Cotyledonary Embryos Research Articles

Induction of somatic embryogenesis in different explants from Ormosia henryi Prain

An efficient in vitro plant regeneration method via somatic embryogenesis has been established in Ormosia henryi Prain, which is an important precious tree in the timber industry. Five types of explants, six types of basic media and different plant growth regulators (PGRs) were used for embryogenic callus (EC) initiation, EC proliferation, somatic embryo (SE) induction and SE germination. The EC and SE induction rates from mature zygotic embryos (MZE) were higher than those from stem segments, cotyledons, leaves and hypocotyls. MZE were used as explants, and the EC induction rate was greater than 30% on B5 medium supplemented with 1.0 mg/l 6-benzylaminopurine (BA) and 0.5 mg/l naphthaleneacetic acid (NAA) or 0.2 mg/l BA and 2.0 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D). The highest induction rate (22.83%) for SE was obtained with B5 medium supplemented with 0.5 mg/l kinetin (KT) and 1.0 mg/l 2,4-D. The EC proliferation rate and SE number in the suspension culture were increased compared to those in the solid culture. Fifty-four percent of SE developed to the cotyledonary stage on B5 medium supplemented with 0.5 mg/l BA and 0.2 mg/l NAA. The number of cotyledon embryos was the highest on medium supplemented with 0.5 mg/l thidiazuron (TDZ) and 0.2 mg/l NAA. Histological analysis confirmed the bipolar organization of the somatic embryos, which were comprised of shoot and root meristems, and showed that SE was characterized by globular, heart-shaped, torpedo-shaped and cotyledon stages in O. henryi, the developmental process of which was similar to that of zygotic embryos. An efficient in vitro plant regeneration method via somatic embryogenesis has been established in Ormosia henryi Prain using mature zygotic embryos as explant.

Protocol for Rescuing Young Cassava Embryos.

Embryo rescue (ER) in cassava breeding has several relevant applications, from the recovery of broad crosses to the recovery of seeds from the standard pollination program. Cassava fruit setting may drop from 100%, during the 1st week after pollination, to less than 40% during the 2nd week after pollination due to the abscission of fruits depending on genotypes. Therefore, the availability of an ER protocol for early stages of embryo development, in particular during the first 2 weeks after pollination (prior the cotyledonary stage), could have practical implications for cassava breeding. Until now, attempts to recover cassava immature embryos at stages of development earlier than the cotyledonary stage failed. The earliest successful rescue reported in cassava is from embryos excised 32–36 days after anthesis (DAA). However, limited information was available regarding embryo development in cassava. This work studied and documented the stage of embryo development in histological sections of hand-pollinated ovules fixed from 1 to 30 days after anthesis (DAA). At 7 DAA, zygotes were just at the first stages of cell division (pro- embryo stage). At 14 DAA, embryos were at the pre-globular stage. Embryos at the early globular stage were observed in sections fixed at 21 DAA, and at the proper globular stage at 24 DAA. Samples at 30 DAA contained cotyledonary embryos that easily developed after ovule culture into viable plants using existing protocols. A second contribution of this work is the development of a protocol for the recovery of fully developed plants from immature embryos rescued and cultured in vitro as early as 7–14 DAA. Since embryos collected at this age are at the pro-embryo to pre-globular stage, ovary/ovule culture was necessary. A method is described whereby ovules were cultured to allow the development of pro-embryos and pre-globular stage embryos into the cotyledonary stage. Subsequently, these mature embryos were excised from the ovules to induce germination and the recovery of fully developed plants.

Differential Influence of Growth Regulators during Somatic Embryogenesis of Gynodioecious Papaya Varieties‘CO.7’ and ‘Red Lady

