Saudi Journal of Biological Sciences (2010) 17,13–22

King Saud University Saudi Journal of Biological Sciences

www.ksu.edu.sa www.sciencedirect.com

ORIGINAL ARTICLE Sporophyte and gametophyte development of Platycerium coronarium (Koenig) Desv. and P. grande (Fee) C. Presl. (Polypodiaceae) through in vitro propagation

Reyno A. Aspiras

Department of Biology, College of Arts and Sciences, Central Mindanao University, University Town, Musuan, Bukidnon, Philippines

Available online 22 December 2009

KEYWORDS Abstract The sporophyte and gametophyte development of Platycerium coronarium and P. grande Propagation techniques; were compared through ex situ propagation using in vitro culture technique and under greenhouse Endangered species; and field conditions. Staghorn ferns; The morphology of the sporophyte and gametophyte, type of spore germination and prothallial Sporophyte; development of P. coronarium and P. grande were documented. Gametophytes of P. coronarium Gametophyte and P. grande were cultured in vitro using different media. The gametophytes were then transferred and potted in sterile chopped Cyathea spp. (anonotong) roots and garden soil for sporophyte forma- tion. Sporophytes (plantlets) of the two Platycerium species were attached on the slabs of anonotong and on branches and trunks of Swietenia macrophylla (mahogany) under greenhouse and field condi- tions. Sporophyte morphology of P. coronarium and P. grande varies but not their gametophyte morphol- ogy. P. coronarium and P. grande exhibited rapid spore germination and gametophyte development in both spore culture medium and Knudson C culture medium containing 2% glucose. Gametophytes of P. coronarium and P. grande transferred to potting medium produced more number of sporophytes while the gametophytes inside the culture media did not produce sporophytes. Sporophytes of P. grande attached on mahogany branches produced more number of leaves with bigger leaf area than those attached on anonotong slabs. Likewise, sporophytes of P. coronarium attached on mahogany branches and anonotong slabs did not develop new leaves during two weeks monitoring and are still in a period of adjustment to its environment. Sporophytes of P. grande grown or attached on the trunk of mahogany trees in the field and under shaded environment favored their growth. ª 2009 King Saud University. All rights reserved.

E-mail address: [email protected]

1319-562X ª 2009 King Saud University. All rights reserved. Peer- 1. Introduction review under responsibility of King Saud University. doi:10.1016/j.sjbs.2009.12.003 Platycerium Desv., commonly known as staghorn fern, stands out as one of the most commonly grown and highly priced Production and hosting by Elsevier ornamental ferns (Darnaedi and Praptosuwiryo, 2003). Stag- horn ferns are becoming threatened in the wild for they are 14 R.A. Aspiras sought – after by plant collectors for their majestic size and more sporophytes (plantlets) of the two species of Platycerium; form (Madulid, 1985). Since staghorn ferns are becoming find out which medium, slab of Cyathea spp. (anonotong) or endangered and having spores that are difficult to germinate branch of Swietenia macrophylla (mahogany), is more effective under natural condition (Amoroso, 1990, 1992; Amoroso for the growth of the sporophytes of the two species of Platyce- and Amoroso, 1998, 2003), it is interesting to find out the dif- rium under greenhouse condition; and observe the growth of ferences of the developmental patterns of their gametophytes the sporophytes of P. coronarium and P. grande attached to and sporophytes which can be utilized in the mass propagation the trunk of S. macrophylla (mahogany) in the field. of these plants. In order to conserve the remaining popula- tions, in vitro technique is necessary to ensure mass production of these species. 2. Materials and methodology Studying the differences in the development of gametophyte and sporophyte of P. coronarium (Koenig) Desv. and P. grande Platycerium coronarium and P. grande are epiphytic ferns (Fee) C. Presl. provide evidences on variation patterns, which growing solitary or in clusters on trunks, branches of trees, is one of the criteria in fern taxonomy (Raghavan, 1989; Joa- and on tree tops and old trees in rainforests at lower altitudes. quin and Zamora, 1996). Part from that, fern spores and These species of ferns were also found cultivated in some gar- gametophytes are excellent biological systems for the analysis dens in the provinces of Bukidnon and Davao (Fig. 1A and D). of physiological and developmental problems (Raghavan, P. coronarium and P. grande are characterized by frond 1989). dimorphism, the formation of a basket of base fronds and More specifically, the study aimed to determine the culture the dichotomously divided pendulous foliage fronds. Base media for rapid spore germination and development of the fronds of P. coronarium forked unequally and its foliage fronds gametophytes of the two species of Platycerium; identify which are asymmetrical. Base fronds of P. grande forked equally and of the two methods of propagation, i.e., (i) transferring the its foliage fronds are symmetrical. gametophytes to potting medium containing sterilized chopped The sori of P. coronarium and P. grande are forming large Cyathea spp. (anonotong) roots and garden soil (1:1) or (ii) soral patches. Sori of P. coronarium cover completely the cen- leaving the gametophytes in the agar culture medium, produce tral fertile lobe. Sporangia have 9 or 10 indurated annular

