Regeneration of haploid and dihaploid plants from protoplasts of supersweet (sh2sh2) corn

Summary Plants were regenerated from maize (Zea mays L.) protoplasts isolated from embryogenic cell suspensions. The donor maize suspension cultures were established from friable callus initiated from microspores of a commercial supersweet hybrid (sh2sh2). The frequency of cell colony formation was higher when protoplasts were cultured on feeder layers of maize cells as compared with a liquid thin layer method. It was demonstrated that haploid and dihaploid soil-grown plants can be regenerated from maize protoplasts isolated from haploid cell cultures.     

Plant regeneration from protoplast-derived callus of rice (Oryza sativa L.)

Protoplasts isolated from cultured rice cells of an A-58 cytoplasmic male sterile line (A-58 MS)(Oryza sativa L.) were used to investigate the regeneration of rice plants. A cultured cell line (T₃) of A-58 MS with a high growth rate and dense cytoplasm was selected. About 10% of the protoplasts prepared from this established cell line plated in RY-2 (a new medium) formed colonies. The calli formed shoots and roots in the regeneration medium and developed into whole plants.Protoplasts also were prepared from suspension cultures of 25 other varieties of rice using the same methods. The protoplasts isolated from two of the 25 varieties, Fujiminori and Toyotama, had high rates of cell division in RY-2 medium. Only protoplastderived calli from Fujiminori, produced whole plants in the regeneration medium.Abbreviations LS Linsmaier and Skoog (1965) - 2,4-D 2,4-dichlorophenoxyacetic acid - BA 6-benzyladenine - MES 2-(N-Morpholino)ethanesulfonic acid, monohydrate     

Effect of NaCl on lipid content of plasma membranes isolated from roots and cell suspension cultures of the dicot halophyte Kosteletzkya virginica (L.) Presl.

Lipid concent was examined in plasma membrane fractions isolated by discontinuous sucrose density-gradient centrifugation from both salinized and unsalinized roots and cell suspension cultures of Kosteletzkva virginica (L.) Presl., seashore mallow, a halophytic dicot. The distribution of marker enzymes along the gradient indicated that plasma membranes of roots and cell cultures accumulated primarily at the 34%/45% interface. Total sterol and phospholipid content increased significantly in plants and cell suspensions grown on salinized nutrient media. In addition, K. virginica plasma membranes were constitutively rich in sterols, and a high sterol-to-phospholipid ratio was maintained or elevated under saline conditions. These results are discussed in relation to membrane composition as a mechanism involved in the cellularly based salt tolerance of K. virginica.     

Biotransformation of eugenol by suspension cultures of transgenic crown galls of <Emphasis Type="Italic">Panax quinquefolium</Emphasis> and suspension cultures of <Emphasis Type="Italic">Nicotiana tabacum</Emphasis>

The biocatalytic ability of transgenic crown galls of Panax quinquefolium was evaluated by using eugenol (1) as a substrate and suspension cultures of Nicotiana tabacum as control system. Three biotransformed products, namely: 2-methoxy-4-(2-propenyl)phenyl-O-β-d-glucopyranoside (2, 67.11%), 2-methoxy-4-(2-propenyl)phenyl-O-β-d-glucopyranosyl (6′ → 1″)-β-d-xylopyranoside (3, 2.85%) and methyl eugenol (4, 14.30%) were obtained after 5 days of administration of eugenol to the suspension cultures of transgenic crown galls of P. quinquefolium. In contrast, only one product, compound 2 (15.41%), was obtained in suspension cultures of N. tabacum after 5 days of incubation. The results indicated that the glycosylation ability of transgenic crown galls of P. quinquefolium was much higher than that of the cultured cells of N. tabacum.     

An assessment of the cultural capabilities of protoplasts of some wild species of linum

Protoplasts of several wildLinum species were isolated enzymatically from roots, hypocotyls and cotyledons of seedlings, and also from theirin vitro grown shoots and cell suspension cultures. When cultured all these protoplasts divided to produce callus but only good plant regeneration capability was evident in the case ofLinum lewissii and to a much lesser extent forL. strictum. Only rhizogenesis was observed withL. alpinum, L. narbonense, L. grandiflorum andL. altaicum. The high plant regeneration capacity ofL. lewissii from protoplast -derived tissues ofin vitro shoots and cell suspension cultures makes this species an attractive experimental system for somatic genetic manipulation.Abbreviations BAP benzylaminopurine - NAA -naphthaleneacetic acid - CPW cell and protoplast wash solution - gFW gram fresh weight On leave from Department of Crop Sciences University of Alexandria Egypt     

