Composition of embryogenic suspension cultures of Ipomoea batatas Poir. and production of individualized embryos

Embryogenic suspension cultures of Ipomoea batatas Poir. contain heterogeneous populations of discrete cellular units. In order to optimize embryo production, a study was conducted to identify the embryogenic fraction of such cultures. Suspension cultures were fractionated with sieves of 1000, 710, 500, 355, 250, 180, 125, 90 and 63m mesh openings and the composition of each fraction was determined. Cellular units larger than 355 m were primarily calli and made up 75% of the total mass of cultures in the stationary phase of growth. These calli were composed of embryogenic and non-embryogenic subunits, and 98% of the embryogenic subunits measured 355–1000 m. Calli and embryogenic calli subunits produced clusters of embryos at various stages of development upon transfer to liquid or solidified media without 2,4-D. The 125–355 m fraction of suspension cultures was composed of cell aggregates of which 20% were embryogenic. The embryogenic cell aggregates produced single globular embryos upon transfer to liquid media containing 0 or 1 M 2,4-D. The 63–125 m fraction of suspension cultures contained only 2% of embryogenic cell aggregates. It can be inferred from our results that the embryogenic fraction of cultures was essentially represented in calli, and that proliferation of the embryogenic fraction occurred through the separation of embryogenic cell aggregates from larger calli when cultures approached their stationary growth phase.Abbreviations and definitions cellular units single cells, cell aggregates, and calli - cell aggregates discrete associations of cells - calli association of cell aggregates - embryogenic cell aggregates yellow aggregates of cytoplasmic cells which have the potential to produce embryogenic calli or embryos [3] - non-embryogenic cell aggregates white aggregates of vacuolated cells [3] - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indoleacetic acid     

Norway spruce somatic embryogenesis: high-frequency initiation from light-cultured mature embryos

Somatic embryos and rooted plantlets have been regenerated from light-initiated embryogenic callus derived from mature embryos of Picea abies. Under a 16 h photoperiod, mature zygotic embryos were cultured on a modified half-strength Murashige & Skoog medium without NH₄NO₃ and supplemented with 5 mM glutamine, 4.5 M N6-benzyladenine and 10.7 M naphthaleneacetic acid or 10 M 2,4-dichlorophenoxyacetic acid. White translucent embryogenic callus, proliferating from the callusing hypocotyl region after 3 weeks incubation, was isolated from the green non-embryogenic tissue and subcultured for over 12 months. Upon transfer of the embryogenic callus through a specific sequence of media, somatic embryos proceeded to mature, elongating and forming rings of cotyledonary leaves similar to those of zygotic embryos. Transferred to medium without growth regulators, the somatic embryos germinated and produced plantlets with green cotyledons, elongated hypocotyls and primary roots.     

Growth and rosmarinic acid accumulation in callus,cell suspension,and root cultures of wild Salvia fruticosa

The accumulation of rosmarinic acid (RA) in Salvia fruticosa callus, cell suspension, and root cultures was studied. For callus induction, leaves excised from microshoots were cultured on MS medium containing thidiazuron (TDZ) (0, 2.3, 4.6, 6.9, 9.2, or 11.5 M) and indole-3-acetic acid (IAA) (0 or 3 M). For root culture, hairy roots were cultured in B5 medium containing 2.7 M -naphthaleneacetic acid (NAA) and different concentrations of sucrose or phenylalanine. Induction of callus was completely inhibited in the absence of both TDZ and IAA and the largest callus (0.79 g) was obtained with a combination of 6.9 M TDZ and 3 M IAA. Culture duration of 5 weeks resulted in maximum callus growth and RA yield (2.12 mg/ 100 mg dry weight). Cell suspension growth and RA yield (5.1 mg/ 100 mg dry weight) were maximum after 20 days of culture. The highest root growth and RA yield (2.62 mg/ 100 mg dry weight) was obtained with 4% (w/ v) sucrose. Incorporation of 10 mg l^–1 phenylalanine in the medium increased RA yield in the roots to 4.68 mg/ 100 mg dry weight after 4 weeks of culture. Amounts of RA extracted from in vivo leaves and roots were 0.21 and 0.72 mg/ 100 mg dry weight, respectively.     

