Direct somatic embryogenesis from shoot apical meristems of pea, and thidiazuron-induced high conversion rate of somatic embryos

Direct somatic embryogenesis from shoot apical meristems of pea is described. Somatic embryos were induced directly (without callus intervention) from meristematic tissues grown on a medium supplemented with 2.5 μM picloram. Within 4 to 5 weeks, fully morphologically developed somatic embryos were obtained. Somatic embryos originated from apical as well as from basal parts of meristem explants. The initiation and development of somatic embryos was asynchronous, basal somatic embryos developed more quickly than apical ones. Abundant secondary embryogenesis was observed after isolation of primary somatic embryos and culturing them on media for germination. Morphologically normal somatic embryos germinated on medium without growth regulators; the conversion rate was increased by application of 10 μM thidiazuron (TDZ). TDZ was also able to induce shoot bud regeneration on embryos without differentiated a shoot apex, allowing to germinate up to 78 % of all harvested somatic embryos with various morphology. The protocol was successfully tested in 47 out of 48 P. sativum and P. arvense cultivars as well as in two wild peas (P. elatius, P. jomardi). This revised version was published online in July 2006 with corrections to the Cover Date.     

几种生长素对木薯体细胞胚发生和植株再生的作用

木薯（ＭａｎｉｈｏｔｅｓｃｕｌｅｎｔａＣｒａｎｔｚ）嫩叶外植体在含２,４－Ｄ（１－１６ｍｇＬ－１）或ＮＡＡ（４０ｍｇＬ－１）的诱导培养基上能直接诱导初生体细胞胚胎发生,而低活性的生长素ＩＢＡ或ＩＡＡ（４０ｍｇＬ－１）或低浓度的２,４－Ｄ（０．１ｍｇＬ－１）则不能。而以木薯初生体细胞胚切段为外植体时,次生体细胞胚的诱导对生长素的活性或浓度的要求降低。降低生长素浓度或活性能缩短体细胞胚诱导时间并促进根的形成,有利于提高体细胞胚的再生频率。体细胞胚外植体在诱导培养基上的培养时间对下一步体细胞胚胎发生的诱导产生影响。通过石腊切片观察,在含２,４－Ｄ诱导培养基上,木薯体细胞胚不能形成芽分生组织。结果表明,２,４－Ｄ等生长素类物质对诱导木薯体细胞胚胎发生是关键因子,但对体细胞胚的进一步发育和植株再生起抑制作用。     

Histological comparison of single somatic embryos of maize from suspension culture with somatic embryos attached to callus cells

An embryogenic suspension culture of Zea mays, genotype 4C1, was obtained from friable callus that was cultured on solid medium and had been obtained from zygotic embryos. The suspension contained non-dividing elongated cells, clusters of dividing isodiametric cells, and globular, ovoid, and polar stages of somatic embryos. The single somatic embryos were blocked in shoot meristem formation: when transferred to regeneration medium they developed a root and, at the shoot side, a green cap with meristematic cells, but a scutellum and leaf primordia were not formed. In medium containing 2,4-dichlorophenoxy acetic acid, somatic embryos formed embryogenic callus aggregates, consisting of globular stage somatic embryos attached to each other via undifferentiated callus cells. These somatic embryos developed into mature embryos with the zygotic histological characteristics, such as scutellum and leaf primordia, in maturation medium, and then regenerated into plants in regeneration medium. By omitting the maturation phase, regeneration occurred via organogenesis. Polyembryos, i. e. embryos attached to each other without callus tissue in between, behaved as single somatic embryos. It is concluded that the attached callus tissue provides a factor that stimulates scutellum and leaf primordia formation.Abbreviations CMM callus maintenance medium - 2,4D 2,4-dichlorophenoxy acetic acid - PCV packed cell volume - MS Murashige and Skoog medium     

Study of aberrant morphologies and lack of conversion of somatic embryos of chickpea (Cicer arietinum L.)

Summary Somatic embryos which originated from mature embryo axes of the chickpea (Cicer arietinum L.) showed varied morphologies. Embryos were classified based on shape of the embryo and number of cotyledons. “Normal” (zygotic-like) embryos were bipolar structures with two cotyledons and a well-developed shoot and root apical meristem, whereas “aberrant” embryos were horn-shaped, had single and multiple cotyledons, and were fasciated. Histological examination revealed the absence of a shoot apical meristem in horn-shaped embryos. Fasciated embryos showed diaxial fusion of two embryos. Secondary embryogenesis was also observed, in which the embryos emerged from the hypocotyl and cotyledonary region of the primary somatic embryo. This report documents the absence of an apical meristem as a vital factor in the lack of conversion of aberrant somatic embryos.     