Effects ofVerticillium albo-atrum culture filtrate on somatic embryogenesis in alfalfa

Cell suspensions derived from young petioles of alfalfa (Medicago sativa L.) were cultured in the presence and absence of aVerticillium albo-atrum culture filtrate (20% v/v) for 6 cycles. The frequency of somatic embryogenesis and the growth rate of the suspension cultures were investigated at each cycle. Somatic embryogenesis in the filtrate-treated cultures declined but was still at a relatively high level after 6 subcultures, compared to controls cultures which virtually lost the capacity for embryo formation in the same period. The decline in the embryogenic capacity of filtrate treated-cultures was accompanied by a six-fold increase in the rate of growth of the cultures.     

Callus formation from protoplasts of a sugarbeet cell suspension culture

Protoplasts of sugarbeet (Beta vulgaris L.) were isolated from cell suspension cultures and cultured in modified PGo medium. Conditions required for the efficient division of the protoplasts were investigated.The optimal combination of phytohormones was found to be 1 mg/l NAA, 0.2 mg/l 2,4-D, 0.5 mg/l zeatin. Protoplast division was also considerably stimulated by the addition of 250 mg/l casein hydrolysate, 200 mg/l yeast extract, and 20% v/v conditioned culture medium to the protoplast culture medium. The highest division rate (up to 35% of the protoplasts) was achieved at a density of 4×10⁴- 1×10⁵ protoplasts/ml. From the colonies callus and suspension cultures were readily obtained.Abbreviations BAP 6-Benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA Indole-3-acetic acid - Kin 6-Furfurylaminopurine (kinetin) - NAA -Naphtalenacetic acid - Zea Zeatin     

Novel biotransformation processes of dihydroartemisinic acid and artemisinic acid to their hydroxylated derivatives by two plant cell culture systems

Novel biotransformation processes of dihydroartemisinic acid (1) and artemisinic acid (2) to their hydroxylated derivatives were investigated using the cell suspension cultures of Catharanthus roseus and Panax quinquefolium crown galls as two biocatalyst systems. Five biotransformation products, 3-α-hydroxydihydroartemisinic acid (3), 3-β-hydroxydihydroartemisinic acid (4), 15-hydroxy-cadin-4-en-12-oic acid (5), 3-α-hydroxyartemisinic acid (6) and 3-β-hydroxyartemisinic acid (7), were isolated by chromatograph methods and identified by the analysis of ¹H NMR, ¹³C NMR, and ESI-MS spectra. Compounds 3–5 were obtained for the first time by biotransformation process. It was also the first time to transform artemisinic acid to yield epimeric 3-hydroxy artemisinic acids in plant cell culture system. The biocatalyst system of C. roseus cell cultures showed a great capacity of regio- and stereo-selective hydroxylation in allyl group of the exogenous substrates. The results also showed that the biocatalyst system of P. quinquefolium crown galls possessed the ability to hydroxylate propenyl group of exogenous substrates in a regio- and substrate-selective manner. Furthermore, the in vitro antitumor activity of the hydroxyl products was evaluated by MTT assay. The result indicated that α-hydroxyl products possessed stronger antitumor activity than β-hydroxyl products against the HepG2 and GLC-82 cell lines.     

The effect of dipicolinic acid on maize tissue culture growth is not solely due to inhibition of lysine biosynthesis

Dipicolinic acid, a known inhibitor of an enzyme (dihydrodipicolinic acid reductase) in the maize (Zea mays L.) lysine biosynthetic pathway, inhibits the growth of maize suspension and callus cultures. Inhibited cultures contain somewhat lower free lysine levels, but the inhibition of suspension culture growth was not reversible with simultaneous addition of L-lysine to the culture medium. It is concluded that dipicolinic acid does not act solely as an analog blocking lysine production. Dipicolinic acid thus appears to be unsuitable as a selection for maize tissue culture mutants with lysine overproduction.Abbreviations FW fresh weight - I₅₀ inhibitor concentration at which cell growth is inhibited by 50% - MS Murashige and Skoog (1962) culture medium - ZM Black Mexican Zea mays suspension culture of Chourey and Zurawski (1981)     

