American Fern Journal 91(4):179-186 (2001) Enhancement Fern of Spore Germination and Gametophyte Growth Media in Artificial Douglas\ HuxHAM^ Wynn E. Sheffield, G. E. Hearne^, and M. S. S. J. J. School of Biological Sciences, 3.614 Stopford Building, University of Manchester, Oxford Road, M13 Manchester 9PL, UK. — Abstract. Spores of braclcen fern, Pteridium aquilinum were sown in medium with and without No emerged agar. clear trend for germination, but in 14 days, significantly more plants passed from the filamentous two-dimensional on medium to state agar-solidified than in liquid shake- flasks. Spores of Pteridium aquilinum, Athyrium Dryopteris expansa and Anemia filix-femina, were sown media phyllitidis in containing sucrose. Percentage germination was of four species all enhanced by significantly the inclusion of sucrose. Fern gametophytes have been described as ideal experimental organisms for Miller Hickok One main et al, 1987). of the attractions of these plants their min- is imal nutritional requirements, and most researchers have exploited them in employing medium cultures media. Agar-solidified has been artificial the sub- used most strate routinely for the artificial culture of fern gametophytes, but disadvantages have been reported. Agar can be toxic to plants Debergh, (e.g., & 1983), can perturb sexual expression of ferns (Rubin and Paolillo, 1983), it Sheffi ame Anemia and phyllitidis, to be significantly higher in liquid than in the same me these higher yields reflected increased germination num- rates (therefore larger bers of individuals), higher rates of growth of similar numbers of individuals, or both. Data in Douglas (1994) indicated differences in germination of Pteri- dium and Anemia between and medium, liquid agar-solidified with germina- medium tion percentage significantly higher in liquid for both species. Sub- work sequent in our laboratory using spores from the same used collections as Camloh in those experiments, has not repeated these findings. (1993) found no germination significant differences in of the epiphytic fern Platycerium bifurcatum between liquid and agar medium, but reported **development" that was enhanced by medium. The aim was liquid first of this investigation there- medium fore to establish whether liquid promotes germination and/or growth of one of the two species used in the study of Douglas and Sheffield, viz. Pteridium aquilinum (bracken fern). UK Museum. Current address: Biodiversity Office, Natural History * Current address: Dept of Animal & Plant Sciences, University of Sheffield. 2 & ^ Current address: Division of Agriculture Horticulture, School of Biological Sciences, University of Nottingham. MISSOURI BOTANICAL MAR 1 8 2002 GARDEN LIBRARY 180 AMERICAN FERN JOURNAL: VOLUME NUMBER 91 4 (2001) Many medium workers have included metabolisable sugars in the artificial used to germinate fern spores, but no clear trends have emerged from such studies. Hurel-Py [1955) reported that sugars inhibited germination of Also- Camloh found phila australis, [1993) that the inclusion of sucrose in liquid medium produced no significant inhibition or stimulation of germination of P. bifurcatum spores. Pilot experiments of Douglas [1994) suggested that sucrose greatly enhanced germination of aquilinum and A. phyllitidis. The second fl aim was whether of this investigation therefore establish the addition of to sucrose enhances or inhibits germination of fern spores. Four species were chosen from different families [Pteridium aquilinum, Athyrium filix-femina, Dryopteris expansa and Anemia phyllitidis] in an attempt to generate broadly , applicable findings. Materials and Methods Spores of Pteridium aquilinum, Athyrium Dryopteris expansa filix-fenuna, and Anemia were and phyllitidis collected stored (dry in glass vials) at 4''C for either one (Pteridium), two [Athyrium, Dryopteris] or twelve years (Anemia). Spores of the first three taxa were harvested from natural populations of British Anemia ferns, and the spores were a kind donation from the glasshouse population of Professor H. Schraudolf [University of Ulm). All spores were sieved mesh through a fine nylon (45|jLm pore size, Lockertex) to separate them