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     

Embryogenic and non-embryogenic cell lines of Daucus carota cloned from meristematic cell clusters: relation with cell ploidy determined by flow cytometry

The presence of totipotent and non-totipotent cells in embryogenic carrot cell suspension cultures was examined by cloning of cell microclusters. Forty clones were isolated and the distribution of their embryogenic potential was studied. Nonembryogenic, weakly and highly embryogenic cell lines were selected. After one year of subculture a second cloning round showed that the highly embryogenic and the non-embryogenic cell lines were homogenous and stable. A measurement of ploidy levels of clones by flow cytometry showed that the embryogenic clones were all diploid whereas the non-embryogenic were diploid or tetraploid. Hence, for our strain, there was a strict relationship between the tetraploid state and the inability to produce somatic embryos.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - PCV packed cell volume - MS Murashige and Skoog medium - MES 2(N-morpholino) ethanesulfonic acid - a.u. arbitrary units     

4-Hydroxybenzyl alcohol accumulates in suspension-cell cultures and inhibits somatic embryogenesis in carrot

Somatic embryogenesis in carrot ( Daucus carota L.) is strongly inhibited by certain factors that accumulate in culture medium of high-density cultures of embryogenic cells. We previously identified 4-hydroxybenzyl alcohol (4HBA) as one of the inhibitory factors. In this study, we analyzed the accumulation pattern of 4HBA in the cultures of carrot suspension cells. When somatic embryogenesis was induced by culturing embryogenic cells in phytohormone-free Murashige and Skoog medium at various initial cell densities, 4HBA accumulated in the culture medium. The concentration of 4HBA in high cell density cultures was higher than in low cell density cultures. The accumulation of 4HBA in high cell density cultures was rapid during the early days of culture. This rapid accumulation of 4HBA in high cell density cultures might result in the strong inhibition of somatic embryogenesis. The production of 4HBA decreased as the somatic embryos developed. In addition, embryogenic cells released larger amount of 4HBA into the culture medium compared with non-embryogenic cells. These results suggest that the production of 4HBA is both related to embryogenic competence and developmentally regulated during somatic embryogenesis.     

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.     

Plant regeneration from embryogenic suspension cultures of Chinese yam (Dioscorea opposita thunb.)

A competent, embryogenic suspension culture of Chinese yam (Dioscorea opposita Thunb. cv. ‘Nagaimo’) has been obtained. Embryogenic callus was induced from stem segments cultured on an agar-solidified MS medium containing 2,4-dichlorophenoxyacetic acid (2,4-D). One month following placement of the embryogenic callus in a liquid medium containing 2,4-D, the embryogenic tissue began to proliferate rapidly. Established suspension cultures consisted almost entirely of early-stage pro-embryos with very little contamination from non-embryogenic tissues. Under optimum conditions, suspension culture packed cell volume increased 2.5-fold per week. Following transfer of the tissue to a hormone-free medium, the embryogenic tissue developed. Globular embryos were formed within 4 weeks and addition of benzyl adenine further enhanced development and germination. Plantlets were regenerated by culturing embryos on a hormone-free agar-solidified medium.     

Purification and immunohistochemical detection of an embryogenic cell protein in carrot

An embryogenic cell protein from carrot (Daucus carota L.), designated ECP31 for embryogenic cell protein and with a relative mass of 31,000, was purified by sequential column chromatographies. Its apparent relative mass was estimated to be 120,000 by gel filtration. Immunoblotting and immunohistochemical studies showed that ECP31 was preferentially localized in the peripheral cells of clusters of embryogenic cells in the presence of 2,4-dichlorophenoxyacetic acid (2,4-D) and disappeared during the course of somatic embryogenesis in the absence of 2,4-D. ECP31 began to accumulate on the 33rd day after initiation of cultures of hypocotyl segments on Murashige-Skoog medium with 2,4-D, when callus began to appear on the segments. In dry seeds, lower amounts of ECP31 were located throughout the entire zygotic embryos but not in endosperm. ECP31 was also detected in provascular tissue of malformed somatic embryos.     

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     

Somatic Embryogenesis in Carrot Cell Suspension: I. PATTERN OF PROTEIN AND NUCLEIC ACID SYNTHESIS

The role of macromolecule synthesis during the early hours ofundifferentiated growth of carrot cell suspension in a mediumcontaining the synthetic auxin, 2,4-dichlorophenoxyacetic acidand during embryogenic growth in an auxin-free medium was investigated.For each set of cultures, total protein and RNA, rate of proteinsynthesis, changes in ATP pool, and rate of RNA synthesis weredetermined. Although no changes in protein and RNA contentsof cells were noted during the first 48 h of their growth infresh media, the rate of protein synthesis in the embryogeniccells markedly increased over that of the non-embryogenic cellsas early as 2 h. ATP pool size in both types of cells stabilizedwithin 1 h and it registered only a slight decrease over a 24h period. Rate of RNA synthesis increased in the embryogeniccells from 4 to 12 h following their transfer to fresh medium,after which it decreased. Transfer of carrot cells of zero embryogenicpotential to an auxin-free medium did not however elicit themacromolecule synthetic pattern characteristic of the highlyembryogenic cells.     

