Ethylene Production and Ethylene-induced Apogamous Bud Formation in Nine Gametophytic Strains of Pteridium aquilinum (L.) Kuhn

A comparative study of the abilities of nine gametophytic strainsof Pteridium aquilinum to produce ethylene and to undergo apogamywas conducted. Each gametophytic strain produced ethylene atits characteristic rate and all the strains, except one, formedapogamous buds in response to evolved ethylene which collectedwithin sealed culture vessels or to exogenous ethylene suppliedin a continuous-flow system. The number of apogamous buds producedby each strain was not directly related to the ability of thestrain to produce ethylene, but rather appeared to be dependenton the ability of the gametophytes to respond to ethylene.     

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     

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     

Increased ethylene and decreased phenolic compounds stimulate somatic embryo regeneration in leaf thin section cultures ofDoritaenopsis hybrid

The effects of endogenous levels of ethylene and phenolic compounds on somatic embryogenesis, medium-browning, and peroxidase activity were evaluated in thin section cultures ofDoritaenopsis. Cultures were maintained for 8 weeks with four different treatments: i) thick leaf segment culture, ii) thin leaf section culture, iii) thin leaf section culture with ventilation, or iv) thin leaf section culture after expiants were first washed. Expiants cultured in closed vessels produced a larger number of somatic embryos than those reared in the ventilated vessels. This enhanced formation confirmed the greater involvement of accumulated ethylene under non-ventilated conditions, because wound-induced tissues from thin leaf sections normally release high level of ethylene. When expiants were washed in the liquid medium and inoculated on the same solid medium, somatic embryo production was 1.7 and 18.5 times higher than in the thin section cultures and thick segment cultures, respectively. Reducing the level of phenolics in expiants at the initial stage of culturing apparently stimulated this embryo regeneration.     

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.     

1-Methylcyclopropene and ethylene as regulators of in vitro organogenesis in kiwi explants

In this paper, we study the influence of ACC, AVG and 1-MCP on in vitro organogenesis of kiwi (Actinidia deliciosa) explants and on ethylene metabolism. Results indicated that increasing ethylene production and accumulation in the head space of the culture vessel by adding ACC to the culture medium inhibited organogenesis, except for rooting, which increased and stimulated ACC oxidase activity threefold, whereas AVG increased the length and number of shoots and leaves and inhibited about 80% ACC synthase activity compared with the reference explants. 1-MCP exerts a stimulatory effect analogous to AVG, increasing ACC synthase activity and organogenesis of kiwi explants. This effect is not reverted by the addition of ACC to the culture medium. Therefore, ethylene production and its accumulation in the headspace of the culture vessels seems to be responsible for the inhibition of shoot development as well as increasing rooting in in vitro cultured kiwi explants.     

Culture of blastomeres from in vitro-matured,fertilized, and cultured bovine embryos

A culture system was devised to study the differentiation of bovine blastomeres. Blastomeres (2–13 per well) from embryos produced by in vitro maturation, fertilization, and culture of oocytes obtained from slaughterhouse ovaries were cultured for 10 days in 24-well culture plates on feeder layers in blastomere culture medium (BCM: equal parts tissue culture medium 199 and low-glucose Dulbecco's modified Eagle's medium with 10% fetal bovine serum). Ovine embryonic fibroblasts and STO cells were superior to bovine and mouse embryonic fibroblasts as mitotically inactivated feeder cells. Over five studies in which four blastomeres from an embryo were added to each culture well, an average of one colony per well formed from the blastomeres. The colonies continued to grow throughout the culture period, and most colonies resembled trophectoderm in their cellular characteristics, although some cultures contained a mixture of trophectoderm and endoderm. When the number of blastomeres cultured in each well was varied from 2–8, the number of colonies formed was proportional to the number of blastomeres added. Blastomeres from day 5 and day 6 embryos produced fewer colonies than did those from day 4 embryos, perhaps as a result of differentiation and tighter blastomere adhesion resulting in damage during their separation. The absence of serum did not alter the number of colonies formed. A number of growth factors, including LIF, OM, PDGFα, and FGF4, had no effect on the number of colonies, the size of colonies, or their alkaline phosphatase staining score beyond that provided by the feeder layer or serum when present. Blastomeres did not form colonies in the absence of feeder layers. Mol. Reprod. Dev. 48:238–245, 1997. © 1997 Wiley-Liss, Inc.     

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.     

