Effect of protoplast source and media on growth and regenerability of protoplast-derived calluses of Solanum tuberosum L

Freshly isolated mesophyll and suspensions-cell protoplasts of S. tuberosum cvs. Desiree and Maris Piper were cultured in different media i.e. modified MS, V-KM and MS-KM. Protoplast plating efficiencies were higher in MS-KM medium. Resulting protoplast-derived calluses were transferred either onto the medium of Bokelmann and Roest (1983) or that of Lam (1977) for shoot regeneration. Calluses derived from mesophyll cell protoplasts differentiated about 2 weeks earlier than calluses derived from suspension-cell protoplasts. Shoot initiation was also about 2 weeks earlier from calluses subcultured onto the former medium as compared to the latter.     

Changes in calcium and magnesium levels during heat-shock synchronized cell division in Tetrahymena

Calcium and magnesium contents were measured in cells of Tetrahymena pyriformis induced to divide synchronously by a multi-heat-shock procedure. During free-running synchronized cell division in complex proteose peptone medium, significant peaks of both calcium and magnesium were observed at points in the cell cycle just prior to division. No such peaks were detected in cells dividing asynchronously in proteose peptone. When synchronized cell division was followed after transfer to an inorganic medium, cell calcium and magnesium levels were observed to decrease in relation to the corresponding cell number increase, indicating that in concentration terms, calcium and magnesium remain fairly constant. This latter result suggests that neither calcium nor magnesium influxes act as triggers for cell division in Tetrahymena and that the fluctuations of these metals seen during the synchronized division cycle in complex medium represent an effect rather than a cause.     

Morphological and molecular characterization of somatic hybrid plants between Lycopersicon esculentum and Solanum nigrum

Summary Mesophyll protoplasts of tomato (Lycopersicon esculentum Mill. var. cerasiforme) and of an atrazine-resistant biotype of black nightshade, (Solanum nigrum L.), were fused by using polyethylene glycol/dimethyl sulfoxide (PEG/DMSO) solution and three somatic hybrid plants, each derived from a separate callus, were recovered. A twostep selection system was used: (1) protoplast culture medium (modified 8E) in which only tomato protoplasts formed calluses; and (2) regeneration medium (MS2Z) on which only S. nigrum calluses produced shoots. These selective steps were augmented by early isozyme analysis of putative hybrid shoots still in vitro. Phosphoglucoisomerase (PGI) and glutamate oxaloacetate transaminase (GOT) mapped to five loci on four chromosomes in tomato confirmed the hybrid nature of the nuclei of regenerated shoots. The somatic hybrid plants had simple leaves, and intermediate flower and bud morphology, but anthesis was reduced to 5% due to premature bud abscission and the pollen grains were non-viable. Southern DNA blot hybridization using a pea 45 S ribosomal RNA gene probe reconfirmed the hybrid nature of the nuclear genome of the three plants. A ³²P-labeled probe of Oenothera chloroplast DNA (cpDNA) hybridized to cpDNA restricted with EcoRI or EcoRV indicated the presence of the tomato cpDNA pattern in all three hybrids. Likewise, the plants were all found to be atrazine sensitive. Analysis with two mitochondrial (mt)DNA-specific probes, maize cytochrome oxidase subunit II and PmtSylSa8 from Nicotiana sylvestris, showed that, in addition to typical mitochondrial rearrangements, specific bands of both parents were present or missing in each somatic hybrid plant.Michigan Agricultural Experiment Station Journal Article No. 12433     

Cell suspension culture and plant regeneration in the latex-producing plant,Calotropis gigantea (Linn.) R. Br.

A procedure is outlined for the establishment of a proliferating cell suspension culture and subsequent plant regeneration of the latex-producing plant,Calotropis gigantea (Linn.) R. Br. Friable calluses were obtained by culturing hypocotyl explants on modified Murashige and Skoog medium with 2.69 μM α-naphthaleneacetic acid and 4.44 μM 6-benzyl-aminopurine. Friable calluses were transferred to modified Murashige and Skoog liquid medium containing 500 mg l⁻¹ casein hydrolysate, 5% (v/v) coconut water and 5% (w/v) sucrose to initiate suspension cultures. Suspensions were subcultured every 10–12 days and supplemented with 13.56 μM 2,4-dichlorophenoxyacetic acid (2,4-D). After 3–4 subcultures, suspensions consisted of small, highly cytoplasmic cell clumps and large vacuolate cells. Plating the suspension clumps on medium containing 4.52 μM 2,4-dichlorophenoxyacetic acid and culturing in darkness produced macroscopic calluses, which subsequently produced a high number of shoots when placed in light and supplemented with 2.22 μM 6-benzyl-aminopurine and 0.45 μM 2,4-dichlorophenoxyacetic acid. Shoots were rooted using Bonner's solution containing 0.49 μM indole-3-butyric acid, and the plants successfully transferred to soil. This revised version was published online in June 2006 with corrections to the Cover Date.     

