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 μM N⁶-benzyladenine (BA) and 2.32 μM 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.     

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.     

Callus formation and plant regeneration from protonemal protoplasts of the mossAnoectangium thomsonii Mitt.

The protoplasts of the mossAnoectangium thomsonii, isolated from protonemal cells underwent divisions in 48–72 hours on modified MS medium enriched with growth regulators, 2,4-D and kinetin, 10 % sucrose and coconut water (5 %). Subculture of protoplast derived cells in a medium of relatively low osmotic potential (5 % sucrose) produced dark green calli which could be maintained completely undifferentiated.     

THE INFLUENCE OF GROWTH SUBSTANCES ON THE INDUCTION OF APOGAMY IN PTERIDIUM GAMETOPHYTES

Experiments employing sequences of three media demonstrated the effect of growth substances on the induction of apogamy. The most effective sequence was 4% sucrose 1–14 days, 4% sucrose plus growth substance 15–28 days, and 0.1% sucrose 29–56 days. In this sequence concentrations of NAA, IAA, and GA promoted apogamous shoot formation. A higher NAA concentration than optimal for shoot formation stimulated apogamous root formation in all medium sequences. Kinetin was without effect or inhibitory to apogamy. Combination of kinetin/GA or GA/NAA concentrations did not increase the apogamous response. One combination of the kinetin/NAA concentrations had a synergistic effect on the apogamous shoot formation. Additions of GA to the synergistic kinetin/NAA combination had an antagonistic effect on the apogamous response.     

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.Abbreviations Kinetin 6-furfurylaminopurine - NAA naphthaleneacetic acid; Murashige and Skoog (1962) medium     

Autonomous endosperm induction by in vitro culture of unfertilized ovules of Viola odorata L.

Autonomous endosperm was found in unfertilized ovules of V. odorata L. cultured on MS medium supplemented with 2,4-D as a sole growth regulator or on media with 2,4-D and BAP or kinetin. Frequency of endosperm induction was approximately 9% in ovules analyzed. The induction rate depended mainly on genotype of the donor plant, and to lesser degrees, on floral stage, flower series and medium type. Multinuclear endosperms consisting of 10–37 nuclei were found in ovules after as few as 4 days of culture. In some ovules at this stage, the egg cell and two polar nuclei were present. The process of endosperm degeneration began after 3 weeks of culture. In some ovules, degenerating autonomous endosperm was observed up to the 7th week. Parthenogenetic development of egg cells or apogamy did not accompany autonomous endosperm, supporting the hypothesis of independent pathways for embryo and endosperm development. Received: 1 December 1998 / Revision accepted: 6 April 1999     

Effect of chemical factors on production of isoflavonoids in Pueraria tuberosa (Roxb.ex.Willd.) DC suspension culture

Suspension cultures of Pueraria tuberosa, a woody legume, have been established and using different concentrations of growth regulators, sucrose, ammonium and nitrate nitrogen, attempts have been made to improve their isoflavonoid content. The cell cultures grew well on all the treatments. Up to approximately 8 folds increased isoflavonoids content was recorded in the cultures grown in MS medium modified with nitrogen and supplemented with 1 mg 1(-1) of kinetin.     

Morphogenetic role of kinetin and abscisic acid in the moss Physcomitrium

Summary In the presence of kinetin, a supposedly gametophytic bud inducing substance, the secondary protonema of the moss Physcomitrium pyriforme Brid., as well as producing leafy gametophytes, continued to exhibit its normal tendency of forming sporophytic buds (i.e. buds with apical cells having two cutting faces). Also remarkable was that callus derived from the secondary protonema, when cultured in a kinetin supplemented liquid medium, formed exclusively apogamous sporophytic buds with a virtual exclusion of gametophytes. In the presence of abscisic acid, the elongation of protonemal cells as well as their differentiation was markedly suppressed. This effect was manifest even when abscisic acid was used in conjunction with kinetin. It is suggested that rather than having a morphoregulatory role, kinetin may be responsible merely for enhancing cell proliferation. The determination of an apical cell with two cutting faces (sporophytic) or one with three cutting faces (gametophytic) is under the control of other factors both external, (e.g. sucrose) and internal. It is proposed that abscisic acid can suppress the usual differentiational capacity of the moss tissue, even in a favourable environment.     

