In Vitro Culture and Regeneration of the Fern Dryopteris Affinis, Species Growing in a Protected Area

In vitro culture and regeneration of the fern Dryopteris affinis, species growing in a protected area

Received for publication, November 18, 2009 Accepted, January 8, 2010

LILIANA CRISTINA SOARE*, EMILIA VIŞOIU**, CODRUŢA MIHAELA DOBRESCU* *University of Pitesti, Faculty of Sciences, 1 Targul din Vale St., 110040, Pitesti, Arges,Romania, E-mail: [email protected], ** National Institute of Research and Development for Biotechnologies in Horticulture, 37 Bucuresti-Pitesti Road, 117715, Stefanesti, Arges, Romania

Abstract 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.

Key words: Dryopteris affinis, in vitro, the Vâlsan Valley, green sporangia (sori), apogamous embryo.

Introduction

The in vitro culture is currently used extensively in order to achieve the multiplication of ornamental ferns (H. FERNÁNDEZ & M.A. REVILLA [1]), of the endangered ferns (G. D. R. RENNER & Á. M. RANDI [2]), or to preserve their gametophytes and sporophytes. 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 (C.R. FRASER-JENKINS [3]). The researches concerning the in vitro culture of this fern demonstrate the fact that the sporophyte is differentiated from the gametophytic tissue. Thus, H. FERNÁNDEZ & al., 1996 [4], cultivating in vitro spore of Dryopteris affinis on the Murashige & Skoog (MS) medium, obtained, after three months, prothalli on which apogamous sporophytes begin to form. The culture of the prothalli obtained on the MS medium with benzyladenine and 2,4-diclorfenoxiacetic acid results in forming morphogenetic callus, out of which the sporophyte is then obtained. The in vitro culture of green sporangia (sori) on the 1/2 MS medium, without hormones, allows one to obtain the gametophyte in pteridofites such as: Dicranopteris linearis, Platycerium 45 In vitro culture and regeneration of the fern Dryopteris affinis, species growing in a protected area coronarium (N. HENSON [5]), Osmunda regalis (L.C. SOARE & M. ANDREI [6]), Cyrtomium falcatum (L.C. SOARE & M. ANDREI [7]), Asplenium ruta-muraria, Cyrtomium falcatum, Dryopteris dilatata (L.C. 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]). Out of the above-mentioned species, the sporophyte forms on the gametophyte only in Dicranopteris linearis, Platycerium coronarium (N. HENSON [5]), Cyrtomium falcatum (L.C. 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. Taking into account the ornamental potential of the species Dryopteris affinis, the aim of the present research was to observe the peculiarities of the process of differentiation of the gametophyte and sporophyte, as well as obtaining the in vitro regeneration of the apogamous species Dryopteris affinis through cultivating green sporangia (sori), followed by the acclimatization of the plants and their transplantation in the soil to form the ex situ collection.

Material and method

The vegetal material used, represented by leaves with green sporangia (sori), was sampled from plants vegetating in the protected area the Vâlsan Valley (Valea Vâlsanului) (C.M. DOBRESCU & L.C. SOARE [11]). The leaves sampled were kept at 4ºC of temperature before the moment the culture was initiated, when they were fragmented. The sterilization of the material was done with calcium hypochlorite 6%, for 5 minutes, after which it was washed in sterile distilled water, three times. The sporangia were taken off the leaves by means of a lanceolate needle, and were inoculated into the half strength MS (1962) medium, having no hormones, recommended in the specialized literature (N. HENSON [5]), (L.C. SOARE & al. [9]). The culture medium was distributed in 10 Petri glass boxes 8 cm in diameter. In each Petri box were inoculated between 5 and 7 sori. The culture pots were kept in the growing room at a temperature of 25±10C, with a photoperiod of 16 hours light, 8 hours of darkness (A.M. BERTRAND & al. [12], H. FERNÁNDEZ & al. [13]). The pH of the medium was adjusted to 5.6 before the autoclaving (N. HENSON [5]). The cultures were analysed periodically, both macroscopically and microscopically. The pictures were taken under the Optika B275 microscope by means of a Canon digital camera. The rapid regeneration of the gametophyte did not require its transfer on a fresh medium. Ten weeks after the initiation of the culture, the groups of prothalli with sporophytes were fragmented and removed into culture vessels of larger dimensions, on the same culture medium. The small plants regenerated in the in vitro culture in the apogamous embryo differentiated at the level of each prothallus in the group of prothalli resulted from the vegetative multiplication of the gametophyte, were acclimatized in order to obtain a larger quantity of biological material for the ex situ collection of various species of pteridophytes. The acclimatization was carried out in keeping with the specific steps of this technological phase, thus: a) the individualization of the small plants in the groups, their planting in Jiffy bags, and securing their protection by means of glass pots in order to keep humidity; b) the gradual removal of individual protection, after 20-25 days, and maintaining atmospheric humidity at about 60-65%, by ensuring group protection of the plants, with polyethylene sheet; c) finalizing the acclimatization by the complete removal of protection after 7-10 days. The cultures were established in the laboratory for Obtaining and conserving the source of vineyard germplasm within the National Institute of Research and Development for Biotechnologies in Horticulture, Stefanesti, Arges.

