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Pinus massoniana planted widely in southern China in recent decades. The pinewood nematode (PWN) causes pine wilt disease (PWD), which threatens pine forests, including masson pine. Resistance breeding to pinewood nematodiasis has become an important way of preventing damage caused by nematodes. In masson pine, somatic embryogenesis (SE) was established, but still restricted by many factors. In this study, whole megagametophyte were used for embryogenic callus induction. The effects of explant maturity and family on the initiation of embryogenic callus, as well as the effects of maltose and long-term subculture on the somatic embryo maturation, were investigated. The maturity of zygote embryos gradually increased as the seed collection date increased, and the initiation rate of embryogenic suspensor masses (ESM) decreased. Four types of extruded ESM were observed, and the extrusion type was related to explant maturity. The megagametophytes had the highest initiation rates when the immature zygotes were mainly concentrated in stage 3. Seed family GX4 was the female parent with highest ESM initiation rate. Long-term subculture significantly affected the somatic embryo yield. Addition of 40.0 g/L maltose to maturation medium yielded 12.7 ± 0.9 somatic embryos per milliliter. We additionally choose 16 regenerated plants and analyzed the genetic stability using SSR molecular marker. The average variation rate of per locus was 2.78%. This report presents a successful protocol for establishing SE of masson pine and provides meaningful suggestions for optimizing the large-scale propagation of masson pine. Key message: We provided meaningful suggestions for optimizing the mass propagation of masson pine and we accelerates the progress of breeding for PWN resistance. [ABSTRACT FROM AUTHOR]

To discuss the effect of different penetrants on maturation and germination of somatic embryo of Sophora davidii. Sucrose, maltose, sorbitol and PEG(6000)were used as penetrant in this paper. The development, maturation, and germination of somatic embryo was determined by different penetrant comparation. The results were as follows: the rate of somatic embryogenesis reached up to 66.21%, and the total number of embryos can achieve 79 when embryonic callus from hypocotyl of Sophora davidii were cultured in MS+ 2,4-D 0.2 mg·L-1 +NAA 1.0 mg·L-1+ 6-BA 2.0 mg·L-1 + TDZ 1.0 mg·L-1+sucrose 40 g·L-1+ glutamine 100 mg·L-1+plant gel 3 g·L-1. Adding 7% sucrose could promote the maturation rate of somatic embryo to 64.36%, meanwhile could also increase the development of polycotyledonous abnormal embryo. The maturation rate of somatic embryo went up as high as 88.89%, and the proportion of abnormal embryo the lowest, when somatic embryo were cultured in 2% maltose +2% sorbitol +4% sucrose combination medium. The maturation rate of somatic embryo was 82.35%, when the concentration of PEG was 30 g·L-1. Torpedo embryo was suitable for transported culture, the formative rate of somatic embryo was up to 90.58%. The rooting rate of mature somatic embryo was high, for 87.47% from three sugar combination culture. This study provides a theoretical reference and a feasible scheme for the development of somatic embryo seedling of Sophora davidii. [ABSTRACT FROM AUTHOR]

A cDNA-sequence-related amplified polymorphism (cDNA-SRAP) approach was used to identify relevant genes expressed at the onset of somatic embryo maturation/desiccation in banana (Musa spp. AAB group, Silk subgroup) cv. Manzano. For this, an embryogenic procedure, starting with cell suspensions, was first improved by including a culture period on a free growth-regulator medium for embryo maturation. This produced an up to 95% germination of the coleoptilar embryos. Gene expression patterns were followed through different stages of the process, including immature male flower explants, embryogenic calli, as well as immature, mature and germinated embryos. A total of 151 differentially expressed transcript-derived fragments (TDFs) were detected, sequenced, resulting in the identification of 71 by their similitude to accessions deposited in different data bases. The participation of four of these genes, putatively involved in the banana somatic embryogenesis, was further analyzed by real-time quantitative PCR (q-PCR). This confirmed their preferential expression at the transition from embryo early development to maturation. Noteworthy, tryptophan aminotransferase relate 2 (TAR2), which is involved in the indole-3-pyruvic acid dependent auxin biosynthesis, corresponded to one of the upregulated four genes, suggesting the requirement of this growth regulator at the transition stage. This study opens the path for future in-depth studies of molecular and biochemical events occurring during in vitro somatic embryogenesis in edible bananas. [ABSTRACT FROM AUTHOR]

