Carrot (Daucus carota) hypocotyl transformation usingAgrobacterium tumefaciens

Summary Daucus carota hypocotyl sections were transformed withAgrobacterium tumefaciens LBA4404 containing CaMV 35S promoter, -glucuronidase coding sequence and the nopaline synthase (Nos) poly adenylation sequences in Bin 19. Sliced sterile seedling hypocotyl segments were preincubated for 2 days, co-cultivated withAgrobacterium for an additional 2 days, and then transferred to medium containing 100ug/ml of kanamycin and 400ug/ml carbenicillin. In 6 weeks kanamycin resistant calli were obtained in 5.8% of the explants from one variety. Calli were subcultured on solid medium, and in 4 weeks introduced into suspension culture. NPTII and Southern blot analysis confirmed that three selected lines were transformed with 1–3 copies of the GUSII construction. GUS activity in transformants was 5 to 250 fold over background.Abbreviations NPT II neomycin phosphotransferase II - Nos nopaline synthase - GUS -glucuronidase - CaMV cauliflower mosaic virus - 2,4-D 2,4 dichlorophenoxyacetic acid - PMSF phenylmethylsulfonyl fluoride     

Influence of enzyme solution on protoplast culture and transient gene expression in maize (Zea mays L.)

The influence of two enzyme solutions, differing only in the presence or absence of Macerozyme, on protoplast yield, colony formation and transient GUS (-glucuronidase) activity was studied. For all parameters tested the presence of Macerozyme during protoplast isolation had a negative influence. Using an enzyme solution without Macerozyme suspension aggregates gave up to 4.4 times higher protoplast yield and plating efficiencies were increased up to 10-fold. Further, protoplasts isolated without macerozyme showed a 5.2-fold higher GUS activity in transient gene expression. Apart from the presence of Macerozyme, longer incubation (3 compared with 1.5 h) of cell aggregates in the enzyme solution also had a negative effect on transient transformation efficiency. These data demonstrate that protoplast isolation conditions have a profound effect on transient gene expression and it is proposed that these factors will also influence stable transformation efficiency.Abbreviations CP cellulase pectolyase - CPM cellulase pectolyase Macerozyme - 2,4-d 2,4-dichlorophenoxyacetic acid     

Anthocyanin accumulation and PAL activity in a suspension culture of Daucus carota L.

Cells of Daucus carota grown in a liquid medium produced large amounts of cyanidin as the only flavonoid aglycon. After inoculation in fresh medium a maximum activity of phenylalanine ammonia lyase (PAL; EC 4.3.1.5) was observed within 24 h. L--aminooxy--phenylpropionic acid (L-AOPP), thought to be a competitive inhibitor of PAL, inhibited cyanidin accumulation up to 80%. In order to study the regulatory role of PAL, the effects of L-AOPP and t-cinnamic acid, the product of the deamination of phenylalanine, were investigated. Cinnamic acid, applied in vivo (10^-4 M), was not able to compensate for the inhibition of cyanidin production caused by L-AOPP (10^-4 M) in the same sample. Carrot cells treated with L-AOPP exhibited a super-induction of PAL already described for gherkin hypocotyls (Amrhein and Gerhardt 1979). This effect was not influenced by t-cinnamic acid. L-AOPP seems to be a very specific inhibitor since it affected neither growth nor soluble protein content, whereas t-cinnamic acid inhibited both. Investigations on the content of soluble amino acids in L-AOPP-treated cells revealed a specific accumulation of soluble phenylalanine, whereas treatment with t-cinnamic acid led to an increase of amino acids in general, thus indicating that the latter compound has a rather unspecific effect on cellular metabolism. In vitro studies with PAL isolated from Daucus carota revealed that L-AOPP inhibited the enzyme at very low doses (K _I=2.4·10^-9), whereas t-cinnamic acid, by comparison, affected the enzyme at high concentrations (K _I=1.8·10^-4).Abbreviations PAL phenylalanine ammonia lyase - L-AOPP L--aminooxy--phenylpropionic acid     

A detailed study of the regulation and evolution of the two classes of patatin genes in Solanum tuberosum L.

