The effect of exogenous and endogenous phytohormones on the in vitro development of gametophyte and sporophyte in <Emphasis Type="Italic">Asplenium nidus</Emphasis> L.

The fern Asplenium nidus L. is in great demand as an ornamental plant. The aim of this work was to investigate the influence of phytohormones in promoting a gametophytic and sporophytic growth in homogenized sporophytes tissue. Exogenous application of 0.5 and 5 μM N ⁶-benzyladenine, 0.05 and 0.5 μM indole-3-acetic acid (IAA), and 0.3 and 3 μM gibberellic acid (GA₃) favoured sporophyte regeneration, whereas gametophyte regeneration took place when plant material was cultured in a hormone-free liquid MS medium. The endogenous contents of the auxin IAA, the cytokinins trans-zeatin, trans-zeatin riboside, dihydrozeatin, dihydrozeatin riboside, isopentenyladenine and isopentenyladenosine, and the gibberellins GA₁, GA₃, GA₄, GA₇, GA₉ and GA₂₀ in growing gametophytes and sporophytes were evaluated. Similar levels of the auxin and cytokinins and qualitative differences in the gibberellins were found between both generations.     

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     

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.     

IAA-induced apogamy in Platycerium coronarium (Koenig) Desv. gametophytes cultured in vitro

Apogamous sporophytes were produced on Platycerium coronarium gametophytes cultured in the presence of indole-3-acetic acid (IAA). The percentage of apogamy as well as the total number of apogamous sporophytes produced per gametophyte clump were highest in the presence of 40 M IAA. When ethylene was allowed to accumulate in the culture vessel in the presence of an optimum level of IAA, the percentage and total number of apogamous sporophyte production decreased significantly. Using light microscope and confocal laser scanning microscope we have shown that nuclear size can be used as a quick parameter to estimate the ploidy level of P. coronarium.Abbreviations CLSM confocal laser scanning microscope - IAA Indole-3-acetic acid - MS Murashige and Skoog     

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.     

THE INFLUENCE OF CULTURAL CONDITIONS ON THE INDUCTION OF APOGAMY IN PTERIDIUM GAMETOPHYTES

Apogamous sporophytes formed on Pteridium gametophytes in response to concentrations of certain sugars which supported gametophytic growth. High osmotic concentration of the medium inhibited apogamy, while variations in the basic medium were not stimulatory. Agar, autoclaving, the ammonium ion, and dry media were not required for apogamy. Renewing the medium during an experiment enhanced the apogamous response. Changing the medium at set intervals facilitated the separation of apogamous plant development into gametophytic, initiative, and developmental phases, thus enabling testing of various factors at each of these stages. Apogamy was light-initiated, while the actual development of apogamous sporophytes was caused by light, succinic acid or sugar.     

Endogenous phytohormone profile during oat (Avena sativa L.) haploid embryo development

The development of embryos requires interaction of many endogenous hormones. The aim of the study was to determine which endogenous phytohormones are involved in the process of oat (Avena sativa L.) haploidization. Oat haploids were obtained by wide crossing with Zea mays L. The hormonal profiles of the ovaries with (OE) and without developed embryo (OWE) were compared. Phytohormone contents were measured by UHPLC coupled with mass spectrometer. The total content of indole-3-acetic acid (IAA), trans-zeatin (tZ), and kinetin (KN) in OE was significantly higher than in OWE. 4-Chloroindole-3-acetic acid was detected only in OWE. There were no differences between OE and OWE in the content of gibberellins (GA₁, GA₃, GA₄, GA₆, GA₇) and stress hormones (abscisic, salicylic, jasmonic acids). Content of endogenous KN was highly negatively correlated with the percentage of haploid embryos, germinated haploid embryos, haploid plants on MS (in vitro), haploid plants in soil (ex vitro), and doubled haploid lines. The tZ content negatively correlated with the frequency of haploid embryo formation, germination, and haploid plants obtained in vitro, as opposed to GA₁, which correlated positively. A positive correlation was found between IAA and tZ in OE, whereas in OWE it was a negative correlation. The profiles of phytohormones in OE and OWE were determined; however, their mode of action needs to be clarified.