Small non-coding RNAs (sncRNAs) are molecules with important regulatory functions during development and environmental responses across all groups of terrestrial plants. In seed plants, the development of a mature embryo from the zygote follows a synchronized cell division sequence, and growth and differentiation events regulated by highly regulated gene expression. However, given the distinct features of the initial stages of embryogenesis in gymnosperms and angiosperms, it is relevant to investigate to what extent such differences emerge from differential regulation mediated by sncRNAs. Within these, the microRNAs (miRNAs) are the best characterized class, and while many miRNAs are conserved and significantly represented across angiosperms and other seed plants during embryogenesis, some miRNA families are specific to some plant lineages. Being a model to study zygotic embryogenesis and a relevant biotechnological tool, we systematized the current knowledge on the presence and characterization of miRNAs in somatic embryogenesis (SE) of seed plants, pinpointing the miRNAs that have been reported to be associated with SE in angiosperm and gymnosperm species. We start by conducting an overview of sncRNA expression profiles in the embryonic tissues of seed plants. We then highlight the miRNAs described as being involved in the different stages of the SE process, from its induction to the full maturation of the somatic embryos, adding references to zygotic embryogenesis when relevant, as a contribution towards a better understanding of miRNA-mediated regulation of SE.

With embryogenic callus of Larix olgensisis, we investigated the effects of inositol, glutamine, casein hydrolysate, carbohydrate, abscisic acid and silver nitrate concentration on the maturation of the somatic embryo. Three dominant factors emerged, and we developed a response surface model based on the Box-Behnken design. We defined the optimal conditions for the maturation of somatic embryos. The contents of abscisic acid, silver nitrate, sucrose and casein hydrolysis significantly affected the amount of maturing embryos, but inositol, maltose and glutamine had no effect. By establishing a response surface model with multiple factors, we predicted that the optimal number of L. olgensis somatic embryos was 204 ± 4 g−1 on basal medium, containing 18.28 mg L−1 abscisic acid, 5.46 mg L−1 silver nitrate and 82.67 g L−1 sucrose. In the verification experiments, the addition of 20 mg L−1 abscisic acid, 5 mg L−1 silver nitrate and 80 g L−1 sucrose to BM yielded an average of 202.06 somatic embryos per gram. These results should guide large-scale breeding of L. olgensis. [ABSTRACT FROM AUTHOR]

Key message: This study provides a novel desiccation indicator for somatic embryos and an efficient system for somatic seedling production in Picea asperata Mast. Abstract: Picea asperata Mast. is a widely distributed native spruce in China with outstanding wood properties and adaptability. In this study, we established an effective system to propagate plantlets through somatic embryogenesis in P. asperata. The effects of cone collection date, seed family, and plant growth regulators on the initiation of embryonal masses (EM) were analyzed. Besides the significant effects of seed family and cone collection date on EM initiation, the highest induction frequency was obtained with modified Litvay medium (MLV) containing 10 µM 2,4-dichlorophenoxyacetic acid and 5 µM 6-benzylaminopurine. The best tissue proliferation in suspension cultures was obtained with an inoculum density of 1:7 (v/v, sedimented cells:liquid medium) after 9 days in culture. The best medium for somatic embryo (SE) maturation was MLV supplemented with 105 µM (±)-abscisic acid, 5% polyethylene glycol 4000, 3% sucrose, 0.1% activated charcoal (AC), 0.05% l-glutamine, 0.1% casein acid hydrolysate, and 0.4% gellan gum. In this medium, 415 ± 103 SEs with well-developed cotyledons were obtained per gram (fresh weight) of EM. After partial desiccation treatment under low light intensity, somatic embryos, depending on their color change, were divided into green cotyledonary embryos (GCEs) and non-green cotyledonary embryos (NGCEs). These embryos, along with non-desiccated SEs as controls, were germinated on 1/2 MLV supplemented with 2% sucrose, 0.2% AC, 0.05% l-glutamine, and 0.6% gellan gum. Only GCEs, with red radicles, produced plantlets at the highest rates (>74.5%) among all of the treatments. This efficient system of somatic seedling production in P. asperata includes screened culture factors and a novel desiccation indicator to ensure SE quality before germination. [ABSTRACT FROM AUTHOR]

