Effect of plumule and radicle on somatic embryogenesis in the cultures of ginseng zygotic embryos

Immature zygotic embryos of ginseng produced somatic embryos on MS medium without growth regulators. However, in the culture of mature zygotic embryos, excision of the embryo was required for somatic embryo induction. Somatic embryos formed only on excised cotyledons without an embryo axis or on excised embryos without the plumule and radicle of the axis. This observation suggests that the axis tip of the embryo might suppress somatic embryo production although the cotyledon tissues have predetermined embryogenic competency. To clarify the role of the embryo axis on somatic embryo formation, excised plumules or radicles were placed in direct contact with the basal cut-ends of cotyledons. The adhesion of plumules or radicles highly suppressed somatic embryo formation from cotyledon explants. When an agar block containing exudate from excised plumules or radicles was placed in contact with the cut end of the cotyledon, a similar inhibition was observed. These results suggest that embryogenic competence is suppressed by endogenous inhibitors present in the axis tip of the zygotic embryo.     

Secondary somatic embryogenesis and plant regeneration in myrtle (Myrtus communis L.)

Immature seeds, as well as hypocotyls and cotyledons excised from seedlings of Myrtus communis L., were cultured on media containing half-strength Murashige and Skoog macronutrients (MS/2) with combinations of auxins and cytokinins, in order to study their morphogenetic competence. Somatic embryogenesis was obtained from cotyledons, hypocotyls and 2-month-old immature seeds with 0.1 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D). The percentage of explants showing this primary somatic embryogenesis ranged from 4% for hypocotyls to 12% for 2-month-old immature seeds. In the latter, somatic embryogenesis was also obtained in media containing 2,4-D plus a cytokinin, and with only a cytokinin. Somatic embryos obtained from hypocotyls, cotyledons or immature seeds were able to develop on MS/2 medium without plant growth regulators. Subculture of primary somatic embryos obtained from immature seeds on MS/2 medium without plant growth regulators gave rise to clusters with secondary somatic embryos and embryogenic calli. These clusters were subcultured every 8 weeks, and they were the source of highly embryogenic cultures. An average of 10% of the secondary somatic embryos developed into plantlets in each subculture. Therefore, the same culture on MS/2 medium without growth regulators yielded both plantlets and de novo secondary embryos. Received: 6 April 1998 / Revision received: 10 July 1998 / Accepted: 21 July 1998     

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.     

Morphology of carrot somatic embryos formed in medium with abscisic acid

Somatic embryogenesis was induced from embryogenie cells derived from cotyledon expiants cultured on MS medium supplemented with 1 mg/L 2,4-D. In order to clarify the effect of abscisic acid (ABA) on the morphology of somatic embryos, embryogénie cell clumps or developing somatic embryos were treated continuously, or briefly, with ABA during culture. When embryogenie cells in MS medium without 2,4-D were treated with 0.04 mg/L ABA for the first week, normal embryos with two cotyledons increased slightly and embryos with anomalous cotyledons decreased. However when cell clumps in 2,4-D-free medium were treated with ABA in the second week normal embryos with two cotyledons decreased prominently and this decrease of normal embryos also occurred in the continuous ABA treatment during culture. Thus the morphological abnormalities in somatic embryogenesis occurred by exogenous ABA treatment beyond globular stage or by continuous treatment. The length of somatic embryos with anomalous cotyledons was larger than that of normal embryos with two cotyledons in control but both the normal and anomalous somatic embryos treated with ABA were almost similar in length. Somatic embryos formed in medium with ABA were larger in size than those in control due mainly to enlarged cotyledons. The enlarged cotyledons were composed of a greater number of cells than those of control. Therefore the enlargement of cotyledon by exogenous ABA seems to be not due to the enlargement of cells in cotyledons.     

TEMPORAL COMPETENCE OF EMBRYONIC PINUS PONDEROSA COTYLEDONS TO FORM MULTIPLE BUDS IN VITRO

To investigate temporal changes occurring during tissue maturation and loss of organogenic competence, embryos of Pinus ponderosa Laws, were excised from seed and placed horizontally on medium in sterile culture. On a hormone-free basal medium, cotyledons differentiated and failed to form multiple buds. When placed on a benzyladenine (BA)-containing medium, the lower cotyledons in contact with the medium formed multiple buds while those elevated above the medium did not. Cotyledons became incompetent to form buds when embryos were initially placed on basal medium for 2 days before being transferred to the BA-supplemented medium. In order to initiate buds, cotyledons of newly excised embryos had to be exposed to BA for at least 3 days. Exposure to BA for longer than 7 days did not significantly increase the number of cotyledonary buds. Cotyledons elevated above the BA-supplemented medium retained organogenic competence to form buds for up to 14 days but did not form buds unless the cotyledons were placed in contact with the medium. The presence of BA is required to retard cotyledon maturation and sustain tissue competence to initiate buds.     

