The influence of pre-treatment on germination of three species of Catasetum (Orchidaceae)

Abstract: Asymbiotic germination techniques are successful in species of the Catasetum genus. The diverse composition of the culture media can be critical; however, viable seeds are required for asymbiotic germination. The reliable method for viability assessment is the tetrazolium test. A decisive step in the efficiency of the tetrazolium test is the seeds preconditioning with sucrose solution, which has the potential to improve germination. Therefore, this work aimed to evaluate the seed germination of three species of the genus Catasetum in culture media, preconditioned or not with sucrose solution. Seeds of the species Catasetum osculatum, Ctsm. galeritum and Ctsm. complanatum were assessed by tetrazolium test and germinated in two different media, pretreated or not with 10% sucrose solution. The statistical analysis showed that the use of sucrose pretreatment significantly increased the germination rates of the species. We conclude that the pretreatment with 10% sucrose for 24 hours, regardless of the species and the culture medium, significantly increases the germination of Ctsm. complanatum, Ctsm. galeritum and Ctsm. osculatum seeds.


INTRODUCTION
The species within the Orchidaceae family have low occurrence in populations and are restricted to small forest fragments (Daviña et al., 2007).The disturbance of these habitats and the indiscriminate collection placed many species at risk (Hirano et al., 2005).The genus Catasetum comprises 121 species, of which 92 are endemic to Brazil (Petini-Benelli, 2022), all suffering from continuous habitat destruction, unbalancing the flowering and fruiting percentages along with other genera (Cardoso et al., 2016;Petini-Benelli, 2016).Most of the Catasetum species have no information on the threat status, making a real assessment of its situation impossible (Petini-Benelli, 2022).
To avoid the over collection of plants from natural populations, since the germination rate in nature is low (< 5%), one solution is to produce high numbers of plants through asymbiotic germination techniques (Arditti and Ernst, 1993;Utami and Hariyanto, 2020) and turn them easily available to the consumers (Seaton and Pritchard, 2011).With this methodology, in vitro germination, it is possible to supply carbon to the embryo to fulfill the energy needs that would be provided in nature by mycorrhizal fungi (Zhang et al., 2013).
Germination occurs differentially among orchid species.For epiphytic species such as Cattleya (Hengling et al., 2020;Fileti et al., 2021), the embryo swells, breaks up the testae, and turns green.For terrestrials, the embryo swells, fulfils the testae before breaking it up and stay white for long, emitting rhizoids and leaf primordia before becoming green (Seaton and Ramsay, 2005).Catasetum species, even an epiphytic genus, follows the germination pattern of the terrestrials.Although many authors show prominent results for asymbiotic germination in the Catasetum genus (Ferreira et al., 2018;Zakizadeh et al., 2019), for the species Ctsm.galeritum, Ctsm.osculatum e Ctsm.complanatum, Brazilian endemics (Petini-Benelli, 2022), which exist in environments subjected to high anthropic pressure, has no data available until now.Some authors point out that the composition of the medium can be critical to achieving high germination rates, as the formulation can be specific to the genus or even the species (Suzuki et al., 2012;Diantina et al., 2020).
The different formulations and compound quantities, as well as the nutrients available (organic are crucial for germination in different species.Determining an ideal medium can improve large-scale production and contribute to the conservation of threatened species (Suzuki et al., 2012).
The culture media are diverse, and the MS medium is the most common (Murashige and Skoog, 1962).Another important medium, recommended especially for terrestrial species, is the MM, Malmgren medium (Malmgren, 1996), which effectiveness was not yet evaluated for the germination of the Catasetum genus.The main difference is the presence of nitric nitrogen in the high concentrations in the MS medium, which has an inhibitory effect in the germination of some terrestrials (Ponert et al., 2013;Figura et al., 2020), in the Malmgren medium nitric nitrogen is absent.
Nevertheless, viable seeds are necessary for asymbiotic germination.The most common, practical, and reliable method is the tetrazolium test, which has the potential to assess seed viability and quality (Hosomi et al., 2011(Hosomi et al., , 2012;;Vudala and Ribas, 2017).A decisive step in the efficiency of the tetrazolium test is the seeds preconditioning with sucrose solution.This stage aims to hydrate evenly and activate the embryo's metabolism (George et al., 2008).To increase seed germination, this step would be of good improvement for the germination rates.
Therefore, this work aimed to evaluate if the pretreatment step with sucrose before seed germination of three species of the genus Catasetum in culture media can increase germination rates.

