Aposporous Gametophytes Research Articles

Gamma radiation-induced in vitro hormetic apogamy in the fern Pityrogramma calomelanos (L.) link

Pityrogramma calomelanos (L.) Link, popularly known as “Silver fern” has significant importance as a medicinal plant used traditionally for its astringent, analgesic, anti-haemorrhagic, anti-hypertensive, anti-pyretic and anthelminthic properties. This fern demonstrates an increased morphogenetic potential towards sporophyte formation, upon exposure to low doses of gamma radiation. Young sporophytic leaf crosier cultures were established in vitro on agar based Knop’s media with and without 20 g/l sucrose. The cultures were subjected to 60Co radiations in the range of 2.5–100 Gy. Apospory (production of gametophytes on sporophytic tissue without spores) was observed on leaf tissue cultured on Knops media with and without sucrose in P. calomelanos, at the end of 60 days. 5 Gy treated explants showed high number of aposporous gametophytes and was comparable to the control. Other tested doses reduced the aposporous gametophyte production significantly. In the second phase of the experimentation, the cultures were retained on the gametophyte induction media for a period of 4 weeks. Aposporous gametophytes were observed to proliferate with occasional development of antheridia. At the end of 4 weeks, morphogenetic development on the gametophytic tissue resulted in a significantly higher number of apogamous sporophytes (production of sporophytes without fusion of gametes) were obtained on 5 Gy treated tissue as compared to control and all the other treated explants. Apogamous sporophytes thus produced were successfully grown in the greenhouse and transferred to the field. Thus the use of gamma radiation in vitro not only reduced the need for sucrose for induction of apospory in P.calomelanos, it also exhibited hormesis at 5 Gy for improved sporophyte production.

The Effects of Sugars and Ethylene on Apospory and Regeneration in <i>Ceratopteris richardii</i>

In land plants, two distinct generations, gametophyte and sporophyte, alternate to complete the life cycle. Sporophytes undergo meiosis to produce spores, from which gametophytes develop. Gametophytes produce gametes, which participate in fertilization to produce the zygote, the first cell of the sporophyte generation. In addition to this sexual reproduction pathway, some fern species can undergo apospory or apogamy, processes that bypass meiosis or fertilization, respectively, to alternate between the two generations without changing the chromosome number. Apospory is inducible in the laboratory in various fern species simply by altering the sugar level in the media. In sporophytes induced to undergo apospory, sporophyte regeneration is also observed. The ratio of aposporous gametophytes to regenerated sporophytes varies, in a manner consistent with being dependent on sugar level. Whereas the sugar signaling pathway is yet to be elucidated in lower plants, in angiosperms it has been shown to play a regulatory role in controlling essential processes including flowering and embryo development, which give rise to the gametophyte and the next sporophyte generation, respectively. Here, we present evidence for the role of different sugar levels on the balance of apospory and regeneration in the fern Ceratopteris richardii. The demonstration of crosstalk between sugar signaling and the hormone ethylene signaling in angiosperms prompted us to test the effects of this hormone in combination with sugar on apospory vs. regeneration. These results provide insight into how a group of redifferentiating cells determines which generation to become and lay the groundwork for further analysis of this asexual pathway.

Sporophyte induction studies in ferns in vitro

Sporophytes induction from spore-derived gametophytes and the morphogenic potential of homogenised sporophytes were performed in several ferns: Adiantum capillus-veneris, Asplenium adiantum-nigrum, Ceterach officinarum, Davallia canariensis, Dicksonia antarctica, Dryopteris dilatata, Dryopteris filix-mas, and Polypodium cambricum. The influence of growth rate, the formation of sexual organs, and the homogenisation of the gametophyte on sporophyte formation were studied. Gametophytic growth and sporophyte development were not antagonistic events and no correlation was found between the proportion of sexual phenotypes and rate of sporophyte formation. A pre-treatment of the rhizome with BAP exhibited swelling and upon transferral to Murashige and Skoog (MS) basal medium developed numerous fronds. Homogenised sporophytes of A. capillus-veneris, D. canariensis, and P. cambricum cultured on MS basal medium regenerated sporophytes while on MS with 4.4 μM BAP favoured formation of aposporous gametophytes and/or dedifferentiated cellular aggregates. In A. adiantum-nigrum, BAP induced either aposporous gametophytes or sporophytes.

Efficient induction of apospory and apogamy in vitro in silver fern (Pityrogramma calomelanos L.)