Figure 1 Habit and gross morphology of Platycerium coronarium and P. grande. (A) P. coronarium found thriving in the lowland dipterocarp forest of Mt. Hamiguitan, Sitio Magum, Barangay Macambol, Mati, Davao Oriental; (B) soral patch of P. coronarium that covers completely the central fertile lobe; (C) sporangium of P. coronarium showing its annular cells and long-stalked receptacular paraphysis; (D) P. grande cultivated in Malagos Garden Resort, Calinan, Davao City; (E) soral patch of P. grande showing its semicircular shape; (F) sporangium of P. grande showing its annular cells and short-stalked receptacular paraphyses. Sporophyte and gametophyte development of Platycerium coronarium 15 cells, long-stalked with 8 spores per sporangium. Sori of Both the germ filament and primary rhizoid elongate along the P. grande are in semicircular shape and located on the equatorial plane of the spore in opposite direction. Both P. undersurfaces of the foliage fronds. The sporangia have 21– coronarium and P. grande showed Drynaria-type of prothallial 26 indurated annular cells, short-stalked with 64 spores per development (Figs. 4A–F and 5A–F). According to Nayar and sporangium (Fig. 1B, C, E and F). Kaur (1971), the establishment of an apical meristematic cell of Gross morphology of P. coronarium was recorded based on this type of prothallial development is far delayed. A broad the mature plant found in the garden of Mr. Romulo Castada spatulate prothallial plate is formed by division of the anterior of Malaybalay City and in Mt. Hamiguitan, Sitio Magum, cells, including the terminal cell of the germ filament by walls Mati, Davao Oriental. For P. grande, it was based from the parallel to the long axis, and by repeated longitudinal and mature plant grown in the fernery of the Department of Biol- transverse divisions in the daughter cells. An obconical meri- ogy, College of Arts and Sciences, Central Mindanao Univer- stematic cell is formed by two oblique divisions in one of the sity, Musuan, Bukidnon and also in Malagos Garden Resort, anterior marginal cells of the prothallial plate when it is 5–10 Calinan, Davao City. Spores of P. coronarium and P. grande or sometimes more cells broad. Further growth results in a were obtained from the same places mentioned. symmetrical cordate prothallus. The experiment on gametophyte development was carried out following a factorial Randomized Complete Block Design 3.2. Propagation techniques (RCBD). The four media (spore culture medium, spore culture medium + 2% glucose, Knudson C culture medium, and 3.2.1. Spore germination and gametophyte development in Knudson C culture medium + 2% glucose) served as factor different culture media A while the two plant species (P. coronarium and P. grande) Stages of gametophyte development of P. coronarium and P. as factor B, and replicated ten times. grande as to the number of days in four culture media The experiment on sporophyte formation was carried out (M1G0 – spore culture medium, M1G1 – spore culture med- using Randomized Complete Block Design (RCBD) with eight ium + 2% glucose, M2G0 – Knudson C culture medium, (8) media and replicated 10 times. The media were: (1) M1G0 – and M2G1 – Knudson C culture medium + 2% glucose) is spore culture medium; (2) M1G0(P) – spore culture medium presented in Table 1. and transferred to potting medium; (3) M1G1 – spore culture medium + 2% glucose; (4) M1G1(P) – spore culture med- 3.2.1.1. Imbibition and swelling of the spores. This gametophyte ium + 2% glucose and transferred to potting medium; (5) developmental stage was first observed in M1G1 and M2G1 in M2G0 – Knudson C culture medium; (6) M2G0(P) – Knudson P. coronarium. This result was obtained for only 6 days after C culture medium and transferred to potting medium; (7) spore inoculation in the media. M1G1 and M2G1 had the M2G1 – Knudson C culture medium + 2% glucose; and (8) significantly earliest imbibition and swelling of the spores com- M2G1(P) – Knudson C culture medium + 2% glucose and pared to the other two media. In P. grande, this gametophyte transferred to potting medium. developmental stage was observed in M1G1 at 10 days after The experiments on leaf formation and leaf area under inoculation and followed by M2G1 at 12 days after inocula- greenhouse condition were carried out using Completely Ran- tion. Among the media used, M1G1 showed significantly the domized Design (CRD) with two media and replicated 10 earliest for imbibition and swelling of the spores, followed by times. The media were: (1) H1 – hanging slab of anonotong; M2G1, and this was then followed by the other two media hav- and (2) H2 – hanging branch of mahogany. Some of the sporo- ing the latest imbibition and swelling period. phytes were then attached to the trunk of mahogany under Imbibition and swelling of the spores in both plant species shaded environment inside the Fernery of the Department of were significantly affected by the media used. It was found that Biology. M1G1 with 8.0 mean number of days for this gametophyte The data gathered were analyzed using the software for developmental stage to occur was significantly the earliest. Analysis of Variance (ANOVA) and medium means were com- This was followed by M2G1 with 9.0 mean number of days. pared using Duncan’s New Multiple Range Test (DNMRT) at These results imply that M1G1 and M2G1 enhance rapid imbi- 5% level of significance. bition and swelling of the spores which could be attributed to the amount of glucose added. The glucose will be transported to the cytoplasm of the spore through facilitated diffusion. In- 3. Results and discussion side the cell, the transported glucose is utilized during respira- tion leading to the production of energy in a form of ATP. The 3.1. Spore morphology, spore germination and prothallial energy yielded will be used for any metabolic processes occur- development ring inside the cell.

