EFFECTIVE AND EASY WAY OF ESTABLISHING IN VITRO CULTURE OF MOSSES

There is today a general unawareness of the fact that the technique of culturing plant tissues and organs under axenic conditions was first established and profitably employed in bryophytes, especially mosses (S e r vet t a z 1913). However, despite such a promising start, bryophytes did not retain for long their rightful place as highly favored research objects, so that most studies of plant morphogenesis are now being done on vascular plants. Apart from the favorable economic aspect, of experimenting on bryophytes, many fundamental and applicative physiological, genetic, morphogenetic, evolutionary and other problems can be tackled more easily by using bryophytes than vascular plants. Bryophytes are the second largest group of higher plants after flowering plants [Magnoliopsiday; with approximately 28.000 species found worldwide. However, most bryophyte species have no commercial value, and are therefore less attractive in a wide range of studies. Some 40% of these tiny species are endangered at present and in urgent need of active protection and conservation. Despite a long history of growing bryophytes in culture and the existing of different-sized collections maintained by some investigators, any newcomer wishing to start their cultures faces a stiff challenge (S a r g e n t 1988). One of the latest ideas is to establish sterile in vitro cultures, then micropropagate plants and later consider methods of their reintroduction into potential native habitats. The first relevant steps in Yugoslavia have already been made and are presented in this communication. Bryum argenteum Hedw. and Bryum capillare Hedw. (Bryaceae) were chosen for this experiment as they are unendangered cosmopolitan species and a model system for further in vitro investigation of mosses. These species are easily accessible because their habitats include urban and suburban areas. Bryum argenteum Hedw. is quite frequent while Bryum capillare Hedw is sporadic. The plants grow up to 5 mm in height and in the form of very small cushions. Reproduction of these species is mostly vegetative, although both are known to produce gametangias. In Bryum capillare Hedw., sporphytes are not usually seen, while in Bryum argenteum Hedw. they are quite rare (S

There is today a general unawareness of the fact that the technique of culturing plant tissues and organs under axenic conditions was first established and profitably employed in bryophytes, especially mosses (S e r vet t a z 1913).However, despite such a promising start, bryophytes did not retain for long their rightful place as highly favored research objects, so that most studies of plant morphogenesis are now being done on vascular plants.Apart from the favorable economic aspect, of experimenting on bryophytes, many fundamental and applicative physiological, genetic, morphogenetic, evolutionary and other problems can be tackled more easily by using bryophytes than vascular plants.
Bryophytes are the second largest group of higher plants after flowering plants [Magnoliopsiday; with approximately 28.000 species found worldwide.However, most bryophyte species have no commercial value, and are therefore less attractive in a wide range of studies.Some 40% of these tiny species are endangered at present and in urgent need of active protection and conservation.
Despite a long history of growing bryophytes in culture and the existing of different-sized collections maintained by some investigators, any newcomer wishing to start their cultures faces a stiff challenge (S a r g e n t 1988).
One of the latest ideas is to establish sterile in vitro cultures, then micropropagate plants and later consider methods of their reintroduction into potential native habitats.
The first relevant steps in Yugoslavia have already been made and are presented in this communication.
Bryum argenteum Hedw.and Bryum capillare Hedw.(Bryaceae) were chosen for this experiment as they are unendangered cosmopolitan species and a model system for further in vitro investigation of mosses.These species are easily accessible because their habitats include urban and suburban areas.Bryum argenteum Hedw. is quite frequent while Bryum capillare Hedw is sporadic.The plants grow up to 5 mm in height and in the form of very small cushions.Reproduction of these species is mostly vegetative, although both are known to produce gametangias.In Bryum capillare Hedw., sporphytes are not usually seen, while in Bryum argenteum Hedw.they are quite rare (S 0 1995).Bryum argenteum Hedw.and Bryum capillare Hedw.are xero-mesophyte and mesophyte plants respectively (I n g e r p u u et al. 1994).
In our experiment bryophyte material consisting of Bryum argenteum Hedw.and Bryum capillare Hedw.has been collected in Kalemegdan Fort Park in Belgrade.The former species was sampled in December, and the latter one in November, 2000.After drying, the plant material was stored in paper bags until the beginning of the experiment.

