Initiation Of Embryogenesis Research Articles

Changes in Protein Synthesis and Phosphorylation during Microspore Embryogenesis in Brassica napus

Changes in protein synthesis and phosphorylation during microspore embryogenesis in Brassica napus were investigated by two-dimensional (2-D) gel electrophoresis. The expression pattern of 6 proteins previously identified as putative markers for embryo initiation in microspore cultures (Cordewener et al., 1994), was studied during later stages of embryo development. All of them were transiently expressed for the induction period (8 h to 2 days) and disappeared on the 5 th day but two of them were expressed again at the later stage of embryogenesis (12 th day). Major changes in the patterns of protein synthesis were observed 5 days after initiation of microspore embryogenesis. In this period of time embryogenic microspores have formed proembryos within their exine. Later stages ofembryo development were not accompanied by major changes in 2-D protein patterns. Comparison of 2-D patterns of phosphorylated proteins revealed minor differences between embryogenic and non-embryogenic cultures, except for the level of phosphorylation of hsp70. A clear difference of extracellular secreted proteins was observed between embryogenic and non-embryogenic microspore cultures. This work provides a biochemical insight for the understanding of microspore embryogenesis at the protein level.

Factors Influencing the Induction and Viability of Somatic Embryos of Quercus Robur L.

To induce somatic embryogenesis in Quercus robur L. immature zygotic embryos at different developmental stages were collected in weekly intervals from June until September in three consecutive years from four open pollinated trees at two Vienna sites. Acorns were surface sterilised and cultured firstly on P24 medium with 5μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.5 μM 6-benzylaminopurine (BAP) or on hormone-free P24 medium and secondly on P24 medium with 0.9 μM BAP. The formation of white-yellow globular structures of somatic embryos started during the fourth week after the induction treatment. High induction frequencies of 30 - 80 % were achieved on 2,4-D/BAP medium, whereas rates on hormone-free medium were below 20 %. The initiation of somatic embryogenesis was favoured in the heartshaped and early cotyledonary stage of the zygotic embryo in all three years and lasted until the acorns reached maximum size in August.

Somatic embryogenesis of Scots pine: cold treatment and characteristics of explants affecting induction

This is the first report on Scots pine (Pinus sylvestris L.) somatic embryo plants regenerated and growing in a greenhouse. The present work focused on improving somatic embryogenesis of the species by studying the factors affecting culture induction. Developmental stage of explants that were immature female gametophytes, including the zygotic embryos with suspensor tissues, was investigated in detail. The genetic background of the material, cold treatments (14 d, 1 or 2 months at +5°C) of cones including explants, as well as the plant growth regulator composition of the initiation medium, were also examined. When initiation of somatic embryogenesis was successful, the zygotic embryos in the explants were either proembryos or early embryos. Cold treatment of the cones had no significant effect on induction, nor were there any differences among the treatments with different duration, thus improving the practical applicability of the culture technique. The explants in cold-stored cones probably retained their initiation capacity due to the conversion of starch to sugars. This was observed as decreased number and size of starch grains in the megagametophytes compared with the controls. The seed family and the medium significantly affected induction success, the medium with auxin (9.1 or 13.6 μM 2,4-dichlorophenoxyacetic acid) and cytokinin (2.2 μM 6-benzylaminopurine) being better than the medium with cytokinin (5 μM 6-benzylaminopurine) alone. The significance of the genetic background of the explants and the initiation medium indicate that it might be possible to improve the initiation rates by using explants from controlled crossings between competent genotypes, and by developing more specific media for important seed families.

