Effects of Carbon Source, Gibberellins and Ethylene on Wall-Bound Invertase Activity in Callus of Convolvulus arvensis

The growth rate of Convolvulus callus on a sucrose-containing medium appeared to be largely independent of the activity of cell wall invertase. The increase of this enzyme activity which occurs upon subculturing was unaffected within the first 24 h by the presence of the substrate sucrose. Neither substitution by glucose or fructose, nor complete deletion of the carbon source had any effect. Gibberellins apparently were not involved in the initiation and/or control of the increase of wall-bound invertase activity occurring upon subculturing. Exogenous ethylene was unable to mimick this effect of subculturing but when applied immediately after subculturing it had a synergistic effect on the increase of invertase. Inhibition occurred, however, when exposure to ethylene was delayed until after 24 h of incubation. These findings suggest a role of ethylene in the control of wall-bound invertase activity.     

Solasodine production from cell culture of <Emphasis Type="Italic">Solanum hainanense</Emphasis> Hance

Stem explants of Solanum hainanense Hance plantlets were cultured on Murashige and Skoog solid medium, containing 3% (w/v) sucrose, supplemented with 0.1 mg/L benzylaminopurine (BAP) and 1.0 mg/L 2, 4-dichlorophenoxyacetic acid (2, 4-D) for callus production. To establish the cell suspension culture, 3 g of fresh callus were cultured in 50 mL of the same medium, but without a solid agent, at an agitation speed of 120 rpm. Every 15 mL of culture was sub-cultured in fresh MS liquid medium for maintenance. The cell biomass of S. hainanense reached a maximum value of 18.47 g after 4 weeks of culture on the same MS medium, but with the sucrose content increased to 4%, at an agitation speed of 150 rpm, with 20 mL of inoculum. Analysis via high performance liquid chromatography (HPLC) showed that the solasodine content in the cell suspension after 4-weeks old (121.01 mg/g) was higher than that of in planta 1-year old roots (20.52 mg/g) by approximately 6-fold.     

Wall-bound enzymes in callus of Convolvulus arvensis

Isolated cell walls of Convolvulus callus contain α- and β-galactosidase, α- and β-glucosidase, α- and β-mannosidase, acid invertase and acid phosphatase activities. No neutral invertase or alkaline phosphatase activities could be detected. Acid invertase activity per mg cell wall increased considerably during incubation of callus fragments in nutrient solution, as opposed to the activities of the other enzymes mentioned.     

Characterization of wall-bound invertase isoforms of Picea abies cells and regulation by ectomycorrhizal fungi

In culture, the ectomycorrhiza-forming fungi Amanita muscaria (Pers. ex Fries) Hock. and Hebeloma crustuliniforme (Bull. ex Fries) Quel. only grow on media with glucose or fructose but not with sucrose as sole carbohydrate source. This is due to their lack of wall-bound invertase activity. Therefore, utilization of sucrose by the fungi within a mycorrhizal association is believed to depend on the wall-bound invertase activity of the host. This enzyme activity was studied in the apoplast of suspension cultured cells of Picea abies (L.) Karst. An ionically and a tightly wall-bound isoform of acid invertase were found that function as β-d -fructofuranoside-fructohydrolases (EC 3.2.1.26). The ionically bound enzyme could be easily released from walls of intact cells with buffer of high ionic strength. In its native form, the ionically bound invertase isoform is a monomeric protein with a molecular mass of 61 kDa, as determined by gel filtration and SDS-PAGE. Glycoprotein nature of the enzyme was demonstrated with antibodies directed against the digoxigenin-labeled protein. The K_m values of both enzymes for sucrose, their natural substrate, are relatively high (ionically bound invertase K_m= 16 mM, tightly bound invertase K_m= 8.6 mM). Activity of both wall-bound invertase isoforms strongly depends on the apoplastic pH. They have a narrow pH-optimum and exhibit highest activity at pH 4.5. with elevated activity between pH 4.5 and 6.0. Furthermore, fructose acts as competitive inhibitor of both isoforms, whereas glucose is not inhibitory. Unloading of sucrose from host cells to the apoplastic interface of the Hartig net in ectomycorrhizae appears to depend on the rate of hydrolysis by the wall-bound invertase of the host. Since the activity of the plant invertase depends on the actual pH value and the fructose concentration in the mycorrhizal interface, we suggest that the fungus can actively influence the activity of the plant invertase by acidification of the cell wall and by fructose uptake. Thus, the fungus itself can regulate its own supply of glucose and fructose.     

