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.     

The effects of silver nitrate and different carbohydrate sources on somatic embryogenesis in Coffea canephora

The response of five Coffea canephora Pierre genotypes with regard to somatic embryogenesis was tested on media containing silver nitrate (AgNO₃) and different carbohydrates (sucrose, fructose, maltose and glucose). The presence of AgNO₃ caused only small modifications to the ionic equilibrium of the media. At concentrations between 30–60 M, AgNO₃ improved embryo yield for the genotypes evaluated, while higher doses negatively affected the regenerative capacity. The substitution of maltose, glucose or fructose for sucrose produced different responses depending on the genotype. Fructose significantly increased somatic embryo production in genotypes N91 and N128, while maltose was highly effective for N75. In addition, more synchronous embryo development was observed in genotype N91 when glucose was used instead of sucrose.     

Recent advances in conifer somatic embryogenesis: improving somatic embryo quality

Somatic embryogenesis of coniferous species was first reported more than 20 years ago. Since then, there has been an explosion of research aimed at developing and optimizing protocols for efficient regeneration of plantlets. Although routinely used both as a means of propagation, as well as a valuable model system for investigating the structural, physiological, and molecular events occurring during embryo development, in vitro embryogenesis is still problematic for some coniferous species. Major problems include: low number of embryos generated; and low frequency of mature embryos able to convert into viable plantlets. Until recent years, despite the fact that embryogenesis is comprised of a sequence of defined steps which include proliferation of embryogenic tissue, embryo maturation, and germination, attempts at improving the whole procedure have been made almost exclusively during the maturation stage. This strategy was based on the assumption that successful regeneration is related to treatments provided during the development of the embryos. Major optimizations of the maturation medium have involved judicious selections of type and concentration of growth regulators, namely abscisic acid, and adjustments of the osmoticum of the culture medium. Extensive work has been conducted in defining the effects of plasmolysing and non-plasmolysing osmoticum agents during maturation, as well as in improving desiccation techniques required for the completion of the maturation program. In the last 2 years, however, work on spruce has clearly demonstrated that the early events in embryogenesis are crucial for the successful completion of the overall embryogenic program. The use of cell tracking techniques, implemented by physiological and molecular studies, has revealed that manipulations of the culture conditions early in the process can increase both number and quality of embryos produced in culture. Additional manipulations of the germination medium can also enhance germination and conversion frequency of somatic embryos matured in a sub-optimal environment. These new findings, together with the unraveling of molecular mechanisms involved in the control/regulation of embryo development hold considerable promise for clonal propagation in conifers.     

Protocols for efficient repetitive and secondary somatic embryogenesis in Helianthus maximiliani (Schrader)

 Indirect somatic embryogenesis was induced on leaf explants of greenhouse-grown Helianthus maximiliani plants. Leaves of the regenerated plants were used as starting explants for the induction of direct somatic embryogenesis. Another cycle of somatic embryogenesis was induced on the leaves of regenerated plants. In both cases, leaf explants were cultured on media containing different auxin/cytokinin ratios. The auxin/cytokinin ratio had an influence on the intensity of embryo formation, germination and the capability to regenerate plants. Somatic embryogenesis was generally more intensive on the medium with lower concentrations of 6-benzylamino-purine. Further, the percentage of regenerated plants was higher when embryos were induced on high-cytokinin, low-auxin medium. Secondary somatic embryogenesis was induced on embryos by culture in liquid hormone-free medium. Similar to direct embryogenesis the efficiency of secondary embryogenesis depended on the medium used for the induction of the primary embryos. In contrast to the mostly low frequencies of conversion of secondary embryos into plants that has been observed in other species, the percentage of regenerated plants from secondary embryos of H. maximiliani was quite high, although slightly lower than that obtained in primary embryos. Received: 28 March 2000 / Revision received: 1 September 2000 / Accepted: 2 October 2000     

Developmental pathways of somatic embryogenesis

Somatic embryogenesis is defined as a process in which a bipolar structure, resembling a zygotic embryo, develops from a non-zygotic cell without vascular connection with the original tissue. Somatic embryos are used for studying regulation of embryo development, but also as a tool for large scale vegetative propagation. Somatic embryogenesis is a multi-step regeneration process starting with formation of proembryogenic masses, followed by somatic embryo formation, maturation, desiccation and plant regeneration. Although great progress has been made in improving the protocols used, it has been revealed that some treatments, coinciding with increased yield of somatic embryos, can cause adverse effects on the embryo quality, thereby impairing germination and ex vitro growth of somatic embryo plants. Accordingly, ex vitro growth of somatic embryo plants is under a cumulative influence of the treatments provided during the in vitro phase. In order to efficiently regulate the formation of plants via somatic embryogenesis it is important to understand how somatic embryos develop and how the development is influenced by different physical and chemical treatments. Such knowledge can be gained through the construction of fate maps representing an adequate number of morphological and molecular markers, specifying critical developmental stages. Based on this fate map, it is possible to make a model of the process. The mechanisms that control cell differentiation during somatic embryogenesis are far from clear. However, secreted, soluble signal molecules play an important role. It has long been observed that conditioned medium from embryogenic cultures can promote embryogenesis. Active components in the conditioned medium include endochitinases, arabinogalactan proteins and lipochitooligosaccharides.     