Somatic embryogenesis of Scots pine: cold treatment and characteristics of explants affecting induction

This is the first report on Scots pine (Pinus sylvestris L.) somatic embryo plants regenerated and growing in a greenhouse. The present work focused on improving somatic embryogenesis of the species by studying the factors affecting culture induction. Developmental stage of explants that were immature female gametophytes, including the zygotic embryos with suspensor tissues, was investigated in detail. The genetic background of the material, cold treatments (14 d, 1 or 2 months at +5 °C) of cones including explants, as well as the plant growth regulator composition of the initiation medium, were also examined. When initiation of somatic embryogenesis was successful, the zygotic embryos in the explants were either proembryos or early embryos. Cold treatment of the cones had no significant effect on induction, nor were there any differences among the treatments with different duration, thus improving the practical applicability of the culture technique. The explants in cold-stored cones probably retained their initiation capacity due to the conversion of starch to sugars. This was observed as decreased number and size of starch grains in the megagametophytes compared with the controls. The seed family and the medium significantly affected induction success, the medium with auxin (9.1 or 13.6 μM 2,4-dichlorophenoxyacetic acid) and cytokinin (2.2 μM 6-benzylaminopurine) being better than the medium with cytokinin (5 μM 6-benzylaminopurine) alone. The significance of the genetic background of the explants and the initiation medium indicate that it might be possible to improve the initiation rates by using explants from controlled crossings between competent genotypes, and by developing more specific media for important seed families.

The influence of osmotic pretreatment and inoculum age on the initiation and regenerability of switchgrass suspension cultures

Experiments were conducted to study the influence of osmotic pretreatment and inoculum (callus) age on the initiation and induction of somatic embryogenesis from suspension cultures of ‘Alamo’ switchgrass (Panicum virgatum L.). Embryogenic 10-, 20-, or 30-d-old calluses (hereafter referred to as inocula), produced from in vitro-cultured inflorescences, were used as explants to initiate the suspensions. Inocula were cultured for 30 h on MS solid medium containing 0.1, 0.2 or 0.3 M each of sorbitol and mannitol as an osmotic pretreatment. They were then transferred to liquid MS medium with 5 µM 6-benzylaminopurine and 9 µM 2,4-dichlorophenoxyacetic acid and cultured for 28 d in 2-ml Multiwell™ plantes. Individual multiwell contents were transferred to 125-ml Erlenmeyer flasks containing 20 ml of the above liquid medium and cultured for an additional 2 wk. Cultures initiated from the 10-d-old inoculum produced a higher embryogenic response than those initiated from 20- or 30-d-old inocula. Cultures initiated with 30-d-old inocula produced nonembryogenic clusters and the number of embryogenic cells was low. More embryos and a higher regeneration frequency were produced by 0.3 M than by 0.1 or 0.2 M of each of the osmostica.

Histological and 2-D protein patterns comparisons between a wild type and a somatic embryogenic mutant of Asparagus officinalis L.

We study the induction of somatic embryogenesis using an original Asparagus model: a mutant type which an important somatic embryogenesis and its non-embryogenic wild type used as control. We describe the histological events and the modifications of protein patterns occurring during the initiation of somatic embryogenesis in cultures of apices. Using the mutant type apices, pro-embryogenic masses and embryogenic cells are observed at the periphery of the callus after transfer on hormone-free medium while in the same culture conditions, the wild type calli never show embryogenic structure. Analysis of the tissular proteins during the culture leads us to classify 116 proteins into 20 groups potentially related to somatic embryogenesis. Two of these groups are particularly interesting as they correspond to polypeptides that are only related to one of the two types: six polypeptides (group 1) are specific to the mutant type apices and could be related to the competence of the mutant tissues with respect to somatic embryogenesis. Eleven proteins (group 7) are detected specifically in the wild type tissues and their presence could be related to an inhibition of the somatic embryogenesis ability.