from sporangial debris. They were weighed and kept in plastic vials containing approximately 10ml distilled water with one drop of detergent (wetting agent) room temperature 18-24 Spores were then with 2.5% at for h. surface sterilised aqueous sodium (10-14% (vol/vol) hypochlorite solution available chlorine, BDH) They for 2 min, then rinsed three times in sterile distilled water. were subsequently suspended in 0.5% (w/vol) high viscosity carboxymethyl sterile (BDH) cellulose to allow even distribution of the spores in suspension for medium inoculation. The basic mineral used throughout was that of Moore pH adjusted (1903) to 6.25. — Agar Medium vs Liquid Medium. containing l.5gl~^ Sigma bacteriological grade agar was dispensed medium was dispensed into petri dishes; liquid as 150ml 250ml and aliquots into conical flasks the flasks sealed with a polyure- thane foam bung. Both media were autoclaved min. at 15lb, 120''C for 15 — Medium With and Without Medium Liquid Sucrose. without and sucrose, medium containing 0.05 and 0.2M sucrose [Sigma) was dispensed into flasks as above, and autoclaved lOlb, 115°C 10 min. at for — Inoculation. Each spore suspension was adjusted to contain 150,000 (± 500) spores using a Sedgewick chamber and inoculum added Rafter aseptically to each dish or flask to give an initial spore concentration of 1000 spores per ca. ml medium. The of snores w^ere RVPnlv nvpr ^o^r cnrfprp ncina di«tribiitprl tV»P moisture unit SHEFFIELD ET SPORE GERMINATION AND GAMETOPHYTE GROWTH AL.: 181 rotary shaker at 96rpin and dishes cukured on the hench in the same room, by illuminated daylight fluorescent tubes providing photosynthetically active nm 400-700 radiation of of 106 (±21) [xEm-^s"^ light at the level of the culture on 16h 8h surface a light dark cycle, 20 (±2)^C. at — Sampling. Cultures were sampled after 7 days' incubation in the above con- Num- and ditions for all treatments, after 14 days for liquid vs agar treatments. bers of germinated and non-germinated spores were counted after 7d, and the numbers had 2D growth and of individuals that established those still filamentous 14d were counted; 250 individuals were counted per after replicate. — were Analyses.^ Individual replicates subjected to analysis of variance, spore 2D germination and growth data were subjected an independent samples to t- test for equality of means, and the means of each different sucrose treatment which subjected to chi-square tests to ascertain sources of variation generated between differences treatments. Results — Agar Medium. was vs Liquid Percentage germination higher Pteridium for sown medium; spores on agar than in liquid the percentage of individuals that had established 2D growth by 14d of culture was considerably higher both for medium The were species in agar than in liquid (see Table treatment effects 1). when was confirmed the experiment repeated, although the overall percentage was same germination and the percentage variation not the for each repeat. from sown medium were more Results obtained spores in liquid routinely far from variable than those obtained agar-solidified cultures. — Medium With and Without was Sucrose. Percentage germination Liquid and was very different for different species (see Tables 2-5) **species tested" the most highly significant factor accounting for differences between treat- = ments 139.51, p<0.001, see Table Germination in different concentra- (F 6). 0<0.05<0.2M tions of sucrose was in the order of for all species except Ane- mia, where germination was slightly less in 0.2 than in 0.05M, but both were OM The between due higher than (see Tables 2-5). differences treatments to ^ was concentration of sucrose significant (F 5.88, p<0.01, see Table 6). Discussion sown The results presented here for germination of spores of Pteridium in medium do not mirror those obtained by Douglas vs agar-solidified liquid The study reported 64(± 4)% germination 42(± 4)% in liquid, (1994], latter — on lower germination agar than in liquid the reverse of the find- in agar, i.e. ings reported herein. Douglas (1994) found less difference for Anemia, but the We upon medium trend was the same. could speculate an interaction between