Establishment and characterization of <Emphasis Type="Italic">Prosopis laevigata</Emphasis> (Humb. &; Bonpl. ex Willd) M.C. Johnst. cell suspension culture: a biotechnology approach for mesquite gum production

This study presents a protocol for the establishment of Prosopis laevigata cell suspension culture as a strategy to obtain an in vitro mesquite gum productive cell line. The callus used for this purpose was obtained with hypocotyls from 15-day-old plantlets, placed on Murashige–Skoog medium with two different plant growth regulators (PGRs), 2,4,5-trichlorophenoxy acetic acid (2,4,5-T; 5.0 μM) and kinetin (KIN; 5.0 μM). With this PGRs treatment, after four subcultures (30 days each) an exuded gum-like substance was observed on the callus surface. The growth kinetics of the cell suspension culture showed a specific cell growth rate (μ) of 0.14 d⁻¹ and doubling time (t _d) of 6.6 days, respectively. The gum-like substance from callus culture and the broth from cell suspension culture were subjected to chemical analysis and compared with the mesquite gum exuded from wild trees. Both, gum-like substance from callus culture and the broth from cell suspension culture showed the presence of Arabinogalactan-proteins, and their polysaccharide fraction presented the same monosaccharides as those isolated from mesquite gum. In addition, the emulsifying properties of gum-like substance from callus culture and the broth from cell suspension culture were compared to those of mesquite gum and all three samples exhibited similar emulsifying capacity and emulsification stability.     

Cell suspension cultures of Populus tremula × P. tremuloides exhibit a high level of cellulose synthase gene expression that coincides with increased in vitro cellulose synthase activity

Summary. Compared to wood, cell suspension cultures provide convenient model systems to study many different cellular processes in plants. Here we have established cell suspension cultures of Populus tremula L. × P. tremuloides Michx. and characterized them by determining the enzymatic activities and/or mRNA expression levels of selected cell wall-specific proteins at the different stages of growth. While enzymes and proteins typically associated with primary cell wall synthesis and expansion were detected in the exponential growth phase of the cultures, the late stationary phase showed high expression of the secondary-cell-wall-associated cellulose synthase genes. Interestingly, detergent extracts of membranes from aging cell suspension cultures exhibited high levels of in vitro cellulose synthesis. The estimated ratio of cellulose to callose was as high as 50 : 50, as opposed to the ratio of 30 : 70 so far achieved with membrane preparations extracted from other systems. The increased cellulose synthase activity was also evidenced by higher levels of Calcofluor white binding in the cell material from the stationary-phase cultures. The ease of handling cell suspension cultures and the improved capacity for in vitro cellulose synthesis suggest that these cultures offer a new basis for studying the mechanism of cellulose biosynthesis. Correspondence and reprints: School of Biotechnology, Royal Institute of Technology, AlbaNova University Centre, 106 91 Stockholm, Sweden. Present address: Department of Biotechnology, Beijing Forestry University, Beijing, People’s Republic of China     

Plant regeneration of Solanum lycopersicoides Dun. from stem explants,callus and suspension cultures

Protocols were established for achieving plant regeneration from stem internode, callus, and cell suspension cultures of Solanum lycopersicoides Dun. Two accessions of S. lycopersicoides exhibited different responses as to callus formation on various media, requirement of gibberellic acid for shoot regeneration, and ability to grow in suspension culture. The optimum medium for initiation and maintenance of cell suspension cultures was Murashige and Skoog [9] medium with 15 mg l^– NAA. For shoot regeneration, of three cytokinins tested, zeatin was found most effective relative to number, rapidity of response and overall quality of shoots. Shoot regeneration from stem explants, callus and suspension cultures was optimum on MS + 3.0 mg l^–1 zeatin + 0.1 mg l^–1 gibberellic acid.Michigan Agricultural Experiment Station Journal Article No. 11589.     

