. Seeds were set to germinate at 25 °C in a germination chamber (Lumistell ICP-19 d-c/iv, with about 38 µmol·m−2·s−1 cool white fluorescent light tubes). Seeds were placed in

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Petri dishes containing agar 1%. Germination (radicle protrusion) was monitored daily for

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‘light’ seeds and at the end of the 30-day incubation period for seeds in the dark. At the end of the incubation period, viability of un-germinated seeds was checked by cutting open

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each seed, to see if an embryo was present and looked healthy.

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2.4.1 Effect of phytohormones

For each phytohormone, gibberellic acid (GA3) and indole-3-acetic acid (IAA), we had

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six concentrations (0, 50, 100, 250, 500 and 1,000 mg l-1).

2.4.2 Effect of removal of mucilage

Rinsing in pure water was not an efficient method to remove the mucilage-forming layer in the seed test. Thus, we used sterilization to remove the mucilage according to López et al. (2011) method: washing with Tween 20 (2%) for 10 minutes in constant 8

agitation and 10 rinses using sterile distilled water, after washing with ethanol (100%) for 1 minute and posteriorly with sodium hypochlorite (6%) during 3 minutes and 5 rinses using sterile distilled water. Seeds were rinsed five times afterwards with sodium thiosulphate (2%) and finally with 10 rinses of sterile distilled water. Unsterilized seeds were used as control. To determine if seeds from the studied species have a secretory layer (mucilage), cresyl blue (1%) to stain the pectin part of the mucilage was used in five seeds per species

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randomly selected (Martini et al., 2008; Barrios et al., 2015). Two hours after, seeds with

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and without mucilage were sectioned to determine the presence of mucilage in the micropyle, in order to know if mucilage functions like a barrier for water entry.

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Unsterilized seeds (with mucilage) were stored in paper bags until used.

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To determine if mucilage was successfully removed, five randomly selected seeds with and without mucilage from each species were mounted on a double-sided adhesive carbon

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tape on metal pins to analyze the testa structure observed under a scanning electron microscope (QuantaTM 200, FEI, OR). Due to lack of seeds, C. maiz-tablasensis was not

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used in this test.

2.4.3 Effect of light treatments

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For light treatments, we used a 12-h daily photoperiod (hereafter ‘light’) and one in

continuous darkness (hereafter ‘dark’) in a germination chamber for 30 days. For incubation in dark conditions, Petri dishes were wrapped in double aluminium foil, as suggested by Baskin and Baskin (2014). The chamber was set at 25 °C following Nobel (1988) as the most suitable temperature for cacti. To reduce temperature fluctuations, fluorescent lamps and air ventilation were used. 9

2.5 Statistical analyses

Data obtained from imbibition test were analyzed using one-way ANOVA to determine differences between intact seeds (with mucilage) and with mucilage removed for each species. Germination percentages were transformed using arcsine of the square root to

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comply with ANOVA requirements (Sokal and Rohlf, 1994). Germination percentage and

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mean germination time (MGT) were obtained with the program GerminaQuant (Marques et al., 2015). A two-way ANOVA was performed for germination percentage and mean

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germination time (MGT), in which factors were GA3 concentrations and mucilage layer.

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Because most seeds in darkness were ungerminated, light was not included in the statistical analyses. IAA concentrations were also not included for the same reason. All ANOVA tests

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determined with Tukey tests.

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were carried out using STATISTICA 8. For all species, differences among treatments were

3. Results

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3.1 Mucilage data

A mucilage layer was found in all species (Fig. 1), and this mucilage covered the

micropyle (Fig. 2). The sterilization treatment removed the mucilage even from the micropyle (Fig. 2). We also observed that seeds without mucilage were internally more colored by cresyl blue than seeds with mucilage, except for F. latispinus (Fig. 1). >>insert Fig. 1 here 10

>>insert Fig. 2 here

3.2 Seed imbibition test

In general, seed imbibition was lower than 10% for four species (Fig. 3), independently of the treatment (with mucilage or without it). However, we found higher imbibition

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percentage in seeds with mucilage than in those without it for F. pilosus (F = 8.049, p =

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0.047) and F. latispinus (F = 20.425, p = 0.01; Fig. 3). Seed imbibition did not differ for different mucilage treatments of E. platyacanthus (F = 0.496, p = 0.52) or S. queretaroensis

>>insert Fig. 3 here

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(F = 0.374, p = 0.57).