The study involved two auxins viz., 2,4-D (2,4-Diclorophenoxyacetic acid) and picloram at three different concentrations (1,2, 3 mg/L) in full strength MS media to study their comparative influence on induction of somatic embryogenesis from immature zygotic embryos of two gynodioecious varieties of papaya ‘CO.7’ and ‘Red Lady’. In papaya cultivar ‘CO.7’, 2,4-D at 2 mg/L gave the highest callus induction frequency of 90.93%, whereas comparatively higher concentration of 3 mg/L 2,4-D was found suitable for ‘Red Lady’ (87.26%). Although there was profuse callus formation, 2 mg/L 2,4-D recorded comparatively higher frequency of embryogenic calli in ‘Red Lady’ (51.67%) when compared to ‘CO.7’ (30.00%). Somatic embryo maturation was achieved upon transfer of embryogenic calli exhibiting globular stage embryos on to maturation medium (MS medium + ABA (Abscisic acid) and BAP (Benzyl amino purine) in different concentrations + glutamine 400 mg/L). In the maturation medium, the combination of 1.5 mg/L ABA and 0.4 mg/L BAP registered better conversion of the globular embryo to cotyledonary embryos than other levels. The frequency of somatic embryo germination was higher in ‘Red Lady’ (50.00%) as compared to ‘CO.7’ (31.67%) on half-strength MS medium devoid of growth regulators.

Changes of cell wall components during embryogenesis of Castanea mollissima.

The Chinese chestnut (Castanea mollissima Blume) 'Huaihuang' was chosen as the experimental material to observe embryogenesis and the dynamic changes of cell wall components during this process. Various developmental stages of embryos, including globular embryos, heart embryos, torpedo embryos and cotyledon embryos, were observed. The results showed that during embryogenesis, cellulose increased, and callose rapidly degraded. In the cell walls of developing embryos, pectic homogalacturonan (HG), especially low-esterified HG, was abundant, suggesting rapid synthesis and de-methyl-esterification of HG. Extensin and galactan increased with the development of the embryos. In contrast, the arabinan epitopes decreased in developing embryos but were more abundant than galactan epitopes at all stages. Xylan epitopes showed explicit boundaries between the outer epidermal wall and the rest of the inner tissues, and the fluorescence intensity of the outer epidermal wall was significantly higher than that of the inner tissues. Furthermore, the results indicated that the outer epidermal wall contained high amounts of cellulose, HG pectin and hemicellulose, especially arabinan and xylan. These results suggested the presence of rapid pectin metabolism, cellulose synthesis, rapid degradation of callose, different distributive patterns and dynamic changes of hemicellulose (galactan, arabinan and xylan) and extensin during embryogenesis. Various cell wall components exist in different tissues of the embryo, and dynamic changes in cell wall components are involved in the embryonic development process.

Manual Isolation of Living Early Embryos from Tobacco Seeds.

Zygotic embryogenesis is one of key processes for fertile seed development and therefore has gained great attention for decades in the field of plant developmental biology. However, this process is deeply embedded in the maternal tissues. The inaccessibility of tiny early embryos has greatly hindered the study of early embryogenesis, especially limits direct observation and accurate omics investigations. In order to investigate the molecular mechanism regulating embryo development with modern technologies, it is necessary to develop a reliable method to isolate living embryos at different stages. For this purpose, plant scientists have been trying to develop different methods for isolating zygotes and early embryos in different plants such as maize, wheat, rice, and tobacco during past decades. Nicotiana tabacum has long been considered as an ideal model eudicot for the study of embryogenesis, which displays a traceable and predictable cell division pattern, spanning from the first zygotic division to the mature embryo formation. Here, we provide a detailed protocol for isolating living embryos from zygote to cotyledon embryo. Isolated living zygotes and early embryos could be used for several important studies such as celltype-specific transcriptome construction and clear GFP observation.

Histocytological Study of Somatic Embryogenesis in the Tree Cinnamomum camphora L. (Lauraceae)