The relationship between polyploidy and organogenetic potential in embryo- and root-derived tissue cultures of Vicia faba L.

The cell cycle was examined in embryo and root explants of Vicia faba in culture to test whether or not polyploidy and aneuploidy affected organogenetic potential. Nuclear DNA contents and the mitotic index were measured in the 0–1 mm apical segment of primary roots of 5-day old seedlings and at various times following transfer to modified MS in darkness or Chu's N6 medium in an 8 h light/16h dark cycle (N6-MS programme) at 20°C. Mature embryos were dissected and cut longitudinally. Each half was cultured on the N6-MS programme. Root explants grown on MS in darkness developed into callus but there was no subsequent organogenesis. Only on the N6-MS programme were new roots initiated from root-derived callus. Using the N6-MS programme, embryo-derived callus became green and after 3 to 4 months, produced roots and shoots. Approximately 40% of these cultures regenerated plantlets. Polyploidy occurred within 24 h of culture irrespective of both tissue source and culture protocol. Variations in chromosome number from 2n=2x=12 were also routinely observed. Thus, calluses had the ability to initiate roots and shoots regardless of persistent polyploidy and aneuploidy. Compared with the baseline of cell cycle data for roots in vivo, the proportions of cells in the different cell cycle phases remained constant. Thus, in V. faba induction of organogenesis seems more related to culture protocols than to specific changes to the cell cycle. The mitotic index was significantly lower in vitro compared with meristems of intact roots.     

Optimization of in vitro multiple shoot clump induction and plantlet regeneration of Kentucky bluegrass (Poa pratensis)

An efficient protocol for Kentucky bluegrass (Poa pratensis L.) in vitro culture was established using shoot apices of seedlings as explants. The optimal procedure of this protocol for majority of the genotypes was that meristematic cell clumps and small calluses were firstly induced from the bases of explants on initial culture medium supplemented with 0.9 μM 2,4-d and 8.9 μM 6-BA for 20 d, then were separated and transferred to shoot clumps induction medium containing 8.9 μM 6-BA for the formation of multiple shoot clumps. The percentage of multiple shoot clumps and numbers of shoots per clump were deeply related with the combinations of different plant growth regulators, duration of initial culture, the intensity of illumination and genotypes. Histological observation of the induced explants revealed that the meristematic cell clumps were produced from repeated division of the cortical cells and original meristematic primodium cells of explants, and the multiple shoots were formed via organogenesis pathway in the meristematic cell regions of cultures on shoot clumps induction medium. In this study, plantlets were efficiently regenerated on large scale from seven cultivars of Kentucky bluegrass. Hence the meristematic cell clumps and small calluses in this protocol could be considered good targets for genetic transformation of Kentucky bluegrass.     

Callus formation from protoplasts of Artemisia sphaerocephala Krasch and some factors influencing protoplast division

Round wormwood (Artemisia sphaerocephala Krasch) seeds were germinated on Murashige & Skoog (1962) medium without plant growth regulators. The hypocotyls of seedlings were sliced and cultured on M1 medium with 2,4-dichlorophenoxyacetic acid (9.05 M) to induce callus. The induced calluses were subcultured on the same medium. Ten day old calluses were used to isolate protoplasts in an enzyme solution with 0.65 M mannitol. Protoplast yield strongly depended upon the state of callus cultures. Certain amount of hemicellulase could improve protoplast isolation. Purified protoplasts were cultured in modified Kao & Michayluk (1975) medium with 0.60 M mannitol as osmoticum, suggesting that protoplasts of A. sphaerocephala need a high initial osmolarity. Protoplasts generally divided evenly and the percentage of first division could reach 10%. Kinetin exhibited a positive effect on initial cell division. Furthermore, we studied the effect of protoplast density and vitamin C on sustained growth of protoplasts. After forty days, 1 mm calluses in diameter formed.Abbreviations CH casein hydrolysate - 2,4-D 2,4-dichlorophenoxyacetic acid - KM8P Kao & Michayluk (1975) protoplast medium - MS Murashige & Skoog (1962) medium - MES-2 (N-morpholino)ethanesulfonic acid     

