Summary Somatic Embryogenesis Research Articles

RNA-Seq screening of differentially-expressed genes during somatic embryogenesis in Fragaria × ananassa Duch. ‘Benihopp’

SUMMARYSomatic embryogenesis is an important model system for studies on embryo development. In order to gain a better understanding of the molecular mechanisms underlying somatic embryogenesis, RNA-Seq data were generated from non-embryogenic callus, embryogenic callus, somatic embryos, early-germinated somatic embryos, and late-germinated somatic embryos of Fragaria X ananassa Duch. ‘Benihopp’. Totals of 21 x 106–34 x 106 high-quality reads were obtained from the various RNA-seq libraries, with average lengths of 87–94 base pairs. A total of 81% of the reads were mapped to the reference Fragaria vesca genome and 946 differentially-expressed genes were identified, most of which were associated with non-embryonic callus. An analysis of the expression data using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database indicated that the gene ontology (GO) classes with the highest representation were ‘metabolic process’ and ‘metabolism’. In total, 38 transcription factors were identified, including members of the AP2/EREBP and MYB families. Three genes related to hormone signalling pathways were identified: one gene encoding the AUXIN RESISTANT 1 (AUX1) protein and two genes encoding pathogenesis- related 1 (PR1) proteins. During somatic embryogenesis, levels of transcripts corresponding to the AUX1 protein increased, while those corresponding to PR1 decreased. Non-embryogenic callus showed the greatest difference in transcriptome profile compared with the other samples, and the GO annotation term ‘metabolism pathways’ was particularly common in the data associated with strawberry somatic embryogenesis. Based on the RNA-seq data, we speculate that the phytohormones auxin and salicylic acid may play important roles during somatic embryogenesis. Our transcriptome dataset represents a valuable resource for researchers studying somatic embryogenesis and has the potential to provide new insights into the underlying mechanisms of molecular regulation.

Massal Induction of Carica papaya L. ^|^lsquo;Golden^|^rsquo; Somatic Embryos and Somaclone Screening by Flow Cytometry and Cytogenetic Analysis

Summary Somatic embryogenesis is a relevant micropropagation technique in Carica papaya �Goldenin view of the difficulties met in conventional seed propagation and the lack of an effective method for early sex determination in this trioecious species. Considering the interest in large-scale production of C. papaya seedlings, we adapted a somatic embryogenesis procedure associating liquid system and immature zygotic embryo explants. This protocol markedly contributed to an increase in the yield of friable embryogenic calli, resulting in a large number of normal somatic embryos at various developmental stages. From these embryos, plantlets were regenerated and evaluated for occurrence of somaclonal variation. Flow cytometry screening revealed diploid (88%), tetraploid (6%), and aneuploid (6%) plantlets. Owing to an interest in the mass propagation of diploid individuals, the karyotype of these plantlets was also analyzed. With this approach, diploidy (2X=18) was confirmed and no structural aberrations were found. Considering the results and the applicability of large-scale production of somatic embryos in C. papaya, the tissue culture protocols shown here could be used for commercial purposes. Moreover, the cytometric and cytogenetic analyses were crucial for a rapid, reliable and conclusive evaluation of the genetic stability of C. papaya plantlets regenerated by somatic embryogenesis.

Temporal regulation of somatic embryogenesis in guava (Psidium guajava L.)