Seed Collection and storage
Seeds of Ctsm.osculatum K.G.Lacerda and V.P.Castro, Ctsm.galeritum Rchb.f. and Ctsm.complanatum F.E.L.Miranda and K.G.Lacerda (Table 1) were produced by crossings, as this genus exhibits sexual dimorphism in Bela Vista Orchids (Assis -SP, -22.67299 -50.41754).At dehiscence, seeds were removed from capsules, put in paper envelopes and these were kept over silica gel for three days at 25 °C.They were then transferred to cryotubes and stored at -18 °C, according to the OSSSU protocol (Orchid Seed Science and Sustainable Use Seed Bank (Seaton and Pritchard, 2008), at the Universidade do Oeste Paulista (UNOESTE -Presidente Prudente -SP).

Seed viability
Initial viability was assessed using the tetrazolium test, according to Hosomi et al. (2011).Portions of 5 mg seeds of each species were placed in microtubes at room temperature for 15 minutes, and then add 1.0 mL of 10% sucrose solution and kept for 24 hours at room temperature.After this period, the liquid was removed, and the seeds were rinsed twice with distilled water, followed by the addition of 1.0 mL of 1% tetrazolium salt (2,3,5 triphenyl tetrazolium chloride) solution and kept in a water bath at 40 °C for 24 hours.Finally, the solution was removed, and the seeds were rinsed with distilled water.
The seed samples were placed on glass slides to obtain the images.The seeds with red embryos were considered as viable, while those with white embryos were non-viable.Empty seeds without an embryo, were not considered.

Seed germination
Each species was divided into two subsamples for germination: one was pretreated for 24 hours in a 10% sucrose solution, and the other did not receive any treatment.Samples were placed in microtubes at room temperature before the pretreatment.All subsamples were disinfected with 3% sodium dichloroisocyanurate solution (DCCA), added with Tween 80 for 10 minutes in a laminar flow chamber, and shaken occasionally (Machado-Neto and Custódio, 2005).After this period, samples were rinsed twice with sterile water and distributed in Petri dishes containing nutrient medium.
The chosen media for germination were the half concentration MS media, with 1% agar and 2% sucrose, and the MM medium described by Malmgren plus MS vitamins, 1% agar and 1% sucrose.The pH for both media was 5.7 before autoclaving at 121 °C for 15 minutes and subsequently were distributed in 60 mm Petri dishes.
Pretreated and not-pretreated seeds were sowed in a laminar flow chamber according to Machado-Neto and Custódio (2005).Three Petri dishes were used per treatment, sealed with PVC film, and transferred to a growth room at 25 ± 3 °C with a 16 h light / 8 h dark.Germination was evaluated weekly in nine selected areas containing at least 30 seeds per area.Only seeds whose embryos were in stage 1 of germination, meaning swelled and rhizoid protrusion (Zettler and Hofer, 1998;Seaton and Ramsay, 2005;Pereira et al., 2011) were counted as germinated; the evaluation continued until germination stabilized.A camera attached to a stereoscope microscope obtained high-definition quality images for evaluation in Gnu Image Manipulation Program.
The germination speed index (GSI), described by Maguire (1962) and modified by Hosomi et al. (2012) for orchids, was also evaluated, as in the following formula: Where G is the number of germinated seeds per period, and N is the time in days after germination.Germination is evaluated weekly; therefore, the time intervals were 7, 14, 21, and so forth until germination stabilization.

Experimental design and statistical analysis
The work was carried out in triplicate in a completely randomized design with a triple factorial arrangement of species x preconditioning x culture medium (3 x 2 x 2).
Normality and homogeneity of the results were initially checked for the germination and GSI data so that it could be subjected to analysis of variance (ANOVA).Then the means were compared by the Tukey test at 5% probability.Statistical analysis was made using the software SISVAR 5.7 (Ferreira, 2011).