Role of 2,4-dichlorophenoxyacetic acid (2,4-D) in somatic embryogenesis on cultured zygotic embryos of Arabidopsis: cell expansion, cell cycling, and morphogenesis during continuous exposure of embryos to 2,4-D

The relationship between cell expansion and cell cycling during somatic embryogenesis was studied in cultured bent-cotyledon-stage zygotic embryos of a transgenic stock of Arabidopsis thaliana harboring a cyclin 1 At:β-glucuronidase (GUS) reporter gene construct. In embryos cultured in a medium containing 2,4-dichlorophenoxyacetic acid (2,4-D), following a brief period of growth by cell expansion, divisions were initiated in the procambial cells facing the adaxial side at the base of the cotyledons. Cell division activity later spread to almost the entire length of the cotyledons to form a callus on which globular and heart-shaped embryos appeared in about 10 d after culture. Anatomical and morphogenetic changes observed in cultured embryos were correlated with patterns of cell cycling by histochemical detection of GUS-expressing cells. Although early-stage somatic embryos did not develop further during their continued growth in the auxin-containing medium, maturation of embryos ensued upon their transfer to an auxin-free medium. In a small number of cultured zygotic embryos the shoot apical meristem was found to differentiate a leaf, a green tubular structure, or a somatic embryo. Contrary to the results from previous investigations, which have assigned a major role for the shoot apical meristem and cells in the axils of cotyledons in the development of somatic embryos on cultured zygotic embryos of A. thaliana, the present work shows that somatic embryos originate almost exclusively on the callus formed on the cotyledons. Other observations such as the induction of somatic embryos on cultured cotyledons and the inability of the embryo axis (consisting of the root, hypocotyl, and shoot apical meristem without the cotyledons) to form somatic embryos, reaffirm the important role of the cotyledons in somatic embryogenesis in this plant.     

Structure and development of somatic embryos formed inArabidopsis thaliana pt mutant callus cultures derived from seedlings

Summary Seeds of theArabidopsis thaliana mutant primordia timing (pt) were germinated in 2,4-dichlorophenoxyacetic acidcontaining liquid medium. The seedlings formed somatic embryos and nonembryogenic and embryogenic callus in vitro in a time period of approximately two to three weeks. Embryogenesis and callus formation were monitored with respect to origin, structure, and development. Ten days after germination globular structures appeared in close vicinity of and on the shoot apical meristem (SAM). Somatic embryos formed either directly on the SAM region of the seedling or indirectly on embryogenic callus that developed at the SAM zone. Globular structures developed along the vascular tissue of the cotyledons as well, but only incidentally they formed embryos. Upon deterioration, the cotyledons formed callus. Regular subculture of the embryogenic callus gave rise to high numbers of somatic embryos. Such primary somatic embryos, grown on callus, originated from meristematic cell clusters located under the surface of the callus. Embryos at the globular and heart-shape stage were mostly hidden within the callus. Embryos at torpedo stage appeared at the surface of the callus because their axis elongated. Secondary somatic embryos frequently formed directly on primary ones. They preferentially emerged from the SAM region of the primary somatic embryos, from the edge of the cotyledons, and from the hypocotyl. We conclude that the strong regeneration capacity of thept mutant is based on both recurrent and indirect embryogenesis.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - DIC days in culture - SAM shoot apical meristem     

Somatic embryogenesis in peach palm using the thin cell layer technique: induction, morpho-histological aspects and AFLP analysis of somaclonal variation

