Proliferation and maintenance of embryogenic capacity in elm embryogenic cultures

Summary This paper investigates maintenance and proliferation of somatic embryogenesis systems for Ulmus minor and U. glabra. Proliferation occurred with subculture of embryogenic calluses. The calluses were mainly formed by friable nodules composed of meristematic cells organized into proembryogenic cell masses (PEMs) and thin-walled vacuolated parenchymatic cells. Cotyledonary embryos, with procambial strands and differentiation of their vascular tissues as well as visible root meristems, were identifiable after 18d of culture on a proliferation medium with 0.44 μM benzyladenine (BA). The shoot meristem was only occasionally well developed. Somatic embryo multiplication from elm embryogenic calluses is a clearly asynchronic system, and PEMs as well as embryos at all stages of development are observed simultaneously at the end of subculture period. Factors affecting the proliferation of elm embryogenic callus, such as culture medium, carbon source and genotype, were studied. Basal medium (MS) or medium supplemented with 0.44 μM BA produced the highest number of somatic embryos. Somatic embryo production was higher with sucrose or glucose than with maltose, and significant differences were also found among the four embryogenic lines tested. The use of liquid medium with filter paper support is an essential step for the survival of isolated somatic embryos during the germination stage. The addition of 0.22 μM BA′ to liquid MS medium was the best treatment for germination and plantlet conversion of elm somatic embryos.     

Genetically stable cell lines of cucumber for the large-scale production of diploid somatic embryos

For the initiation of embryogenic cucumber ( Cucumis sativus L.) cell lines, from excised radicles, directly in liquid medium, the culture regime, explant density and type and concentration of hormones were adjusted so that pro-embryogenic masses (PEMs) were formed within about 8 weeks. The established cucumber cell lines were maintained tor several years without loss of embryogenic and genetic stability. The ploidy level of somatic embryos from different cucumber eell lines was either diploid or tetraploid and depended on the ploidy level of Ihe cell line. Cucumber cell lines that produced only diploid embryos were obtained by selecting completely diploid explant material and growing it in the dark during the initiation phase. Mixoploid explains could lead to tetraploid or mixoploid ceil lines. Isolation and additional selection and subculturing of single PEMs resulted in either completely diploid or tetraploid cell lines, indicating that all cells of individual PEMs are either diploid or tetraploid. The ernbryogenic cucumber cell Imes, differing only in ploidy level, were indistinguishable in growth rate and embryogetiic potential and were genetically stable over several years.     

High-efficiency somatic embryogenesis and plant regeneration from suspension cultures of grapevine

Embryogenic suspensions of grapevine (Vitis vinifera L.) were initiated from somatic embryos of `Thompson Seedless' and `Chardonnay'. Suspension cultures consisted of proembryonic masses (PEM) that proliferated without differentiation in a medium containing 2,4-dichlorophenoxyacetic acid (2,4-D). `Chardonnay' somatic embryos developed fully from PEMs following subculture in medium without 2,4-D; however, somatic embryo development did not advance beyond the heart stage in `Thompson Seedless' suspension cultures. Highly synchronized development of somatic embryos was obtained by inoculating <960-μm PEMs into liquid medium without 2,4-D. Somatic embryos were also produced in large numbers from suspension-derived PEMs of both cultivars on semisolid medium lacking 2,4-D. Somatic embryos matured and regenerated into plants in MS basal medium containing 3% sucrose. Using this method more than 60% of the somatic embryos regenerated plants. More than 90% of the regenerated plants were successfully transferred to the greenhouse. Received: 27 July 1998 / Revision received: 15 October 1998 / Accepted: 27 October 1998     

Scale-up of somatic embryogenesis in alfalfa (Medicago sativa L.) I subculture and indirect secondary somatic embryogenisis