composition and spore age, as our spores oi Pteridium were only one year old, and Douglas were two years old Pteridium and seven years old those of for for 182 FERN JOURNAL Table The mean 1. percentage of P. aquilinum spores that germinated, and the mean percentage of individuals that had progressed to 2D growth after 14 d in liquid and agar-solidified Moore's medium (n = 4 for in parentheses). all, s.e. Agar P Liquid ' Germination 85.2 60.0 0.026* (1.1) (16.6) P 2D growth 94.8 34.2 (1.2) (13.6) 0.01 * Less than 0.05, therefore differences between treatments significant Table 2. The mean percentage germination of spores sown in liquid medium containing different con- = centrations of sucrose for P. aquilinum (n 4). % Sucrose concentration germination OM 30.4 (8.0) M 0.05 39.3 (10.2) M 0.2 43.3 (18.1) < Between P concentrations chi-square 145.94 0.001 Table The mean percentage germination of spores sown medium 3. in liquid containing different con- ^ centrations of sucrose for A. phyllitidis (n 4). % Sucrose concentration germination OM 54.0 (3.1) M 0.05 57.8 (8.6) M 0.2 56.4 (8.6) < Between P concentrations chi-square 7.54 0.05 Table The mean 4. percentage germination of spores sown medium in liquid containing different con = OM = centrations of sucrose for D. expansa (n 4 for and 0.05M sucrose; n 0.2M 3 for sucrose). % Sucrose concentration germination OM 57.8 (3.3) M 0.05 61.1 (4.6) M 0.2 66.0 (4.5) Between < concentrations chi-square 33.34 P 0.001 Table The mean 5. percentage germination of spores sown medium in liquid containing different con- = OM = centrations of sucrose for Athyrium filix-femina (n 4 for and 0.05M sucrose; n for 0.2M 1 sucrose). % Sucrose concentration germination OM 87.2 (L3) M 0.05 91.2 (2.6) M 0.2 94.7 (na) < Between P concentrations chi-square 50.61 0.001 1 SHEFFIELD ET SPORE GERMINATION AND GAMETOPHYTE GROWTH AL.: 183 Table Chi-square of differences examined. 6. tests attributable to the variables Source of variation: Chi-square Probability < Species 4952.35 P 0.00 < Concentrations within species 237.45 P 0.001 we Anemia, and obtained higher germination percentages than Douglas overall in our experiments. However, seems no safer to state that there are predict- it able differences in germination of Pteridium in liquid and agar-solidified medium. This suggests that the higher yields of aquilinum and A. phyllitidis P. medium tissue in liquid than in agar-solidified observed by Douglas & Shef- may field [1992) have been due to higher growth rates of individuals in medium Our in liquid form. finding that fewer of the individuals growing in medium liquid go on to develop into 2-dimensional plates than on agar medium mirrors the data of Douglas (1994) relating to cell number. The latter study reported significantly fewer cells per gametophyte in 28 day-old indi- same viduals of the species in liquid, than on agar-or agarose-solidified medium. This indicates that although early growth rate enhanced, subsequent is differentiation into the form usually adopted by Pteridium perturbed by is medium. liquid This the reverse of the situation reported Platycerium is for may mean bifurcatum (Camloh, 1993). This that the response to liquid spe- is cies- or ecotype-specific (as P. bifurcatum is an epiphytic species, and spores would conceivably land in more aqueous situations on tree bark than in the colonised by Pteridium). Yields of aquilinum and A. terrestrial sites pttyl- P. are enhanced by using immobilised, rather than shake culture (Douglas litidis & Sheffield, 1992) and perturbation of the development of these two species in liquid can be circumvented by the use of immobilised or cultures air-lift (Sheffield 1997). et al., Differences in percentages of germinating spores from one population, ex- common periment or culture to another are in fern experiments Pangua (e.g. 1999), and large differences in overall germination percentage the rule et al., is in simultaneous experiments on different taxa in one set of conditions (e.g. No was made Ranal, 1999). attempt to identify the source of the variation made between the species tested herein, because no effort had been to refine medium growth the basic mineral or find preferred