The ultrastructure of somatic embryo development in pearl millet (Pennisetum glaucum; Poaceae)

The ultrastructure, morphology, and histology of somatic embryogenesis in pearl millet (Pennisetum glaucum) were examined using light and electron microscopic techniques. Somatic embryogenesis was initiated from zygotic embryo explants cultured 8 d after pollination. Formation of a ridge of tissue began 3–4 d after culture (DAC) by divisions in the epidermal and subepidermal cells of the scutellum. Ridge formation was accompanied by a decrease in vacuoles, lipid bodies, and cell size, and an increase in endoplasmic reticulum (ER). Proembryonic cell masses (proembryoids) formed from the scutellar ridge by 10 DAC. Proembryoid cells had abundant Golgi bodies and ER while the amounts of lipids and starch varied. Somatic embryos developed from the proembryonic masses 13 DAC and by 21 DAC had all the parts of mature zygotic embryos. Although shoot and root primordia of somatic embryos were always less differentiated than those of zygotic embryos, scutellar cells of somatic and zygotic embryos had similar amounts of lipids, vacuoles, and starch. Somatic scutellar epidermal cells were more vacuolated than their zygotic counterparts. In contrast, somatic scutellar nodal cells were smaller and not as vacuolated as in zygotic embryos. Somatic embryogenesis was characterized by three phases of cell development: first, scutellar cell dedifferentiation with a reduction in lipids and cell and vacuole size; second, proembryoid formation with high levels of ER; and third, the development of somatic embryos that were functionally and morphologically similar to zygotic embryos.     

Comparative study of reserve lipid accumulation during somatic and zygotic <Emphasis Type="Italic">Acca sellowiana</Emphasis> (O. Berg.) Burret embryogenesis

Somatic embryogenesis is an in vitro morphogenetic route in which isolated cells or a small group of somatic cells give rise to bipolar structures resembling zygotic embryos. Lipids, carbohydrates, and proteins are major compounds in plant and animal metabolism. Comparative analysis along different developmental stages of Acca sellowiana (Myrtaceae) zygotic and somatic embryos, revealed a progressive increase in levels of total lipids. A high degree of similarity could be found in the total lipids composition between A. sellowiana somatic and zygotic embryos. High lipid levels were found in zygotic embryos in the torpedo and cotyledonary stages, and these levels increased according to the progression in the developmental stages. Somatic embryos obtained through direct embryogenesis route showed higher levels of lipids than in indirect somatic embryogenesis. The compounds most frequently were linoleic acid (C18:2), palmitic (C16:0) and oleic (C18:1). These results indicate a high similarity degree of accumulation of total lipids, regardless of zygotic or somatic embryogenesis.     

Morphology and Anatomy of Pisum Sativum Somatic Embryos

The morphological and anatomical aspects of direct and indirect somatic embryogenesis in pea were described. Direct embryos were induced from shoot apical meristems of 3 to 5-d-old pea seedlings, embryogenic callus originated from immature pea zygotic embryos or shoot apices. Auxin (picloram, 2,4-dichlorophenoxyacetic acid) was necessary to induce somatic embryos. The developmental stages typical for pea zygotic embryos were detected. Globular and heartshaped somatic embryos were morphologically similar to their zygotic counterparts; in contrast, torpedo and cotyledonary somatic embryos displayed great morphological variation, which affected mainly cotyledons (size, shape, number). Based on anatomical sections, possible ways of somatic embryo formation and localization of initiation sites within primary explant tissue have been proposed. The multicellular origin of somatic embryos is supposed in both systems of pea somatic embryogenesis under investigation.     

The ultrastructure of zygotic embryo development in pearl millet (Pennisetum glaucum; Poaceae)

The ultrastructure, morphology, and histology of zygotic embryogenesis in pearl millet (Pennisetum glaucum) were examined using light and electron microscopic techniques. Embryogenesis was initially characterized by the presence of a vacuolated egg cell and zygote. The increased presence of Golgi bodies in the zygote suggested it was metabolically more active than the egg cell. The first zygotic division resulted in a densely cytoplasmic apical cell and a highly vacuolated basal cell. The club-shaped proembryo displayed a large amount of endoplasmic reticulum (ER) and ribosomes, very few lipids, and a continuous gradient of vacuoles from the highly vacuolated basal suspensor cells to the densely cytoplasmic apical cells. The embryo had well-defined parts by 8 days after pollination, including shoot and root meristems, coleoptile, scutellum, provascular system, and the first leaf primordium. Large increases in ER, lipids, starch, and vacuoles occurred in the scutellum during the maturation of the embryo, except in the provascular cells. Throughout zygotic embryogenesis, embryo cells were connected by plasmodesmata except where intercellular spaces occurred. Ultrastructural, morphological, and histological observations of zygotic embryogenesis in pearl millet are in agreement with previous reports for other grass species.     