RESULTS AND DISCUSSION
Viability was assessed utilizing the tetrazolium test by the methodology proposed by Hosomi et al. (2011Hosomi et al. ( , 2012) ) (Figure 1).We could observe seeds with viable embryos stained in red (Figure 1, red arrows); non-viable embryos were not stained (white arrow, Figure 1) and empty testa (Figure 1, black arrows).The observed results shown that viability was 44.13% for Ctsm.complanatum, 21.13% for Ctsm.galetirum and 21.95% for Ctsm.osculatum.Although not considered for the viability test, we detected in the total amount of seeds 53.64% of empty testae for Ctsm.osculatum, 28.07%for Ctsm.complanatum, and 5.73% for Ctsm.galeritum (Table 2 and Figure 1).Journal of Seed Science, v.45, e202345010, 2023 The analysis of the triple factorial of species, pretreatment, and culture medium showed no interaction between them (Table 3).Only the use of sucrose pretreatment significantly increased the germination rate (Table 3 and Figure  2).However, there was no difference in comparing the performance of the culture media MS and MM, as observed in not pre-treated seeds of Cyrtopodium aliciae (Oliveira et al., 2023).
Even though there is a difference between MS and MM media in not treated seeds (1.13% and 8.84%), the observed germination in pretreated seeds does not differ in both used media (Figure 3).The GSI supports the germination and media average, as the observed Speed Index from the pretreated seed is higher than those form the not pretreated seeds (Table 4).
Actual germination occurred from the second week of incubation, when seeds reach stage 1 of germination according to Pereira et al. (2011) and Zettler and Hofer (1998) in pretreated seeds of Catasetum galeritum, third week in Ctsm.complanatum and fourth week in Ctsm.osculatum.After the fourth week of cultivation, it was possible to observe germinated seeds in both pretreated and not pretreated seed of Ctsm.complanatum, fifth week of Ctsm.galeritum and seventh week of Ctsm.osculatum.Development was followed until protocorms reached stage 3 proposed by Zettler and Hofer (1998).These results imply that regardless of the species and the culture medium, the    use of preconditioning with a 10% sucrose solution for 24 hours before sowing was of great value for the germination process (Figure 2).The GSI corroborates these results, exhibiting higher values in pretreated seeds.Deconninck and Gerakis (2021) presoaked Anacamptis laxiflora seeds with 10% sucrose solution, but for microorganism growth and increase sterilization susceptibility not for germination.
Comparing the pretreated seeds to not pretreated seeds, the observed increase in germination was 6.8 times for Ctsm.osculatum seeds, 9 times for Ctsm.galeritum up to 12.2 times for Ctsm.complanatum.This may be due to the osmoregulatory activity of the sucrose solution, reducing the osmotic potential and delaying the absorption of water by the seeds, avoiding damage to the embryo during hydration and activating the germination metabolism even before contact with the nutrient medium (Li et al., 2005;George et al., 2008;Hosomi et al., 2011).
According to Manning and Van-Staden (1987), a simple sugar exogenous source from the culture medium, would facilitate the energy cell machinery, supplying the enzyme system to activate the embryo germination.The pretreatment with sucrose 10% probably initiates the energy cascade, as imbibition occurs.
Usually, culture media with amino acids as a source of nitrogen are indicated for the germination of terrestrial orchids (Ponert et al., 2013;Figura et al., 2020) and some authors suggest that seeds and protocorms assimilate nitrogen more easily from the amino acids form than from nitric nitrogen which must be converted into amino acids before plant absorption (Malmgren, 1996;Majerowicz et al., 2000;Kauth et al., 2008;Gupta, 2016).Studies with Ctsm.fimbriatum demonstrated its preference for ammoniacal nitrogen form over nitric form or amino acids (Majerowicz et al., 2000).However, the results here are promising, with no difference in average germination rates between MM and MS medium.Comparing the usage of pretreatment, the observed germination comparison shows an increase in 4.5 times for MM and 39.5 times in MS medium, demonstrating that the use of sucrose 10% independent of the species was efficient.Ferreira et al. (2018) proposed that the culture media are specific to the Ctsm.macrocarpum development stage.The ease of having seeds and plants available to evaluate the different asymbiotic conditions increase the capacity of reintroduced species in nature (Kitsaki et al., 2004).
The tetrazolium test presented inferior results than those observed in actual germination using pretreatment with 10% sucrose solution, which may indicate that despite being a good predictor of viability, the test does not substitute quantification for in vitro asymbiotic germination, or it might need adjustments to the Catasetum genus.

CONCLUSIONS
The pretreatment with 10% sucrose for 24 hours, regardless of the culture medium, significantly increases the germination potential of Ctsm.complanatum, Ctsm.galeritum and Ctsm.osculatum seeds.

Table 1 .
Catasetum species, their place of origin and conservation status.

Table 2 .
Tetrazolium test (TZ) results and empty testae percentages of Catasetum species.Values expressed as means and standard error.
*Empty testae percentages were based in the total count of seeds.

Table 3 .
Germination average (%) of Catasetum in MS and MM media, with and without pretreatment and their interactions.
Means followed by equal letters do not differ by Tukey's test at 5% probability.*Significant at 1% probability by the F test.CV: coefficient of variation.

Table 4 .
Germination (G%) and germination speed index (GSI) of Catasetum species with and without pretreatment in MS and MM media.Values expressed as means and standard error.Means followed by the same letter, lowercase for column, uppercase for line, do not differ in Tukey's test at 5% probability.