The class-specific expression of patatin genes was investigated by analysing four new patatin genes. A class I patatin gene from cv. Berolina as well as a class I and two class II patatin genes from the monohaploid cultivar AM 80/5793 were isolated and partially sequenced. Sequence comparison indicates rearrangements as the major source for the generation of diversity between the different members of the classes. The expression of single genes was studied in potato plants transformed with chimaeric genes where the putative patatin promoters were fused to the GUS reporter gene. A detailed histochemical analysis reveals that both class I genes are expressed as the previously described class I patatin gene B33 from cv. Berolina [1], i.e. in the starch-containing cells of potato tubers and in sucrose-induced leaves. The class II gene pgT12 shows the same pattern as the previously described class II gene pgT2 [2], i.e. expression in root tips and in the vascular tissue of tubers, whereas no activity was detectable for pgT4. Thus the expression pattern of both classes of genes seems to be stable at least within or even between different cultivars.     

Genetic transformation of Norway spruce (Picea abies (L.) Karst) using somatic embryo explants by microprojectile bombardment

Stable transformation of Norway spruce tissue has been obtained following bombardment of mature somatic embryos with pRT99gus, a plasmid that contains neo coding for NPTII, and gusA, coding for -glucuronidase, both fused to the CaMV 35S promoter. At least 8 lines have been stably transformed (over 15 months in culture) following bombardment and selection on kanamycin. Polymerase chain reaction analyses showed a high frequency of cotransformation of the gusA and neo genes. The frequency of coexpression of the selected and unselected markers was 100%. DNA/DNA hybridization of one transformed line provided conclusive evidence of stable integration and showed copy numbers of over 10 plasmid sequences per genome. None of the transformed lines has remained embryogenic.     

cDNA cloning and expression of a potato (Solanum tuberosum) invertase

A cDNA clone encoding an invertase isoenzyme has been isolated from a potato leaf cDNA library. The deduced amino acid sequence shows significant similarities to previously characterised invertases. The highest degree of overall similarity, including the signal peptide sequence, is to carrot cell wall invertase, suggesting that the potato gene encodes an apoplastic enzyme. Expression of the gene, as determined by RT-PCR, is detected in stem and leaf tissue, and at lower levels in tuber, but is absent from roots.     

Analysis of Erythrocyte and Platelet Membrane Proteins in Various Forms of β-Thalassemia

Major membrane proteins have been quantitatively analyzed in erythrocytes and platelets from patients with homozygous (splenectomized and non-splenectomized) and heterozygous forms of beta-thalassemia depending on severity of clinical manifestation of this disease. Quantitative analysis of erythrocyte membrane proteins revealed increase in alpha- and beta-spectrin. (In non-splenectomized patients with homozygous beta-thalassemia the amount of this protein was lower than in corresponding controls.) Besides spectrin, the increase of 2.1-2.3 fractions of ankyrin, and the decrease of band 3 protein (anion-transport protein), 4.1, palladin, and glyceraldehyde-3-phosphate dehydrogenase were also found. Analysis of major platelet membrane proteins revealed significant increase in gelsolin. This increase was found in all forms of beta-thalassemia irrespective of gender. Significant changes in platelet membrane protein fractions were found in patients (especially non-splenectomized) with homozygous beta-thalassemia. These included significant decrease in myosin, profilin, and gamma-actin and increase in actin-binding protein in both male and female patients. The content of other protein fractions (alpha-actinin, tubulin, tropomyosin) remained unchanged. Changes in protein fractions of erythrocytes and platelets correlated with severity of clinical manifestation of the disease.     

Expression of one of the members of the Arabidopsis chaperonin 60β gene family is developmentally regulated and wound-repressible

To study the pattern of gene regulation of the plastid chaperonin 60 gene family a chimaeric gene was constructed fusing the 5-flanking region of the chaperonin 60 B3 gene to the -glucuronidase reporter gene. Histochemical and fluorometric analysis of the GUS activity present in transgenic plants harbouring this gene construct showed that the B3 promoter is expressed in leaves, stem, petioles and several flower tissues. The pattern of cell type-specific expression in stems and flowers was found to be developmentally regulated. Expression of the B3 promoter was found not to be heat-inducible, but highly repressed by wounding. The rapid decay in GUS activity upon wounding indicates that, at least under some physiological conditions, the gene product of this reporter gene is not as stable as has been previously thought.     

A repeated chromosomal DNA sequence is amplified as a circular extrachromosomal molecule in rice (Oryza sativa L.)

