T REPRO N DU LA C The International Journal of Reproductive Biology 9(2) Jul., 2017, pp.89-92 P T I F V O E

Y B

T I

DOI 10.14787/ijprb.2017 9.2.89-92 O E

I L

O C

G O

S

I

S

E T

S H Studies on the Gametophyte Development and Reproductive Behavior of T aureum L. Meenam Bhatia, Priti, P. L. Uniyal1 and P.V. Madhusoodanan* Department of Botany, University of Delhi, Delhi-110007, India *Malabar Botanical Garden and Institute for Plant Sciences, Kozhikode-673014, India 1Corresponding author: [email protected] Received: 11.01.2017; Revised: 13.02.2017; Accepted and published online: 01.03.2017 ABSTRACT is a pioneer species of mangrove sites growing rapidly and colonizing in disturbed habitats especially in the wetland areas. of this species are useful in the mangrove restoration. This paper presents the study on the gametophyte development of A. aureum by light microscopy and scanning electron microscopy, including spore germination, development of the gametophyte and reproductive behavior. Spore is tetrahedral, trilete, and laesura is tenuimarginate. Spore germination is of Vittaria type. Germinal filament is short and uniseriate and prothallial development is type. Gametophytes attained two-dimensional stage in 7-10 days and spathulate stage in about 15-20 days and became asymmetric chordate in 50-60 days. Gametophytes became bisexual after 50 days of sowing. In isolated populations 25% gametophytes and in composite population 68% produced sporophytes indicating the predominance of intragametophytic selfing. The length of unisexual phase before the attainment of bisexuality is sufficient to facilitate intergametophytic mating. It is further supported from observations on high numbers of sporophyte production and time of appearance of sporophyte in isolate and composite cultures. Gametophyte morphology and gametangial ontogeny also suggest that intergametophytic mating should be prevalent in gametophyte populations of A. aureum. Keywords: Mangrove, out-breeding, saline , restoration, trilete, tenuimarginate. Acrostichum belonging to the family (Smith used for bioremediation. The in vitro propagation protocol et al.2006, PPGI 2016), is the only fern genus of mangroves. It may be useful for the extraction of alkaloids for medicinal consists of at least three species. A. danaeifolium Langsd. & uses. Fisch. is a New World endemic and widely distributed in fresh MATERIAL AND METHODS water and slightly saline swamps (Adams and Tomlinson Collection and culture: Fertile fronds of A. aureum were 1979). A. speciosum Willd. is distributed in tropical Asia and collected from a single plant from Malabar Botanical Garden Australia and grows abundant in mangroves throughout and Research Institute, Calicut, Kerala in October after North- Malaya in areas frequently inundated by tides (Holttum 1954). east monsoon. Sporophylls were kept in brown paper packets A. aureum L. is distributed in tropical regions and abundant in and stored in a desiccator for the release of the spores. For mangrove habitats where it can withstand partial tidal culture, spores were sterilized with 2 % sodium hypochlorite immersion (Holttum 1954, Small 1938). It is a pioneer species solution for two minutes and rinsed thrice with double distilled of mangrove areas and grows rapidly during ecological water. Spores were sown on sterile Parker's macro and succession in destroyed mangrove habitat (Page 2002). Thompson's micronutrient culture medium (P&T) solidified Acrostichum aureum L. is the only species that occurs in with 1% agar in petri-plates. The pH of the medium was Indian coastline of Kerala in association with mangroves. It is maintained to 5.8. Cultures were kept in a culture room under a large herbaceous, perennial plant with pinnately compound controlled conditions of light intensity range between 47.3- fronds which are fertile only near the tips. It is rich in many 56.8 μmol m-2 sec-1 at 23 ±2oC for 16 hour photoperiod active compounds as alkaloids, saponin, stanins, sterols, followed by 8 hour dark period (Klekowski 1969). triterpenoids, patriscabratine, tetracosane, ponasterone, Light Microscopy: Periodically the percentage of spore pterosterone, kaempferol, quercetin and sulphated protein-C germination, subsequent gametophyte growth and (Raja and Ravindranadh 2014). Whole plant is used for differentiation, and gametangial ontogeny were observed anthelmintic and styptic purposes. Powdered rhizome paste is under Olympus CX21 microscope and photographed using used to treat wounds, ulcers and boils. Fronds are used as Olympus camera EP-1. Gametophytes in culture were antimicrobial agent and used to stop bleeding (Lobo and observed at regular intervals and were isolated in separate Krishnakumar 2014, Thomas 2012). Soniya and petri-plates containing P & T medium before initiation of Krishnakumar (2015) reported its suitability as a bio-sorbent gametangia. The ratios of gametophyte bearing male, female, for the removal of heavy metals like Cu(II), Pb(II) and Zn(II) bisexual or neuter conditions were recorded. from the aqueous solution. The present study deals with the Set 1: 25 petri-plates with single gametophyte in each (isolate aspects of nutritional requirements for the gametangial culture) initiation, reproduction and sporophyte development so that Set 2: 10 petri-plates with 25 gametophytes in each the species may be introduced in the degraded habitats and (composite culture). 90 The International Journal of Plant Reproductive Biology 9(2) July 2017, pp.89-92