Histocytological studies were conducted on primary, secondary, and malformed embryos produced during somatic embryogenesis of Cinnamomum camphora L. to better understand its development. Exploring its callus types and structures provided a theoretical basis for clarifying the mechanism of somatic embryogenesis, which may shed light on the mechanism of zygotic embryogenesis. We used immature zygotic embryos as explants to induce somatic embryos, forming many embryogenic calli that differentiated into mature somatic embryos. Our results showed that somatic embryogenesis of C. camphora was similar to that of zygotic embryos. We have been dedifferentiated four types of callus. Compared with non-embryogenic cells, embryogenic cells had a closer arrangement, larger nucleus, thicker cytoplasm, more starch granules and easier to stain into black. Somatic embryogenesis had two pathways: direct (predominate) and indirect (rare). Embryogenic cells of C. camphora could have either an internal or external origin, the latter being primary, for which occurrence sites include epidermis and near-epidermis (little internally). Mostly arising from single cells, C. camphora follows two developmental pathways: single-cell equal as opposed to unequal, wherein both divide to form multi-cell proembryos. However, multicellular origins can occasionally occur and feature physiological isolation during somatic embryo development. This development has four embryo stages: globular, heart-shaped, torpedo, and cotyledon, with procambium cells apparent in globular embryos and late cotyledons forming “Y-shaped” vascular bundles. Secondary embryos were present in all stages, directly occurring on primary embryo’s germ and radicle end surfaces. We conclude that secondary and primary embryos of C. camphora undergo similar developmental processes. At the same time, conjoined cotyledon embryos and morphological abnormal embryos were found, with an internal origin more likely to generate abnormal embryos.
 
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 In press - Online First. Article has been peer reviewed, accepted for publication and published online without pagination. It will receive pagination when the issue will be ready for publishing as a complete number (Volume 47, Issue 4, 2019). The article is searchable and citable by Digital Object Identifier (DOI). DOI link will become active after the article will be included in the complete issue.
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Studies on single and double layered biocompatible encapsulation of somatic embryos in Albizia lebbeck and genetic homogeneity appraisal among synseed derived lines through ISSR markers

Embryogenic synseeds were prepared in Albizia lebbeck by encapsulating cotyledon stage somatic embryos derived from in vitro maintained embryogenic cultures in different types of Ca-alginate beads. The germination rate of somatic embryos was affected significantly by the bead type, matrix composition and germination substrate. A matrix made of 3% Na2-alginate complexed with 100 mM CaCl2·2H2O for a hardening period of 20 min provided uniform encapsulation of somatic embryo. Among different types of synseeds, type IIA, wherein somatic embryos encapsulated in a single layer of Ca-alginate matrix composed of MS medium supplemented with 2 g L−1 activated charcoal and 1.0 µM gibberellic acid (GA3) as reconstituted endosperm, was found to be the most efficient type having maximum germination rates (88.6 ± 0.51%). Incorporation of GA3 in the alginate beads stimulated greater germination of somatic embryos as against GA3 supplementation in the germination substrate. Further, viability studies on short term cold (4 °C) storage of different types of embryogenic synseeds revealed that double layered synseeds (DLS) were found comparatively more robust to withstand longer storage durations than single layered synseeds as evident by greater germination rates of the former after 4–8 weeks of refrigerated storage. Also, the elevated levels of antioxidative enzymes (superoxide dismutase, catalase, ascorbate peroxidase and glutathione reductase) and leaf proline content in the plantlets derived from DLS reveals the possible role of alginate coatings in conferring alleviation to low temperature stress generated during different storage durations. Similar Inter simple sequence repeat profiles of embryogenic synseeds derived plantlets and mother tree nullifies the possible occurrence of somaclones, thereby establishing the efficacy of synseed technology for clonal propagation of A. lebbeck germplasm. Encapsulation of cotyledonary stage somatic embryos combined with high in vitro germination rates provides a feasible tool for interim storage and exchange of A. lebbeck germplasm.

Dormancy breaking in Fagus sylvatica seeds is linked to formation of abscisic acid-glucosyl ester

Seed dormancy is an adaptive mechanism that allows seed germination under suitable environmental conditions. Germination of stored dormant seeds proceeds after dormancy breaking induced by stratification. To improve understanding of dormancy breaking in beechnuts, we: investigated effects of moisture content and temperature during storage; analysed contents of abscisic acid, abscisic acid metabolites and indole-3-acetic acid in embryonic axes during storage and stratification; and histochemically localized storage proteins in embryogenic axes and cotyledons of beechnut embryos. The results show that storage conditions can affect the nuts’ abscisic acid contents, but not the stratification process. In addition, dormancy breaking is linked to a reduction in abscisic acid contents and depth of dormancy is not influenced by either of these storage factors. Detected changes that correlated most strongly with dormancy breaking were a big increase in abscisic acid-glucosyl ester levels and accompanying reduction in the abscisic acid-glucosyl ester to abscisic acid ratio. We detected extremely low concentrations of other abscisic acid metabolites-neophaseic, phaseic and dihydrophaseic acids—in non-dormant stored beechnuts, but somewhat higher concentrations during dormancy. No relationships between changes in indole-3-acetic acid levels with either storage conditions or dormancy breaking were detected. Changes in distributions of storage proteins were related to the seeds’ moisture content during storage and stratification rather than seed dormancy. We conclude that increases in endogenous abscisic acid-glucosyl ester levels and abscisic acid-glucosyl ester to abscisic acid ratios are good markers of depth of dormancy and/or effectiveness of stratification in beechnuts.