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. Received: 3 March 1997 / Revision received: 11 April 1997 / Accepted: 3 June 1997     

Rooting and the Metabolism of Nicotine in Tobacco Callus Cultures

The usefulness of exogenous nicotine as a factor in the induction of morphogenesis in a tobacco tissue culture medium has been demonstrated. Nicotiana rustica callus cell cultures were grown on a modified Murashige and Skoog medium with 2 mg/l indoleacetic acid (IAA) and 0.2 mg/l kinetin (MMS). Root morphogenesis was induced in roller tube callus cell cultures and solid callus cell cultures grown on MMS without kinetin supplemented with 10–100 mg/l nicotine. Optimal nicotine concentration for root induction was 50 mg/l. Other tests using varying combinations of IAA, kinetin and nicotine produced no obvious morphogenesis, although some changes in the amount of callus growth and endogenous protein concentration did correlate with nicotine concentration relative to the presence of IAA and/or kinetin. In liquid MMS medium, ¹⁴C-nicotine was primarily incorporated into the protein fraction of cultured cells while primarily incorporated into the cell wall and/or cell membrane fraction of cells cultured on MMS without kinetin in the medium. In MMS without IAA and MMS without both IAA and kinetin, there was incorporation, but to a lesser extent in both the protein and the cell wall and/or cell membrane fractions.     

Regulation of a Sub-sexual Life Cycle in a Moss: Evidence for the Occurrence of a Factor for Apogamy in Physcomitrium

As demonstrated in our earlier studies, the differentiationof apogamous sporophytes on the secondary protonema of the mossPhyscomitrium pyriforme Brid. requires an exogenous supply ofsucrose in the medium. In the present work, similar differentiationwas observed in the leaf cells of aged gametophytes. The experimentsindicate an accumulation in the leaves of a sporophytic factorwhich initiates a de novo differentiation of sporophytes fromleaf cells without the intervention of sexual reproduction.In the absence of sucrose, the factor for apogamy was not present.Highlight intensity (5000–6000 lx) also inhibited itsproduction. There was no evidence that its presence interferedwith or inhibited production of gameto-phores. Growth regulatorssuch as IAA and kinetin altered only the effectiveness of thissporophytic factor, demonstrating that it was endogenous. Sporogenesisin the apogamous sporophytes took place without orthodox meiosis. Results obtained by using different exogenous environments forthe in vitro differentiation of callus into gametophytes orsporophytes are also reported. These support our contentionthat there is an accumulation of a sporophytic factor in thegametophytic callus cells, which is diluted during the processof differentiation. The morpho-regulatory influence of lightin the differentiation of apical cells with three cutting facesfrom unorganized callus is also considered.     

Induction of apogamy inEquisetum arvense

InEquisetum arvense, apogamous sporophytes were produced on medium containing 5×10^?6–5×10^?8 g/ml kinetin. NAA, IAA, GA₃, glucose and saccharose were ineffective for the induction of apogamy. On medium containing 5×10^?7–5×10^?8 g/ml kinetin, the gametophytes passed into sporophytic structures directly. On medium containing 5×10^?6 g/ml kinetin, some gametophytes passed into sporophytic structures directly, and others became a callus-like cell mass from which an apogamoun shoot arose. The results of the morphological observations on them were reported and compared with the sexually produced sporophyes. The apogamous sporophytes induced by 5×10^?7 g/ml kinetin were haploid in their nuclear phase and some of those induced by 5×10^?6 g/ml kinetin had a tendency to become diploid.     

Plant regeneration from tissue cultures of Persian buttercup (Ranunculus asiaticus L.)

Different plant explants of Persian buttercup (Ranunculus asiaticus L.) were screened for callus induction and adventitious shoot regeneration on different media to establish totipotent cultures. Murashige & Skoog (MS) medium was used, supplemented with different concentrations of the following growth regulators: kinetin, benzyladenine (BA), naphthaleneacetic acid (NAA) and indoleacetic acid (IAA). Callus was induced and adventitious buds regenerated only from cotyledonary explants after 4–5 weeks. Subculture of the regenerated buds on the same basal medium in presence of gibberellic acid (GA₃) and BA produced well-organized shoots. Rooting was obtained by transferring shoots to growth regulator-free MS medium. A high rate of shoot multiplication has been achieved on medium with high concentration of kinetin and long-day photoperiod. Finally the plants were successfully transferred to soil and grown in a greenhouse.     

