Spores germination and gametophytes of Alsophila odonelliana (Cyatheaceae) in different sterile media

Resumen. El helecho arbóreo Alsophila odonelliana crece desde el sur de Bolivia hasta el Noroeste de Argentina, en la selva tucumanoboliviana. Se evalúa la germinación de las esporas en tres medios nutritivos estériles, Dyer (D) Murashige & Skoog (MS) y Knudson (K), y el efecto sobre el desarrollo gametofítico de la adición al medio de cultivo Dyer, de dos reguladores de crecimiento, BAP y 2,4 D. El efecto del 2,4-D fue evaluado en dos concentraciones: 0,1 y 2,0 mg/L, y para BAP se utilizaron tres concentraciones, 0,01; 0,1 y 1,0 mg/L. No se encuentran diferencias significativas en la germinación de las esporas en los tres medios de cultivo. Se observa que el suplemento del 2,4 D conduce al desarrollo de gametofitos filamentosos cortos y formas laminares amerísticas; el suplemento de BAP afecta el crecimiento en longitud de los gametofitos filamentosos y la proliferación de ramificaciones desde etapas tempranas. Ambos reguladores de crecimiento afectan la estructura de los gametangios masculinos. El efecto del 2,4 D no produce embriogénesis somática como en otras plantas.


INTRODUCTION
Cyathaeceae is a family of tree ferns with approximately 500 species (Korall et al., 2006), growing in mountainous regions of the Pelotropic and Neotropics.Alsophila odonelliana (Alston) Lehnert is found in southern Bolivia and Northwestern Argentina (Lehnert, 2005) in the Tucuman-Bolivian forests, where it grows in humid ravines, on gullies, margins of rivers, etc.
Sexual reproduction in Cyatheaceae has been confirmed by Conant (1990), Khare et al. (2005), Cheng et al. (2008), and Chen et al. (2008).However, during three years we have tried to grow Alsophila odonelliana sporophytes on in vitro cultures, unsuccessfully.The lack of positive results prompted us to experiment different culture media to assess spore germination, and the response of gametophytes to growth regulators.
The addition of growth regulators to sterile culture media for the study of fern gametophytes is not a usual practice.The results of these treatments show that 2,4-Dichlorophenoxiacetic ammonium (2,4-D) inhibits the growth of gametophytes in Pteris longifolia L. (Strickler, 1946); gibberellin and benzylaminopurine (BAP) accelerate germination of stored spores of Pteris inermis (Rosenst.)de la Sota (Tanco et al. 2009).Menéndez et al. (2009) found that exogenous and endogenous cytokinins of female gametophytes of Blechnum spicant (L.) Roth in the culture media induce the formation of male gametophytes in this species; Furelli & García (1987) obtained calli of Pteris cretica L. from explants of young leaves by adding 2 mg/L 2,4-D and 2 mg/L BAP to the culture medium.
The aims of this work were to evaluate (1) the germination of A. odonelliana spores in three culture media, Dyer (D), Murashige & Skoog (MS) and Knudson (K) with the purpose of determining which of these three substrates is the optimal, and (2) the effect of the addition of two growth regulators (BAP and 2, to the culture medium, on the development of the gametophytic phase of this fern.
We hope to provide information on the action of BAP cytokinin and 2,4-D auxin on the gametophyte development of A. odonelliana, and the use of different culture media in the germination of fern spores, as a practical contribution for fern conservation projects.
Spores were collected after natural dehiscence of sporangia in stove at 30°C, and stored for 16 months.The dimensions of the spores were calculated from a 50-spore sample from mature sporangia.Equatorial and polar diameters were measured and standard deviations (SD) calculated.
For in vitro cultures, spores were cold-stored at 4 ± 2°C, sterilized using 5% sodium hypochlorite plus 2 drops of Tween for 2 minutes under continuous stirring, and then washed three times with sterile distilled water.
The germination of spores is a gradual process taking several weeks according to the species; therefore, we made observations at 60 and 120 days to compare and evaluate the germination progress.The emission of a rhizoid was considered a positive germination.
In order to study the effect of different culture media on the germination of spores, we used three nutritive media, Dyer (D), Knudson (K) and Murashige & Skoog (MS), solidified through the addition of 7 g/L Britania agar.Cultures were kept in a growth chamber at 25 ± 3 °C under a 12:12 L/D photoperiod.
We evaluated the effect of growth regulators on the morphogenesis of gametophytes using Dyer medium supplemented with 2,4-D in two concentrations: 0.1 and 2.0 mg/L, and BAP in three concentrations, 0.01, 0.1 and 1.0 mg/L.We employed a completely randomized factorial design.One of the factors studied was each medium, with growth regulators at their respective concentrations.Gametophytes were grown in 12 flasks, 5 cm high by 4 cm diameter, for each treatment.The other study factor was the time for spore germination.Cultures were maintained during twenty months.
The variable analyzed was the percentage of germination.For each observation four quadrants were considered per flask, and the records were the average of ten counts per quadrant.The effect of the culture medium and the time of germination, as well as their interactions, were analyzed using ANOVA, and the significance level was set at 5%.Data were analyzed using InfoStat ver.2008.
For photographing under a light microscope, gametophytes were dyed with aceto-carmine chloral hydrate (Edwards & Miller, 1972) and mounted in water; microphotography samples were fixed with glutaraldehyde in phosphate solution.All Gametophytes of Alsophila odonelliana observations, illustrations and photographs were made using a Hitachi S-3000 and a Standard 16 Zeiss light microscopes, and a JEOL JSM 6480LL Scanning Electron Microscope, property of Universidad Nacional de Salta (Argentina).
From a 300-spore sample, we found that 94% were viable, whereas the remaining 6% lacked cellular content or were very small-sized.Effect of the culture medium on the germination of spores.The germination of spores began 9-12 days after sowing.Although sixty days after sowing we found a 26% germination in MS medium, 34% in D and 43% in K, after 120 days we found that all three media had the same effect on the germination of spores, with differences of less than 9% among them (Fig 2).
At the end of the experiment, no significant differences among the culture media Dyer (D), Murashige & Skoog (MS) and Knudson (K) were found.