After the initiation of the gametangia in cultures, watering of all the isolate and composite populations was done from above with sterile distilled water twice a week to facilitate fertilization. Percentage of sporophytes was recorded in both of the above mentioned sets (Srivastava et al. 2014). Scanning Electron Microscopy (SEM): Oven dried spores were mounted on aluminum stab using sticky tape and placed in Sputter coater (JFC-1600 autofinecoater, JEOL Japan) for gold palladium coating. For SEM, gametophytes were fixed in 2.5 % glutaraldehyde in 0.1M sodium phosphate buffer, pH 7.2 and then dehydrated in a graded alcohol series. They were subsequently subjected to critical point dryer using Polaron Critical Point drier (D-09-R-711/A) and mounted and coated with gold and palladium as done with spores. Scanning was done under Scanning Electron Microscope (JSM-6610LV- JEOL, Japan) installed in the Department of Botany, University of Delhi, Delhi. RESULTS Fig. 1—Developmental stages of gametophyte of Acrostichum aureum The spores of A. aureum were homosporous, trilete and (a) Spore, (b) Filamentous thallus, (c) Spathulate thallus, (d) tetrahedral-globose, radially symmetrical and had smooth Prothallus showing antheridia, (e) Prothallus showing archegonia, (f) Bisexual prothallus bearing both antheridia and archegonia, (g) rounded broad corners with slightly concave edges (Fig. 1a, Antheridia -An (h) Archegonia- Ar, (i) Sporophyte in isolate population, Fig. 2a). Spore surface was minutely tuberculate (Fig. 2b). The (j) and (k) Sporophtye in composite population. polar outline was triangular to rounded-triangular. The Fully developed gametophytes were produced in 63-70 proximal face was low pyramidal while the distal face was days of sowing and they were more or less symmetrical (Fig. hemispherical. The laesura was prominent, tenuimarginate 1e, Fig. 2d). Prothallial development was of Ceratopteris and triradiate with ridges extending two third of the spore type. The mature gametophytes were dark green, glossy, width (Fig. 2a). The spore size was found 50-53 µm. The exine cordate, lopsided and generally one side larger with a deeply was 4.0-4.5 μm thick and clearly distinguished into a sexine notched apex and a broad midrib bearing gametangia and and nexine. The sexine was densely granulose and rhizoids (Fig. 1f). conspicuously thicker than nexine. Spore germination was initiated after 4-6 days of sowing and there was 95% germination. Spore germination was of Vittaria type as the pattern reported by Nayar and Kaur (1971). The spores turned pale green and germinated by rupturing the spore coat at laesura region. Rhizoid emerged first as a hyaline protrusion. The germ filament produced 8-10 cells in a one dimensional structure in about 7-10 days (Fig. 1b). The two-dimensional growth started with a longitudinal division in a central or basal cell of the one dimensional prothallus. In both cases, the prothallus expanded and developed into 4-6 cells wide spathulate gametophyte in about 15-20 days after sowing (Fig. 1c). Subsequent division of cells towards one of the lateral margins of the prothallus developed a lateral meristematic zone located near the basal part of the gametophyte (Fig. 1c, Fig. 2c). The Fig. 2- Spores in different views. (a) Triradiate laesura of spore, (b) Enlarged view of spore showing tuberculate exine, (c) Gametophyte meristematic region became notched and further growth showing development of apical notch, (d) Asymmetric cordate resulted in an asymmetrical ovate prothallus with different gametophyte showing gametangial development. sized lobes (Fig. 1d). Mid rib formation started when the Gametangia were initiated within 35-40 days exhibiting a prothallus was nearly of 30- 40 days. female to hermaphroditic sequence of development (Fig. 2d). 2017 Observations on the reproductive biology of Solanum torvum Swartz ...... 91