Plant regeneration from callus cultures ofLithospermum erythrorhizon

We previously studied the production of shikonin derivatives by cell lines ofLithospermum erythrorhizon. As a result, we have obtained a cell line LE 87, which exhibited high cell growth and high shikonin production. In the present study, the effects of auxins (2,4-D, IAA, picloram, and NAA) and cytokinins (BAP and kinetin) on organogenesis and somatic embryogenesis in this shikonin-producing cell line were investigated. The highest organogenic and embryogenic efficiency was obtained on MS medium supplemented with 10 µM NAA and 0.3 µM kinetin. Subcultured calli showed different morphogenic frequencies depending on the NAA and kinetin concentration. Morphologically normal plants have been regenerated via mostly organogenesis. Shoots subsequently produced roots on plant growth regulator-free MS medium and developed into plantlets. In most cases, a few thin roots were formed at the bases of the shoots after four weeks on the rooting medium. More than fifty green plantlets were transplanted to soil in pots and developed into phenotypically normal plants 8 weeks after being transferred to soil. The regenerated plants grew to maturity, flowered, and set seeds by only artificial pollination.Abbreviations BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - NAA -naphthalene-acetic acid - MS Murashige and Skoog (1962) medium Communicated by S. Gleddie     

Protonemal Morphogenesis of the Moss Tetraphis pellucida Hedw. in Culture and in the Wild

In axenic culture, the protonemal filaments of Tetraphis pellucidaderived from either spores and gemmae, or excised stems andleaves, share a mixture of attributes of chloronemata and caulonemata.Indirect immunofluorescence reveals that the tip cells containcortical and endoplasmic arrays of microtubules at interphase,and phragmoplasts associated with cell plate formation, butpre-prophase bands are absent. Protonemal plates originate fromthe same sites as filamentous protonemal side branches or directlyfrom young gemmae or excised stem fragments. These plates havea cylindrical base, the latter producing a single gametophorebud, and a unistratose lamina. The gametophores produce gemmacups in culture with the vegetative life cycle taking approximately28 d. Gibberellic acid (GA₃) has no visible effect on protonemal morphogenesiswhereas the auxin naphthalene acetic acid (NAA) suppresses plateand side branch formation. In the presence of kinetin the platesare callus-like and produce supernumerary buds. Abscisic acid(ABA) induces malformed plates and filaments with swollen cells,similar to those found in ageing cultures. Rhizoids are produced in abundance from gametophores and protonemalplates in nature but were never seen in culture. In the wild,rhizoids produce numerous protonemal plates and occasional gametophorebuds. The former are the main source of new shoots. The filamentousprotonemal phase in nature mainly comprises upright filamentscontaining one or more abscission cells. The protonemal plates in Tetraphis are homologous with thosein the allied genus Tetrodontium but are very different fromthose in Diphyscium and Sphagnum. Differences between cultureand nature are attributed to lower nutrient levels and irradiancesin the wild. Tetraphis pellucida, protonema, moss, morphogenesis, immunocytochemistry, gemmae, tip growth, vegetative reproduction     

Effects of genotype,explant source and growth regulators on organogenesis in carnation callus

For callus induction, shoot tips and nodal or internodal stem segments of carnation cultivars (Coral, Jaguar, Salome and Sarinah) were grown on MS basal medium with 2,4-dichlorophenoxyacetic acid and kinetin. To achieve organogenesis, calli were transferred onto MS medium without or with growth regulators (indoleacetic acid, naphthaleneacetic acid, indolebutyric acid, kinetin, benzyladenine) in different combinations. Shoot primordia emerged from the subsurface meristemoids of calli, roots developed from the inner callus cells. The effects of genotype, explant source and growth regulators on callus-mediated organogenesis differently manifested themselves in caulogenesis and rhizogenesis, respectively. The number of root-forming calli most of all depended on genotype and least of all on explant source. Unlike rhizogenesis, caulogenesis essentially depended on explant source: internodal calli of all the tested cultivars practically missed the shoot formation ability. The number of caulogenetic calli from apical-nodal segments significantly depended on genotype, but was also affected by growth regulators. This revised version was published online in June 2006 with corrections to the Cover Date.     

