Production of human lactoferrin in transgenic cell suspension cultures of sweet potato

Shoot apical meristem-derived calli were transformed with a hLF cDNA in an attempt to produce human lactoferrin (hLF) in transgenic cell suspension cultures of sweet potato [Ipomoea batatas (L.) Lam.]. Calli were bombarded with tungsten particles coated with the binary vector pLSM1 containing a hLF cDNA under the control of the 35S promoter and the neomycin phosphotransferase gene as a selection marker. Calli were then transferred to Murashige and Skoog (MS) medium supplemented with 4.52 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 100 mg dm⁻³ kanamycin. Kanamycin-resistant calli were selected at four-week intervals and subcultured. Cell suspension cultures were established in liquid MS medium with 4.52 μM 2,4-D. Southern and Northern blot analyses confirmed that hLF cDNA was incorporated into the plant genome and was properly expressed in the cells. ELISA analysis showed that transgenic cells produced hLF up to 3.2 μg mg⁻¹ (total protein).     

Induction of umbelliferone in sweet potato cell suspension culture using mercuric chloride

HgCl₂ was used at up to 10 mg l^–1 as an elicitor of phytoalexins in sweet potato (Ipomoea batatas (L.) Lam. cv Centennial) cell suspension cultures. Maximum stimulation of a coumarin compound was after one day of exposure using 1 mg HgCl₂ l^–1. The compound was identified by HPLC and GC-MS analyses as 7-hydroxycoumarin (umbelliferone).     

Solanidine metabolism in potato tuber tissue slices and cell suspension cultures

Two metabolites have been isolated from potato tuber tissue slices or cell suspension cultures that have been incubated with labeled solanidine. Initially glucosyl solanidine is formed which subsequently is glycosylated to a diglucosyl solanidine.     

Occurrence,location and development of anthocyanoplasts

Intensely pigmented organelles (anthocyanoplasts) have been found in anthocyanin-producing cells of more than 70 species representing at least 33 families of angiosperms. When fully developed these structures are typically spherical and normally only one is present in each pigmenented cell. The development of the anthocyanoplast has been studied in both light and dark-grown red cabbage seedlings and the location of the mature organelle has been shown, by the use of isolated protoplasts and vacuoles, to be within the main cell vacuole. Evidence is presented which suggests that the anthocyanoplast is membrane-bounded and that it is the site of anthocyanin biosynthesis.     

The effect of water stress on the vacuole-extravacuole compartmentation of proline in potato cell suspension cultures

Solute compartmentation in cells is an important component of metabolic regulation. There is only little information on how stress treatment of cells effects this component. Therefore, the effect of water stress [10% (w/v) PEG 6000] on the vacuolar-extravacuolar proline compartmentation was studied in a cell suspension culture of Svlanum tuberosum L, cv, HH258, In non-stressed cells 34% of the total cellular proline was located in the vacuole. After 20 h of water stress the proline pool of the cells was increased 4-6 fold and only t6% of it was found in the vacuole. A negative correlation between the total cellular proline content and its percentage in the vacuole was observed, irrespective of the culture method (stress or non-stress culture). The stress-induced changes in proline compartmentation are discussed.     

Aspects of resistance to sweet potato virus disease in sweet potato

In field trials during the first and the second rainy season of 1996 in Uganda, whiteflies were similarly abundant and aphids were absent on three clones of sweet potato (NIS-93–63, cv. Tanzania and cv. New Kawogo) although the three clones differed considerably in their resistance to sweet potato virus disease (SPVD), a complex disease resulting from infection by both the aphid-borne sweet potato feathery mottle virus (SPFMV) and the whitefly-borne sweet potato chlorotic stunt virus (SPCSV). This suggests that vector resistance does not determine the relative SPVD resistance of these genotypes. SPFMV alone had only a low virus titre in sweet potato cvs Tanzania and New Kawogo, became increasingly difficult to detect in plants of these cultivars and was seldom acquired by aphids. However, this resistance to SPFMV was not apparent in plants which were also infected with SPCSV. Plants then had a high SPFMV titre, appeared unable to eliminate SPFMV and provided good sources for aphids to acquire it.     