Effect of liquid pulses with 6-benzyladenine on the induction of somatic embryogenesis from coffee (Coffea arabica L.) callus cultures

We investigated the effect of the physical state of the nutrient medium on the induction of somatic embryogenesis on cell cultures derived from coffee (Coffea arabica L.). Non-embryogenic callus tissues were pulsed initially with 50 μM 6-benzyladenine (BA) for 6, 24 or 48 h in half-strength liquid Murashige and Skoog (MS) medium. After pretreatment, calli were transferred to agar-solidified half-strength MS medium supplemented with 50 μM BA (‘standard induction medium’). Control callus tissues were incubated directly on the solid standard induction medium. Callus growth was promoted by longer pretreatment periods. Formation of globular somatic embryos was observed on callus tissues pretreated with BA for 24 or 48 h, which developed fully to cotyledonary-stage within only 2 weeks after transfer to agar-solidified medium supplemented with BA. No embryo formation occurred in control cultures. Pretreatment with BA in liquid medium was associated with changes in the redox status of cultured cells, such as alterations of the ascorbate–glutathione redox systems and the accumulation of free radicals and oxidized lipids, as well as the possible reduction of cytochrome c-mediated apoptotic pathways. In particular, the induction of somatic embryogenesis was highly positively correlated (r ² = 0.822) with the accumulation of protein carbonyls. The physiological role of BA as an inducer of both embryonic differentiation and cellular death is discussed.     

Induction,maturation and germination of holm oak (Quercus ilex L.) somatic embryos

Cotyledon explants of Panax ginseng produced somatic embryos directly on solid hormone-free MS medium containing 3% (w/v) sucrose while high concentration of NH₄NO₃ (60 mM) induced embryogenic callus. Ten subcultures of the embryogenic callus on hormone-free MS medium with 40 mM NH₄NO₃ gave hormone-independent proliferation of callus, which exhibited proliferation potential even on MS medium with a standard level of NH₄NO₃ (20 mM). Pulse treatment of callus with exogenous auxin or cytokinin (1.0 mg 1^–1 2,4-D, 1.0 mg 1^–1 kinetin) resulted in the loss of the hormone-independent characteristic and caused the callus to brown. For the suspension culture, embryogenic callus was transferred to MS liquid medium containing 3% (w/v) sucrose in an 500 ml Erlenmyer flask. Embryogenic cell clumps in full-strength MS liquid medium discharged toxic substances, resulting in strong suppression of cell growth. In 1/3-strength MS medium, exudation of toxic material did not occur. Embryogenic cell clumps were mass-grown on a large-scale in a bioreactor (20-1), showing a 7.1 increase of fresh weight in 1/3-strength MS medium with 3% (w/ v) sucrose after 5 weeks of culture. Total ginsenoside content of cultured embryogenic cell clumps was low and 6 times below naturally-cultivated ginseng roots.     

Formation of callus and somatic embryos from protoplasts of a commercial hybrid of maize (Zea mays L.)

Summary Protoplasts isolated from a totipotent embryogenic cell suspension culture of Zea mays L. (cultivar Dekalb XL82) underwent sustained cell divisions when cultured in liquid as well as agarose media. Optimal colony formation (5%) occurred in a liquid medium containing 0.5 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D). A soft and unorganized callus was formed when the protocolonies were transferred to agar solidified suspension maintenance medium. Compact, organized and yellow to pale green folded structures and somatic embryos were formed upon subsequent transfer of this callus to a low 2,4-D medium. Clusters of somatic embryos germinated precociously but no plants were recovered.     

Acid and alkaline invertases in suspension cultures of sugar beet cells

Alkaline invertase was induced during the initiation of suspension cultures of single cells from leaf explants of sugar beets in Murashige-Skoog liquid medium which contained benzyladenine. This activity was barely detectable in the leaves themselves. In suspension cultures, the presence of both acid and alkaline invertases was detected; alkaline invertase was only present in the cytoplasm of the cultured cells, whereas acid invertase was present in the cytoplasm and cell walls, and was also detected in the culture medium. The cell wall contained at least three types of acid invertase; two of these activities were solubilized by saline (saline-released) and EDTA (EDTA-released), respectively, and the third remained tightly associated with the cell wall. Saline-released and EDTA-released invertases from the cell wall showed the significant differences in their properties: the saline-released enzyme had the highest affinity for sucrose among the invertases tested, and was easily bound to cell walls, to DNA, and to a cation exchanger, unlike the EDTA-released enzyme. Sucrose is the source of carbon for plant cells in suspension culture and is probably degraded in the cell wall by the saline-released invertase, which had the highest activity and the highest affinity for sucrose. Hexose products of this degradation would be transported to cytoplasm. Soluble invertase, EDTA-released invertase from the cell wall, and one of two extracellular invertases behaved similarly upon chromatography on DEAE-cellulose. They had similar activity profiles with changing pH, and similar K_m values for sucrose. Thus it appears that they are identical. Two extracellular invertases found in the growth medium of the suspension cultures were probably identical with those in the soluble fraction of callus and seedlings of sugar beets, because they showed similar behaviors during chromatography on DEAE-cellulose, and had similar activity profiles with changing pH and K_m values for sucrose.     