Neo-formation of flower buds and other morphogenetic responses in tissue cultures of Melia azedarach

Melia azedarach has great interest because of its insecticidal properties. Recently, the occurrence of precocious flowering in tissue cultures of this species was reported. This paper describes some in vitro morphogenetic responses using hypocotyl segments as explants and MS basal medium. Amongst the results we report are: (a) in basal medium, 5% of the explants neo-formed floral buds and flowers, and 80% formed vegetative shoots; (b) flower neo-formation could not be controlled or increased by addition of benzyladenine, or lowering the nitrogen level; (c) benzyladenine increased the regeneration of vegetative shoots; (d) compact green calluses were eventually formed in basal medium, and vigorous friable calluses can be easily induced with 0.5 mM 2,4-D; (e) green calluses could be subcultured and regenerated into plants, and, from friable calluses, cell suspensions were started; (f) histological studies showed that neo-formations originate in the wound tissue or from the inner tissue of the hypocotyl.     

Inherited chilling tolerance in somatic hybrids of transgenic Hibiscus rosa-sinensis x transgenic Lavatera thuringiaca selected by double-antibiotic resistance

Summary Improvement of Hibiscus rosa-sinensis for increased frost tolerance has been attempted through somatic hybridization with the frost tolerant Lavatera thuringiaca. Cell suspensions from Hibiscus and Lavatera were transformed with A. tumefaciens harboring plasmids containing selectable genes coding for kanamycin and hygromycin resistance, respectively. We provided evidence that H. rosa-sinensis and L. thuringiaca were transformed by strong selection of transformed calluses in medium containing antibiotics, by GUS activity determination in protein extracts and by molecular confirmation of chromosomal integration and expression of the selectable genes. Protoplasts isolated from a kanamycinresistant Hibiscus callus and from a hygromycin-resistant Lavatera callus were fused and selected in medium containing both antibiotics. We determined unambiguously that the regenerated double-antibiotic resistant clones obtained are indeed somatic hybrids through analysis of acid phosphatase zymograms and nuclear DNA content. Plant regeneration through somatic embryogenesis was accomplished from both isolated protoplasts and transgenic calluses of L. thuringiaca. However, regeneration from the double-antibiotic resistant fusant calluses was unsuccessful. Analysis of the somatic hybrids at the callus level showed that chilling and freezing tolerance are governed by independent genetic components. The somatic hybrids displayed significant improvement for chilling tolerance at conditions lethal to H. rosa-sinensis, although frost tolerance was not expressed.     

Proliferation and maintenance of embryogenic capacity in elm embryogenic cultures

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

Transgenic sweet potato plants obtained byAgrobacterium tumefaciens-mediated transformation

Stable expression of foreign genes was achieved in sweet potato (Ipomoea batatas (L.) Lam) plants using anAgrobacterium tumefaciens mediated system. Embryogenic calluses produced from apical meristems of cultivar White Star were multiplied and cocultivated withA. tumefaciens strain EHA101 harboring a binary vector containing the -glucuronidase (GUS) and neomycin phosphotransferase (NPT II) genes. The calluses were transferred to selective regeneration medium and kanamycin resistant embryos were recovered which developed into morphologically normal plants. Histochemical and fluorimetric GUS assays of plants developed from the kanamycin resistant embryos were positive. Amplified DNA fragments were produced in polymerase chain reactions using GUS-specific primers and DNA from these plants. Transformation was confirmed by Southern analysis of the GUS gene. With the developed method, transgenic sweet potato plants were obtained within 7 weeks. This method will allow genetic improvement of this crop by the introduction of agronomically important genes.Florida Agricultural Experiment Station Journal Series N-02231. This research was partially supported by CNP_q/RHAE (Brazil).     

Somatic embryogenesis and plant regeneration in zygotic embryo cultures of balloon flower

Mature zygotic embryos of balloon flower (Platycodon grandiflorum) formed embryogenic calluses at a frequency of 43% when cultured on Murashige and Skoog medium supplemented with 4.52 μM 2,4-dichlorophenoxyacetic acid (2,4-D). Cell suspension cultures were established from embryogenic calluses using MS liquid medium with 4.52 μM 2,4-D. Following transfer to solid MS basal medium, cell suspension cultures gave rise to somatic embryos, which then developed into plantlets. Plantlets were transplanted to potting soil and grown to maturity. This revised version was published online in June 2006 with corrections to the Cover Date.     