Plant regeneration from cultured protoplasts of the cooking banana cv. Bluggoe (Musa spp., ABB group)

Summary Suspensions of embryogenic cells of a triploid banana (Musa spp., cv. Bluggoe) were initiated from the uppermost part of meristematic buds, and used as protoplast source. After 20 weeks in culture, the suspension contained a mixture of globular structures or globules and embryogenic cell clusters, as well as single cells. Two types of protoplasts were obtained from embryogenic suspension culture: small (20–30 m) and larger (30–50 m) protoplasts with a dense cytoplasm and large starch grains respectively. The small protoplasts probably originated from embryogenic cell clusters, and also from pseudocambial cells of globules, while larger protoplasts were probably released from oval starchy cells and those of the globule peripheral area. In co-culture with a suitable feeder, consisting of suspensions of diploid banana cells, the protoplasts of triploid banana reformed the cell wall within 24 h and underwent sustained divisions leading to the formation of small clusters of 2–3 cells within 7 days. The latter developed directly into embryos without passing through an apparent callus phase. 10% of such embryos gave rise to plantlets when subcultured in 2.2 M 6-benzylaminopurine and 2 M 4 amino-3,5,6-trichloropicolinic acid for 1 week, before transfer to MS medium containing 10 M 6-benzylaminopurine. The rest of the embryos underwent intensive direct secondary embryogenesis which could lead to the formation of plantlets with a frequency of up to 50% upon further transfer to hormone-free medium.Abbreviations BAP 6-benzylaminopurine - MS Murashige and Skoog (1962) medium - 2,4-D dichlorophenoxyacetic acid - UV ultraviolet light - FDA fluorescein diacetate - MES 2-(N-morpholino)ethanesulfonic acid - Picloram 4 amino-3,5,6-trichloropicolinic acid     

Effect of plant growth regulators on the cellular growth and levels of intracellular protein,starch and polyamines in embryogenic suspension cultures of Pinus taeda

Somatic embryogenesis is the most important in vitro culture system for conifer propagation. However, Pinus taeda has been considered recalcitrant to somatic embryogenesis in commercial scale-up. The study of biochemical and physiological aspects of cell growth could lead to a better understanding of somatic embryogenesis in this species. In the present work, we investigated the cell growth dynamics, intracellular levels of proteins, starch and polyamines in suspension cultures of Pinus taeda established in plant growth regulator-free medium (BM0) and in medium supplemented with 2 M 2,4-dichlorophenoxyacetic acid, 0.5 M 6-benzylaminopurine and 0.5 M Kinetin (BM2). Cell cultures growing in BM0 medium showed an increase in the sedimented cell volume from 3.77 to 17.73 ml after 24 days of culture. Those cultured in BM2 medium showed an increase in the sedimented cell volume from 4.23 to 25.17 ml after 20 days of culture. Intracellular proteins levels increased during the exponential growth phase and starch levels decreased until the exponential phase, followed by a synthesis up to the stationary phase, in both BM0 and BM2 media. Highest putrescine levels occurred in cultures growing in BM0 medium and this was associated with the low cellular growth.     

Factors affecting transient gene expression in electroporated black spruce (Picea mariana) and jack pine (Pinus banksiana) protoplasts

Summary Methods were developed for transient gene expression in protoplasts of black spruce (Picea mariana) and jack pine (Pinus banksiana). Protoplasts were isolated from embryogenic suspension cultures of black spruce and from non-embryogenic suspensions of jack pine. Using electroporation, transient expression of the chloramphenicol acetyltransferase (CAT) gene was assayed and shown to be affected by the cell line used, by voltage, temperature, and by the plasmid concentration and conformation. Increasing the plasmid DNA concentration (0–150g ml^–1) resulted in higher levels of transient CAT expression. In jack pine, linearized plasmid gave 2.5 times higher levels of CAT enzyme activity than circular. Optimal voltage varied for each cell line of the two species within the range 200–350 V cm^–1 (960 F). A heat shock treatment of protoplasts for 5 min at 45 °C resulted in enhanced CAT gene expression for both species.NRCC No. 30491     