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percentage

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3.3 Effect of phytohormones, removal of mucilage and light treatments on germination

Coryphanta maiz-tablasensis seed germination was affected by the interaction of mucilage × GA3 (Table 1). Seeds with mucilage at 50 mg/L of GA3 had lower germination percentage than all other combined treatments (Fig. 4A). Seeds germinated higher than

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90% with and without mucilage (Fig. 5). Seeds did not germinate in darkness and GA3 did not promote seed germination in darkness (data not shown). >>insert Table 1 here >>insert Fig. 4 here For E. platyacanthus germination percentage differed by mucilage layer and GA3, but the interaction of these factors was not significant (Table 1). Seed germination (%) was 11

about four times higher in seeds with mucilage than in seeds without it (Fig. 5) and higher at 500 and 1000 mg l-1 than at 50 and 100 mg l-1 (Table 2). Seeds did not germinate in darkness and GA3 did not promote seed germination in darkness (data not shown). >>insert Table 2 here Germination percentage for F. latispinus differed by mucilage layer and GA3, and the interaction of these factors was significant (Table 1). Seed germination percentage was

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higher in seeds with mucilage than in seeds without it for this species (Fig. 5) and higher at

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1000 mg l-1 of GA3 than at the control and at the other concentrations (Table 2). Seeds with mucilage had higher germination at 0, 50, 100 and 250 mg l-1 of GA3 than seeds without

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mucilage, but at 500 and 1000 mg l-1 germination was high with and without mucilage (Fig.

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6). Seeds did not germinate in darkness and GA3 did not promote seed germination in

>>insert Fig. 5 here

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darkness (data not shown).

Germination percentage for F. pilosus was only affected by GA3 (Table 1). Gibberellic

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acid did promote seed germination in light, because seed germination was higher at 1000 mg l-1 of GA3 than at control and 50 mg l-1 (Table 2). Seeds with and without mucilage had similar germination percentage (Fig. 5). Seeds did not germinate in darkness and GA3 did not promote seed germination in darkness (data not shown).

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For S. queretaroensis, germination percentage differed by mucilage layer, but not for

GA3 or the interaction of these factors (Table 1). Seed germination percentage was about 50 times higher in seeds with mucilage than in seeds without it for this species (Fig. 5). GA3 did not promote seed germination in light, but in dark conditions there was higher germination (above 60%) at 1000 mg l-1 of GA3 (data not shown). Seeds without mucilage of this species had very low germination. 12

For all species, most un-germinated seeds were viable after experiment.

3.4 Effect of phytohormones, removal of mucilage and light treatments on mean germination time (MGT)

MGT for C. maiz-tablasensis differed by mucilage layer and GA3 treatment and the

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interaction of these factors was significant (Table 1). Removal of mucilage resulted in

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faster germination in the presence of GA3 compared to control (Fig. 4B). Coryphanta maiztablasensis presented shorter MGT at 250 and 500 mg l-1 of GA3 than at the other

>>insert Table 3 here

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treatments (Table 3).

For E. platyacanthus and F. latispinus MGT was only affected by mucilage layer

>>insert Fig. 6 here

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removed (Fig. 7).

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(Table 1) in that seeds with mucilage germinated faster than those where this layer was

MGT for F. pilosus differed by mucilage layer and GA3 treatment, but the interaction of these factors was not significant (Table 1). This species presented faster MGT at 1000 mg l1

of GA3 than at the other treatments (Table 3). Seeds with mucilage germinated faster than

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seeds without mucilage (Fig. 7).

>>insert Fig. 7 here

For S. queretaroensis, MGT differed by mucilage layer, but not for GA3 or the

interaction of these factors (Table 1). Seeds without mucilage showed very low germination (Fig. 7).

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4. Discussion Seeds from the five studied species have a surrounding secretory layer of mucilage that has the ability to retain water (seeds had higher imbibition) in two species, F. pilosus and F. latispinus. Thus, our results partially support Bregman and Graven (1997) who found that mucilage layer in the cactus seed coat can function retaining water and, therefore, facilitating the passage of water into the seed.