In silico and in vivo analysis of ABI3 and VAL2 genes during somatic embryogenesis of Coffea arabica: competence acquisition and developmental marker genes

The employment of biotechnology-based approaches such as somatic embryogenesis has been applied to several plants including Coffea sp. Despite the economic importance of this genus, few information about the role of key regulatory genes in somatic embryogenesis in coffee is available. This work provides information about ABI3 and VAL2 genes performance by RT-qPCR during indirect somatic embryogenesis of Coffea arabica. To achieve this, bioinformatics analysis was performed to identify the genes of the B3 superfamily in the coffee genome. The cell suspensions lines presented similar histological and regeneration patterns, yielding of up to 6.6 embryos per 1 mg of embryogenic aggregates at 7 months. We have identified possible orthologs for VAL2 (Cc06g00410) and ABI3 (Cc01g17380) as well as the other members belonging to superfamily B3. The CaABI3 expression was higher in dedifferentiated competent cells for somatic embryogenesis as compared with non-embryogenic calli. Whereas the expression of VAL2 gene is more active in cotyledonary embryos and plantlets, showing its clear performance in the embryogenesis late stages. The present study suggests that CaABI3 gene could be potentially used as a biomarker for embryogenic process improvement. The good plantlets development obtained from the protocol used may be a reflection of the high expression of CaVAL2 in cotyledonary embryos and plantlets. The activity of CaABI3 is correlated to embryogenic potential with highly expressed in embryogenic masses and expression of the VAL2 gene is increased at the end of the embryogenic process.

Pectin De-methylesterification and AGP Increase Promote Cell Wall Remodeling and Are Required During Somatic Embryogenesis of Quercus suber.

Somatic embryogenesis is a reliable system for in vitro plant regeneration, with biotechnological applications in trees, but the regulating mechanisms are largely unknown. Changes in cell wall mechanics controlled by methylesterification of pectins, mediated by pectin methylesterases (PMEs) and pectin methyl esterase inhibitors (PMEIs) underlie many developmental processes. Arabinogalactan proteins (AGPs) are highly glycosylated proteins located at the surface of plasma membranes, in cell walls, and in extracellular secretions, with key roles in a range of different processes. In this study, we have investigated changes in two cell wall components, pectins and AGPs, during somatic embryogenesis in Quercus suber, a forest tree of high economic and ecologic value. At early embryogenesis stages, cells of proembryogenic masses showed high levels of esterified pectins and expression of QsPME and QsPMEI genes encoding a PME and a putative PMEI, respectively. At advanced stages, differentiating cells of heart, torpedo and cotyledonary embryos exhibited walls rich in de-esterified pectins, while QsPME gene expression and PME activity progressively increased. AGPs were detected in cell walls of proembryogenic masses and somatic embryos. QsLys-rich-AGP18, QsLys-rich-AGP17, and QsAGP16L1 gene expression increased with embryogenesis progression, as did the level of total AGPs, detected by dot blot with β-glucosyl Yariv reagent. Immuno dot blot, immunofluorescence assays and confocal analysis using monoclonal antibodies to high- (JIM7, LM20) and low- (JIM5, LM19) methylesterified pectins, and to certain AGP epitopes (LM6, LM2) showed changes in the amount and distribution pattern of esterified/de-esterified pectins and AGP epitopes, that were associated with proliferation and differentiation and correlated with expression of the PME and AGP genes analyzed. Pharmacological treatments with catechin, an inhibitor of PME activity, and Yariv reagent, which blocks AGPs, impaired the progression of embryogenesis, with pectin de-esterification and an increase in AGP levels being necessary for embryo development. Findings indicate a role for pectins and AGPs during somatic embryogenesis of cork oak, promoting the cell wall remodeling during the process. They also provide new insights into the regulating mechanisms of somatic embryogenesis in woody species, for which information is still scarce, opening up new possibilities to improve in vitro embryo production in tree breeding.

