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

Abstract

The sporophyte and gametophyte development of Platycerium coronarium and P. grande were compared through ex situ propagation using in vitro culture technique and under greenhouse and field conditions.

The morphology of the sporophyte and gametophyte, type of spore germination and prothallial development of P. coronarium and P. grande were documented. Gametophytes of P. coronarium 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 formation. 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 conditions.

Sporophyte morphology of P. coronarium and P. grande varies but not their gametophyte morphology. 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.

1. Introduction

Platycerium Desv., commonly known as staghorn fern, stands out as one of the most commonly grown and highly priced ornamental ferns (Darnaedi and Praptosuwiryo, 2003). Staghorn ferns are becoming threatened in the wild for they are sought – after by plant collectors for their majestic size and form (Madulid, 1985). Since staghorn ferns are becoming endangered and having spores that are difficult to germinate under natural condition (Amoroso, 1990, 1992; Amoroso and Amoroso, 1998, 2003), it is interesting to find out the differences of the developmental patterns of their gametophytes and sporophytes which can be utilized in the mass propagation of these plants. In order to conserve the remaining populations, in vitro technique is necessary to ensure mass production of these species.

Studying the differences in the development of gametophyte and sporophyte of P. coronarium (Koenig) Desv. and P. grande (Fee) C. Presl. provide evidences on variation patterns, which is one of the criteria in fern taxonomy (Raghavan, 1989; Joaquin and Zamora, 1996). Part from that, fern spores and gametophytes are excellent biological systems for the analysis of physiological and developmental problems (Raghavan, 1989).

More specifically, the study aimed to determine the culture media for rapid spore germination and development of the gametophytes of the two species of Platycerium; identify which of the two methods of propagation, i.e., (i) transferring the gametophytes to potting medium containing sterilized chopped Cyathea spp. (anonotong) roots and garden soil (1:1) or (ii) leaving the gametophytes in the agar culture medium, produce more sporophytes (plantlets) of the two species of Platycerium; find out which medium, slab of Cyathea spp. (anonotong) or branch of Swietenia macrophylla (mahogany), is more effective for the growth of the sporophytes of the two species of Platycerium under greenhouse condition; and observe the growth of the sporophytes of P. coronarium and P. grande attached to the trunk of S. macrophylla (mahogany) in the field.

2. Materials and methodology

Platycerium coronarium and P. grande are epiphytic ferns growing solitary or in clusters on trunks, branches of trees, and on tree tops and old trees in rainforests at lower altitudes. These species of ferns were also found cultivated in some gardens in the provinces of Bukidnon and Davao (Fig. 1A and D).

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.

P. coronarium and P. grande are characterized by frond dimorphism, the formation of a basket of base fronds and the dichotomously divided pendulous foliage fronds. Base fronds of P. coronarium forked unequally and its foliage fronds are asymmetrical. Base fronds of P. grande forked equally and its foliage fronds are symmetrical.

The sori of P. coronarium and P. grande are forming large soral patches. Sori of P. coronarium cover completely the central fertile lobe. Sporangia have 9 or 10 indurated annular cells, long-stalked with 8 spores per sporangium. Sori of P. grande are in semicircular shape and located on the undersurfaces of the foliage fronds. The sporangia have 21–26 indurated annular cells, short-stalked with 64 spores per sporangium (Fig. 1B, C, E and F).

Gross morphology of P. coronarium was recorded based on the mature plant found in the garden of Mr. Romulo Castada of Malaybalay City and in Mt. Hamiguitan, Sitio Magum, Mati, Davao Oriental. For P. grande, it was based from the mature plant grown in the fernery of the Department of Biology, College of Arts and Sciences, Central Mindanao University, Musuan, Bukidnon and also in Malagos Garden Resort, Calinan, Davao City. Spores of P. coronarium and P. grande were obtained from the same places mentioned.