Comparison of NAA and 2,4-D induced somatic embryogenesis in Cassava

NAA and 2,4-D were compared for their ability to induce somatic embryogenesis in cassava (Manihot esculenta Crantz). In all seven cultivars tested, only 2,4-D had the capacity to induce primary somatic embryos from leaf explants, however, both NAA and 2,4-D were capable of inducing secondary somatic embryos. More secondary somatic embryos were formed in NAA than in 2,4-D medium. Furthermore, the maturation period for secondary somatic embryos was shorter in NAA medium than in 2,4-D medium. In some cultivars, repeated subculture of secondary somatic embryos in NAA medium resulted in a gradual shift from somatic embryogenesis to adventitious root formation. This shift could be stopped and reversed by subculture of the material in 2,4-D medium. In NAA medium the most secondary somatic embryos were formed when they were subcultured every 15 days whereas in 2,4-D a 20 day subculture interval was optimal. Subculture of secondary somatic embryos at a high inoculum density (>1.5 g jar⁻¹) in NAA medium did not result in the formation of secondary somatic embryos, whereas in 2,4-D it lead to the formation of globular secondary somatic embryos. With 2,4-D the newly induced secondary somatic embryos were connected vertically to the explant and with NAA medium horizontally. For all cultivars tested, desiccation stimulated normal germination of NAA-induced somatic embryos. However, the desiccated, secondary somatic embryos required a medium supplemented with BA for high frequency germination. The concentration of BA needed for high frequency germination was higher when the desiccated secondary somatic embryos were cultured in light instead of dark. In only one cultivar desiccation enhanced germination of 2,4-D induced secondary somatic embryos and in three other cultivars it stimulated only root formation. This revised version was published online in June 2006 with corrections to the Cover Date.     

Organization of initial stages of somatic embryogenesis in tissue culture of Citrus sinensis cv. Tarocco at the organismal level

Four-step protocol was established for the in vitro regeneration of Citrus sinensis cv. Tarocco somatic embryos that were morphologically similar to small somatic embryos in vivo. The regeneration procedure comprises a mechanical destruction of embryogenic culture to obtain proembryogenic cell masses (PEMs) (step 1) followed by culturing on three different media (steps 2–4). The approach developed allows in vitro simulating somatic embryogenesis by dividing this process into three partially independent steps: PEM → globular somatic embryo → heart-shaped somatic embryo → somatic embryo with developed cotyledons. The highest frequency of morphogenetic stage transition was 64, 40, and 26%, respectively. It was shown that the first step (PEM → globular embryo) was associated with the formation of heterogeneous population of spherical bodies 50–500 μm in diameter, among which about 40% were somatic embryos at globular stage. The scheme is offered of alternative pathways for the development of spherical bodies in vitro, and interrelations between their sizes and ability to direct morphogenesis are discussed.     

Improving tolerance of somatic emrbyos of Picea glauca to flash desiccation with a cold treatment (desiccation after cold acclimation)

Summary Controlled mild desiccation of mature white spruce somatic embryos prior to germination improves the quality of the germinated embryos. More severe desiccation results in increased injury and death but is desirable for long-term storage of embryos and production of desiccated artificial seed. A method was developed to improve desiccation tolerance in somatic embryos using a temperature treatment. Culture plates with embryos at four stages of development were subjected to temperatures of 1, 5, 10, or 20°C for periods of 0, 1, 2, 4, or 8 wk duration. After the temperature treatment, the embryos were harvested and air-dried for 2h under a laminar flow hood. Dried embryos were placed directly on germination medium and the quality of the germinants was assessed after 4 wk. The initial maturation stage of the embryo and the temperature and duration of the treatment had a significant effect on the quality of the germinants. Most treatments caused marked differential survival of organs. The optimal response was obtained with embryos that had been grown for 51 d (cotyledonary stage) on maturation medium and that were subsequently exposed to a temperature of 5°C for 8 wk prior to air drying. This treatment produced 58% undamaged germinants with normal cotyledons, hypocotyls, and roots. Only 1% of the untreated air-dried embryos germinated normally.     

