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.     

Cell wall proteins from sugar beet cells in suspension culture

Several proteins were extracted from the purified cell walls of suspension-cultured sugar beet cells with 0.5% EDTA (pH 6.8) after prior extraction of the walls with 0.5% deoxycholate and then with 2 molar NaCl. Two abundant proteins (P-I and P-II protein) were separately purified to homogeneity by procedures that included fractionation with ammonium sulfate, column chromatography on DEAE-cellulose and butyl Toyopearl, and preparative polyacrylamide electrophoresis. P-I exists as a dimer of identical subunits, and P-II is composed of four different subunits. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that quite different polypeptides are present in the culture medium and in the NaCl and EDTA extracts of the wall.     

Purification and properties of glucoamylase from sugar beet cells in suspension culture

Glucoamylase and α-amylase are present in callus and suspension cultures of sugar beets (Beta vulgaris L.) as well as in mature roots. The subcellular localization of glucoamylase differed in callus and suspension-cultured cells: in callus, glucoamylase was present together with α-amylase in the soluble fraction of cells, but in suspension cultures, it was present predominantly in the extracellular fraction while most of the α-amylase activity remained in cells. Glucoamylase activity was considerably lower in callus protoplasts relative to the activities of α-mannosidase and α-galactosidase and the suspension of callus in Murashige-Skoog liquid medium or in mannitol by brief agitation resulted in the release of glucoamylase to the medium. These findings suggest that glucoamylase in callus may be present in a soluble form in the free space in the cell wall. Both mature roots and callus contained α-amylase and glucoamylase in the soluble fraction. Glucoamylases in the soluble fraction of callus and in the medium of suspension cultures were purified separately to homogeneity by the same four-step purification procedure, which included fractionation with ammonium sulfate, column chromatography on carboxymethyl cellulose, gel filtration on Bio-Gel P-150, and preparative disc electrophoresis. The identity of the glucoamylases from the two sources was confirmed by a comparison of chromatographic behavior during purification, mobility during gel electrophoresis, M_r (83,000 D by SDS PAGE), and enzymic and kinetic properties of the catalytic reaction, such as optimal pH and temperature, heat stability, and K_m value for soluble starch. Glucoamylase from suspension cultures was one of the major proteins that were secreted into the medium. Dedifferentiation of leaves of young plants to callus was accompanied by induction of glucoamylase and repression of some α-amylases and the debranching enzyme.     

Rearrangements in sugar beet mitochondrial DNA induced by cell suspension,callus cultures and regeneration

Structural alterations in mitochondrial DNAs (mtDNAs) from a plant of a sterile sugar beet line, callus derived from it, suspension-cultured cells and plants regenerated from the callus were studied. BamHI restriction analysis revealed that structural alterations between the mtDNAs of the callus and the control plant had occurred. Multiple rearrangements were also demonstrated in the mtDNA from the suspension culture, of which some were similar to those appearing in the callus, and others had arisen de novo. Rearrangements were also identified by means of blot hybridization of BamHI-digested mtDNA from suspension-cultured cells with the genes encoding subunit II of cytochrome oxidase (cox II) and subunit 1 of NADH-dehydrogenase (Nd1). No alterations were observed in the mitochondrial genome of the callus and regenerants. The location of the genes for the -subunit of F1-ATPase (atpA) and apocytochrome b (cob) in the mtDNA remained unchanged.Our salient finding was of a plant with an altered mitochondrial genome as judged by EcoRI and BamHI restriction analysis. This exceptional plant had retained the sterile phenotype like all of the other regenerants and the parent. The set of plasmid-like molecules of mtDNA remained the same as that in the control plant and in all of the regenerants, callus and suspension-cultured cells. The only type of plasmid-like molecule found in all of the DNAs was the 1.6-kbp minicircle, which is a feature of sterile cytoplasms. These structural changes in mtDNA were obviously a consequence of somaclonal variation during the in vitro cultivation of the sugar beet cells.     