High frequency somatic embryogenesis and plant regeneration in petiole and leaf explant cultures and petiole-derived embryogenic cell suspension cultures of Hylomecon vernalis

Culture conditions are described for high frequency somatic embryogenesis and plant regeneration in petiole and leaf explant cultures and petiole-derived embryogenic cell suspension cultures of Hylomecon vernalis Max. Petiole explants formed embryogenic calluses at a frequency of 53% when cultured on B5 medium supplemented with 13.6 M 2,4-dichlorophenoxyacetic acid (2,4-D) alone. Leaf explants formed embryogenic calluses at a frequency of 21% when cultured at a combination of 4.52 M 2,4-D and 2.22 M 6-benzyladenine. Cell suspension cultures were established with petiole-derived embryogenic calluses using liquid B5 medium with 4.52 M 2,4-D. Upon plating onto B5 basal medium, cell suspension cultures produced numerous somatic embryos, which then developed into plantlets. Regenerated plantlets were transplanted to potting soil and grown to maturity in a greenhouse.     

Formation of giant protoplasts from protoplasts of Pleurotus cornucopiae by the cell wall lytic enzyme

Summary When purified protoplasts of Pleurotus cornucopiae IFO9614 were incubated with a mixture of cell wall lytic enzymes, they were found to increase their size. Their average diameter increased from 4.3 m to 31 m after 65 h incubation at 24° C. The presence of cellulase ONOZUKARS in the enzyme mixture had a significant effect on the formation of giant protoplasts. Regeneration frequency of giant protoplasts in a medium containing 0.5 M sucrose was 3.5%, approximately six times that of normal protoplasts.     

Clonal propagation of Callistemon by tissue culture techniques

Callus development in Callistemon viminalis was readily achieved when axillary buds derived from nodal tissue were placed in a medium containing macro- and micro-nutrients, sucrose (0.06 M), inositol (300 M), nicotinic acid (20 M), pyridoxine hydrochloride (3 M), thiamine hydrochloride (2 M), riboflavin (10 M), cytokinins (5 M) and auxins (0.1 M). The presence of benzylaminopurine (5 M) and p-chlorophenoxyacetic acid (0.1 M) promoted the most vigorous callus development and sprout formation. Rooting of nodal material was rare but occurred readily following the transference of sprouts developed on callus to a basal medium containing sucrose and salts. Root initiation was stimulated, however, by the presence of auxins. Chlorophenoxyacetic acid while stimulating root initiation repressed root growth. Indole butyric acid stimulated both root initiation and shoot growth at concentrations of 0.005 to 0.1 M. The treatment of choice for rooting and shoot growth was the addition of indole butyric acid at a concentration of 0.01 M.     

Initiation and growth characteristics of suspension cultures of Calendula officinalis cells

Callus cultures of marigold (Calendula officinalis L.) were induced on Murashige and Skoog medium with different concentrations of auxin (dichlorophenoxyacetic acid (2,4-D) or indole-3-acetic acid (IAA) and cytokinin (kinetin or 6-(,-dimethylallylamino)purine (2iP). Of all hormone combinations used in the medium, two were the most efficient in promoting callus development: 1.81 M (0.4 mg l^–1) 2,4-D and 1.85 M (0.4 mg l^–1) kinetin (0.4d–0.4k culture) or 0.45 M (0.1 mg l^–1) 2,4-D and 2.02 M (0.5 mg l^–1) 2iP (0.1d–0.5p culture). These combinations were selected to induce cell suspension cultures. The suspension cultures were maintained under light or dark conditions. The light stimulated cell aggregation in the cultures. In both cultures cells were undifferentiated under darkness, whereas in the light, rhyzogenesis was observed in 0.1d–0.5p culture. The cell growth and protein and oleanolic acid contents were determined. Initially, biomass production was similar under light and dark conditions, but after 7–8 months from the induction the cell growth was reduced by approximately 30% in the light, whereas the cell growth of the cultures maintained under darkness did not reveal any changes. The presence of oleanolic acid was detected in the suspension cultures kept in darkness. This compound reached two quantitative peaks: in the lag and stationary phases –- beyond the active growth phase of the culture cycle and its concentration was several times higher in 0.1d–0.5p culture than that in 0.4d–0.4k culture. It was for the first time that callus and suspension cultures were induced from the marigold plant.     