Starch deposition and amylase accumulation during somatic embryogenesis in bamboo (Dendrocalamus hamiltonii)

In monocots, the zygotic embryo is protected and nourished by an endosperm. In the present study starch deposition and amylase accumulation was noticed during somatic embryogenesis in stem callus of a bamboo, Dendrocalamus hamiltonii. SEM studies revealed that starch grains were clearly visible in the scutellum during the maturation stage of the somatic embryo. As the somatic embryo developed further, the scutellum got reduced with corresponding increase in amylase. The amylase activity was tested periodically at different developmental stages of embryos. The role of scutellum in somatic embryos for starch deposition and amylase accumulation is discussed.     

Comparative ultrastructural analysis of the in vitro microspore embryoids and in vivo zygotic embryos of wheat as a basis for understanding of cytophysiological aspects of their development

Ultrastructures of in vitro microspore embryoids and in vivo zygotic embryos of spring wheat have been analyzed and compared. Along with the similarity of ultrastructural characteristics of embryoid and embryo cells at the corresponding developmental stages, some differences have been revealed. Unlike embryos, embryoid cells are characterized by lipid inclusions and numerous mitochondria with well-developed internal membranes. According to our hypothesis, lipids represent an alternative energy source required for active cell divisions in the forming embryoids. Unlike embryos, since the earliest developmental stages, embryoid cells accumulate a significant amount of starch and then utilize it during the organogenesis and germination. A conclusion has been made that embryoid cells create their own reserve of carbohydrates, which is then mobilized during their development. The concept of T.B. Batygina (1987, 1997, 2014) about the universal character of the plant morphogenesis in vivo, in situ, and in vitro has been confirmed. The prospects for the use of microspore embryoidogenesis in vitro as a model to study cytophysiological aspects of zygotic embryogenesis in vivo are discussed.     

Similarity of expression patterns of knotted1 and ZmLEC1 during somatic and zygotic embryogenesis in maize ( Zea mays L.)

Expression of knotted1 ( kn1) and ZmLEC1, a maize homologue of the Arabidopsis LEAFY COTYLEDON1 ( LEC1) was studied using in situ hybridization during in vitro somatic embryogenesis of maize ( Zea mays L.) genotype Hi-II. Expression of kn1 was initially detected in a small group of cells (5-10) in the somatic embryo proper at the globular stage, in a specific region where the shoot meristem is initiating at the scutellar stage, and specifically in the shoot meristem at the coleoptilar stage. Expression of ZmLEC1 was strongly detected in the entire somatic embryo proper at the globular stage, gradually less in the differentiating scutellum at the scutellar and coleoptilar stages. The results of analyses show that the expression pattern of kn1 during in vitro somatic embryogenesis of maize is similar to that of kn1 observed during zygotic embryo development in maize. The expression pattern of ZmLEC1 in maize during in vitro development is similar to that of LEC1 in Arabidopsis during zygotic embryo development. These observations indicate that in vitro somatic embryogenesis likely proceeds through similar developmental pathways as zygotic embryo development, after somatic cells acquire competence to form embryos. In addition, based on the ZmLEC1 expression pattern, we suggest that expression of ZmLEC1 can be used as a reliable molecular marker for detecting early-stage in vitro somatic embryogenesis in maize.     

A hormonal misunderstanding in Acca sellowiana embryogenesis: levels of zygotic embryogenesis do not match those of somatic embryogenesis

Endogenous levels of IAA, ABA and four types of CKs were analyzed in zygotic and indirect (ISE) and direct somatic embryogenesis of Acca sellowiana. Zygotic and somatic embryos at different developmental stages were sampled for morphological and hormonal analysis. Both embryo types showed substantial asymmetry in hormone levels. Zygotic embryos displayed a conspicuous peak of IAA in early developmental stages. The results outlined the hormonal variations occurring during zygotic and somatic embryogenesis regarding the timing, nature and hormonal status involved in both processes. The short transient pulse of IAA observed on the 3rd day in culture was suggested to be involved with the signaling for the induction of somatic embryogenesis. Fertilized ovule development was associated with increased IAA levels 21?C24?days after pollination, followed by a sharp decrease in the cotyledonary stage, both in zygotic and somatic embryos. There was a prominent increase in ABA levels in cultures which generated ISE 24?C30?days after pollination, a period that corresponds to the heart and torpedo stages. The levels of total CKs (Z, [9R]Z, iP and [9R]iP) were also always higher in zygotic than in somatic embryogenesis. While zygotic embryogenesis was dominated by the presence of zeatin, the somatic process, contrarily, was characterized by a large variation of the other cytokinin forms and amounts studied. The above results, when taken together, could be related to the previously observed high frequency formation of anomalous somatic embryos formed in A. sellowiana, as well as to their low germination ability.     