Chromatin- and nuclei-directed ribonucleic Acid synthesis in sugar beet root

The synthesis of RNA by chromatin-bound RNA polymerase prepared from sugar beet (Beta vulgaris) root tissue is completely dependent on the presence of a divalent metal (Mg²⁺ or Mn²⁺) and the presence of four ribonucleoside triphosphates. Accumulation of labeled acid-insoluble product is inhibited by the addition of RNase and actinomycin D to the reaction. When beet root slices are washed for 25 hours, chromatin-associated RNA polymerase activity increases 7-fold over that of unwashed tissue. This enzyme activity declines with further washing. DNA template availability, as measured by saturating levels of added Escherichia coli RNA polymerase, was also found to follow a pattern similar to that for RNA polymerase. Nearest neighbor frequencies of the RNA synthesized by chromatin isolated from unwashed and washed tissue are different.     

Cell wall invertases of sugar cane

A study of the cell wall invertases in the different organs of a sugar cane cultivar has been undertaken. The enzymes could not be separated from the cell wall. They are compared on the basis of optimum pH, K_m, the effect of various chemicals and the substrate specificities of the preparations. According to the results each organ appears to possess a set of cell wall invertases with predominance of a different activity in each case.     

Foaming and enzyme activity in fungal cultures grown on sugar beet cosette

Summary Enzyme synthesis, foaming behaviour and its effects were studied using two common cellulolytic fungi;Trichoderma reesei andSporotrichum pulverulentum in a medium containing sugar beet cosette as a cellulosic substrate. Cellulase enzyme activities in the culture broth were found to be higher than the enzyme activities in natural and experimentally forced foam layers.     

Acid and alkaline invertases in roots and nodules of Lupinus angustifolius infected with Rhizobium lupini

Summary Both acid and alkaline invertase activity were found in tips and cortical tissue of Lupinus angustifolius L. roots infected with Rhizobium lupini NZP 2257. Only the alkaline invertase was detected in the nodule cytoplasm. Weak invertase activity found in the bacteroids was probably a contamination from plant invertase. The alkaline invertase activity in the nodule cytoplasm was 250 times that detected in the bacteroids and 8 times that detected in cortical tissue. No intracellular or extracellular invertase was detected in R. lupini cultured in liquid medium containing sucrose.     

Characterization and inhibition of invertases in sugar cane juice

(NH₄)₂SO₄ fractionation followed by Sephadex G-200 chromatography of sugar cane juice gave an acid invertase with MW of 380 000 and 23.5% carbohydrate and a neutral invertase with MW of 66 000 and 22% carbohydrate. For acid invertase, K_m is 2.8 mM and V_max is 2.7 μmol sucrose hydrolysed/hr/mg protein. For neutral invertase, K_m and V_max are 0.32 mM and 2.8 μmol hydrolysed/hr/mg protein, respectively. Inhibition of both invertases by either lauryl sulfate or metasilicate is not competitive.     

Abscisic Acid metabolism by source and sink tissues of sugar beet

The fate of exogenously applied, labeled abscisic acid (±)-(ABA) was followed in source leaves and taproot sink tissues of sugar beet (Beta vulgaris cv AH-11). The objective was to determine if differential pathways for ABA metabolism exist in source and sink tissues. Tissue discs were incubated for up to 13 hours in a medium containing 1 micromolar labeled ABA. At various time intervals, samples were taken for metabolite determination by reverse-phase high performance liquid chromatography. The labeled metabolites were identified by retention times using an online scintillation counter.

