Suppression of somatic embryogenesis in Citrus cell cultures by extracellular proteins

Nucellar-derived cell cultures of sour orange (Citrus aurantium L.) proliferate as proembryogenic masses. By a change in the carbon source of the medium from sucrose to glycerol they are induced to undergo synchronous embryogenesis forming embryo initials that develop into globular embryos. The proembryogenic masses released glycoproteins to the medium. Exogenous addition of the glycoproteins to cells in glycerol-containing medium modified the course of embryo development in a dose-dependent manner. Addition of 20 g · ml^–1 of glycoproteins blocked embryogenesis and resulted in an accumulation of embryo initials. When glycoproteins were added to cultures containing advanced globularstage embryos further development was suppressed. The inhibitory component of the glycoproteins was found to be a family of polypeptides with apparent molecular masses of 53–57 kDa. While these proteins normally accumulated only in cultures of proembryogenic masses, they could be induced to accumulate in glycerol-containing medium by the addition of the glycoproteins. Thus, their accumulation was not a direct consequence of the type of growth medium used or the developmental state of the cultures. The results indicate that the 53-to 57 kDa glycoproteins could play a regulatory role in in-vitro embryogenesis in sour orange. The normal progression of embryo development appears to depend, in an obligatory manner, on the absence of these glycosylated extracellular proteins from the medium.Abbreviations kDa kilodalton - PEM proembryogenic masses - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis - 2D-PAGE Two-dimensional polyacrylamide gel electrophoresis We thank Dr. S. Satoh (Institute of Biological Sciences, Tsukuba, Japan) for sending protein samples of the purified 57-kDa glycoprotein. This research was supported by a grant from the Charles H. Revson Foundation for Basic Research in the Life Sciences of the Israel Academy of Sciences. R.F. is a recipient of the Jack and Florence Goodman Career Development Chair.     

Somatic embryogenesis in pumpkin (Cucurbita pepo L.): control of somatic embryo development by nitrogen compounds

Embryogenic cultures of pumpkin (Cucurbita pepo L.) were initiated from mechanically wounded mature zygotic embryos on 2,4-D-containing MS medium, and on hormone-free, semisolid modified MS medium containing NH4Cl as the sole source of nitrogen. The habituated line was derived from the embryogenic tissue induced with 2,4-D and maintained on medium without growth regulators. Sustained subculturing of the three embryogenic lines on a medium with NH4Cl as the sole source of nitrogen enabled the establishment of highly uniform cultures in which no further development into mature embryo stages occurred. The tissue consisting of proembryogenic globules or globular stage embryos was maintained, without decline, for over six years. Globular embryos proceeded to maturity when a combination of reduced (NH4) and unreduced (NO3) forms of nitrogen was provided in the medium. Different nitrogen sources in the medium caused changes of medium pH during subculture in the pH range of 4.0-6.5. The tissue growth and embryo development were blocked on medium with pH adjusted and stabilized at 4.0 or at 3.2.     

Proteins related with embryogenic potential in callus and cell suspensions of sugarcane (Saccharum sp.)

Summary Previous results have shown that some proteins secreted in the culture medium are involved with the formation of embryogenic cells and can modify somatic embryo differentiation. Undifferentiated cell suspensions grown in the presence of 13 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and obtained from embryogenic and non-embryogenic callus were used to study these events in sugarcane plants (cv.PR-62258). The cell suspension growth curves were determined and soluble proteins were extracted from embryogenic and non-embryogenic callus and culture medium from cell suspensions. In embryogenic callus we detected 1.43 times more protein than in non-embryogenic callus and the electrophoretic protein patterns show specific polypeptides for both callus types. In embryogenic callus we detected a cluster of four polypeptides in the range of 38–44 kDa and another polypeptide of 23 kDa that were not observed in non-embryogenic callus. In nonembryogenic callus there is a 35-kDa polypeptide that was not detected in embryogenic callus. In the case of extracellular proteins, the medium from embryogenic cell suspensions contained four polypeptides of 41, 38, 34 and 28 kDa that were slightly detected in the medium from non-embryogenic cell cultures; we also detected a band at 15 kDa that could not be observed in the medium from non-embryogenic cell suspensions. These results suggest that the development of embryogenic callus and cell suspensions is related to the type and amount of intracellular proteins in the callus cells and to the secreted proteins from these cells into the medium.     