Plant regeneration from protoplasts of embryogenic suspension cultures of orchardgrass (Dactylis glomerata L.)

An embryogenic suspension culture of orchardgrass (Dactylis glomerata L.) consisting of small, embryogenic cell clusters was obtained from callus formed on basal sections of young leaves through a process of selective enrichment. These suspensions were used as a source of protoplasts. The isolated protoplasts divided at a frequency of 0.5–10% when plated in an agarose solidified culture medium. Conditioned medium, in which embryogenic Dactylis suspension cultures had been grown, was found to increase the rate of cell colony formation. Protoplast-derived colonies grew rapidly in a bead-type culture system of floating agarose slabs in liquid medium. New suspension cultures formed as the colonies grew out of the agarose. These cultures were embryogenic and formed green plantlets when plated on a solid medium lacking auxin. The plantlets were established in soil and grown to mature plants.Abbreviations B5 medium according to Gamborg et al. (1968) - SH-x medium according to Schenk and Hildebrandt (1972) supplemented with x M dicamba - dicamba 3,6-dichloro-o-anisic acid - KM-8p medium 8p of Kao and Michayluk (1975)     

Establishment of oil palm cell suspensions and plant regeneration

Primary globular callus from immature zygotic embryos and friable embryogenic tissue derived from mature zygotic embryos were used to establish suspension cultures. Callus cultures were established either on modified Y3 or MS medium containing 475–500 M 2,4-D or 250 M picloram and 0.3% (w/v) activated charcoal. Suspension cultures of both cell lines were established in modified Y3 medium containing 10 M 2,4-D. The establishment of cell suspensions from friable embryogenic tissue took only 2 months, in contrast with suspensions from primary globular callus which took 3–5 months to establish. Embryo differentiation was observed only in cell suspensions derived from the friable embryogenic tissue after plating aliquots on regeneration medium. Germinated embryos were recovered and plantlets were successfully established under greenhouse conditions.Abbreviations CET compact embryogenic tissue - FET friable embryogenic tissue - CIM callus induction medium - PGC primary globular callus - 2,3-D 2,4-dichlorphenoxyacetic acid Y3-Eeuwens' medium - MS Murashige & Skoog medium - PVP-40 polyvinylpyrrolidone - KM Kao & Michayluk vitamins - ABA abscisic acid     

Ultrastructural analysis ofSpinacia oleracea sperm cells isolated from mature pollen grains

Summary In isolated condition, the sperm cells ofSpinacia oleracea are no longer arranged in pairs as in the pollen grain. The vegetative membrane, which surrounds a sperm cell pair in a mature pollen grain, is lost during the isolation procedure. The sperm cells become spherical in shape.The isolated sperm cell is surrounded by an intact plasma membrane. The heterochromatic or euchromatic sperm cell nucleus is located in the cell center. Mitochondria are round to oval and have distinct cristae. Often they are clustered in groups of 5 to 10 mitochondria. Dictyosomes are present in the cytoplasm and consist of 4 to 5 cisterns. Endoplasmatic reticulum is mostly situated at the sperm cell periphery, as single cisterns very near the plasma membrane.From diameters of sectioned sperm cells in electron micrographs, it is possible to calculate the average diameter of the whole sperm cell. This average diameter is 3.66 m with a variation of 3.0 m to 4.2 m, resulting in an average volume of 25.6 m³. The nuclear volume is 12.8 m³ (50.0% of the whole cell) and the mitochondrial volume is 0.7 m³ (2.5% of the whole cell). The frequency distribution of the isolated sperm cells diameters shows only one peak with a normal distribution, indicating that there is no dimorphism in volume.     

