Scientia Hor,k,dtume, 51 (1992) 343-346 Elsevier Science Publishers B.V., Amsterdam

Short Comrnunication

343

In vitro culture of Platycerium bijurcatum garnetophytes

Marjana Camloh a and Nada Gogala b ainstitute of Biology, University of Ljubljana, Karlovgka 19, 61000 Ljubljana. Slovenia bDepartment of Biology, Biotechnical Faculty, University of Ljubljana, Agker?eva 12,

61000 Ljubljana, Slovenia

(Accepted 7 February 1992 )

ABSTRACT

Camloh, M. and Gogala, N., 1992. In vitro culture of Piatycerium bifurcatum gametophytes. Scientia Hortic.. 5 !: 343-346.

Clumps ofPlatycerium bifurcatum (Cav.)C.Chr. gametophytes initiated in vitro from spores were used to determine the effects of sucrose, agar and pH of the medium on their growth. Optimal growth ofgametophytes was obtained on modified Miller medium supplemented with 4% sucrose, 0 or 0.6% agar, and the pH adjusted to 5.5. Gametophytes cultured for 2 months in liquid medium and poured on sterilized soil demonstrated a successful sporophyte development.

Keywords: gametophytes; in vitro culture; Platycerium bifitrcatum.

Abbreviations: BM = basal medium.

INTRODUCTION

Fern gametophytes have been hailed as a convenient experimental orga- nism for various basic and applied investigations (Hickok et al., 1987 ). Since studies on in vitro culture of gametophytic tissue of the fern Platycerium bi- furcatum (Cav.)C.Chr. (staghorn fern) are very rare (Thentz and Moncou- sin, 1984; Camloh and Gogala, 1988), more complete information on the optimal growth conditions of these gametophytes should provide better un- derstanding of their in vitro requirements. The stagharn fern is an ornamen- tal plant traditionally propagated sexually by spores arid asexually through root bud development (Richards et al., 1983). Phytopathological problems

Correspondence to: Marjana Camloh, Institute of Biology, University of Ljubljana, Karlovgka 19, 61000 Ljubljana, Slovenia.

© 1992 Elsevier Science Publishers B.V. All rights reserved 0304-4238/92/$05.00

344 M. CAMLOH AND N, GOGALA

are very common with extra vitro fern propagation from spores (Lane, 1981 ). Therefore, the aim of the present study was to determine optimal conditions for in vitro growth of gametophytes in order to develop a simple system for fern propagation, thus avoiding biological contamination.

MATERIALS AND METHODS

Spores of greenhouse-grown P. bifurcaturn were collected prior to use, then surface-sterilized for 5 s with 70% ethanol followed by 15 min in a solution of commercial bleach (sodium hypochlorite) diluted (3: 7 v/v) with H20 giv- ing 1.2% active chlorine. Spores were collected on filter paper and rinsed at least three times with sterile double-distilled water.

Approximately 1500 sterilized spores were sown on the surface of l0 ml modified Miller medium (Miller and Miller, 1961 ) in which Fe citrate was substituted by NaFeEDTA. Difco Bacto agar at 0.8% and 3% sucrose were added. The pH was adjusted to 5.7-5.8 before autoclaving. This represents our basal medium (BM) which was also used for the liquid cultures.

Gametophytic clumps, 6 to 7 weeks old, approximately 0.5 cm in diameter, were used to test the effects of sucrose, agar and pH of the medium on the growth of the gametophytes: ( l ) sucrose, 0, l, 2, 3, 4 or 5% of sucrose was added to BM; (2) agar, explants were placed on filter paper bridges in liquid medium or on BM solidified with 0.6, 0.8 or 1% agar; (3) pH ofthe medium, BM was adjusted to pH 5.0, 5.5, 6.0, 6.5 or 7.0. Fresh and dry weight mea- surements were taken after 4 weeks on different media. For each treatment 15-20 explants were used, and all experiments were repeated two or three times.

Two months after initiating the liquid culture from spores the gameto- phytes were poured onto sterilized soil to observe sporophyte development. Cultures were incubated at 25 _ 2 °C under a 16 h photoperiod at 5.5-12.3 W m- 2 (Sylvania GroLux F 40 T 12 and fluorescent LV 20 lamps). Student's t- test was used to calculate th~ levels of statistical significance (P) between the data obtained in control and other media.