46 Romanian Biotechnological Letters, Vol. 15, No. 1, Supplement (2010) LILIANA CRISTINA SOARE, EMILIA VIŞOIU, CODRUŢA MIHAELA DOBRESCU

Results

The initiation of the gametophyte (prothallus) in the explants cultivated in vitro takes place, very much as in the case of other analysed species, after approximately two weeks (Fig. 1).

Along the process of the differentiation, the gametophyte passes through the characteristic stages, i.e. prothallic filament, spatulated prothallic blade, and cordate prothallus. A colony of prothalli was obtained from each inoculum, and that colony included approximately 100 gametophytes (Fig. 2).

1 2

Fig. 1-2. Dryopteris affinis: 1. gametophyte initiation; 2. gametophyte colony.

Thus, in each Petri box 500 to 700 prothalli were obtained. A characteristic feature frequently noticed in the in vitro cultures, in various species of pteridophytes, is represented by the ramification of the gametophyte (Fig. 3), which led to its vegetative multiplication. In Dryopteris affinis, the branching out occurred in the region of the prothallic wings, and at its base.

After 45 days since the initiation of the in vitro culture, on each prothallus a more or less circular, rather light-coloured area, made up of small cells could be noticed near the cordate region (Fig. 4,6). That is the area where, on each prothallus, an apogamous embryo is differentiated. The different colour of the apogamous embryo, contrasting to that of the adjacent prothallian tissue, is due to the absence of the chloroplasts, a characteristic trait specific to embryo tissues. Around the apogamous embryo, secreting unicellular trichomes can be noticed (Fig. 5).

Romanian Biotechnological Letters, Vol. 15, No. 1, Supplement (2010) 47 In vitro culture and regeneration of the fern Dryopteris affinis, species growing in a protected area

4 5

6 7

Fig.3-7. Dryopteris affinis: 3-ramification of the gametophyte (oc.10x, ob.10, zoom 4; orig.); 4-gametophyte with apogamous embryo and secreting unicellular trichomes (oc.10x, ob.4, zoom 4; orig.); 5-secreting unicellular trichomes around of apogamous embryo (oc.10x, ob.10, zoom 4; orig.); 6-apogamous embryo from which the first leaf is differentiating (oc.10x, ob.10, zoom 4; orig.); 7-secreting unicellular trichomes of gametophyte (oc.10x, ob.40, zoom 4; orig);

After 60 days from the initiation of the culture, the first leaf (F1), was differentiated out of the apogamous embryo, a leaf which, originally, is shaped like a flattened crest. The leaf presents numerous trichomes, both unicellular, and pluricellular; they are either uni- series, or ramified, the latter making the passage towards the paleae (fig. 8-11).