Three methods of increasing the productivity of somatic embryogenesis in Medicago sativa L. were investigated. In the basic procedure, somatic embryos were initiated from young petioles and carried through several phases: callus formation, suspension culture, selection of the embryogenic fraction by sieving, development, maturation, desiccation and storage. The suspensions were normally separated into three fractions by sieving. Fraction I (<200 m) containing nonembryogenic cells or cell clusters was discarded. Fraction II (200–500 m) consisting of embryogenic cell clusters was collected for embryo development and maturation. Fraction III (over 500 M) containing the mixture of petiole residues with large pieces of calli and globular somatic embryos was usually discarded. Several methods to scale-up the suspension phase were unsuccessful. Direct subculture of the entire suspension by the addition of fresh liquid medium resulted in the loss of embryogenic capacity by the third subculture. Subculture of fraction II decreased embryogenic cell mass, and hence reduced total productivity. The recycling of fraction III back to fresh B₅g liquid medium resulted in high productivity in the first culture but further subculture of this fraction resulted in a rapid decline in the embryogenic capacity.As an alternative, somatic embryos from the first tissue culture cycle were also used as explants for the initiation of secondary embryogenic callus. The embryogenic capacity of these somatic embryo explants declined rapidly as they matured. More than 100 secondary somatic embryos could be induced from embryo explants removed from development medium at 10 days after sieving the suspension, but only 40 somatic embryos were produced from each mature somatic embryo explant, and 13 from desiccated embryos. The secondary somatic embryos were comparable to the primary embryos in quality according to germination tests. The implications of the results to the efficiency of somatic embryo production of Medicago are discussed.Abbreviations ABA abscisic acid - 2,4-d 2,4-dichlorophenoxyacetic acid - DAS days after sieving - PPF photosynthetic photon flux density - SE somatic embryo     

Somatic embryogenesis and plant regeneration from suspension cultures of Acanthopanax koreanum Nakai

High-frequency somatic embryogenesis was achieved from an embryogenic cell suspension culture of Acanthopanax koreanum Nakai. Stem segments were cultured on Murashige and Skoog (MS) medium containing auxins and cytokinins. Opaque and friable embryogenic callus formed on MS medium with 4.5 μm 2,4-dichlorophenoxyacetic acid (2,4-D) and 2.0 μm kinetin or zeatin, but was highest on medium containing 4.5 μm 2,4-D alone. Embryogenic calli were transferred to MS liquid medium containing 4.5 μm 2,4-D and maintained by subculture at 2-week intervals. Initiation of somatic embryogenesis and development up to the globular stage from embryogenic cell clumps occurred in medium containing 0.45 μm 2,4-D, whereas maturation and germination of somatic embryos occurred in MS medium lacking 2,4-D. Cytokinin treatment suppressed the normal growth of embryos, but stimulated secondary somatic embryogenesis from the surfaces of primary embryos. Plants from somatic embryos were acclimatized in a greenhouse. Received: 14 January 1997 / Revision received: 17 June 1997 / Accepted: 5 July 1997     

植物激素对棉花体细胞胚胎发生的诱导及调节作用

选用11种激素研究了外源激素对棉花胚性愈伤组织增殖、胚胎发生和发育的调控作用。结果表明不同激素对棉花胚性愈伤组织增殖、胚胎发生与发育的影响不同。除2,4-D和BA对棉花胚性愈伤组织的增殖影响不大外,其他激素对棉花胚性愈伤组织的增殖均具有抑制作用,且具有一定的时间效应,同时还受基因型的影响。激素对棉花体细胞胚的形成和发育的影响极大,2,4-D既抑制了体细胞胚的形成,又抑制了体细胞胚的发育;TDZ的作用与2,4-D相似,显抑制了体细胞胚的形成,且诱导获得的体细胞胚均停留在球形胚阶段;GA也抑制了体细胞胚的形成,且不利于体细胞的成熟与萌发;BU-30对棉花体细胞胚形成与发育的影响不大。其他7类生长素类物质和细胞分裂素类物质对棉花体细胞胚的形成均具有促进作用,且依IBA、ABA、IAA、BA、KT、ZT、2iP序增强,其总胚数为对照的1．193—3．852倍;其中2iP的促进作用最大,可使产生的体细胞胚数提高2．852倍。     