Spaceflight reduces somatic embryogenesis in orchardgrass (Poaceae)

Somatic embryos initiate and develop from single mesophyll cells in in vitro cultured leaf segments of orchardgrass ( Dactylis glomerata L.). Segments were plated at time periods ranging from 21 to 0·9 d (21 h) prior to launch on an 11 d spaceflight (STS-64). Using a paired t -test, there was no significant difference in embryogenesis from preplating periods of 14 d and 21 d. However, embryogenesis was reduced by 70% in segments plated 21 h before launch and this treatment was significant at P = 0·0001. The initial cell divisions leading to embryo formation would be taking place during flight in this treatment. A higher ratio of anticlinal:periclinal first cell divisions observed in the flight compared to the control tissue suggests that microgravity affects axis determination and embryo polarity at a very early stage. A similar reduction in zygotic embryogenesis would reduce seed formation and have important implications for long-term space flight or colonization where seeds would be needed either for direct consumption or to grow another generation of plants.     

Improved somatic embryogenesis from Cocos nucifera (L.) plumule explants

Summary Coconut is one of the most recalcitrant species to regenerate in vitro. Although previous research efforts using plumule explants have resulted in reproducible somatic embryogenesis, efficiency is only 4 or 10 somatic embryos per plumule without or with a brassinolide treatment, respectively. In order to increase the efficiency of somatic embryogenesis in coconut, two different approaches were evaluated and reported here: secondary somatic embryogenesis and multiplication of embryogenic callus. Primary somatic embryos obtained from plumule explants were used as explants and formed both embryogenic callus and secondary somatic embryos. The embrogenic calluses obtained after three multiplication cycles were capable of producing somatic embryos. The efficiency of the system was evaluated in a stepwise process beginning with an initial step for inducing primary somatic embryogenesis followed by three steps for inducing secondary somatic embryogenesis followed by three steps for embryogenenis callus multiplication, and finally production of somatic embryos from callus. The total calculated yield from one plumule was 98 000 somatic embryos. Comparing this to the yield obtained from primary somatic embryogenesis results in about a 50 000-fold increase. When compared to the yield previously reported in the literature with the use of a brassinolide treatment, it is about a 10 000-fold increase in yield. The present protocol represents important progress in improvement in the efficiency of coconut somatic embryo production.     

Callus culture of Coronilla varia L. (crownvetch): plant regeneration through somatic embryogenesis

Callus culture and plant regeneration through somatic embryogenesis have been obtained in Coronilla varia. Media used were UM (25) supplemented with 2 mg/l 2,4-D followed by subculture on MS (18) containing 1 mg/l 2-iP and 0.1 mg/l IAA. Embryoids developed into complete plantlets on filter paper saturated with hormone-free MS medium.     

Regulation of somatic embryogenesis in celery cell suspensions

Embryogenic suspension cultures of celery (Apium graveolens L.) were established from petiole and leaf callus. Suspensions were routinely subcultured in a maintenance medium (with 2.3 M 2,4-D and 0.88 M BA). Somatic embryogenesis occurred in this medium, but was considerably improved in a regeneration medium (2.3 M kinetin, without 2,4-D). Cultures thus maintained, contained embryogenic clumps, aggregated somatic embryos, and few free-floating singular somatic embryos. Addition of mannitol (3–4% w/v) prevented cell lysis, greatly increased the number of singular somatic embryos, improved their normal differentiation, and accelerated torpedo embryo development. Experiments to reveal the nature of the mannitol effect demonstrated that the decreased osmotic potential was an important factor, but not the only one: iso-molar solutions of sucrose alone were not as effective. The mannitol effect could be manifested after a short (2–3 days) exposure period, suggesting a trigger (induction) mechanism. Several pathways of somatic embryogenesis in celery and its regulation by subculturing, with the addition of mannitol, are outlined. Cultures thus maintained resulted in a high rate of normal somatic embryogenesis and production of normal transplantable celery plants.     

Regeneration via somatic embryogenesis and organogenesis in different cultivars of cotton (Gossypium SPP.)

Summary In vitro studies related to somatic embryogenesis and organogenesis were performed in different cultivars of cotton. Gossypium hirsutum cultivars SH-131, LH-900, Hybrid H8, Khandwa-2, and Gossypium arboreum cultivars BD-1, BD-6, Sarvottam, and Jawahar Tapti were screened for their ability to regenerate in vitro. Cotyledonary leaves and hypocotyls were used as explants. Vigorous callusing was observed in G. arboreum cultivars. Globular somatic embryos were formed in BD-1, BD-6, Sarvottam, Jawahar Tapti, SH-131, and LH-900. Heart-shaped and torpedo stages were also observed. Embryos of BD-1 and BD-6 germinated and formed distincts shoot and root poles. 2-Isopentenyladenine (2iP) was effective in the induction of somatic embryos. Hybrid H8 and Khandwa-2 regenerated by directly forming shoots when cultured on Murashige and Skoog (MS) medium supplemented with 6-benzyladenine and formed roots on hormone-free MS medium     