Dihydrophaseic acid (DPA) aldopyranoside, DPA, phaseic acid (PA), ABA glucose ester (ABA-GE), and two unidentified compounds were recovered from both tissues. An additional unidentified metabolite was also present in root tissue. Leaf tissue discs exhibited a higher capacity for ABA conjugation, and root discs showed a greater preference for ABA catabolism to PA and DPA. After 4 to 5 hours, ABA incorporation into the various metabolites was proportional to the external ABA concentration in both tissues. But the internal ABA pool size was independent of external concentrations below 10⁻⁶ molar. These results suggested that rates of ABA metabolism was proportional to the rates of uptake in both tissues.

     

Phenotypic polymorphism and allele differentiation of isozymes in fodder beet,multigerm sugar beet and monogerm sugar beet

Summary Thirteen enzymes (MDH, SDH, LAP, PGM, PX, IDH, GPI, 6PGD, APH, GOT, GDH, ME and SOD) of 3 cultivated beet (B. vulgaris L.) gene pools, comprising 12 accessions of fodder beet, 11 of old multigerm sugar beet and 10 of modern monogerm sugar beet, were investigated using horizontal starch gel electrophoresis. Eleven accessions of primitive or wild B. vulgaris were also included for the comparison of isozymes. Variation in isozyme phenotypes was investigated to detect diversity in the three cultivated forms of beet. Phenotypic variation was observed in all except ME and SOD, which were monomorphic. A high degree of phenotypic polymorphism (Pj) was found in GDH, PGM, IDH, APH and MDH. Differences in phenotypic polymorphism in MDH, GPI and PX were recognized between fodder beet and both sugar beet groups. Average polymorphism for 13 enzymes in both sugar beets was significantly higher than that in fodder beet. For 13 enzymes, the existence of high isozyme diversity in both sugar beet gene pools was revealed. Allele frequencies in 13 alleles of five enzyme-coding loci, Lap, Px-1, Aph-1, Got-2 and Gdh-2, were investigated. New alleles, Px-1 ¹ and Got-2 ¹, were found in fodder beet accessions. No significant differences of average allele frequencies of five loci between fodder beet and both sugar beets were recognized. Several unique alleles and different isozyme phenotypes were observed in the accessions of B. vulgaris ssp. macrocarpa and ssp. adanensis. Future utilization of cultivated beet gene pools for sugar beet breeding is discussed from the viewpoint of genetic resources.     

Genetic variability of somatic embryogenesis in tissue cultures of sugar beet breeding lines

Genetic variability of callus initiation and plant regeneration has been investigated among three sugar beet genotypes. It was found that TDZ has a genotype-independent effect on callus initiation and is responsible for more than a two-fold increase in the friable callus induction rate and more than a three-fold increase in the shoot regeneration rate from this callus. Along with the genotype-independent organogenesis, regeneration from callus occasionally went through the process of somatic embryogenesis in a highly genotype-specific manner. Despite fast and uncontrollable conversion of embryos to normal plants, it was possible to select and maintain repetitive embryogenic culture without loosing regeneration and root formation capabilities. Extensive experimenting with medium composition and culture conditions resulted in an optimal medium for maintenance of repetitive embryos. Comparing with BAP, low concentrations of TDZ provide higher level of adventitious shoot formation and do not induce vitrification of tissues.     

Salt tolerance in suspension cultures of sugar beet : induction of na/h antiport activity at the tonoplast by growth in salt

Cell suspension cultures of sugar beet were grown at various salinities (0-200 millimolar NaCl). Their tolerance to Na⁺ was comparable to that of the intact plant. Tonoplast vesicles were prepared by sucrose density gradient centrifugation of microsomal membranes and shown to be highly purified. The vesicles were subjected to a pH jump in the presence of acridine orange and the rate of recovery of fluorescence after addition of Na⁺ was used as a measure of Na⁺-dependent H⁺ efflux. In the presence of K⁺ and valinomycin, the Na⁺/H⁺ antiport showed saturation kinetics. Increasing Na⁺ in the growth medium did not change the apparent K_m for Na⁺, but increased V_max to about twice the control value, suggesting a specific induction of antiport synthesis by salt.     

