Somatic Embryogenesis and Protoplast Isolation and Culture in Papaya (Carica Papaya L.)

This study was carried out with the main objective of establishing somatic embryo production in papaya that may be used for further genetic improvement of the crop. The specific objectives were to induce somatic embryogenesis from immature zygotic embryos, to establish cell suspension culture of somatic embryos and to isolate and culture protoplasts from in vitro leaves. The first experiment studied the effect of growth regulators on the induction and regeneration of somatic embryos from immature zygotic embryos. The combinations of 2,4-dichlorophenoxyacetic acid (2,4-0) and 6-benzylaminopurine (BAP) at various concentrations were assessed in order to determine the best combination for somatic embryogenesis. The experiment was conducted in a Completely Randomized Design. The results showed that higher 2,4-D with or without BAP in the medium increased the percentage of somatic embryo formation and the number of somatic embryos per explant. MS medium supplemented with 5.0 mg/L 2,4-D promoted the highest percentage of somatic embryogenesis (74.55%), while the combinati on of 1.0 mg/L 2,4-0 and 0.01 mg/L BAP produced the highest percentage of callus formation (52. 58%). The highest number of somatic embryos per explant (66.61) was obtained when 5.0 mg/L 2,4-D and 0.01 mg/L BAP were added into MS medium. For germinati on of somatic embryos, the result was very inconsistent; nevertheless, the best treatment for plantlet formation was MS medium without growth hormone (NoBo). The second experiment was the establishment of cell suspensi on culture of somatic embryos. Four weeks after the transfer of somatic embryos into liquid MS medium containing 2.0 mg/L 2,4-0, pro-embryogenic masses (PEMs) were formed in the suspension. Maturation of embryos was achieved on transferring the heart-shaped embryos to liquid MS medium without growth regulator. Germination of somatic embryos occurred following the subculture of cotyledonary embryos from liquid MS medium with 0.2 mg/L BAP and 0.02 mg/L naphthalene acetic acid (NAA) to solid hormone-free MS medium

THE BIOTECHNOLOGY OF EMBRYOGENIC CELL LINES OBTAINING AND PLANTLETS OF CONIFEROUS SPECIES IN SIBERIA IN CULTURE IN VITRO

Experiments of culturing the immature isolated embryos and megagamethophytes of Siberian coniferous species were carried out on different modified media: ½ LV medium for Pinus sibirica and Pinus pumila, MSG and AI media (patent № 2431651) for Larix sibirica and Larix gmelinii, DCR medium for Picea ajanensis. For induction of embryogenic callus every species needs the optimized medium supplemented with L-glutamine, casein hydrolysate, ascorbic acid and hormones with different concentrations and their different proportions. The active proliferation of embryonal masses is observed on the same medium with reduced concentration of cytokinins. The proliferation of embryonal masses was significantly improved when they were subcultured after dispersing in liquid medium. The somatic embryos from embryonal masses are matured on basal medium with ABA (60-120 mM) and PEG. In spite of species specificity the embryogenesis of morphogenic structures had the same scheme: elongation and asymmetric division of somatic cells, formation of initial cells and embryonal tubes, development of globular, torpedo and bipolar somatic embryos, embryos maturation and germination. However, not all donor-plants of coniferous species can form the embryogenic cell lines and somatic embryos. The active development of embryonal masses and formation of somatic embryos are observed from zygotic embryo of hybrid seeds of P. sibirica and L. sibirica. The obtained embryogenic lines were characterized by different proliferative activity. During 10 months cultivation the value of embryonal masses in different lines was 140-570 g. The number of somatic embryos varies from 210 to 410 per 100 mg of callus fresh weight. Decreasing proliferation activity did not observed during 24-45 months cultivation. However, development of somatic embryos in long cultivated lines decreased. Maturation of somatic embryos and development of plantlets were established in L. sibirica and P. pumila 50-60 somatic embryos were matured per 1g of callus fresh weight. Somatic embryogenesis passes over the strong genetic control. Only donor tree genotypes with high reproductive potential form embryogenic cell lines and somatic embryos. The maternal affect was very strong relative to paternal and other effects. The studying molecular mechanisms involved in the control regulation of embryo development (embryo maturation, desiccation and germination) allows to understand many aspects of molecular biology of gymnosperms