Comparison of NAA and 2,4-D induced somatic embryogenesis in Cassava

NAA and 2,4-D were compared for their ability to induce somatic embryogenesis in cassava (Manihot esculenta Crantz). In all seven cultivars tested, only 2,4-D had the capacity to induce primary somatic embryos from leaf explants, however, both NAA and 2,4-D were capable of inducing secondary somatic embryos. More secondary somatic embryos were formed in NAA than in 2,4-D medium. Furthermore, the maturation period for secondary somatic embryos was shorter in NAA medium than in 2,4-D medium. In some cultivars, repeated subculture of secondary somatic embryos in NAA medium resulted in a gradual shift from somatic embryogenesis to adventitious root formation. This shift could be stopped and reversed by subculture of the material in 2,4-D medium. In NAA medium the most secondary somatic embryos were formed when they were subcultured every 15 days whereas in 2,4-D a 20 day subculture interval was optimal. Subculture of secondary somatic embryos at a high inoculum density (>1.5 g jar⁻¹) in NAA medium did not result in the formation of secondary somatic embryos, whereas in 2,4-D it lead to the formation of globular secondary somatic embryos. With 2,4-D the newly induced secondary somatic embryos were connected vertically to the explant and with NAA medium horizontally. For all cultivars tested, desiccation stimulated normal germination of NAA-induced somatic embryos. However, the desiccated, secondary somatic embryos required a medium supplemented with BA for high frequency germination. The concentration of BA needed for high frequency germination was higher when the desiccated secondary somatic embryos were cultured in light instead of dark. In only one cultivar desiccation enhanced germination of 2,4-D induced secondary somatic embryos and in three other cultivars it stimulated only root formation. This revised version was published online in June 2006 with corrections to the Cover Date.     

High Frequency of Plant Production via Somatic Embryogenesis from Callus or Cell Suspension Cultures inEleutherococcus senticosus

Hypocotyl segments ofEleutherococcus senticosuscultured on Murashigeand Skoog's (MS) medium with 4.5 µM2,4-D produced somaticembryos directly from the surface of explants without interveningcallus formation. When these somatic embryos were subculturedto the same MS medium with 4.5 µM2,4-D, friable embryogeniccalli were formed mainly from radicle tips of somatic embryos,but at a low frequency (5%). Selected embryogenic calli weremaintained on MS agar or liquid medium with 4.5 µM2,4-D.To induce somatic embryo development, embryogenic calli andcell clumps were transferred to MS medium lacking 2,4-D. Thefrequency of somatic embryo formation differed between culturetypes with 1570 embryos formed per Petri dish from callus cultureand 5514 embryos formed per flask from cell suspension cultures.Somatic embryos formed on agar medium had larger cotyledonsthan those of embryos formed in liquid medium. GA₃treatmentwas necessary to induce germination from somatic embryos. Therate of plant conversion was 97% in somatic embryos from callusculture and 76% in embryos from liquid culture. Regeneratedplantlets were successfully acclimatized in the glasshouse.Copyright1999 Annals of Botany Company Eleutherococcus senticosus, micro propagation, somatic embryogenesis.     

Long-term optimized embryogenic cultures in durum wheat (Triticum durum Desf.)