Accumulation of isoflavones and pterocarpan phytoalexins in cell suspension cultures of different cultivars of chickpea (Cicer arietinum)

Cell suspension cultures of chickpea (Cicer arietinum L.) were established from cultivars ILC 3279 and ILC 1929, resistant and susceptible towards the chickpea pathogenic fungus Ascochyta rabiei. The two cell culture lines possess identical growth properties and show high accumulation of the isoflavones biochanin A and formononetin together with their glucoside and malonylglucoside conjugates. The cultures of the two cultivars, however, significantly differ in their accumulation of the phytoalexins medicarpin and maackiain essentially as previously demonstrated for the plant genotypes. Phytoalexin formation was elicited by using yeast extract as an inducing agent.     

Steroidal alkaloids in tissue cultures and regenerated plants of Solanum dulcamara

Callus and cell suspension cultures were established with shoots of the soladulcidine variety of the bittersweet Solanum dulcamara L. Plantlets were regenerated from undifferentiated callus. From mixotrophic callus as well as mixotrophic suspension cultures soladulicidine, solasodine and the corresponding neutral spirostanes tigogenin and diosgenin were isolated and identified by thin layer chromatography and mass spectrometry. Total alkaloid concentrations were about 0.2 mg/g dry weight (callus) and 0.1 mg/g dry weight (green suspension cultures). In the heterotrophic cell line only the neutral sapogenins could be detected. Alkaloid accumulation in callus of Solanum dulcamara could be enhanced by the induction of organogenesis. The shoots of the regenerated plants from the mixotrophic callus contained soladulcidine (1.6 mg/g dry weight) and tigogenin. Thus, in concentration and composition the regenerated plants equalled the source plant.Abbreviations 2.4-D 2.4-dichlorophenoxyacetic acid - NAA naphthylacetic acid - HPLC high performance liquid chromatography - TLC thin layer chromatography     

Cell division and differentiation in protoplasts from cell cultures of Glycine species and leaf tissue of soybean

Protoplasts were isolated from cell cultures of G. soja and G. tabacina, respectively. The isolation procedure employed Percoll for the separation and concentration of protoplasts. The cultured protoplasts formed cells which developed into embryo-like structures. Protoplasts also were isolated from leaf tissue of soybean cv. Williams 82. Upon culture, the protoplasts regenerated cell walls and divided to form cell cultures.Abbreviations 2,4-D 2,4-Dichlorophenoxyacetic acid BA|Benzyladenine - BA Benzyladenine     

Epchrosine — a new indole alkaloid isolated from plant cell cultures of Ochrosia elliptica Labill

From plant cell suspension cultures of Ochrosia elliptica Labill. an indole alkaloid of the apparicine type has been isolated, which is not known to occur in differentiated plants. The structure of the new compound, named epchrosine, was established by UV, MS and high resolution two-dimensional ¹H NMR (COSY and NOESY) as (19R,20R)-epoxyapparicine.     

Extracellular accumulation of high specific-activity peroxidase by cell suspension cultures of cowpea

Cell suspension cultures of cowpea (Vigna sp.) were able to produce extracellular peroxidase. Different growth regulator concentrations induced different peroxidase activity in callus. The crude extracellular medium after four weeks of culture showed higher (6 times) specific peroxidase activity and higher thermo stability than commercial horse-radish peroxidase. The commercial production of peroxidase enzyme from cowpea by utilizing plant cell cultures is discussed.     

A technique for bulk production of cytoplasts and miniprotoplasts from suspension culture-derived protoplasts

Protoplasts isolated from suspension cultures of atrazine resistant black nightshade (Solanum nigrum L.) a weed biotype, were enucleated by centrifugation through a stepwise mannitol/sucrose gradient. Two cytoplast, enucleated subprotoplast, bands were routinely formed: one, a minor band at the 6.4%/18.2% mannitol border containing highly vacuolate cytoplasts with 95%+ enucleation; secondly a major cytoplast band at the 18.2% mannitol/33% sucrose border containing 90%+ enucleated protoplasts in quantities up to 4 million per 50 ml gradient tube. Efficient production of cytoplasts depended on the subculture procedures used for the cell suspensions. Optimal cytoplast yield (44%) occurred for protoplasts isolated three days after subculture. The vigor of the donor suspension cultures as visually monitored had to be controlled in order to obtain consistently high enucleation percentages.Abbreviations CPW Cell and Protoplast Wash Solution - 2,4-D 2,4-dichlorophenoxyacetic acid - DMSO Dimethylsulfoxide - FDA Fluorescein diacetate - MS Murashige and Skoog medium (1962) - UM Uchimiya and Murashige medium (1976)     