The spores of P. coronarium and P. grande are bean-shaped, 3.2.1.2. Emergence of the rhizoid at the lower posterior end of monolete and bilaterally symmetrical. Both of them showed the spore. A rupture at the lower end of the spore caused by Equatorial-Gleichenia type of spore germination (Figs. 2A–F spore swelling led to the emergence of the rhizoid and a germ and 3A–F). According to Nayar and Kaur (1971), this type papilla emerged next at the upper end. This gametophyte of spore germination is found in the Gleicheniaceae, Dipterid- developmental stage was first observed in M1G1 and M2G1 aceae, Loxogrammaceae, and many of the Polypodiaceae to in P. coronarium at 9 days after inoculation. M1G1 and which family Platycerium belongs. At germination a rhizoid M2G1 had the significantly earliest emergence of the rhizoid initial is cut off laterally by a wall parallel to the polar axis at the lower posterior end of the spore compared to the other of the spore. A series of divisions in the prothallial initial cell two media. In P. grande, this gametophyte developmental by walls parallel to the first result in a uniseriate germ filament. stage was observed in M1G1 at 14 days after inoculation 16 R.A. Aspiras

Figure 2 Spore morphology and Equatorial-Gleichenia type of spore germination of P. coronarium. (A) Bean-shaped, monolete and bilateral spore (·200); (B) swollen spore showing its chloroplasts (·200); (C) emergence of chlorophyllous papillate structure or germ filament and the development of the rhizoid from the spore (·200); (D–F) spores with 7-, 8-, 9-celled germ filaments (·100).

Figure 3 Spore morphology and Equatorial-Gleichenia type of spore germination of P. grande. (A) Bean-shaped, monolete and bilateral spore (·200); (B) swollen spore showing its chloroplasts (·200); (C) emergence of chlorophyllous papillate structure or germ filament and the development of the rhizoid from the spore (·200); (D–F) spores with 7-, 9-, 11-celled germ filaments (·100).