Plant regeneration through callus initiation from anthers and ovules of Scabiosa columbaria

Anthers and ovules of Scabiosa columbaria L. were cultured in vitro to determine whether gametophytic cells would proliferate and/or a protocol for plant regeneration could be developed. Several factors were tested, including explant type, donor plant, cold pre-treatment, and medium composition. Callus induction frequency varied among treatments, indicated by significant effects of explant type, medium composition, and their interactions. Histological analysis revealed numerous sites of callus induction, however, gametophytic cells did not proliferate. Stepwise removal of growth regulators and simultaneous lowering of sucrose from the nutrient medium, resulted in initiation of embryogenesis or shoot organogenesis, and allowed plant regeneration. Under the conditions tested, regeneration capacity was donor related, because only material of one donor responded. Regenerants were diploid (except one mixoploid individual), but showed various types of flower heads. They were probably of sporophytic origin.

Carbon metabolism in in vitro cultures of date palm: the role of carboxylases (PEPC and RubisCO)

While describing major trends of carbon metabolism during the initiation and expression of somatic embryogenesis in date palm (Phoenix dactylifera L., cv. Deglet Nour), we have investigated the role of two carboxylases, namely PEPC (Phosphoenolpyruvate carboxylase, EC 4.1.1.31) and RubisCO (Ribulose 1,5-bisphosphate carboxylase/oxygenase, EC 4.1.1.39), in embryogenic and non-embryogenic cultures. The detection of PEPC activity on polyacrylamide native gels after electrophoresis revealed the presence of 3 active isoforms in crude extracts from the embryogenic (E) callus strain, whereas only a single band was present in the non-embryogenic (NE) one. The level of PEPC specific capacity was of the same order (3.9 ± 1.2 μmol CO2 h−1 mg−1 TSP) in both types of cultures. Further changes in carboxylase (PEPC and RubisCO) activities during the growth and development of somatic embryo–derived plantlets were also analysed. The PEPC/RubisCO ratio was found to progressively decrease (from 17.7 to 0.2) throughout the in vitro development of plantlets, due to a substantial depletion of PEPC activity, which decreased from 5.3 to 1.2 μmol CO2 h−1 mg−1 TSP. Concomitantly, RubisCO assumed greater importance (from 0.3 to 5.3 μmol CO2 h−1 mg−1 TSP ) and became the main route for inorganic carbon fixation. Western blot analysis using polyclonal antibodies raised against PEPC and RubisCO purified from tobacco leaves confirmed this trend in terms of relative enzyme abundance.

Somatic embryogenesis and plant regeneration from suspension cultures of Acanthopanax koreanum Nakai.

High-frequency somatic embryogenesis was achieved from an embryogenic cell suspension culture of Acanthopanax koreanum Nakai. Stem segments were cultured on Murashige and Skoog (MS) medium containing auxins and cytokinins. Opaque and friable embryogenic callus formed on MS medium with 4.5 µM 2,4-dichlorophenoxyacetic acid (2,4-D) and 2.0 µM kinetin or zeatin, but was highest on medium containing 4.5 µM 2,4-D alone. Embryogenic calli were transferred to MS liquid medium containing 4.5 µM 2,4-D and maintained by subculture at 2-week intervals. Initiation of somatic embryogenesis and development up to the globular stage from embryogenic cell clumps occurred in medium containing 0.45 µM 2,4-D, whereas maturation and germination of somatic embryos occurred in MS medium lacking 2,4-D. Cytokinin treatment suppressed the normal growth of embryos, but stimulated secondary somatic embryogenesis from the surfaces of primary embryos. Plants from somatic embryos were acclimatized in a greenhouse.