The experiment on gametophyte development was carried out following a factorial Randomized Complete Block Design (RCBD). The four media (spore culture medium, spore culture medium + 2% glucose, Knudson C culture medium, and Knudson C culture medium + 2% glucose) served as factor A while the two plant species (P. coronarium and P. grande) as factor B, and replicated ten times.

The experiment on sporophyte formation was carried out using Randomized Complete Block Design (RCBD) with eight (8) media and replicated 10 times. The media were: (1) M₁G₀ – spore culture medium; (2) M₁G₀(P) – spore culture medium and transferred to potting medium; (3) M₁G₁ – spore culture medium + 2% glucose; (4) M₁G₁(P) – spore culture medium + 2% glucose and transferred to potting medium; (5) M₂G₀ – Knudson C culture medium; (6) M₂G₀(P) – Knudson C culture medium and transferred to potting medium; (7) M₂G₁ – Knudson C culture medium + 2% glucose; and (8) M₂G₁(P) – Knudson C culture medium + 2% glucose and transferred to potting medium.

The experiments on leaf formation and leaf area under greenhouse condition were carried out using Completely Randomized Design (CRD) with two media and replicated 10 times. The media were: (1) H₁ – hanging slab of anonotong; and (2) H₂ – hanging branch of mahogany. Some of the sporophytes were then attached to the trunk of mahogany under shaded environment inside the Fernery of the Department of Biology.

The data gathered were analyzed using the software for Analysis of Variance (ANOVA) and medium means were compared using Duncan’s New Multiple Range Test (DNMRT) at 5% level of significance.

3. Results and discussion

3.1. Spore morphology, spore germination and prothallial development

The spores of P. coronarium and P. grande are bean-shaped, monolete and bilaterally symmetrical. Both of them showed Equatorial-Gleichenia type of spore germination (Figs. 2A–F and 3A–F). According to Nayar and Kaur (1971), this type of spore germination is found in the Gleicheniaceae, Dipteridaceae, Loxogrammaceae, and many of the Polypodiaceae to which family Platycerium belongs. At germination a rhizoid initial is cut off laterally by a wall parallel to the polar axis of the spore. A series of divisions in the prothallial initial cell by walls parallel to the first result in a uniseriate germ filament. Both the germ filament and primary rhizoid elongate along the equatorial plane of the spore in opposite direction. Both P. coronarium and P. grande showed Drynaria-type of prothallial development (Figs. 4A–F and 5A–F). According to Nayar and Kaur (1971), the establishment of an apical meristematic cell of this type of prothallial development is far delayed. A broad spatulate prothallial plate is formed by division of the anterior cells, including the terminal cell of the germ filament by walls parallel to the long axis, and by repeated longitudinal and transverse divisions in the daughter cells. An obconical meristematic cell is formed by two oblique divisions in one of the anterior marginal cells of the prothallial plate when it is 5–10 or sometimes more cells broad. Further growth results in a symmetrical cordate prothallus.

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).

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. Propagation techniques

3.2.1. Spore germination and gametophyte development in different culture media

Stages of gametophyte development of P. coronarium and P. grande as to the number of days in four culture media (M₁G₀ – spore culture medium, M₁G₁ – spore culture medium + 2% glucose, M₂G₀ – Knudson C culture medium, and M₂G₁ – Knudson C culture medium + 2% glucose) is presented in Table 1.

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 swelling of the spore	Emergence of the rhizoid at the lower posterior end of the spore	Germ filament formation	Spatulate prothallial plate formation	Lopsided prothallus formation	Cordate prothallus formation	Emergence of germ filament from primary gametophyte	Elongation of secondary 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.

M₁G₀ – spore culture medium; M₂G₀ – Knudson C culture medium; M₁G₁ – spore culture medium + 2% glucose; M₂G₁ – Knudson C culture medium + 2% glucose.

^⁎⁎– Highly significant.

