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     

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.     

Morphogenetic plasticity of callus reinitiated from cell suspension cultures of the fern Platycerium coronarium

Cell suspension cultures were initiated from gametophyte-derived callus of the fern Platycerium coronarium. Two distinct types of callus masses, distinguished by their colouration, were obtained when the cells from suspension culture were plated on semisolid Murashige and Skoog (MS) medium containing 10 M kinetin. The two types of callus masses had distinct morphogenetic capacities despite their common origin. Morphogenesis into either gametophytes or sporophytes occurred when these callus masses were cultured on phytohormone-free MS medium depending on the type of callus used. The dark-green gametophytic callus showed a faster rate of growth and morphogenesis as compared to the pale-green sporophytic callus. Total chlorophyll content and autofluorescence and size of chloroplasts of the sporophytic callus and cell suspension cultures were lower than that of the gametophytic callus. Observations from confocal laser scanning microscopy were in agreement with the physiological parameters measured. The availability of cell cultures of the same ploidy level, but with two distinct pathways of development will be useful for comparative studies of developmental plasticity.     

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.     

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     

Integration of semaphorin-2A/MAB-20, ephrin-4, and UNC-129 TGF-beta signaling pathways regulates sorting of distinct sensory rays in C. elegans

Semaphorins and ephrins are axon guidance cues. In C. elegans, semaphorin-2a/mab-20 and ephrin-4/efn-4/mab-26 also regulate cell sorting to form distinct rays in the male tail. Several erf (enhancer of ray fusion) mutations were identified in a mab-20 enhancer screen. Mutants of plexin-2 (plx-2) and unc-129, which encodes an axon guiding TGF-beta, were also found to be erfs. Genetic analyses show that plx-2 and mab-20 function in the same pathway, as expected if PLX-2 is a receptor for MAB-20. Surprisingly, MAB-20 also signals in a parallel pathway that requires efn-4. This signal utilizes a non-plexin receptor. The expression of plx-2, efn-4, and unc-129 in subsets of 3-cell sensory ray clusters likely mediates the ray-specific cell sorting functions of the ubiquitously expressed mab-20. We present a model for the integrated control of TGF-beta, semaphorin, and ephrin signaling in the sorting of cell clusters into distinct rays in the developing male tail.     

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