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In vitro cultures of B. capillare and B. argenteum were initiated from apical shoots of gametophytes and spores, respectively.Small shoots of B. capillare were separated from soil and the rest of substratum, and the material was placed in glasses covered with cheese cloth and rinsed with tap water for 30 min.As most mosses have filoids with one-cell layer and not protection layers, surface sterilization with 70% ethanol was inappropriate, because it damages plants.
Variously dilute solutions of commercial NaOCI bleach (8% active chlorine) were used for sterilization: 0.5; 1; 2; 3; 5; 7; 9; 11; 13%.Rinsed plant material was kept for 5 min in a bleach solution containing a few drops of liquid detergent and rinsed again with sterile distilled water.This method was effective for surface sterilization.
Immature capsules of B. argenteum with operculum or both with operculum and calyptra, and undamaged sporophytes were separated from the gametophytes and washed in distilled water.If calyptra was present, it was carefully separated from the capsule, taking care not to separate or damage operculum.Capsules prepared this way were kept in 9, 11, 13, 15% solution of commercial bleach for 5 min and than rinsed 3 times with sterile distilled water.
Capsules were opened by sterilized needle and the spores transferred onto a solid medium.As spores are sterile in undamaged and immature capsules we made advantage of that fact to avoid complex procedures for sterilization of tiny spores invisible to the naked eye.Small shoots of B. capillare (about 3 mm long) were isolated aseptically and cultured with tip side up on a solid nutrient medium.
The basal medium (BM) contained MS (M u r as h i g e and S k 0 0 g 1962) mineral salts and vitamins, 100 mg Vl myo-inozitol, 30 g Vl sucrose, 0.70% agar (Torlak purified, Belgrade), and was supplemented with 1.0 mg L-l 2, 4-dichlorophenoxyacetic acid (2,4-D) and 2.0 mg Vl kinetin (KIN).The medium pH was adjusted to 5.8 prior to autoclaving, at 115°C for 20 min.The cultures were grown at 25±2°C under white fluorescent light (47 pmolm-2 s-l irradiance) and a day/night regime of 16/8 h.The plants were subcultured in one month intervals.
Surface moss sterilization with commercial bleach (8% active chlorine) gives the best results.We found that depending on plant morphology of gametophyte (position of filoids, density, etc.) the concentration of bleach solution should be varied from 0.5 to 15% in order to find out the one that provides the best sterilization without tissue damage.The results acquired in this experiment indicate that the best concentration of bleach solution for sterilization of this plant material is 9% both for gametophytes and sporophyte.It was observed that the addition of a few drops of liquid detergent to the solution of NaOCI bleach solution improves surface sterilization of plant material and enables development in axenic culture.This we may explain by the fact that detergent decreases surface pressure and enables filoids to spread, so tl:e solution of Na-OCI bleach can reach most of the shoots.
After 10 to 14 days some changes were observed in the cultures of B. capillare, i.e secondary protonema developed.After a month, the plants gave the caulonema, but even six months after the establishing of in vitro culture, plants still failed to form protonemal and caulonemal buds.C hop r a and R ash i d (1969) reported that in some pottiateous mosses (gen.Anoectangium, fam.Pottiaceae) bud formation could be induced by substituting Fe-EDTA with a natural iron source (ferric citrate).
This method seems to be simple and much easier than those reported by Bas i I and Bas i I (1988) -surface sterilization by the washing machine methods, and by Can 0 et al. (1996) -ultrasound surface sterilization.
Another simple method that we used to establish B. argenteum culture was to collect plants with immature sporophytes, but not too young (preferably in early spring).In this way, the samples can be kept in dry and cold place for quite a long period of time.Our plants were kept for 10 days before culturing them in vitro.
Considering that capsules have better protection than filoids and young capsules are usually smooth, not plicate as filoids, all concentrations of NaOCI bleach used in this experiment showed good sterilization results.
The germination of spores was obtained 7 days after their transfer to the medium, followed by visible protonem a formation within the next 8 days.Development of caulonema and buds, as well as regeneration of the whole gametophytes was obtained 3 days after the beginning of spore germination.Small leafy shoots appeared one month after the germination onset.The MS medium supplemented with with 1.0 mg VI 2, 4-D and 2.0 mg VI KIN was very convenient for spontaneous regeneration of B. argenteum, although bryological literature suggest that Knudson solution (K n u d son 1946) is much appropriate for bryophyte growing.
C hop r a and K u m r a (1988) reported that some mosses could not grow or even germinate without a symbiosis with fungi and bacteria.
According to D 0 Y I e (1967), F r a h m and Nordh o r n -Ric h tel ' (1984) andVi t t et al. (1985) any plant of the Bryaceae family and certainly not any of the Bryum genus has yet been grown in the axenic culture.Very good knowledge of the biology of bryophyte appears therefore to be a considerable advantage in the process of establishing their in vitro culture.
We have shown in this experiment a very easy, effective and convenient method both for surface sterilization and in vitro culturing of two moss species.