Somatic embryogenesis from callus cultures of <Emphasis Type="Italic">Terminalia chebula</Emphasis> Retz.: an important medicinal tree

Somatic embryogenesis was obtained from cotyledon and mature zygotic embryo callus cultures of Terminalia chebula Retz. Callus cultures of cotyledon and mature zygotic embryo were initiated on induction medium containing Murashige and Skoog (MS) nutrients with 1.0 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) either 0.01 or 0.1 mg/l Kinetin and 30 g/l sucrose. Induction of somatic embryogenesis, proliferation and development was obtained through different culture passages. Embryogenic cotyledon callus with globular somatic embryos was obtained on MS basal medium supplemented with 50 g/l sucrose. Globular somatic embryos were observed from mature zygotic embryo callus on induction medium. Different stages of somatic embryo development from cotyledon and mature zygotic embryo calluses were observed on MS basal medium supplemented with 50 g/l sucrose after 4 weeks of culture. Histological studies have revealed the developmental stages of somatic embryos. A maximum of 40.3±1.45 cotyledonary somatic embryos/callus was obtained from mature zygotic embryo compared to 7.70±0.37 cotyledonary somatic embryos/callus initiated from cotyledons. Germination of somatic embryos and conversion to plants were achieved. Highest frequency of germination (46.66±0.88) of somatic embryos was obtained on MS basal medium containing benzyladenine (0.5 mg/l) with 30 g/l sucrose.     

Developmental anatomy and ultrastructure of early somatic embryos in European black pine (Pinus nigra Arn.)

Summary Embryogenic callus cultures of European black pine (Pinus nigra Arn.) were established on megagametophytes containing zygotic embryos in early developmental stage. In addition to many elongated cells and disorganized growing clumps they contained early somatic embryos at various stages of development. At all stages of embryogenesis the embryos were organized as bipolar structures. Cell pairs composed of one isodiametric cell with dense cytoplasm and a second large vacuolated cell were the simplest bipolar system. The vacuolated cell underwent senescence. The cytoplasm-rich cell and its derivates divided transversally, resulting in several cytoplasmic cells arranged in row. An early embryonal cylindrical mass was formed by longitudinal division of the cells in a filament. Proximally localized cells in the early embryonal mass became vacuolized and elongated gradually giving rise to the secondary suspensor. Distal cells remained cytoplasmic in character and formed an embryonal mass along the axis of long early somatic embryos. Differences in the proportion of organelles and heterochromatin clumps, thickness of cell walls and number of plasmodesmata between cells at various stages of early somatic embryogenesis were described.     

Patterns of somatic embryo formation in Siberian larch: Embryological aspects

Somatic embryogenesis was induced in Siberian larch by in vitro culturing zygotic embryos at different developmental stages. Cultures were grown in modified Murashige and Skoog medium supplemented with hormones 2,4-dichlorophenoxyacetic acid (2 mg/l) and 6-benzylaminopurine (0.5–1 mg/l). The success of somatic embryogenesis in this species depended on the tree genotype and developmental stage of embryos used for culturing. Somatic embryogenesis from immature zygotic embryos at the stage of cotyledon initiation was most active. After 5–10 days, such embryos formed the embryogenic tissue including two cell types—elongated highly vacuolated embryonic tubes and small embryonic cells. Somatic embryos were isolated from proliferating embryogenic tissues after 2 months of culture.     

Patterns of somatic embryo formation in Siberian larch: embryological aspects

Somatic embryogenesis was induced in Siberian larch by in vitro culturing zygotic embryos at different developmental stages. Cultures were grown in modified Murashige and Skoog medium supplemented with hormones 2,4-dichlorophenoxyacetic acid (2 mg/l) and 6-benzylaminopurine (0.5-1 mg/l). The success of somatic embryogenesis in this species depended on the tree genotype and developmental stage of embryos used for culturing. Somatic embryogenesis from immature zygotic embryos at the stage of cotyledon initiation was most active. After 5-10 days, such embryos formed the embryogenic tissue including two cell types--elongated highly vacuolated embryonic tubes and small embryonic cells. Somatic embryos were isolated from proliferating embryogenic tissues after 2 months of culture.     