Heparin Promotes Suspension Adaptation Process of CHO–TS28 Cells by Eliminating Cell Aggregation

While heparin has been shown to eliminate cell aggregation in suspension adaptations of insect and HEK293 cells for virus-based cell cultures, the role of heparin in long period serum-free suspension adaptation of the anchorage-dependent Chinese hamster ovary (CHO) cell lines remains inconclusive. In this paper, we explore the potential application of heparin in suspension adaptation of CHO cell line which produces an anti-human chimeric antibody cHAb18. Heparin showed a concentration-dependent inhibition of CHO–TS28 cell-to-cell adhesion, with a significant inhibitory effect occurring when the concentration exceeded 250 μg/ml (P < 0.001). Heparin also exhibited a cell aggregation elimination role at all concentrations (P < 0.001). Furthermore, heparin promoted cell growth and antibody secretion, with the highest cell density ((99.83 ± 12.21) × 10⁴ cells/ml, P = 0.034) and maximum antibody yield ((9.46 ± 0.94) mg/l, P < 0.001) both occurring at 250 μg/ml heparin. When agitated, cell aggregates were effectively dispersed by 250 μg/ml heparin and a single-cell suspension culture process was promoted. In suspension adapted CHO–TS28 cells, cell growth rates and specific antibody productivity were maintained; while antigen-binding activity improved slightly. Together, our results show that heparin may promote suspension adaptation of anchorage-depended CHO cells by resisting cell aggregation without reducing cell growth, antibody secretion, and antigen-binding activity.     

Chromosomal variation in dividing protoplasts derived from cell suspensions of barley (Hordeum vulgare L.)

Summary Numerical and structural chromosome variation was analysed in dividing protoplasts isolated from suspension cells of barley. Five cell lines exhibited distribution patterns in chromosome number with different peaks and ranges. Embryogenic/morphogenic cell lines showed a peak at 2n = 14 (ca. 50%) after 6–7 months in culture, while older non-embryogenic cell lines had peaks at aneuploid or polyploid chromosome numbers. Culture duration had a clear effect on numerical and structural chromosome variation in embryogenic cell lines. With ageing of the cultures chromosome variation accumulated and the proportion of 2n = 14 cells decreased. The effect of protoplast isolation and culture on chromosome variation was examined; more cells with normal chromosome sets (12%) were maintained in protoplast-derived colonies than in source suspension cells (4%) of the same culture age.Abbreviations DC Dicentric - F fragment - T telocentric     

Establishment and characterization of Pinus pinaster suspension cell cultures

We report the establishment of a Pinus pinaster (Ait.) cell suspension culture in a modified MS medium supplemented with 2 mg ml⁻¹ 2,4-D and 1 mg ml⁻¹ BA. Calli were obtained from seedling root segments and established a friable isodiametric cell suspension, suitable for in vitro studies of maritime pine at the cellular level. Growth (dry weight), cell viability, pH, and nutrient consumption: carbon source (sucrose, fructose and glucose), nitrogen source (ammonia and nitrate) and phosphate were monitored over 24 h. Suspension cells exhibited a 15-day exponential growth stage, during which a biphasic consumption profile was observed for all nutrients. Phosphate was the first limiting nutrient and preferable consumption was observed for glucose over fructose and nitrate over ammonium.     

Selection of paraquat-resistant variants of tobacco from cell cultures

Summary Paraquat-resistant cell lines and plants were isolated from cell cultures ofNicotiana tabacum var. Wisconsin 38. The resistant phenotype was successfully obtained from both callus and plated suspension cultures; however, recovery of phenotypically stable, resistant cell lines was higher among those derived from suspension cultures. The use of X-rays increased the recovery of paraquat-resistant cell lines. Stepwise selection procedures did not increase recovery of the resistant phenotype. Only 15 of 43 plants regenerated from paraquat-resistant cell lines retained the resistant phenotype; however, calli derived from leaves of both resistant and nonresistant plants exhibited either partial or complete resistance. This paper is a contribution from the Botanical Laboratory, N. Ser. 531. This material is based in part upon work supported by the U.S. Department of Agriculture, Agreement 5901-0410-9-0347-0.     