• PA levels increased during maturation of grapevine somatic embryos over a semipermeable membrane. • The increase in PAs was mostly due to free putrescine. • Accumulation of free putrescine was successively supported by new synthesis and spermidine degradation. • VvADC and VvPAO gene expression contributed to explain the increase in free putrescine. • Results support the involvement of PA and ABA metabolisms in somatic embryo maturation. The endogenous content of polyamines and the expression of genes involved in their metabolism were analyzed in grapevine (Vitis vinifera L. 'Mencía') somatic embryo aggregates to determine the effect of a semipermeable membrane on their maturation in differentiation medium. The endogenous polyamine content in the somatic embryo aggregates was higher in those cultured over the semipermeable membrane and significantly increased with culture time due to an increase in the free polyamine fraction. Free putrescine represented more than 95% of the total free polyamine fraction and significantly peaked in the second week of culture of the somatic embryo aggregates over the semipermeable membrane. This finding appears to be supported by active expression of the VvADC gene and the low free spermidine level. Another significant peak of free putrescine was detected at the end of culture over the membrane, in which free spermidine level remained low despite the V vSPDS2 gene was upregulated. Hence, it is advisable that this increase in free putrescine was supported by back conversion from spermidine through VvPAO expression. As the semipermeable membrane successfully avoided precocious germination of the grapevine somatic embryos, the results support that polyamine metabolism, particularly putrescine metabolism, is involved in their correct maturation. [ABSTRACT FROM AUTHOR]

Key message: This paper provides an important direct proembryogenic mass initiation and plant regeneration method that might be supportive and stable for gene transformation and commercial production of Torreya grandis. Abstract: Torreya grandis is an important and commercially valuable tree that is utilized as a source of essential oil and nuts. Propagation researches of T. grandis have been focused on micropropagation and somatic embryogenesis which derived from callus but limited data regarding direct somatic embryogenesis and plant regeneration are available in the literature. This study investigated the effects of plant growth regulators combinations on direct proembryogenic masses initiation and plant regeneration. Proembryogenic masses were initiated from cotyledons and hypocotyls of immature zygotic embryos directly. The frequency of direct proembryogenic masses initiation (52.6 %) was significantly greater than other treatments when the explants were cultured on Schenk and Hildebrandt medium which supplemented with 0.1 mg l N6-benzylaminopurine, 0.1 mg l Kinetin and 0.1 mg l 2,4-dichlorophenoxyacetic acid. Combined application of Abscisic acid and Polyethylene glycol 6000 stimulated cotyledons formation and resulted in morphologically superior somatic embryos. Schenk and Hildebrandt medium supplemented with 10 mg l Abscisic acid and 20 mg l Polyethylene glycol 6000 was optimum (88 %) for somatic embryos maturation. Highest germinated somatic embryos percentage (35.6 %) was obtained on Schenk and Hildebrandt medium supplemented with 2000 mg l activated charcoal, with root and shoot lengths of 2.3 and 1.9 cm, respectively. As proembryogenic masses were initiated without the formation of callus, this study will aid to establish stable genetic transformation and will also be useful in improving understanding of the somatic embryogenesis of T. grandis. [ABSTRACT FROM AUTHOR]

Summary: The levels of abscisic acid (ABA), its conjugate ABA-GE, and IAA were determined in embryogenic calli of Vitis vinifera L. (cv. Mencía) cultured in DM1 differentiation medium, to relate them to the maturation process of somatic embryos. To achieve this goal, we developed an analytical method that included two steps of solid-phase extraction, chromatographic separation by HPLC, ABA-GE hydrolysis, and sensitive ELISA quantification. Because the ABA immunoassay was based on new polyclonal antibodies raised against a C4′-ABA conjugate, the assay was characterized (detection limit, midrange, measure range, and cross-reaction) and validated by a comparison of the ABA data obtained with this ELISA procedure and with a physicochemical method (LC–ESI–MS/MS). Radioactive-labeled internal standards were initially added to callus extracts to correct the losses of plant hormones, and thus assure the accuracy of the measurements. The endogenous concentration of ABA in the embryogenic callus cultured in DM1 medium was doubled at the fifth week of culture, concurring with the maturation process of somatic embryos, as indicated by the accumulation of carbohydrates observed through histological analysis. The ABA-GE content was higher than ABA, decreasing at 21 days of culture in DM1 medium but increasing thereafter. The data suggest the involvement of the synthesis and conjugation of ABA in the final stages of development in grapevine somatic embryos from embryogenic callus. IAA levels were low, suggesting that auxin plays no significant role during the maturation of somatic embryos. In addition, the lower ABA levels in calli cultured in DM differentiation medium with PGRs, a medium presenting high precocious germination and deficiencies in somatic embryo development indicate that an increase in ABA content during the development of somatic embryos in grapevine is necessary for their correct maturation. [ABSTRACT FROM AUTHOR]