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Because we also found that mucilage covers the micropyle and that seeds without

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mucilage were internally more colored than seeds with mucilage in most species, we suggest that mucilage layer in the micropyle can function like a barrier regulating the

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passage of water to the inner seed.

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The first hypothesis that seeds with mucilage have higher germination percentage and lower mean germination time than seeds without mucilage was in part corroborated. Seeds

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with mucilage germinated in higher percentage for E. platyacanthus and S. queretaroensis. However, for these species mucilage made no difference to imbibition. Perhaps mucilage

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might contain micro-organisms involved in promoting seed germination, as found for Opuntia spp. (Cactaceae) testa seeds by Delgado et al. (2011, 2013). Attack by microorganisms reduces mechanical resistance of the testa of Opuntia seeds, making it easier for the embryo to emerge (Delgado et al., 2011, 2013). In other desert plants, seed mucilage

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and soil microbial community interactively promote successful establishment of seedlings (Hu et al., 2019). Intact seeds of E. platyacanthus, F. latispinus and F. pilosus germinated faster than

seeds without mucilage, while S. queretaroensis did not show germination in seeds without mucilage. Our findings for these species are in agreement with Bregman and Graven (1997) who found intact seeds from two South American species (Echinopsis thionantha and 14

Gymnocalycium gibbosum) to take up significantly more water and germinate better than seeds for which the hydrophilous layer had been artificially removed. In contrast, seed germination percentage from C. maiz-tablasensis was not reduced by removal of the mucilage layer, except in GA3 at a concentration of 50 mg l-1, which had a slightly lower germination in seeds with mucilage. However, this low germination was due to fungal contamination. In addition, shorter MGT in seeds without mucilage than in seeds

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with it was found. Perhaps C. maiz-tablasensis seeds do not need to retain water in the

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mucilage, because this species inhabits flat areas subject to flooding (Dicht and Lüthy, 2005) and seeds from this species have more access to water than seeds from non-flooded

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areas.

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The second hypothesis that cactus seeds are positively photoblastic (they need light to germinate), but phytohormones, as gibberellic acid (GA3) and indole-3-acetic acid (IAA)

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promote germination in the dark was not supported by our findings. Although all species were positively photoblastic, phytohormones did not promote germination in the dark,

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except for S. queretaroensis, in which GA3 did not promote seed germination in light, but in dark conditions there was higher germination (above 60%) at 1000 mg l-1 of GA3. These results are in agreement with findings of GA3 promoting seed germination in darkness for several cacti as Gymnocalycium oursellianum, Hamatocactus setispinus, Polaskia chende,

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Pachycereus hollianus, Rebutia minuscula and R. marsoneri (Brencher et al., 1978). Indeed, the effect of phytohormones did depend on the phytohormone type and on the species. We found that GA3 did promote seed germination in three species, but IAA did not affect them. Our results of no germination with IAA coincide with findings by AmadorAlférez et al. (2013) for Ferocactus histrix and F. latispinus.

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Ferocactus latispinus was the only species for which seeds without mucilage increased germination with high concentrations of GA3 (500 and 1000 mg l-1). Perhaps sterilized seeds lost microorganisms involved in promoting seed germination and high concentrations of GA3 replaced the effect of microbes, promoting germination. We also found that GA3 was an important factor under light conditions in two species. For F. latispinus, seed germination was higher in light at 500 and 1,000 mg l-1 of GA3 than

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at other concentrations. For F. pilosus, seed germination was higher in light at 1,000 mg l-1

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of GA3 than at other concentrations. Our results for GA3 in light agree with findings for other cacti, e.g. McDonough (1964) for Carnegiea gigantea and Stenocereus thurberi,

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Sánchez-Venegas (1997) for Opuntia joconostle, Brencher et al. (1978) for Gymnocalycium

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oursellianum and Polaskia chende, Zimmer and Buttner (1982) for Myrtillocactus geometrizans, Mammillaria ritteriana, Arequipa erectocylindrica, Eulychnia longispina

wislizeni.