Monoclonal antibody against a cell wall marker protein for embryogenic potential of <Emphasis Type="Italic">Dactylis glomerata</Emphasis> L. suspension cultures

We identified and isolated a monoclonal antibody (MAb 3G2) raised against extracellular proteins from microcluster cells of orchard grass (Dactylis glomerata L.) embryogenic suspension culture. MAb 3G2 recognized with high specificity an antigen ionically bound within the primary cell wall and in the culture medium of microcluster cells. Two-dimensional polyacrylamide gel analysis and blotting of proteins on PVDF membrane showed that MAb 3G2 detected a single polypeptide of apparent molecular mass of 48 kDa and an isoelectric point (pI) of 5.2, designated EP48. A transient expression during somatic embryogenesis was observed for EP48. Indirect immunofluorescence showed that this protein highly accumulated in the cell walls of some single cells, microclusters and partly in proembryogenic masses (PEMs), but not in globular embryos of the embryogenic cell line and microclusters from the non-embryogenic cell line. Signal intensity varied between individual cells of the same population and in successive stages of somatic embryo development. Screening of several D. glomerata L. embryogenic and non-embryogenic cell lines with MAb 3G2 indicated the presence of ECP48 in only embryogenic suspension cultures at early stages of embryo development long before morphological changes have taken place and thus it could serve as an early marker for embryogenic potential in D. glomerata L. suspension cultures.     

Extracellular proteins and early embryo development in Citrus nucellar cell cultures

Highly efficient synchronous embryogenesis was induced in suspension cultures of sour orange ( Citrus aurantium L.) by a change in the carbon source of the growth medium from sucrose to glycerol. In liquid culture the embryos developed into globular structures during a three week period. Embryo development showed an absolute requirement for the continued presence of glycerol. The embryo cell cultures turned green in the light, but light did not affect the course of development. The profiles of soluble cellular protein extracts of embryo and proembryogenic (PEM) cells were very similar as judged by two-dimensional polyacrylamide gel electrophoresis. However, major differences were detected in the profiles of extracellular proteins. PEM cells accumulated extracellular glycoproteins of 53–57 kDa mass. Upon subculture in glycerol containing medium, the accumulation of these proteins ceased within two days. Developing embryos accumulated at least 4 new extracellular polypeptides of 41–42 kDa mass. In addition to these polypeptides, stage specific peroxidases and proteases were found. The relatively extended duration and synchrony in which these early developmental events take place make Citrus cultures an especially useful system for the study of early events in plant embryogenesis.     

Influence of extracellular proteins,proteases and protease inhibitors on grapevine somatic embryogenesis

An embryogenic grapevine rootstock cell suspension, continuously grown in the presence of auxin, was predominantly composed of proembryogenic masses. When transferred to an auxin-free medium, grapevine somatic embryos developed but were rapidly blocked at the heart stage. This inhibition has been related to the presence of extracellular macromolecules (Coutos-Thévenot et al., 1992a). In this study, the initial cell population density has been found to influence markedly embryo development. Inoculations below 5·10³ cells per ml were required to obtain fully grown cotyledonary embryos. Interestingly, extracellular proteins of molecular weights of 32, 34, 48 and 52 kDa accumulated in cultures grown at high population cell densities and disappeared in cultures inoculated at densities below 5·10³ cells per ml. Protein fractions partially purified by ion exchange chromatography caused both an early inhibition of embryogenesis and a stimulation of secondary embryogenesis. Moreover, to test for the possibility of modulating embryo development through alterations of extracellular proteins, cultures were supplemented with proteases and protease inhibitors. The addition of trypsin increased the rate of embryo development only in cultures inoculated at a low cell population density. Conversely, the protease inhibitor aprotinin inhibited development, arresting embryos at globular and heart stages. Together, these results provide evidence that extracellular proteins modulate somatic embryogenesis and suggest that an extracellular proteolitic mechanism could be implicated in development. This revised version was published online in June 2006 with corrections to the Cover Date.     