Cytotaxonomic study of genusHymenasplenium (Aspleniaceae) in Xishuangbanna, Southwestern China

The gametic chromosome numbers of sevenHymenasplenium (Aspleniaceae) species from Xishuangbanna, Yunnan Prov., China, were investigated. All the examined individuals ofH. obscurum, H. cheilosorum andH. latipinnum were sexual diploids with n=39 chromosomes. Intraspecific cytological variation was found inH. excisum, which has a sexual diploid (n=39) and a tetraploid (n=78). Only a triploid apogamous cytotype (n=ca.117) was found inH. laterepens. Hymenasplenium apogamum showed the most complicated intraspecific variation and included a sexual diploid (n=39), a sexual tetraploid (n=78) and an apogamous triploid (n=ca.117). This work reports for the first time the sexual diploids ofH. cheilosorum andH. apogamum, which are only apogamous elsewhere in east Asia, Himalayas and Indochina. These results may indicate that this area is one of the diversity centers ofHymenasplenium. Most of the above species have chromosome numbers based on x=39. In contrast,H. costarisorum contains a sexual diploid (n=36) and a sexual tetraploid (n=72), indicating that its basic number is x=36.     

Physical separation of hemopoietic stem cells from cells forming colonies in culture

Mouse bone marrow cells in suspension were separated into a number of fractions on the basis of cell density by equilibrium density gradient centrifugation, or on the basis of cell size by velocity sedimentation. After each type of separation, the cells from the various fractions were assayed for their ability to form macroscopic spleen colonies in irradiated recipient mice, and for their ability to form colonies in a cell culture system. The results from either separation technique demonstrate that cells in some fractions formed more colonies in vivo than in the culture system, while cells in other fractions formed more colonies in culture than in the spleen. The results of control experiments indicate that this separation of the two types of colony-forming cells was not an artifact of the separation procedures. From these experiments it was concluded that the population of cells which form colonies in culture under the conditions used is not identical to the population of cells detected by the spleen colony assay.     

Ethylene and in vitro rooting of rose shoots

Effects of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC), ethylene biosynthesis inhibitor, (CoCl₂), and inhibitor of ethylene binding to receptors, 1-methylcyclopropene (1-MCP), on ethylene production and rooting in shoot culture of Rosa hybrida L. cv. Alba meidiland were studied. Additionally, effect of ethylene removal by KMnO₄ and HgClO₄ on rooting was tested. ACC increased ethylene production and delayed root formation, decreased the number of roots per shoot and inhibited root growth. In contrast, inhibition of ethylene production by CoCl₂ accelerated root emergence, and increased the number of roots per shoot. Likewise, removing ethylene from the ambient atmosphere improved root emergence and, increased root number of per shoot and markedly inhibited root growth. Blocking the ethylene receptors by 1-MCP increased ethylene level in the ambient atmosphere and increased both emergence and root formation. Both ethylene biosynthesis and action are involved in the control of rooting. Ethylene concentration in glass jars was too high for root emergence and formation, but was appropriate for root growth. CoCl₂ or 1-MPC can be recommended for regulation of rooting in rose shoot culture, since both emergence and number of roots were improved but root growth was not inhibited.     

Relationship of queen number and worker size in polygyne colonies of the fire antSolenopsis invicta

Summary We examined the relationship between queen number and worker size in colonies of the fire antSolenopsis invicta. Worker size in monogyne colonies was significantly greater than in polygyne colonies; furthermore, polygyne colonies snowed a strong negative linear relationship between queen number and worker size. Higher queen pheromone level and/or decreased food availability accompanying an increase in queen number likely play important roles in producing the observed patterns.     

Evaluation of a closed system,diffusive and humidity-induced convective throughflow ventilation on the growth and physiology of cauliflower in vitro