48 Romanian Biotechnological Letters, Vol. 15, No. 1, Supplement (2010) LILIANA CRISTINA SOARE, EMILIA VIŞOIU, CODRUŢA MIHAELA DOBRESCU

8 9

10 11

Fig. 8-11. Dryopteris affinis: 8-apogamous embryo with he first leaf (F1) and differentiation of the first root (R1)(oc.10x, ob.4, zoom 4; orig.); 9-11 trichomes and paleae on F1 (oc.10x, ob.4, zoom 4; orig.);

The unicellular hairs and the apical cell of the bicellular ones contain chloroplasts, and the substance the latter produce is eliminated across the outer cell wall (Fig. 12-14).

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12 13

14 15

Fig. 12-15. Dryopteris affinis: 12-unicellular trichoma on the F1 (oc.10x, 0b 40, zoom 4; orig.); 13- chloroplast division (arrow) in F1 cells (oc.10x, 0b 40, zoom 4; orig.); 14-secreting bi-cellular trichoma on F1 lamina (oc.10x, 0b 40, zoom 4; orig.); 15-initiation of asymmetric division on F1 lamina (arrow) (oc.10x, 0b 10, zoom 4; orig.).

The pluricellular trichomes present a curving apical cell of dense contents. The secreting apical cell does not eliminate the substances produced to the outside; they remain inside it. The formation of the trichomes is preceded by an asymmetric division of a prothallic or sporophytic cell (Fig. 15). The smaller resulting cell is the mother-cell of the trichome. In

50 Romanian Biotechnological Letters, Vol. 15, No. 1, Supplement (2010) LILIANA CRISTINA SOARE, EMILIA VIŞOIU, CODRUŢA MIHAELA DOBRESCU both the prothallic cells, and the cells of the juvenile leaves the process of chloroplastotomy could be observed (Fig. 13). The first root (R1) was differentiated, very much as in the case of other apogamous species, at the stage where the sporophyte had 2-3 leaves, i.e. after approximately three months from the initiation of the culture. Its absence is supplanted by the rhizoids that become differentiated around the apogamous sporophyte. Since one sporophyte is formed on each prothallus, each colony of prothalli originating from an explant differentiated approximately 100 young plants.

Fig. 16-18. Dryopteris affinis: 16-pluricellular trichomes on F1; 17-18-two-month-old plants transplanted from the culture pots into Jiffy nutrient pots. 17 18

The two-month-old plants were transplanted from the culture pots into Jiffy nutrient pots (Fig. 17-18). Ten months after the initiation of the culture, the acclimatized plants were transplanted into the soil.

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Discussions

According the explant used, the differentiation of the gametophyte begins, very much as in the case of other analysed species, after approximately two weeks (L.C. SOARE & al. [9]), when the greening of the explant can be observed macroscopically. The process of differentiation evolves in the manner characteristic of leptosporangiate ferns (F. EHRENDORFER [14]). A specific feature the in vitro culture of the gametophyte is its vegetative multiplication, which was frequently noticed within this group of plants (H. FERNÁNDEZ & M.A. REVILLA [1]). This is done through gemmae or though branching out; in the latter case, the initial stage of the formation of a new gametophyte is represented by a prothallic filament. The ramification of the prothallus, which can also be met within Dryopteris affinis, is the most frequent way through which the gametophyte propagates itself in vitro. That process of gametophyte multiplication leads to obtaining a large amount of vegetative material starting from a small quantity of explant. In contradistinction to the cultures obtained from spores, where only one prothallus is produced from each spore, in a culture of sori, a colony is obtained from each sorus, which is made up of approximately 100 prothalli. Apogamy, known for approximately 10% of the ferns, can also be found in an unknown percentage of other pteridophytes (E. SHEFFIELD & P.R. BELL [15]); it was also described in Dryopteris affinis (H. FERNÁNDEZ & al., 1996 [4]). Unlike other apogamous species, such as Pellaea viridis, P. flexuosa, Notholaena sinuata, in which the differentiation begins after two months (B.K. NAYAR & N. BAJPAI [16]), in Dryopteris affinis the process occurred within a shorter period (45 days); H. FERNÁNDEZ et al. [4] report a longer period of time for the formation of the apogamus embryo in Dryopteris affinis on the MS medium, i.e. three months from the initiation of the sporus culture. The first leaf (F1) differentiated from the apogamous embryo originally assumes the shape of a flattened crest, very much like other apogamous leptosporangiate ferns, e.g. Pellaea viridis (B.K. NAYAR & N. BAJPAI [16]), Cyrtomium falcatum (L.C. SOARE & M. ANDREI [7]). The unicellular trichomes on the leaves and those on the gametophyte produce the wax that has an important role in protecting against dehydration (M.A. LINGLE & al. [17]); apogamy is considered an adaptation to the xerophytic habitats were some fern species vegetate (H. FERNÁNDEZ & M.A. REVILLA [1], W. H. WAGNER & A.R. SMITH [18]). The ability to form trichomes that protect the gametophyte and the sporophyte recalls one of the bryophytes (K.S. RENZAGLIA & al. [19]). The pluricellular trichomes with a curving apical cell, having a dense content, were also described on the juvenile leaves of Cyrtomium falcatum (L.C. SOARE & M. ANDREI [7]). The asymmetric division, which results in the initial cell of the trichomes, is also met in the formation of the rhizoids of the gametophyte. The smaller cell appearing as a result of such a division inherits factors that determine it to differentiate in the trichome (B. ALBERTS & al. [20]). The process of chloroplast division was confirmed in the developing vegetal cells as early as the 1960s (J.V. POSSINGHAM & W. SAURER [21]). The process of binary fission of the chloroplasts, considered morphologically similar to that of division in the bacterium cell, suggests the possibility of a similar genetic control of the two processes (K.A. PYKE [22]). The discovery of the genes that are homologic to those of bacterium cell division suggests the fact that the division of the plastids in the superior plants is based on a system that evolved from that used by the prokaryote cells, thus supporting the endosymbiotic origin of plastids (L. MARGULIS [23], M.W. GRAY [24]).