Plant regeneration from embryogenic suspension cultures of dune reed

Embryogenic callus, derived from mature seeds of dune reed (Phragmites communisTrinius) was used to establish suspension culture. Green shoot-forming type and albino shoot-forming type embryogenic callus of dune reed were selected carefully by the difference of shape and color of callus growing under light and mechanically dispersed before suspending in liquid MS medium supplemented with 1.0 mg l^–12,4-D. They were subcultured every 5 days to remove mucilaginous material in the early culture stage. Both fine albino and green shoot-forming cell suspension lines of dune reed were composed of rapidly growing small cell aggregates that were densely cytoplasmic and potentially embryogenic. Globular somatic embryos were continuously produced in each liquid medium containing 1.0 mg l^–1 2,4-D. The cell aggregates in fine albino cell suspension line (size below 300 m) were smaller than that of green shoot-forming cell suspension line (size between 300 and 800 m). Following transfer to a differentiation medium, both suspension cultures formed regenerating plants with normal roots and albinotic or green shoots, respectively.     

TDZ-induced somatic embryogenesis in non-responsive caryopses of rice using a short treatment with 2,4-D

Caryopses cultures of rice on an auxin medium (2,4-D, 20 M) formed slow-growing tissues that failed to regenerate. The embryogenic tissue possibly lost its regeneration potential on the auxin medium. Reproducible regeneration, however, could be achieved by a short treatment with 20 M 2,4-D for 3 days. Transfer of these caryopses to the medium containing TDZ or BA at 10 M resulted in regeneration of somatic embryos and shoots in 30% of the cultures within 10–15 days. TDZ was better than BA for inducing shoot regeneration. Short treatment for 3 days at higher concentrations of auxin (40–80 M) and subsequent transfer to TDZ or BA medium resulted in an increased frequency (up to 50%) of regeneration.     

Establishment and characterization of Rubus tissue culture systems for in vitro bioassays against phytotoxins from Rubus fungal pathogens

The Rubus species R. parviflorus, R. spectabilis and R. strigosus interfere with conifer seedling establishment on forest regeneration sites in Canada and the United States. As a first step towards microbial metabolite-based control, callus and cell suspension cultures of the Rubus species were developed as a bioassay system to detect phytotoxic compounds that may have relevance in a vegetation control context. Rapidly growing friable callus and suspension cultures were obtained from leaf disks of the three weedy Rubus species using similar culture media conditions (modified Murashige and Skoog) but required different plant growth regulators (R. parviflorus, 4.5 M 2,4-D; R. spectabilis, 26.9 M NAA/0.5 M zeatin; and R. strigosus, 12.4 M picloram). Cell growth and health attributes including callus circumference, degree of browning and suspension culture cell viability as measured by the TTC vital stain assay were developed and were rapid and convenient to use. We have established Rubus tissue culture systems that will make it possible for large scale screening of phytotoxic metabolites.     

Development of an L6 myoblast in vitro model of moniliformin toxicosis

L6 myoblasts were used as an in vitro model to investigate the role of moniliformin and its interaction with monensin in turkey knockdown syndrome and sudden death syndromes in poultry. Cell viability and microscopic and ultrastructural alterations noted in L6 myoblasts cultured in the presence of moniliformin (0.0–0.3 g/l) were compared to those observed in parallel cultures also containing one of the following compounds: selenium (0–0.004 ng/l), thiamine (0–0.3 g/l), or pyruvate (0–0.46 g/l). Marked dilation of the RER, membranous whorls, glycogen deposition, membrane-bound cytoplasmic inclusions and necrosis were observed in myoblasts exposed to 0.03/2-0.30 g moniliformin/l medium. Supplementation of medium with thiamine and pyruvate, or selenium, provided significant protection to cells exposed to 0.0–0.3 g/l or 0.0–0.15 g moniliformin/l, respectively. Dose-dependent differences in protein and ATP production were not detected. Myoblasts grown in medium containing 0–0.15 g moniliformin/l and 7.5–50.0 M A23187, beauvericin or monensin had degrees of cytotoxicity similar to parallel cultures receiving only an ionophore. L6 myoblasts were a useful model of moniliformin toxicosis. The findings of this study suggest cytotoxicity due to moniliformin in L6 myoblasts may be due in part to oxidative damage and altered pyruvate metabolism, and that moniliformin does not predispose myoblasts to ionophore toxicosis. This study supports the results of in vivo investigations in poultry that moniliformin and monensin do not act synergistically to induce knockdown or monensin toxicosis.     

