Developmental Morphology of Platycerium coronarium (Koenig) Desv

The growth habit and certain developmental aspects of Platyceriumcoronarium (stag's horn fern) are described. Starting from theprimordial stage, the nest and pendulous leaves develop to maturityin 90 and 80 days respectively. The fertile lobe, which is partof the pendulous leaf, reaches its maximum size in 40 days.The morphogenesis of the nest leaf is more variable, and itmatures and deteriorates earlier than the pendulous leaf. Theacrostichoid sorus formation is completed in 3 weeks from inceptionand spore dispersal takes place when the fertile lobe is about100 days old. The area of the fertile lobe and number of sporesproduced were determined. On Knop's agar medium the gametophytesdevelop in 2 months and 85 per cent of them are unisexual (bothmale and female) and 15 per cent bisexual. Less than 1 per centof the gametophytes give rise to sporophytes. The juvenile leavesare simple, displaying open dichotomous venation; the firstnest and pendulous leaves are produced 24 months after the dateof spore germination. Platycerium coronarium, stag's horn fern, leaf development, morphogenesis, spore production     

Sporophyte and gametophyte development of Platycerium coronarium (Koenig) Desv. and P. grande (Fee) C. Presl. (Polypodiaceae) through in vitro propagation

The sporophyte and gametophyte development of Platycerium coronarium and P. grande were compared through ex situ propagation using in vitro culture technique and under greenhouse and field conditions.The morphology of the sporophyte and gametophyte, type of spore germination and prothallial development of P. coronarium and P. grande were documented. Gametophytes of P. coronarium and P. grande were cultured in vitro using different media. The gametophytes were then transferred and potted in sterile chopped Cyathea spp. (anonotong) roots and garden soil for sporophyte formation. Sporophytes (plantlets) of the two Platycerium species were attached on the slabs of anonotong and on branches and trunks of Swietenia macrophylla (mahogany) under greenhouse and field conditions.Sporophyte morphology of P. coronarium and P. grande varies but not their gametophyte morphology. P. coronarium and P. grande exhibited rapid spore germination and gametophyte development in both spore culture medium and Knudson C culture medium containing 2% glucose. Gametophytes of P. coronarium and P. grande transferred to potting medium produced more number of sporophytes while the gametophytes inside the culture media did not produce sporophytes. Sporophytes of P. grande attached on mahogany branches produced more number of leaves with bigger leaf area than those attached on anonotong slabs. Likewise, sporophytes of P. coronarium attached on mahogany branches and anonotong slabs did not develop new leaves during two weeks monitoring and are still in a period of adjustment to its environment. Sporophytes of P. grande grown or attached on the trunk of mahogany trees in the field and under shaded environment favored their growth.     

Establishment and physiological analyses of photoautotrophic callus cultures of the fern Platycerium coronarium (Koenig) Desv. under CO2 enrichmen

Gametophyte-derived callus cultures of Platycerium coronariumcould be maintained under photoautotrophic conditions on Murashigeand Skoog medium supplemented with 2µM 2,4-dichlorophenoxyaceticacid (2,4-D) and with CO₂ enrichment. Progressive reductionof sucrose from the medium resulted in a reduction in growth,but an increase in total chlorophyll content. When subculturingwas delayed beyond 2 weeks, callus cells differentiated intogametophytes on the medium with 0.2 sucrose and no CO₂ enrichment.Enriching the photoautotrophic cultures on 2µM 2, 4-Dwith 1% CO₂ resulted in about 1.7-fold increase in fresh weightwithin 42 d. Total chlorophyll content was generally higherwith 1% CO₂ enrichment than with 10%. F_v/F_m ratio was higherfor callus on low levels of sucrose (>0.5%) than that onsucrose 1.0%. An increase in autofluorescence of chloroplasts,but not the size, was observed with decreasing sucrose levelsin the medium. Autofluorescence decreased with increase in CO₂from 0.03%. Our data are in agreement with the view that long-termexposure to high levels of decrease in photosynthetic capacity. Key words: Platycerium coronarium, stag's horn fern, autofluorescence of chloroplasts, confocal laser scanning microscope, F_v/F_m ratio, photoautotrophic callus     

Role of ethylene in the production of sporophytes from Platycerium coronarium (Koenig) desv. frond and rhizome pieces cultured in Vitro