Five varieties of durum wheat: Appulo, Ofanto, Latino, Creso, and Castello (Triticum durum Desf.) adapted to the semi-arid mediterranean environment have been tested for their in vitro response. Compact, embryogenic, highly regenerable calli originated from primary callus derived through proliferation of the scutellum of immature embryos explanted in the presence of 2,4 dichlorophenoxyacetic acid. Selective subculture of the white, compact, embryogenic sectors led to the establishment of long-term cultures. Regeneration occurred on hormone-free medium either via germination of somatic embryos, or via multiple-shoot formation probably due to precocious germination of somatic embryos. The three varieties, Ofanto, Creso and Appulo, were the best responding genotypes. Callus fragmentation and two subsequent transfers onto fresh medium at 7-day intervals yielded a frequency of plant regeneration of some 25–40 plantlets per gram fresh weight callus in 21 days on Murashige and Skoog's hormone-free medium. Plantlets could be efficiently established in soil, thus confirming the possibility of biotechnological approaches with varieties of this crop species.Abbreviations E embryogenic - NE non embryogenic - MS Murashige and Skoog's (1962) medium - 2,4-D 2,4 dichlorophenoxyacetic acid - DAA days after anthesis - FWT fresh weight tissue     

影响籼稻体细胞胚胎发生几个因素的研究

以 IR36、IR50、IR52及 IR54等品种的幼穗及成熟种子为材料,研究了蔗糖浓度、2,4-D、NAA、激动素及脱落酸对体细胞胚胎发生、结构的保持及植株分化的影响。6%蔗糖有利于胚性愈伤组织的诱导;3%的有利于胚性结构的保持及植株分化。当培养基中不含2,4-D,而含激动素与 NAA 时,幼穗直接出芽;当不含激动素而含2,4-D与 NAA 时,外植体产生非胚性愈伤组织;当不含 NAA 而含2,4-D 与激动素时,外植体产生胚性愈伤组织。认为,2,4-D与激动素是籼稻体细胞胚胎发生的基本因素,而 NAA 的作用是不明显的。不同外植体(幼穗与成熟种子)的体细胞胚胎发生,对2,4-D 与激动素的反应略有不同,幼穗更为敏感。在继代培养基中,加入低浓度的脱落酸有利于胚性结构的保持。随着继代世代的延续,分化培养中愈伤组织所表现出的绿色生长点状物不能发育成完整植株。     

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     

Maturation and germination of oak somatic embryos originated from leaf and stem explants: RAPD markers for genetic analysis of regenerants

Experiments were performed to determine the influence of maturation medium carbohydrate content on the rates of germination and plantlet conversion (root and shoot growth) of somatic embryos from four embryogenic lines derived from leaf or internode explants of Quercus robur L. seedlings. The conversion rate was favoured by high carbohydrate content as long as the maturation medium contained at least 2% sucrose, which was necessary for healthy embryo development. Given this, sorbitol and mannitol favoured the conversion rate more efficiently than sucrose, the highest rate, 32%, being achieved by medium with 6% sorbitol and 3% sucrose. Maturation treatment did not affect the root or shoot lengths of converted embryos. In supplementary experiments, 2 weeks of gibberellic acid treatment between maturation and germination treatments did not improve germination rates, but did reduce root length and the number of leaves per regenerated plantlet. In the four embryogenic lines tested, plant recovery rate was enhanced by inclusion of benzyladenine into the germination medium following culture of the embryos on maturation medium with 6% sorbitol and 2-3% sucrose. In embryogenic systems it is important to assess the uniformity of the regenerants. Random amplified polymorphic DNA (RAPD) analysis using 32 arbitrary oligonucleotide primers was performed to study variability in DNA sequences within and between four embryogenic lines. No intraclonal nor interclonal polymorphism was detected between embryogenic lines originating from different types of explant from the same seedling, but every one of the primers detected enough polymorphism among clones originating from different plants to allow these three origins to be distinguished. No differences in DNA sequences between regenerated plantlets and their somatic embryos of origin were detected, but a nodular callus line that had lost its embryogenic capacity was found to be mutant with respect to three other clones originating from the same plantlet. This study shows that high carbohydrate levels in the maturation medium significantly increase plant conversion of oak somatic embryos, which exhibit no variation in DNA sequences when proliferated by secondary embryogenesis.     