3.2.1.1. Imbibition and swelling of the spores

This gametophyte developmental stage was first observed in M₁G₁ and M₂G₁ in P. coronarium. This result was obtained for only 6 days after spore inoculation in the media. M₁G₁ and M₂G₁ had the significantly earliest imbibition and swelling of the spores compared to the other two media. In P. grande, this gametophyte developmental stage was observed in M₁G₁ at 10 days after inoculation and followed by M₂G₁ at 12 days after inoculation. Among the media used, M₁G₁ showed significantly the earliest for imbibition and swelling of the spores, followed by M₂G₁, and this was then followed by the other two media having the latest imbibition and swelling period.

Imbibition and swelling of the spores in both plant species were significantly affected by the media used. It was found that M₁G₁ with 8.0 mean number of days for this gametophyte developmental stage to occur was significantly the earliest. This was followed by M₂G₁ with 9.0 mean number of days. These results imply that M₁G₁ and M₂G₁ enhance rapid imbibition 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. Inside the cell, the transported glucose is utilized during respiration leading to the production of energy in a form of ATP. The energy yielded will be used for any metabolic processes occurring inside the cell.

3.2.1.2. Emergence of the rhizoid at the lower posterior end of the spore

A rupture at the lower end of the spore caused by spore swelling led to the emergence of the rhizoid and a germ papilla emerged next at the upper end. This gametophyte developmental stage was first observed in M₁G₁ and M₂G₁ in P. coronarium at 9 days after inoculation. M₁G₁ and M₂G₁ had the significantly earliest emergence of the rhizoid at the lower posterior end of the spore compared to the other two media. In P. grande, this gametophyte developmental stage was observed in M₁G₁ at 14 days after inoculation followed by M₂G₁ at 17 days after inoculation. M₁G₁ showed the significantly earliest emergence of the rhizoid at the lower posterior end of the spore, followed by M₂G₁, and this was then followed by the other two media having the latest emergence of the rhizoid at the lower posterior end of the spore.

The number of days for emergence of the rhizoid at the lower posterior end of the spore between two plant species was significantly different. The gametophytes of P. coronarium required 11.50 days for this gametophyte developmental stage to occur while P. grande required 16.75 days.

3.2.1.3. Germ filament formation

The elongation of the germ papilla and its continuous division gave rise to a protonema consisting of a uniseriate germ filament with 2–8 or more barrel-shaped and densely chlorophyllous cells, and at the basal end form the rhizoids. This gametophyte developmental stage was first observed in M₁G₁ and M₂G₁ in P. coronarium at 14 days after inoculation and found to have the significantly earliest germ filament formation compared to the other two media. In P. grande, this gametophyte developmental stage was observed in M₁G₁ at 18 days after inoculation followed by the other three media 21 days after inoculation. Among the media used, M₁G₁ showed the significantly earliest germ filament formation.

The number of days for germ filament formation between two plant species was significantly different. The gametophytes of P. coronarium required 15.50 days for germ filament formation to occur while P. grande required 20.25 days.

3.2.1.4. Spatulate prothallial plate formation

A broad spatulate prothallial plate is formed by division of the anterior cells, including the terminal cell, of the germ filament by walls parallel to the long axis, and by repeated longitudinal and transverse divisions in the daughter cells. This gametophyte developmental stage was first observed in M₁G₁ and M₂G₁ in P. coronarium at 17 days after inoculation and had the significantly earliest spatulate prothallial plate formation compared to the other two media. In P. grande, this gametophyte developmental stage was observed in M₁G₁ at 21 days after inoculation followed by the other three media 25 days after inoculation. Among the media used, M₁G₁ showed the significantly earliest spatulate prothallial plate formation.

The number of days for spatulate prothallial plate formation between two plant species was significantly different. The gametophytes of P. coronarium required 19 days for this gametophyte developmental stage to occur while P. grande required 24 days.