The effect of ethylene on in vitro plant regeneration from frond and rhizome expiants of Platycerium coronarium was investigated. Ethylene levels in the culture vessels increased with time, resulting in a decrease in the percentage of sporophytes produced. Addition of the ethylene action inhibitor silver thiosulfate resulted in an increase in the percentage of plants regenerated, indicating an inhibitory effect of ethylene on regeneration. However, the presence of 2,5-norbornadiene was not effective in reversing the effect of ethylene. Inhibitors of ethylene biosynthesis, such as cobalt chloride, salicylic acid, benzylisothiocyanate, and aminoethoxyvinylglycine, were also ineffective in increasing sporophyte regeneration. 1-Aminocyclopropane-1-carboxylic acid, the ethylene precursor, was ineffective in increasing the level of ethylene in the culture vessels. Therefore, the biosynthetic pathway of ethylene in the fern P. coronarium appears to be different from that of higher plants but similar to that of some other ferns.Abbreviations SA salicylic acid - AVG aminoethoxyvinylglycine - BITC benzylisothiocyanate - STS silver thiosulfate - ACC 1-aminocyclopropane-1-carboxylic acid     

Ribulose-1,5-bisphosphate carboxylase and phosphoenolpyruvate carboxylase activities of photoautotrophic callus of Platycerium coronarium (Koenig ex O.F. Muell.) Desv. under CO2 enrichment

The in vitro activities of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) and phosphoenolpyruvate carboxylase (PEPC) were measured in cell-free extracts of Platycerium coronarium callus cultured for up to 42 days under photoautotrophic conditions with CO₂ enrichment. With an increase in CO₂ in the culture environment to 10% (v/v) at low light, the apparent photoautotrophic fixation of CO₂ by Rubisco declined, whereas the non-photoautotrophic CO₂ fixation by PEPC activity was enhanced. Hence, photosynthesis appears to play a lesser role in providing carbon skeletons and energy with prolonged culture in a CO₂-enriched environment. Instead, the anaplerotic supply of C-skeletons by PEPC may be important under such a situation. Short-term H¹⁴CO₃-fixation experiments indicated that photoautotrophic callus cultured for 3 weeks with 10% CO₂ enrichment assimilated less ¹⁴CO₂ than the control (0.03% CO₂). Analyses of ¹⁴C-metabolites indicated that about 50% of the total soluble ¹⁴CO₂ fixed was in the organic acid fraction and 35% in the amino acid fraction. Despite the changes in the in vitro Rubisco/PEPC activity-ratio, no significant change in the ¹⁴C distribution pattern was apparent in response to increasing sucrose or CO₂ concentrations. The suppression of Rubisco activity and total chlorophyll content in high sucrose or elevated CO₂ concentrations suggests an inhibition of the capacity for photoautotrophic callus growth under these conditions. This revised version was published online in June 2006 with corrections to the Cover Date.     

Micropropagation of Hedychium coronarium J. Koenig through rhizome bud

An optimized protocol was developed for in vitro plant regeneration of a medicinally important herb Hedychium coronarium J. Koenig using sprouted buds of rhizomes. The rhizomes with sprouted bud were inoculated on Murashige and Skoog (Physiol Plant 15:473–497, 1962) medium (MS) supplemented with either N⁶-benzyladenine (BA) alone (1.0–4.0 mg L⁻¹) or in combination with 0.5 mg L⁻¹ naphthalene acetic acid (NAA). Of these combinations, MS supplemented with a combination of 2.0 mg L⁻¹ BA and 0.5 mg L⁻¹ NAA was most effective. In this medium, best shoots (3.6) and roots (4.0) regeneration was observed simultaneously with an average shoot and root length of 4.7 cm and 4.2 cm respectively. Regeneration of shoots and roots in the same medium at the same time (One step shoot and root regeneration) reduced the time for production of in vitro plantlets and eliminates the media cost of rooting. Cent-percent (100 %) success in plant establishment was observed in both gradual acclimatization process as well as when plants were directly transferred to outdoor in clay pots containing a mixture of garden soil and sand (2:1) without any sequential acclimatization stage.     

白姜花不同开花时期的香味组分及其变化

采用顶空固相微萃取和气相色谱-质谱(GC-MS)技术,鉴定处于花蕾期、始花期、盛花期和衰老期的白姜花香味组分的结果表明,白姜花的香味组分共有49种,主要成分为单萜类、倍半萜类、酯类、酮酚类。花蕾期有30种,始花期有43种,盛花期和衰老期均为37种。单萜类化合物含量随着花的发育和衰老而逐渐增多,衰老期达到最高。酯类中,除了戊酸甲基苯甲酯外,其他酯类含量随着花的发育成熟而升高。初步确认,L-沉香醇、1,8-桉油醇、3,7-二甲基-1,3,7-辛三烯、乙酸月桂酯、苯甲酸甲酯为白姜花的特征香气。     

Ammonium and nitrate uptake and nitrate reductase activity of photoautotrophic callus cultures of the fernPlatycerium coronarium (Koenig) DESV