Somatic embryogenesis in the commercial papaya hybrid UENF/Caliman 01 relying on plantlet production from sexed adult hermaphrodite donor plants.

Somatic embryogenesis from explants from hermaphrodite papaya mother plants is an alternative for the production of true-to-type plants without the need for sexing. This study aimed to analyze hormonal and osmotic inducers in different somatic embryogenesis stages in the commercial hermaphrodite hybrid papaya UENF/Caliman 01. Leaf disks from in vitro shoots originated from ex vitro hermaphrodite plants were cultured in induction medium supplemented with different concentrations of 2,4-D (6, 9, 12, 15, and 18 μM) and 4-CPA (19, 22, 25, 28, and 31 μM). After 90 days, the formation of somatic embryos was verified. The 2,4-D induced the formation of light brown calli with low frequency (20%) of somatic embryogenesis. However, 4-CPA (25 μM) induced 96% of embryogenic calli, which were transferred to maturation medium (MM) and cultured for 30 days. The MM contained ABA (0.5 μM) and AC (15 g L-1) and produced 36.6 somatic embryos callus-1, mainly on cotyledonary stage. Cotyledonary embryos were transferred to germination medium supplemented with gibberellic acid (GA3) (0.0, 1.44, 2.88, and 4.32 µM), and the conversion into plantlets was enhanced with GA3 at 2.88 µM.

High-Efficiency Somatic Embryogenesis from Seedlings of Koelreuteria paniculata Laxm.

Research Highlights: In the current study, we established a method for plant regeneration via somatic embryogenesis (SE) in Koelreuteria paniculata Laxm. for the first time. Background and Objectives: K. paniculata is an important ornamental and medicinal plant in China. However, the plant has difficulty with asexual reproduction, which imposes a limitation on large-scale propagation. Materials and Methods: Embryogenic calluses were induced from stems of aseptic seedlings on induction media. The effects of different media types and concentrations of N6-benzyladenine (BA), α-naphthaleneacetic acid (NAA), and 2,4-dichlorophenoxyacetic acid (2,4-D) on callus induction were examined. Embryogenic calluses were then transferred to Driver-Kuniyuki Walnut (DKW) media containing NAA (0.1–0.2 mg L−1) or 2,4-D (0.5–2.0 mg L−1) to develop somatic embryos. Cotyledon embryos were cultured on DKW media containing NAA (0.1–0.2 mg L−1) until maturation, and were then transferred to 1/2 DKW medium supplemented with 1.0 mg L−1 indole-3-butyric acid (IBA) to produce complete plants. The effects of IBA and NAA on rhizogenesis were then examined by clonal culture. Results: The maximum callus induction frequency (80.25%) was obtained on DKW medium supplemented by 0.5 mg L−1 BA, 0.25 mg L−1 NAA, and 1.5 mg L−1 2,4-D. NAA had a more pronounced effect on somatic embryo growth than did 2,4-D, with a maximum SE frequency (54.75%) observed with 0.1 mg L−1 NAA added to DKW medium. For clonal culture, the highest rooting rate (52%) was observed on 1/4 DKW medium containing 1.5 mg L−1 IBA. Histology studies confirmed the presence of embryogenic calluses and somatic embryos in different stages. Conclusions: This protocol provides a novel method for large-scale propagation of K. paniculata, and creates opportunities for genetic engineering in this species.

Histology of somatic embryogenesis in Coffea arabica L.