3.2.1.5. Lopsided prothallus formation

The continuous division of the anterior and terminal cells of the prothallial plate and by repeated longitudinal and transverse divisions of the daughter cells led to the formation of a lopsided prothallus. In this stage, an obconical meristematic cell is formed by two oblique divisions in one of the anterior marginal cells of the prothallial plate when it is 5–10 or sometimes more cells broad. This gametophyte developmental stage was first observed in M₁G₁ and M₂G₁ in P. coronarium at 21 days after inoculation and had the significantly earliest lopsided prothallus formation compared to the other two media. In P. grande, this gametophyte developmental stage was observed in M₁G₁ at 25 days after inoculation followed by the other three media 30 days after inoculation. Among the media used, M₁G₁ showed the significantly earliest lopsided prothallus formation.

The number of days for lopsided prothallus formation between two plant species was significantly different. The gametophytes of P. coronarium required 23 days for this gametophyte developmental stage to occur while P. grande required 28.75 days.

3.2.1.6. Cordate prothallus formation

Further growth of the lopsided prothallus resulted to the formation of symmetrical cordate prothallus. This gametophyte developmental stage was first observed in M₁G₁ and M₂G₁ in P. coronarium at 25 days after inoculation. M₁G₁ and M₂G₁ had the significantly earliest cordate prothallus formation compared to the other two media. In P. grande, this gametophyte developmental stage was observed in M₁G₁ at 30 days after inoculation followed by the other three media 35 days after inoculation. Among the media used, M₁G₁ showed the significantly earliest cordate prothallus formation. It was found that the 30–35 days cordate prothallus formation is contrary to the result of the study conducted by Delfin (1998) on which this gametophyte developmental stage was observed 60 days after inoculation in spore culture medium.

The number of days for cordate prothallus formation between two plant species was significantly different. The gametophytes of P. coronarium required 27.50 days for this gametophyte developmental stage to occur while P. grande required 33.75 days.

3.2.1.7. Emergence of germ filament from primary gametophyte

Most of the primary gametophytes produced uniseriate outgrowths from the basal areas. This gametophyte developmental stage was first observed in M₁G₁ and M₂G₁ in P. coronarium at 35 days after inoculation. M₁G₁ and M₂G₁ had the significantly earliest emergence of germ filament from primary gametophyte compared to the other two media. In P. grande, this gametophyte developmental stage was observed in M₁G₁ at 35 days after inoculation followed by the other three media 42 days after inoculation. Among the media used, M₁G₁ showed the significantly earliest emergence of germ filament from primary gametophyte.

The number of days for emergence of germ filament from primary gametophyte between two plant species was significantly different. The gametophytes of P. coronarium required 38.50 days for this gametophyte developmental stage to occur while P. grande required 40.25 days.

3.2.1.8. Elongation of secondary gametophyte from primary gametophyte

Uniseriate outgrowths from the basal areas later developed into secondary gametophytes, following the same process of cell division from the primary gametophyte. This gametophyte developmental stage was first observed in M₁G₁ and M₂G₁ in P. coronarium at 42 days after inoculation. It was then followed by M₁G₀ and M₂G₀ 49 days after inoculation. M₁G₁ and M₂G₁ had the significantly earliest elongating secondary gametophyte from primary gametophyte compared to the other two media. In P. grande, this gametophyte developmental stage was observed in M₁G₁ at 49 days after inoculation followed by the other three media at 56 days after inoculation. Among the media used, M₁G₁ showed the significantly earliest elongating secondary gametophyte from primary gametophyte.

The number of days for the elongation of secondary gametophyte from primary gametophyte between two plant species was significantly different. The gametophytes of P. coronarium required 45.50 days for this gametophyte developmental stage to occur while P. grande required 54.25 days. Since P. coronarium was first to show the imbibition and swelling of the spores and emergence of the rhizoid at the lower posterior end of the spore, it indicates that all gametophyte developmental stages will be first observed as to the number of days in P. coronarium. This is the main cause why almost same results in statistical analysis were obtained in all gametophyte developmental stages.