BACKGROUND AND AIMS: The thin cell layer (TCL) technique is based on the use of very small explants and has allowed enhanced in vitro morphogenesis in several plant species. The present study evaluated the TCL technique as a procedure for somatic embryo production and plantlet regeneration of peach palm. METHODS: TCL explants from different positions in the shoot apex and leaf sheath of peach palm were cultivated in MS culture medium supplemented with 0-600 microM Picloram in the presence of activated charcoal. The production of primary calli and embryogenic calli was evaluated in these different conditions. Histological and amplified fragment length polymorphism (AFLP) analyses were conducted to study in vitro morphogenetic responses and genetic stability, respectively, of the regenerated plantlets. KEY RESULTS: Abundant primary callus induction was observed from TCLs of the shoot meristem in culture media supplemented with 150-600 microM Picloram (83-97%, respectively). The production of embryogenic calli depends on Picloram concentration and explant position. The best response observed was 43% embryogenic callus production from shoot meristem TCL on 300 microM Picloram. In maturation conditions, 34+/-4 somatic embryos per embryogenic callus were obtained, and 45.0+/-3.4% of these fully developed somatic embryos were converted, resulting in plantlets ready for acclimatization, of which 80% survived. Histological studies revealed that the first cellular division events occurred in cells adjacent to vascular tissue, resulting in primary calli, whose growth was ensured by a meristematic zone. A multicellular origin of the resulting somatic embryos arising from the meristematic zone is suggested. During maturation, histological analyses revealed bipolarization of the somatic embryos, as well as the development of new somatic embryos. AFLP analyses revealed that 92% of the regenerated plantlets were true to type. The use of TCL explants considerably improves the number of calli and somatic embryos produced in comparison with previously described protocols for in vitro regeneration of peach palm. CONCLUSIONS: The present study suggests that the TCL somatic embryogenesis protocol developed is feasible, although it still requires further optimization for in vitro multiplication of peach palm, especially the use of similar explants obtained from adult palm trees.     

Somatic embryogenesis in tamarillo (<Emphasis Type="Italic">Cyphomandra betacea</Emphasis>): approaches to increase efficiency of embryo formation and plant development

Somatic embryogenesis induction and somatic embryo development of the solanaceous tamarillo tree were previously established and successfully used for plant regeneration from different explants and varieties. Somatic embryogenesis was induced in Murashige and Skoog medium containing 2,4-dichlorophenoxyacetic acid (2,4-D) or picloram and high sucrose concentrations (0.25 M). The embryogenic tissues were transferred to an auxin-free medium, with reduced sucrose levels, to permit embryo development and conversion into plantlets. This two-step protocol is often impaired by an ineffective transition from the proembryogenic masses to embryo development. In this work, attempts to optimize the somatic embryogenesis system of tamarillo by improving the quality of somatic embryo and embryo conversion were carried out. The results showed that the presence of a high number of abnormal somatic embryos did not significantly inhibit plant conversion, hence indicating that shoot apical meristem development was not affected in abnormal somatic embryos. It was also shown that the manipulation of sucrose concentration in the development medium (0.11 M) and dark conditions before conversion increased the number of morphologically normal somatic embryos. The comparison between mature cotyledonary zygotic and somatic embryos showed an inefficient accumulation of storage compounds, mainly lipids, in somatic embryos. These reduced levels of lipid storage could be responsible for the abnormal patterns of embryo development found in tamarillo somatic embryos.     

Development of white spruce somatic embryos: II. Continual shoot meristem development during germination

Summary This study compares the development of shoot apical meristems of white spruce somatic and zygotic embryos during germination. In mature somatic embryos, the functional part of the shoot apical meristem was bi-layered. After partial drying, a normal shoot meristem was formed from these two cell layers during germination. Other cells within the meristem were vacuolated and separated by intercellular air spaces. In the absence of the partial drying treatment, somatic embryos enlarged in size primarily due to vacuolation of cells and the formation of large intercellular air spaces. A majority of these somatic embryos failed to form a functional shoot apical meristem. Compared with somatic embryos, the shoot apical meristem of a mature zygotic embryo was well organized with a densely cytoplasmic apical layer. The cells within the meristem were tightly packed. Judging from the cell profiles during germination, all cells within the meristem of the zygotic embryo took part in the formation of the vegetative shoot apical meristem.     

Improvement of Citrus somatic embryo development by temporary immersion

Liquid medium improves and facilitates somatic embryo development from Citrus deliciosa Ten. suspension cultures. Three different culture conditions were compared to determine a means of overcoming poor somatic embryo development. Somatic embryos derived from suspension cultures were plated on solid medium, maintained in suspension culture or temporarily immersed. About 60% of somatic embryos plated on solid medium developed to the cotyledonary stage, but were hyperhydric. Continuous growth in suspension culture at 100 rpm hindered cotyledon and protoderm formation, and somatic embryos were unable to develop beyond the globular stage. Temporary immersion promoted somatic embryo development, i.e. 66% of the somatic embryos produced were cotyledonary, and were morphologically similar to nucellar embryos. This latter culture system also improved regeneration synchronization by hampering secondary embryogenesis at the onset of germination. Irrespective of the culture system used, most cotyledonary somatic embryos studied had no caulinary meristem or starch and protein reserves, thus explaining the low germination rates obtained. This revised version was published online in June 2006 with corrections to the Cover Date.     