     

THE INITIATION OF SPOROPHYTES BY OBLIGATE APOGAMY IN CHEILANTHES CASTANEA

The initiation of apogamous sporophytes in Cheilanthes castanea was recorded by daily photography of individual gametophytes. Whereas an ordinary embryo arises from a zygote, apogamous embryos of C. castanea originate from one to three initial cells which occur just behind the apical region of the prothallus. The initial (or initials) produce cells with small chloroplasts behind the sinus of the gametophyte. The appearance of cells with smaller chloroplasts than those normally found in gametophytes is the first indication that apogamy is occurring. The cells with small plastids produce a group of densely-cytoplasmic meristematic cells. The size of the meristematic mass increases until shoot and root apices of the apogamous embryo are organized.     

Hormones in zygotic and microspore embryos of Brassica napus

A number of studies have used microspore-derived embryos (MDEs) as amodel for examining a range of processes, including hormonal regulation ofembryo development. We examined the hormonal physiology of MDEs with theprimaryobjective of testing the validity of using the MDE system as a model forhormonally-regulated development in zygotic embryos, through late stages. To dothis we identified and quantified endogenous levels of abscisic acid (ABA),indole-3-acetic acid (IAA) and a number of gibberellins (GAs), includingGA₁₉, GA₂₀, GA₁ and GA₈ in bothMDEsand zygotic embryos. The presence of GA₁₉, together with itsC₁₉ metabolites indicates that the early-13 hydroxylation pathway isoperative in both embryo systems. Gibberellins A₄ and GA₉were also identified, thereby confirming the presence of the earlynon-hydroxylation pathway in B. napus MDEs and zygoticembryos. In general, the pattern of change of hormone (ABA, IAA, GA₁and GA₂₀) content per embryo through embryogenesis was similar forMDEs and zygotic embryos. Indole-3-acetic acid and GA₁ increased toamaximum at day 30 after culture (DAC) before decreasing. Abscisicacid levels increased to a maximum at day 35, and declined in zygoticembryos but not in MDEs. GA₂₀ increased to the final harvest atmaturity, or day 40. The absolute content (g/embryo) of each hormone, howeverwas appreciably lower (5- to 15- fold) in the MDEs. This was not the result ofdilution into surrounding medium for ABA or IAA; GA₁, however, didaccumulate in the medium. Although there were absolute quantitative differencesin the levels of IAA and ABA found in the two embryo systems, the similaritiesin the pattern of hormone changes suggests that the MDE system can serve as auseful model for examining the physiological roles of hormones duringembryogenesis.     

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 GA_1, 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.     

The Relationship between Levels of Endogenous Gibberellins and the Response of Vigna Hypocotyls to Exogenous Indole-3-Acetic Acid

Segments excised from the upper and the lower parts of cowpea(Vigna unguiculata L.) hypocotyls were compared in terms oftheir responses to exogenous indole-3-acetic acid (IAA) in relationto their endogenous levels of gibberellin. Growth of the segmentswas measured continuously during xylem perfusion with a lineardifferential transformer. IAA induced a burst of elongationin the upper segments but only slight promotion of growth inthe lower segments. Treatment with uniconazole, a potent inhibitorof the biosynthesis of gibberellins, reduced the responsivenessof the upper segments to exogenous IAA to about one half ofthe control value. Pre-perfusion with GA₃ of such segments fortwo hours prior to application of IAA, partially restored theresponsiveness to IAA. Analysis by GC/MS identified GA₁, GA₄,GA₉ GA₂₀ and GA₅₁ as native gibberellins in the hypocotyls ofcowpea seedlings. Analysis by GC/SIM also showed that the physiologicallyactive gibberellins (GA₁ and GA₄) were located mainly in theupper part of the hypocotyl and the treatment with uniconazolemarketly reduced the endogenous level of gibberellins thereto less than 11% of the control level. These results suggestthat levels of endogenous gibberellins possibly control theresponse to IAA in these segments. (Received May 12, 1994; Accepted November 15, 1994)     

In vitro propagation of caper (Capparis spinosa L.)

Summary A twenty fold multiplication per twenty days of caper was achieved by culturing nodal shoot segments in the presence of BAP (4 μM) plus IAA (0.3 μM) and GA₃ (0.3 μM). The use of a modified MS medium facilitated this response. Plantlet regeneration was induced on single shoots taken from proliferating clusters subcultured for 20 days on a reduced BAP (2 μM) without auxin and gibberellin Higher rooting responses (70%) were obtained after a 20-day incubation period in darkness on solid half-strength MS1 medium plus IAA (30 μM), followed by a subsequent 20 day culture period on half-strength MSI basal medium. Proliferation was mainly due to axillary shoot-bud development as revealed by histological studies. The extensive meristematic activities observed indicated the enormous morphological potential of this species.     

