Polyethylene glycol and abscisic acid improve maturation and regeneration of<Emphasis Type="Italic"> Panax ginseng</Emphasis> somatic embryos

Embryogenic culture was initiated from mature zygotic embryos of Panax ginseng. Multiple somatic embryos formed and proliferated on Murashige and Skoog medium supplemented with 2,4-dichlorophenoxyacetic acid (2.26 M) and kinetin (0.046 M). Mature as well as immature somatic embryos grew into plantlets lacking roots on the same media. Histomorphological analysis of somatic embryos treated with abscisic acid (ABA) and polyethylene glycol (PEG 4000) showed a slight improvement in the root meristem organization of torpedo-stage embryos (embryos were more compact and their cells exhibited a lower degree of vacuolation). Shoot regeneration of non-treated somatic embryos was 31% while that for somatic embryos treated with PEG 4000 and ABA was 70%. Moreover, 75% of plants regenerated from PEG- and ABA-treated embryos formed roots while plants from non-treated embryos did not form roots.Abbreviations ABA (±)-Abscisic acid - BAP N ⁶-Benzyladenine - 2,4-D 2,4-Dichlorophenoxyacetic acid - GA ₃ Gibberellic acid - Kin Kinetin - MS Murashige and Skoog medium - PEG 4000 Polyethylene glycol 4000 - PGR Plant growth regulators Communicated by H. van Onckelen     

Somatic embryogenesis of Panax ginseng in liquid cultures: a role for polyamines and their metabolic pathways

A callus with embryogenic capacity was generated fromroot sections of Panax ginseng and used as aninoculum source for embryogenic liquid cultures in athree-step process: – a suspension culture of cellaggregates in the presence of an auxin/cytokininmixture, – an induction medium containing auxin only(for 5 to 30 days), – a regeneration medium containingcytokinin only (for one month). Up to 25 embryos wererecovered per 2.5 g of aggregates in these conditions.Incorporation of polyamines or their precursorsarginine and ornithine into either the induction orregeneration media increased the number of embryosproduced by up to 4 times. Inhibitors of bothbiosynthesis and biodegradation of polyamines reducedthe number of embryos. These results support earlierfindings of the role of polyamines in the process ofsomatic embryogenesis. The success of these liquidcultures opens up the possibility of producing somaticembryos of Panax ginseng in bioreactors.     

Callus and embryoid formation from protoplasts of Helianthus annuus

Sunflower hypocotyl protoplasts have been isolated and cultured. Optimum plating density for cell division and colony formation was in the range of 5 to 7×10⁴ cells/mi in an agarose medium supplemented with BAP (1 mg/l) and NAA (1 mg/l). Plating efficiency was 60% after 21 days of culture. In the resultant culture a mixed population of calli and embryoids was observed. Thirty seven percent of the cell clusters exhibited a developmental pattern similar to an embryoid. Many stages of embryogenesis were observed in the same cultures.Abbreviations 2,4-D 2,4 dichlorophenoxyacetic acid - NAA 1-naphtaleneacetic acid - IAA Indole-3-acetic - BAP 6-benzylamino purine - GA₃ Gibberellic acid     

Plant regeneration through somatic embryogenesis in root-derived callus of ginseng (Panax ginseng C. A. Meyer)

Summary Callus culture was initiated from expiants of mature root tissues of ginseng (Panax ginseng C.A. Meyer) on MS medium enriched with 2,4-D. The ageing callus produced numerous embryoids in this medium. Reculture of these embryoids in media (1/2 MS or B5) supplemented with benzyladenine and gibberellic acid resulted in profuse plantlet regeneration.     