Dynamics of morphological and anatomical changes in leaf tissues of an interspecific hybrid of oil palm during acquisition and development of somatic embryogenesis

Oil palm is an economically important plant species due to its high oil production per unit area. Large-scale clonal propagation of the species’s elite specimens is only possible through somatic embryogenesis, although methodology is partially still unknown and insufficiently understood. Current study characterizes in morphological and anatomical terms the acquisition and development stages of somatic embryogenesis of the oil palm’s immature leaves. The respective embryogenic stages were analyzed and characterized: immature leaves (initial explants); leaves with calli formation; leaves which failed to respond to calli formation; leaves with formation of root structures; primary calli; primary calli with differentiation of embryogenic calli; embryogenic calli; pro-embryogenic calli; calli with differentiated somatic embryos; somatic embryos at globular and torpedo stage; and mature fruit zygotic embryos. Cell masses emerged after approximately 60 days of cultivation through the proliferation of cells associated to initial explants´ vascular bundles. Consequently, the formation of two different types of calli was identified, namely, primary and embryogenic, respectively consisting partially and completely of meristematic cell clusters. After 420 days of cultivation, the propagules formed somatic embryos with no connection to source tissues, initially composed (globular stage) of a very characteristic ground meristem and protoderm. After 480 days of cultivation, as the cultures matured (torpedo stage), procambial strands, a structural characteristic also observed in mature zygotic embryos, were reported. The results provide an in-depth understanding of somatic embryogenesis of immature leaves of oil palm. Further, current analysis develops morphological markers at different stages of development obtained during the process.     

Improvement of somatic embryogenesis and plant recovery in cassava

Methods for improving the efficiency of plant recovery from somatic embryos of cassava (Manihot esculenta Crantz) were investigated by optimizing the maturation regime and incorporating a desiccation stage prior to inducing germination. Somatic embryos were induced from young leaf lobes of in vitro grown shoots of cassava on Murashige and Skoog medium with 2,4-dichlorophenoxy acetic acid. After 15 to 20 days of culture on induction medium, the somatic embryos were transferred to a hormone free medium supplemented with activated charcoal. In another 18 days mature somatic embryos became distinctly bipolar and easily separable as individual units and were cultured on half MS medium for further development. Subsequent desiccation of bipolar somatic embryos resulted in 92% germination and 83% complete plant regeneration. The plants were characterized by synchronized development of shoot and root axes. Of the non-desiccated somatic embryos, only 10% germinated and 2% regenerated plants. Starting from leaf lobes, transplantable plantlets were derived from primary somatic embryos within 70 to 80 days.Abbreviations 2,4-D 2,4 dichlorophenoxyacetic acid - BA Benzyl aminopurine - GA Giberellic acid - MS Murashige and Skoog - NAA Naphthalene acetic acid     

Maturation,germination, and conversion of norway spruce (Picea abies L.) somatic embryos to plants

Summary Quantitative data are presented on the efficiency of three stages of plant regeneration from somatic embryos of Norway spruce (Picea abies L.): 1) Maturation, the development of immature embryos to the cotyledonary stage; 2) Germination, primary root growth; and 3) Conversion, plantlet survival and continued growth in nonaxenic conditions. Maturation frequency was calculated relative to the number of immature somatic embryos induced to develop on the basal medium of von Arnold and Eriksson (1981). The average number of immature somatic embryos was 700 per gram of embryogenic callus, on medium supplemented with ABA and IBA (1 μM each). Maturation was the least efficient stage of regeneration; an average of 3% of the embryos induced to develop reached the cotyledonary stage. Mean germination frequencies were improved on treatments which avoided immersion of the radicle in medium solidified with agar. Whereas, 27% of the somatic embryos germinated when radicles were immersed in agar medium, 45% germinated when placed on the surface of the medium, and 56% germinated when cotyledons were immersed in agar medium and the culture vessel inverted. Twenty-nine percent of the somatic embryos germinatedin vitro were converted to plants. Under greenhouse conditions these plants set dormant buds, subsequently survived overwintering (to −5°C), and renewed vegetative growth synchronously with seedlings grown under the same conditions. Our results verified long-term (2 year) growth and development potential of conifer somatic embryo plants.     

