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Embryogenic cultures of Norway spruce () were cultivated either on solidified media, in liquid media, or on polypropylene membrane rafts (LifeRaft). The cultivation on rafts was found to be the most successful way: the number of developed somatic embryos increased, synchronization of the development was enhanced, and the time necessary for embryo development and maturation was shortened. It was shown that the process could be further improved by insertion of a pre-maturation phase on PGR-free medium between proliferation and maturation steps. Germination frequency remained unchanged. PEG 4000 added to the maturation medium increased the number of developed somatic embryos. PEG in lower concentration (1.87 % (w/v)) still had the significant beneficial effects on embryo numbers and development, but a decrease of germination frequency or increased aberrations of developing root and shoot were not found. On the other hand, PEG in the concentration 5 and 7.5 % (w/v) had a negative effect on the germination of somatic embryos and, in sensitive cell line, 7.5 % (w/v) PEG decreased somatic embryo yield.

Embryogenic suspensor mass derived from a zygotic embryo cotyledon of (L.) Karst. was propagated either in suspension culture or on agar-based ‘proliferation medium’ (Vagner, 1998), then transferred to agar-based ‘maturation medium’ for the development of somatic embryos. The ‘maturation’ medium was supplemented with silver nitrate at either 0, 1, 10 or 100µmol. Cultures were transferred to fresh medium weekly, for 10 weeks, during which numerical and photographic records of somatic embryo development were kept for each culture (n = 14 for each treatment). For cultures originating from both suspension and agar-based systems, there was approximately a two to three-fold increase in the number of Stage IV somatic embryos in media that had been supplemented with silver nitrate.

Embryos were obtained using liquid medium culture of sunflower hypocotyl epidermis layers according to the Pélissier et al. (1990) method. In the present work we identified genetic factors controlling somatic embryogenesis and we evidenced the role of ionic channels in embryogenic tissues. Two traits, the number of embryogenic explants (EE) aand the number of embryos (EM) were scored in 74 recombinant inbred lines (RILs) from a cross between lines PAC-2 and RHA-266. Analysis of variance indicated the existence of highly significant differences among the parental genotypes and their RILs. Heritability for the somatic embryogenesis traits studied were high (0.64 for EE and 0.77 for EM). Four quantitative trait loci (QTLs) for EE and seven for EM were detected using composite interval mapping. The QTLs for EE explained 48% of the phenotypic variation while the QTLs for EM explained about 89% of the variation, thus revealing several genomic regions related to somatic embryogenesis control in sunflower. In order to study the distribution of ion channels in somatic embryos as compared to zygotic ones, we used a fluorescent-labelled phenylalkylamine, DM-Bodipy PAA, as a probe. Fluorescence labelling was determined by confocal microscopy. The probe intensively labelled the protoderm and epidermis cells in both zygotic and somatic embryos. Callus exhibited labelling on sites where somatic embryos developed. Considering that the location of phenylalkylamine (PAA) binding sites is related to the distribution of ion channels, the high intensity in the protoderm and epidermis of embryos, point to similar properties and functions and their key role in embryo development.

Somatic embryogenesis offers the promise of a cost-effective, large-scale propagation method and is considered as a unique alternative technique to overcome some of the limitations of conventional clonal propagation methods. Production of somatic embryos from cell, tissue and organ cultures may occur directly which involves the formation of an asexual embryo from a single cell or a group of cells on a part of the explant tissue without an intervening callus phase. In this study, the photosynthetic ability of different stage coffee () somatic embryos and the development of photoautotrophy are reported. Results revealed that cotyledonary and converted somatic embryos have the ability to photosynthesise and can be grown under photoautotrophic conditions (with no supply of sugar from the culture medium). The development of photosynthetic ability can be accelerated by placing the somatic embryos in a photosynthetic photon flux of 100 µmol ms for at least 14 days. Cotyledonary stage somatic embryos were cultured under photoautotrophic conditions in three different growing systems to develop an optimized protocol for a large-scale embryo-to-plantlet conversion and propagation system. Our results demonstrated that the use of a newly developed temporary root zone immersion bioreactor is effective for the embryo-to-plantlet conversion and enhanced growth under photoautotrophic conditions.

Cell suspensions are the material of choice for rapid multiplication and for genetic engineering strategies such as mutagenesis and genetic transformation. Effective use of cell suspension cultures relies on the knowledge of several key parameters, which include genetic stability, kinetics of the cell cycle and a mode of plant regeneration. Here we report on the use of DNA flow cytometry for quality monitoring of banana cell suspension cultures. The method facilitates detection of ploidy changes and the occurrence of aneuploidy, which result in somaclonal variation of cell-suspension-derived plants. Flow cytometry could also be used to analyse the cell cycle kinetics by calculating the ratio of cells in the G2 and G1 phase of the cell cycle. This is important to determine the most appropriate moment for mutagenic treatment or for genetic transformation but also as an indicator on the proportion of cycling cells. In addition, the unicellular origin of somatic embryos was verified by treating embryogenic cell suspensions with colchicine and by determining the ploidy of regenerated plants by flow cytometric analysis. None of the plants regenerated from colchicine-treated embryogenic cell suspensions were mixoploid (chimeric). The application of flow cytometry will be discussed in relation to (a) the monitoring of genetic instability in DNA content of cell suspensions (b) the analysis of cell cycle and (c) the origin of somatic embryos of bananas and plantains.