conditions for each fern. known well that surface sterilisation reduces percentage germination, and It is that spore age, storage conditions and culture conditions all have a great in- fluence on fern spore viability Miller, 1968; Raghavan, 1989; Camloh, (e.g. & 1993 1999; Sheffield, 1996). is therefore unsurprising that the greatest It How- germination observed were taxon differences in attributable to the tested. ever, clear that for all four species tested here, whatever the background it is and level of spore viability after collection, storage surface sterilisation, the medium enhances germination addition of sucrose in liquid (see Fig. This 1). who P Camloh was bifurcatum by found no promotive not reported for (1993), may on effect of sucrose germination. Again, therefore, this reflect species- 184 AMERICAN FERN JOURNAL: VOLUME NUMBER 91 4 (2001) 14 * + t * 12 O 1 » * * + * 10 * £ 9 ,- , • A 4 » I I P * V « * 4 8 I ^ ^ 4 * 4 4 4 A I 1*444 4 4 4 W I * * I I « V + 4 P 4 * T Ip frdp ^fr^ t4p ^ r « 4 A * * 4 L4 +I ^44^^ * * t_ b * p 4 * P 4 V 4 * 4 * * P 4 4 P * * * * t4V 4P« 4 4 » P • 4 • P 4 » » + - 4 P * ^ 4 ^ ***•*+•* * p + * ^ » .5 * 4 t * * + * t 4 4 m P > P P « * u h 4 ri P ^ * » 4PP 4 4 B 4 p « 4P^ * 4 V44 ^4 « 4 * 4 *' 4 4 4 44 4•I 4 4 ^4 4 th « 4 V b # .at » P • > 4 «4*« 44 4 ^ T- 4 P * 4 4 4 Q '• ,> *' .. * P ^44mA+*44+*«mA#**P44I I * 4 * 4* pp* #i*t ppP fPPe •»* 4444 *^4 4PP 4^ ^ «4 4 4 w 4 P4 « • * • * K 4- h * A 4 4 P 4 - . 4 * 4 4 4 4 4 » i p fe 4 P _ 4 4 4 4 4 * H4 4 h PV4 *m P4 P4 I4*4P*+* *+«P4**44» 4 4 44 4 4#4 w # 4 ^•VPP44 » ht ^ * 4 VI P 4*4 * 4 • * * • 4 4 * 4 4 4 4 4 4 w 4 * P p * 4 P * • 4 4 4 * 4 4 tt * * * ^k P '4 4 *1 * iJ X t 1_ 4 4 ' ' P. aquilinum D. expansa A. femina A. filix phyllitidis species % Fig. 1. Histogram of spore germination plotted as differences between sucrose treatments — — 2M DM (0.05M diamond and dotted; shading) sucrose Order controls. of species reflects in- creasing age of spores, youngest far left, oldest far right. may specific processes, or again be significant that Platycerium epiphytic, it is and therefore very from The different the terrestrial ferns tested in this study. much spores of Platycerium are also larger than those of the species tested in may the present study, so this difference reflect different levels of storage carbohydrate. All the spores of Platycerium tested by Camloh may have had suf- ficient storage carbohydrate to germinate, in which case no increase in germination with added would may sucrose be expected. be also that a different It medium sugar or component would promote The germination in this species. Camloh spores used were and known stored before use, that levels of is it proteins as well as soluble sugars decline with & storage of fern spores (Beri could be Bir, 1993). It speculated, therefore, that the spores of Platycerium that medium germinate fail to in basal including might germinate provided sugar, if ammo 4 Our finding that sucrose did not promote germination Anemia of spores as may strongly as the other species also reflect storage-related decline [see Fig. The Anemia spores used here had been many 1). stored for years longer than A the other species. pilot experiment carried out with the same spore sample showed five years earlier far greater promotion with sucrose (Douglas, 1994). In that experiment the germination = percentages n in parentheses, (s.e. 4) OM were as follows: sucrose 0.05M 53.1(6.7); sucrose 72.5 0.2M sucrose (6.3); The 74.8 percentage germination without was (3.4). sucrose very similar to that seen here (54.0), but the promotion of germination by sucrose was far more may pronounced. This suggest that during storage another com- cellular ponent had declined by to limiting levels the time the spores were retested. SHEFFIELD ET SPORE GERMINATION AND GAMETOPHYTE GROWTH AL.: 185 The interaction between promotive effects of sucrose and length of time in storage should clearly be tested for individual spore samples, growth condi- and However, promotes tions species. the results indicate that sucrose germi- many nation in ferns and would therefore be worth adding to media used to initiate cultures of a previously untested species. This