Acquisition of embryogenic potential in carrot cell-suspension cultures

Embryogenic suspension cultures of domesticated carrot (Daucus carota L.) are characterized by the presence of proembryogenic masses (PEMs) from which somatic embryos develop under conditions of low cell density in the absence of phytohormones. A culture system, referred to as starting cultures, was developed that allowed analysis of the emergence of PEMs in newly initiated hypocotyl-derived suspension cultures. Embryogenic potential, reflected by the number of FEMs present, slowly increased in starting cultures over a period of six weeks. Addition of excreted, high-molecular-weight, heat-labile cell factors from an established embryogenic culture considerably accelerated the acquisition of embryogenic potential in starting cultures. Analysis of [³⁵S]methionine-labeled proteins excreted into the medium revealed distinct changes concomitant with the acquisition of embryogenic potential in these cultures. Analysis of the pattern of gene expression by in-vitro translation of total cellular mRNA from starting cultures with different embryogenic potential and subsequent separation of the [³⁵S]methionine-labeled products by two-dimensional polyacrylamide gel electrophoresis demonstrated a small number of abundant in-vitro-translation products to be present in somatic embryos and in embryogenic cells but absent in nonembryogenic cells. Several other in-vitro-translation products were present in explants, non-embryogenic and embryogenic cells but were absent in somatic embryos. Hybridization of an embryoregulated complementary-DNA sequence, Dc3, to RNA extracted from starting cultures showed that the corresponding gene is expressed in somatic embryos and PEMs but not in non-embryogenic cells.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - cDNA complementary DNA - PAGE polyacrylamide gel electrophoresis - PEM proembryogenic mass     

Influence of culture conditions on embryo formation and maturation in auxin-induced embryogenic cultures of Digitalis obscura

Somatic embryogenesis was induced in hypocotyls of Digitalis obscura using indoleacetic acid or 2,4-dichlorophenoxyacetic acid with different culture and subculture conditions. Indoleacetic acid-induced embryogenic cultures were used to investigate the effects of amino acids, polyamines and growth regulators on embryo differentiation and maturation. Supplementation of the media with amino acids, polyamines or abscisic acid did not influence or had an adverse effect on embryogenic response. Gibberellic acid at 1.4 M in either culture (30 days) or subculture medium was effective in promoting both differentiation and normal embryo development. The efficiency of somatic embryogenesis was greatly enhanced when isolated indoleacetic acid-induced proembryogenic masses were subcultured in liquid medium with reduced auxin content.Abbreviations ABA abscisic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - GA gibberellic acid - IAA indoleacetic acid - Ptr putrescine - Spd spermidine - Spn spermine     

Relation of the developmental stage of zygotic embryos of yellow-poplar to their somatic embryogenic potential

The goal of the study was to characterize the optimal developmental stage of zygotic embryo expiants of the hardwood forest tree species yellow-poplar (Liriodendron tulipifera L.) for the initiation of embryogenic cultures, using morphological measurements and polypeptide profiles of the embryos. Developing zygotic embryos from seeds of six full-sib families, collected every two weeks from 4 weeks postpollination until seed maturity (18 weeks postpollination) were divided into 2 subsamples for each collection date. One group was used to initiate tissue cultures. Embryos in the other group were measured (total length, cotyledon length and hypocotyl thickness) and soluble polypeptide profiles of the embryos were analyzed by SDS-polyacrylamide gel electrophoresis. Potential of an expiant to produce an embryogenic culture peaked during the eighth week following pollination, with an average of 28% of the expiants producing proembryogenic masses, and declined to near zero for mature zygotic embryos. The maximum embryogenic potential corresponded to the globular stage of embryo developmet. Soluble protein profiles of zygotic embryos from 5 sampling dates indicated that decline in embryogenic potential appeared to parallel an increase in the level of a polypeptide of approximately 55 kDa, possibly a storage protein.Abbreviations BA Benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - CH Casein hydrolysate - SDS-PAGE Sodium dodecylsulfate polyacrylamide gel electrophoresis - PEM Proembryogenic mass     

Somatic embryogenesis and plant regeneration from cell suspension culture of niger (Guizotia abyssinica Cass.)