Biosynthesis of furano-terpenes by sweet potato cell culture

Callus was induced from sweet potato root tissue on an agarmedium containing Heller's minerals, vitamins, 2,4-D, yeastextract and sucrose. Furano-terpenes were scarcely detectedin the callus. However, when the callus was transferred to aliquid culture medium and incubated with reciprocal shaking,furano-terpenes were rapidly produced mainly in the culturemedium. Furano-terpene production by the cell culture was suppressedby addition of Ceratocystis fimbriata spores or HgCl₂ to theculture medium. Yeast extract and sucrose in the culture mediumwere important for furano-terpene production. 3-Hydroxy-3-methylglutarylcoenzyme A (HMG-CoA) reductase activity increased in the cells,followed by the production of furano-terpenes. The TLC patternof furano-terpenes produced by the cell culture was essentiallythe same as that produced by sweet potato root tissue infectedby C. fimbriata or treated with HgCl₂, but the quantitativeproportion of the individual furano-terpenes in the former differedmarkedly from that in the latter. (Received January 11, 1979; )     

Frost tolerance in cell cultures of potato

Different methods of freezing and of estimating frost damage in cell cultures of Solanum tuberosum L. and a number of wild Solanum species were compared. Frost-killing temperatures (FKT, i.e. the temperature resulting in 50% of the maximum possible frost damage) in leaves of these species were -6°C ( S. acaule ), 5°C ( S. me-gistacrotobum ), -4.5°C ( S. commersonii ) and -3°C ( S. polytrichon and S. tubero-sum ) No appreciable species differences were found in FKT when cells were submerged in either buffer or medium and frozen. However, differences did exist when cells were frozen in a non-submerged condition: S. acaule and S. commersonii callus were more sensitive to frost than suspensions, whereas suspensions of the other species were the most sensitive. Measurement of freezing damage by either electrolyte leakage or by 2,3,5-triphenyltetrazolium chloride viability assays revealed similar FKT values. Cell cultures of S. acaule showed better frost tolerance than S. tuberosum (FKT values were -4.5 to -6°C and -2.5°C. respectively), however, frost tolerance of S. megistacrolobum and S. commersonii was only poorly expressed at the cell level (FKT values were between -2 and -3°C). Variant cell lines previously selected for resistance to the amino acid analogues hydroxyprotine, aminoethylcysteine and 5-methyhryptophan appeared to be more tolerant to frost than the wild type S. tuberosum clone.     

Multiphasic uptake systems in cell suspension cultures

The concentration dependent uptake of copper by cell suspension cultures of bush beans could be shown to be multiphasic. Whereas uptake periods of 1 and 4 h resulted in triphasic patterns, the third phase disappeared when the uptake period was extended to 24 h.     

Paclitaxel production in suspension cell cultures of Taxus

Five separate cell lines, three of Taxus canadensis Marsh. and two of Taxus cuspidata Sieb. et Zucc., were used to test the effect of carbohydrates and plant growth regulators on the growth of cells and production of paclitaxel in culture. There was no significant correlation between growth of cells and paclitaxel production. While no single medium was developed that was optimal for all cell lines, it was possible to develop a medium for each species that represented a superior combination of growth and paclitaxel production. A combination of NAA and thidiazuron produced the best combination of growth and paclitaxel production in cell lines of T. canadensis, while IAA and BA produced the best results in cell lines of T. cuspidata. A mixture of sucrose and fructose gave the best combination of growth and paclitaxel production. The addition of carbohydrates midway through the growth cycle increased the rate at which paclitaxel accumulated in the culture medium. The highest paclitaxel concentration obtained was 14.78±0.86 mg 1^–1 (n=3).Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - 2ip 6-(,-dimethylamino)-purine - BA 6-benzyladenine - IAA indole-3-acetic acid - IBA indole-3-butyric acid - kinetin 6-furfurylaminopurine - NAA -napthaleneacetic acid - picloram 4-amino-3,5,6-trichloropicolinic acid - thidiazuron 1-phenyl-3 (1,2,3-thiadiazol-5-yl)urea     

Anthraquinones in cell suspension cultures of Morinda citrifolia

From cell suspension cultures of Morinda citrifolia five known anthraquinones, rubiadin, lucidin, morindone, lucidin-3-β-primeveroside and morindone-6-β-primeveroside, and seven new anthraquinones were isolated. Six of the seven new quinones were characterized as 2-methyl-3,5,6-trihydroxyanthraquinone, 3-hydroxymorindone, 5,6-dihydroxylucidin, 2-methyl-3,5,6-trihydroxyanthraquinone-6-β-primeveroside, 3-hydroxymorindone-6-β-primeveroside and 5,6-dihydroxylucidin-3-β-primeveroside, respectively.     