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.     

Endogenous Gibberellins in the Developing Liquid Endosperm of Tea

Changes in the kind and level of endogenous gibberellins (GAs) in the developing liquid endosperm of tea (Camellia sinensis L.) were investigated. Gibberellin A₁ (GA₁), GA₈, GA₁₉, GA₂₀, and GA₄₄ were identified by GC-MS or GC-SIM. Besides these early C-13 hydroxylated GAs, GA₃, iso-GA₃, and GA₃₈ were also identified. Of these GAs, GA₁ and GA₃ were the major gibberellins. The levels of these GAs were at a maximum in the globular embryo stage and then decreased rapidly during embryo maturation.     

Thermomorphogenic and Photomorphogenic Control of Stem Elongation in Fuchsia Is Not Mediated by Changes in Responsiveness to Gibberellins

Stem elongation in Fuchsia × hybrida was influenced by cultivation at different day and night temperatures or in different light qualities. Internode elongation of plants grown at a day (25°C) to night (15°C) temperature difference (DIF+10) in white light was almost twofold that of plants grown at the opposite temperature regime (DIF−10). Orange light resulted in a threefold stimulation of internode elongation compared with white light DIF−10. Surprisingly, internode elongation in orange light was similar for plants grown at DIF−10 and DIF+10. Flower development was accelerated at DIF−10 compared with DIF+10 in both white and orange light. To examine whether the effects of DIF and light quality on shoot elongation were related to changes in gibberellin metabolism or plant sensitivity to gibberellins (GAs), the stem elongation responses of paclobutrazol-treated plants to applied gibberellins were determined. In the absence of applied gibberellins paclobutrazol (>0.32 μmol plant⁻¹) strongly retarded shoot elongation. This inhibition was nullified by the application of about 10–32 nmol of GA₁, GA₄, GA₉, GA₁₅, GA₁₉, GA₂₀, GA₂₄, or GA₄₄. The results are discussed in relation to possible effects of DIF and light quality on endogenous gibberellin levels and gibberellin sensitivity of fuchsia and their effects on stem elongation. Received October 4, 1997; accepted December 17, 1997     

Effect of Gibberellin on Growth, Protein Secretion, and Starch Accumulation in Maize Endosperm Suspension Cells

The physiologic effect of gibberellins (GA) in seed development is poorly understood. We examined the effect of gibberellic acid (GA₃) on growth, protein secretion, and starch accumulation in cultured maize (Zea mays L.) endosperm suspension cells. GA₃ (5 and 30 μm) increased the fresh weight, dry weight, and protein content of the cultured cells, but the effect of GA₃ at 50 μm was not significantly different. However, the protein content in the culture medium was increased by these three concentrations of GA₃. The effect of GA₃ on the amount of cellular structural polysaccharides was not significant, but GA₃ had a dramatic effect on the starch content. At 5 μm, GA₃ caused an increase in the starch content, but at 50 μm the starch accumulation was reduced. Chlorocholine chloride (CCC), an inhibitor of GA biosynthesis, significantly increased the starch content and decreased the structural polysaccharide content of the cultured cells. The effects of CCC at 500 μm on the starch and polysaccharide content were partially reversed by 5 μm GA₃ applied exogenously. Based on these results we suggest that GA does not favor starch accumulation in the cell cultures and that the addition of lower concentrations of GA₃ in the medium may provide an improved balance among the endogenous GA in the cultured cells. Received October 31, 1995; accepted March 25, 1997     

GA3和IAA对慈竹综纤维和叶绿素含量动态积累的调控效应

以慈竹为材料,研究不同浓度和配比的GA₃和IAA对综纤维和叶绿素含量动态积累的调控效应,为纸浆用竹的遗传改良研究提供理论依据。研究结果表明, GA₃和IAA处理对慈竹综纤维动态积累调控作用具有差异。处理前30 d内,GA₃50IAA200处理慈竹综纤维积累强度高于GA₃200IAA50处理,40~50 d时,则反之。GA₃和IAA处理的综纤维最终水平明显高于空白对照,差异达极显著水平。GA₃和IAA处理能提高慈竹叶绿素含量,GA₃200IAA50处理慈竹叶绿素含量高于GA₃50IAA200处理。相关分析结果表明,叶绿素含量与综纤维含量间呈不显著正相关关系。     