Induction of somatic embryos in pea,Pisum sativum L.

Using low concentrations of picloram (0.06 mg/l), embryoids were formed on the surface of leaf-derived callus of pea, Pisum sativum L. (c.v. Dippes Gelbe Victoria) upon transfer to liquid medium. After some days in culture, embryoids spontaneously separated from the calli, and developed into torpedo-shaped embryos, which were transferred to solid medium. In a second series of experiments, embryos were also formed by mutant 489C and a genetic line of Pisum arvense, which additionally exhibited embryogenesis also from epicotyl-derived callus. Some of the embryos showed root formation, but no shoot morphogenesis occurred. In a limited number of cases, an additional root was formed in the apparent shoot apical region after 2–5 days.     

Influence of osmotic stress on the respiration of potato tuber callus

Potato tuber ( Solanum tuberosum L. cv. Bintje) callus shows a decrease in fresh weight and an increase in dry weight upon transfer to nutrient medium supplemented with 0.3 or 0.5 M mannitol. The osmolarity of the intracellular fluid increases simultaneously. Probably mannitol is taken up from the medium till the osmolarity of the tissue is in equilibrium with that of the medium. After osmotic adaptation, on a medium with 0.5 M mannitol, growth is negligible, although the tissue retains its viability.
Respiration increases upon transfer to medium with extra mannitol, especially when expressed on a fresh weight basis. On this basis cytochrome and alternative pathway capacities do not change appreciably. The respiratory increase is exclusively caused by an increased engagement of the alternative pathway. The participation of this pathway in uninhibited respiration increases from about 10 to 90% upon transfer to medium with extra mannitol. The increase in respiration is partly correlated with the decrease in fresh weight upon transfer. Per disc, the capacities of the cytochrome and alternative pathway decline. Yet, total respiration per disc significantly increases due to the increased participation of the alternative pathway. This results in an almost equal ATP-production per disc before and after transfer. We suggest, that the alternative pathway functions as a reserve capacity in potato callus, which is switched on when ATP-production coupled to the cytochrome pathway is impaired.     

Experiments on tissue culture in the genus Lycopersicon miller

Callus cultures from cotyledon explants were established and maintained in culture for more than two years. After several months callus cultures were transferred into liquid medium and cultured as cell suspensions. Protoplasts were isolated from these cell suspension cultures and cultured in a liquid medium. After formation of new cell walls the cells were further cultured in liquid medium and afterwards transferred to an agar-solidified medium to give a vigorously growing callus culture. In the case of the cultivar Lukullus shoots were recovered from callus. All efforts to root these shoots failed and this, in addition to variations in appearence, suggests that the shoots are changed genetically possibly due to the prolonged culture period.Abbreviations IAA Indoleacetic acid - IBA indolebutyric acid - 2,4-D 2,4-dichlorophenoxyacetic acid - BA benzyladenine - GA₃ gibberellic acid - MS Murashige and Skoog (1962) medium - B5 Gamborg et al. (1968) medium - EV Evans et al. (1972) washing solution Dedicated with sincere compliments to Professor Dr.Dr.h.c.mult. Hans Stubbe on the occasion of his 80th birthday     

Asiaticoside production from centella (Centella asiatica L. Urban) cell culture

Petiole explants of centella plants (Centella asiatica L. Urban) were cultured on Murashige and Skoog (MS) solid medium containing 20 g/L sucrose, supplemented with 1.0 mg/L benzylaminopurine and 1.0 mg/L naphthaleneacetic acid for callus production. To establish a cell suspension culture, 2 g of fresh callus was cultured in 50 mL of the same medium but without solid agent at a 100 rpm agitation speed. Every 2 g of culture was subcultured in fresh MS liquid medium for maintenance. After 24 days of culture at a 120 rpm agitation speed, the centella cell biomass reached a maximum of 9.03 g/50 mL on the same MS medium with 30 g/L sucrose and a 3 g inoculum size. A high performance liquid chromatography analysis showed that asiaticoside content in 24-day old suspension cultured cells (45.35 mg/g dry weight) was significantly higher (4.5 fold) than that of in planta leaves (10.55 mg/g dry weight).     