1 Paternal Effects in Drosophila: Implications for Mechanisms of Early Development

The study of paternal effects on development provides a means to identify sperm-supplied products required for fertilization and the initiation of embryogenesis. This review describes paternal effects on animal development and discusses their implications for the role of the sperm in egg activation, centrosome activity, and biparental inheritance in different animal species. Paternal effects observed in Caenorhabditis elegans and in mammals are briefly reviewed. Emphasis is placed on paternal effects in Drosophila melanogaster. Genetic and cytologic evidence for paternal imprinting on chromosome behavior and gene expression in Drosophila are summarized. These effects are compared to chromosome imprinting that leads to paternal chromosome loss in sciarid and coccid insects and mammalian gametic imprinting that results in differential expression of paternal and maternal loci. The phenotypes caused by several early-acting maternal effect mutations identify specific maternal factors that affect the behavior of paternal components during fertilization and the early embryonic mitotic divisions. In addition, maternal effect defects suggest that two types of regulatory mechanisms coordinate parental components and synchronize their progression through mitosis. Some activities are coordinated by independent responses of parental components to shared regulatory factors, while others require communication between paternal and maternal components. Analyses of the paternal effects mutations sneaky, K81, paternal loss, and Horka have identified paternal products that play a role in mediating the initial response of the sperm to the egg cytoplasm, participation of the male pronucleus in the first mitosis, and stable inheritance of the paternal chromosomes in the early embryo.

REVIEW ARTICLE

This review will summarize molecular and genetic analyses aimed at identifying the mechanisms underlying the sequence of events during plant zygotic embryogenesis. These events are being studied in parallel with the histotogical and morphological analyses of somatic embryogenesis. The strength and limitations of somatic embryogenesis as a model system will be discussed briefly. The formation of the zygotic embryo has been described in some detail, but the molecular mechanisms controlling the differentiation of the various cell types are not understood. In recent years plant molecular and genetic studies have led to the identification and characterization of genes controlling the establishment of polarity, tissue differentiation and elaboration of patterns during embryo development. An investigation of the developmental basis of a number of mutant phenotypes has enabled the identification of gene activities promoting (1) asymmetric cell division and polarization leading to heterogeneous partitioning of the cytoplasmic determinants necessary for the initiation of embryogenesis (e.g.GNOM), (2) the determination of the apical-basal organization which is established independently of the differentiation of the tissues of the radial pattern elements (e.g.KNOLLE, FACKEL, ZWILLE), (3) the differentiation of meristems (e.g.SHOOT-MERISTEMLESS), and (4) the formation of a mature embryo characterized by the accumulation of LEA and storage proteins. The accumulation of these two types of proteins is controlled by ABA-dependent regulatory mechanisms as shown using both ABA-deficient and ABA-insensitive mutants (e.g.ABA, ABI3). Both types of embryogenesis have been studied by different techniques and common features have been identified between them. In spite of the relative difficulty of identifying the original cells involved in the developmental processes of somatic embryogenesis, common regulatory mechanisms are probably involved in the first stages up to the globular form. Signal molecules, such as growth regulators, have been shown to play a role during development of both types of embryos. The most promising method for identifying regulatory mechanisms responsible for the key events of embryogenesis willcome from molecular and genetic analyses. The mutations already identified will shed light on the nature of the genes that affect developmental processes as well as elucidating the role of the various regulatory genes that control plant embryogenesis.

A mutation that allows endosperm development without fertilization.

The mechanisms that initiate reproductive development after fertilization are not understood. Reproduction in higher plants is unique because it is initiated by two fertilization events in the haploid female gametophyte. One sperm nucleus fertilizes the egg to form the embryo. A second sperm nucleus fertilizes the central cell to form the endosperm, a unique tissue that supports the growth of the embryo. Fertilization also activates maternal tissue differentiation, the ovule integuments form the seed coat, and the ovary forms the fruit. To investigate mechanisms that initiate reproductive development, a female-gametophytic mutation termed fie (fertilization-independent endosperm) has been isolated in Arabidopsis. The fie mutation specifically affects the central cell, allowing for replication of the central cell nucleus and endosperm development without fertilization. The fie mutation does not appear to affect the egg cell, suggesting that the processes that control the initiation of embryogenesis and endosperm development are different. FIE/fie seed coat and fruit undergo fertilization-independent differentiation, which shows that the fie female gametophyte is the source of signals that activates sporophytic fruit and seed coat development. The mutant fie allele is not transmitted by the female gametophyte. Inheritance of the mutant fie allele by the female gametophyte results in embryo abortion, even when the pollen bears the wild-type FIE allele. Thus, FIE carries out a novel, essential function for female reproductive development.