Interaction of Brassinosteroids with Light Quality and Plant Hormones in Regulating Shoot Growth of Young Sunflower and <Emphasis Type="Italic">Arabidopsis</Emphasis> Seedlings

Sunflower hypocotyls elongate as light quality changes from the normal red to far-red (R/FR) ratio of sunlight to a lower R/FR ratio. This low R/FR ratio-induced elongation significantly increases endogenous concentrations of indole-3-acetic acid (IAA) and also of three gibberellins (GAs): GA₂₀, GA₁, and GA₈. Of these, it is likely GA₁ that drives low R/FR-induced growth. Brassinosteroids are also involved in shoot growth. Here we tested three R/FR ratios: high, normal, and low. Significant hypocotyl elongation occurred with this stepwise reduction in R/FR ratio, but endogenous castasterone concentrations in the hypocotyls remained unchanged. Brassinolide was also applied to the seedlings and significantly increased hypocotyl growth, though one that was uniform across all three R/FR ratios. Applied brassinolide increased hypocotyl elongation while significantly reducing (usually) levels of IAA, GA₂₀, and GA₈, but not that of GA₁, which remained constant. Given the above, we conclude that endogenous castasterone does not mediate the hypocotyl growth that is induced by enriching FR light, relative to R light. Similarly, we conclude that the hypocotyl growth that is induced by applied brassinolide does not result from an interaction of brassinolide with changes in light quality. The ability of applied brassinolide to influence IAA, GA₂₀, and GA₈ content, yet have no significant effect on GA₁, is hard to explain. One speculative hypothesis, though, could involve the brassinolide-induced reductions that occurred for endogenous IAA, given IAA’s known ability to differentially influence the expression levels of GA20ox, GA3ox, and GA2ox, key genes in GA biosynthesis.     

Induction of somatic embryogenesis in cashewnut (Anacardium occidentale L.)

Summary Somatic embryogenesis was induced in callus cultures derived from nucellar tissue of cashewnut (Anacardium occidentale L.). Callus was obtained from nucellar tissue after 3 wk of culture on semisolid Murashige and Skoog (MS) basal medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D, 5 μM)+gibberellic acid (GA₃, 15 μM)+N⁶-benzyladenine (BA, 5 μM). This callus gave rise to an embryogenic mass after 9 wk on maintenance medium containing 2,4-D (10 μM)+GA₃ (15 μM)+4% sucrose +0.5% activated charcoal +10% coconut water (CW) +0.05% casein hydrolysate (CH). The embryogenic mass, after transfer to medium supplemented with 2,4-D (5 μM)+GA₃ (30 μM)+4% sucrose +0.5% activated charcoal +10% CW +0.05% CH, gave rise to somatic embryos. The developmental stages of somatic embryos were observed using light and stereo microscopes. Histological study of somatic embryo development was also carried out. The present study would be useful for clonal propagation, and variety improvement in cashewnut, which is essential due to its increasing demand and export potential.     

Improvement of in vitro propagation of apricot cultivar ‘Bebecou’

Factors affecting successful establishment in vitro, rapid proliferation and rooting of apricot cultivar ‘Bebecou’ were studied. Ethanol and NaOCl were applied in several combinations for disinfection; chilling, plant growth regulators BA, IAA and GA₃, antibiotics, different culture vessels and systems of subculture were evaluated for the optimization of shoot proliferation and the auxins NAA and IBA were assessed for root induction. The highest number of new microshoots/explant (18.7) was obtained in a culture medium supplemented with 2.2 μM BA+0.57 μM IAA after 300 h of chilling. The effect of GA₃ (11.4 μM) on shoot proliferation was positive in combination with 4.4 or 8.9 μM BA. Shoot length and productivity were highest at 2.2 μM BA+11.4 μM GA₃+0.57 μM IAA and at 2.2 μM BA+0.57 μM IAA, respectively and decreased as cytokinin concentration increased. The antibiotic ‘Na-cefotaxime’ had a minimal impact on shoot growth when used at the lowest concentration (250 mg l⁻¹). Subculture every 2 weeks in a medium supplemented with 2.2 μM BA and 0.57 μM IAA was more efficient for shoot induction than alternation of 20 days culture in a propagation medium supplemented with 2.2 μM BA and 10 days culture in an elongation medium supplemented with 1.1 μM BA and 5.71 μM IAA. The highest number of roots/shoot (8.1) was recorded at 19.6 μM IBA.