Extractability of polyhydroxyalkanoate synthesized by <Emphasis Type="Italic">Bacillus flexus</Emphasis> cultivated in organic and inorganic nutrient media

Bacillus flexus was isolated from local soil sample and identified by molecular methods. In inorganic nutrient medium (IM) containing sucrose as carbon source, yield of biomass and polyhydroxyalkanoate (PHA) were 2 g/l and 1 g/l (50% of biomass), respectively. Substitution of inorganic nitrogen by peptone, yeast extract or beef extract resulted in biomass yields of 4.1, 3.9 and 1.6 g/l, respectively. Corresponding yields of PHA in biomass was 30%, 40% and 44%. Cells subjected to change in nutrient condition from organic to inorganic, lacked diaminopimelic acid in the cell wall and the concentration of amino acids also decreased. Under these conditions the extractability of the polymer from the cells by hot chloroform or mild alkali hydrolysis was 86–100% compared to those grown in yeast extract or peptone (32–56%). The results demonstrated that growth, PHA production and the composition of cell wall of B. flexus are influenced by the organic or inorganic nutrients present in the growth medium. Cells grown in inorganic medium lysed easily and this can be further exploited for easier recovery of the intracellular PHA.     

Phytase production by high cell density culture of recombinant Bacillus subtilis

Bacillus subtilis BD170, harboring a plasmid pGT44[phyC] carrying the phytase gene (phyC) and a phosphate-depletion inducible pst-promoter, was grown in a 2 l bioreactor. Using a controlled feeding of glucose, high cell densities of 32 and 56 g dry cell weight l^–1 were achieved with peptone and yeast extract, respectively, as the complex nitrogen sources in a semi-defined growth medium. The fed-batch protocol was applied to production of recombinant phytase and a high extracellular phytase activity (48 U ml^–1) was reached with peptone. Although the yeast extract feeding resulted in a higher cell density, it was unsuitable as a medium component for phytase expression due to its relatively high phosphate content.     

Transformation of recalcitrant turfgrass cultivars through improvement of tissue culture and selection regime

Tissue culture techniques, medium composition, pH value and targeted tissues, agroinfection and co-culture conditions, selection process were optimized for efficient turfgrass transformation. A highly regenerable callus lines were produced in callus induction medium modified from N6 basal medium. Six-week-old calluses were cultured on Pre-regeneration medium I for 4 days and then subjected to Agrobacterium tumefaciens. After co-cultivation at 20±1 °C in a 16 h light/8 h darkness for 3 days, the calluses were cultured on non-selective Pre-regeneration medium II supplemented with 400 mg l⁻¹ l-cysteine for 7 days. Plantlets were regenerated on the Regeneration medium without selection pressure. A selection pressure was given to the regenerated plantlets when they were rooted on the Plantlet rooting medium. Roots appeared within 8–12 days in putative transformed plantlets. Resistant plants obtained were phenotypically normal and fully fertile. Chemical and molecular analyses confirmed that foreign genes were successfully introduced into the genome of perennial ryegrass or tall fescue. The transformation efficiency can attain 23.3% in perennial ryegrass.     

<Emphasis Type="Italic">Organogenic callus</Emphasis> as the target for plant regeneration and transformation via <Emphasis Type="Italic">Agrobacterium</Emphasis> in soybean (<Emphasis Type="Italic">Glycine max</Emphasis> (L.) Merr.)

A regeneration and transformation system has been developed using organogenic calluses derived from soybean axillary nodes as the starting explants. Leaf-node or cotyledonary-node explants were prepared from 7 to 8-d-old seedlings. Callus was induced on medium containing either Murashige and Skoog (MS) salts or modified Finer and Nagasawa (FNL) salts and B5 vitamins with various concentrations of benzylamino purine (BA) and thidiazuron (TDZ). The combination of BA and TDZ had a synergistic effect on callus induction. Shoot differentiation from the callus occurred once the callus was transferred to medium containing a low concentration of BA. Subsequently, shoots were elongated on medium containing indole-3-acetic acid (IAA), zeatin riboside, and gibberellic acid (GA). Plant regeneration from callus occurred 90 ∼ 120 d after the callus was cultured on shoot induction medium. Both the primary callus and the proliferated callus were used as explants for Agrobacterium-mediated transformation. The calluses were inoculated with A. tumefaciens harboring a binary vector with the bar gene as the selectable marker gene and the gusINT gene for GUS expression. Usually 60–100% of the callus showed transient GUS expression 5 d after inoculation. Infected calluses were then selected on media amended with various concentrations of glufosinate. Transgenic soybean plants have been regenerated and established in the greenhouse. GUS expression was exhibited in various tissues and plant organs, including leaf, stem, and roots. Southern and T₁ plant segregation analysis of transgenic events showed that transgenes were integrated into the soybean genome with a copy number ranging from 1–5 copies.     