Regeneration of plantlets through isolated scutellum culture of durum wheat

Four durum wheat cultivars, viz. ‘Renville’, ‘Medora’, ‘Monroe’ and ‘Vic’, were selected for studies on rapid regeneration of plantlets through in vitro culture of isolated immature scutella. Effects of two concentrations of sucrose (either 2% or 3%) and three gelling agents, viz. agar (0.8%), agarose (0.8%), and phytagel (0.4%), were studied for callus induction. All cultivars responded to the MS induction medium supplemented with 2 mg/l, 2,4-D, 3% sucrose and 0.8% agar, and initiation of somatic embryogenesis was evident within 2–3 days of culture. The embryogenic calli developed into clusters of small, dense globular somatic embryos within 3–4 weeks of culture and subsequently developed into distinct somatic embryos and plantlets after transfer to the regeneration medium. An average of up to 16 plantlets per cultured scutellum were recovered from two regeneration media studied, i.e. half-strength hormone-free MS medium (for cultivar ‘Monroe’) and MS medium with BA and IAA (for cultivars ‘Renville’, ‘Medora’ and ‘Vic’). The plantlets were then transferred to pots in the greenhouse, where they developed into mature, fertile plants.

Specific nutrient uptake during initiation of somatic embryogenesis in coconut calluses

Changes in the concentration of major nutrients and sugars in the media were measured for two embryogenic strains of coconut calluses, L1 and L7, after 0, 14, 28, 42 and 56 days of culturing on multiplication medium and embryogenesis induction medium. A histological study was made on day 0, 28 and 56. The initation of somatic embryogenesis was achieved in these two strains through two different pathways: one (strain L1), of pluricellular origin, was obtained by decreasing the 2,4-D concentration in the medium, and the other (strain L7), of unicellular origin, by increasing the level of 2,4-D. In spite of these differences, both kinds of embryogenesis initiation were linked to specific requirements: higher uptake per g of dry matter of NH 4 +, Ca 2+, Mg 2+ and sucrose.

Free Amino Acid Composition of Coconut ( Cocos nucifera L.) Calli under Somatic Embryogenesis Induction Conditions

Summary The amino acid composition of coconut calli under somatic embryogenesis induction conditions was compared with that of calli under multiplication conditions. Six amino acids were detected: proline, valine, leucine, serine, alanine and threonine. Calli were classified into five groups according to their amino acid composition by a clustering method taking into account the morphogenetic stage of each callus. A discriminant analysis and an analysis of variance revealed that the groups had been classified correctly. The first group comprised all calli sampled at the beginning of culture (TO). Thereafter, two major phases were detected. The first phase concerned analysis times T8 and T15 and corresponded to a latency phase as regards growth and adaptation to culture conditions. Calli on the multiplication medium were significantly different from calli on the induction medium, characterized by a drop in proline, valine and serine contents. This drop was an initial reaction to the increase in 2,4-D and mineral element concentrations in the media to initiate unicellular type embryogenesis. The second phase, regrouping analysis times T28, T42 and T60, corresponded to callus growth. Heterogeneity was detected in calli on the induction medium and was confirmed by a histological study. Some calli adapted to the changes in the medium by remaining at the meristematic stage; their amino acid composition became similar to that of calli on the multiplication medium. The others tended well towards embryogenesis and were characterized by increases in the proline, valine and leucine contents and an unchanged alanine content; these increases were concomitant with the formation of storage proteins observed histologically at the time of embryogenesis initiation.