Pulse treatments of penicillin-G and streptomycin minimise internal infections and have post-treatment effects on the morphogenesis of ginseng root culture

This study was initiated to determine whether antibiotic pulse treatments (APT) could effectively eliminate internal infections of ginseng (Panax ginseng) root explants containing vascular tissue, and subsequently have post-treatment effects on changing explant behaviors in callus induction and organogenesis or somatic embryogenesis. For contamination control, a treatment of 40 min with an antibiotic solution consisting of 1000 mg/1 of penicillin-G and 1000 mg/1 of streptomycin immediately following Na-hypochlorite sterilisation significantly decreased contamination rate. Extending treatment time to 2–3 h further lowered the contamination rate to 30–40%. On the other hand, explants treated with antibiotics for 20 min or less were all contaminated. APT also had post-treatment effects; it delayed callus induction for 1–12 months depending on pulse duration and stimulated the generation of more hardand darker looking than fragile- and lighter looking callus. The induced callus proliferated at a depressed rate, increasing subculture intervals from 1 to several weeks, and not until after five subcultures did it fully recover. The regeneration ability of the recovered callus was also affected by APT: the regeneration of adventitious roots was promoted, whereas somatic embryos were not observed.Abbreviations APT Antibiotic pulse treatments - 2,4-D 2,4-dichlorophenoxyacetic acid     

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

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

2,4-Dichlorophenoxyacetic acid affects mode and frequency of regeneration from hypocotyl protoplasts ofBrassica oleracea

Summary The effect of 2,4-dichlorophenoxyacetic acid (2,4-D) on the regeneration from hypocotyl protoplasts ofBrassica oleracea was studied by varying the 2,4-D concentration in the protoplast culture medium, 8 p, and the callus proliferation medium, K3. When hypocotyl protoplasts of the inbred line BL12 were cultured in the complete absence of 2,4-D, they divided and produced embryogenic calli. Moreover, these calli generated somatic embryos which were easily recognized by red cotyledons due to the presence of anthocyanin. When 2,4-D was present either in 8p medium or K3 medium the formation of somatic embryos was reduced. On the other hand, the number of shoot-forming calli increased considerably. We therefore conclude that 2,4-D directs the mode of regeneration by suppressing somatic embryogenesis in favour of shoot regeneration. Secondly, 2,4-D increases the regeneration efficiency. Furthermore, the callus proliferation phase on K3 medium is most important with respect to the determination of either somatic embryogenesis or shoot regeneration.Abbreviations BA benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole acetic acid - NAA naphthalene acetic acid - PE plating efficiency     

Developmental anatomy and ultrastructure of early somatic embryos in European black pine (Pinus nigra Arn.)

Summary Embryogenic callus cultures of European black pine (Pinus nigra Arn.) were established on megagametophytes containing zygotic embryos in early developmental stage. In addition to many elongated cells and disorganized growing clumps they contained early somatic embryos at various stages of development. At all stages of embryogenesis the embryos were organized as bipolar structures. Cell pairs composed of one isodiametric cell with dense cytoplasm and a second large vacuolated cell were the simplest bipolar system. The vacuolated cell underwent senescence. The cytoplasm-rich cell and its derivates divided transversally, resulting in several cytoplasmic cells arranged in row. An early embryonal cylindrical mass was formed by longitudinal division of the cells in a filament. Proximally localized cells in the early embryonal mass became vacuolized and elongated gradually giving rise to the secondary suspensor. Distal cells remained cytoplasmic in character and formed an embryonal mass along the axis of long early somatic embryos. Differences in the proportion of organelles and heterochromatin clumps, thickness of cell walls and number of plasmodesmata between cells at various stages of early somatic embryogenesis were described.     

Chromatin reorganization and endogenous auxin/cytokinin dynamic activity during somatic embryogenesis of cultured cotton cell

We conducted a systematic assessment and comparative study on the biochemical and cellular characteristics of cultured cotton cells during the entire process of somatic embryogenesis (SE). All staged cultures were widely investigated in this assay. Cell and tissue ectogenesis manipulation combined with flow cytometry (FCM) was employed to cellular study during the whole totipotency process of dedifferentiation and redifferentiation. We identified two phases of chromatin decondensation during the dedifferentiation and redifferentiation. At the same time, sharp increase in the ratio of indoleacetic acid (IAA), isopentenyladenosine group (iPAs) at the same stage of cell dedifferentiation and redifferentiation process serve as distinct biochemical maker of dedifferentiation and SE initiation with the unique feature. Our results suggest the two phases of chromatin reorganization associated with endogenous auxin/cytokinin dynamic activity may underlie dedifferentiation and redifferentiation during the entire SE process in cotton.     