Selection of host cell lines for scale-up growth conditions of a recombinant baculovirus vector expressing the measles virus nucleocapsid protein

Summary Various insect cell lines were grown as suspension cultures in spinner vessels and infected with a recombinant baculovirus vector expressing the measles virus nucleoprotein. The highest yields of recombinant protein production were achieved using Trichoplusia ni (BTI-Tn 5B1-4) cells growing as natural aggregates in suspension and cell line Mb as a single cell suspension culture.     

Variation in the growth and biosynthetic activity of cloned cell cultures of Capsicum frutescens and their response to an exogenously supplied elicitor

Twenty clones established from single cells of a suspension culture of Capsicum frutescens were maintained as callus and in suspension over a sixteen week culture period. These clones exhibited marked differences in growth, chlorophyll and chloroform-soluble phenolic content which became more apparent with increasing time in culture. Clones in suspension exhibited a more rapid change in morphology and biosynthetic activity than those cultured as callus. Elicitation increased PAL activity, reduced the incorporation of L-[U-¹⁴C] phenylalanine into the chloroform-soluble fraction of the culture medium and increased incorporation into the methanol-soluble fraction of the cells in ten suspension clones. Differences to elicitation were observed among clones; in particular the faster growing isolates incorporated more radioactive label into soluble phenolics that remain in the cells than those that are released into the medium. The implications of these results are discussed.Abbreviations SH Schenk & Hildebrandt - PAL phenylalanine ammonia-lyase - RGR relative growth rate - TCC total chlorophyll content - HPLC high performance liquid chromatography     

Development and Characterization of a Nonmorphogenetic Cell Line of Wheat (Triticum aestivum)

This study was conducted to compare characteristics of a wheat (Triticum aestivum L.) cell line to those of the maize (Zea mays L.) black Mexican sweet (BMS) cell line and to compare protoplasts isolated from suspension cells of these cell lines. The wheat cell line was established from immature-embryo derived callus of the experimental line ‘ND7532’ and was conditioned for growth in suspension culture. For both cell lines, measurements of packed cell volume (PCV), fresh weight (FW), and dry weight (DW) were taken at 3 day intervals from suspension cultures. Measurements of FW of calluses cultured from suspension cells of both cell lines were taken at 6 day intervals. The morphogenetic potential of the wheat ND7532 cell line was tested in both callus and suspension cultures using media promoting regeneration and/or organogenesis. Growth rates of ND7532 cells in suspension culture were comparable to those of BMS cells. However, relative growth rates of calluses recovered from ND7532 suspension cells were slower than those of calluses recovered from BMS suspension cells. The ND7532 cell line has very limited morphogenetic potential and has been maintained as rapidly growing callus tissue for 11 years. Yields of protoplasts from suspension cells of the two cell lines were comparable, though ND7532 protoplasts were typically smaller. The wheat cell line has is now designated ND7532-NM (nonmorphogenetic) and is available for cellular and molecular biology research.     

Regeneration of plants from cryopreserved embryogenic cell suspension and callus cultures of cotton (Gossypium hirsutum L.)

Successful regeneration of cotton (Gossypium hirsutum L.) plants from cryopreserved embryogenic callus and cell suspension cultures is described. The cryoprotectant mixture consisting of a modified Murashige and Skoog (1962) medium with sucrose (5% w/v), DMSO (5% v/v) and glycerol (5% v/v) gave the highest survival rate (70%) from cell suspension cultures cryopreserved in liquid nitrogen after slow cooling (0.5 to 1.0°C/min). A cooling rate of 0.5°C/min provided a satisfactory recovery rate (30%) from cryopreserved embryogenic callus cultures and was superior to a cooling rate of 1°C/min. Regenerated plants from cell suspension and embryogenic callus cultures cryopreserved for more than four years exhibited normal morphology, growth and boll set upon transfer to soil.Abbreviations DMSO dimethylsulfoxide - MS Murashige and Skoog (1962) - MMS modified MS - NAA -naphthaleneacetic acid     

