Growth conditions influence regeneration of encapsulated carrot somatic embryos into plantlets

Summary Carrot somatic embryos were encapsulated in calcium alginate beads, with or without a maturation step, to produce synthetic seeds. Germination and plantlet regeneration frequencies were compared for liquid or solid nutritive media, and with sucrose supply and sterilization or not. Germination rates were greater than 80% in all treatments except in non-sterilized sand. No conversion occured on a liquid MS medium. Normal plantlets were 7–22% on agar and 4–40% on sand. Abnormal plantlets were only observed on agar due to secondary embryogenesis. A sterilized sand growth support supplied with sucrose appears to be most appropriate to assess morphologically normal plantlet regeneration.Corresponding author     

Germination and growth of encapsulated somatic embryos of carrot for mass propagation

Summary Shoot growth and root development of plantlets germinated from encapsulated somatic embryos of carrot were promoted by transferring the embryos from a culture medium containing sucrose (1.5%) to a culture vessel with a similar medium without sucrose, which was kept under a constant relative humidity condition (90%) during the culture period. The length and dry weight of plantlet shoots placed on medium supports (ceramic wool) containing a liquid culture medium without sucrose from three weeks after placing encapsulated somatic embryos were approximately two times greater than those placed on medium supports containing a culture medium with sucrose throughout the five-week culture period.     

Effects of capsule hardness on germination frequency of encapsulated somatic embryos of carrot

Summary Somatic embryos of carrot were encapsulated in calcium alginate beads to provide artificial carrot seeds. Alginate capsules with a hardness of 0.2 to 0.5 kg/cm² were found to be suitable for germination of encapsulated somatic embryos. The germination frequencies were more than 95%, when grown aseptically on polyester fiber supports loaded with hormone-free Murashige-Skoog medium.     

Isolation of protoplasts from somatic embryos of carrot

Protoplasts were isolated enzymatically from synchronously induced globular somatic embryos from a carrot suspension culture. Among the macerating enzymes tested, Driselase was the most effective for release of protoplasts from embryos. A higher medium osmolarity was required for the isolation of protoplasts from embryos than from undifferentiated cells. Protoplasts from embryos were smaller than protoplasts from undifferentiated cells. On step gradients of Ficoll, protoplasts from embryos gave one major band. Protoplasts from undifferentiated cells gave two major bands, one lighter and the other heavier than the protoplasts from embryos.     

Production of carrot somatic embryos in a bioreactor

Somatic carrot embryos were grown as batch cultures in a stirred 10-l bioreactor. Embryo production in the bioreactor was comparable to that obtained in shake-flasks. A production of about 50·10³ embryos/l per day was commonly achieved with an inoculum density of 0.1% volume of tissues/volume of medium. Regularly changing of the medium increased embryo viability. A filtering unit coupled to the bioreactor was developed in order to calibrate embryos. The characteristics of the population of harvested embryos are described.Correspondence to: J.-P. Ducos     

Stage-specific nitrogen metabolism in developing carrot somatic embryos

The physiology of individual somatic embryo developmental stages otDaucus carota L. was examined by in vivo nuclear magnetic resonance (NMR) spectroscopy, amino acid analysis and ¹⁴C-labeling. ¹⁵N NMR spectroscopy was used to examine the uptake and incorporation of ¹⁵N isotopically labeled inorganic nitrogen sources. NMR spectra of proembryogenic masses (PEMs) contained resonances for histidine, amino sugars, glutamine, arginine, urea, alanine. α-amino nitrogen, serine, aliphatic amines and several unknowns. Similar resonances were found in various embryo developmental stages. However, resonances for arginine and aliphatic amines peaked during globular and torpedo stages and substantially decreased in germinating stage embryos. The dominant resonances observed in non-embryogenic cells and germinating embryos were glutamine and α-amino nitrogen. Amino acid analysis of the various embryo stages showed that glutamate, glutamine and arginine were the major contributors to the soluble amino acid profiles. During development, glutamate and glutamine continued to increase in concentration whereas arginine and its related metabolites (i.e. ornithine and y-aminobutyric acid [GABA]) were biphasic; increasing in globular and torpedo stage embryos and decreasing in germinating embryos. Carbon-14 labeling indicated that labeled glutamine pools in non-embryogenic and germinating embryos were greatest compared to other embryo stages, whereas labeled GABA pools were greatest in globular and torpedo stage embryos. Taken together, these data indicate that the physiology of each embryo developmental stage is distinct. They also suggest that during somatic embryo development, a switch takes place in metabolism whereby the glutamine synthetase/glutamate synthase (GS/GOGAT) pathway is predominant in non-embryogenic cells and germinating stage embryos. Furthermore, during early to mid-embryo development (PEMs, globular and torpedo stage embryos), metabolism utilizing the omithine cycle is enhanced and predominant.     