Inoculum size and auxin concentration influence the growth of adventitious roots and accumulation of ginsenosides in suspension cultures of ginseng ( Panax ginseng C.A. Meyer)

The effect of the root-inoculum size and axuin concentration on growth of adventitious roots and accumulation of ginsenosides were studied during suspension cultures of ginseng (Panax ginseng C.A. Meyer). Of the various concentrations of indole-3-butyric acid (IBA) and γ-naphthaleneacetic acid (NAA) used as supplementary growth regulators along with Murashige and Skoog medium, 25 μM IBA was found suitable for lateral root induction and growth, as well as accumulation of ginsenosides. Inoculum size of 5 g L⁻¹ was found suitable for optimal biomass (10.5 g L⁻¹ dry biomass) and ginsenosides (5.4 mg g⁻¹ DW) accumulation. Of the various length of root inocula tested (chopped to 1–3, 4–6, 7–10 mm and un-chopped), root inocula of 7–10 mm was found suitable for biomass and ginsenoside accumulation.     

The Agrobacterium rhizogenes rolC-gene-induced somatic embryogenesis and shoot organogenesis in Panax ginseng transformed calluses

Expression of the Agrobacterium rhizogenes rolC gene in Panax ginseng callus cells results in formation of tumors that are capable to form roots. The selection of non-root forming tumor clusters yielded the embryogenic 2c3 callus line, which formed somatic embryos and shoots independently of external growth factors. Although the 2c3 somatic embryos developed through a typical embryogenesis process, they terminated prematurely and repeatedly formed adventitious shoot meristems and embryo-like structures. A part of the shoots and somatic embryos formed enlarged and fasciated meristems. This is the first indication of the rolC gene embryogenic effect and, to our knowledge, the first indication that a single gene of non-plant origin can induce somatic embryogenesis in plants.     

A morphological and histological comparison of the initiation and development of pecan (Carya illinoinensis) somatic embryogenic cultures induced with naphthaleneacetic acid or 2,4-dichlorophenoxyacetic acid

Summary Somatic embryos produced in vitro may exhibit structural abnormalities that affect their subsequent germination and conversion into plants. To assess the influence of auxin type on embryo initiation and development, a morphological and histological comparison was made of pecan (Carya illinoinensis) somatic embryogenic cultures induced on media with naphthaleneacetic acid or 2,4-dichlorophenoxyacetic acid (2,4-D), using light and scanning electron microscopy. Both auxins promoted enhanced cell division, particularly in subepidermal cell layers. However, notable differences were observed in mitotic activity, location of embryogenic cell proliferation, epidermal continuity, callus growth, and embryo morphology. Cultures induced on naphthaleneacetic acid had embryogenic regions composed of homogeneous, isodiametric, meristematic cells. Embryos derived from these cultures generally had a normal morphology, were single, and had a discrete apical meristem. In contrast, tissues induced on media with 2,4-D had more intense and heterogeneous regions of cell division. Proliferating cell regions were composed of meristematic cells interspersed with callus and involved more extensive regions of the mesophyll. Marked callus proliferation caused epidermal rupture in some areas. Embryos induced on medium with 2,4-D had a higher incidence of abnormalities that included fasciated, fan-shaped, and tubular embryos. Defined apical meristems were often lacking or partially obliterated due to callus proliferation. The heterogeneous, often intensive proliferation of cells in cultures induced with 2,4-D may interfere with normal patterns of embryo development.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - BAP 6-benzylaminopurine - NAA naphthaleneacetic acid - SEM scanning electron microscopy     

Isolation and functional analysis of a set of auxin genes with low root-inducing activity from an Agrobacterium tumefaciens biotype III strain