Silver fern (Pityrogramma calomelanos L.) is a terrestrial or lithophytic herbaceous fern used for ornamental and medicinal purposes. In its farina it produces the cytotoxic and anticancer compound dihydrochalcone. In vitro induction of apospory and apogamy, and direct field establishment of aposporous gametophytes and subsequent sporophyte development has been accomplished. Half-strength Murashige and Skoog (MS) medium with 3.33 microM N(6)-benzyladenine (BA) and 2.32 microM kinetin (Kn) showed earlier development and produced higher numbers of aposporous gametophytes than half-strength MS basal medium. Crozier explants developed higher numbers (mean value 29.2) of gametophytes, but were slower than frond explants (mean value 23.2). The gametophytes originated from the epidermal hairs progressed from uniseriate filamentous to cordate through bi-, tri- and multiseriate and spatulate stage with the development of antheridia. Reduction in the nutrient and sucrose concentrations in the media favoured apogamy. Sucrose-free 1/10 strength MS medium and agar plates developed a mean of 30.4 and 29.9 sporophytes, respectively in the light. The greenhouse-established gametophytes developed sporophytes. The established sporophytes ex vitro showed 95% survival rate. Apogamous sporophytes and the source plant showed the same chromosome numbers (2n=116). The established protocol accomplishes apogamy and apospory in silver fern, and the aposporous gametophytes can be used for genetic transformation and development of transgenic silver fern.

Apospory in leaf culture of staghorn fern (Platycerium bifurcatum)

The induction, origin, morphology, and ploidy of aposporous gametophytes produced on juvenile leaves of the fern Platycerium bifurcatum (Cav.) C. Chr. were studied. Leaf explants were grown on modified Murashige and Skoog medium with 0%, 0.01%, 0.1%, 1%, or 2% sucrose. A low sucrose concentration (0.01%) and wounding of the adaxial side of the leaf significantly increased the induction of aposporous gametophytes (90% of leaves produced gametophytes). Regeneration began as a proliferation of mainly epidermal cells on both sides of the leaf; subsequent development was similar to that shown by gametophytes originating from spores. Flow cytometric analysis of sporophytes and aposporous gametophytes revealed that both forms had the same ploidy level. On the basis of these findings, we propose a set of conditions which regularly and reproducibly induces apospory on most of the leaf explants of the fern P. bifurcatum.

In vitro regeneration patterns of Platycerium bifurcatum leaf cell suspension culture.

A simple suspension culture system of Platycerium bifurcatum was developed where sporophytes could be regenerated directly from leaf cells or indirectly through an aposporous gametophyte stage under the same culture conditions. Single cells and aggregates of up to 100 cells developed aposporous gametophytes which later gave rise to sporophytes. Such gametophytes started apogamy when they were mostly less than 0.7 mm in length, bearing only rhizoids. In most cases, only one sporophyte was regenerated from one gametophyte. Aggregates of 500-1000 or more cells, on the other hand, regenerated sporophytes directly. Intercellular interaction was considered to be the physiological cause, and the separation of leaf cells to a certain degree drove the cells to embark on different regeneration paths. It is suggested that the possible existence of a threshold size of cell aggregates separates the two regeneration patterns.

Gametophytic and Sporophytic Regeneration from Bud Scales of the FernPlatycerium bifurcatum(Cav.) C.Chr.In Vitro

Abstract Regeneration from Platycerium bifurcatum scales in vitro was observed for the first time. Regeneration was obtained from scales detached from in vitro grown juvenile shoots. Regeneration began as rhizoids or as a proliferation of one or more scale cells into undifferentiated outgrowths distinguishable from adjacent cells by their shape and colour. Outgrowths developed into adventitious buds, aposporous gametophytes, or remained undifferentiated. Numerous outgrowths formed rhizoids on their surfaces. Aposporous gametophytes produced gametangia. Sucrose enhanced scale viability and regeneration, and promoted rhizoid development on outgrowths, but sucrose concentrations greater than 0.1% tended to inhibit apospory.

Parthenogenesis and apospory in the Laminariales: A flow cytometry analysis

Tissue explants and unisexual cultures of Macrocystis pyrifera, Laminaria saccharina and L. digitata regenerated into various forms, including gametophyte-like filaments, callus-like structures, stipe-like structures and either abnormal or normal sporophytes. The ploidy of the various forms of growth was assessed using flow cytometry analysis of isolated nuclei. Unisexual cultures of female gametophytes of L. digitata gave rise to abnormal parthenogenetic sporophytes, with nuclei exhibiting 2C, 4C and 8C ploidy levels. Aposporous gametophyte-like filaments grown from the tissue explants of M. pyrifera developed into abnormal sporophytes and both forms were diploid. Development of stipe medullary explants of L. saccharina led to the complete regeneration of functional sporophytes, involving the outgrowth of aposporous gametophytes. The regenerated sporophytes were either diploid or tetraploid but only diploid sporophytes were found to be fertile. The developmental and biotechnological significance of these...