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

Current applications of coffee (Coffea arabica) somatic embryogenesis for industrial propagation of elite heterozygous materials in Central America and Mexico

Of ail the possible micropropagation techniques, it is widely accepted that vegetative propagation by somatic embryogenesis is by far the most promising for rapid, large-scale dissemination of elite individuals. Yet, to date, examples of somatic embryogenesis processes applied on an industrial scale are very few and far between. There are many complications. They usually involve a major genotypic effect, particularly for obtaining embryogenic tissues, or are related to the quality of regenerated somatic embryos, the incidence of somaclonal variation and, more generally, a lack of reproducibility and efficiency at certain stages of the process, leading to production costs that are prohibitive. Research on coffee somatic embryogenesis began at the end of the 1970s at various institutes, including CIRAD. Between 1995 and 2001, CIRAD moved the technique forward from a research laboratory scale to a technique enabling industrial dissemination of extremely promising Coffea arabica F1 hybrids. Over that period, two technological innovations made technology transfer economically feasible: mass production of somatic embryos in temporary immersion bioreactors and the possibility of sowing them directly in the nursery. At the same time, reassuring data were obtained on the genetic conformity of regenerated plants (somaclonal variation frequency < 3%). In 2002, in partnership with the ECOM group, CIRAD decided to transfer the somatic embryogenesis method on an industrial scale to Central America so that four Arabica hybrid clones, that were selected for agroforestry-based farming systems, could be disseminated throughout that part of the world. This article describes the different stages and the difficulties we had to overcome before successful technology transfer could occur in 2010. . It describes one of the first examples of somatic embryogenesis technology applied on a commercial scale. Keywords: Somatic embryogenesis, micropropagation, technological transfer, coffee tree, production costs, clonai conformity, somaclonal variations, in vitro plantlet, nursery (Résumé d'auteur

Evidence of somatic embryogenesis from root tip explants of the rattan Calamus manan

Somatic embryogenesis of #Calamus manan#, a single-stemmed rattan species, in tissue culture was scientifically demonstrated for the first time. Root tips of #in vitro# plantlets produced friable callus when the explants were cultivated for several mo. on a Murashige and Skoog induction medium containing 7.5 mg Picloram per 1 (31.1 =M). Histological analyses established the presence of proembryos within the callus which differentiated subsequently into somatic embryos using the same culture medium. Histological examination revealed that these somatic embryos completely lacked starch and protein reserves, which did not prevent them, however, from germinating, and showing bipolar development. These somatic embryos further developed into young plants, similarly to zygotic embryos

Possibilities and limitations of vegetative propagation in breeding and mass propagation of Norway spruce

The use of vegetative mass propagation in practical forestry with Norway spruce (Picea abies (L.) Karst.) is limited at present, although its potential to deliver high genetic gains is obvious. The objective of this thesis was to study possibilities and limitations of vegetative propagation when applied in different parts of a breeding/mass propagation system for Norway spruce. Two vegetative propagation methods were studied: somatic embryogenesis and cutting propagation. Somatic embryogenesis was accompanied by losses of genotypes during the propagation process. The embryogenic response at proliferation and maturation was under family control, while germination was obtained for all families. Parental effects on proliferation and maturation were found for male parents but not for female. However, no correlations between embryogenic characters and breeding goal traits could be detected on parental level. Shortening of treatment with abscisic acid (ABA) during somatic embryo development gave pronounced positive effects on height growth of regenerated plants. An improved protocol, including five weeks ABA treatment and root development in liquid medium significantly improved performance of the resulting plants. The number of plants with lateral roots at the time of ex vitro transfer increased substantially with this protocol. Lateral roots at ex vitro transfer were shown to be a marker for good height growth and clonal uniformity during the next two years. Selection for height of cutting propagated clones in the nursery resulted in low responses in height after six years in field. The likely reason for this was low correlations between nursery traits and field traits. Genotype x environment interactions in the studied clonal test series varied from close to zero to more than 50% of the clone component. A tendency towards increased interaction components with age was obtained in one of the series. In situations with large genotype x environment interactions, clonal stability over sites should be included in the selection criteria

Interspecific somatic hybrids between Solanum bulbocastanum and S. tuberosum and their haploidization for potato breeding.