Histology of Organogenic and Embryogenic Responses in Cotyledons of Somatic Embryos of Quercus Suber L

In cork oak (Quercus suber L.), recurrent embryogenesis is produced in vitro through autoembryony without exogenous plant growth regulators (PGRs); secondary embryos appear on the embryo axis but seldom on cotyledons. Focusing mainly on the histological origin of neoformations, we investigated the influence of the embryo axis and exogenous PGRs on the embryogenic potential of somatic embryo cotyledons. Isolated cotyledons of somatic embryos became necrotic when cultured on PGR-free medium but gave secondary embryos when cultured on media containing benzyladenine and naphthaleneacetic acid. Cotyledons of cork oak somatic embryos are competent to give embryogenic responses. Isolated cotyledons without a petiole showed a lower percentage of embryogenic response than did those with a petiole. In petioles, somatic embryos arose from inner parenchyma tissues following a multicellular budding pattern. Joined to the embryo axis, cotyledons did not show morphogenic responses when cultured on PGR-free medium but revealed budlike and phylloid formations when cultured on medium with PGRs. The different morphogenic behavior displayed by somatic cotyledons indicates an influence of the embryo axis and indicates a relationship between organogenic and embryogenic regeneration pathways.     

Characterization of immature embryos of interior spruce by SDS-PAGE and microscopy in relation to their competence for somatic embryogenesis

SDS-PAGE analysis of total proteins from cotyledonary embryo explants reveals that their competence to form somatic embryos is limited to a specific stage of development prior to the accumulation of storage proteins. When protein profiles of embryo explants of different open pollinated families from the same collection date are compared, there is a close relationship between the absence of storage proteins and their ability to produce embryogenic callus. In addition, the appearance of storage proteins in embryos from subsequent collections is associated with their loss of competence. Light microscopy combined with staining for total protein demonstrates that competent immature embryos have cotyledons but do not contain protein bodies.Abbreviations SDS-PAGE Sodium dodecylsulfate polyacrylamide gel electrophoresis - 2,4-D 2,4 dichlorophenoxyacetic acid - ABA Abscisic acid - BA Benzyladenine - EDTA Ethylenediaminetetraacetic acid     

Enhancing the frequency of somatic embryogenesis following <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of immature cotyledons of soybean [<Emphasis Type="Italic">Glycine max</Emphasis> (L.) Merrill.]

Summary A characteristic phenotype of highly embryogenic explants along with the location of embryogenesis- and transformation-competent cells/tissues on immature cotyledons of soybean [Glycine max (L.) Merrill.] under hygromycin selection was identified. This highly embryogenic immature cotyledon was characterized with emergence of somatic embryos and incidence of browning/necrotic tissues along the margins and collapsed tissues in the mid-region of an explant incubated upwards on the selection medium. The influences of various parameters on induction of somatic embryogenesis on immature cotyledons following Agrobacterium tumefaciens-mediated transformation and selection were investigated. Using cotyledon explants derived from immature embryos of 5–8 mm in length, a 1∶1 (v/v; bacterial cells to liquid D40 medium) concentration of bacterial suspension and 4-wk cocultivation period significantly increased the frequency of transgenic somatic embryos. Whereas, increasing the infection period of explants or subjecting explants to either wounding or acetosyringone treatments did not increase the frequency of transformation. An optimal selection regime was identified when inoculated immature cotyledons were incubated on either 10 or 25 mgl⁻¹ hygromycin for a 2-wk period, and then maintained on selection media containing 25 mgl⁻¹ hygromycin in subsequent selection periods. However, somatic embryogenesis was completely inhibited when inoculated immature cotyledons were incubated on a kanamycin selection medium. These findings clearly demonstrated that the tissue culture protocols for transformation of soybean should be established under both Agrobacterium and selection conditions.     

AtSERK1 expression precedes and coincides with early somatic embryogenesis in Arabidopsis thaliana.