Occasionally some gametophyte precociously produced Table 2—Breeding behavior of different populations. antheridia. The length of the unisexual stage varied from Population Number of Number of Percentage of gametophyte sporophytes sporophytes culture to culture. But in all cases the prothalli finally became observed produced produced hermaphroditic within 60-70 days. Archegonia were produced Isolation 20 5 25 continuously on the tissue just behind the young lateral Composite 75 48 68 meristem till the gametophyte became cordate (Fig 2d). The neck was 4-5 cells long (Fig 3a, b). Antheridia were initiated in DISCUSSION the cordate prothallus and were restricted to the midrib region The surface of spores is densely granulated and on the lower surface (Fig. 1g). Antheridia were large elongate- tuberculate and spore wall is thick (4.0-4.5μm) indicating that globose on maturity with a bluntly tapering apex slightly tilted spores can withstand in water for longer period (Nayar and to one side (Fig. 1h, Fig. 3c, d). Kazmi 1964, Lloyd 1980). The percentage of spore germination after one week of sowing was 95% which suggest the species is a successful colonizer. Zhang et al. (2013) reported 79.8% spore germination when allowed to germinate directly on the fertile fronds placed in petri dishes containing distilled water only. The mature gametophytes are large enough and generally one side larger with a deeply notched apex and a broad midrib. The antheridia are restricted to the midrib region on the lower surface as also documented by Nayar and Kazmi (1964) but they were not found on apical wings as reported by Lloyd (1980). Such variations may be related to the culture conditions. High density of the gametophyte increases the chances of inter-gametophytic mating, leading to the variability. The gametangial ontogeny observed in the present study suggests that intergametophytic mating should be prevalent in Figs. 3- Gametangia of Achrostichum aureum (a) Archegonia, (b) Details of archegonium, (c) Antheridia, (d) Single antheridium. gametophyte populations of A. aureum. The probability for Archegonia appeared first about 35 days while antheridia intergametophytic mating is strongly supported by specific developed about 42 days after sowing. The first bisexual stage factors exhibited by the gametophyte generation including the was found after 49 days of sowing (Table 1). Gametophytes female to bisexual gamentangial sequence in most became bisexual after 70 days of sowing. In isolated population, gametophyte studied, the dioecious condition and the 25% of the gametophyte produced sporophytes (Fig-1i), sequential functional unisexuality expressed in some whereas, 68% of the gametophyte produced sporophytes in gametophytes (Lloyd 1980). The initial sex ontogeny was composite population (Fig 1j, Table 2). The first young female to bisexual or dioecious as described by Lloyd (1980). sporophyte leaf appeared in composite culture in 89 days after Gametangia appeared about 35 to 42 days after sowing while sowing and in isolate culture in 114 days. Fully developed bisexual stage developed after 49 days of sowing. Thus the sporophytes (Fig 1k) were then hardened to acclimatize in length of unisexual phase before the attainment of bisexuality natural conditions to see their survival so that the protocol can is sufficient to facilitate intergametophytic mating. It is further be used for restoration purpose. supported from observations on percent sporophyte Table 1– Chronological changes in the Sex Ratio of a composite production and time of appearance of sporophyte in isolate culture (N=20) and composite cultures. Days Neuter Male Female Bisexual Llyod and Gregg (1975) reported high variations of sex after (Numbers) (Number) (Number) (Number) ontogeny in gametophytes of A. danaeifolium grown in sowing cultures and in soil. The first young sporophyte appeared in 28 20 0 0 0 composite culture up to 89 days, whereas up to 114 days in 35 19 0 1 0 isolated cultures. Thus sporophyte production in composite 42 7 1 12 0 cultures occurred 25 days earlier than in isolate cultures. The 49 11 2 6 1 percentage of sporophyte production in composite population 56 2 4 8 6 was 64% whereas in isolated was 25 %. The massive 63 0 0 2 18 70 0 0 0 20 production of spores increases the probability of inbreeding. Population size, spore output per plant, influence of specific 92 The International Journal of Plant Reproductive Biology 9(2) July 2017, pp.89-92

