The aim of this study is to compare physiological development of Heliconia bihai cv. Lobster Claw Two plants derived from in vitro culture of zygoticembryos and conventional propagation. Heliconias obtained from rhizomes and from in vitro multiplication were evaluated every 30 days during ten months under greenhouse conditions. The experimental design was completely randomized, with ten repetitions, and the 2x10 factorial arrangement consisted of two plant multiplication methods and ten evaluations performed at different times. The analyzed biometric parameters were plant height, number of leaves, number of tillers, leaf area, and color intensity in the bracts. Plants derived from in vitro culture showed significant differences in the development of the evaluated physiological parameters in comparison to plants derived from rhizomes, and they also showed early flowering. Although the in vitro cultured plants were derived from zygoticembryos, no morphological changes were found in the vegetative and reproductive parts (inflorescence) of the plants or in the colorimetry. It shows that the in vitro cultures of zygoticembryos may be used as a technique to produce seedlings on a large-scale, thus allowing the floriculture sector to grow in the region and all over the country.
The period of 4 to 8 hours of imbibition can be considered promising for conductivity analysis in coconut zygoticembryos with low values. Vieira (1994) reported that the lower the electrolyte leakage, the lower the possibility of damage to the cell membrane and, consequently, the lower the conductivity. Souza et al. (2009) concluded that from 6 hours of incubation it was possible to separate batches of two castor bean cultivars, with significant reduction in the incubation period of the seed in comparison to the 24‑hour period, which is adopted as a standard for most species.
The interspecific oil palm hybrid BRS Manicoré (E. guineensis x E. oleifera) has superior agronomic characteristics. However, the germination rate is low (30%) and the process is slow when the seeds are sown in a conventional form. The purpose of this study was to optimize the in vitro germination of zygoticembryos of this hybrid comparing seed lots. The viability of zygoticembryos was evaluated by the tetrazolium test (0.075%) for 4 h. The embryos were cultured on MS and Y3 culture media, with and without the addition of NaH 2 PO 4 , as well as on MS, MS½ and N6 medium. In MS medium containing NaH 2 PO 4 , the germination rate was increased from 40 to 70% in comparison with the medium without sodium phosphate. The comparison between the culture media MS, MS ½, N6 and Y3 showed that 75% of zygoticembryos cultured in the Y3 medium formed whole plants (with roots and shoots defined), a higher percentage than embryos cultured on MS, MS ½ and N6 media (46, 35 and 17% respectively). In the same Y3 culture medium, the embryos were larger (36% ≥ 2 cm and 30% ≥ 5 cm) than in the other media. Results obtained by the tetrazolium test were similar to those of germination, showing the effect of the genotype of each seed lot. For the germination and development of plantlets it is essential to add NaH 2 PO 4 to a culture medium containing no phosphate or with a low phosphate concentration.
In response to a growing interest in improving seedling production of oilseed species (like macaw palm), a fruit drying protocol for facilitating seed extraction was proposed. This enabled the production of macaw palm seedlings, but the temperature most suitable for seed extraction without losing its physiological quality is unknown. The goal of this study was to evaluate the effects of different drying temperatures on the physiological quality of macaw palm zygoticembryos to improve previously published drying methods. Fruits were dried in a forced-air drying oven at 57 or 37°C at different time periods (zero, two, four, six and eight days). Following each drying period, the fruits were removed from the drying oven, and the water content of the fruits and seeds were measured in addition to embryo viability and in vitro germination. Seed water content could be estimated based on fruit water content at both drying temperatures, eliminating the need to remove the seeds from the fruit. Drying at 57°C decreased the drying time by 50% compared to drying at 37°C; however, it was detrimental to embryo viability and germination. Therefore, drying of fruit at 37°C is recommended. Embryos dried at this temperature were still able to germinate after 16-day drying period, which corresponded to a decrease of 24.8% in the initial fruit water content.
Figure 2. Percentage of regeneration (a), number of shoots (b), buds (c) and leaves (d), multiplication rate (e), shoot length (f), number of roots (g) and callus formation (h) obtained from zygoticembryos of Caesalpinia ferrea after 30 days of culture in vitro in MS medium supplemented with different concentrations of BAP and NAA. Means followed by the same letter do not differ statistically among themselves by the Duncan’s test (p < 0.05).