Somatic embryogenesis was achieved from cell suspension cultures of niger (Guizotia abyssinica Cass.). Initially, friable embryogenic calluses were induced from cotyledonary leaves of niger on Murashige and Skoog (MS) agar medium containing 5 μM 2,4-Dichlorophenoxyacetic acid (2,4-D) and 0.5 μM kinetin (KIN). Cell suspension cultures were established by using embryogenic calluses in MS liquid medium containing 5 μM 2,4-D and 0.5 μM KIN. Initiation of somatic embryogenesis and development up to globular stage from embryogenic cell clumps occurred in the liquid medium itself. Thereafter embryogenic cell aggregates were transferred to MS agar medium supplemented with 3 μM KIN for embryo differentiation, whereas maturation of somatic embryos occurred in MS agar medium containing 10 μM abscisic acid.     

Somatic embryogenesis and plant regeneration in Cyphomandra betacea (Cav.) Sendt

Callus cultures with globular proembryogenic structures were induced from zygotic embryos and hypocotyl segments of Cyphomandra betacea on MS medium supplemented with 2,4-D. Proembryogenic structures produced somatic embryos and plantlets on regulator-free basal medium. Pieces of embryogenic callus subcultured on medium with the same original composition gave rise to new globular structures and the potential for plantlet regeneration has been maintained for over a year. The histological examination of these proembryogenic structures suggested that somatic embryos arise from single cells. Regenerated plants are phenotypically normal, having diploid chromosome numbers (2n = 24).     

Changes in DNA methylation during somatic embryogenesis in <Emphasis Type="Italic">Cucurbita pepo</Emphasis> L.

Three pumpkin embryogenic lines were initiated on wounded zygotic embryos cultured on medium with or without 2,4-dichlorophenoxyacetic acid (2,4-D). Somatic embryo development was controlled by the availability of various compounds in the medium: presence/absence of 2,4-D, nitrogen sources. The highest rate of DNA methylation was in the early embryo stages, predominantly on MSC medium with 2,4-D and on auxin-free medium supplemented with 1.0 mM NH₄Cl. DNA methylation was correlated with early embryo development in a manner that was not exclusively dependent on the presence/absence of exogenous auxin. DNA methylation decreased during embryo maturation on auxin-free MSC medium and on auxin-free MSC supplemented with 12.3 M 5-azacytidine (5-azaC). The embryogenic features of the pumpkin tissue were preserved, even after a 2-month treatment with 5-azaC.Abbreviations 5-azaC 5-Azacytidine - CRED-RA Coupled restriction enzyme digestion and random amplification - 2,4-D 2,4-Dichlorophenoxyacetic acid - DNMRT Duncans new multiple range test - IAA Indole-3-acetic acid - 5-mC 5-Methylcytosine     

Variation in endoplasmic-reticulum-associated glycoproteins of carrot cells cultured in vitro

Glycoproteins extracted from microsomes of in-vitro-cultured cells of Daucus carota L. cv. US-Harumakigosun were analysed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and detected by peroxidase-conjugated concanavalin A. The appearance of a glycoprotein with M_r 31 000 (GP 31) was correlated with the ability of cells to form somatic embryos. GP 31 appeared in embryogenic cells cultured in 2,4-dichlorophenoxyacetic acid (2,4-D)-containing medium, but not in somatic embryos and non-embryogenic cells; it disappeared when the cultures were transferred to auxin-free medium. Another glycoprotein with M_r 32 000 (GP 32) was detected only in non-embryogenic cells, regardless of the presence or absence of 2,4-D. Both glycoproteins, GP 31 and GP 32, were associated with the rough endoplasmic reticulum and were extractable with 0.05% deoxycholate.Abbreviations Con A concanavalin A - 2,4-D 2,4-dichlorophenoxyacetic acid - ER endoplasmic reticulum - GP 31, GP 32 a glycoprotein with an apparent molecular mass of 31 or 32 kdalton - kDa kilodalton - MS Murashige and Skoog - M_r apparent molecular mass - SDS-PAGE sodium dodecylsulfate-polyacrylamide gel electrophoresis     

Low external pH replaces 2,4-D in maintaining and multiplying 2,4-D-initiated embryogenic cells of carrot