Initiation of embryogenesis from cultured barley microspores: a further investigation into the toxic effects of sucrose and glucose

The toxic effects of sucrose and glucose upon Hordeum vulgare L. ev Igri microspore cultures were investigated. It was concluded from this study that: -microspores could be cultured in the presence of low concentrations of glucose without any deleterious effects upon cell viability, but the microspores did not form embryos or calluses. -microspores died when incubated in the presence of 40 mM glucose during the first 2 of days of incubation, but, if glucose was added after this period, cells went on to produce embryos or calluses. -the toxic effects of sucrose upon cultured microspores were irreversible after 6 h from the start of incubation. Implications of these results on underlying causes of cell death in the presence of sucrose and glucose are discussed.

The effect of different carbohydrate sources upon the initiation of embryogenesis from barley microspores

Isolated microspores of Hordeum vulgare L. cv. Igri were incubated in the presence of different sugars. In the presence of maltose, the optimum concentration for the development of embryoids or calluses from the microspores was 175 mM. At this concentration 0.2% of the cells developed into embryoids or calluses. Microspores cultured without a carbohydrate source died after three days' incubation. In contrast microspores incubated in the presence of sucrose, glucose or fructose died within three days. Moreover, microspores also died when incubated in the presence of a combination of 175 mM maltose with varying concentrations of either sucrose, glucose or fructose. It is concluded that incubation of microspores in the presence of sucrose, glucose or fructose results in the death of the cells via some unknown mechanism. In contrast to this, maltose can sustain development of embryoids and calluses from cultured microspores.

Analysis of the genotypic effect on different developmental pathways in wheat gametophyte cultures

Seven wheat cultivars and one wild subspecies Triticum aestivum ssp. spelta were compared for their in vitro fertility and androgenetic capacity by studying their anther culture response and in vitro seed production. Both haploid embryogenesis and in vitro seed set showed very wide genotype dependent variability in accordance with previous observations. At the same time, an analysis of the data showed a significant negative correlation between the in vitro androgenetic ability and the in vitro fertilization potential, which was especially obvious in the case of highly embryogenic genotypes. The reason for this inverse correlation might be the different genetic regulation of the two quantitative traits. Presumably, alleles which increase the stability of the gametogenetic developmental programme hinder the initiation of haploid embryogenesis, while alleles with the opposite effect promote the sporophytic type of growth.

Biotechniques for improving acid aluminum tolerance in alfalfa

Alfalfa (Medicago sativa L.), cultivars ARC, Regen Y and Saranac were selected in vitro in a recently developed acid/aluminum toxic media. The new media produced higher initiation rates and higher fresh callus weights than those obtainable with the media described by Meredith and Connor for the selection of aluminum resistant variants in Nicotiana plumbaginofolia. Both rescue and direct initiation yielded adequate amounts of healthy callus for the initiation of embryogenesis. The toxic effect of the acid/aluminum media is expressed in both the percent of explants initiating callus and in the fresh-weights obtained during initiation and two subsequent subcultures.

Initiation of somatic embryogenesis in white spruce (Picea glauca): genetic control, culture treatment effects, and implications for tree breeding

The degree of genetic control and the effects of cultural treatments on somatic embryogenesis (SE) in white spruce were investigated with material derived from six-parent diallel crosses, including reciprocals. Thirty zygotic embryos from both immature and mature cones of each family were cultured in media with either 2,4-D or Picloram immediately after the collection of cones and after 2 months of cold storage. There were significant differences in SE initiation between immature and mature explants, and fresh and cold-stored seeds, but there was no significant differences with culture media effect. Significant variances due to families and to family x treatment interactions were found. The mean percentage of explants that initiated SE in each family ranged from 3.3% to 54.6%, with an overall average of 30.5%. The partitioning of family variance revealed that 21.7% was due to general combining ability effects, 3.5% was due to maternal effects, and 5.5% was due to reciprocal effects, but that the specific combining ability (SCA) was negligible. Variance due to interactions of family x treatments collectively accounted for 32.6%, while the remaining 37.8% of variation was accounted for by random error. However, when comparing the responses obtained with the treatment combinations, the SE response for freshly excised immature embryo explants showed comparatively large SCA variance, whereas the SCA variance was very small in the other treatment combinations.