RESULTS

The results of fresh and dry weight measurements indicate similar features ofgametophytic tissue growth, so only dry weights are represented (Table 1 ).

Effect o f sucrose. - The dry weights of gametophytic tissue significantly in- creased in all applied concentrations of sucrose (Table 1 ). The optimal su- crose level was 4%. Explants grown on media supplemented with different sucrose levels differ somewhat in their morphology. At higher concentrations

IN VITRO CULqIURE OF P. BIFURCATUM GAMETOPHYTES 345

TABLE 1

Effects of sucrose, agar and pH of the medium on the dry weight (DW) ofP. bifurcatum gametophytic tissue

Sucrose DW Agar DW pH DW (%) (%)

0 3.9+0.2 0' 14.6+ !.1 5.0 13.1 +0.7 1 8.6+0.6*** 0.6 12.9_+0.7 5.5 15.1 _+0.6* 2 11.5+0.5"** 0.8 11.8_+0.4" 6.0 11.7+0.5 3 12.9 +_ 0.7*** 1.0 11.8 -+ 0.7* 6.5 11.3 -+ 0.5" 4 15.7 _+ 1.3"** 7.0 9.7 + 0.4*** 5 13.1 +0.6***

Mean values + SE (mg) are shown. The levels of significant differences were calculated between con- trol media (the first value in each column ) and other media. 'Explants were grown on filter paper bridges in liquid medium. *P< 0.05; **P< 0.01; ***P< 0.001.

the tissue was more firmly packed together and changed colour from light to slightly darker green (data not shown).

Effect ofagar. - Concentrations higher than 0.6% significantly decreased dry weights ot ~explants. The best results were obtained when explants were placed on filter paper bridges in liquid medium (Table 1 ).

Effect o f the pH. - Growth of gametophytic tissue is pH-dependent. Dry weights were significantly greater when the pH ofthe medium was 5.5 (Table 1).

Sporophyte development. - Tw~ months after initiating the liquid gameto- phyte culture and I month after growth on sterilized soil, the first sporophytes appeared. After another 2 months they already had two leaves measuring 0.6- 0.8 cm.

DISCUSSION

Our results, showing the optimal growth of P. bifurcatum gametophytes at 4% sucrose, are similar to those previously described for gametophytes of Ne- phrolepis exaltata (L~, 1983 ). The report by Thentz and Moncousin (1984) differs in part from our results. They observed the best growth of P. bifurca- turn gametophytes at 1-2% sucrose; they determined growth 30 days after initiation of gametophyte cultures from spores, while at the end of our exper- iment the tissue was 70-77 days old, so we may attribute the difference to the age of the tissue. A similar phenomenon is known for gametophytes of Botry-

346 M. CAMLOH AND N. GOGALA

chium dissectum where the optimal sucrose concentration is 4% for older tis- sue and 2% for younger ~i~,c (Whittier, 1984 ~.

Our observations, indicating that higher agar concentrations decrease growth, may be a result of agar impurities (Debergh, 1983). Douglas and Sheffield (1990) also noted that fern gametophyte cultures cannot grow op- timally on agar-soli6ified medium. Thentz and Moncousin (1984) reported that lower concentrations of agar (0.3-0.5%) are favorable for P. bifurcatum gametophytes, which is similar to our results showing optimal growth of ex- plants on filter paper bridges (liquid medium) or on medium supplemented with 0.6% agar. The same culture method was successfully used for N. exal- rata and Asplenium nidus avis.

ACKNOWLEDGMENTS

We thank Dr. Jana Zel for constructive comments on the manuscript and Dr. Zdravko Podlesekfor his help with the statistical analysis of the data. We are also grateful to Nada Hojnik and Vanda Golobinek for technical assistance.

REFERENCES

Camloh, M. and Gogala, N., 1988. In vitro culture of gametophytic and sporophytic tissue of the fern Plat.vcerium b~[urcatum. 6th Congress of the European Societies of Plant Phy3ioiogy, 4- I 0 September ! 988, Split, Yugoslavia.

Debergh, P.C., 1983. Effects of agar brand and concentration on the tissue culture medium. Physiol. Plant., 59: 270-276.

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