A mixed culture comprised of both embryonic globules and nonembryogenic callus. was derived from seedling hypocotyls of Daucus carota cv. Scarlet Nantes on 2,4-D-containing medium using well-established methods. Then the mixed cultures were transferred to, and serially subcultured on, a hormone-free medium near pH 4. The medium contained 1 m M NH+ as the sole nitrogen source. When cultured in this way, embryonic globules were able to multiply without development into later embryo stages Nonembryogenic callus did not survive. Continuous culture of embryonic globules on this low pH hormone-free medium yielded cultures consisting entirely of preglobular stage proembryos (PGSPs). PGSP cultures have been maintained as such with continuous multiplication for nearly 2 years without loss of embryogenic potential. These hormone-free-maintained PGSPs continue their development to later embryo stages when cultured on the same hormone-free medium buffered at pH 5.8. We show that hormone-free medium near pH 4 can replace 2.4-D in its ability to sustain multiplication of 2,4-D-initiated embryogenic cells of carrot at an acceptable growth rate without their development into later embryo stages. This procedure provides selective conditions that do not permit the growth of nonembryogenic cells while providing an adequate environment for embryogenic cell proliferation and should prove invaluable in studying habituation.     

Synthesis of extracellular proteins in embryogenic and non-embryogenic cell cultures of alfalfa

Extracellular proteins, released into the culture medium from alfalfa cells grown in embryogenic and non-embryogenic conditions, were ³⁵S-methionine labelled at different days of culture. SDS-PAGE analysis showed significant differences between the patterns of extracellular proteins secreted into the medium devoid of 2,4-d, in which cells formed somatic embryos, or in presence of 2,4-d, in which undifferentiated cell proliferation took place. Some proteins, evident in 2,4-d-supplied cultures, disappeared when cells were subcultured in the embryogenic conditions. Western analysis with antibodies against the carrot extracellular proteins EP1 and EP2 showed the presence of homologous alfalfa proteins. In 2,4-d depleted alfalfa cells, an EP1-like protein disappeared and another one was reduced, while the presence of the EP2-like protein was, in the same conditions, strongly enhanced.Abbreviations 2,4-d 2,4-dichlorophenoxyacetic acid - EP extracellular proteins - ns-LTP non specific lipid transfer protein - SDS-PAGE Sodium dodecyl sulphate polyacrilamide gel electrophoresis     

Propagation of Lirlodendron hybrids via somatic embryogenesis

Propagation of hybrids between the Chinese tuliptree (Liriodendron chinense) and the North American yellow-poplar (Liriodendron tulipiferea) could be greatly accelerated with a highly productive somatic embryogenesis system. Flowers were collected from a single Chinese tuliptree and the anthers used for controlled pollinations of 4 yellow-poplar mother trees. Aggregates of samaras resulting from the pollinations were harvested 8 weeks post-pollination. Following surface disinfestation, samaras were dissected and embryos and endosperm were cultured together on a semisolid induction medium containing 9.0 M 2,4-dichlorophenoxyacetic acid and 1.1 M benzyladenine. Following 2–3 months on induction medium, an average of 15.6 percent of the explants produced either somatic embryos or proembryogenic masses. Compared to pure yellow-poplar embryogenic cultures, putative hybrid cultures tended not to maintain growth as proembryogenic masses while exposed to auxin, instead proliferating via repetitive embryogenesis as globular-stage embryos. Four to six weeks following transfer of globular embryos to basal medium, mature embryos were produced from the putative hybrid lines. Mature embryos germinated following transfer to basal medium lacking casein hydrolysate. Plantlets survived transfer to potting mix and acclimatization to greenhouse conditions. Verification of the hybrid nature of the embryogenic lines and somatic embryo-derived plantlets was accomplished by Southern hybridization analysis with a species-specific DNA marker.Abbreviations BA benzyladenine - 2,4-d 2,4-dichlorophenoxyacetic acid - PEM proembryogenic mass - CH casein hydrolysate - RFLP restricted fragment length polymorphism - CTAB hexadecyltrimethylammonium bromide - TBE Tris-borate-EDTA - SDS sodium dodecylsufate - SSC sodium citrate/chloride     

Somatic embryogenesis in <Emphasis Type="Italic">Schisandra chinensis</Emphasis> (Turcz.) Baill.