followed by M2G1 at 17 days after inoculation. M1G1 showed rel-shaped and densely chlorophyllous cells, and at the basal the significantly earliest emergence of the rhizoid at the lower end form the rhizoids. This gametophyte developmental stage posterior end of the spore, followed by M2G1, and this was was first observed in M1G1 and M2G1 in P. coronarium at then followed by the other two media having the latest emer- 14 days after inoculation and found to have the significantly gence of the rhizoid at the lower posterior end of the spore. earliest germ filament formation compared to the other two The number of days for emergence of the rhizoid at the low- media. In P. grande, this gametophyte developmental stage er posterior end of the spore between two plant species was sig- was observed in M1G1 at 18 days after inoculation followed nificantly different. The gametophytes of P. coronarium by the other three media 21 days after inoculation. Among required 11.50 days for this gametophyte developmental stage the media used, M1G1 showed the significantly earliest germ to occur while P. grande required 16.75 days. filament formation. The number of days for germ filament formation between 3.2.1.3. Germ filament formation. The elongation of the germ two plant species was significantly different. The gametophytes papilla and its continuous division gave rise to a protonema of P. coronarium required 15.50 days for germ filament forma- consisting of a uniseriate germ filament with 2–8 or more bar- tion to occur while P. grande required 20.25 days. Sporophyte and gametophyte development of Platycerium coronarium 17

Figure 4 Prothallial development of P. coronarium. (A) Early spatulate stage (·100); (B) mid-spatulate stage (·100); (C) late-spatulate stage (·100); (D) early lopsided prothallus (·100); (E) cordate prothallus (·40); (F) secondary gametophyte (·40).

Figure 5 Prothallial development of P. grande. (A) Early spatulate stage (·100); (B) mid-spatulate stage (·100); (C) late-spatulate stage (·100); (D) early lopsided prothallus (·40); (E) cordate prothallus (·40); (F) secondary gametophyte (·40).

3.2.1.4. Spatulate prothallial plate formation. A broad spatulate 25 days after inoculation. Among the media used, M1G1 prothallial plate is formed by division of the anterior cells, showed the significantly earliest spatulate prothallial plate including the terminal cell, of the germ filament by walls par- formation. allel to the long axis, and by repeated longitudinal and trans- The number of days for spatulate prothallial plate forma- verse divisions in the daughter cells. This gametophyte tion between two plant species was significantly different. developmental stage was first observed in M1G1 and M2G1 The gametophytes of P. coronarium required 19 days for this in P. coronarium at 17 days after inoculation and had the sig- gametophyte developmental stage to occur while P. grande re- nificantly earliest spatulate prothallial plate formation com- quired 24 days. pared to the other two media. In P. grande, this gametophyte developmental stage was observed in M1G1 at 3.2.1.5. Lopsided prothallus formation. The continuous division 21 days after inoculation followed by the other three media of the anterior and terminal cells of the prothallial plate and by 18