Experiments to characterise long-term embryogenic suspension cultures of (L.) and (King) are reported. Cell suspensions of both species differed in the percentage of five selected fractions of cell aggregates, as well as in fresh and dry mass during three years of culture. In the ratio of cells in phase G to G was higher than in . The response of suspension cultures to GA3 (at 0, 1.49 or 2.89 µmol), kinetin (at 0, 2.32, 4.64 or 9.28 µmol) and adenine sulphate (at 0 or 434 µmol) was studied. The increase of kinetin concentration stimulated embryo production in suspensions of . Somatic embryo production in was significantly higher than in . In , the aggregate fraction >450 µm was at least four times more productive than the same fraction in suspensions.

Bulbing has been induced in somatic embryos formed in ovary tissue cultures of ‘Apeldoorn’. A high frequency of adventitious bulb formation took place on the stolons developing from embryos converted into plantlets in the liquid medium lacking growth regulators and containing 6% of sucrose. Number of bulblets per explant and individual fresh weight significantly increased in liquid medium (160 mg per bulblet) as compared to control (49 mg per bulblet). Plantlets must be chilled at 5†C in dark during 12 weeks period before formation and growth of adventitious bulbs. Plantlets, which were exposed to white light and cultured at temperature of 25†C (before chilling period), resulted in a higher crop of bigger bulblets (149 mg per bulblet, an average fresh weight) as opposed to embryos cultivated in darkness or irradiated by red light (59 mg per bulblet, an average fresh weight). After chilling period bulbs grew well in darkness and a temperature of 25†C. The temperature of 20†C of the liquid medium was less suitable for bulblet formation and growth, resulting in a lower percentage of large 200 mg bulblet (2.2%) in comparison to a medium temperature of 25†C (25.4%). Small bulbs, weighing less then 200 mg per bulblet, did not survive during the acclimatisation. Analysis of the level of ploidy of regenerated tulip plants did not reveal any changes.

is a monocotyledonous bulbous plant native of the South African Cape Region grown commercially as an ornamental plant for the production of flower stems. Commercial propagation is by the vegetative production of corms, which may suffer from a number of serious phytosanitary problems. In order to increase production uniformity and quality and to produce virus-free plants, a study of an alternative vegetative propagation system was investigated. Corms of two cultivars: &quote;Panorama&quote; and &quote;Rose Emperor&quote; were surface-sterilised and the meristems of the sprouting buds were excised and multiplied in semisolid medium in the presence of BA at 2.22 µmol. For liquid culture initiation, the explants were transferred to glass jars containing the liquid media supplemented with BA (at 0, 2.22 or 4.44 µmol) in the presence or in absence of ANC (3.90 µmol). At the end of the second subculture, in the presence of BA at either 2.22 or 4.44 µmol, the biomass was at least doubled and significantly increased when compared to the control treatment. The addition of ANC appeared to inhibit biomass proliferation although in the medium containing 2.22 µmol BA + ANC, a high number of bulblets was counted per biomass (callus) unit. At the end of the second subculture (60 days) all the material was transferred to hormone-free medium and, after 30 additional days, the culture parameters were evaluated. In the cultures of both genotypes, several shoot primordia and complete small plantlets developed from the callus and the cell aggregates. The histological analysis of the calli and the behaviour of the neoformed propagules indicated the activation of the embryogenetic pathway in the presence of ANC. The plantlets were successfully acclimatised in the greenhouse.

A liquid culture system using nodal segments was used for shoot proliferation and root induction in and , two commercially-important species of scented rose. For efficient and large scale induction of roots in microshoots, a rooting vessel was designed and developed to facilitate the micropropagation protocol. The present work highlights the significance of osmotic potential in relation to enhanced growth and development in liquid cultures, vis-à-vis agar-gelled cultures, especially in relation to root induction during micropropagation. An additional significant feature of the protocol developed, was the high success rate of hardening the micropropagated plants in low-cost hardening chambers, up to ca. 96.7% for and 100% for .

At Weyerhaeuser Company, somatic embryo production in liquid medium and manufactured seed delivery has been developed to reduce labor costs and increase efficiency of mass clonal propagation. We have scaled-up embryonal suspensor masses (ESM) of Douglas-fir in 1-litre liquid medium flasks. Over 10,000 somatic embryos have been produced from a single flask. We have cryostored ESM of over 700 genotypes in liquid nitrogen. Somatic embryos of Douglas-fir have also been produced from liquid medium in a bioreactor. Different types of bioreactors are required for embryo multiplication and for cotyledonary embryo development. Over 250,000 Douglas-fir somatic seedlings from a large number of genotypes have been produced for clonal field tests.