The objective of this study was to characterize the histodifferentiation of somatic embryogenesis obtained from leaf explants of C. arabica. Therefore, we histologically analyzed the respective stages of the process: leaf segments at 0, 4, 7, 15 and 30 days of cultivation, Type 1 primary calli (primary calli with embryogenic competence) and 2 (primary calli with no embryogenic competence), embryogenic calli, globular, torpedo and cotyledonary embryos, and mature zygotic embryos. Callus formation occurred after seven days of culture, with successive divisions of procambium cell. In this cultivation phase, it was found that Type 1 primary calli are basically formed by parenchymal cells with reduced intercellular spacing, whereas Type 2 primary calli are predominantly composed of parenchymal cells with ample intercellular spaces and embryogenic calli composed entirely of meristematic cells. After 330 days, it was evident from the differentiation of somatic embryogenesis that there was formation of globular somatic embryos, consisting of a characteristic protoderm surrounding the fundamental meristem. With the maturation of these propagules after 360 days, torpedo-stage somatic embryos arose, in which tissue polarization and early differentiation of procambial strands were verified. After 390 days, cotyledonary somatic embryos were obtained, where the onset of vessel elements differentiation was verified, a characteristic also observed in mature zygotic embryos. We concluded that somatic embryogenesis obtained from C. arabica leaves initiates from procambium cell divisions that, in the course of cultivation, produce mature somatic embryos suitable for regenerating whole plants.

Female gametogenesis and early seed development in Amaranthus hypochondriacus L.

Background: Attention to amaranth grains has increased in recent years due to the nutritional value of their seed proteins, which have high levels of the amino acid lysine. However, there is no detailed study describing the stages of seed development in Amaranthus hypochondriacus. Question: How are the developmental patterns of the female gametophyte and young seed in Amaranthus hypochondriacus ?Species studied: Amaranthus hypochondriacus L ’Revancha’ (Amaranthaceae).Study site and years of study: Plants were growth and collected from 2014 to 2016, in a greenhouse at Instituto Tecnológico de Celaya, Guanajuato, Mexico.Methods: Glomerules were collected before pollination and two weeks after anthesis. The ovules at different development stages were fixed and cleared and were analyzed by light microscopy. A clearing protocol was used to observe the developmental stages during female gametogenesis and embryogenesis.Results: We observed that the Amaranthus hypochondriacus ovule has a campylotropous form. The female gametophyte showed a Polygonum -type pattern of development. We were also able to identify all the stages from the megaspore mother cell to the cotyledon embryo stage. After meiosis, the micropylar megaspore differentiates into the functional megaspore. The embryo did not show symmetric divisions, although the final pattern is similar to that of in eudicotyledons. The suspensor showed additional longitudinal divisions, giving rise to a 2-rowed suspensor, while the endosperm showed a helobial development.Conclusions: These results will be used as baseline to identify morphological changes during seed development and to develop new strategies to improve seed quality or increase the yield.

Regeneration of plantlets through somatic embryogenesis from root derived calli of Hibiscus sabdariffa L. (Roselle) and assessment of genetic stability by flow cytometry and ISSR analysis.

Induction of somatic embryogenesis and complete plantlet regeneration from callus culture of Hibiscus sabdariffa L. var. HS4288 has been made. Leaf and root explants were cultured on Murashige and Skoog (MS) and Driver–Kuniyuki Walnut (DKW) basal media supplemented with different concentrations of synthetic auxins and cytokinins. Root explants on DKW medium supplemented with 2.26μM 2, 4-Dichlorophenoxyacetic acid (2, 4-D) and 4.65μM kinetin (KIN) induced highest percentage (70%) of embryogenic calli. Average number of globular embryos per root derived callus produced within 6 weeks of culture initiation on MS media with different plant growth regulators (PGRs) ranged from 2.27±0.12 to 8.80±0.17 and that of cotyledonary embryos ranged from 0.00 to 2.53±0.20. On DKW medium comparatively more globular embryos (2.70±0.15 to 14.53±0.23) and cotyledonary embryos (0.00 to 8.90±0.17) were produced than that of MS medium. Regeneration of complete plantlets was highest (76.67%) when embryogenic calli with mature somatic embryos were grown on DKW medium containing 2.32μM KIN and 2.22μM 6-Benzyladenine (BA). Plants were primarily hardened in humidity, temperature and light controlled chamber and finally in a greenhouse showed 70% survival ability. Different stages of somatic embryogenesis process in the root derived embryogenic calli were elaborated in detail by morphological, histological and SEM study. The data were statistically analyzed by Duncan Multiple range test (p ≤ 0.05) and Principal component analysis (PCA). Flow cytometry and Inter-simple sequence repeats (ISSR) marker analysis confirmed that there was no genetic variation within the regenerated plants.