Table 1 Mean number of days for the gametophyte developmental stages of the two species of Platycerium on different culture media. Media Mean number of days Imbibition and Emergence of the Germ filament Spatulate prothallial Lopsided Cordate Emergence of Elongation of swelling of the rhizoid at the lower formation plate formation prothallus prothallus germ filament from secondary spore posterior end of the spore formation formation primary gametophyte gametophyte from primary gametophyte P. coronarium M1G0 9.0 d 14.0 c 17.0 c 21.0 b 25.0 b 30.0 b 42.0 a 49.0 b M1G1 6.0 e 9.0 d 14.0 d 17.0 c 21.0 c 25.0 c 35.0 b 42.0 c M2G0 10.0 c 14.0 c 17.0 c 21.0 b 25.0 b 30.0 b 42.0 a 49.0 b M2G1 6.0 e 9.0 d 14.0 d 17.0 c 21.0 c 25.0 c 35.0 b 42.0 c P. grande M1G0 14.0 a 18.0 a 21.0 a 25.0 a 30.0 a 35.0 a 42.0 a 56.0 a M1G1 10.0 c 14.0 c 18.0 b 21.0 b 25.0 b 30.0 b 35.0 b 49.0 b M2G0 14.0 a 18.0 a 21.0 a 25.0 a 30.0 a 35.0 a 42.0 a 56.0 a M2G1 12.0 b 17.0 b 21.0 a 25.0 a 30.0 a 35.0 a 42.0 a 56.0 a Plant species means P. coronarium 7.75 b 11.50 b 15.50 b 19.00 b 23.00 b 27.50 b 38.50 b 45.50 b P. grande 12.50 a 16.75 a 20.25 a 24.00 a 28.75 a 33.75 a 40.25 a 54.25 a Media means M1G0 11.5 b 16.00 a 19.00 a 23.00 a 27.50 a 32.50 a 42.00 a 52.50 a M1G1 8.0 d 11.50 c 16.00 c 19.00 c 23.00 b 27.50 c 35.00 b 45.50 c M2G0 12.0 a 16.00 a 19.00 a 23.00 a 27.50 a 32.50 a 42.00 a 52.50 a M2G1 9.0 c 13.00 b 17.50 b 21.00 b 25.50 b 30.00 b 38.50 b 49.00 b F-Test Plant sp. ** ** ** ** ** ** ** ** Medium ** ** ** ** ** ** ** ** P · M ** ** ** ** ** ** ** ** CV, % 6.61 4.68 3.49 2.73 2.55 2.19 1.75 1.24 Means within each column having a common letter are not significantly different at 5% level based on Duncan’s New Multiple Range Test. P · M – plant · medium. ** – Highly significant. M1G0 – spore culture medium; M2G0 – Knudson C culture medium; M1G1 – spore culture medium + 2% glucose; M2G1 – Knudson C culture medium + 2% glucose. ..Aspiras R.A. Sporophyte and gametophyte development of Platycerium coronarium 19 repeated longitudinal and transverse divisions of the daughter gametophyte developmental stage was first observed in cells led to the formation of a lopsided prothallus. In this stage, M1G1 and M2G1 in P. coronarium at 42 days after inoculation. an obconical meristematic cell is formed by two oblique divi- It was then followed by M1G0 and M2G0 49 days after inocu- sions in one of the anterior marginal cells of the prothallial lation. M1G1 and M2G1 had the significantly earliest elongat- plate when it is 5–10 or sometimes more cells broad. This ing secondary gametophyte from primary gametophyte gametophyte developmental stage was first observed in compared to the other two media. In P. grande, this gameto- M1G1 and M2G1 in P. coronarium at 21 days after inoculation phyte developmental stage was observed in M1G1 at 49 days and had the significantly earliest lopsided prothallus formation after inoculation followed by the other three media at 56 days compared to the other two media. In P. grande, this gameto- after inoculation. Among the media used, M1G1 showed the phyte developmental stage was observed in M1G1 at 25 days significantly earliest elongating secondary gametophyte from after inoculation followed by the other three media 30 days primary gametophyte. after inoculation. Among the media used, M1G1 showed the The number of days for the elongation of secondary game- significantly earliest lopsided prothallus formation. tophyte from primary gametophyte between two plant species The number of days for lopsided prothallus formation be- was significantly different. The gametophytes of P. coronarium tween two plant species was significantly different. The game- required 45.50 days for this gametophyte developmental stage tophytes of P. coronarium required 23 days for this to occur while P. grande required 54.25 days. Since P. corona- gametophyte developmental stage to occur while P. grande re- rium was first to show the imbibition and swelling of the spores quired 28.75 days. and emergence of the rhizoid at the lower posterior end of the spore, it indicates that all gametophyte developmental stages 3.2.1.6. Cordate prothallus formation. Further growth of the will be first observed as to the number of days in P. coronari- lopsided prothallus resulted to the formation of symmetrical um. This is the main cause why almost same results in statisti- cordate prothallus. This gametophyte developmental stage cal analysis were obtained in all gametophyte developmental was first observed in M1G1 and M2G1 in P. coronarium at stages. 25 days after inoculation. M1G1 and M2G1 had the signifi- cantly earliest cordate prothallus formation compared to the other two media. In P. grande, this gametophyte developmen- 3.2.2. Sporophyte (plantlet) formation tal stage was observed in M1G1 at 30 days after inoculation Developmental details of sporophytes of two species of Platy- followed by the other three media 35 days after inoculation. cerium are presented in Table 2 and Figs. 6A–C and 7A–F. Among the media used, M1G1 showed the significantly earliest Sporophytes of P. coronarium started to develop from the cordate prothallus formation. It was found that the 30–35 days gametophytes 7 weeks after transferring the gametophytes to cordate prothallus formation is contrary to the result of the potting media containing sterile chopped Cyathea spp. (anono- study conducted by Delfin (1998) on which this gametophyte tong) roots and garden soil (1:1). For P. grande, sporophytes developmental stage was observed 60 days after inoculation started to develop from the gametophytes 9 weeks after trans- in spore culture medium. ferring the gametophytes to potting media. Gametophytes of The number of days for cordate prothallus formation be- the two species of Platycerium transferred to the potting media tween two plant species was significantly different. The game- produced more number of sporophytes up to the last week of tophytes of P. coronarium required 27.50 days for this monitoring, and when compared to the culture media it gametophyte developmental stage to occur while P. grande re- showed significant difference for there was no sporophyte for- quired 33.75 days. mation in the culture media which could be attributed to the scanty water inside the culture bottle. This result implies that 3.2.1.7. Emergence of germ filament from primary gametophyte. the gametophytes remained in the culture medium and the Most of the primary gametophytes produced uniseriate out- gametophytes transferred to potting medium significantly af- growths from the basal areas. This gametophyte developmen- fected the formation of sporophytes. tal stage was first observed in M1G1 and M2G1 in P. The details of the number of leaves formed and leaf area of coronarium at 35 days after inoculation. M1G1 and M2G1 the transplanted sporophytes of P. grande in the two media had the significantly earliest emergence of germ filament from (H1 – hanging slab of anonotong, and H2 – hanging branch primary gametophyte compared to the other two media. In P. of mahogany) under greenhouse condition are presented in Ta- grande, this gametophyte developmental stage was observed in ble 3 and Fig. 7A–F. Sporophytes of P. grande attached to the M1G1 at 35 days after inoculation followed by the other three hanging branch of mahogany (H2) showed higher mean num- media 42 days after inoculation. Among the media used, M1G1 ber of leaves produced than in the sporophytes attached to the showed the significantly earliest emergence of germ filament hanging slab of anonotong (H1). However, statistical analysis from primary gametophyte. revealed no significant difference. The number of days for emergence of germ filament from The two substrate media significantly affected the leaf area primary gametophyte between two plant species was signifi- of the sporophytes of P. grande. Sporophytes attached to the cantly different. The gametophytes of P. coronarium required hanging branch of mahogany (H2) showed larger mean leaf 38.50 days for this gametophyte developmental stage to occur area than those sporophytes attached to the hanging slab of while P. grande required 40.25 days. anonotong roots (H1). The larger leaf area and the higher number of leaves formed in H2 could be attributed to the tex- 3.2.1.8. Elongation of secondary gametophyte from primary ture of the two media. It was observed that after 4 hours from gametophyte. Uniseriate outgrowths from the basal areas later watering, water on the slabs of anonotong already disappeared developed into secondary gametophytes, following the same for it can easily be evaporated from the slabs due to its poros- process of cell division from the primary gametophyte. This ity. High rate of transpiration and the absence of water in the 20 R.A. Aspiras