The effects of ethylene inhibitors (silver nitrate – AgNO₃ and silver thiosulphate – Ag₂S₂O₃ as inhibitors of ethylene activity, cobalt chloride – CoCl₂ as inhibitor of ethylene biosynthesis) and ethylene stimulator (aminocyclopropane-1-carboxylic acid – ACC) were studied on the growth of cauliflower (Brassica oleracea L.) seedlings cultured in closed vessels (60 cm³). The addition of ethylene inhibitors have significant stimulatory effects on the growth and development of seedlings and the effects were greatest with 10 μM AgNO₃, the fresh weight of leaves was 2.6×, and the leaf area 2.8× those of the control (no additives). The effects of various methods of ventilation (humidity-induced convective through-flow ventilation, diffusive ventilation and sealed condition) on the growth and physiology of in vitrocauliflower seedlings were also investigated. The seedlings were cultured either in the presence or absence of AgNO₃ (inhibitors of ethylene activity) and ACC (a precursor). Ethylene and CO₂ levels in the head-space of the culture vessels were monitored. The humidity-induced through-flow ventilation system has shown to be effective for improving growth, leaf chlorophyll content and the rate of net photosynthesis and preventing symptoms of hyperhydricity, such as leaf epinasty, and franginess, reduction of leaf area etc. In contrast, the results also indicated that the sealing of culture vessels could have serious inhibitory effects on growth and development, induce hyperhydricity and reduce leaf chlorophyll content. In the light period, CO₂ depletion occurred in the head-space of the sealed vessels (ca. 40 μl l^-1), the CO₂ concentration increased with increasing efficiency of the ventilation. No ethylene accumulation was noticed in the head-space of the culture vessels when humidity-induced throughflow ventilation was applied; however, high ethylene accumulation occurred in sealed vessels. This revised version was published online in June 2006 with corrections to the Cover Date.     

Ethylene influences in vitro regeneration frequency in the FR13A rice harbouring the SUB1A gene

Many studies have examined the effects of ethylene on in vitro plant growth and development, often with controversial results. Ethylene accumulates in culture vessels due to both the release from the tissues and the physical entrapment due to the need for closed containers. This hormone has several effects on plant regeneration, depending on the plant species and even the cultivar. A prerequisite for ethylene use for in vitro culture is thus to formulate a specific protocol for the genotype of interest. In rice, ethylene is a key regulator of adaptation strategies to low oxygen environments. In particular, the SUBMERGENCE1A (SUB1A) gene, when present, drives the acclimation response which when activated by ethylene produced by submerged plants leads to adaptation through reduced plant growth and ethanolic fermentation enhancement. This gene is restricted to a limited number of rice for which a very specific response to ethylene is expected, whatever the source. This paper reports the regeneration differences between a SUB1A rice landrace (indica-aus, FR13A) and a non-SUB1A variety (japonica, Nipponbare). Our results suggest that regeneration protocols with exogenous ethylene precursors supply are required for the FR13A rice harbouring the SUB1A gene to overcome the problem of low regeneration efficiency.     

The effect of the gaseous state on bud induction and shoot multiplication in vitro in eastern white cedar

The effect of vessel type and the gaseous phase on the morphogenic response of Thuja occidentalis L. explants in vitro was studied. Explants were cultured in container types that varied in their degree of gas exchange. Traps for ethylene and CO₂ were employed. During shoot bud induction from embryonic explants, the number and elongation of shoot buds improved significantly when gastight, serum-capped flasks were used compared to the foam bung-capped flasks or the regularly used Petri dishes. Elimination of the two gases from the headspace of the flasks either singly or together reduced shoot bud induction and especially elongation of shoots. A similar response was seen during axillary bud development from cultured shoots. Ethylene and CO₂ accumulation promoted development and elongation of axillary shoots. An increase in the zeatin concentration in the medium produced a greater number of axillary shoots and higher levels of ethylene in the culture vessels. Removal of CO₂ caused gradual death of the shoots, while removal of ethylene alone reduced axillary shoot lengths significantly. Inclusion of aminoethoxyvinylglycine in the medium combined with ethylene traps produced an effect similar to the use of ethylene traps alone.     

Ecological and phylogenetic approaches for diversification of apogamous ferns in Japan

It has long been argued that related asexual and sexual taxa have different distribution patterns. In general, apomictic angiosperms are believed to occur preferentially at higher latitudes and elevations compared to their sexual relatives. It is thus expected that the frequency of apomixis increases with latitude or from warmer to colder climatic regions. However, despite the significant role played by apogamy in fern evolution and diversification, the distribution pattern of apogamous ferns and lycophytes has received little attention. To clarify the ecological diversity pattern and evolutional diversification of apogamous ferns, we analysed a variety of apogamous fern species with reference to latitude, elevation and climatic factors, and reconstructed the ancestral state and estimated the divergence time of Japanese apogamous ferns. Our results on the distribution of apogamous ferns along these two gradients suggest a decline in the proportion of apogamous ferns towards high latitudes and elevations. Temperature was correlated with the proportion of apogamous ferns along both gradients, and the seasonality of precipitation was correlated with the proportion of apogamous ferns along latitude. Reconstruction of ancestral state and estimates of divergence time showed that the crown groups of apogamous ferns diversified less than 15 Ma. The results of our ecological and phylogenetic approaches reinforce the hypothesis based on previously reported phylogenetic results in which the apogamous ferns appears to be correlated with strongly seasonal climates such as the Asia monsoon.     

