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Phegopteris connectilis (Michx.) Watt -along the Valley of the Vâlsan River (orig.) RESULTS AND DISCUSSIONS Differentiation of the gametophyte. It starts two weeks after the culture is initiated, and follows the characteristic stages of the gametophyte differentiation in Leptosporangiatae, namely the prothallic filament, the prothallic blade, and the ± cordate prothallus (Ehrendorfer, 1999). Chloroplastotomy was evinced in the prothallic chlorocytes, which is an important process in the development of vegetable cells (Pyke, 1999). The prothalli display secretory one-cell trichomes on the rim, similar to those observed and described in the case of Cyrtomium falcatum and Dryopteris dilatata (Soare and Andrei, 2005), as well as various other species (Nayar & Chandra, 1963; Nayar & Kaur, 1963, 1964, 1965; Lingle et al., 2004, etc.). The trichomes are differentiated in the apical region of the gametophyte, as a result of an asymmetric division (Alberts et al., 2002). This type of division is also found during the differentiation of the trichomes characteristic to angiosperms sporophyte and stomata (Larkin et al., 1997), etc. The waxlike substance produced by the trichomes is eliminated outwardly, forming a kind of cap covering their tops (Figure 2). Lingle et al. (2004) consider that these secretory trichomes, which are differentiated on the fern gametophyte, partially protect it against de-hydration, being noticed that trichome density augments in water scarcity conditions. For instance, in Phegopteris, trichome density on the gametophytes is much lower in the case of the cultures realized on the liquid nutrient medium
Source publication
Phegopteris connectilis is an apogamous species, endangered in certain regions. The ex situ conservation, through the in vitro production of the gametophyte, as well as the embryos and the plants, followed by their cryo-stocking, is necessary for ensuring the survival of the endangered species, while at pace with international methodology. The diff...
Contexts in source publication
Context 1
... vegetable material was collected, along the Valley of the Vâlsan River in June 2006 ( Figure 1). The research technique employed for this material was previously described for other species ). ...
Context 2
... trichomes (Plate I, Figure 3) are formed on its rim are, as well as uniserial pluricellular ones (Figure 6), the latter representing the initial stage of the palea differentiation. The lamina of the first leaf is trilobate, having a nervation of a dichotomic type (Plate I, Figure 1). The rim of the lamina is mono-stratified, as the cells contain few chloroplasts. ...
Citations
... and Pteris multifida Poir. did not form roots in parallel to the first leaf (Kawakami et al. 1995;Soare et al. 2007;Gabancho et al. 2010). Moreover, the first sporophyte leaf possessed stomata, multicellular glandular hairs, and scales (Gabancho et al. 2010). ...
Key message
Somatic embryogenesis has never been reported in ferns. The study showed that it is much easier to evoke the acquisition and expression of embryogenic competence in ferns than in spermatophytes.
Abstract
We discovered that the tree fern Cyathea delgadii offers an effective model for the reproducible and rapid formation of somatic embryos on hormone-free medium. Our study provides cyto-morphological evidence for the single cell origin and development of somatic embryos. Somatic embryogenesis (SE) in both primary and secondary explants was induced on half-strength micro- and macro-nutrients Murashige and Skoog medium without the application of exogenous plant growth regulators, in darkness. The early stage of SE was characterized by sequential perpendicular cell divisions of an individual epidermal cell of etiolated stipe explant. These resulted in the formation of a linear pro-embryo. Later their development resembled that of the zygotic embryo. We defined three morphogenetic stages of fern somatic embryo development: linear, early and late embryonic leaf stage. The transition from somatic embryo to juvenile sporophyte was quick and proceeded without interruption caused by dormancy. Following 9 weeks of culture the efficiency of somatic embryogenesis reached 12–13 embryos per responding explant. Spontaneous formation of somatic embryos and callus production, which improved the effectiveness of the process sevenfold in 10-month-long culture, occurred without subculturing. The tendency for C. delgadii to propagate by SE in vitro makes this species an excellent model for studies relating to asexual embryogenesis and the endogenous hormonal regulation of that process and opens new avenues of experimentation.
... while full strength MS medium showed average growth of protonemal length (394.40µm) with protonemal width (644.37µm). In earlier reports, induction of gametophytes and its significant growth was noticed on half strength MS medium in spores of Pityrogramma calomelanos (Martin et al., 2006), Osmunda regalis, Cyrtomium falcatum, Asplenium ruta-muraria, Dryopteris dilatata (Soare and Andrei, 2005;2007b), Dryopteris fortunei (Chang et al., 2007), Pteris vittata (Yang et al., 2007), Phegopteris connectilis (Soare et al., 2007a), D. varia (Ouyang et al., 2008), Pteris wallichiana (Zhang et al., 2008), Pyrrosia lingua , D. affinis (Soare et al., 2010) and some selected Blechnum sp., Cibotium sp., Cyathea sp., Dicksonia sp. (Goller and Rybczynski, 2007). ...