Micropropagation and phase change in Blechnum spicant and Pteris ensiformis

Rhizomes of juvenile sporophytes of Blechnum spicant L. and Pteris ensiformis L. cultured on MS medium with N6-benzyladenine alone (0.44–4.4 μM) or in combination with naphthaleneacetic acid (0.053–0.53 μM), respectively, gave rise to several proliferation centres located at the epidermal and inner parenchyma of this organ after one month in culture. Subculture of these rhizomes for one month in growth regulator-free medium allowed organization of internal proliferation centres, and regeneration of a large number of sporophytes. Subculture in the above-mentioned proliferation medium, induced phase change and development of aposporous gametophytes from surface-localized proliferation centres. Addition of naphthaleneacetic acid to the culture medium promoted proliferation of green globular bodies (GGB) in B. spicant and both rhizogenesis and callogenesis processes in P. ensiformis. GGB derived from rhizomes of B. spicant produced numerous sporophytes and from the point of view of micropropagation it is a good system to regenerate sporophytes in this species.

In vitro regeneration of Asplenium nidus L. from gametophytic and sporophytic tissue

Gametophytic and sporophytic tissues of Asplenium nidus arising from spores, were mechanically homogenized to test their regeneration capacity in MS liquid medium containing 3% sucrose. After 2 months of culture, the homogenated gametophytic tissue regenerated only gametophytes whereas the sporophytic tissue formed both sporophytes and aposporous gametophytes. It was possible to obtain a new system good enough to carry out the micropropagation of this species, as well as to increase the ploidy levels of both generations involved in the life cycle of this fern.

Production of aposporous gametophytes and calli from Pteris vittata L. pinnae strips cultured in vitro

Murashige and Skoog's modified medium in 1% Difco Bacto-agar supplemented with sugar alcohols (sorbsitol, mannitol), growth regulators (1-naphthalenacetic acid, 2,4-dichlorophenoxyacetic acid, benzyladenine, kinetin) and sugars (fructose, glucose, sucrose) induced aposporous gametophytes from pinnae of Pteris vittata cultured in vitro at lower concentrations of all the mentioned components. Aposporous gametophytes and vegetative calli were produced at higher concentrations. The calli regenerated sporophytes when cultured on MS medium without growth regulators. The gametophytes grew vegetatively on MS medium but produced sporophytes when transferred into 0.1 strength MS medium. This is the first report of simultaneous production of calli and gametophytes from fern explants.

Production of first and second generations aposporous gametophytes fromPyrrosia piloselloides (L.) Price frond strips cultured in vitro

Second generation aposporous gametophytes were obtained from sporophytes derived from first generation aposporous gametophytes, which in turn came from the mature fronds grown from spores in the laboratory. Murashige and Skoog modified medium in 1% agar supplemented with sugar alcohols (sorbitol, mannitol), auxins (NAA, 2,4-D) and cytokinin (BA) promoted a higher percentage of aposporous development from mature fronds ofPyrrosia piloselloides derived from aseptically cultured spores as compared with those obtained from plants in the field. A method using 4'6-diamidino-2-phenyl indole and fluorescence microscopy correlated the deoxyribonucleic acid contents of the aposporous gametophytes and sporophytes derived from them with their ploidy level.

Production of aposporous gametophytes from Drymoglossum piloselloides (L.) Presl. frond strips

Strips of Drymoglossum piloselloides fronds in a modified Murashige and Skoog medium in 1% agar produced aposporous gametophytes in about 2 months. Young fronds showed a greater ability to develop aposporous gametophytes than older fronds. The addition of kinetin to the medium improved the ability of older fronds while the presence of 1-naphthaleneacetic acid enhanced the effect of kinetin.

C-Glycosylxanthones in Diploid and Tissue Culture-Induced Autotetraploid Davallia fejeensis