important medicinal and economical properties. The development of improved micropropagation techniques is necessary to provide rapid and efficient clonal propagation of elite genotypes with high resistance and uniform production, as well as a system that can be used for genetic transformation. For this reason, we focused on establishing a protocol for somatic embryogenesis in P. ligularis from mature zygoticembryos. Our results demonstrate that the highest frequencies of somatic embryo formation was observed on a culture medium supplemented with 27.2 µM 2,4- diclorophenoxyacetic acid plus 4.5 µM 6-benzyladenine. Histological analyses of somatic embryogenesis were performed every 7 days after induction over 60 days of exposure to the medium. We present clear evidence for the precise origin of de-differentiation. Initial cell divisions occurred from a group of cells (multicellular origin) on the abaxial surface of the cotyledon in the periphery of the epidermal tissues of mature zygoticembryos after 14 days of incubation. After 21 days, internal segmenting divisions resulted in the embryogenic character of the tissue. Globular embryos contain a protoderm surrounding a mass of vacuolated parenchymatous cells and meristematic regions with an observable procambium zone after 45 days. The complete independence of somatic embryos from the adjacent tissues was histologically confirmed by the absence of vascular continuity between them, after 60 days. We describe for the first time the development of somatic embryos in P. ligularis and demonstrate that somatic embryos develop into plants that can later on be acclimatized.
somatic embryogenesis in interspecific zygoticembryos collected 100 days after pollination. Three progenies were cultivated in an induction medium developed for Tenera (E. guineensis tp. dura x pisifera) embryos. The number of embryos bearing calli and germinating was recorded and submitted to the Z test. Calli were weighted and submitted to histological analysis. Progenies differed in the number of embryos presenting plumules and calli simultaneously. By the ninth month, the apices of incompletely developed somatic embryos were observed protruding from the surfaces of nodular calli. Highly embryogenic and friable secondary calli producing globular somatic embryos were not observed.
Cryopreservation, however, has been used for the conservation of genetic resources in many species, including coconut. Early studies on the cryopreservation of coconut were carried out by Assy- Bah & Engelmann (1992), who used a vitrification technique on mature zygoticembryos of the PB 121 hybrid coconut (Malayan Yellow Dwarf x West African Tall) and of the Cameroon Red Dwarf, Indian Tall, and Rennel Tall varieties. This technique is based on the exposure of explants to glycerol-based vitrification
has been heavily affected by the exploitation of natural resources. Thus, the aim of this study was to evaluate the induction of buds from nodal segments of in vitro germinated plants, aiming at the conservation of E. velutina. In the experiment I, intact seeds and zygoticembryos were inoculated in WPM culture and the number of buds, leaves and roots, germination percentage, shoot length and percentage of normal plants were analyzed. In the experiment II, the nodal segments of plants from germination of zygoticembryos in vitro were inoculated in WPM culture supplemented with BAP and CIN [0.0, 10.0, 20.0, 30.0 and 40.0 µM] and percentage of responsive explants to form buds, number of shoots and leaves, shoot length and the percentage of callus formation were analyzed. The cultivation of zygoticembryos provided more vigorous and uniform plants, with more gems than those obtained from the germination of intact seeds. The concentration of 20 µM of BAP is indicated for the regeneration of plants from zygoticembryos of E. velutina; with this concentration, an average of 2.78 shoots/explant was obtained.
Data from the literature show that after the formation of embryogenic calli, the reduction of auxin or its elimination from the culture medium is recommended, as it may have an inhibitory effect on the formation of somatic embryos. According Fehér (2015), in order to allow the establishment of cell or tissue polarity and embryonic development, 2,4-D should be removed from the culture medium. Fehér (2015) reports studies with Arabidopsis showing that 2,4-D removal induces the establishment of auxin synthesis and the transport of polar auxins is a key step in the formation of the meristem underlying embryonic development. According to Guerra & Handro (1998), successive subcultures in culture medium with high concentrations of auxin for the multiplication of embryogenic calli can affect negatively and even prevent the development of somatic embryos. Balzon et al. (2013) observed that the reduction of the auxin concentration from 450 to 40 μM was important to establish repetitive cycles of cell division, thus enabling the multiplication of the embryogenic callus of E. guineensis. The same observations were described when the auxin is reduced in
In spite of the major role conifers are bound to play in reforestation strategies, current research on their vegetative propagation is not sufficiently developed (Sutton 2002). In part, this is due to the slow progress in propagation methods, mainly because of rooting problems associated with the tree maturation phase, an age-related develop- mental process that affects reproductive competence, morphology, and growth rate (Greenwood and Hutchison 1993). This notwithstanding, commercial scale propagation through rooted cuttings of young trees has been reported for radiata pine (Pinus radiata D. Don.), Norway spruce (Picea abies [L.] Karst.), Sitka spruce (Picea sitchensis [Bong.] Carr.), black spruce (Picea mariana [Mill.] B.S.P.) and sugi (Cryptomeria japonica D. Don) (Menzies et al. 2001). For a few economically important forest conifer species, an alternative vegetative, large-scale in vitro propagation technology has been developed, called somatic embryogenesis that utilizes mature or immature seed embryos as starting explants (reviewed by Klimaszewska et al. 2007). The advantages of somatic embryogenesis over rooted cuttings are: unlimited number of clonal