Changes in soluble carbohydrates and starch amounts during somatic and zygotic embryogenesis of Acca sellowiana (Myrtaceae)

Comparative analysis of zygotic and somatic embryogenesis of Acca sellowiana showed higher amounts of sucrose, fructose, raffinose, and myo-inositol in zygotic embryos at different developmental stages than in corresponding somatic ones. These differences were mostly constant. In general, glucose levels were significantly lower than the other soluble carbohydrates analyzed, showing minor variation in each embryo stage. Despite the presence of sucrose in the culture medium, its levels conspicuously diminished in somatic embryos compared with the zygotic ones. Raffinose enhanced parallel to embryo development, regardless of its zygotic or somatic origin. Analysis of the soluble carbohydrate composition of mature zygotic cotyledon used as explant pointed out fructose, glucose, myo-inositol, sucrose, and raffinose as the most important. Similar composition was also found in the corresponding somatic cotyledon. Total soluble carbohydrates varied inversely, decreasing in zygotic embryos and increasing in somatic embryos until the 24th d, at which time they increased rapidly about sixfold in zygotic embryos until the 27th d, a period coinciding with the zygotic proembryos formation. Such condition seems to reflect directly the variation of endogenous sucrose level, mainly because glucose and fructose diminished continuously during this time period. This means that, in terms of soluble sugars, zygotic embryo formation occurred under a situation represented by high sucrose amounts, simultaneously with low fructose and glucose levels, while in contrast, somatic embryo formation took place under an endogenous sugar status characterized by a substantial fructose enhancement. Starch levels increased continuously in zygotic embryos and decreased in somatic ones, the reverse to what was found in fructose variation. Starch accumulation was significantly higher in somatic torpedo and cotyledonary embryos than in the corresponding zygotic ones.     

油菜花蜜腺发育过程的超微结构变化与泌蜜机理的研究

油菜花蜜腺由２枚侧蜜腺和２枝中蜜腺组成,其基本结构类似。在蜜腺发育过程中,产蜜组织细胞内的内质网、高尔基体、质体和线粒体以及液泡都发生有规律变化。泌蜜前,细胞器的数量增加。其中,质体内积累淀粉,此过程与蜜腺内初皮部的分化并和线粒体的增加相关。泌蜜时,内质网数量增多,并产生小泡．小泡向质膜移动。泌蜜后,细胞液泡化,细胞器数量减少,细胞萎缩。根据观察结果分析,其原蜜汁来源于韧皮部,转运至产蜜组织细胞的质体、内质网和高尔基体内加工成蜜汁,最后通过胞吐和渗透相结合的方式泌出。     

Synthetic seed production from somatic embryos of Pinus radiata

Pinus radiata is one of the most important forestry species in the southern hemisphere. This work describes the regeneration of this plant via somatic embryogenesis from immature zygotic embryos. To improve this process, somatic embryogenic cell suspensions were established in liquid media for the generation of material for embryo maturation. Each developmental stage of these suspensions was characterized by microscopy and their growth phases quantified. An alginate-containing medium was used as an encapsulation method for the somatic embryos that were then germinated as artificial seeds in vitro. The protocols described in this work are both useful and reliable for industrial purposes.     

Histocytological changes and reserve accumulation during somatic embryogenesis in Eucalyptus globulus

We recently described a protocol for Eucalyptus globulus somatic embryogenesis (SE). For its immediate use at industrial levels, some stages of the process require better control. In particular, SE germination rates are variable, decreasing SE efficacy. As reserves may play a central role in embryogenic processes, we followed histocytological changes and reserve fluctuations, during SE. For SE induction, explants of mature zygotic embryos were grown on Murashige and Skoog (MS) medium with 3 mg l⁻¹ α-naphthalene acetic acid and later transferred to MS without growth regulators (MS_WH). Samples of zygotic embryo cotyledons (explants), of globular and dicotyledonar somatic embryos, and of embling leaves were analysed for reserve accumulation and histocytological profiles. Cotyledon cells of zygotic embryos were rich in lipid and protein bodies, having almost no starch. After 3 weeks of induction, starch grain density increased in differentiated mesophyll regions, while in meristematic regions their occurrence was diffuse. In globular somatic embryos, starch accumulation increased with time (in amyloplasts), but protein bodies were absent. Cotyledonary somatic embryos had lower density of starch grains and absence of lipid and protein bodies. Embling leaves showed typical histological organisation. This is the first comprehensive study on histological and cytological changes during Eucalyptus SE with emphasis in reserve accumulation. With this work we demonstrate that the presently available SE protocol for E. globulus leads to reserve fluctuations during the process. Moreover, the reserves of somatic embryo cotyledons differ from those of their zygotic embryo counterparts, which reinforce the importance of reserves in the embryogenic process and suggests that manipulating external conditions, SE may be optimised giving suitable emblings production for industrial purposes.     