Synthesis and Secretion of Proteinases by Bacillus intermedius in the Late Stages of Sporulation

In the late stages of sporulation, cells of Bacillus intermedius 3-19 secreted into the medium two proteinases, glutamyl endopeptidase and subtilisin, whose maximum activities were recorded in the 40th and 44th hours of growth, respectively. By estimating -galactosidase activity as a marker of cytoplasmic membrane integrity, it was revealed that the accumulation of these proteinases in the medium was a result of their secretion and not of lysis of the cell envelope. Concentrations of peptone and inorganic phosphate ensuring the maximum production of the enzymes were established. Ammonium ions were shown to inhibit the production of proteinases by the mechanism of repression by nitrogen metabolites.     

Organogenesis and somatic embryogenesis from callus cultures in Muscari armeniacum Leichtl. ex Bak.

Summary Establishment of fast-growing, highly regenerable callus cultures was examined in Muscari armeniacum Leichtl. ex Bak. in order to develop an efficient genetic transformation system. High-frequency callus formation was obtained from leaf explants of cv. Blue Pearl on media containing 2,4-dichlorophenoxyacetic acid (2,4-D), α-naphthaleneacetic acid (NAA) or 4-amino-3,5,6-trichloropicolinic acid (picloram, PIC). Fast-growing, yellowish nodular callus lines and white friable callus lines containing a few somatic embryos were established on initiation medium supplemented with 4.5 μM 2,4-D and with 54 μM NAA, respectively. The yellowish nodular calluses vigorously produced shoot buds after transfer to media containing 0.44–44 μM 6-benzyladenine (BA), whereas the white friable calluses produced numerous somatic embryos upon transfer to plant growth regulator-free (PGR-F) medium. Histological observation of shoot buds and somatic embryos indicated that the former consisted of an apparent shoot meristem and several leaf primordia, and the latter had two distinct meristematic regions, corresponding to shoot and root meristems. Both shoot buds and somatic embryos developed into complete plantlets on PGR-F medium. Regenerated plants showed no observable morphological alterations. High proliferation and regeneration ability of these calluses, were maintained for over 2 yr.     

<Emphasis Type="Italic">In vitro</Emphasis> selection and regeneration of chrysanthemum (<Emphasis Type="Italic">Dendranthema grandiflorum</Emphasis> Tzelev) plants resistant to culture filtrate of <Emphasis Type="Italic">Septoria obesa</Emphasis> Syd

Callus cultures derived from leaf segments of chrysanthemum cultivar ‘Snow Ball’ which was susceptible to Septoria obesa were successfully used for in vitro selection for resistance to this pathogenic fungus. Resistant cell lines were selected by culturing callus on growth medium containing various concentrations of S. obesa filtrate. Resistant calluses obtained after two cycles (30 d each cycle) of selection were used for plant regeneration. About 30% of the plants regenerated from the resistant calluses and 70–80% of the plants raised from cuttings had acquired considerable resistance against the pathogen in the field. No phenotypic variation was observed in the selected regenerates.     

Efficient plant regeneration from leaves of rapeseed (Brassica napus L.): the influence of AgNO3 and genotype

Factors influencing reliable shoot regeneration from leaf explants of rapeseed (Brassica napus L.) were examined. Addition of AgNO₃ to callus induction medium was significantly effective for shoot regeneration in all three genotypes initially tested. When 48 genotypes subsequently were surveyed, a large variation of shoot regenerability was observed, ranging from 100 to 0% in frequency of bud formation and from 7.5 to 0 in the number of buds per explant. A significant correlation (r=0.84) was observed between the frequency of bud formation and the number of buds per explant. The shoot regenerability from leaf explants was not related to that from cotyledonary explants (r=0.28). Histological observations showed that an organized structure developed from calluses produced at vascular bundle tissues after 7 days of culture on callus induction medium, and they developed shoot apical meristems one week after transfer onto shoot induction medium. Regenerated plantlets were obtained 2 months after the initiation of culture and they normally flowered and set seeds. No alterations of morphology or DNA contents were observed in regenerated plants and their S1 progenies.