Several studies have been conducted to commercial- ize in vitro propagation of oil palm that could help to over- come the limitations associated with conventional asexual propagation techniques and to increase the number of plants produced in a short time. Two main sources of explants were chosen for the in vitro culture; young leaf (Ahee et al., 1981; Pannetier et al., 1981) and zygoticembryos (Teixeira et al., 1993). In these two explants, embryo explants are more convenient because fruit are readily available, have a high degree of physiological uniformity, and can be shipped to a long distances. Recently, secondary somatic embryogenesis (SSE) was reported to be an efficient technique for plant propagation from both sources of explants (Hilae and Te- chato, 2007). Multiple shoot formation in in vitro culture is a disadvantage by using single shoot in this palm. Induction of embryo-derived shoots (EDS) through somatic embryo- genesis is now recognized as a useful technique for propa- gation and in vitro conservation of oil palms. The whole
Our studies also indicated that the zygoticembryos of A. sellowiana develop via similar stages to those reported for in vitro somatic embryos by Canhoto & Cruz (1996b), Canhoto et al. (1996), Canhoto et al. (1999) and R. Pescador (unpublished). However, zygotic and somatic embryos can show structural differences. At the cotyledonary stage since the zygotic embryo becomes spiral whilst the somatic embryo is always erect. It is also important to mention that during zygotic embryogenesis the nucellus and endosperm are formed, structures which are absent in somatic embryogenesis. According to Dodeman et al. (1997), comparison between zygotic and somatic embryogenesis can be made from the globular stage.
detection, were mounted in synthetic resin. The sections were analyzed and photographed in a U-photo system light microscope, camera and microcomputer with the Spot-Basic software. For scanning electron microscopy, somatic embryos were fixed in 3% glutaraldehyde in 0.1 M sodium cacodylate buffer at 4 ºC, dehydrated through a graded ethanol series, followed by liquid CO2 critical point drying. The samples were mounted on the supports, coated with gold, observed and photographed in a scanning electronic microscope. The histological examination confirmed the development of pro- embryos, from which somatic embryos morphologically similar to zygoticembryos were produced, however the zygoticembryos presented a well developed procambium, a large amount of storage reserves and were tenfold larger than the somatic embryos. Histological analyses of somatic embryos showed several anomalies, which hindered the conversion of those embryos to plantlets. Besides the anomalies, the lack of apical meristem and the much less procambium developed than in the zygoticembryos also contribute to the low conversion to plantlets. The histochemical analyses of somatic and zygoticembryos proved that the storage reserves in both is starch, however, in somatic embryos, the amount of starch is smaller and of sporadic occurrence, being one of the causes of the low conversion to plantlets. Histological analyses showed that somatic embryos progress through different stages of development such as globular, heart shape, torpedo and cotyledonary, similar to zygoticembryos. The morphology of somatic and zygoticembryos is similar, both developing a pair of cotyledons and reduced embryonic axis.
Regulation of seed development by small non-coding RNAs (sRNAs) is an important mechanism controlling a crucial phase of the life cycle of seed plants. In this work, sRNAs from seed tissues (zygoticembryos and megagametophytes) and from somatic embryos of Pinus pinaster were analysed to identify putative regulators of seed/embryo development in conifers. In total, sixteen sRNA libraries covering several developmental stages were sequenced. We show that embryos and megagametophytes express a large population of 21-nt sRNAs and that substantial amounts of 24-nt sRNAs were also detected, especially in somatic embryos. A total of 215 conserved miRNAs, one third of which are conifer-specific, and 212 high-confidence novel miRNAs were annotated. MIR159, MIR171 and MIR394 families were found in embryos, but were greatly reduced in megagametophytes. Other families, like MIR397 and MIR408, predominated in somatic embryos and megagametophytes, suggesting their expression in somatic embryos is associated with in vitro conditions. Analysis of the predicted miRNA targets suggests that miRNA functions are relevant in several processes including transporter activity at the cotyledon-forming stage, and sulfur metabolism across several developmental stages. An important resource for studying conifer embryogenesis is made available here, which may also provide insightful clues for improving clonal propagation via somatic embryogenesis.
cotyledonary embryos difered in the diferent culture media (Table 2). All treatments produced embryos in each of the three size categories: small, medium, and large. The producion of cotyledonary embryos of diferent sizes is commonly observed in cylindrical bioreactors and may be due to the decreased light in the interior of the embryo mass inside the bioreactor (Ducos et al. 2007a). Barry-Eienne et al. (2002) obtained 86% germinaion of somaic embryos using RITA bioreactors, but these embryos exhibited heterogeneous anatomical features. Similarly, for Cofea canephora, Ducos et al. (2007b) reported heterogeneity in cotyledonary embryos grown in large (8 L) cylindrical temporary immersion bioreactors. The use of horizontal bioreactors reduces the heterogeneity of embryo size by exposing the embryo mass to more incident light (Ducos et al. 2007a).