... SOARE & M. ANDREI [8]), Phegopteris connectilis (L.C. SOARE & al. [9]), as well as Asplenium ruta-muraria, Dryopteris dilatata, Polypodium vulgare (L.C. SOARE [10]). ...
... SOARE & M. ANDREI [7]), and Phegopteris connectilis (L.C. SOARE & al. [9]), in the last two through apogamy. Through the in vitro culture of green sporangia (sori) in Phegopteris connectilis, the sporophyte starts to form very rapidly, after only seven weeks, which means that fewer resources are used to obtain the plant. ...
... HENSON [5]), (L.C. SOARE & al. [9]). The culture medium was distributed in 10 Petri glass boxes 8 cm in diameter. ...
Dryopteris affinis is an apogamous species of European sub-Atlantic and sub-paleo-Mediterranean distribution, ranging from South West Norway to North Africa, and from Macaronesia to Caspian Iran. It vegetates in the protected area of the Vâlsan Valley, and it is similar to the common fern, Dryopteris filix-mas, both species having an ornamental potential. The aim of the present research was to observe the peculiarities of the process of differentiation of the gametophyte and sporophyte, and to obtain the in vitro regeneration of the apogamous species Dryopteris affinis by means of cultivating green sporangia, the acclimatization of the plants and their transplantation in the soil to make up the ex situ collection. The green sporangia were cultivated on the half strength Murashige & Skoog medium, without hormones; the culture pots were kept in the growing room at a temperature of 25±10C, with a photoperiod of 16 hours of light, and 8 hours of darkness. A colony of prothalli was obtained from each inoculum, and that colony included approximately 100 gametophytes, i.e. 100 sporophytes. Unlike other methods used for the in vitro culture of this species, through the culture of green sporangia the formation of the embryo occurred rapidly, within only 45 days after the initiation of the in vitro culture. The first root was differentiated in the stage where the sporophyte had 2-3 leaves. The in vitro culture of green sporangia made it possible to transplant the acclimatized plants into the soil, only ten months after the initiation of the culture, in order to initiate the ex situ collection. The biotechnological method used to differentiate and regenerate this species starting from green sporangia can be recommended for the rapid multiplication of the plant with a view to obtaining biological material with a commercial purpose, or to conduct fundamental or experimental researches.
Rumohra adiantiformis, also known as “leatherleaf fern”, is an ornamental species that, because of its long display life, is widely used in floral arrangements. In this study, a new protocol for in vitro regeneration of the leatherleaf fern was established. For spore germination, two culture media (MS and Knop) were assessed with presence or absence of 1g L−1 activated charcoal (AC) under different light and dark conditions. Frond, frond microcuttings, and prothallus explants were evaluated on Knop regeneration medium supplemented with 1g L−1 AC, 0.5% agar, pH 5.0 in combination with 2,4 dichlorophenoxyacetic acid (2,4-D: 0.0, 0.1, 0.5 and 1.0 mg L−1) and 6-benzylamino purine (BA: 0.0, 0.1, 0.5 and 1.0 mg L−1). For rooting, four levels of α-naphthaleneacetic acid (NAA: 0.0, 0.01, 0.1 and 0.2 mg L−1) were tested. After 18 days of culture, spore germination rate was 100% on Knop medium with AC and 8 h light/16 h dark. After 120 days of culture, sporophytes 1.7 ± 0.4 cm in length developed on Knop medium, while those spore-cultured on MS medium never produced sporophytes. From those germinated sporophytes, prothallus explants cultivated on Knop medium with AC and 0.5 mg L−1 BA showed the highest regeneration rate with 235.7 gametophytes. The best sporophyte rooting response was obtained with 0.01 mg L−1 NAA. Complete, regenerated sporophytes were obtained 183 days after culture initiation. By this procedure it would be possible to obtain up to 2 million sporophytes from one fertile frond. To determine the origin of the regenerated gametophytes, a histological analysis was performed with scanning electron microscopy (SEM). The analysis revealed that the gametophytes were regenerated from explant epidermal tissues on either the adaxial or abaxial surface.
Asplenium auritum Sw. is a sexual fern that produces 64 spores per sporangium, while A. monodon Liebm. is an apogamous species that produces 32 spores. The hybrid between them, A.×lellingerianum Sánchez & Regalado, also shows an apogamous life cycle, with mainly abortive spores. The scope of this work was to study
the sexual and apogamous behavior in these taxa. We studied spore germination and gametophyte development of the three species,
a process that followed the Adiantum type. Afterward, we observed morphological apogamous characters in A. monodon and A.×lellingerianum. Apogamous sporophytes arose from apical and basal regions of gametophytes, lacking feet and roots in the first instance,
but developing other normal sporophytic structures, such as tracheids, stomata, glandular hairs, and scales. Finally, we studied
the gametangia production in all three taxa, finding that the scarce production of antheridia in A. monodon is indicative of the Braithwaite apogamous life cycle scheme, and this scheme has probably been inherited by A.×lellingerianum.
Keywords
Asplenium
-Sexuality-Apogamy-Cuba