α-Glucosidase Activity Located at the Cell Surface in Callus of Convolvulus arvensis

Cell wall preparations of Convolvulus callus were found to contain α-glucosidase activity, the bulk of which could be solubilized by solutions of high ionic strength. Callus tissue incubated in 0.5 M KC1 released α-glucosidase activity into the washing medium as distinct from tissue incubated in 1.0 M sorbitol. The wall-bound activity of KCl-treated tissue was found to be less than that of sorbitol-treated tissue, while the difference between both activities proved to be equal to the enzyme activity found in the washing medium of KCl-treated tissue. Since no trace of cell leakage was observed, it is concluded that α-glucosidase activity is located at the cell surface. The level of this surface-located enzyme was not affected by the presence of maltose in the nutrient medium.     

Phenylalanine ammonia-lyase activity in callus cultures of Pinus elliotii

Phenylalanine ammonia-lyase (PAL) activity increased 8- to 12-fold in pine (Pinus elliotii Engelm.) callus tissue within 2 days after subculturing on fresh medium. Factors such as increasing the sucrose content of the media, imposing additional tissue in jury or subculturing more frequently did not cause additional stimulation of PAL activity. The rapid increase in PAL activity appeared to be due to enzyme activation, since cycloheximide did not appreciably reduce the stimulation of PAL activity. The subsequent loss of increased PAL activity with age was reduced by cycloheximide and a cool growth environment.     

Ultrastructural characteristics of an achlorophyllous callus line ofLotus corniculatus

Summary An achlorophyllus isoline originated spontaneously from a green callus line derived from a genotype ofLotus corniculatus L. cv. Leo. This isoline is characterized by significantly faster callus growth than the green line and by differentiating significantly fewer plants per gram of callus when subcultured on a shoot differentiating medium. The isoline does, however, develop loci of meristematic cells when transferred from a medium containing 2,4-D to one containing benzyl adenine. After this transfer, plastids, mitochondria, dictyosomes, and endoplasmic reticulum appear to be more prevalent in many cells. Nuclei and nucleoli become more prominent in these cells. Ingrowths, lined with plasmalemma, develop along the outer tangential cell wall of many cells in contact with the culture medium and along various walls of cells found in the interior of the callus. These cells have characteristics of transfer cells. A few cells at the periphery of meristematic groups of cells elaborate lamellar structures similar to suberin lamellae in their cell walls.     

Further studies on the growth and differentiation of single,isolated cells of tobacco in vitro

Summary Single cells of hybrid tobacco callus, isolated from germinating seeds, were grown individually in microchambers in the absence of any other cells, in fresh, liquid, coconut milk-medium. These produced a conlony of 50–75 cells in 10–15 days. In all instances the plane of the first cell division, and in most cases also that of the second division, were at right angles to the long axis of the cell. There was more variation in the size, shape and pattern of the second and subsequent divisions in single cells isolated from fresh stem pith and seed callus than those from old stem callus. Upon transfer from the microchambers to agar medium the colony of cells gave rise to a huge mass of callus tissue in about 3 months. In no instance did the cell colonies or any stages preceding them resemble or simulate any stages of normal embryogeny of tabacco. Single-cell clones obtained by this method from seed callus or fresh pith callus produced shoots with leaves and roots in synthetic medium with various indoleacetic acid-kinetin combinations. Normal plants established from the above cultures in the greenhouse flowered in due course of time. This method offers the possibility of producing a very large number of clones or identical plants in species where vegetative propagation is not otherwise possible, apart from its use in studies on the genetics and morphogenesis in higher plants.     

Effect of culture medium and illumination on the acid invertase activity in callus of Medicago strasseri

The different acid invertase activity (total, soluble, wall-bound and extracellular) in calli induced on explants (cotyledon, petiole, hypocotyl and leaf) originated from Medicago strasseri seedlings were evaluated. In cultures subjected to 16 h photoperiod, the highest total, soluble and extracellular activities were found in calli from leaves cultured in medium 12 (MS with 0.01 mg·dm⁻³ (0.045 μM) of TDZ), elevated amounts of total and wall-bound invertase being found in calli induced on petioles in 12G medium (MS with 0.01 mg·dm⁻³ (0.045 μM) TDZ and 3.104 mg·dm⁻³ glycerol). In cultures maintained in darkness, the activity detected was lower than that observed in cultures under light conditions. The highest amounts of enzyme was bound in calli cultured on medium 12 (total and extracellular invertase) -leaves- and medium 12D (MS with 0.001 mg·dm⁻³ (0.0045 μM) TDZ) (soluble invertase) -using hypocotyls. In general, the different forms of invertase activity studied seem to appear in greatest amounts in calli induced under light conditions using leaves as explant and TDZ as growth regulator.     