Growth, morphology and maturation ability of Pinus pinea embryogenic suspension cultures

Stone pine (Pinus pinea L.) is a Mediterranean forest species used for the commercial harvesting of its edible seeds, known as pine nuts. Plant regeneration by somatic embryogenesis on semi-solid medium has been achieved in stone pine, but suspension culture systems for the large-scale production of somatic embryos need to be developed. Suspension cultures were established from several embryogenic lines. Growth parameters as settled cell volume (SCV) and dry weight (DW) in suspension cultures were influenced by genotype, initial inoculum density, orbiting speed and type of carbohydrate in the culture medium. A higher linear growth (DW) up to week four was obtained using 20 mg mL−1 inoculum. The orbiting speed affected the SCV as well as the morphology of embryogenic cultures. Growth parameters decreased when orbiting speed was increased from 50 rpm to 150 rpm. Cultures at 50 rpm most commonly grew as clusters of embryonal cell masses, whereas at 150 rpm there were large numbers of single cells. Cultures grew slowly in medium with maltose instead of sucrose, but growth was not affected when plant growth regulators (PGRs) concentration was decreased. A sustainable suspension culture could be maintained by transferring 250 mg of filtered cell mass to 25 mL of liquid medium at 3-week intervals. Culture scaling-up in liquid medium resulted in a 23-fold DW increase in 6 weeks, compared with 12 fold increase on semi-solid medium. Cotyledonary embryos were obtained on semi-solid maturation medium from lines proliferated in suspension cultures. Increased somatic embryo production was obtained when proliferation was performed at 100 rpm orbiting speed in liquid medium with low PGRs concentration and sucrose instead of maltose.

Cryopreservation of Pinus radiata embryogenic tissue: effects of cryoprotective pretreatments on maturation ability

The goal of the study was to quantify the effect of different cryoprotective pretreatments on the maturation ability of cryopreserved Pinus radiata embryogenic tissue. Fourteen cell lines were evaluated under five pretreatments: (1) Dimethyl sulfoxide 5% (v/v); (2) Dimethyl sulfoxide 10% (v/v); (3) Dimethyl sulfoxide 5% (v/v) and 0.09 M of l-proline; (4) Dimethyl sulfoxide 10% (v/v) and 0.09 M of l-proline; (5) 0.09 M of l-proline and compared to embryogenic tissue that was not cryopreserved (control). The cell lines were thawed at 37 °C to evaluate the recovery percentage and determine the maturation ability. The recovery rate of all cryopreserved material was significantly lower than the control and material treated with 0.09 M of l-proline, in absent dimethyl sulfoxide, showed the lowest levels of recovery. Regarding the number of embryos, an interaction between pretreatments and the cell lines under evaluation was identified. Moreover, morphological differences in the plants being treated with the most concentrated Dimethyl sulfoxide pretreatment was observed. At the microscopic level, no damage or morphological changes in proembryogenic masses were observed during the post-cryopreservation evaluation. Therefore, the best pretreatment was 5% Dimethyl sulfoxide supplemented with 0.09 M l-proline, allowing the most growth recovery of thawed samples and more cotyledonary embryos (maturation ability). The evaluated responses in presence of cryoprotective elements were genotype dependent.

Repetitive somatic embryogenesis induced cytological and proteomic changes in embryogenic lines of Pseudotsuga menziesii [Mirb.