The Arabidopsis thaliana primordia timing (pt) mutant was transformed with an AtSERK1::GUS construct. Liquid cultures of this line were used to study the relationship between somatic embryogenesis and the expression of SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE (AtSERK1) as a marker for cells competent to form embryos. In order to search for the expression of AtSERK1::GUS during early stages of somatic embryogenesis, histochemical as well as immunochemical approaches were used for the detection of beta-glucuronidase (GUS). Four sites of AtSERK1 expression were found in the embryogenic cultures: in embryogenic callus, where primary somatic embryos developed; in the basal parts of primary somatic embryos; in the outer layers of cotyledons of primary somatic embryos where secondary embryos were formed; and in provascular and vascular strands of developing somatic embryos. The in vitro expression of AtSERK1::GUS coincides with embryogenic development up to the heart-shaped stage. Prior to the expression in embryos, AtSERK1 was expressed in single cells and small cell clusters, indicating that AtSERK1 indeed marks embryogenic competence. Its expression in (pro)vascular strands, suggests that embryogenic cells in tissue culture retain at least in part their original identity.     

Thidiazuron-induced somatic embryogenesis in intact seedlings of peanut (Arachis hypogaea): Endogenous growth regulator levels and significance of cotyledons

The regulatory role of thidiazuron (TDZ) and explant factors in imparting somatic embryogenic potential was assessed in relation to endogenous growth regulator levels in peanut ( Arachis hypogaea L. cv. Tango). TDZ induced somatic embryogenesis over a range of concentrations (0.5 to 10 μ M ). Culture of seedlings for just 2 days on TDZ-supplemented medium was sufficient to induce somatic embryogenesis. Seedling age and retention or removal of cotyledons during culture influenced morphogenic potential significantly. The younger the seedlings, the better was the embryogenic response to TDZ treatment. The embryogenic potential of seedlings was limited for explants with no cotyledons and they did not respond to increasing levels of TDZ. In contrast, retention of at least one or both the cotyledons resulted in increased response to TDZ. Endogenous levels of cytokinins, auxins and abscisic acid were influenced by TDZ treatment, while TDZ itself was detected only in the cotyledons. The cytokinin N⁶-(2-iso-pentenyl)adenine (2iP) fluctuated on a 10-day cycle in the cotyledons; TDZ treatment suppressed this change and caused an overall decrease in the pool of available 2iP in the tissue. It appeared that TDZ induced somatic embryogenesis in peanut by influencing endogenous levels of both auxin and cytokinins.     

Genetic improvement of the somatic embryogenesis and regeneration in soybean and transformation of the improved breeding lines

Somatic embryos of soybean [Glycine max (L.) Merrill] have been used to generate transgenic plants by particle bombardment. The induction and proliferation of somatic embryos from immature cotyledons are dependent on the genotype of the cultivar. Whereas somatic embryogenesis and plant regeneration are inefficient in most cultivars, they are efficient in the cultivar Jack. We previously established a breeding line, QF2, by the integration of null mutations of each subunit of the major seed storage proteins glycinin and β-conglycinin, but the embryogenic response of this line is insufficient to allow efficient transformation. We have now backcrossed QF2 to cultivar Jack in order to combine the null traits with competence for somatic embryogenesis. The backcrossed breeding lines selected on the basis of the absence of the major storage proteins exhibited an improved capacity for the induction and proliferation of somatic embryos compared with that of QF2. The induced somatic embryogenic tissue of these breeding lines was successfully used for the production of transgenic plants by particle bombardment. These results also indicate that somatic embryogenesis in soybean is genetically controlled and inherited in a manner independent of the null traits of the major seed storage proteins.     

Endogenous hormonal content and somatic embryogenic capacity of Corylus avellana L. cotyledons

Endogenous indole-3-acetic acid (IAA), abscisic acid (ABA) and cytokinins [zeatin (Z) zeatin riboside, dihydrozeatin, dihydrozeatin riboside, N⁶-isopentenyl adenine (iP) and N⁶-isopentenyladenine riboside] were evaluated in hazelnut (Corylus avellana L.) cotyledons of different developmental stage and genetic source for their somatic embryogenic capacity. There was an inverse correlation between the embryogenic potential of cotyledons and the degree of maturity of zygotic embryos, the first characteristic being associated with iP-type cytokinins and the second with Z-type cytokinins. Although the differences in total cytokinin, ABA and IAA contents between the cotyledons were small, the IAA/ABA and, mainly, the iP-type/Z-type cytokinin ratios were found to be two good indexes of the embryogenic competence of explants, suggesting that the endogenous hormonal balance is a very important factor defining the in vitro potential of hazelnut cotyledons. Received: 6 January 1997 / Revision received: 3 March 1997 / Accepted 1 April 1997     

The effects of reduced and oxidized glutathione on white spruce somatic embryogenesis