habitat and the genetic system are other factors that have Page CN 2002. Ecological strategies in fern evolution: a significant effect on genetic load. neopteridological overview. Rev. Palaeobotany and Acknowledgment - Authors thank the University of Delhi, Palynology 119 1-33. Delhi for providing financial assistance in the form of R & D PPGI 2016. A community-derived classification for extant Grant (No.RC/2015/9677). lycophytes and . J. Syst. Evol. 54(6) 563– 603. REFERENCES Raja S and Ravindranadh K 2014. A complete profile on Adams DC and Tomlinson PH 1979. Acrostichum in Florida. Acrostichum aureum – Traditional uses, pharmacological Am. Fern J. 69 42-46. activities and phytoconstituents. World J. Pharmaceutical Res. 3(10) 624-630. Chan HT, Huse N and Chong PF 1987. Is there a need to eradicate prior to planting Small JK 1938. Ferns of the Southeastern States. Descriptions Rhizophora mucronata in logged-over mangrove forest of Fern-Plants, Growing Naturally in the States South of area? FRIM occasional Paper. Forest Research Institute, Virginia-Kentucky State Line and East of the Mississippi Kuala Lumpur, Malaysia.7 River, Facsimile edition 1964. Hafner Publishing Co., New York. Holttum RE 1954. Ferns of Malaya. Govt. Printing office, Singapore. Smith RA, Kathleen MP, Schuettpelz E, Korall P, Schneider H and Wolf PG. 2006. A classification for extant ferns. Klekowski EJ Jr. 1969. Reproductive biology of the Taxon 55(3) 705–731. Pteridophyta. II. Theoretical considerations. Bot. J. Linnean Soc. 62 347-359. Soniya ML and Krishnakumar G 2015. Biosorption of heavy metals from aqueous solution using mangrove fern Lloyd RM 1980. Reproductive biology and gametophyte Acrostichum aureum L. leaf biomass as a Sorbent. Int. morphology of New World populations of Acrostichum Res. J. Environ. Sci. 4(11) 25-31. aureum. Am. Fern J. 70(3) 99-110. Srivastava R, Uniyal PL and Kholia BS 2014. Studies on Lloyd RM and Gregg TL 1975. Reproductive biology and reproductive biology of Microsorum alternifolium gametophyte morphology of Acrostichum danaeifolium (Willd.) Copel. Int. J. Plant Repro. Biol. 6(1) 15-19. from Mexico. American Fern J. 65(4) 105-120. Stockey AG and Atkinson LR 1952. Then gametophyte of Lobo SM and Kishnakumar G 2014. Studies on ecological Achrostichum speciosum Willd. Phytomorphology 2 105- anatomy of the mangrove fern Acrostichum aureum L. 113. Int. J. Plants, Animals and Environ. Sci. 4(1)195-200. Thomas T. 2012. In vitro evaluation of antibacterial activity Nayar BK and Kazmi F 1964. The gametophyte of of Acrostichum aureum Linn. Indian J. Natural Products Acrostichum aureum L. Proc. Indian Acad. Sci. Section and Resources 3(1) 135-138. B. 59(4) 185-194. Zhang R, Lui T, Wu W, LiY, Chao L, Huang Y, Shi and Zhou R Nayar BK and Kaur S 1971. Gametophytes of homosporous 2013. Molecular evidence for natural hybridization in the ferns. Bot. Rev. 37 295-396. mangrove fern Acrostichum. BMC Plant Biol. 13 74.