3.2.2. Sporophyte (plantlet) formation

Developmental details of sporophytes of two species of Platycerium are presented in Table 2 and Figs. 6A–C and 7A–F. Sporophytes of P. coronarium started to develop from the gametophytes 7 weeks after transferring the gametophytes to potting media containing sterile chopped Cyathea spp. (anonotong) roots and garden soil (1:1). For P. grande, sporophytes started to develop from the gametophytes 9 weeks after transferring the gametophytes to potting media. Gametophytes of the two species of Platycerium transferred to the potting media produced more number of sporophytes up to the last week of monitoring, and when compared to the culture media it showed significant difference for there was no sporophyte formation in the culture media which could be attributed to the scanty water inside the culture bottle. This result implies that the gametophytes remained in the culture medium and the gametophytes transferred to potting medium significantly affected the formation of sporophytes.

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.

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) – gametophytes 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.

^⁎⁎– Highly significant.

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.

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.

The details of the number of leaves formed and leaf area of the transplanted sporophytes of P. grande in the two media (H₁ – hanging slab of anonotong, and H₂ – hanging branch of mahogany) under greenhouse condition are presented in Table 3 and Fig. 7A–F. Sporophytes of P. grande attached to the hanging branch of mahogany (H₂) showed higher mean number of leaves produced than in the sporophytes attached to the hanging slab of anonotong (H₁). However, statistical analysis revealed no significant difference.

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.

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

^⁎⁎– Highly significant.

The two substrate media significantly affected the leaf area of the sporophytes of P. grande. Sporophytes attached to the hanging branch of mahogany (H₂) showed larger mean leaf area than those sporophytes attached to the hanging slab of anonotong roots (H₁). The larger leaf area and the higher number of leaves formed in H₂ could be attributed to the texture of the two media. It was observed that after 4 hours from watering, water on the slabs of anonotong already disappeared for it can easily be evaporated from the slabs due to its porosity. High rate of transpiration and the absence of water in the slabs mostly during afternoon led to water stress which cause decreased growth.

Sporophytes (plantlets) of P. grande grown and attached to the main stem of mahogany and exposed under shaded environment inside the Fernery of the Department of Biology showed better growth (Fig. 7G–I).

4. Conclusions

Based on the results of the study the following conclusions are drawn:

• (1)P. coronarium and P. grande are characterized by frond dimorphism. Base fronds of P. coronarium forked unequally and its foliage fronds are asymmetrical. The sori cover completely the central fertile lobe. Sporangia have 9 or 10 annular cells and are long-stalked with 8 spores per sporangium. Base fronds of P. grande forked equally and its foliage fronds are symmetrical. The sori are in semicircular shape and located on the undersurfaces of the foliage fronds. The sporangia have 21–26 annular cells and are short-stalked with 64 spores per sporangium. The spores of P. coronarium and P. grande are bean-shaped, monolete and bilaterally symmetrical. Both of them showed Equatorial-Gleichenia type of spore germination and Drynaria-type of prothallial development.
• (2)The use of spore culture medium + 2% glucose and Knudson C culture medium + 2% glucose are the culture media that enhanced rapid spore germination and gametophyte development of P. coronarium. For P. grande, the use of spore culture medium + 2% glucose enhanced rapid spore germination and gametophyte development.
• (3)Transferring the gametophytes of P. coronarium and P. grande to potting medium containing sterilized chopped Cyathea spp. (anonotong) roots and garden soil (1:1) produced more sporophytes (plantlets).
• (4)The use of the branch of S. macrophylla (mahogany) as a medium is more effective for the growth of the sporophytes (plantlets) of P. grande under greenhouse condition than the slab of Cyathea spp. (anonotong).
• (5)Attaching the sporophytes (plantlets) of P. grande to the trunk of S. macrophylla (mahogany) in the field and exposing them under shaded environment favored growth of the sporophytes.