In vitro response of encapsulated somatic embryos of camellia

Plant regeneration via somatic embryogenesis was achieved in leafbase and leaf tip explants derived from 10-day-oldin vitro-grown seedlings ofEchinochloa colona. Somatic embryogenesis was induced in the callus on Murashige and Skoog (1962) medium supplemented with 4.44 μM 6-benzyladenine, 4.64 μM kinetin and 8.05 μM 1-naphthaleneacetic acid after 4 weeks of culture incubated for 14 days in continuous dark and subsequently under a 14-h photoperiod. The incidence of somatic embryogenesis was greater in leafbase- than in leaf tip-derived calluses. Histological observations revealed various stages of development of somatic embryogenesis. The embryos matured and germinated on fresh medium lacking growth regulators. The somatic embryo-derived plantlets were established in soil.     

Successful long-term preservation of abscisic-acid-treated and desiccated carrot somatic embryos

We have previously reported that strong desiccation tolerance in carrot somatic embryos can be achieved by treatment with abscisic acid (ABA). In this study, we examined the possibility of long-term preservation of ABA-treated and desiccated somatic embryos. Somatic embryos that had been desiccated after treatment with ABA survived for at least 169 weeks at –25 °C. By contrast, somatic embryos that had not been desiccated after treatment with ABA survived for at least 24 weeks at +5 °C but died at –25 °C. Received: 11 July 1998 / Revision received: 20 October 1998 / Accepted: 20 November 1998     

Tissue-specific localization of mRNA for carrot homeobox genes, CHBs, in carrot somatic embryos

We examined spatial and temporal expression patterns of four carrot HD-Zip I homeobox genes in somatic embryos. The mRNAs for CHB3, CHB4 and CHB5 were accumulated preferentially in the innermost cortical cell layers of the embryo axis in the torpedo-shaped embryo. In contrast, the accumulation of CHB6 mRNA was restricted to procambial cells of the heart- and torpedo-shaped embryos. In the embryonic cotyledons and the hypocotyl of the seedlings, all of the mRNAs for the four genes were located in the vascular tissues. These findings indicate that different HD-Zip I homeobox genes may be involved in the differentiation of specific tissues during somatic embryogenesis.     

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.     

In vitro response of encapsulated somatic embryos of Lagerstroemia indica L

A method to produce encapsulatable units for synthetic seeds was developed in L. indica. Somatic embryos were harvested from leaf derived embryogenic callus on Murashige and Skoog's basal medium supplemented with 2, 4-dichlorophenoxy acetic acid (2, 4-D, 0.5 mg/l), 6-benzyl amino purine (BAP, 1 mg/l) and ascorbic acid (AA, 50 mg/l). The embryos were encapsulated in alginate beads and dehydrated. Germination ability of the artificial seeds were investigated. The frequency of regeneration from the encapsulated embryos was significantly affected by (i) the concentration of alginate (ii) the duration of storage, and (iii) the effect of different types of media. A 2% sodium alginate concentration on MS salts resulted in significantly higher germination frequencies than at other concentrations. L. indica showed maximum germination on MS medium (93.84%) after 6 weeks of culture. The germinated synthetic seeds with well developed roots and shoots were transferred successfully to green house. This is the first report on artificial seeds in Lagerstroemnia indica.     