Summary The uptake of nitrate and ammonium by callus ofPlatycerium coronarium from the culture medium was examined. Nitrate reductase activity of photoautotrophic callus cultures under CO₂ enrichment was significantly lower compared to the cultures without CO₂ enrichment, but higher than that of heterotrophic callus cultured on medium with 2% (wt/vol) sucrose. When sucrose concentration of the heterotrophic culture was lowered to 0.2%, nitrate reductase activity increased. The level of nitrate reductase activity increased by about 25% in the heterotrophic callus with an increase in 2,4-D from 2 μM to 10 μM, despite a decline in fresh weight gain. However, photoautotrophic cultures with 1% CO₂ enrichment showed 20% decline in nitrate reductase activity and 45% decline in fresh weight gain with a similar increase in 2,4-D level. The rate of uptake of nitrate from the culture medium was unrelated to the level of nitrate reductase activity in the callus. For photoautotrophic callus under CO₂ enrichment, the presence of 1% (vol/vol) CO₂ generally resulted in the highest rate of nitrate uptake. The rate of uptake of ammonium was higher for callus cultured on 2 μM 2,4-D compared to that on 10 μM 2,4-D.     

Gemmation in cultured gametophytes ofOsmunda 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.Abbreviations MS Murashige and Skoog medium (1962) - KD Knudson medium (1946) - KP Knop medium (1865)     

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.     

Testing two chromosome doubling agents for in vitro tetraploid induction on ginger lilies,Hedychium gardnerianum Shepard ex Ker Gawl. and Hedychium coronarium J. Koenig

In Vitro Cellular & Developmental Biology - Plant - Polyploidization can be a way to produce new varieties in vegetatively propagated species where options on increasing genetic variability are...     

Spore-bearing structures in Salvinia nymphellula Desv. (Salviniaceae)

Fructifications of Salvinia nymphellula Desv., recently discovered from two places in Ghana, are recorded and described here for the first time. Two particularly interesting features have been noticed in the specimens examined: (1) the occurrence of amphisporangiate sporocarps between the proximal megasporangiate and the distal microsporangiate ones of each fertile segment, and (2) a difference between the specimens collected from the two places in the relative sizes of the proximal and distal sporocarps of their fertile segments. These features have also been detailed and commented on in the text.     

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

Summary 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 46-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.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - DAPI 46-diamidino-2-phenyl indole - DNA deoxyribonucleic acid - MS Murashige and Skoog medium - BA benzyladenine - NAA 1-naphthaleneacetic acid     

Micropropagation of Madhuca longifolia (Koenig) MacBride var. latifolia Roxb.

Bud break and multiple shoots were induced in apical and axillary meristems derived from 10-d old seedlings of Madhuca longifolia var. latifolia on Murashige and Skoog (MS) medium supplemented with 1.0 mg/l N⁶-benzyladenine (BA) singly or in combinatiobn with 1-naphthalene acetic acid (NAA), indole-3-acetic acid (IAA) and indole-3-butyric acid (IBA). Excised shoots were rooted on half-strength MS with IBA (1.0 mg/l) after 18d of culture. Regenerated plantlets were acclimatized and successfully transferred to soil.Abbreviations BA N⁶ benzyladenine - KN kinetin - ADS adenine sulphate - IBA indole-3-butyric acid - IAA indole3-acetic acid - NAA 1-naphthaleneacetic acid - MS Murashige and Skoog (1962) medium     

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.     

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.     

姜花(Hedychium coronarium)花部维管束系统解剖学研究

姜花(Hedychium coronarium Koen.)花梗横切面整体轮廓呈椭圆形,可分为表皮、基本组织和维管束。维管束在基本组织中呈内、外两部分排列。内部维管束联结成网,形成明显外移的三束心皮背束和内方与心皮背束相间的三束隔膜束。至子房室区,心皮背束继续外移,其中主支进入花萼中脉;小分支内移,与内方一轮维管束联结,后来进入唇瓣中央及2枚侧生附属物。在花萼形成的同时,远轴面的两个隔膜中各形成一个上位腺体;同时两束远轴面隔膜束向外、两侧分别形成3束大分支,外方大分支继续外移成为2枚远轴面花瓣中脉,两侧大分支与原外方内移的子房壁维管束集合成一相连的环状维管束网,后进入唇瓣两侧;近轴面隔膜束形成3枚分支,外方分支成为近轴面花瓣中脉,两侧分支进入可育雄蕊。探讨了侧生附属物和唇瓣的来源,支持子房延长部形成的腺体为隔膜蜜腺的变异结构的观点。