SummarySomatic embryogenesis was induced in immature zygotic embryos of guava (Psidium guajava L.) at 10-weeks post anthesis using 2 – 38 d pulses and continuous treatments with different concentrations (0.001 – 3.0 mg l–1) of 2,4-dichlorophenoxyacetic acid (2,4-D). Subsequent development of somatic embryo was observed by sub-culturing onto plant growth regulator (PGR)-free, full-strength Murashige and Skoog (MS) agar medium containing 3% (w/v) sucrose. The maximum responses were shifted gradually from 0.01 mg l–1 2,4-D (continuous or 38 d treatment), to 0.05 mg l–1 2,4-D (28 – 18 d treatment), to 0.1 mg l–1 2,4-D (14 – 12 d treatment) and, finally, to 0.5 mg l–1 2,4-D (10 – 8 d treatment).The highest frequency of embryogenesis (68.8%) and embryogenic intensity (69.2 embryos per explant per culture), as well as moderate frequencies of convertible elongated and short torpedo-stage somatic embryos (15% and 42.9%, respectively), with the highest efficiency (27.5) achieved in the 8 d treatment compared to continuous treatment of zygotic embryo explants with 0.5 mg l–1 2,4-D in MS agar medium containing 3% (w/v) sucrose at both the induction and development phases. Cellular pools of total, free, conjugated, and bound forms of various polyamines (e.g., putrescine, spermidine, and spermine) showed slow increases in the presence of 2,4-D during the 8 d induction phase. This was followed by rapid increases immediately after sub-culturing explant tissue onto the PGR-free MS development medium. The concentrations of various polyamines declined gradually over time on MS development medium. The production, metabolism, and use of different forms of various polyamines at various times (d) after culture initiation correlated with their temporal regulation during the induction and development phases of somatic embryogenesis. Exogenous application of different polyamines during the induction phase resulted in slightly increased frequencies, intensities, and efficiencies of embryogenesis, but with no observable change in the frequencies of the various categories of somatic embryos. The present study indicated that the process of somatic embryogenesis in guava may be less sensitive to PGRs and was induced even in the continuous presence of 2,4-D. Furthermore, somatic embryogenesis in guava was temporally regulated as a function of the exogenous concentration of 2,4-D. In addition, the temporal regulation of somatic embryogenesis was also associated with high levels of production, metabolism, and use of cellular polyamines during the different phases. Hence, improvements in the process of somatic embryogenesis in guava could be regulated by timed applications of 2,4-D alone, or in combination with polyamines, thereby modulating endogenous levels of the latter.

Differential gene expression in embryogenic and non-embryogenic clusters from cell suspension cultures of Coffea arabica

Summary Somatic embryogenesis (SE) is a very useful system for studying the differentiation process in plants and involves gene regulation at several levels. During SE induction in Coffea arabica cv. Catura Rojo two types of cell clusters, embryogenic (EC) and non-embryogenic (NEC), were observed. The goal of this work was to compare the most relevant characteristics between EC and NEC for a better understanding of the mechanism driving SE. Morphohistological observations indicated a correlation between the morphological features of clusters and their embryogenic competence. On the other hand, no variation at the DNA level, studied by AFLP, were found to explain the disparity in embryogenic competence of clusters, but gene expression, observed by RNA differential display, and SDS-PAGE showed differences that can explain that disparity. Our results lead us to propose that differential gene expression can modulate the embryogenic capacity of coffee cells and that the number of genes turned off in somatic cells to allow for the change from a somatic to an embryogenic state, is higher than those genes that are turned on.

Continuous Somatic Embryogenesis in Japanese Maple ( Acer palmatum Thunb.)

Summary Somatic embryogenesis was induced in immature zygotic embryos of Japanese maple ( Acer palmatum Thunb.) cultured on modified Murashige and Skoog medium supplemented with different combinations of 2,4-D and BAP. The most effective combinations were 10.0 μmol/L BAP and 0.1 μmol/L 2,4-D and/or 5.0 μmol/L BAP without 2,4-D. The presence of BAP proved essential for somatic embryogenesis. Somatic embryos were formed directly on zygotic embryos or on developed calluses. Continuous somatic embryogenesis was achieved after transfer of cultured tissues on MS medium with 0.05 μmol/L BAP and 0.5 μmol/L NAA. New somatic embryos developed directly on somatic embryos (secondary somatic embryogenesis), from a microcallus formed on the root tip of a somatic embryo or from an embryogenic callus. Somatic embryos with normal and also aberrant morphology developed, matured and converted into plantlets on the same medium. The conversion continued after transfer onto half strength MS medium without growth regulators. The rooted plants were grown in a greenhouse. The selected cultures of Japanese maple were able to produce somatic embryos continuously and this ability has not been declining for more than 2 years.

Somatic Embryogenesis and Plant Regeneration from Roots of Bamboo ( Bambusa beecheyana Munro var. beecheyana)

Summary Somatic embryogenesis and subsequent formation of plantlets was achieved from callus cultures derived from the roots of bamboo ( Bambusa beecheyana Munro var. beecheyana). Embryogenic callus was initiated on Murashige and Skoog's medium (MS) supplemented with 2 mg/L kinetin, 3 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D), and 6 % sucrose. Prolonged culture of embryogenic callus on the same medium resulted in embryoid formation, eventually germination, and establishment of rooted plantlets that were successfully transferred to soil.

