Endogenous levels of reducing sugars, free amino acids and phenols during various stages of in vitro culture of cotton (Gossypium Spp.)

Somatic embryogenesis is widely preferred as the regeneration route for in vitro studies in cotton. However, the regeneration efficiency through this approach is low; a problem that is believed to be as a result of the biochemical properties of the plant. The objective of this study was to investigate possible relationships between three biochemical factors (reducing sugars, phenols, and free amino acids) and somatic embryogenesis. In vitro cultures of the different embryogenic and non-embryogenic cultivals were established. The levels of reducing sugars, phenols and free amino acids were determined at different developmental stages of the cultures. Higher levels of reducing sugars and lower level of phenol were observed in embryogenic cultivars compared to their non-embryogenic counterparts. There was a general increase in the levels of free amino acids, which decreased with time in the highly embryogenic cultivars, whereas the levels remained high in the poorly embryogenic and non-embryogenic cultivars. The higher content of phenols and free amino acids may be implicated in the poor somatic embryogenic response. The data show that there are factors that may serve as markers of somatic embryogenesis in cotton, which need to be empirically determined for any particular cultivar chosen for genetic improvement through embryogenesis

Analysis of protoplasts and somatic embryogenesis in Medicago truncatula

This thesis examined protoplast proliferation and somatic embryogenesis, by comparing a highly with a poorly embryogenic Medicago truncatula line through microscopic, proteomic and in situ hybridization analysis. Proteome analysis of M. truncatula was used to identify proteins involved in protoplast proliferation and the initiation of somatic embryogenesis. Furthermore, an in situ hybridization study was done to compare the expression of genes known to be involved in zygotic embryogenesis with the expression during somatic embryogenesis. A large number of proteins were up-, and down-regulated during the first 5 days of protoplast culture indicating that cellular reorganization took place. An up-regUlation of PR 1 O-like proteins and flavonoid synthesis proteins and a down-regulation of energy metabolism proteins were observed, indicating an initiation of a stress response. The observed stress response in protoplasts was down-regulated before the first cell divisions at 5-7 d. A stress-inducing bioassay on protoplasts showed that the ability of protoplasts to overcome stress and to proliferate under stress conditions depended on the level of stress and density of the protoplast culture, whereby more stress or a lower culture density resulted in higher levels of cell death. Proteomic analysis of the initiation of somatic embryogenesis showed that similar metabolic pathways were involved in the initiation of somatic embryogenesis and protoplast proliferation. By using a highly embryogenic (2HA) line, and a poorly embryogenic (A 17) line of M. truncatu!a, it was shown that particular proteins were specifically accumulated during the initiation of somatic embryogenesis. A high accumulation of a peroxidase was observed only in At7 tissue at the time of initiation of somatic embryogenesis and might be the reason why the initiation of somatic embryogenesis is inhibited in A 17 tissue. The specific accumulation of flavonoid synthesis proteins might also indicate that flavonoids are involved during the initiation of somatic embryogenesis. In situ hybridization with probes to genes known to be involved in zygotic embryogenesis, showed that M. truncatula somatic and Arabidopsis thaliana zygotic embryogenesis both followed similar developmental pathways. However, a few genes showed distinct patterns of gene expression in M. truncatula somatic embryos.ARC Centre of Excellence for Integrative Legume Research for funding and scholarshi

Somatic embryogenesis in higher plants

Somatic embryogenesis is an illustration of plant totipotency. There are many factors involved in causing development switching during somatic embryogenesis. These include combination of plant growth regulators, media, pretreatments and culture environments, which relate to various molecular events encompassing gene expression and signal transduction pathways.The present review collates information on various aspects of somatic embryogenesis focusing on genes involved, proteins and metabolites that have been identified during the last few years. Future work on integrating various data on somatic embryogenesis using the computational or systems biology approach is suggested

Tissue culture of oil palm : finding the balance between mass propagation and somaclonal variation