Slow vacuolar channels of non-embryogenic and embryogenic cultures of winter wheat

Slowly activating vacuolar channels (SV), were examined in embryogenic and non-embryogenic cultures of winter wheat using a patch-clamp technique. Four different types of cultures were examined: embryogenic and non-embryogenic calli from embryos, embryogenic and non-embryogenic calli from inflorescences. In a cell-attached mode single SV channel events were recorded. Unitary conductance of single SV channels was between 37 pS and 48 pS and did not significantly depend on the kind of the culture, although it was a tendency that SV channels of embryogenic calli possessed lower unitary conductance than those of non-embryogenic. 2,4-D caused significant lowering of unitary conductance from 48±6 pS in the control culture of embryogenic embryos to 28±6 pS in vacuoles treated. The SV channel density was estimated as 0.34 μm⁻².     

Somatic embryogenesis and plant regeneration from cultured mature caryopses of bahiagrass (Paspalum notatum Flugge)

Callus cultures were initiated from mature excised caryopses of bahiagrass (Paspalum notatum Flugge) on Murashige & Skoog medium supplemented with 20 gl^–1 sucrose and 2 mg l^–1 2,4-D. Excised mature caryopses readily germinated and callus developed at the base of coleoptiles. There was considerable variation in the amount of non-embryogenic callus among the cultures. Most of the explants produced non-embryogenic translucent callus consisting of thin-walled cells and unorganized tissue. Some of these calli gave rise only to roots. Other explants formed embryogenic calli which were distinguished morphologically as white, globular and friable. Somatic embryos developed and germinated precociously when embryogenic calli were transferred to a 2,4-D-free medium. Somatic embryogenesis was confirmed by histological sections and scanning electron microscopy. Of the 300 cultures, 35 were embryogenic but only 10 produced plants that were successfully grown to maturity.     

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.     

Ethanol production and toxicity in suspension-cultured carrot cells and embryos

The process of carrot (Daucus carota L.) somatic embryogenesis is highly sensitive to exogenously added ethanol, since 5 mM ethanol inhibits this process by 50%, whereas the growth of proliferating carrot cells is inhibited to the same extent by 20 mM ethanol. This is consistent with the fact that proliferating cultures produce ethanol and release it into the medium at concentrations up to 20 mM, whereas embryogenic culture medium contains less than 1 mM ethanol. Data are presented showing the influence of cell density and 2,4-dichlorophenoxyacetic acid on ethanol production and on the presence of an alcohol-dehydrogenase (EC 1.1.1.1.) inactivator in carrot embryos.Abbreviations ADH alcohol dehydrogenase - 6-BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - DTT dithiothreitol - FW fresh weight     

Detection of an Embryogenic Cell Antigen in Carrot

Monoclonal antibodies were raised against proteins in a microsomalfraction from carrot embryogenic cells. The presence of a 31-kDaembryogenic cell antigen detected by one antibody (ID 11) wasdemonstrated in embryogenic cells but not in other plant materials,such as non-embryogenic cells, somatic embryos, crown gallsand hairy roots. The antigen was also present in organ segmentsthat carried somatic embryos having been induced by exposureto various stresses. In non-embryogenic cells, the antibodyrecognized a small amount of a 32-kDa antigen. Both the 31-and the 32-kDa antigens accumulated in carrot seeds during theirdevelopment and then disappeared after germination. (Received April 16, 1990; Accepted July 16, 1990)     

苜蓿体细胞胚胎发生过程中DNA、RNA和蛋白质的合成动态

苜蓿(Medicago sativa L.)下胚轴切段产生的愈伤组织经2,4-D短时间诱导后,在无激素液体培养基中可形成大量体细胞胚胎。经2,4-D诱导后的愈伤组织在转入无激素培养基1天后,其DNA、RNA和蛋白质的合成即进入活跃合成状态,并在体细胞胚胎发育过程中保持逐步升高的趋势。在苜蓿体细胞胚胎发生过程中,有些蛋白质组分含量减少或消失,但绝大部分蛋白质组分的含量明显增加,并且有若干新蛋白的出现,其中24 KD和46 KD蛋白质为体细胞胚胎发生早期所特有。     

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     

The effects of glutamine of the maintenance of embryogenic cultures of Cryptomeria japonica

Summary Embryogenic tissues of sugi (Cryptomeria japonica) were induced on a modified Campbell and Durzan (CD) medium containing 1 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 600 mg l⁻¹ glutamine, and subcultured in the medium of the same composition for over 1 yr. This resulted in a mixed culture of embryogenic and non-embryogenic cells. When embryogenic cells were isolated and cultured independently, their capacity to form embryogenic aggregates was lost. Thus, the non-embryogenic cells present within a mixed culture system were essential to the formation of embryogenic aggregates. When embryogenic tissues were isolated and cultured independently on a high glutamine-containing (2400 mg l⁻¹) medium, dry weights and endogenous levels of glutamine increased, and the tissue could generate a large number of embryogenic aggregates. Amino acid analysis of embryogenic and non-embryogenic cells from the maintenance culture indicated a higher level of glutamine was present in the latter. The high endogenous level of glutamine in the non-embryogenic portion of mixed cell masses may be the supplier of glutamine for maintaining the embryogenic property of the tissues.     