Polyamine metabolism and gene regulation during the transition of autonomous sugar beet cells in suspension culture from quiescence to division

Sugar beet cells grown in batch suspension culture have been used to study the regulation of polyamine levels during the transition from a quiescent to a proliferating state. The quiescent state was achieved by maintenance of the phytohormone autonomous cells in the stationary phase of the batch culture cycle. After subculture into fresh medium there was an increase in DNA synthesis which was accompanied by a dramatic increase in cellular polyamine levels. The levels of both free and bound cellular putrescine and spermidine within the cells reached a peak before the onset of the first synchronous division. The levels of putrescine, spermidine and to some extent spermine in the culture medium also increased dramatically shortly after subculture. The increase in polyamines was preceded by a rapid but transient increase in omithine decarboxylase (EC 4.1.1.17) and S -adenosylmethionine decarboxylase (EC 4.1.1.50). Arginine decarboxylase (EC 4.1.1.19) and S -adenosylmethionine synthetase (EC 2.5.1.6) activity did not show the same pattern of cell division-related variation. Inhibition of S -adenosylmethionine biosynthesis with methylglyoxal bis-(guanylhydra-zone) (MGBG) reduced cell division in the suspension culture. Inhibitors of ornithine decarboxylase and arginine decarboxylase individually had little effect on cell division, but in combination led to a reduction in cell division. Addition of polyamines and their precursors to cells in the stationary phase of a batch culture cycle led to the induction of expression of a mitotic cyclin sequence ( BvcycII ).     

Polyamine metabolism and gene regulation during the transition of autonomous sugar beet cells in suspension culture from quiescence to division

Sugar beet cells grown in batch suspension culture have been used to study the regulation of polyamine levels during the transition from a quiescent to a proliferating state. The quiescent state was achieved by maintenance of the phytohormone autonomous cells in the stationary phase of the batch culture cycle. After subculture into fresh medium there was an increase in DNA synthesis which was accompanied by a dramatic increase in cellular polyamine levels. The levels of both free and bound cellular putrescine and spermidine within the cells reached a peak before the onset of the first synchronous division. The levels of putrescine, spermidine and to some extent spermine in the culture medium also increased dramatically shortly after subculture. The increase in polyamines was preceded by a rapid but transient increase in omithine decarboxylase (EC 4.1.1.17) and S -adenosylmethionine decarboxylase (EC 4.1.1.50). Arginine decarboxylase (EC 4.1.1.19) and S -adenosylmethionine synthetase (EC 2.5.1.6) activity did not show the same pattern of cell division-related variation. Inhibition of S -adenosylmethionine biosynthesis with methylglyoxal bis-(guanylhydra-zone) (MGBG) reduced cell division in the suspension culture. Inhibitors of ornithine decarboxylase and arginine decarboxylase individually had little effect on cell division, but in combination led to a reduction in cell division. Addition of polyamines and their precursors to cells in the stationary phase of a batch culture cycle led to the induction of expression of a mitotic cyclin sequence ( Bvcycll ).     

The permeability of electroporated cells and protoplasts of sugar beet

A simple method has been developed to determine the changes in permeability of protoplasts and intact cells when electroporated. Cells and protoplasts of sugar beet, Beta vulgaris L., were subjected to electric pulse treatments of different field strengths, pulse number and pulse duration, and the ability to accumulate and retain the hydrophilic dye phenosafranine was determined spectrophotometrically. Results of timecourse studies of phenosafranine accumulation and retention indicated that pores are formed or enlarged rapidly in the plasmamembrane and remain permeable to phenosafranine for relatively long periods; the half-life of the pores was temperaturedependent. Both cells and protoplasts retained the highest levels of phenosafranine when supplied with a series of five rectangular pulses of 50 s duration and of field strength 2500 V·cm^-1. If these parameters were exceeded, The phenosafranine content was reduced, concomitant with a decline in viability as indicated by fluorescein-diacetate staining, indicating the loss of the integrity of the plasmamembrane. The pattern of accumulation and retention by protoplasts of radioactivity from [³H]pABD1, a modified bacterial plasmid, was similar to that of phenosafranine, but uptake of the plasmid by cells was not demonstrated. The mothod can be used to determine conditions for the optimum permeabilization of protoplasts for direct gene transfer.     