β -Galactosidase ( β -gal) activity was detected by a histochemical assay. Twenty-four hours after bombard- ment, embryos were fixed in a 4% paraformaldehyde so- lution for 5 min at 4 ° C, followed by staining for 4 h at 37 ° C with a chromogenic substrate containing 100 mM sodium phosphate, 1.3 mM MgCl 2 , 3 mM K 4 Fe(CN) 6 , 3 mM K 3 Fe(CN) 6 and 1 mg X-gal (5-bromo-4-chloro-3- indolyl- β -D-galactopyranoside)/ml. β -Galactosidase expres- sion was detected histochemically by the hydrolysis of X- gal to produce insoluble indigo. The resulting blue color indicated the localization of β -gal activity in transformed embryos. Transformation was verified by the absence or presence of blue points (expression units) representing gene expression in a cell or group of cells. All embryos with blue points were considered in the analyses, independently of the number of units expressed. Embryos that showed β -gal activity were classified into two expression categories: 1) expression in embryo body and extra-embryonic tissue (to- tal expression), 2) expression in embryo body.
heterogeneous combination of genes, zebrafish aid and human MBD4 elicited DNA demethylation in zebrafish embryos, which was promoted by the additional expression of gadd45a . Based on these findings, together with other supporting data, they proposed a mechanism for demethylation that is exerted by the concerted action of the three products: the conversion of 5mC to T via deamination by AID, followed by thymine base excision repair by MBD4, can occur in zebrafish embryos, and is facilitated by Gadd45a, which may serve as a scaffold to physically and/or functionally couple AID and MBD4. To the best of our knowledge, this was the first evidence for the involvement of T-G mismatches in vertebrate DNA in global demethylation and provided the rationale for the three factors to be investigated for active demethylation in mammalian cells; however, questions were raised on their model . Following this study, Rai et al. suggested that the proposed demethylation machinery may be involved in regulating intestinal cell fating .
The goal for this phase is to simulate the events that would occur in the oviduct and uterus, so that putative zygotes obtained after fertilization would undergo optimal development into blastocysts. For this reason, culture can be performed in vivo using surrogate oviducts . IVC, on the contrary, is achieved in a controlled atmosphere at 38.5–39°C with 5% O2, 5% CO2, and 90% N2. This medium is usually composed of synthetic oviductal fluid supplement‐ ed with amino acids, bovine serum albumin (BSA), and/or serum [112,122], although the use of some media with oviduct cells or fetal fibroblasts was also reported . In this stage, it is important to keep an oxygen low concentration to prevent oxidation  and the deleterious effects of reactive oxygen species (ROS). Some low molecular weight thiol compounds are also added to IVM and IVC media, such as cysteamine, β-mercaptoetha‐ nol, and cysteines, to increase the synthesis of glutathione (GSH), which in turn reduces the oxidative stress . The embryos spend 6 to 7 days in culture before being transferred in fresh or after cryopreservation.
Further studies of murine ESCs revealed that there are distinct states of pluripotency (naïve and primed) that differ both morphologically and functionally (De Los Angeles et al., 2012). Naïve murine pluripotent stem cells are derived from the inner cell mass (ICM) or early epiblast cells, proliferate in culture as packed dome-like colonies, are maintained in the undifferentiated state by LIF and BMP4 signaling, readily contribute to germline transmitting chimeric embryos, maintain two active X chromosomes (in female cells) and are relatively resistant to differentiation into primordial germ cells (PGCs) and extra-embryonic lineages (Kuijk et al., 2011). In contrast, primed pluripotent stem cells are derived from the epiblast of post-hatching murine blastocysts, are termed epiblast stem cells (EpiSCs), are molecularly and epigenetically different from murine ESCs (Brons et al., 2007; Tesar et al., 2007), have a more flattened colony morphology, depend on bFGF or TGFa/activin signaling, exhibit a limited ability to contribute to chimeras and have undergone X-chromosome inactivation (Brons et al., 2007). Human ESCs were first derived in 1998 (Thomson et al., 1998) and, surprisingly, they exhibit characteristics more like those of primed murine EpiSCs than their naïve murine ESC counterparts (Thomson et al., 1998).