Comparative analysis of cell cycle events in zygotic and somatic embryos of Cyclamen persicum indicates strong resemblance of somatic embryos to recalcitrant seeds

Embryo development and germination of Cyclamen persicum have been comparatively characterized for zygotic and somatic embryos with regard to mitotic activity and morphology in order to identify developmental abnormalities in somatic embryogenesis. Zygotic embryo development proved to be highly synchronous with distinct periods of cell division, cell elongation and embryo maturation within a total period of 17 weeks of seed development. Somatic embryo development was accomplished within only 3 weeks, resulting in a mixture of morphologically highly variable embryos. No distinct developmental periods could be identified and no reduction of the mitotic activity was discovered for non-desiccated somatic embryos. Controlled desiccation of somatic embryos severely reduced their germination rate, demonstrating resemblance of somatic embryos to recalcitrant seeds, whereas zygotic Cyclamen seeds could be characterized as typically orthodox.     

Ultrastructural changes and the distribution of arabinogalactan proteins during somatic embryogenesis of banana (Musa spp. AAA cv. 'Yueyoukang 1')

A better understanding of somatic embryogenesis in banana (Musa spp.) may provide a practical way to improve regeneration of banana plants. In this study, we applied scanning electron microscopy (SEM) and transmission electron microscopy (TEM) to visualize the ultrastructural changes during somatic embryogenesis of banana (Musa AAA cv. 'Yueyoukang 1'). We also used histological and immunohistochemical techniques with 16 monoclonal antibodies to study the spatial distribution and cellular/subcellular localization of different arabinogalactan protein (AGP) components of the cell wall during somatic embryogenesis. Histological study with periodic acid-Schiff staining documented diverse embryogenic stages from embryogenic cells (ECs) to the late embryos. SEM revealed a mesh-like structure on the surface of proembryos which represented an early structural marker of somatic embryogenesis. TEM showed that ECs were rich in juvenile mitochondria, endoplasmic reticulum and Golgi stacks. Cells in proembryos and early globular embryos resembled ECs, but they were more vacuolated, showed more regular nuclei and slightly more developed organelles. Immunocytochemical study revealed that the signal of most AGP epitopes was stronger in starch-rich cells when compared with typical ECs. The main AGP component in the extracellular matrix surface network of banana proembryos was the MAC204 epitope. Later, AGP immunolabelling patterns varied with the developmental stages of the embryos. These results about developmental regulation of AGP epitopes along with developmental changes in the ultrastructure of cells are providing new insights into the somatic embryogenesis of banana.     

Cytological features of developing embryogenic callus and somatic embryos of Iris pumila L. and Iris setosa Pall

Abstract

Cytological examination during somatic embryogenesis in Iris pumila L. and Iris setosa Pall, were performed using light and electron microscopy. The first sign of the cellular differentiation in the initial embryogenic callus (EC; stage 1) of both Iris species was the formation of short and elongated cell types. After the onset of embryogenesis, short cells divided producing a mass of densely packed meristematic cells, closely connected with numerous plasmodesmata. Further differentiation into globular embryos (GE) led to a loss of plasmodesmata and cell separation. In vacuolated elongated cells, cytoplasm was located near the wall and around the nucleus. In both cell types amyloplasts and small mitochondria with poorly developed crystae were abundant.

Cell of GE (stage 2) contained an increased number of mitochondria and plastids comparing to those from stage 1, indicating further differentiation. Thylakoids and starch grains were observed within the plastids, while the number of cristae within the mitochondria was increasing.

In cells of embryos with coleoptile (ECl) (stage 3), plastids differentiated into chloroplasts with thylakoids. In all stages of cell differentiation, short and long cisternae of endoplasmic reticulum with ribosomes were seen. Activity of dictyosomes was increased in stages 1 and 2, then reduced in stage 3.

Ultrastructure of EC cells was identical to that of proembryogenic cells, i.e. of early GE. Ultrastructural appearance of GE cells was identical in both Iris species, but evident, and increasing, differences in mitochondria and plastids were observed between GE and ECl embryos.The presence of bi-, three- and eight- cell proembryos demonstrates that they originate from a single cell in both Iris species.