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and Eulychnia castanea, and Deno (1994) for Ferocactus acanthodes and Ferocactus

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In conclusion, seeds from the five studied species have a surrounding secretory layer of mucilage and the effect of mucilage on germination depends on the species. Mucilage resulted in higher germination percentage in E. platyacanthus, F. latispinus and S. queretaroensis, as well as a lower mean germination time in E. platyacanthus, F. pilosus

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and F. latispinus. The effect of GA3 on cactus seed germination also depends on the species. Since seeds without mucilage were internally more colored than seeds with mucilage in most species, we suggest that the mucilage layer in the micropyle can function like a barrier regulating the passage of water to the inner seed. This is the first study including the effect of mucilage on germination of North American cactus species. The

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relationship between mucilage, seed microbiota and seed degradation in these species remains to be tested.

CRediT (Contributor Roles Taxonomy)

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Joel Flores: Conceptualization, Supervision. Ernesto Mascot-Gómez: Investigation, Formal analysis, Writing- Original draft preparation. Nguyen E. López-Lozano: Resources, Writing- Reviewing and Editing. Laura Yáñez-Espinosa: Methodology, Visualization

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Declaration of interests

Acknowledgements

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☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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This research was supported by Secretaría de Educación Pública – Consejo Nacional de Ciencia y Tecnología (No. CB-254406) and FORDECYT – Consejo Nacional de Ciencia y

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Tecnología (296354). E. Jurado made useful suggestions to this manuscript.

References

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Figure legends

Figure 1. (A) Mucilage formation and staining results with cresyl blue around seeds from five cactus species. (B) Cactus washed seeds (without mucilage) showing that cresyl blue did enter to inner seeds. (C) Cactus unwashed seeds (with mucilage) showing that cresyl blue did not enter to inner seeds and that mucilage layer can function in retaining water

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and, therefore, facilitating the passage of water at the inner seed. Arrows indicate the more

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colored area by cresyl blue. Bar is 1 mm.

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Figure 2. Photomicrographs of mucilage removed from the micropyle in Echinocactus platyacanthus (A), Ferocactus latispinus (C), Ferocactus pilosus (E) and Stenocereus queretaroensis (G); and mucilage covering the seed micropyle in E. platyacanthus (B), F. latispinus (D), F. pilosus (F) and S. queretaroensis (H). Photomicrographs were obtained with an environmental scanning electron microscope.

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Figure 3. Seed imbibition percentage of four species (Echinocactus platyacanthus, Ferocactus latispinus, Ferocactus pilosus and Stenocereus queretaroensis) without mucilage (black column) and with mucilage (gray column). Different letters indicate

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statistical differences (p < 0.05) between means within each species.

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Figure 4. (A) Seed germination and (B) Mean germination time (MGT) of Coryphantha

maiz-tablasensis under combined mucilage and GA3 treatments. Different letters indicate

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statistical differences (p < 0.05) between means of all treatment combinations.

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Figure 5. Germination percentage (%) of five species (Coryphantha maiz-tablasensis, Echinocactus platyacanthus, Ferocactus latispinus, Ferocactus pilosus and Stenocereus

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queretaroensis) without mucilage (black column) and with mucilage (gray column). For each species, different letters indicate statistical differences (p < 0.05) between means

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within each species.

Figure 6. Seed germination of Ferocactus latispinus under combined mucilage and GA3 treatments. Different letters indicate statistical differences (p < 0.05) between means. Figure 7. Mean germination time (MGT) of five species (Coryphantha maiz-tablasensis,

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Echinocactus platyacanthus, Ferocactus latispinus, Ferocactus pilosus and Stenocereus queretaroensis) without mucilage (black column) and with mucilage (gray column). For each species, different letters indicate statistical differences (p < 0.05) between means within each species. Asterisk indicates no seed germination.

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Table 1. F and p values from ANOVA for five cactus species under combined mucilage and GA3 treatments. Mean Germination Germination (%) Time (MGT) Coryphantha maiz-tablasensis F(5,24) = 2.10, p = 0.10