Induction of somatic embryogenesis in Azadirachta indica

A modified culture protocol has been developed for the induction of somatic embryogenesis in Azadirachta indica (neem). Embryogenic calluses were initiated from cotyledons or hypocotyls using a Murashige and Skoog (MS) agar medium supplemented with 0.5 mg l⁻¹ α-napthaleneacetic acid (NAA), 1 mg l⁻¹ 6-benzylaminopurine (BA), 1 g l⁻¹ casein hydrolysate, and 50 g l⁻¹ sucrose. The calluses, when transferred to a liquid medium similar to the agar medium but with NAA replaced by 0.5 mg l⁻¹ indole-3-acetic acid (IAA), formed globular structures which further developed a rudimentary root, after 4 to 5 weeks incubation. Subsequently, these highly differentiated tissues when transferred into a hormone-free MS medium containing 1 g l⁻¹ casein hydrolysate and 50 g l⁻¹ sucrose, active embryo masses started to appear after 1 to 2 weeks. The embryo production was found to improve more than 2 fold by adding 0.2 mg l⁻¹ zeatin to the medium. This revised version was published online in June 2006 with corrections to the Cover Date.     

Improvements of cyclic somatic embryogenesis of cassava (Manihot esculenta Crantz)

Summary In cassava a cyclic system of somatic embryogenesis was developed. Primary (torpedo shaped or germinated) embryos, originating from leaf lobes, could only be obtained after culture on solid medium. Cyclic embryos, originating from embryos, could be obtained in both liquid and on solid medium. The production of embryos in liquid medium was distinctly higher, faster and more synchronized than on solid medium. Lower densities and fragmentation of starting embryos improved the production significantly. The highest production found was 32.1 embryos per initial embryo. In all treatments the explants initiated multiple embryos. The production of single embryos was achieved by pressing starting embryos through a fine meshed sieve, indicating that embryos can be produced from a piece of tissue with a restricted number of cells. The shoot conversion rate of embryos from liquid medium was comparable with that of embryos from solid medium.Abbreviations BM Basal Medium - MIE medium volume per initial embryo - E/IE number of Embryos per Initial Embryo     

Noninvasive evaluation of somatic embryogenesis

Callus Suspension Cultures of Ipomea batates Poir. cv. White Star were grown in an airlift bioreactor. A machine vision system was used to monitor nondestructively callus growth during a 10 day culture period. Growth data obtained with this system included the overall reactor population and population estimates for the 200-1200-mum fractions at 200-mum intervals. A model of callus growth was developed to explain the mechanics of callus enlargement. The model was based on the assumptions that (1) the calli could not subdivide or shrink, (2) there was a fixed percentage of the initial population for each fraction that was nonviable, and (3) growth rates did not vary with time during the culture period. It was determined that the growth rates and nonviable ratios decreased as fraction size increased.     

Morphohistological analysis and histochemistry of Feijoa sellowiana somatic embryogenesis

Summary. Morphohistological analysis and histochemical studies were carried out during the induction and development of Feijoa sellowiana somatic embryos. Zygotic embryos were cultured on LPm medium containing 2,4-dichlorophenoxyacetic acid (20µM) and glutamine (8mM). Somatic embryogenesis could be induced from embryogenic cells that originated in meristematic centers or from clusters of cells. The presence of few starch grains and abundant protein bodies was observed in the globular and early torpedo stages, while in torpedo and cotyledonary-stage somatic embryos an enhanced synthesis of starch grains was associated with the accumulation of reserves to be used in the conversion of the embryos to plantlets. Proteins were predominantly observed in protoderm cells, as well as in the meristematic apical region of torpedo and cotyledonary-stage somatic embryos.Correspondence and reprints: Departamento de Fitotecnia, Centro de Ciências Agrárias, Universidade Federal de Santa Catarina, C.P. 476, 88034-001 Florianópolis, SC, Brazil.     

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.     

Secondary somatic embryogenesis of carnation (<Emphasis Type=Italic>Dianthus caryophyllus</Emphasis> L.)

This is the first report describing culture conditions necessary to induce secondary embryogenesis in two carnation cultivars, Nelson and Spirit. In the first step, embryogenic calli were induced on petal explants followed by development of primary somatic embryos from the calli. In the second stage, secondary somatic embryos were obtained when precotyledonary and cotyledonary primary embryos were isolated and transferred onto a series of culture media all containing MS basal salt mixture, and supplemented with different concentrations of 2,4-D, BA, sucrose and mannitol. The highest rate of secondary embryogenesis occurred on mannitol containing media. Secondary somatic embryos were converted into plantlets when they were transferred onto growth regulator-free half-strength MS medium and successfully acclimated in the greenhouse.