Protoplast fusion between incongruent Solanum bulbocastanum and S. tuberosum haploids was accomplished to produce hybrids combining elite traits from both parents. We identified 11 somatic hybrids out of 42 regenerants analyzed through ISSR markers. Some hybrids had loss or gain of fragments compared to the parents, likely due to rearrangements and deletions of chromosome segments after fusion, and/or to somaclonal variation during hybrid regeneration. Increased heterotic vigor for some traits as well as high diversity was observed as the effect of both ploidy and fusion combination. Microsporogenesis analysis indicated the occurrence of multivalent configurations and several meiotic abnormalities, such as chromosomes bridges and various spindle orientations. Since all hybrids were sterile, in vitro anther culture was employed for haploidization as a possible strategy to overcome barriers to hybridizations. Haploids were obtained from all the tetraploid S. bulbocastanum (+) S. tuberosum somatic hybrids tested, although with differences in both the number of embryos per 100 anthers cultured and the number of differentiated green plantlets. This is the first report on the successful production of haploid plants from S. bulbocastanum (+) S. tuberosum hybrids

Effect of cryopreservation and post-cryopreservation somatic embryogenesis on the epigenetic fidelity of Cocoa (Theobroma cacao L.)

While cocoa plants regenerated from cryopreserved somatic embryos can demonstrate high levels of phenotypic variability, little is known about the sources of the observed variability. Previous studies have shown that the encapsulation-dehydration cryopreservation methodology imposes no significant extra mutational load since embryos carrying high levels of genetic variability are selected against during protracted culture. Also, the use of secondary rather than primary somatic embryos has been shown to further reduce the incidence of genetic somaclonal variation. Here, the effect of in vitro conservation, cryopreservation and post-cryopreservation generation of somatic embryos on the appearance of epigenetic somaclonal variation were comparatively assessed. To achieve this we compared the epigenetic profiles, generated using Methylation Sensitive Amplified Polymorphisms, of leaves collected from the ortet tree and from cocoa somatic embryos derived from three in vitro conditions: somatic embryos, somatic embryos cryopreserved in liquid nitrogen and somatic embryos generated from cryoproserved somatic embryos. Somatic embryos accumulated epigenetic changes but these were less extensive than in those regenerated after storage in LN. Furthermore, the passage of cryopreserved embryos through another embryogenic stage led to further increase in variation. Interestingly, this detected variability appears to be in some measure reversible. The outcome of this study indicates that the cryopreservation induced phenotypic variability could be, at least partially, due to DNA methylation changes. Key message: Phenotypic variability observed in cryostored cocoa somatic-embryos is epigenetic in nature. This variability is partially reversible, not stochastic in nature but a directed response to the in-vitro culture and cryopreservation

Establishment of Hevea brasiliensis lines overexpressing genes involved in ethylene signalling pathway