Summary The glutathione-glutathione disulfide redox pair was utilized to improve white spurce somatic embryo development. Mature cotyledonary-stage somatic embryos were divided into two groups (A and B) based on morphological normality and the ability of the mature somatic embryos to convert into plantlets. Group A embryos had four or more cotyledons and converted readily upon germination after a partial drying treatment. Group B embryos had three or fewer cotyledons with a low conversion frequency. The addition of reduced glutathione (GSH) at a concentration of 0.1 mM resulted in an increase in embryo production (total population) with a mean total number of 64 embryos per 100 mg embryogenic tissue as well as an increase in post-embryonic root growth. However, at a higher concentration (1 mM), GSH inhibited embryo formation. The manipulation of the tissue culture environment via the inclusion of glutathione disulfide (GSSG), at concentrations of 0.1 and 1.0 mM, enhanced the development of better-quality embryos. This quality was best exemplified when embryos forming four or more cotyledons increased by at least twofold to 73.9% when treated with 1.0 mM GSSG, compared to 38% in control. Furthermore, this improved quality was reflected by an increased conversion frequency. A 20% increase in the ability of the somatic embryo to produce both root and shoot structures during post-embryonic development was noted when embryos were matured on maturation medium supplemented with 1.0 mM GSSG over the control.     

Relationship between auxin-induced cell proliferation and somatic embryogenesis in culture of carrot cotyledons

We elucidated the relationship between cell proliferation and somatic embryogenesis in the culture of carrot cotyledons. Fresh weights of the cotyledon expiants were determined every five days while being cultured on a medium containing 2,4-D. Callus production increased exponentially from Day 20 to Day 25, showing a two-fold rate of proliferation. To examine the embryogenic potential of the callus, we pre-cultured cotyledon explants on an MS medium with 2,4-D, then transferred them to an MS basal medium at five-day intervals. Somatic embryos formed most frequently when the cotyledons were pre-cultured for 20 days on an MS medium that contained 5 μ2,4-D. The frequency of somatic embryo formation was 81%, while that of normal embryos with two cotyledons was 51% among those formed on a hormone-free medium. We used FACScan analysis to relate the embryogenic potential of the callus to the S phase in the cell cycle of cultured cells. The S phase was high after 25 days of culture on the medium with 5 μM 2,4-D. In contrast, the frequency of normal embryogenesis was higher at Day 20 of the pre-culture period. Culturing embryogenic calli on a medium with 5 μM 2,4-D was most favorable for producing somatic embryos with two cotyledons. We verified that active somatic embryogenesis was apparently related to cell division activity; somatic embryos induced from actively dividing cells were apt to accompany cotyledonary abnormality.     

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.     

Proliferation,maturation and germination of Castanea sativa Mill. Somatic embryos originated from leaf explants

Experiments were performed to determine the influence of proliferation medium on the maintenance of embryogenic competence and on repetitive embryogenesis in Castanea sativa Mill. somatic embryos derived from leaf explants. Somatic embryo proliferation was carried out by both direct secondary embryogenesis and by the culture of nodular callus tissue originated from cotyledons of somatic embryos. Both systems led to the production of cotyledonary somatic embryos on Murashige and Skoog proliferation medium supplemented with 0.1 mg l-1 benzyladenine and 0.1 mg l-1 naphthaleneacetic acid. Carbon source and concentration had a marked influence on maturation and subsequent germination ability of chestnut somatic embryos. Plantlet conversion was achieved in embryos matured on media with 6 % sucrose, and on 3 or 6 % maltose, whereas mean shoot length, root length and leaf number of produced plants were not significantly affected by these maturation media. Overall, the best results were obtained with 3 % maltose-matured somatic embryos, giving rise to 6 % plant recovery in addition to 33 % of embryos exhibiting only shoot development. The application of a 2-month cold treatment at 4 degrees C to somatic embryos matured on medium with 3 % maltose was necessary for achieving plant conversion, while partial desiccation did not appear to influence this response. A total of 39 % of embryos eventually produced plants either through conversion to plantlets or indirectly through rooting of shoots. Shoots formed by somatic embryos could be excised, multiplied and rooted following the micropropagation procedures previously developed for chestnut.     