Involvement of ethylene in the rooting of seedling shoot cultures of <Emphasis Type="Italic">Bixa orellana</Emphasis> L.

Using seedlings derived from the shoot apex of annatto (Bixa orellana L. cv. Bico-de-Pato) we observed the rooting frequency of B. orellana, the number and length of roots and the rate of ethylene production during 30 d in culture. The rhizogenesis response was affected by auxins (NAA or IBA) and by both the ethylene biosynthesis precursor 1-aminocyclopropane-1-carboxylic acid (ACC) and the inhibitor 2-aminoethoxyvinylglycine (AVG). Auxin supplementation to the medium resulted in root induction, ethylene production, and an undesirable callusing in the epidermal and cortical tissues. Irrespective of the presence of auxins, supplementing the medium with ACC promoted ethylene biosynthesis and callusing, which resulted in increased cell proliferation mainly in the cortical and vascular tissues, while the epidermis was mostly unaltered. In both ACC and auxin-supplemented medium, increased ethylene production and callusing occurred, suggesting a synergistic effect between these two responses. ACC was capable of inducing adventitious root formation, but the roots produced had a wrinkled appearance when compared to normal roots. Conversely, AVG reduced ethylene production and callusing, while the epidermis, cortex, and inner tissues remained unaltered, regardless of the presence of auxins. AVG was beneficial in these aspects, although its application led to a reduction in the number of roots and in the average root length. In conclusion, it was not possible to establish a direct relation between ethylene and rooting, but we hypothesize that, under the experimental conditions described, ethylene may enhance tissue sensitivity to auxin. However, ethylene did not seem essential to the rhizogenesis process in annatto.     

Inhibition of ethylene biosynthesis enhances embryogenesis of cultured microspores of Brassica napus

Procedures that induce microspore embryogenesis have been described for a range of Brassica species, but embryo yield remains low for a number of genotypes. We have carried out experiments with the microspores from a weakly responsive line of B. napus to determine the culture conditions that optimize their in vitro embryogenesis by treating them with effectors of ethylene synthesis and action. The results revealed that isolated microspores subjected to an initial heat stress in a medium supplemented with inhibitors of ethylene synthesis such as AVG and CoCl₂ exhibited significantly increased embryo yields. This suggested that regulatory effects are exerted by the ethylene produced by the isolated microspores on the early processes of gametogenesis. As a consequence, treatment of microspores with SAM, an ethylene synthesis precursor, or with the ethylene-releasing agent ethephon, led to decreases in embryo yield. A special response to ethylene during the early stages of microspore development was finally shown to occur through experiments where isolated microspores were treated for increasing periods of time with CoCl₂. Collectively, our data demonstrated that the induction of embryogenesis induced by heat stress can be enhanced by inhibitors of ethylene biosynthesis.     

Ethylene-associated phase change from juvenile to mature phenotype of daylily (Hemerocallis) in vitro

Hemerocallis plantlets maintained in vitro for extended periods of time in tightly closed culture vessels frequently show a phenotype, albeit on a miniaturized scale, typical of more mature, field-grown plants. The positive relationship of elevated ethylene in the headspace of such vessels to the phase shift from juvenile to mature form is established. Rigorous restriction in air exchange with the external environment by means of silicone grease seals hastens the phase change and improves uniformity of response. Although some plantlets may take longer to accumulate enough ethylene in sealed jars to undergo change, added ethylene and ethylene-releasing agents promote it. Ethylene adsorbants (e.g. mercuric perchlorate) block the shift of juvenile to mature form. Critical ambient ethylene level for the shift is ca 1 μl l⁻¹. Levels up to 1000 μl l⁻¹ do not hasten the response but are not toxic. The phase change is fully reversible when air exchange permits ethylene to drop below 1 μl l⁻¹. At least 1 μl l⁻¹ ethylene is required to sustain the mature phenotype. The ethylene synthesis inhibitor aminoethoxyvinylglycine (AVG) prevents the phase change, while the ethylene biosynthesis intermediate 1-aminocyclopropanecarboxylic acid (ACC) improves it. KOH, as a CO₂ absorbant, does not prevent the phase change. Histology sections demonstrate subtle changes in the form of shoot tips of plantlets undergoing phase change.