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The forming of the first root (R1) at the stage where the sporophyte has several leaves is described in terms similar to those used for other apogamous species such as Pellaea viridis, P. flexuosa, Notholaena sinuata (B.K. NAYAR & N. BAJPAI [16]), Cyrtomium falcatum (L.C. SOARE & M. ANDREI [7]). The absence of the root in the first development stages of the apogamous sporophyte backs up the assertion that the embryo of the fern is not structured in a two-polar manner like that of the spermatophytes, but in a unipolar manner (P.A. GROFF & D.R. KAPLAN [25]), the first root being an endogenous formation, lateral cauligenous (F. EHRENDORFER [14]). The formation of the sporophyte in Dryopteris affinis conduces to the cessation of the growth of the gametophyte, and eventually to its deterioration, in a similar manner as in Asplenium nidus, while in Woodwardia virginica, the growth of the gametophyte continues even after the formation of the sporophyte. In Blechnum spicant, Osmunda regalis and Pteris ensiformis the formation of the sporophyte and the growth of the gametophyte occur simultaneously (H. FERNÁNDEZ & al., [13]). The small plants, acclimated in a proportion of 80-100%, proved to be morphologically identical to the mother-plant from which they sprang out, and were used in making up the collection of fern species in the protected area of the Vâlsan Valley (Valea Vâlsanului).

Conclusions

In the species Dryopteris affinis, very much as in other pteridophytes cultivated in vitro, the vegetative multiplication of the gametophyte can be noticed, a process that ensures the obtaining of a large amount of vegetable material starting from a small amount of explant. Unlike other biotechnological methods used for the multiplication of this species, through the culture of green sporangia (sori) the rapid differentiation was noticed of the apogamous embryo, only 45 days after the initiation of the in vitro culture and the obtaining of the acclimatized and fortified plants after only ten months; after that, the plants were transplanted into the soil in order to form the ex situ collection. The biotechnological method used for the differentiation and regeneration of this species starting from green sporangia (sori) can be recommended for the rapid multiplication of the plant with a view to obtaining commercially used biological material or as a support for conducting research, either fundamental or experimental. It can also be tested for the multiplication of a number of endangered species of the genus Dryopteris.

Aknowledgements

The research was funded by the Ministry of Education, Research and Youth, through the programme Partnerships in Priority Domains, within contract no. 32-174/2008 concluded with the National Centre of Programme Management.

References

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