Plant regeneration through somatic embryogenesis on Holostemma ada-kodien,a rare medicinal plant

Plant regeneration through indirect somatic embryogenesis has been established on Holostemma ada-kodien Schult. Type of auxin significantly influenced somatic embryogenesis. Friable callus, developed from leaf, internode and root explants on Murashige and Skoog (MS) medium supplemented with 2,4-D (1.0 mg l^–1), was most effective for the induction of somatic embryos. Subculture of the friable callus developed on 2,4-D (1.0 mg l^–1) onto solid or liquid 1/2 MS medium with 0.1 or 0.5 mg l 2,4-D turned the callus embryogenic. Suspension cultures were superior to static cultures (solid medium) for the induction of somatic embryos. Transfer of embryogenic callus to liquid 1/2 or 1/4 MS medium with lower levels of 2,4-D (0.05–0.1 mg l^–1) induced the highest number of somatic embryos. An average of 40 embryos were obtained from 10 mg callus. Fifty per cent embryos exhibited maturation and conversion upon transfer to 1/10 MS basal solid medium. Plantlets were established in field conditions and 90 per cent survived.     

Optimizing embryogenic callus production and plant regeneration from `Tifton 9' bahiagrass seed explants for genetic manipulation

Bahiagrass (Paspalum notatum Flügge) is a warm season forage grass widely cultivated in southeastern U.S. and South America. The cultivar Tifton 9 has several desirable characteristics such as high forage yield, more vigor at the seedling stage, etc.; but its forage quality is very low. As an initial step for future genetic manipulations to improve its forage characteristics, we have optimized in vitro culture conditions for plant regeneration. In this report, we describe an efficient method for embryogenic callus induction and plant regeneration from bahiagrass (cv. Tifton 9) seed explants, which are readily available and easy to manipulate, compared to other explant sources reported in the literature.Murashige and Skoog (MS) medium containing 30 M dicamba and 5 M 6-benzyladenine (BA) was optimal for callus induction and growth. Out of 9734 seeds cultured, 65.7% germinated and 21.4% produced embryogenic callus on this medium. Shoot formation was best when embryogenic calluses induced in this medium were transferred to MS medium supplemented with 5 M BA and 1 M gibberellic acid with 1640 plantlets formed per gram fresh weight of callus tissue. When transferred to hormone-free SH medium, shoot systems produced well-developed root systems. The resulting plantlets grew normally produced viable seeds when transferred to soil in the greenhouse. Histochemical staining for GUS activity arising from transient expression of the introduced uidA (-glucuronidase) gene indicated that bahiagrass embryogenic callus produced by this method is suitable for gene transfer via biolistic bombardment; and it can serve as a good target tissue for future genetic manipulations to improve the forage quality of bahiagrass (cv. Tifton 9).     

Relationship between endogenous auxin and cytokinin levels and morphogenic responses inActinidia deliciosa tissue cultures

Summary Thein vitro culture ofActinidia deliciosa petioles results in a decline of cytokinin content and an increase of auxin levels. The addition of plant growth regulators (PGRs) to the medium lead to recovery of the initial auxin content, and callus induction occurs at the basal end of the explants. Endogenous auxin/cytokinin ratio was higher at this side than in the apical one, due to unequal distribution of endogenous PGRs in the cultured petioles. Some of the induced calluses showed shoot formation when they were transferred to proliferation medium. Most important differences found in hormonal content between organogenic and non-organogenic callus concerned benzyladenine levels. In this paper the relationships between explant behaviour and their hormonal content is discussed.Abbreviations BM basal medium - BA 6-benzyladenine - CIM callus induction medium - CPM callus proliferation medium - (diH)Z dihydrozeatin - DW dry weight - ELISA enzyme linked immunoassay - FW fresh weight - HPLC high performance liquid chromatography - IAA indole-3-acetic acid - iP N⁶-(²-isopentenyl)adenine - NAA 1-naphthaleneacetic acid - PBS phosphate buffer - PGR plant growth regulator - (diH)[9R]Z 9--D-ribofuranosyl-(diH) - [9R]iP 9--Dribofuranosyl-iP - [9R]Z 9--D-ribofuranosylzeatin - TBS trishydroxymethyl-aminomethane buffer - Z zeatin