Rheological properties of <Emphasis Type="Italic">Chlorella pyrenoidosa</Emphasis> culture grown heterotrophically in a fermentor

Rheological properties of Chlorella pyrenoidosa culture grown heterotrophically in a 14 L fermentor were investigated. It was found that the fluid viscosity was rather low and remained almost unchanged during the cultivation, implying that no (or very few) viscous substances were excreted into the medium. Investigation of the condensed suspension of C. pyrenoidosa showed that for biomass concentration under 150 g.L⁻¹, the suspension of C. pyrenoidosa exhibited Newtonian behavior, and the fluid viscosity was rather low (about 40 mPa·s) and increased very slowly with the increase in cell concentration. With further increase in biomass concentration however, the fluid rheological behavior changed to non-Newtonian, and the fluid viscosity increased rapidly with the increase in cell concentration. From the viewpoint of rheology, C. pyrenoidosa is an excellent organism for high-cell-density culture, and there will be no rheological problems at cell densities under 150 g.L⁻¹.     

Selection <Emphasis Type="Italic">in vitro</Emphasis> and accumulation of phytochelatins in cadmium tolerant cell line of cucumber (<Emphasis Type="Italic">Cucumis sativus</Emphasis>)

Cucumber (Cucumis sativus L.) cells from suspension culture were selected for their ability to grow and divide rapidly in toxic concentration of cadmium. As a result of selection a cell suspension tolerant to 100 M cadmium chloride (CdCl₂) was initiated. The selected tolerant line exhibited stable and repeatable increase in fresh and dry weight of cells in the presence of cadmium. The accumulated level of phytochelatins in cadmium sensitive (unselected) and tolerant cell line was measured by high performance liquid chromatography (HPLC) after 3, 24 h and 5 days of cadmium treatment. It was shown that in both cell lines Cd induced accumulation of phytochelatins and simultaneous glutathione depletion occurred. No distinct changes were found after 3 and 24 h of cadmium treatment whereas after 5 days of exposure to the metal, the level of phytochelatins was two times higher in the sensitive cell line as compared to the tolerant one. The accumulation of phytochelatins was correlated with cadmium concentration that increased in both cell lines during the course of cell exposure to metal. However, the level of cadmium was always lower in the tolerant cell line. The results showed no direct correlation between the tolerance of cucumber cells to Cd and the accumulated level of phytochelatins. Other mechanisms responsible for the increased tolerance of cucumber cells exposed to Cd are discussed.     

Drought,heat and salt stress induce the expression of a citrus homologue of an atypical late-embryogenesis Lea5 gene

In a search for genes that are induced in citrus cell suspension in response to salt stress, a cDNA clone with high homology to cotton Lea5 gene was isolated. Data base analysis of the protein deduced from the nucleotide sequence indicates that, like in cotton, the protein from citrus contains regions with significant hydropathic character. The gene, designated C-Lea5, is expressed in citrus leaves as well as cell suspension. The steady-state level of C-Lea5 is increased in cell suspension that is grown in the presence of 0.2 M NaCl. This phenomenon is also observed in leaves of citrus plants irrigated with NaCl and in citrus seedlings which are exposed to drought and heat stress. We suggest that the osmotic stress resulted from elevated level of salt is responsible for the increase in the level of C-Lea5.     