Summary Regeneration of plants via somatic embryogenesis was achieved from zygotic embryo explants isolated from mature seeds of Schisandra chinensis. Merkle and Sommer's medium, fortified with 2,4-dichlorophenoxyacetic acid (2,4-D; 9.04 μM) and zeatin (0.09 μM), was effective for induction of embryogenic callus. The development of a proembryogenic mass and somatic embryos occurred on Murashige and Skoog medium (MS) free of plant growth regulators. The embryogenic callus induced on Merkle and Sommer's medium supplemented with 2,4-D (9.04 μM) and zeatin (0.09 μM) showed development of the maximum number of somatic embryos when transferred to MS medium free of plant growth regulators. The maximum maturation and germination of cotyledonary somatic embryos (46.3%) occurred on MS medium supplemented with 2,4-D (0.45 μM) and N⁶-benzyladenine (1.11 μM). The somatic embryo-derived plants were successfully hardned, with a survival rate of approximately 67%, and established in the field.     

Expression of auxin and light-regulated arrestin-like proteins,G proteins and nucleoside diphosphate kinase during induction and development of wheat somatic embryos

The modulation of three signal transduction elements: arrestin-like proteins, G proteins and NDPK was assessed during the induction of wheat (Triticum aestivum L.) somatic embryogenesis under different auxin (2,4-D) and light conditions. Immunological approaches using specific antibodies, kinase activity measurement and [α-³²P]-GTP-binding assay were performed. The induction of embryogenic capacity by 2,4-D was characterised both by the increased expression of the classical 40-kDa arrestin-like form and by the appearance of an additional arrestin-like protein of 29 kDa. The 40-kDa arrestin-like soluble form was unaffected by light stimuli. On the other hand, the 29-kDa arrestin-like form, specific of the embryogenic tissue culture, was found to be light regulated. From embryogenic cultures grown under light or dark, different soluble G proteins from 22 to 48 kDa were detected by probing polyvinylidene fluoride (PVDF) blots with [α-³²P]-GTP. In addition, in the microsomal fraction from light-grown cultures, a polypeptide of 20 kDa was heavily labelled. Under light conditions, cell proliferation induced by 2,4-D stimulated the appearance of a 32-kDa nucleoside diphosphate kinase (NDPK) form in addition to the classical 16–18-kDa protein, without a significant change in the NDPK activity. The modulated expression of plant arrestin-like proteins, G proteins and NDPK molecules in response to auxin and light support the view that they play key roles in signalling cascades participating in plant development.     

Repetitive somatic embryogenesis from peanut cultures in liquid medium

Summary A regeneration system based on repetitive somatic embryogenesis was developed for peanut (Arachis hypogaea L.). Embryogenic suspension cultures were initiated using individual somatic embryos induced from immature cotyledons cultured on a modified Murashige and Skoog medium containing 40 mg/l 2,4-D for 30 days. After transfer to a modified MS liquid medium, the somatic embryos produced masses of secondary and tertiary embryos which continued to proliferate following manual separation and subculture of the embryogenic clumps. The cultures exhibited exponential growth, and have been maintained for over one year without apparent loss of embryogenic potential. Further embryo development, germination, and conversion were achieved by placing embryo clumps onto hormone-free, solid medium. The inclusion of a desiccation period during embryo development enhanced conversion four-fold. Plants have been established in soil and appear to be phenotypically normal.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphthaleneacetic acid - BA 6-benzylaminopurine - MSO Modified Murashige and Skoog basal medium - EM embryogenic masses     

High frequency somatic embryogenesis in peanut (Arachis hypogaea L.) using mature,dry seed

Peanut (Arachis hypogaea L.) somatic embryos were produced from the embryo axes of mature, dry seeds of cultivar GK-7. Percent embryogenic explants ranged from 88–100% using 10–40 mg/1 of 2,4-D in the induction medium. Neither 2,4-D concentration nor photoperiod during the induction period had a large effect on percent embryogenesis, mean number of embryos per explant, or embryo morphology. However, embryos obtained from cultures grown in the dark were easier to remove from the explant than those under a 16-h photoperiod. Somatic embryos developed on the epicotyl portion of the embryo axis, primarily on the young, expanding leaves. A survey of 14 genotypes indicated that genotype had a large influence on embryogenic capacity, with all genotypes being embryogenic to some extent. The ability to recover somatic embryos from axes of harvested, stored seeds represents significant advantages for the establishment of peanut embryogenic cultures, including the use of simple sterilization procedures and a constant source of explant tissue.Abbreviations B5 medium of Gamborget al. (1968) - 2,4-D 2,4-dichlorophenoxyacetic acid - MS Murashige and Skoog (1962) salts medium