The oil palm (Elaeis guineensis Jacq.) is typically propagated in vitro by indirect somatic embryogenesis, a process in which somatic cells of an explant of choice are, via an intermediate phase of callus growth, induced to differentiate into somatic embryos. The architecture of the oil palm, lacking axillary shoots, does not allow for vegetative propagation. Therefore, somatic embryogenesis is the only alternative to seed propagation, which is hampered by long germination times and low germination rates, for the production of planting material. The current oil palm somatic embryogenesis procedure is associated with several difficulties, which are described in this review. The limited availability of explants, combined with low somatic embryo initiation and regeneration rates, necessitate the proliferation of embryogenic structures, increasing the risk for somaclonal variants such as the mantled phenotype. Several ways to improve the efficiency of the tissue culture method and to reduce the risk of somaclonal variation are described. These include the use of alternative explants and propagation techniques, the introduction of specific embryo maturation treatments and the detection of the mantled abnormality in an early stage. These methods have not yet been fully explored and provide interesting research field for the future. The development of an efficient oil palm micropropagation protocol is needed to keep up with the increasing demand for palm oil in a sustainable way. Mass production of selected, high-yielding palms by tissue culture could raise yields on existing plantations, reducing the need for further expansion of the cultivated area, which is often associated with negative environmental impacts

Programmed cell death and genetic stability in conifer embryogenesis

Somatic embryogenesis, the generation of embryos from somatic cells, is a valuable tool for studying embryology. In addition, somatic embryos can be used for large-scale vegetative propagation, an application of great interest for forestry. A critical event during early embryo differentiation in conifers is the apical basal polarization, which proceeds through the establishment of two embryonic parts: the proliferating embryonal mass and the terminally differentiated suspensor. The development of both parts is strictly coordinated and imbalance causes embryonic defects. The suspensor cells are eliminated by programmed cell death (PCD). In animals, caspase family proteases are the main executioners of PCD. In this work we have used synthetic peptide substrates containing caspase recognition sites and corresponding specific inhibitors to analyse the role of caspase-like activity during early embryo differentiation in Norway spruce (Picea abies L. Karst.). We found that VEIDase is the principal caspase-like activity. This activity is localized specifically in suspensor cells, and its inhibition prevents normal embryo development by blocking the suspensor differentiation. The in vitro VEIDase activity was shown to be highly sensitive to pH, ionic strength, temperature and zinc concentration. In vivo studies with Zinquin, a zinc-specific fluorescent probe, revealed a high accumulation of intracellular free zinc in the embryonal masses and an abrupt decrease in the suspensor. Increased zinc concentration in the culture medium suppresses terminal differentiation and PCD of the suspensor. In accordance, exposure of early embryos to TPEN, a zinc-specific chelator, induces ectopic cell death affecting embryonal masses. This establishes zinc as an important factor affecting cell fate specification during plant embryogenesis. Before somatic embryos can be accepted for clonal propagation it is important to show that the regenerated plants have similar growth to that of seedlings and are genetically uniform. The genetic integrity during zygotic and somatic embryogenesis in Norway spruce and Scots pine (Pinus sylvestris L.) was investigated by comparing the stability of variable nuclear microsatellite loci. The stability varied significantly among families in both species during somatic embryogenesis. Scots pine families showing low genetic stability during establishment of embryogenic cultures had a higher embryogenic potential than those that were genetically more stable. In contrast, embryo development was suppressed in genetically unstable families. The stability of microsatellites was in general higher in zygotic embryos than in somatic embryos. No deviation in growth was observed in somatic embryo plants of Norway spruce carrying mutated microsatellites

Genotype effect on somatic embryogenesis and plant regeneration of Vitis spp.

The effect of genotype on grapevine embryogenic culture was studied on eight Vitis spp. Cultures were obtained from excised anthers, isolated from flower buds. Somatic embryogenesis was achieved from 6 of the 8 varieties at various frequencies. Embryos were germinated either on solid or in liquid medium in the dark before being exposed to light. The regeneration rate ranged from 8 to 81 %. For induction of recurrent somatic embryogenesis, torpedoes and early cotyledonary stage somatic embryos were employed applying two plant growth regulator treatments. Results demonstrate that grape cultivars are highly variable in their response to induce somatic embryogenesis, recurrent somatic embryogenesis and germination of embryos