A new type of root-inducing iaa gene set was cloned from the Ti plasmid of the biotype III Agrobacterium tumefaciens strain Tm-4. These iaa genes are characterized by a very low DNA homology with the well-characterized iaa gene set, iaaM and iaaH, of the common DNA region of the biotype I strain Ach5 and by a low root-inducing activity.The biological activities of both iaa gene sets were compared by transferring each into a disarmed Ti vector and by testing the resulting strains on Nicotiana rustica leaf discs, decapitated Datura stramonium stems, tomato plants and Kalanchoë daigremontiana. Tm-4 iaa genes have a reproducibly weaker root-inducing ability on Nicotiana rustica, induce very little tumour growth on decapitated Datura plants or on tomato plants and do not induce roots on Kalanchoë daigremontiana. The Tm-4 iaa region was mapped by :: Tn5 transposon mutagenesis and tested on Nicotiana rustica. These tests combined with complementation experiments map the iaa genes to a 4.5-kb region.The Tm-4 iaa genes were able to complement the corresponding Ach5 iaa genes on Nicotiana rustica, indicating that the differences between these genes are quantitative rather than qualitative. Complementation experiments on Kalanchoë showed the iaaM gene of Tm-4 responsible for the overall weak auxin activity of the intact iaa set. In view of the observed structural and functional differences we propose to call the Tm-4 iaa genes TB-iaaM and TB-iaaH and the Ach5 iaa genes A-iaaM and A-iaaH.     

Effects of light conditions and 2,4-D concentration in soybean anther culture

The morphogenic response of anther walls and connective tissue is the greatest obstacle to androgenesis in soybean anther culture. Whereas induction to microspore embryogenesis occurs in the dark in almost all plant species, soybean anthers have been cultured under light. In an attempt to establish culture conditions that simultaneously stimulate microspore embryogenesis and inhibit epidermal and connective cell proliferation, the effect of light and two 2,4-dichlorophenoxyacetic acid (2,4-D) concentrations (2 and 10 mg l^–1) on the induction process was investigated. Higher 2,4-D concentration speeded up microspore plasmolysis and did not improve androgenesis. Callogenesis and embryogenesis induction from sporophytic cells were significantly lower in the dark, and some microspores showed major alterations in the sporoderm. Auxin 2,4-D and induction under light contributed to the morphogenic response of the anther walls and connective tissue under the conditions previously recommended to trigger microspore embryogenesis.     

Somatic embryogenesis and plant regeneration from seeds of wild<Emphasis Type="Italic"> Dicentra spectabilis</Emphasis> (L.) LEM

Regeneration via somatic embryogenesis from callus was studied in Dicentra spectabilis. To obtain somatic embryogenic callus, we cultured D. spectabilis seeds on MS basal media supplemented with various concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D). The highest percentage of embryogenic callus formation was observed on media containing 1.0 mg/l 2,4-D under dark conditions. Somatic embryogenesis was studied by transferring the callus onto MS basal medium containing different concentrations (0.0, 0.1, 0.5, 1.0, 2.0 mg/l) of KIN (kinetin) and/or BAP. Somatic embryogenesis on MS basal media with 1.0 mg/l of KIN was excellent under light conditions. Somatic embryos were rooted by transferring them to half-strength MS basal media containing 2 g/l Phytagel. About 64.2% of the somatic embryos converted to rooted plantlets, 4% showed secondary embryogenesis and 31.8% did not develop and died. Rooted plantlets showed a 46% survival rate when acclimatized ex vitro.Abbreviations BAP 6-Benzylaminopurine - 2.4-D 2,4-Dichlorophenoxyacetic acid - KIN Kinetin - SEM Scanning electron microscopyCommunicated by H. Lörz     

Histological study of organogenesis and embryogenesis inCyclamen persicum Mill. tissue cultures: Evidence for a single organogenetic pattern

Summary Evidence is given thatCyclamen persicum callus culture give rise to structures such as shoot-buds, roots, unipolar tubers, bipolar tubers and embryos. Observations made on their structure by means of morphological and histological analyses are reported and discussed in relation to the anatomy of someCyclamen structures such as zygotic embryos and seedling tubers. Such analyses demonstrate that the bipolar structures including somatic embryos and bipolar tubers lack vascular connection with the callus whereas unipolar structures establish vascular strands with the callus. Here, the relationship between these regenerated structures is depicted. The organogenetic pattern inCyclamen persicum callus culture could well be a derived pathway for somatic embryogenesis. A theoretical diagram concerning the morphogenetic program is presented.     