Somatic embryogenesis and metabolic differences between embryogenic and non-embryogenic structures in mangosteen

Somatic embryogenesis in mangosteen (Garcinia mangstana L.) was investigated using seed and leaf segments cultured on Murashige and Skoog medium with treatments of 6-benzyladenine (BA) [2.0, 3.0, 4.0 µM] and 2,4-diclorophenoxyacetic acid (2,4-D) [4.5, 9.0, 13.5 µM]. There were four types of structures (globular, nodular compact, friable and spongy) formed. Two treatments resulted in embryogenic characteristics from seed cultures; the highest percentage 46.67?% of globular structure (resembling somatic embryos) grown on 3.0 µM BA and 80?% of nodular compact structures on 4.0 µM BA?+?13.5 µM 2,4-D. For the leaf culture, highest percentage, 93.33?% produced nodular compact structures on 2.0 µM BA?+?4.5 µM 2,4-D. Histological analysis showed that the globular structure has well-defined protoderm and separated from the original explant. Nodular compact structure also showed the presence of densely cytoplasmic meristematic cells with a high nucleoplasmic ratio. These characteristics observed in globular and nodular compact structure indicates somatic embryo formation. The globular structures which were converted into shoots and roots (60.00?%) showed atypical somatic embryogenesis in mangosteen. Metabolite fingerprinting was carried out using gas chromatography–mass spectrometry. Amino acids, carbohydrates, organic acids and fatty acids were found in both the embryogenic structures and non-embryogenic structures tested. Multivariate discriminant analyses of the metabolic data revealed significant metabolites (P?≤?0.05) for both types of structures. Principle component analysis suggested that amino acids and carbohydrates were the major compounds distinguishing embryogenic and non-embryogenic structures. Ornithine and mannose were present at significant level in embryogenic structures as compared to non-embryogenic ones while fructose was significantly higher in non-embryogenic structures.     

Selective enhancement of Ipomoea batatas Poir. embryogenic and non-embryogenic callus growth and production of embryos in liquid culture

Embryogenic callus cultures of Ipomoea batatas Poir. produce fast growing non-embryogenic material which soon dominates the cultures. Our objective was to selectively enhance the proliferation of the embryogenic fraction. For this, the effect of BAP and 2,4-D concentrations on growth of embryogenic and non-embryogenic callus were studied and consequently, nutrient media for the production and indefinite maintenance of embryogenic callus without embryo formation were defined. Selective proliferation of embryogenic callus was obtained on solid media with 10 M 2,4-D and 1 M BAP and in liquid media with 5 M 2,4-D. Selective proliferation of non-embryogenic callus occurred in liquid medium with 1 M 2,4-D. In embryogenic liquid culture, embryos were produced with 0–2 M 2,4-D. Increasing 2,4-D concentration from 0 to 2 M in these cultures restricted embryo development.Abbreviations 2,4-D = 2,4-dichlorophenoxyacetic acid - BAP = 6-benzylaminopurine     

The influence of phytohormones on zeta potential and electrokinetic charges of winter wheat cells

The zeta potential measurements of protoplasts obtained from winter wheat cell culture and phospholipid liposomes were performed to determine the electrokinetic charge in a medium containing various phytohormones (kinetin, 2,4-D and zearalenone) in absence and in presence of 2 x 10(-5) MCa2+. Calli were induced from immature inflorescences (inf) and embryos (emb) and cultured to obtain non-embryogenic (NE) and embryogenic (E) cell tissues. All investigated phytohormones indicate ability to adsorb to the negatively charged surfaces (latex, L88 - model negative adsorption site) both in water solutions and at the presence of mannitol and buffer (MES). In biological systems (protoplasts and liposomes - prepared from phospholipids of protoplasts) the electrokinetic charges were dependent on the phospholipid and protein composition of cells. The influence of protein groups on electrokinetic charge was calculated from charge values of protoplasts and liposomes, assuming additivity of surface charges. The comparison of calculated charges for protoplasts and liposomes indicate that 2,4-D is better adsorbed to the phospholipid and proteins of NE cells whereas kinetin is bound to the phospholipid and protein sites of E calli. This effect may be connected with embryogenesis process, where non-embryogenic culture of wheat requires 2,4-D in the medium, and embryogenic culture requires cytokinin rather. Zearalenone binding is especially dependent on the kind of explant.     

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.