The gaseous plant hormone ethylene has a wide variety of applications in agriculture and horticulture. Ethylene Response Factors (ERF) are the last transcription factors of the ethylene signalling pathway and control a large number of ethylene-responsive genes. Two Hevea brasiliensis ERF, HbERF-IXc4 and HbERF-IXc5, are orthologs to ERF1 a key regulator at the crosstalk of ethylene and jasmonate signalling pathways. These genes were suggested to play an important role in regulating latex cell metabolism in response to tapping and ethephon stimulation. In this study, transgenic lines overexpressing HbERF-IXc4 and HbERF-IXc5 under control of 35S CaMV and HEV2.1 promoter have been conducted. Transgenic Hevea lines were obtained by Agrobacterium tumefaciens-mediated genetic transformation. The somatic embryogenesis process was affected by these modifications. Agrobacterium tumefaciens genetic transformation procedure has been developed from friable callus line for clone PB260. Hevea callus was sub-cultured as small aggregates on paromomycin selection medium. Transgenic callus lines were established from sub-aggregates showing full GFP activity. Ten transgenic lines were confirmed as transgenic by Southern blot hybridization. This result showed successfully establishment of H. brasiliensis transgenic lines. Further plant regeneration and characterization were necessary to understand the function HbERF-IXc4 and HbERF-IXc5 in latex. (Résumé d'auteur

Padrões de acúmulo de proteínas e carboidratos durante a embriogênese somática de Acca sellowiana

The aim of this work was to quantify the protein, starch and total sugars levels during histodifferentiation and development of somatic embryos of Acca sellowiana Berg. For histological observations, the samples were dehydrated in a battery of ethanol, embedded in historesin and stained with toluidine blue (morphology), coomassie blue (protein bodies) and periodic acid-Schiff (starch). Proteins were extracted using a buffer solution, precipitated using ethanol and quantified using the Bradford reagent. Total sugars were extracted using a methanol-chloroform-water (12:5:3) solution and quantified by a reaction with anthrone at 0.2%. Starch was extracted using a 30% perchloric acid solution and quantified by a reaction with anthrone at 0.2%. During the somatic embryogenesis' in vitro morphogenesis and differentiation processes, the total protein levels decreased and the soluble sugars levels increased during the first 30 days in culture and remained stable until the 120th day. On the other hand, total protein levels increased according to the progression in the developmental stages of the somatic embryos. The levels of total sugars and starch increased in the heart and cotyledonary stages, and decreased in the torpedo and pre-cotyledonary stages. These compounds play a central role in the development of somatic embryos of Acca sellowiana. © 2009 Embrapa Informação Tecnológica.O objetivo deste trabalho foi quantificar os teores de proteína, amido e açúcares totais durante a histodiferenciação e desenvolvimento dos embriões somáticos em Acca sellowiana Berg. Para as observações histológicas, as amostras foram desidratadas em uma bateria de etanol, emblocadas em historesina e coradas com azul de toluidina (morfologia), azul de coomassie (corpos proteicos) e reativo ácido periódico de Schiff (amido). As proteínas foram extraídas usando uma solução tampão, precipitadas usando etanol e quantificadas por meio do reativo de Bradford. Os açúcares totais foram extraídos usando uma solução metanol-clorofórmioágua (12:5:3) e quantificados pela reação com antrona a 0,2%. O amido foi extraído usando uma solução de ácido perclórico a 30% e quantificado pela reação com antrona a 0,2%. Durante a diferenciação e morfogênese in vitro da embriogênese somática, os teores de proteínas totais decresceram e os açúcares solúveis aumentaram durante os 30 primeiros dias em cultura e permaneceram constantes até os 120 dias. Por outro lado, os teores das proteínas totais apresentaram incremento de acordo com a progressão nos estádios de desenvolvimento dos embriões somáticos. Os teores de açúcares totais e de amido aumentaram nos estádios cordiforme e cotiledonar e diminuíram nos estádios torpedo e pré-cotiledonar. Esses compostos exercem papel central no desenvolvimento de embriões somáticos de Acca sellowiana.Fil: Cangahuala-Inocente, Gabriela Claudia. Universidade Federal de Santa Catarina; BrasilFil: Steiner, Neusa. Universidade Federal de Santa Catarina; BrasilFil: Maldonado, Sara Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Biodiversidad y Biología Experimental y Aplicada. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Biodiversidad y Biología Experimental y Aplicada; ArgentinaFil: Guerra, Miguel Pedro. Universidade Federal de Santa Catarina; Brasi