Enhanced production and germination of somatic embryos by temporary starvation in tissue cultures of Daucus carota

Embryogenic callus was induced from cotyledonary explants of Daucus carota L. cultured on solidified MS medium supplemented with 1 mg l^-1 2,4-D. Following callus initiation somatic embryos were developed from the callus on MS medium without 2,4-dichlorophenoxyacetic acid. To stimulate the production and germination of somatic embryos we cultured the callus under physically and chemically modified conditions during subculture. When the embryogenic callus was cultured on half-strength MS medium or MS medium without sucrose or cultured under conditions of reduced humidity (69.3%), the production of embryos increased 3.4- to 4.5-fold compared to culture on MS medium containing 3% sucrose (control). Embryogenic callus cultured on MS medium after 5 days of starvation (by being placed in empty 12-well tissue culture plates) showed a 20-fold increase in somatic embryo production and enhanced maturation and germination of embryos. An important point is that the germination of somatic embryos with cup-shaped cotyledons, after a period in culture without medium, was remarkably improved (92%) compared to that of the controls (23%).Thus, we were able to show that stress by starvation without medium led to the enhanced production and increased germination of somatic embryos.     

A method for quantification of the level of somatic embryogenesis among Norway spruce callus lines

A method for quantitative determination of the level of somatic embryogenesis in Norway spruce embryogenic callus is described. Embryogenic callus was dispersed in liquid by agitation and plated in a thin layer of medium containing 0.6% low melting point agarose. The number of embedded somatic embryos per mg of callus ranged from 0.2 to 1.5 among 11 embryogenic callus lines surveyed. Each callus line was derived from an individual immature embryo explant. Further development occurred as somatic embryos grew out of the agarose layer. This method was useful for identifying highly embryogenic callus lines among phenotypically similar lines, and should be useful for quantitatively determining the effect of medium and growth regulator modifications on somatic embryo density and developmental capacity.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - BAP 6-benzylaminopurine - IBA indole-3-butyric acid - ABA abscisic acid     

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

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

<Emphasis Type="Italic">In vitro</Emphasis> plant regeneration via somatic embryogenesis through cell suspension cultures of horsegram [<Emphasis Type="Italic">Macrotyloma uniflorum</Emphasis> (Lam.) verdc.]

Summary In vitro regeneration of plants via somatic embryogenesis through cell suspension culture was achieved in horsegram. Embryogenic calluses were induced on leaf segments on solid Murashige and Skoog (MS) medium with 9.0 μM 2,4-dichlorophenoxyacetic acid (2,4-D). Differentiation of somatic embryos occurred when the embryogenic calluses were transferred to liquid MS medium containing 2,4-D. Maximum frequency (33.2%) of somatic embryos was observed on MS medium supplemented with 7.9 μM 2,4-D. Cotyledonary-torpedo-shaped embryos were transferred to liquid MS medium without growth regulators for maturation and germination. About 5% of the embryos germinated into plants, which grew further on solid MS medium. The plants were hardened and established in soil. Effects of various auxins, cytokinins, carbohydrates, amino acids, and other additives on induction and germination of somatic embryos were also studied. A medium supplemented with 7.9 μM 2,4-D, 3.0% sucrose, 40 mg l⁻¹ L-glutamine, and 1.0 μM abscisic acid was effective to achieve a high frequency of somatic embryo induction, maturation, and further development.     