Table 2 Mean number of sporophytes (plantlets) formed from the gametophytes of P. coronarium and P. grande in different culture media, and from the gametophytes transferred to potting medium containing sterile chopped Cyathea spp. (anonotong) roots and garden soil (1:1). Media Mean number of sporophytes formed P. coronarium P. grande 7th week 8th week 9th week 9th week 10th week 11th week 12th week 13th week 14th week 15th week 16th week

M1G0 0.00 0.00 0.00 b 0.00 0.00 0.00 c 0.00 00.0 00.0 00.0 00.0 b M1G0(P) 0.00 0.50 3.10 a 0.00 0.00 0.70 b 9.30 17.2 23.7 29.1 30.5 a M1G1 0.00 0.00 0.00 b 0.00 0.00 0.00 c 00.0 00.0 00.0 00.0 00.0 b M1G1(P) 0.00 0.40 3.60 a 0.00 0.10 1.90 b 10.5 17.8 23.4 25.5 29.1 a M2G0 0.00 0.00 0.00 b 0.00 0.00 0.00 c 00.0 00.0 00.0 00.0 00.0 b M2G0(P) 0.20 1.20 4.00 a 0.10 2.60 6.60 a 10.9 15.7 20.5 24.7 30.2 a M2G1 0.00 0.00 0.00 b 0.00 0.00 0.00 c 00.0 00.0 00.0 00.0 00.0 b M2G1(P) 0.00 1.10 3.00 a 0.40 2.30 6.60 a 10.6 16.1 19.4 22.1 27.4 a F-Test Replication NS NS NS Medium ** ** ** Means within each column having a common letter are not significantly different at 5% level based on Duncan’s New Multiple Range Test. NS – not significant. ** – Highly significant.