Establishment of <Emphasis Type="Italic">Cyperus aromaticus</Emphasis> cell suspension cultures for the production of Juvenile hormone III

Cell suspension cultures of Cyperus aromaticus were established from the yellow friable callus derived from the root explants of in vitro plantlets. Four callus cell lines were selected based on their growth index from two populations of callus cultures originated from the mother plants grown in two different locations. The selected four cell lines (Z1, Z6, P4, P9) showed uniform cell growth but produced different amounts of juvenile hormone III (JHIII). The Z1 cell line possessed fast-growing characteristics, produced a high JHIII content, and was chosen as the elite cell line for an optimization study of C. aromaticus cell suspension cultures. An inoculum cell mass of 0.3 g from 12-d cultures in 30 ml culture medium was found to be the optimum inoculum size and culture age for establishing the cell suspension culture of C. aromaticus. MS basal medium supplemented with 4.5 mg/l 2,4-D and 5.5 mg/l NAA was found to be the best medium for production of maximum cell biomass and JHIII. These results indicated that JHIII can be produced from suspension culture of C. aromaticus using a single-stage cell-culture system.     

Analysis of mitochondrial DNA from somatic hybrid cell lines of Saccharum officinarum (sugarcane) and Pennisetum americanum (pearl millet)

Mitochondrial DNA (mtDNA) from cell suspension cultures of two intergeneric somatic hybrids of Pennisetum americanum (pearl millet) + Saccharum officinarum (sugarcane) was examined by restriction endonuclease digestion and hybridization with sorghum mtDNA cosmids. The mtDNA of one somatic hybrid was indistinguishable from that of pearl millet, while the second exhibited a combination of parental mtDNAs, suggesting mitochondrial fusion. Several novel, possibly recombinant, mtDNA restriction fragments were detected in this hybrid, which may have resulted from intergenmic recombination.Florida Agriculture Experiment Station Journal Series No: 8090.     

Application of31P nuclear magnetic resonance spectroscopy to investigate plasmid effects onEscherichia coli metabolism

Summary Glucose metabolism inE. coli strain HB101, as a plasmid-free cell and as a host to two plasmids of different copy numbers, has been characterized using³¹P NMR. While the low-copy-number strain was found to behave very similarly to the plasmid-free strain, dramatically different behavior was exhibited by the high-copy-number strain. This strain maintained a nearly constant intracellular pH after addition of glucose to a starved suspension while intracellular pH of the other strains dropped considerably. The inorganic phosphate level in the high-copy-number strain was substantially higher than in the other strains, and the NTP level was much lower. Glycolytic rates of all three strains, however, were nearly identical. The trend in glycolytic rate strongly suggests that transport of glucose into the cell is the rate-limiting step under these conditions.     

Effects of abiotic and biotic elicitors on growth and isoflavonoid accumulation in Pueraria candollei var. candollei and P. candollei var. mirifica cell suspension cultures

This study demonstrates the effects of various concentrations of abiotic and biotic elicitors on the cell growth and isoflavonoid accumulation of P. candollei var. mirifica (PM) and P. candollei var. candollei (PC) cell suspension cultures. The two plant varieties exhibited different growth responses and varied isoflavonoid accumulation after the addition of elicitors. Copper sulfate, methyl jasmonate (MeJA), and yeast extract did not significantly affect the growth of either plant variety, whereas oligosaccharide and the biotic elicitors used in this study [i.e., 50 mg l⁻¹ chitosan and all concentrations of laminarin (LAM)] suppressed the growth of PM. The addition of MeJA to the medium principally induced an effect on the isoflavonoid content in both PM and PC, with 2.0 μM MeJA inducing the highest isoflavonoid content, as indicated by the induction index—4.41 in PM and 9.62 in PC cells on the 12th and ninth day of culture, respectively. A maximum total isoflavonoid content of 40.49 mg g⁻¹ dry weight was achieved in PM 21 days after elicitation with 2.0 μM MeJA. LAM elicited the PM cell suspension culture to produce puerarin, which was not found in the unelicited culture. The results of this study provide information that will be useful for enhancing the accumulation of isoflavonoids in P. candollei cell suspension cultures.