Gametophytes of Alsophila odonelliana
The addition of growth regulators to the culture media did not affect the germination of spores.None of the growth regulators in any of the study concentrations induced a significantly different average percentage of germination (p=0.73).The interaction between growth regulators and time was also not significant (p=0.83).However, the ANOVA showed that the average percentage of germination was significantly higher at 120 days (p<0.0001)(Fig 3).

Effect of growth regulators on the gametophytic morphogenesis.
The qualitative results showed different effects of the growth regulators on the gametophytes.

Gametophytes of Alsophila odonelliana
Laminar phase: growth was slow, and only 50% achieved chordate form.Some laminar forms emitted projections from the margin (Fig. 4 D, E).Most mature gametophytes, about 80% do not form notches, lost chlorophyll over time, turning pale to ocher in color, with few gametangia, and most by poor antheridia.
Laminar phase: BAP produced a fast longitudinal development, each ramification of the filamentous gametophyte originated a laminar gametophyte, forming colonies of gametophytes (Fig. 4 F, 5 A-C).The mature gametophytes originate numerous submarginal proliferations that in turn developed other laminar gametophytes (Fig. 6).The maintenance of meristematic activity led to the formation of lobules in the wings (Fig. 7 A).
The mature gametophytes, in BAP, showed increased volume of the central ridge (Fig. 7 B-C), sometimes generating 1-2 ramifications, each emitting marginal proliferations.The lamellae of mature gametophytes exhibit a characteristic bright green color.Male gametophytes dominated over those bisexual and sterile ones.The antheridia evidence certain deformations compared to those originated in control media (Fig. 7 D-F).Male and female gametangia maintained their structure, although their cells noticeably increased in size, leading to deformations.Twenty months after sowing of spores, no calli or sporophytes were observed.

DISCUSSION
Spore germination in ferns is a process that extends through time according to the age of the spores, the time and conditions of storage (humidity, temperature, light, etc), and the taxonomic group they belong to (Lloyd & Klekowski, 1970;Smith & Robinson, 1975;Beri & Bir, 1993;Camloh, 1999).Spores of Alsophila odonelliana germinate 7-8 days after sowing (Martínez, 2011;unpublished data), whereas the spores used in this work, stored for 16 months, took longer to germinate, likely due to the biochemical changes produced during storage (Bery & Bir, 1993).
Germination time of the spores was relatively similar in all three nutritive media D, K and MS, despite MS contained a greater amount of micro and macronutrients than D and K. Sixty days after sowing, we observed a slightly higher percentage of germination in the K medium compared with D and MS, although the statistical analysis showed no significant differences; therefore, the amount of nutrients in the substrate would not be a limiting factor in the germination of A. odonelliana spores.
The formation of 1-2 ramifications in filamentous gametophytes and the production of buds in laminar gametophytes are frequent during the gametophytic development in the Cyatheaceae family in the absence of growth regulators (Conant, 1990;Chen et al., 2008).Our results indicate that 0.1 mg/L BAP massively activated cellular proliferation, which led to the formation of filamentous gametophytes of up to 20 cells which ramified 17-20 times.In concentrations of 1 mg/L, BAP caused less ramifications in filamentous gametophytes.Supplementing the culture media with BAP induced A. odonelliana to develop long filamentous gametophytes, and proliferation of ramifications from early developmental stages until the adult stage.
González-Santos ( 2009) noted that BAP possessed the property of maintaining the concentration of chlorophyll and total soluble proteins for longer periods in plants, so that the foliar senescence is decreased; therefore, this growth regulator is likely responsible for maintaining the amount of chlorophyll in the laminar gametophytes developing in media with BAP.BAP was efficient from a morphological viewpoint, since it induced massive proliferation of gametophytes from the ramifications or the production of vegetative seedlings.
Supplementing the culture medium with 2,4-D led to the formation of short filamentous gametophytes, which originated laminar gametophytes with certain peculiarities: ameristic forms, furcated gametophytes and decreases in pigmentation, particularly in those for which the concentration of this regulator was 2 mg/L.In higher plants such as Cephaelis ipecacuanha A. Richard and Aloe vera L., this growth regulator, at the same concentrations, induces the formation of calli for obtaining embryos (Roya & Sarkara, 1991;Routa et al., 2000); in Alsophila odonelliana, we did not observe formation of calli in the gametophytes.
None of the growth regulators, BAP or 2,4-D, induced cell differentiation in the pluristratified ridges of the mature gametophytes for the formation of calli, nor apogamic sporophytes in A. odonelliana, in this first experience.Further testing is needed, with more repetitions, a wider range of growth regulator concentrations and longer observation times, to determine whether the growth regulators fail to produce embryos of tree ferns from this methodology.

Fig. 2 .
Fig. 2. Percentage of germination of Alsophila odonelliana spores in MS, K and D culture media without growth regulators.The curves correspond to records obtained at 60 and 120 days after sowing.

Fig. 5 .
Fig. 5. Laminar gametophytes of Alsophila odonelliana in BAPsupplemented medium.(A) colony of gametophytes developing in medium with 0.1 mg/L BAP 50 days after germination of spores (x 60); (B) laminar gametophytes without notch developing in medium with 0.01 mg/L BAP at 60 days (x 55); (C) four gametophytes with marked notch in medium with 1 mg/L BAP at 70 days (x 45).