Somatic Embryogenesis and Plant Regeneration from Seedling Cultures of Tomato ( Lycopersicon esculentum Mill.)

Summary Somatic embryogenesis was induced in several cultivars of tomato (Lycopersicon esculentum Mill.) using a simple, one-step method. Seeds were germinated in the presence of 50 or 80 μmol · L-1 N6-benzylaminopurine, and the resultant seedlings were maintained for six weeks on the same medium. Somatic embryogenesis occurred directly without an intervening callus proliferation phase from the meristematic centres formed in the subepidermal region of the nodular tissues developed at the basal ends of induced seedlings. Regenerated somatic embryos were successfully grown into whole plants.

Isolation and Characterization of Long-term Embryogenic Lines in Asparagus officinalis L.

Summary Somatic embryogenesis was obtained from 14 Asparagus officinalis L. genotypes by in vitro culture of different explant types such as cladophylls, apices and isolated mesophyll cells. Explants first induced on a culture medium containing growth regulators produced calli and developed few somatic embryos when transferred on a hormone-free medium. During this development phase, we isolated a new type of friable tissues that produced abundant somatic embryos. The frequencies of emergence of these highly embryogenic tissues were estimated between 10 −5 and 10 −6 . These tissue were habituated; being established as long-term embryogenic lines by repeated subcultures on hormone-free medium, continuously they produced numerous somatic embryos, some of them converting to whole plants. Histological data showed these long-term embryogenic lines to grow by recurrent embryogenesis arising, from single epidermal cells of preexisting embryos.

Plant Regeneration of Aesculus hippocastanum L. (Horse Chestnut) through Somatic Embryogenesis

Summary Somatic embryogenesis and subsequent formation of plantlets was obtained from callus cultures derived from immature embryos of horse chestnut (Aesculus hippocastanum L.) cultured on Murashige and Skoog's medium supplemented with 5 % sucrose, 2,4-D (3 mgl-1), kinetin (1 mgl-1), CH (250 mgl-1) and proline (250 mgl-1). Proline was a necessary factor for this process and in its presence, whitish, compact globular embryogenic and non-embryogenic calli were simultaneously formed. Reduction of 2,4-D to 1 mgl-1 in MS solid medium resulted in an increase of both callus mass and number of somatic embryos. Germination of somatic embryos and their further development into plantlets was achieved by short-term culture of the embryos on Murashige and Skoog's liquid medium supplemented with glutamine (400 mgl-1), IAA or IBA, and GA3 (1 mgl-1). Addition of cytokinins to the culture medium inhibited plantlet formation. Regenerated horse chestnut plantlets had a diploid chromosome number (2x = 2n = 40).

Somatic Embryogenesis and Plantlet Regeneration in Cultured Immature Embryos of Picea glauca

Summary Somatic embryogenesis and plantlet regeneration was obtained from cultured immature embryos of Picea glauca (Moench) Voss (white spruce) on a modified von Arnold and Eriksson’s (1981) medium supplemented with N 6 -benzyladenine and one of the auxins: 2,4-dichlorophenoxyacetic acid, 2-methyl-4-chlorophenoxyacetic acid or 4-amino-3, 5, 6-trichloropicolinic acid (picloram). Picloram at 5 or 10 μM was the most effective auxin. The optimum size of immature embryo for initiation of embryogenic callus and somatic embryos was found to be 1.5–2 mm. Increasing the osmolarity of the medium and reducing the concentration of 2,4-D enhanced development and maturation of the somatic embryos. Comparison of the growth of plantlets on different basal media showed that the best growth occurred on one-third strength Schenk and Hildebrandt’s (1972) medium.

Regeneration of Plantlets By Embryogenesis from Callus Cultures of Carica Candamarcensis

Summary Somatic embryogenesis was induced on callus derived from hypocotyl sections of Carica candamarcensis growing on several nutrient media in the presence of naphthaleneacetic acid (NAA) (1-5 mg/l) and kinetin (1 mg/l) within 3 months culture in the light. Embryoids developed into plantlets when subcultured on a half-strength Murashige and Skoog medium (MS) supplemented with indoleacetic acid (IAA) (0.5 mg/l) and casein hydrolysate (200 mg/l), Callus derived from immature pericarp tissue or peduncles did not show morphogenetic responses.