BackgroundTo explore poorly understood differences between primary and subsequent somatic embryogenic lines of plants, we induced secondary (2ry) and tertiary (3ry) lines from cotyledonary somatic embryos (SEs) of two Douglas-fir genotypes: SD4 and TD17. The 2ry lines exhibited significantly higher embryogenic potential (SE yields) than the 1ry lines initiated from zygotic embryos (SD4, 2155 vs 477; TD17, 240 vs 29 g− 1 f.w.). Moreover, we observed similar differences in yield between 2ry and 3ry lines of SD4 (2400 vs 3921 g− 1 f.w.). To elucidate reasons for differences in embryogenic potential induced by repetitive somatic embryogenesis we then compared 2ry vs 1ry and 2ry vs 3ry lines at histo-cytological (using LC-MS/MS) and proteomic levels.ResultsRepetitive somatic embryogenesis dramatically improved the proliferating lines’ cellular organization (genotype SD4’s most strongly). Frequencies of singulated, bipolar SEs and compact polyembryogenic centers with elongated suspensors and apparently cleavable embryonal heads increased in 2ry and (even more) 3ry lines. Among 2300–2500 identified proteins, 162 and 228 were classified significantly differentially expressed between 2ry vs 1ry and 3ry vs 2ry lines, respectively, with special emphasis on “Proteolysis” and “Catabolic process” Gene Ontology categories. Strikingly, most of the significant proteins (> 70%) were down-regulated in 2ry relative to 1ry lines, but up-regulated in 3ry relative to 2ry lines, revealing a down-up pattern of expression. GO category enrichment analyses highlighted the opposite adjustments of global protein patterns, particularly for processes involved in chitin catabolism, lignin and L-phenylalanine metabolism, phenylpropanoid biosynthesis, oxidation-reduction, and response to karrikin. Sub-Network Enrichment Analyses highlighted interactions between significant proteins and both plant growth regulators and secondary metabolites after first (especially jasmonic acid, flavonoids) and second (especially salicylic acid, abscisic acid, lignin) embryogenesis cycles. Protein networks established after each induction affected the same “Plant development” and “Defense response” biological processes, but most strongly after the third cycle, which could explain the top embryogenic performance of 3ry lines.ConclusionsThis first report of cellular and molecular changes after repetitive somatic embryogenesis in conifers shows that each cycle enhanced the structure and singularization of EMs through modulation of growth regulator pathways, thereby improving the lines’ embryogenic status.

<b>Cultivation of immature <i>Capsicum</i> spp. embryos for incompatible-crossing embryo rescue

The in vitro culture of embryos is an important technique to enable the rescue of embryos from incompatible crosses. Studies analyzing the factors that affect the in vitro culture of zygotic embryos are scarce. This study evaluated the effect of the genotype (Capsicum baccatum and C. frutescens), the composition of the culture medium (MS and ½ MS medium with different concentrations of sucrose, IAA and GA3) and the stage of development (globular, cordiform, torpedo and cotyledonary) in the in vitro culture of immature embryos. Embryo germination was influenced primarily by the stage of development of the embryo and the composition of the culture medium. Regardless of the species, the most suitable culture medium for the germination of globular and cordiform embryos was ½ MS with 0.05 mg L-1 of GA3 and IAA and 40 g L-1 of sucrose. For torpedo and cotyledonary embryos, ½ MS culture medium with 20 g L-1 of sucrose without phytoregulators is recommended for germination. These results were better than those described in the literature for all development stages in the two species. The results in the present study will be useful for geneticists and genetic enhancers interested in applying germination techniques conducted in vitro for immature Capsicum embryos.

Improved germination conditions for Norway spruce somatic cotyledonary embryos increased survival and height growth of emblings

Norway spruce is one of the most cultivated tree species in Nordic countries. However, intermittent shortages of improved seeds occur. As a powerful vegetative propagation technology, somatic embryogenesis (SE) could provide an alternative solution for this problem and also shorten the time required to obtain breeding gains. However, there are still large bottlenecks in SE, e.g. in the germination and acclimatization phases, which greatly affect the final outcome of somatic embryo plants (emblings). In this work, we examined the effects of in vitro embryo storage and germination treatments and ex vitro growing techniques on the survival and growth of emblings. The study comprised 32 genotypes from 18 full-sib families in four experiments, testing two different cold storage methods, three durations of in vitro germination, lower inorganic nitrogen content in the germination medium, and two plant-growing techniques. The best treatment combination—cold storage on filter paper, lower nitrogen content in the germination medium and one-week in vitro germination—resulted in an 88% higher survival and 28% higher growth compared to the poorest, reference treatment in the same test year. These emblings could be planted after a nursery period one year sooner than that of the control emblings. The results indicate that Norway spruce emblings germinated for one week in vitro can be transplanted and grown in nurseries without any additional treatments or environmental control differing from seedlings, which is a prerequisite to reach standards for forest regeneration material.