Direct somatic embryogenesis and plant regeneration from single cell suspension cultures of Elymus giganteus Vahl

Summary A yellowish, nodular callus was induced from mature embryos of Elymus giganteus Vahl on MS medium containing 2.0 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.5 mg/l kinetin, from which a cell suspension culture was initiated in liquid MS medium supplemented with 0.5 mg/l 2,4-D, 1.0 mg/l kinetin and 0.2 mg/1 naphthaleneacetic acid (NAA). By filtering through a series of sieves with decreasing mesh sizes and collecting the resultant filtrate, a suspension culture composed mainly of single embryogenic cells was established. In a medium containing 0.3 mg/l 2,4-D, 1.0 mg/l 6-benzylaminopurine (6-BAP) and 500 mg/l casein hydrolysate (CH), the single cells underwent direct somatic embryogenesis resulting in the formation of proembryos. These proembryos developed into mature embryos when placed in a double-layer liquid overlay culture. Intact plants were developed from somatic embryos when they were transferred onto solidified MS medium without added growth regulators.     

Plantlet regeneration from protoplast-derived embryogenic calli of Crocus cancellatus

Plant regeneration from protoplast culture of Crocus cancellatus was investigated using regenerable embryogenic calli obtained from shoot meristem culture on LS (Linsmaier and Skoog, 1965) medium containing 4 mg l⁻¹ kinetin and 1 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D). Protoplasts were isolated directly from embryogenic calli. The best protoplast growth was found on those embedded in Ca-alginate beads and cultured with nurse cells in MS (Murashige and Skoog, 1962) medium supplemented with 2 mg l⁻¹ kinetin, 1 mg l⁻¹ 2,4-D and 100 mg l⁻¹ ascorbic acid at 25 °C in darkness. After 4–5 weeks of culture, microcalli appeared on the surface of the Ca-alginate beads, but the protoplasts without immobilization in Ca-alginate beads showed very low cell division. Growth of the microcalli in the medium with nurse cells was much better than in the medium without nurse cells. Transferring beads onto half strength MS medium supplemented with 0.2 mg l⁻¹ kinetin and 0.1 mg l⁻¹ 2,4-D, increased the growth of embryogenic calli. Somatic embryo development was observed either on half strength MS medium growth regulator free or with 1 mg l⁻¹ abscisic acid. Matured embryos germinated on half strength MS medium containing 25 mg l⁻¹ of gibberelic acid. Plantlet formation was obtained on half strength MS medium containing 1 mg l⁻¹ 6-benzyladenine and 1 mg l⁻¹ α-naphthaleneacetic acid at 20 °C in a 16/8 h light/dark cycle. This revised version was published online in June 2006 with corrections to the Cover Date.     

Callus induction and plantlet regeneration inCichorium intybus L.: II. Effect of different hormonal treatments

Summary Expiants ofCichorium intybus L. storage roots were grownin vitro on a modified Heller's medium lacking auxins and cytokinins, or supplemented with auxins (either 2,4-D or NAA) alone or with a cytokinin (kinetin) or auxin and kinetin combinations in different concentrations. The morphogenetic responses of root explants varied with the different hormonal treatments. The best response for callus growth was obtained in presence of 2,4-D. On the contrary, kinetin alone was very effective for shoot induction, increasing the formation of adventitious buds (up to 100% of the explants) in respect to control (hormone-free medium). NAA induced either shoot differentiation (in a medium frequency) or root formation. Expiants excised from root zones near to apex, which showed on hormone-free medium a very low regenerative capacity (lower than proximal zones of the root), responded to kinetin by increasing significantly the number of shoots from adventitious buds.Cytological analyses in developing primary calli showed, in all media, high incidence of amitotic phenomena confirmed by DNA cytophotometry in calli at different growth stages. The histological analysis demonstrated the formation of meristematic growth centers on the organogenesis inducing media and the subsequent development of these meristemoids as shoot (or root) apices in the callus mass.The results are discussed in comparison with previous observations of the authors inCichorium intybus (Caffaro et al. 1982) and in relation to the action of hormonal treatments on callus formation and organogenesis. The cytological and histological results are also discussed in relation to the hormonal composition of the medium.     

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 M) and naphthalene acetic acid (0.53 M) 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.Abbreviations BA benzyladenine - 2,4-d 2,4-dichlorophenoxyacetic acid - F.W. fresh weight - MS Murashige & Skoog medium - NAA 1-naphthalene acetic acid - SE standard error