The influence of temperature on glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase and the regulation of these enzymes in a mesophilic and a thermophilic cyanobacterium

The activities and kinetics of the enzymes G6PDH (glucose-6-phosphate dehydrogenase) and 6PGDH (6-phosphogluconate dehydrogenase) from the mesophilic cyanobacterium Synechococcus 6307 and the thermophilic cyanobacterium Synechococcus 6716 are studied in relation to temperature. In Synechococcus 6307 the apparent K _m's are for G6PDH: 80M (substrate) and 20M (NADP⁺); for 6PGDH: 90M (substrate) and 25M (NADP⁺). In Synechococcus 6716 the apparent K _m's are for G6PDH: 550M (substrate) and 30M (NADP⁺); for 6PGDH: 40M (substrate) and 10M (NADP⁺). None of the K _m's is influenced by the growth temperature and only the K _m's of G6PDH for G6P are influenced by the assay temperature in both organisms. The idea that, in general, thermophilic enzymes possess a lower affinity for their substrates and co-enzymes than mesophilic enzymes is challenged.Although ATP, ribulose-1,5-bisphosphate, NADPH and pH can all influence the activities of G6PDH and 6PGDH to a certain extent (without any difference between the mesophilic and the thermophilic strain), they cannot be responsible for the total deactivation of the enzyme activities observed in the light, thus blocking the pentose phosphate pathway.Abbreviations G6PDH glucose-6-phosphate, dehydrogenase - 6PGDH 6-phosphogluconate dehydrogenase - G6P glucose-6-phosphate - 6PG 6-phosphogluconate - RUDP ribulose-1,5-bisphosphate - Tricine N-Tris (hydroxymethyl)-methylglycine     

Effect of abscisic acid and cytokinins on the development of somatic embryos in Hevea brasiliensis

Addition of liquid medium, conditioned by an embryogenic suspension, to MH1 solid medium (3,4-dichlorophenoxyacetic acid 9 M, 6-benzyladenine 9 M) permitted the frequent induction of highly embryogenic calli from slices of internal integument of immature seeds of Hevea brasiliensis Müll. Arg. The proliferation of embryogenic cell clusters was achieved in MH1 liquid medium. Abscisic acid (ABA), cytokinins and adenine were tested for their ability to affect development of somatic embryos to plantlets. The transfer of embryogenic cell clusters on auxin-free solid medium with 10^-5M ABA for 2 months stimulated embryo development. When torpedo-shaped embryos were transferred to medium with adenine or cytokinins they turned green in 1 month. Green embryos produced secondary embryos when they were collected and placed on medium without growth regulators.Abbreviations ABA abscisic acid - BA 6-benzyladenine - IBA indole-3-butyric acid - NOA -naphthoxyacetic acid - 2iP 2-iso-pentenyladenine - 3,4-D 3,4-dichlorophenoxyacetic acid     

Callus and cell suspension cultures of Rudgea jasminoides,a tropical woody Rubiaceae

Rudgea jasminoides is a woody Rubiaceae that produces phytoalexins in response to fungal inoculation, the response being dependent of the seasonal conditions. With the aim of studying phytoalexin induction under controlled conditions, callus cultures were established from petiole explants of R. jasminoides on a modified basal MS medium supplemented with picloram alone or in combination with kinetin. The highest frequency of callus formation was observed in solid medium containing 2.22 M kinetin and 2.07 M picloram. Development of fast-growing friable white callus was achieved in the absence of kinetin, in cultures supplemented only with 8.28 M picloram. Cell suspension cultures were established from this friable callus by transferring pieces directly to the same medium without agar. Preliminary experiments revealed that cell suspension cultures of R. jasminoides represent a useful system to analyse induced defensive metabolites produced by this Rubiaceae species.