F(5,24) = 4.42, p < 0.01

Mucilage layer

F(1,24) = 0.50, p = 0.48

F(1,24) = 139.87, p < 0.01

GA3 × Mucilage layer

F(5,24) = 2.90, p = 0.03

F(5,24) = 6.78, p < 0.01

GA3

F(5,24) = 7.71, p < 0.01

F(5,24) = 2.45, p = 0.06

Mucilage layer

F(1,24) = 662.47, p < 0.01 F(1,24) = 87.98, p < 0.01

GA3 × Mucilage layer

F(5,24) = 2.19, p = 0.08

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GA3

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Echinocactus platyacanthus

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Ferocactus latispinus

F(5,24) = 1.75, p = 0.15

F(5,24) = 16.18, p < 0.01

Mucilage layer

F(1,24) = 216.02, p < 0.01 F(1,24) = 46.07, p < 0.01

GA3 × Mucilage layer

GA3

F(5,24) = 22.58, p < 0.01

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Ferocactus pilosus

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GA3

F(5,24) = 0.38, p = 0.85

F(5,24) = 0.68, p = 0.63

F(5,24) = 4.39, p < 0.01

F(5,24) = 5.77, p < 0.01

Mucilage layer

F(1,24) = 1.95, p = 0.17

F(1,24) = 8.10, p < 0.01

GA3 × Mucilage layer

F(5,24) = 1.29, p = 0.29

F(5,24) = 1.30, p = 0.29

GA3

F(5,24) = 0.64, p = 0.66

F(5,24) = 7.70, p = 0.01

Mucilage layer

F(1,24) = 756.28, p < 0.01 F(1,24) = 0.77, p = 0.57

GA3 × Mucilage layer

F(5,24) = 0.02, p = 0.99

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Stenocereus queretaroensis

F(5,24) = 0.59, p = 0.70

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(p < 0.05) between means within each species according to Tukey’s HSD test.

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Table 2. Seed germination under different concentrations of GA3 in the study species. Different letters indicate statistical differences

Coryphantha GA3 (mg

l-1)

maiz-tablasensis

Echinocactus platyacanthus

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________________________________________________________________________________________

Ferocactus latispinus

Ferocactus pilosus

Stenocereus queretaroensis

100 ± 0.0 a

54.44 ± 17.67 ab 61.11 ± 16.64 c 55.00 ± 6.71 b 95.55 ± 2.22 a

50

97.22 ± 1.26 a

46.67 ± 16.06 b 54.44 ± 13.71 c 56.66 ± 9.55 b 97.77 ± 2.22 a

100

98.61 ± 1.39 a

43.33 ± 15.56 b 57.78 ± 14.47 c 71.66 ± 3.07 ab 97.77 ± 2.22 a

250

100 ± 0.0 a

56.66 ± 16.58 ab 67.78 ± 9.49 c 73.33 ± 4.22 ab 95.55 ± 2.22 a

500

100 ± 0.0 a

1000

100 ± 0.0 a

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60.00 ± 14.4 a

67.77 ± 11.85 a

80.00 ± 3.44 b 66.66 ± 3.33 ab 93.33 ± 6.66 a 88.89 ± 4.77 a

85.00 ± 3.42 a 97.77 ± 2.22 a

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________________________________________________________________________________________

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Table 3. Mean germination time (MGT) under different concentrations of GA3 in the study species. Different letters indicate statistical

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differences (p < 0.05) between means within each species according to Tukey’s HSD test.

_________________________________________________________________________________________________________________

100 4.61 ± 0.54 a

250 3.64 ± 0.67 b

500 3.68 ± 0.68 b

1000 3.76 ± 0.58 ab

13.46 ± 1.59 a 16.60 ± 2.58 a 17.04 ± 2.84 a 13.87 ± 2.33 a 15.09 ± 3.07 a 12.10 ± 1.93 a

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Echinocactus platyacanthus

50 4.61 ± 0.47 a

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Species / Treatment (mg l-1) 0 Coryphantha maiz-tablasensis 4.08 ± 0.24 ab

10.85 ± 1.62 a 10.60 ± 1.33 a 11.78 ± 2.13 a 11.64 ± 2.17 a 10.50 ± 1.72 a 10.02 ± 1.22 a

Ferocactus pilosus

17.33 ± 1.51 ab 18.24 ± 1.17 a 15.93 ± 1.07 ab 14.37 ± 0.93 b 13.71 ± 0.88 bc 11.90 ± 1.05 c

Stenocereus queretaroensis

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Ferocactus latispinus

6.67 ± 0.65 a

6.72 ± 0.24 a

6.14 ± 0.14 a

6.00 ± 0.60 a

5.71 ± 0.16 a

6.27 ± 0.60 a

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