Production of chrysanthemic acid and pyrethrins by tissue cultures ofChrysanthemum cinerariaefolium

Tissue cultures ofChrysanthemum cinerariaefolium were established, and then used to study the production of pyrethrin insecticides, and their precursor chrysanthemic acid. Callus cultures and root-differentiated cultures did not contain pyrethrins whereas shoot differentiated callus was found to produce the pyrethrins. Chrysanthemic acid was isolated by extraction from callus cultures, and feeding¹⁴C-labelled chrysanthemic acid to a cell suspension ofC. cinerariaefolium established that the acid accumulates largely as a glucoside ester.Abbreviations MS Murashige and Skoog (1962) medium - 2,4-D 2,4-dichlorophenoxyacetic acid - 1AA Indoleacetic acid - BAP 6-Benzylaminopurine - GC-MS Gas chromatography - mass spectrometry     

Cell wall synthesis and salt (saline) sensitivity in cultured colt cherry (Prunus avium × pseudocerasus) protoplasts

In order to investigate the cellular basis of salt tolerance, Colt cherry (Prunus avium ×pseudocerasus) protoplasts from mesophyll tissues and root cell suspension cultures were cultured in the presence of NaCl, KCl or Na₂SO₄, at normalities of 25, 50, 100 or 200 mN for each salt and with or without 2,6-dichlorobenzonitrile, an inhibitor of cell wall synthesis. Results showed that the acquisition of salt tolerance was concomitant with the onset of cell wall regeneration, with protoplasts exhibiting a greater salt tolerance than cells.Abbreviations DBN 2,6-dichlorobenzonitrile - FDA fluorescein diacetate     

Direct somatic embryogenesis from protoplasts of Citrus mitis Blanco

Protoplasts isolated from embryogenic suspension cultures of Citrus mitis were cultured in a medium without any plant growth substances. Somatic embryos developed directly from protoplasts without an obvious intervening callus phase. As many as 1,800 somatic embryos developed from 4 ml of protoplast suspension (density 2×10⁶/ml) cultured for 35 days. Upon transferring the embryoids to medium with 1 mgl^–1 GA₃, they developed into plant-lets. Rooted plantlets were obtained in 3 months after protoplast isolation.Abbreviations BAP Benzylaminopurine - GA₃ Gibberellic acid - MT Murashige and Tucker medium (1969) - FDA Fluorescein diacetate     

REGENERATION OF LIRIODENDRON TULIPIFERA (FAMILY MAGNOLIACEAE) FROM PROTOPLAST CULTURE

Protoplasts were isolated enzymatically from suspension cultures of two embryogenic lines of yellow-poplar (Liriodendron tulipifera L.) and cultured in droplets of a regeneration medium supplemented with 1 mg/1 2,4-D and 0.25 mg/1 6BA and solidified with agarose. Suspension cultures grown in the light yielded substantial numbers of protoplasts, while insignificant numbers were isolated from cultures grown in the dark. The protoplasts reformed cell walls within three days, and 75 percent of them divided at least once within one week in culture. Embryogenic suspension or callus cultures were regenerated by placing agarose droplets containing protoplast-derived microcalli directly into liquid conditioning medium or onto plates of conditioning medium solidified with agar. Embryogenesis was obtained by transferring the callus to a hormone-free induction medium.     

Biochemical differences between carrot inbreds differing in plant regeneration potential

Cultures derived from domestic carrot (Daucus carota L.) inbreds were found to vary with respect to regeneration potential as measured by the production of somatic embryos in suspension cultures. A number of biochemical parameters previously reported to distinguish embryogenic from non-embryogenic cultures of other species were measured in these carrot cell lines. Ethylene production was found to be inversely related to regeneration potential. The cell line producing the greatest number of somatic embryos exhibited the lowest rate of ethylene biosynthesis, even when grown on 2, 4-D-containing maintenance medium. A specific isozyme of acid phosphatase was associated with embryogenic calli. Proteins visualized by SDS-PAGE did not discriminate between embryo-forming and proliferating calli in all inbreds.