Effects of ABA on secondary embryogenesis from somatic embryos induced from inflorescence culture ofAralia cordata Thunb

In order to investigate the effect of ABA on secondary embryogenesis from somatic embryos inAralia cordata Thunb., embryogenic callus and somatic embryos were induced from inflorescence on solid MS basal medium supplemented with 1.5 mg/L 2,4-D after eight weeks without subculture. For mass production of somatic embryos, embryogenic cell clumps were maintained in liquid MS medium supplemented with 1.0 mg/L 2,4-D, and then transferred to 2, 4-D-free medium. When developing embryos at various stages were cultured separately in liquid medium with ABA (0 to 2.0 mg/L) for three weeks, and then cultured in ABA-free liquid medium for two weeks, torpedo-shaped embryos exhibited secondary embryogenesis of 65.9% in only 0.2 mg/L ABA pretreatment. Cotyledonary embryos in cultures by 0.2, 0.5 and 1.0 mg/L ABA pretreatment also exhibited secondary embryogenesis (73%, 9.4% and 6.0%, respectively). However, globular and heart-shaped somatic embryos treated with ABA did not form secondary embryos on their hypocotyl surfaces. When cotyledonary embryos were cultured in ABA-free medium or 0.2 mg/L ABA treated medium for three weeks, and then in ABA-free liquid medium for 6 weeks, the germination frequency was lower in medium with 0.2 mg/L ABA (45.9%) than in hormone-free medium (56.8%). This result seems to be related to the high frequency of secondary embryogenesis. It is suggested that secondary embryogenesis by ABA application depends upon the stage of embryo cultured and the ABA concentration.     

High frequency plant regeneration via somatic embryogenesis in cell suspension cultures of coriander (Coriandrum sativum L.)

Hypocotyl segments and zygotic embryos of coriander formed embryogenic calli at frequencies of up to 75% when cultured on MS medium supplemented with 1 mgl^–1 2,4-D. Calli were transferred to MS liquid medium with 1 mgl^–1 2,4-D to initiate cell suspension cultures. Embryogenic cells became finely dispersible in the medium as the subculture proceeded. Cultures were transferred to a nitrogen compound enriched liquid MS medium containing 2% sucrose and 0.1 mgl^–1 2,4-D, and cultured two weeks before plating on MS basal medium. Approximately 75% of cell aggregates (1 to two mm in diameter) underwent development into globular to cotyledonary somatic embryos after two weeks of plating. Most of the embryos were subsequently regenerated into plantlets. Regenerants were successfully transplanted to potting soil and grown to maturity in a phytotron.Abbreviations MS Murashige and Skoog - MS1D MS medium + 1 mgl^–1 2,4-D     

High-frequency somatic embryogenesis from germinated zygotic embryos of <Emphasis Type="Italic">Schisandra chinensis</Emphasis> and evaluation of the effects of medium strength,sucrose, GA<Subscript>3</Subscript>, and BA on somatic embryo development

We developed a new protocol for highly efficient somatic embryogenesis and plantlet conversion of Schisandra chinensis. Friable embryogenic callus was induced from cotyledonary leaves and hypocotyls of germinated zygotic embryos on Murashige and Skoog (MS) agar medium containing 2,4-dichlorophenoxyacetic acid (2,4-D). Preculture of zygotic embryos on 2,4-D-containing medium increased embryogenic callus induction efficiency. The highest embryogenic callus induction frequency of 56.7% was obtained from shoot apical meristem-containing hypocotyl explants from 1-week-old germinated embryos on MS medium containing 4.0 mg l⁻¹ 2,4-D. Embryogenic callus proliferation, somatic embryo (SE) formation, and subsequent plantlet conversion occurred under optimal culture conditions. The effects of MS medium strength, sucrose, gibberellic acid (GA₃), and 6-benzyladenine (BA) on SE formation and plantlet conversion were evaluated. Low MS medium strength (1/4 to 1/2) was necessary for SE formation, and the optimal sucrose concentration was 2.0%. Supplementing medium with GA₃ negatively impacted SE formation and subsequent development. BA significantly increased the number of SEs and the plantlet conversion capacity. One-third-strength MS medium with 1.0% sucrose and 0.5 mg l⁻¹ BA produced the highest number of SEs (309 embryos from 9 mg embryogenic callus) and the highest frequency of plantlet conversion from germinated SEs (52.6%). When transplanted to soil, 90% of the regenerated plants developed into normal plants.     

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