Global gene expression analysis of bovine somatic cell nuclear transfer blastocysts and cotyledons

Low developmental competence of bovine somatic cell nuclear transfer (SCNT) embryos is a universal problem. Abnormal placentation has been commonly reported in SCNT pregnancies from a number of species. The present study employed Affymetrix bovine expression microarrays to examine global gene expression patterns of SCNT and in vivo produced (AI) blastocysts as well as cotyledons from day‐70 SCNT and AI pregnancies. SCNT and AI embryos and cotyledons were analyzed for differential expression. Also in an attempt to establish a link between abnormal gene expression patterns in early embryos and cotyledons, differentially expressed genes were compared between the two studies. Microarray analysis yielded a list of 28 genes differentially expressed between SCNT and AI blastocysts and 19 differentially expressed cotyledon genes. None of the differentially expressed genes were common to both groups, although major histocompatibility complex I (MHCI) was significant in the embryo data and approached significance in the cotyledon data. This is the first study to report global gene expression patterns in bovine AI and SCNT cotyledons. The embryonic gene expression data reported here adds to a growing body of data that indicates the common occurrence of aberrant gene expression in early SCNT embryos. Mol. Reprod. Dev. 76: 471–482, 2009. © 2008 Wiley‐Liss, Inc.     

Histochemical studies on mobilization of storage components in cotyledons of germinatingPhaseolus mungo seeds

Phaseolus mungo seeds were allowed to germinate in the dark at 27 C, and time-sequence changes of mobilization of protein and starch reserves in cotyledons were observed by histochemical techniques. The distributions of amylase and protease activities in cotyledon sections were also examined during germination by use of the starch-polyacrylamide gel film and India ink-gelatin film methods, respectively. Amylolytic and proteolytic processes occurred more or less simultaneously during the germination. At the day 2 stage, low levels of hydrolytic enzyme activities were observed throughout cotyledon sections. At day 4, both amylase and protease activities appeared to increase in tissue areas farthest from vascular bundles, and the mobilization of starch and protein reserves also proceeded in these areas. At day 6, the reserves were found to remain only in the cells around vascular bundles. When cotyledons were detached from axis organs, allowed to imbibe water and incubated for 4 days at 27 C, the breakdown of reserves was markedly retarded and the patterns of enzyme localization in cotyledon sections appeared not as conspicuous as those in the sections from intact cotyledons. These histochemical results are discussed with reference to the previous results ofin vitro experiments.     

Developmental and structural aspects of somatic embryos formed on medium containing 2,3,5-triiodobenzoic acid

Cotyledon explants of ginseng (Panax ginseng C.A. Meyer) zygotic embryos produced somatic embryos at a high rate (68%) on medium without any growth regulators. Under this culture condition, apparent polar somatic embryogenesis occurred near the basal-excised portion of the cotyledons. When the cotyledon explants were cultured on medium containing 2,3,5-triiodobenzoic acid (TIBA), an auxin polar-transport inhibitor, the frequency of somatic embryo formation markedly decreased and was completely inhibited on medium containing 20 μM TIBA. On medium containing 5–10 μM, somatic embryos developed sporadically on the surface of the cotyledons and had a normal embryo axis but jar-shaped cotyledons. Embryos with jar-shaped cotyledons were also observed to occur at a high frequency when the early globular embryos formed on hormone-free medium were transferred to medium containing 20 μM TIBA. From these results, it was deduced that endogenous auxin in the cotyledon explants plays an important role in the induction of somatic embryos and that the cotyledon development in somatic embryos is also related to the polar transport of endogenous auxin. Received: 11 October 1996 / Revised version received: 8 January 1997 / Accepted: 26 January 1997     

Microsurgery reveals regional capabilities for pattern reestablishment in somatic carrot embryos

The extent to which regions of a somatic embryo were committed to a particular developmental fate was explored by surgically removing portions of somatic embryos and observing patterns of regeneration. Through a variety of excisions that resulted in tissue slices ranging from less than 10% to nearly 90% of the original embryo mass, we observed only a few cases where such isolates completely abandoned preexisting patterns of organized growth. Instead, most subcultured portions of the embryonic axis restored all, or part of, a missing complement of the organism. At the shoot apex, a single lost cotyledon was replaced by new cotyledonary structures, although these usually occurred as multiple pairs of cotyledons. If both cotyledons were removed, secondary axes, each with its own cotyledons, typically formed at the embryo midlength. When embryos were divided into shoot and root pieces, the shoot pole usually regenerated a new root, while the original root and rapidly elongated and matured days earlier than uncut controls. Surprisingly, cotyledon regeneration from excised root sections occurred at much greater frequency when the root piece comprised only 10-25% of the embryo mass; larger portions of the root pole rarely produced recognizable shoot structures. These studies indicate that several discrete regions of the embryo are committed to specific types of patterned growth, and that continuity between certain of these regions is required for the maintenance of axial polarity.