Developmental pattern formation of somatic embryos induced in cell suspension cultures of cowpea [Vigna unguiculata (L.) Walp]

The sequence of events in the functional body pattern formation during the somatic embryo development in cowpea suspensions is described under three heads. Early stages of somatic embryogenesis were characterized by both periclinal and anticlinal cell divisions. Differentiation of the protoderm cell layer by periclinal divisions marked the commencement of somatic embryogenesis. The most critical events appear to be the formation of apical meristems, establishment of apical-basal patterns of symmetry, and cellular organization in oblong-stage somatic embryo for the transition to torpedo and cotyledonary-stage somatic embryos. Two different stages of mature embryos showing distinct morphology, classified based on the number of cotyledons and their ability to convert into plantlets, were visualized. Repeated mitotic divisions of the sub-epidermal cell layers marked the induction of proembryogenic mass (PEM) in the embryogenic calli. The first division plane was periclinally-oriented, the second anticlinally-oriented, and the subsequent division planes appeared in any direction, leading to clusters of proembryogenic clumps. Differentiation of the protoderm layer marks the beginning of the structural differentiation in globular stage. Incipient procambium formation is the first sign of somatic embryo transition. Axial elongation of inner isodiametric cells of the globular somatic embryo followed by the change in the growth axis of the procambium is an important event in oblong-stage somatic embryo. Vacuolation in the ground meristem of torpedo-stage embryo begins the process of histodifferentiation. Three major embryonic tissue systems; shoot apical meristem, root apical meristem, and the differentiation of procambial strands, are visible in torpedo-stage somatic embryo. Monocotyledonary-stage somatic embryo induced both the shoot apical meristem and two leaf primordia compared to the ansiocotyledonary somatic embryo.     

Cellular dynamics during maturation‐related decline of adventitious root formation in forest tree species

Adventitious root formation is a process in which roots are induced, from determined or differentiated cells that have not been specified to develop a root, at positions where they do not normally occur during development. In forest tree species, a decline in the capacity to form adventitious roots from similar cell types in stem cuttings is associated with tree age and maturity. This decline limits the success of vegetative propagation of selected adult trees. The joint action of local signals and a dynamic cascade of regulatory changes in gene expression, resulting in stereotypical cell division patterns, regulate cell fate changes that enable a somatic differentiated cell to reactivate meristem programs toward the induction of an adventitious root meristem.     

Genotypic control of peanut somatic embryogenesis

The protocol for obtaining a high frequency of plant development via somatic embryogenesis from mature zygotic embryo-derived leaflets of peanut (Arachis hypogaea L.) involves multiple stages; these include the induction of embryogenic masses, development of embryos, radicle emergence/conversion of embryos and the development of plants from rooted abnormal embryos. Sixteen genotypes were subjected to this protocol by exposing mature zygotic embryo-derived leaflets to the common media sequence and comparing responses. Although the protocol was effective for all the genotypes, variation in frequency of response at each stage of development indicated that, with the exception of root meristem differentiation and subsequent radicle emergence, the whole process of somatic embryogenesis depended on the genotypic constitution of the original plant. The failure of somatic embryos to undergo conversion to plantlets could be a genotype-dependent characteristic. Received: 5 June 1997 / Revision received: 2 December 1997 / Accepted: 12 December 1997     

Expression of the QrCPE gene is associated with the induction and development of oak somatic embryos

Somatic embryogenesis is a powerful tool for plant regeneration and also provides a suitable material for investigating the molecular events that control the induction and development of somatic embryos. This study focuses on expression analysis of the QrCPE gene (which encodes a glycine-rich protein) during the initiation of oak somatic embryos from leaf explants and also during the histodifferentiation of somatic embryos. Northern blot and in situ hybridization were used to determine the specific localisation of QrCPE mRNA. The results showed that the QrCPE gene is developmentally regulated during the histodifferentiation of somatic embryos and that its expression is tissue- and genotype-dependent. QrCPE was strongly expressed in embryogenic cell aggregates and in embryogenic nodular structures originated in leaf explants as well as in the protodermis of somatic embryos from which new embryos are generated by secondary embryogenesis. This suggests a role for the gene during the induction of somatic embryos and in the maintenance of embryogenic competence. The QrCPE gene was highly expressed in actively dividing cells during embryo development, suggesting that it participates in embryo histodifferentiation. The localised expression in the root cap initial cells of cotyledonary somatic embryos and in the root cap of somatic seedlings also suggests that the gene may be involved in the fate of root cap cells.