Plant regeneration from encapsulated somatic embryos of Carica papaya L.

Carica papaya L. (papaya) single somatic embryos (2.0 mm diameter) produced in a high-frequency liquid production system were encapsulated in two different synthetic encapsulation compounds. The frequency of regeneration from encapsulated embryos was significantly affected by (1) the concentration of sodium alginate, (2) the presence or absence of nutrient salts in the capsule, and (3) the duration of exposure to calcium chloride. A 2.5% sodium alginate concentration in a half-strength MS salts base resulted in significantly higher germination frequencies than other treatments. A relatively short (10 min) exposure to CaCl₂ provided uniform encapsulation of embryos and the highest frequencies of successful germination (77.5%). Germinated artificial seeds produced normal plantlets. Received: 12 March 1997 / Revision recieved: 24 June 1997 / Accepted: 18 July 1997     

Plantlet regeneration from encapsulated somatic embryos of hybrid Solanum melongena L

High frequency somatic embryogenesis was induced from leaf expiants of F1 hybrid Solanum melongena L. on Murashige and Skoog's medium supplemented with 8.0 mg/1 NAA and 0.1 mg/1 Kn. The somatic embryos were encapsulated in various concentrations (2–6%) of sodium alginate and complexed with calcium chloride (25–100mM): 3% sodium alginate and 75 mM calcium chloride were found to be optimal for encapsulation. The encapsulated somatic embryos were transferred to various conversion media in vitro and in vivo. The frequency of plantlet regeneration varied from 27.0–49.7% in vitro and 2.0–4.5% in vivo.Abbreviations IAA indole-3-acetic acid - IBA indole-3-butyric acid - Kn Kinetin - MS Murashige and Skoog (1962) - NAA naphthalene acetic acid     

Plant regeneration from alginate encapsulated somatic embryos of Asparagus cooperi baker

Somatic embryos of Asparagus cooperi were encapsulated as single embryos approximately 4–6 mm in diameter to produce individual synthetic seeds. The frequency of conversion of artificial seeds to plants was 34%. This frequency was affected by the concentration of calcium chloride, the commercial sources of sodium alginate, and the nutrient medium. The conversion frequency of artificial seeds to seedling plants was 8.3% after storage for 90 days at 2°C.Abbreviations NAA -Naphthaleneacetic acid - ABA Abscisic acid - GA₃ Gibberellic acid - Kn 6-Furfurylaminopurine - MS Murashige and Skoog (1962) medium - SH Schenk and Hildebrandt (1972) medium - WM White (1963) medium     

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.     

Physico-chemical properties of the encapsulation matrix and germination of carrot somatic embryos

Carrot somatic embryos were encapsulated in alginate gel beads. To improve the quality of a "synthetic seed" coating, the rheology and dehydration properties of different matrices were tested. By increasing alginate and CaCl(2) concentrations, additional mineral elements were shown to increase resistance to rupture, and to depress the germination of somatic embryos. A polysaccharide addition was found to slow the alginate matrix dehydration; alginate-gellan gum and alginate-kaolin matrices could preserve the viability of somatic embryos at low relative humidities (30% to 35% germinations at 50% relative humidity) to a greater extent than other matrices. (c) 1995 John Wiley & Sons, Inc.     