M1G0 – gametophytes grown and remained in the spore culture medium; M1G0(P) – gametophytes grown in the spore culture medium and transferred to potting medium; M1G1 – gametophytes grown and remained in the spore culture medium + 2% glucose; M1G1(P) – gameto- phytes grown in the spore culture medium + 2% glucose and transferred to potting medium; M2G0 – gametophytes grown and remained in the Knudson C culture medium; M2G0(P) – gametophytes grown in the Knudson C culture medium and transferred to potting medium; M2G1 – gametophytes grown and remained in the Knudson C culture medium + 2% glucose; M2G1(P) – gametophytes grown in the Knudson C culture medium + 2% glucose and transferred to potting medium.

Figure 6 Gametophytes and sporophytes of P. grande. (A–B) Non-sporophyte formation from gametophytes inside the culture media; (C) sporophytes produced from the transferred gametophytes to potting medium containing sterilized chopped Cyathea (anonotong) roots.

slabs mostly during afternoon led to water stress which cause sori cover completely the central fertile lobe. Sporangia decreased growth. have 9 or 10 annular cells and are long-stalked with 8 Sporophytes (plantlets) of P. grande grown and attached to spores per sporangium. Base fronds of P. grande forked the main stem of mahogany and exposed under shaded envi- equally and its foliage fronds are symmetrical. The sori ronment inside the Fernery of the Department of Biology are in semicircular shape and located on the undersur- showed better growth (Fig. 7G–I). faces of the foliage fronds. The sporangia have 21–26 annular cells and are short-stalked with 64 spores per 4. Conclusions sporangium. The spores of P. coronarium and P. grande are bean-shaped, monolete and bilaterally symmetrical. Based on the results of the study the following conclusions are Both of them showed Equatorial-Gleichenia type of drawn: spore germination and Drynaria-type of prothallial development. (1) P. coronarium and P. grande are characterized by frond (2) The use of spore culture medium + 2% glucose and dimorphism. Base fronds of P. coronarium forked Knudson C culture medium + 2% glucose are the cul- unequally and its foliage fronds are asymmetrical. The ture media that enhanced rapid spore germination and Sporophyte and gametophyte development of Platycerium coronarium 21

Figure 7 Developmental stages of P. grande sporophytes (plantlets). (A–C) Plantlets attached to the slabs of Cyathea (anonotong); (D– F) plantlets attached to the branch of Swietenia macrophylla (mahogany); (G–I) plantlets attached to the trunk of mahogany.

Table 3 Mean number of leaves formed and mean leaf area from outplanted P. grande sporophytes (plantlets), 2–8 weeks after transplanting to two media under greenhouse condition. Media Mean number of leaves formed Mean leaf area (cm2) 2nd week 4th week 6th week 8th week 2nd week 4th week 6th week 8th week

H1 0.375 a 1.000 a 1.375 a 2.000 a 0.28 b 3.05 b 12.56 b 29.08 b H2 0.500 a 1.000 a 1.600 a 2.300 a 0.54 a 7.09 a 30.55 a 58.81 a F-Test Replication NS NS NS NS NS NS NS NS Medium NS NS NS NS NS ** ** ** Means within column row having a common letter are not significantly different at 5% level based on Duncan’s New Multiple Range Test. NS – not significant. ** – Highly significant.

H1 – hanging slab of anonotong; H2 – hanging branch of mahogany.

gametophyte development of P. coronarium. For P. (4) The use of the branch of S. macrophylla (mahogany) as a grande, the use of spore culture medium + 2% glucose medium is more effective for the growth of the sporo- enhanced rapid spore germination and gametophyte phytes (plantlets) of P. grande under greenhouse condi- development. tion than the slab of Cyathea spp. (anonotong). (3) Transferring the gametophytes of P. coronarium and P. (5) Attaching the sporophytes (plantlets) of P. grande to the grande to potting medium containing sterilized chopped trunk of S. macrophylla (mahogany) in the field and Cyathea spp. (anonotong) roots and garden soil (1:1) exposing them under shaded environment favored produced more sporophytes (plantlets). growth of the sporophytes. 22 R.A. Aspiras

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