Histological observations of morphogenesis in petiole derived callus of Amorphophallus rivieri Durieu in vitro

The initiation and development of somatic embryos and organogenic shoots and corm-like structures (CLSs) from petiole-derived calli of Amorphophallus rivieri Durieu were observed histologically. The petioles were cultured on Murashige and Skoog (MS) medium supplemented with 5.37 μM α-naphthaleneacetic acid (NAA) and 4.44 μM N⁶-benzylaminopurine (6-BA) for callus induction. The shoot and corm organogenesis occurred from the compact calli when they were transferred to a medium containing 0.54 μM NAA and 4.44 μM 6-BA. A combination of 13.57 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 8.88 μM 6-BA or 24.18 μM NAA and 6.66 μM 6-BA was optimum for induction of somatic embryos, which failed to produce plantlets because of their structural abnormalities. Shoot regeneration predominantly happened through organogenesis although somatic embryogenesis infrequently occurred. The subepidermal cells of the compact callus converted to competent cells and started divisions, which resulted in formation of the meristemoids. The meristemoid cells continued division to develop into bud primordia. Subepidermal cells could also form the globular structures. Subsequently, these globoids developed into CLSs from which plantlets regenerated during subculture. Meanwhile, the CLSs were capable to form cormels, which could be a promising way for the propagation of A. rivieri.     

Dispersal and size fractionation of embryogenic callus increases the frequency of embryo maturation and conversion in hybrid tea roses

Plant regeneration from embryogenic cells of two Rosa hybrida cultivars, Kardinal and Classy, was increased by dispersing embryogenic callus in liquid medium for 3 h followed by size-fractionation to isolate proembryogenic masses that were smaller than 530 m. Dispersed callus of three cultivars, Kardinal, Classy, and Tineke, produced 61–135 cotyledonary-stage embryos/100 mg fresh weight (FW) as compared to intact callus that had not been dispersed, which produced only zero to three cotyledonary-stage embryos/100 mg FW. Over 500 cotyledonary-stage embryos/100 mg FW callus developed from proembryogenic masses of Kardinal, Classy, and Tineke following 2 months of culture on solidified Murashige and Skoogs basal salts medium supplemented with 0.25% activated charcoal. Cotyledonary-stage embryos of Classy that developed from both dispersed callus and fractionated cells of various sizes showed a significantly higher conversion frequency to plants (28%) than cotyledonary-stage embryos isolated from intact callus (9%). The highest conversion frequencies for Kardinal (50–58%) occurred from cotyledonary-stage embryos that developed from dispersed callus and from the fraction of cells smaller than 850 m.Abbreviations BAP 6-Benzyladenine - FeEDDHA Ferric ethylenediamine di(o-hydroxyphenylacetate) - FW Fresh weight - PEMs Proembryogenic masses - PGRs Plant growth regulatorsCommunicated by S.A. Merkle     

Increase of embryogenic callus formation in cucumber by initial culture on high concentration of 2,4-dichlorophenoxyacetic acid

Cucumber (Cucumis sativus L.) leaf explants were cultured either continuously on standard medium containing 4.5 µM 2,4- dichlorophenoxyacetic acid (2,4-d) and 4.4 µM benzylaminopurine, or first cultured for various periods at different levels of 2,4-d, picloram or naphthaleneacetic acid (NAA), and then transferred to standard medium. When cultured continuously on standard medium, less than 10% of the explants formed embryogenic callus. Initial culture on picloram or NAA, or on 2,4-d at a low concentration (1.4 µM) did not result in any embryogenic callus formation. Embryogenic callus formation increased to 40% if during the initial phase of the culture (10 days), the 2,4-d concentration was raised to 14 µM. Prolonged culture on 14 µM 2,4-d resulted in less embryogenic callus formation.Abbreviations BA benzylaminopurine - 2,4-d 2,4-dichlorophenoxyacetic acid - NAA naphthaleneacetic acid