Biosynthesis of cytokinins by potato cell cultures

Potato cells grown in liquid culture incorporated mevalonic acid lactone-[2-¹⁴C] into free cytokinin (zeatin riboside and zeatin and the cytokinin of RNA (zeatin riboside). The cytokinin liberated by catabolism of RNA can account for no more than 40% of the free cytokinins.     

Cryopreservation of photosynthetic plant cell suspension cultures

Attempts were made to cryopreserve in liquid nitrogen six different photomixotrophic suspension cultured lines of five different species:Amaranthus powellii Wats.,Datura innoxia Mill.,Glycine max (L.) Merr.,Gossypium hirsutum L. andNicotiana tabacum xNicotiana glutinosa L. fusion hybrid. Only theD. innoxia line, DAT, and theG. max line, SB1, could be successfully recovered as viable, growing, dark green cultures. The successful method utilized a preculture treatment of from 2 to 8 days in a medium containing 3% starch and 3% sorbitol for DAT and 3% sucrose and 3% sorbitol for SB1 cells. The cells survived if frozen with 10% dimethylsulfoxide (DMSO) and 9.1% sorbitol or with 10% DMSO and 8% sucrose. Following a programmed slow-cooling, the cells were thawed in a 40° C bath and could be recovered directly when added to fresh liquid medium. Cryostorage of these lines will save labor and prevent further genetic changes from occurring in these unique suspension cultures.     

Transformation of sugar beet cell suspension cultures

Summary A sugar beet transformation method was developed using particle bombardment of short-term suspension cultures of a breeding line FC607. Highly embryogenic suspension cultures derived from leaf callus were bombarded with the uidA (gusA) reporter gene under the control of either the osmotin or proteinase inhibitor II gene promoter, and the npt II selectable marker gene. Transient uidA expression was visualized as 500–4000 blue units per 200 mg of bombarded cells 2 d after bombardment. Stably-transformed calluses were recovered on both kanamycin and paromomycin media. The greatest number of GUS (+) calluses was obtained when 50 or 100 mgl⁻¹ of kanamycin was applied 2 d after transformation for 3–5 wk, followed by either no selection or reduced levels of the antibiotic. PCR analyses of the GUS (+) callus lines revealed the expected size fragment for uidA and npt II genes. Stable incorporation of the uidA gene into the genome was confirmed by Southern blot analyses. Several transformed embryos were detected by histochemical β-glucuronidase (GUS) staining.     

NaCl-resistant variant cells isolated from sweet potato cell suspensions

Salt-resistant cells of sweet potato (Ipomoea batatas L.) were selected by subculturing cell suspensions (11 transfers at 15-day intervals) in MS medium supplemented with 1% NaCl (170.9 mM NaCl).Selected cells showed a brownish pigmentation, and exhibited morphological changes (they were smaller and rounder than non-selected cells). The change in coloration was reversible when the selected cells were subcultured in medium without NaCl. The reduction in size was partially reversed but the change in form was not reversible when selected cells were subcultured 5 times at 15-day intervals in the absence of NaCl.Selected cells exhibited NaCl-tolerance when they were cultured in medium with 1% NaCl and subsequently transferred to NaCl free medium for 3 passages. This finding suggests that the acquired trait is stable for at least 3 passages.     

Temporary chromosome number stabilization in potato cell cultures

Cell cultures of Solanum chacoense (monohaploid) and Solanum tuberosum (tetraploid cultivars and parthenogenetically derived dihaploid clones) were found to be highly mixoploid.Relative stabilization of chromosome number at the ploidy level of the original plant material was achieved in microcalli obtained from single cells or small cell colonies (up to about 5 cells) of stock callus lines. This relative stabilization was maintained over three subcultures, which is sufficient for selection procedures. It has been shown that the stabilization can be maintained during a number of further subcultures. Division centers were repeatedly observed in calli characterized by high mitotic activity. As has been shown for the first time there exist significant differences in the ploidy levels of several division centers within one and the same callus. This is of particular importance to callus subculture.