Rhizome tips (about 2 cm long) of diploid Davallia fejeensis were collected in the Enid A. Haupt Conservatory of the New York Botanical Garden, cleaned, rinsed in tap water and further trimmed. Voucher specimens are at NY. Explants (6-8 mm long) were cultured on 1% agar-gelled sterile nutrient medium of Knudson as modified by Steeves et al. (1955) and supplemented with 2% sucrose in order to provide control diploid sporophytes growing in axenic culture. Roots, leaves, and rhizomes of the resulting plants were excised and induced to differentiate into aposporous diploid gametophytes. Stock cultures of diploid gametophytes were multiplied and further grown on liquid media supplemented with 0.6% agar to effect fertilization and raise tetraploid sporophytes. Tetraploid gametophytes were isolated from this tissue culture-induced tetraploid material by repeating the procedure followed at the diploid level. In this way, both diploid and tetraploid sporophytes and gametophytes were made available for this study. Phenolic compounds were isolated by two-dimensional paper chromatography of an 80% methanol extract of green material in t-BuOH-HOAc-H20, 3:1:1 (TBA) and 15% aqueous acetic acid (HOAc), followed by one-dimensional paper chromatography in water. C-glycosylxanthones appeared as orange compounds in ultraviolet light and turned yellow when fumed with ammonia. Both purified compounds were co-chromatographed with mangiferin and isomangiferin isolated from Asplenium montanum Willd. (Bozeman & Radford 11552, NY). Rf values in TBA, BAW, HOAc, and H20 were: mangiferin, 0.31, 0.43, 0.43, 0.12; isomangiferin, 0.19, 0.29, 0.23, 0.04; Asplenium mangiferin, 0.34, 0.42, 0.42, 0.11; Asplenium isomangiferin, 0.21, 0.32, 0.23, 0.03; and rutin standard, 0.43, 0.45, 0.56, 0.25. The BAW and HOAc values are very similar to those of Smith and Harborne (1971), but the TBA and HOAc values differ from those of Markham and Wallace (1980). Absorption spectra (MeOH, nm) were: mangiferin, 232, 260, 270sh, 310, 364; isomangiferin, 240, 258, 270sh, 312, 365. The compounds were unaffected by acid hydrolysis (1 hr, 2N HC1, 100?C).

THE DEVELOPMENT OF APOSPOROUS GAMETOPHYTES AND REGENERATED SPOROPHYTES FROM EPIDERMAL CELLS OF EXCISED FERN LEAVES: AN ANATOMICAL STUDY

Three different types of outgrowths develop from epidermal cells of excised juvenile leaves of Microgramma vacciniifolia: aposporous gametophytes, intermediates, and regenerated sporophytic plantlets. The gametophytes and intermediates arise from derivatives of epidermal cell divisions which are developed to the exterior of the leaf surface, whereas the sporophytic regenerants originate from derivatives produced by cell divisions to the interior of the leaf. Anatomical observations of excised leaves grown in vitro demonstrate that only the epidermal cells are stimulated to divide and give rise to the various types of outgrowths. Incorporation of tritiated thymidine by the nuclei of leaf epidermal cells gives further evidence for the metabolic activity of these cells.

The Development of Aposporous Gametophytes and Regenerated Sporophytes from Epidermal Cells of Excised Fern Leaves: An Anatomical Study

Three different types of outgrowths develop from epidermal cells of excised juvenile leaves of Microgramma vacciniifolia: aposporous gametophytes, intermediates, and regenerated sporophytic plantlets. The gametophytes and intermediates arise from derivatives of epidermal cell divisions which are developed to the exterior of the leaf surface, whereas the sporophytic regenerants originate from derivatives produced by cell divisions to the interior of the leaf. Anatomical observations of excised leaves grown in vitro demonstrate that only the epidermal cells are stimulated to divide and give rise to the various types of outgrowths. Incorporation of tritiated thymidine by the nuclei of leaf epidermal cells gives further evidence for the metabolic activity of these cells.

The Effect of Sucrose on the Differentiation of Excised Fern Leaf Tissue into Either Gametophytes of Sporophytes

Excised juvenile leaves of Microgramma vacciniifolia (Polypodiaceae) develop sporophytic regenerants when grown on mineral agar with sucrose. The ratio of sporophytes to gametophytes produced from the leaf tissue increases with higher percentages of sucrose such that at 4% sucrose, the induction of aposporous gametophytes is a rare occurrence. Experiments varying the osmotic potential with sorbitol and those holding the osmotic potential of the culture medium constant while varying the sucrose level indicate that the effect of sucrose on the differentiation of fern leaf tissue into either gametophyte or sporophyte is nutritional rather than osmotic. A significant effect of sucrose in altering the differentiation of fern leaf tissue is the increased rate of senescence promoted by high sucrose concentrations.

Natural Apospory in Pteridium?

From a population of the bracken fern in New Hampshire, Farlow (1889) described prothalloid outgrowths from the marginal sori. He concluded that natural apospory, i.e. the formation of a gametophyte vegetatively from a sporophyte under normal environmental conditions, had occurred as reported by Druery (1884) and Bower (1884). Steil (1949) studied prothalloid outgrowths on Pteridium leaves from Wisconsin. He reported that these outgrowths never produced rhizoids or sex organs nor became cordate under natural or experimental conditions. Since gametophytic characters were absent, Steil concluded that the outgrowths were not aposporous gametophytes. In recent years I have studied this abnormality of the leaves of Pteridium aquilinum (L.) Kuhn var. latiuscilum (Desv.) Underw. ex Heller from three localities in Massachusetts: Bos-