Regeneration of avocado via somatic embryogenesis is difficult due to poor embryo maturation, resulting in low frequencies of germination. In this study, the influence of semi-permeable cellulose acetate membranes and culture media, containing high levels of sucrose along with coconut water, on maturation and germination of somatic embryos of avocado have been evaluated. The culture of embryogenic calli on top of cellulose acetate membranes significantly increased the number of mature, white-opaque embryos that were recovered after 5 weeks of culture. These embryos showed a much more normal appearance and better quality compared with the control embryos, although the embryo size was significantly reduced. To increase the embryo size and to complete maturation, several two-step maturation treatments were tested. The culture of white-opaque somatic embryos in a modified MS medium with B5 macronutrients gelled with 10 g L agar (B5m10A medium) over a 5-week period, followed by 5 additional weeks in B5m10A with 45 g L sucrose and 20 % coconut water, yielded the best results, reducing the percentage of necrotic embryos and the number of calli formed. The beneficial effects of this maturation treatment were enhanced when using embryos that were pre-matured on cellulose acetate membranes. Following this two-step maturation treatment, the germination rate of the control somatic embryos, which were not cultured on cellulose membranes, was lower than 10 %, but it significantly improved when the embryos had been pre-matured on cellulose acetate membranes for 5 weeks, reaching a germination rate close to 40 %. The water availability was significantly reduced when somatic embryos were cultured on cellulose membranes, and after this pre-maturation treatment, the white-opaque embryos showed lower water potential and ABA content compared with the control embryos. These results suggest that culturing over cellulose membranes causes a controlled embryo desiccation that enhances the recovery of plants. [ABSTRACT FROM AUTHOR]

Suspension cultures have been established from embryogenic tissues of Pinus nigra initiated from immature zygotic embryos. The growth of tissues in liquid medium has been influenced by initial tissue weight used for the establishment of the cultures as well as by genotype. In most of the cases initial tissue weight 0.5 g was insufficient and the cultures showed poor growth and later degeneration. Higher amount of initial tissues (1 or 2.5 g) was more efficient for the establishment and proliferation of somatic embryos in liquid medium. The growth of suspension cultures was also cell line dependent. Somatic embryo maturation in liquid medium was very limited and no plantlet regeneration occurred. Cotyledonary somatic embryos developed and produced emblings when the suspension was plated on filter paper discs and cultured on solid maturation medium. Based on our experiments we can state that the embryogenic tissues are able to grow and proliferate in liquid medium but somatic embryo maturation and plantlet regeneration occur only on solid medium. [ABSTRACT FROM AUTHOR]

Maturation and germination of somatic embryos of hybrids A. alba × A. alba and A. alba × A. nordmanniana were followed by protein analysis of single embryogenic -suspensor masses (ESM) and analysis of storage protein accumulation during somatic embryo development. Very important step was one week pre-cultivation of ESM on medium with polyethylene glycol (PEG) and abscisic acid (ABA). Low osmotic potential of maturation medium and addition of ABA supported development of somatic embryo. Also partial drying of somatic embryo during following three weeks was needed for its normal development. In spite of morphologically fully developed, the somatic embryos were not physiologically ready for germination at least in terms of storage protein accumulation. [ABSTRACT FROM AUTHOR]

Changes in cellular protein accumulation and in in vivo and in vitro protein synthesis, in somatic embryo tissues of white spruce during a 42 d maturation period were followed by two-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis (2-D SDS-PAGE). These investigations were complemented by an analysis of uptake and fate of fed abscisic acid (ABA) in somatic embryo tissues grown on maturation medium. When Stage 1 somatic embryos were cultured on ABA-containing maturation medium, many changes were observed in patterns of gene expression and in protein synthesis and accumulation which could be associated with embryo development. The polypeptides observed could be categorized as constitutive, embryo-abundant, embryo maturation-related and embryo stage-related, as well as those with non-specific changes. Accumulation of label from fed 3H-(+)-ABA in embryo tissues reached a plateau 3 d after Stage 1 somatic embryos were placed on maturation medium. ABA taken into tissues was rapidly metabolized, and 40% of radioactivity in tissues after 1 d of culture resulted from ABA metabolites. This value increased to 90% after 3 weeks culture. Conjugated ABA and oxidized ABA (phaseic acid and dihydrophaseic acid) were major forms of ABA metabolites in spruce embryo tissues. Using a single 42 d culture period following transfer to medium with ABA, the conditions that stimulate the sequence of developmental changes of somatic embryo maturation during the first 21 d do not reoccur during the second 21 d. Unless greater synchronization of Stage 1 cultures can be achieved, it is therefore unlikely that yields of mature somatic embryos will be increased by this method. [ABSTRACT FROM PUBLISHER]

