H. Fernández

Biological and nutritional aspects involved in fern multiplication

Gametophytes of several species of ferns were mechanically triturated and the resulting homogenates cultured in vitro for propagation purposes. Differences in the time period from spore culture to sporophyte development were perceivable between species. For those species with a fast life cycle and high sporophyte production such as Woodwardia virginica and Dryopteris affinis sp. affinis, homogenization of gametophytes can be considered to be excellent method for propagation, yielding hundreds of sporophytes in a short period of time. Sporophyte formation was inhibited in O. regalis by the succesive application of homogenization to gametophytes regenerated by this technique. The effect of the culture medium composition on fern production was also studied in O. regalis and P. ensiformis gametophytes. In these species, sporophyte formation increased when the gametophytes were cultured in a medium containing water+0.7% agar. Addition of sucrose inhibited gametophyte development and induced their necrosis. The 1/2 dilution of Murashige and Skoog basal medium, without sucrose, favoured leaf expansion in P. ensiformis sporophytes.

Influence of tissue culture conditions on apogamy in Dryopteris affinis sp. affinis

The naturally-occurring apogamy of some ferns can be modified by culture conditions and growth regulators. Gametophytes of the apogamic fern Dryopteris affinis sp. affinis L., were cultured on Murashige and Skoog (MS) basal medium. Changes in concentration of MS medium components, sucrose, agar and different pH values were tested. The addition of benzyladenine (4.43 μM) and naphthalene acetic acid (0.53 μM) enhanced sporophyte proliferation on the gametophytes. After one month in culture, the gametophytes formed callus with a high morphogenic capacity. Culture of calli on medium without growth regulators yielded about 10,000 sporophytes per 1 g fresh weight of callus. This pattern of differentiation slowed with time to a point where only gametophyte regeneration was observed.

Gemmation in cultured gametophytes of Osmunda regalis

Gametophytes ofOsmunda regalis were culturedin vitro to determine the optimal conditions for growth and development. Culture media with low ammonium concentration (Knop, Knudson or 1/4 Murashige and Skoog dilution) were the most effective for growth of this organism. Addition of sucrose and mannitol to the culture medium at the same osmoticum, inhibits or enhances, respectively, growth and development of the gametophytes, which show a strong autotrophyin vitro. In darkness, both two-dimensional growth and sexual organ formation took place. Gemmae formation is described for the first time in this species and this process does not occur without sucrose in the culture medium or in darkness.

Gibberellins and antheridiogen on sex in Blechnum spicant L.

The role of gibberellins (GAs) in determining sex in the gametophyte of the fern Blechnum spicant L. was studied through (a) the effect of exogenous GA4+7 and GA3 (b) quantitation of the endogenous levels of GA1, GA3, GA4, GA7, GA9, and GA20 in male and female gametophytes, and (c) the effect of flurprimidol, a GAs biosynthesis inhibitor of the steps of oxidation of ent-kaureno to ent-kaurenoic acid. Our results show that GA4+7 had a slight effect of inducing either male or female sexual organs, antheridia and archegonia, respectively. The endogenous GAs content was not significantly different between sexes, but the GA4, GA7, and GA20 levels were raised above those of the other GAs in both sexes. Neither antheridiogen biosynthesis nor antheridia formation was inhibited by flurprimidol. Gametophytes regenerated from homogenized mature gametophytes (HG) show a different physiological behavior than spore-derived gametophytes. In the first case, gametophytes are males and synthesize antheridiogen before they attain maturity, in contrast to what occurs in spore-derived gametophytes which are females and synthesize antheridiogen when mature.

In vitro organogenesis of Polypodium cambricum

Sporophyte regeneration through green globular bodies (GGB) is affected by the supply of growth regulators as well as by the source of the donor explant. Rhizome, frond, petiole or root tip explants from juvenile sporophytes of Polypodium cambricum were cultured on media containing N6-benzyladenine (BA) or Kinetin alone or in combination with α-naphthaleneacetic acid (NAA). GGB production took place even in growth regulator-free medium. Addition of BA into the culture medium was very effective to induce formation of GGB in rhizome explants. Root tip explants exhibited the lowest organogenic capacity. Homogenization of BA-pretreated frond or rhizome was a powerful system to improve fern propagation.

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

Abstract 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.

Plantlet regeneration in Asplenium nidus L. and Pteris ensiformis L. by homogenization of BA treated rhizomes

Abstract Whole sporophytes of Asplenium nidus L. and Pteris ensiformis L. were cultured on Murashige and Skoog (MS) medium supplemented with 4.4 μM 6-benzylaminopurine (BA). After 1 month, the rhizomes became swollen due to bud proliferation induced by cytokinin. These rhizomes were isolated, homogenized and plated on MS hormone-free medium. In Pteris ensiformis, the rhizome expiants developed into sporophytes, and from the expiants of Asplenium nidus both gametophyte and sporophyte regeneration took place. Approximately 500 sporophytes were produced from homogenates of 0.5 g of BA treated rhizomes of A.nidus or P.ensiformis.

Gametophyte culture in vitro and antheridiogen activity in Blechnum spicant

Growth of Blechnum spicant gametophytes was optimal in MS liquid medium, a 16-h photoperiod, and it was unaffected by variation of the pH between 4.7 and 8.7. Antheridia were observed during all developmental stages of the gametophyte: filamentous, spatulate or cordate and their formation was induced by compounds excreted into the culture medium by mature gametophytes. This antheridiogen activity was found in the fractions corresponding to free and apolar esters of gibberellins. IBA at 5 µM and 50 µM, and BA at 50 µM inhibited antheridiogen. Exogenous application of GA3 allowed spore germination but strongly inhibited gametophyte development; the two dimensional state was not reached.

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.

Endogenous Hormonal Profiles in Hop Development

Changes in gibberellins (GAs), indole-3-acetic acid (IAA), and cytokinins associated with the transition from vegetative growth to reproductive growth in Humulus lupulus L. buds and leaves harvested at fortnight intervals were studied. During vegetative growth, GA1 increased gradually and the lowest content was observed during flower development. Both GA3 and GA4 showed a dramatic increase in the samples taken from the apical part of axillary branches from plants 4–5 m high, which corresponds to the maximum vegetative development prior to macroscopically visible inflorescences. Notable increases in the cytokinins trans-zeatin (t-Z), isopentenyladenine (iP), and the riboside and ribotide forms of iP were also obtained. The auxin, indole-3-acetic acid, was the most abundant plant hormone, and its content was highest during vegetative growth. These results show for the first time a relationship between endogenous hormone profiles and both vegetative and reproductive development in hop plants, which may be relevant for future research on the control of the flowering by exogenous hormone applications.