study examined the effects of sucrose on germination only. Effects of sucrose on subsequent growth of the species studied were not tested and ap- Camloh pear to differ from one species to another. (1993) reported a stimula- medium tion of growth (in terms of cell number) using sucrose in used to culture Platycerium, while Douglas (1994) found evidence of growth inhibition numbers 2D Anemia and terms of achieving growth) in Pteridium. Fernan- (in dez (1997) reported that sucrose inhibited growth (fresh weight and gem- et al. ma Osmunda formation) in gametophytes of regalis. Further experimentation may some with a wide range of species reveal reliable trends, but the current on on growth weight of evidence the side of deleterious effects of sucrose is of terrestrial species. This, coupled with the enhancement of growth of micro- which media prompts contaminants attends the use of containing sugars, bial us suggest a 2-stage cultivation process in order to optimise yield/growth to of cultured fern gametophytes. Germination should be effected in media con- subsequent growth should employ immobilised taining sucrose, or cul- air-lift media with no sucrose. ture in Acknowledgments GED We are grateful to Sue Rumsey and Dave Robson for technical support. was in receipt of a BBSRC studentship, JMW is in receipt of a NERC CASE studentship. We thank Bob Callow and Russ Drakeley for their help with statistical analysis, and an anonymous referee for suggestions which improved manuscript. vastly this Literature Cited and 1993. Germination of stored spores of Fteris vittata. Amer. Fern. 83:73-78. Beri, a., S. S. Bir. J. Camloh, M. 1993. Spore germination and early gametophyte development of Platycerium bifurcatum. Amer. Fern. 83:79-85. J. 1999. Spore age and sterilisation affects germination and early gametophyte development . Amer. 89:124-132. Platycerium bifurcatum. Fern. oi J. Debergh, 1983. Effects of agar brand and concentration on the tissue culture medium. Phy- C. p. 59:270-276. siol Plant. An development and Douglas, 1994. investigation into the growth, ultrastructure of fern G. E. PhD Man- gametophytes in existing and novel culture systems. dissertation, University of UK. chester, Douglas, G. and E. Sheffield. 1992. The investigation of existing and novel artificial growth E., systems the production of fern gametophytes. Pp. 183-187, in M. Ide, A. C. Jermy, and for J. and M. Paul Fern Horticulture: post, present future perspectives. Intercept Andover, A. (eds). Dyer, A. 1979. The culture of fern gametophytes for experimental investigation. Pp 253-305, F. Academic Dyer The Experimental Biology of Ferns. Press. A. (edj. in F. Fernandez, A. M. Bertrand and R. Sanches-Tames. 1997. Germination in cultured gameto- H., Osmunda Plant Cell Rep. 16:358-362. phytes of regalis. Wakne HiCKOCK, C, K. and M. K. Slocum. 1987. Ceratopteris ricbardii: appUcations for L. L. Amer. 74:1304-1316. experimental plant biology. Bot. J. VOLUME 186 Hurley-Py, G. 1955. Action de quelques sucres sur la germination des spores d'AIsopbila australis. Compt. Rend. Acad. Sci. Paris 241:1813-1815. Miller, H. 1968. Fern gametophytes experimental as material. Bot. Rev. (Lancaster] 34:361- J. 440. Moore, Methods G, T. 1903. for growing pure cultures of algae. Appl. Microscop. Lab. Meth. J. 2309-2314. 6: Pangua, E., L. Garcia-Alvarez and Parajon. 1999. Studies on Cryrptogramma S. crispa spore germination. Amer. Fern. 89: 159-170. J. Raghavan, Developmental V. 1989. biology gametophytes. Cambridge of fern University Press. 361 pp. Ranal, M. a. 1999. Effects of temperature on spore germination in some fern species from semi- deciduous mesophytic Amer. Fern 89:149-158. forest. J. Rubin, G., and D. Paolillo. 1983. Sexual development of Onoclea sensibilis on agar and soil J. media without the addition of antheridiogen. Amer. Bot. 70:811-815. J. From Sheffield, E. 1996. pteridophyte spore to sporophyte in the natural environment. Pp 541- Pteridology Gardens, Kew. game an in airlift fermenter. Plant Cell Rep. 16:561-564.

Enhancement of Fern Spore Germination and Gametophyte Growth in Artificial Media

Similar Enhancement of Fern Spore Germination and Gametophyte Growth in Artificial Media