Small non-coding RNAs (sncRNAs) are molecules with important regulatory functions during development and environmental responses across all groups of terrestrial plants. In seed plants, the development of a mature embryo from the zygote follows a synchronized cell division sequence, and growth and differentiation events regulated by highly regulated gene expression. However, given the distinct features of the initial stages of embryogenesis in gymnosperms and angiosperms, it is relevant to investigate to what extent such differences emerge from differential regulation mediated by sncRNAs. Within these, the microRNAs (miRNAs) are the best characterized class, and while many miRNAs are conserved and significantly represented across angiosperms and other seed plants during embryogenesis, some miRNA families are specific to some plant lineages. Being a model to study zygotic embryogenesis and a relevant biotechnological tool, we systematized the current knowledge on the presence and characterization of miRNAs in somatic embryogenesis (SE) of seed plants, pinpointing the miRNAs that have been reported to be associated with SE in angiosperm and gymnosperm species. We start by conducting an overview of sncRNA expression profiles in the embryonic tissues of seed plants. We then highlight the miRNAs described as being involved in the different stages of the SE process, from its induction to the full maturation of the somatic embryos, adding references to zygotic embryogenesis when relevant, as a contribution towards a better understanding of miRNA-mediated regulation of SE. [ABSTRACT FROM AUTHOR]

This report presents an efficient protocol of the stable genetic transformation of coffee plants expressing the Cry10Aa protein of Bacillus thuringiensis. Embryogenic cell lines with a high potential of propagation, somatic embryo maturation, and germination were used. Gene expression analysis of cytokinin signaling, homedomains, auxin responsive factor, and the master regulators of somatic embryogenesis genes involved in somatic embryo maturation were evaluated. Plasmid pMDC85 containing the cry10Aa gene was introduced into a Typica cultivar of C. arabica L. by biobalistic transformation. Transformation efficiency of 16.7% was achieved, according to the number of embryogenic aggregates and transgenic lines developed. Stable transformation was proven by hygromycin-resistant embryogenic lines, green fluorescent protein (GFP) expression, quantitative analyses of Cry10Aa by mass spectrometry, Western blot, ELISA, and Southern blot analyses. Cry10Aa showed variable expression levels in somatic embryos and the leaf tissue of transgenic plants, ranging from 76% to 90% of coverage of the protein by mass spectrometry and from 3.25 to 13.88 μg/g fresh tissue, with ELISA. qPCR-based 2−ΔΔCt trials revealed high transcription levels of cry10Aa in somatic embryos and leaf tissue. This is the first report about the stable transformation and expression of the Cry10Aa protein in coffee plants with the potential for controlling the coffee berry borer. [ABSTRACT FROM AUTHOR]

Shoot-tip explants obtained from offshoots of adult date palms are an excellent source for callus induction and subsequent somatic embryogenesis. In this protocol, the shoot-tip explants are transferred sequentially to a series of media containing gradually reduced concentrations of plant growth hormones: (a) 10 mg/L 2,4-dichlorophenoxy acetic acid (2,4-D) and 3 mg/L 2-isopentenyl adenine (2iP), (b) 7 mg/L 2,4-D and 1 mg/L 2iP, (c) 5 mg/L 2,4-D and 1 mg/L 2iP, and (d) 3 mg/L 2,4-D and 1 mg/L 2iP. Embryogenic callus differentiates into somatic embryos upon transfer to MS medium containing 0.5 mg/L abscisic acid (ABA) and 0.1 mg/L naphthalene acetic acid (NAA). Well-matured somatic embryos germinate on a medium containing 0.1 mg/L NAA. Repeated, multiple, and secondary somatic embryos are induced to produce normal well-developed somatic embryos upon transfer to MS medium containing 0.1 mg/L NAA and 0.05 mg/L benzyladenine (BA). This protocol is potentially applicable for commercial micropropagation of date palm.