Isolation and sugar uptake characteristics of protoplasts from maize endosperm suspension cultures

The isolation and sugar uptake characteristics of protoplasts from maize ( Zea mays L.) endosperm-derived suspension cultures are described. In contrast with protoplasts from intact developing endosperm, which by virtue of their large size and high starch content are too fragile for sugar uptake experiments, suspension cultures yielded protoplasts capable of withstanding the necessary handling and centrifugations. Intactness of the protoplasts was demonstrated by dye exclusion or accumulation and latency of malate dehydrogenase activity. Uptake of radioactivity from [³H]-inulin did not increase with time, but that from [¹⁴C]-sugars increased over a wide range of external concentrations. Kinetics of fructose, glucose and sucrose uptake were biphasic, and the saturable components of uptake were eliminated by p -chloromercuribenzene sulfonate (PCMBS). Rates of uptake of sucrose and 1'-fluorosucrose were similar, confirming that hydrolysis by cell wall invertase contributes to sucrose uptake by the suspension cultures. The isolation of protoplasts from this tissue source will enable experimental access to plasma membrane sugar carriers which may exist in the intact maize endosperm.     

Transient gene expression in electroporated protoplasts and intact cells of sugar beet

Factors influencing the transient expression of introduced foreign DNA in electroporated protoplasts and intact cells of sugar beet were determined by assaying for the activity of chloramphenicol acetyltransferase (CAT), using a rectangular pulse generating system. Extractable CAT activity depended upon 1) applied plasmid DNA concentration, 2) protoplast density, 3) the interaction between pulse field strength, duration, number, time interval between pulses and the resultant effect on culture viability, and 4) the physiological state of the protoplasts. Mesophyll protoplasts were more susceptible to damage by electroporation, and were more specific in their requirement for electroporations which allowed CAT expression, than were protoplasts derived from suspension culture cells. CAT activity was also demonstrated, at low levels, after electroporation of intact suspension culture cells, and could be increased by pectinase treatment of the cells before electroporation.     

Morphological and proteomic analyses of sugar beet cultures and identifying putative markers for cell differentiation

Normal (N), habituated nonorganogenic (HNO), and tumour (T) sugar beet cell lines were analysed in order to establish specific patterns of extracellular proteins and identify protein markers that might explain the distinct phenotypical characteristics. Electron microscopy showed that the ultrastructure of N cells corresponds to that of parenchyma cells, and that these cells contain plastids with large starch grains. HNO and T cells had enlarged, lobed nuclei with an increased number of nucleoli; the number of nuclei in HNO cells was greater than in T cells. The T plastids were elongated, with reduced thylakoids and abundant phytoferritin deposits, while HNO plastids were small and vacuolated, with an irregular, underdeveloped thylakoid system. The extracellular proteome of the cells was separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis. Greater differences in protein expression were observed between the HNO and N lines than between the T and N lines. Sixteen of the most prominent bands differentially expressed among the cell lines were cut out from the gel and analyzed by mass spectrometry. Cell wall-modifying enzymes were identified, including a peroxidase whose expression was twofold higher in N and T tissue than in HNO tissue; pectinesterase, which was expressed at a level threefold lower in the T line than in the other cell lines; and xyloglucan endotransglucosylase, which was expressed at a level sixfold higher in HNO and T tissue. Three proteins belonged to the chitinase gene family and their expression was higher in HNO and T tissue than in N tissue. The differential expression of these proteins suggests that these play a role in cell line-specific cell wall composition and cell-to-cell adhesion.