Calcium increases the yield of somatic embryos in carrot embryogenic suspension cultures

An upward shift in the concentration of calcium present in the medium during somatic embryogenesis increased the number of embryos produced approximately two-fold. This was observed when embryogenic suspension cells grown in 2,4-D medium with the normal calcium concentration of 10^–3 M were transferred to hormone-free medium containing 10^–2 M calcium and when embryogenic suspension cells grown in 2,4-D medium containing 10^–4 M calcium were transferred to hormone-free medium with 10^–3 M calcium. At calcium concentrations between 6·10^–3 and 10^–2 M globular stage somatic embryos were found in cultures supplemented with 2·10^–6 M of 2,4-D indicating that elevated calcium counteracts the inhibitory effect of 2,4-D on somatic embryogenesis. No qualitative changes were found in the pattern of extracellular polypeptides as a result of growth and embryogenesis in media with different calcium concentrations.     

Possible involvement of abscisic acid in the induction of secondary somatic embryogenesis on seed-coat-derived carrot somatic embryos

When seed coats (pericarps) were picked from 14-day-old carrot (Daucus carota) seedlings and cultured on agar plates, embryogenic cell clusters were produced very rapidly at a high frequency on the open side edge. Embryo induction progressed without auxin treatment; indeed treatment caused the formation of non-embryogenic callus. The embryogenic tissues (primary embryos) developed normally until the torpedo stage; however, after this a number of secondary somatic embryos were produced in the hypocotyl and root regions. Tertiary embryos were formed on some of the secondary embryos, but many developed into normal plantlets. The primary embryos contained significantly higher levels of abscisic acid (ABA) than the hypocotyl-derived normal and seed-coat-derived secondary embryos. Fluridone inhibited the induction of secondary embryogenesis, while exogenously supplied ABA induced not only tertiary embryogenesis on the seed-coat-derived secondary embryos, but also secondary embryos on the hypocotyl-derived normal somatic embryos. These results indicate that ABA is one of the important endogenous factors for the induction of secondary embryogenesis on carrot somatic embryos. Higher levels of indole-3-acetic acid (IAA) in primary embryos also suggest the presence of some concerted effect of ABA and IAA on the induction of secondary embryogenesis in primary embryos.     

Measurement of endogenous ABA levels in chilled somatic embryos of carrot by immunoassay

Somatic embryos of carrot, Daucus carota L. Royal Chantenay, were chilled at 4°C for the last 3 days of development in order to harden torpedo stage embryos to increase embryo survival during desiccation. ABA levels in chilled and non-chilled embryos were measured using a polyclonal radioimmunoassay and a monoclonal enzyme-linked immunosorbent assay (ELISA). The monoclonal ELISA is the preferred technique due to superior sensitivity and specificity. ABA levels, measured by either technique, were similar in chilled and non-chilled embryos. The relative water content was lower in chilled embryos than in non-chilled embryos and chilling altered protein secretion of one cell line.List of abbreviations ABA abscisic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - BHT 2,5-di-tert-butyl-4-methyl phenol - BSA bovine serum albumin - ELISA enzyme-linked immunosorbent assay.     

Characterzation of chitinases able to rescue somatic embryos of the temperature-sensitive carrot variantts11

To characterize the acidic endochitinase EP3, able to rescue somatic embryos of the carrot cell linets11, the enzyme was purified from the medium of wild-type suspension cultures. Peptide sequences, deduced amino acid sequences of corresponding PCR-generated cDNA clones, serological relation and biochemical properties showed that there were at least five closely related chitinases, four of which could be identified as class IV EP3 chitinases with an apparent size of 30 kDa. Two other proteins were identified as a serologically related class I acidic chitinase (DcChitI) of 34 kDa, and a serologically unrelated 29 kDa class II acidic chitinase (DcChitII), respectively. Additional cDNA sequences, Western and Southern analysis showed the presence of a least two, but possibly more, highly homologous class IV EP3 genes in the carrot genome. Two class IV EP3 chitinases were tested and found to be able to increase the number ofts11 globular embryos formed under non-permissive conditions. One of the class IV EP3 chitinases as well as the class I chitinase DcChitI promoted the transition from globular to heart-stagets11 embryos. The class II endochitinase and a heterologous class IV chitinase from sugar-beet were not active onts11. This suggests that there are differences in the specificity of chitinases in terms of their effect on plant somatic embryos.