Summary The regulation in tobacco of the rolB and rolC promoters of Agrobacterium rhizogenes pRi 1855 T_L-DNA was studied by using the -glucuronidase (GUS) reporter system in transgenic plants. A 20- to 100-fold increase of GUS activity was selectively induced by auxin in rolB-GUS transformed mesophyll protoplasts, whereas this auxin-dependent increase was only 5-fold in rolC-GUS protoplasts. Moreover, both gene fusions exhibited similar tissue-specific expression in aerial parts but different patterns in roots. The spatial pattern of rolBGUS expression could be strongly modified by the addition of exogenous auxin, further suggesting that auxin plays a central role in the regulation of the rolB promoter in tobacco. The tissue-specific and auxin-dependent regulation of the rolB promoter is discussed in relation to the effects of the rolB gene on rhizogenesis and on cellular responses to auxin.Abbreviations BA benzoic acid - 6-BAP benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - GUS -glucuronidase - 2,4,5-T 2,4,5,-trichlorophenoxyacetic acid - 2,4,6-T 2,4,6-trichlorophenoxyacetic acid - IAA indoleacetic acid - NAA naphthaleneacetic acid - MU 4-methyl umbelliferone - 35S CaMV cauliflower mosaic virus 35S (promoter) - TCA trichloroacetic acid - X-Glu 5-bromo-4chloro-3-indolyl -d-glucuronic acid     

Transformation of the endospermous legume guar (Cyamopsis tetragonoloba L.) and analysis of transgene transmission

A procedure for transformation of the large-seeded endospermous legume guar (Cyamopsis tetragonoloba L.) and a study on transmission of the transgenes to offspring generations are presented. Using Agrobacterium tumefaciens with a T-DNA construct harbouring a -glucuronidase gene (uidA) and a neomycin phosphotransferase gene (nptII), maximum transformation frequencies of cotyledonary explants were obtained using 145 mg/l kanamycin sulfate as selective agent. Carbenicillin and cefotaxime, used for the elimination of Agrobacterium after co-culture, displayed considerable toxicity to guar tissues but replacing most of these -lactams by the non-phytotoxic -lactamase inhibitor sulbactam as well as addition of thidiazuron and silver thiosulfate increased transformation frequencies up to 10-fold in total. The presence of the transgenes in the primary transformants was demonstrated by genomic DNA analysis of GUS-positive shoots. Chimaeric plants (5–10%) were identified by GUS analysis at the flowering stage and were discarded. Analysis of the R₁ offspring from 17 independent transformants showed that in 41% of those, the uidA gene(s) was expressed and stably inherited consistent with Mendelian genetics. This was also found for the R₂ and R₃ generations of single copy transformants. On the other hand, a large proportion (47%) of the primary transformants gave R₁ offspring in which 100% of the plants were GUS-negative. Analysis of these plants by PCR revealed that, at least, most of the transgene sequences were absent, suggesting that they had not been transmitted from the parent transformants. This occurred at similar high frequencies (40–50%) irrespective of the estimated copy number of the transgenes. Thus, major parts of the transgenes, even when present in multiple copies, displayed aberrant transmission, at a high frequency, in the process of going from the primary transformants to the first offspring generation.     

New 2-naphthyloxyacetates for trunk sucker growth control on grapevine (Vitis vinifera L.)

Eighteen new hexyl esters of 2-naphthyloxyacetic acid (-NOA) were synthesized and their efficiency on sucker control of grapevine was investigated. Different solutions (0.5, 1, and 2%, w/w concentrations) of each compound were sprayed on suckers and the wilting effect after 4, 10, and 30 days after treatment was evaluated. Esters derived from primary and secondary alcohols gave a rapid control of suckers, whereas tertiary alcohol derivatives showed milder activity. Moreover, all compounds proved to cause a long-lasting inhibition effect on the growth of new suckers even 1 year after treatment, especially when applied at a 2% concentration.     

Secondary structural changes in the intact and the disulfide bridges cleaved beta-lactoglobulin A and B in solutions of urea, guanidine hydrochloride, and sodium dodecyl sulfate

The relative proportions of -helix, -sheet, and unordered form in -lactoglobulin A and B were examined in solutions of urea, guanidine, and sodium dodecyl sulfate (SDS). In the curve-fitting method of circular dichroism (CD) spectra, the reference spectra of the corresponding structures determined by Chen et al. (1974) were modified essentially according to the secondary structure of -lactoglobulin B predicted by Creamer et al. (1983), i.e., that the protein has 17% -helix and 41% -sheet. The two variants showed no appreciable difference in structural changes. The reduction of disulfide bridges in the proteins increased -sheet up to 48% but did not affect the -helical proportion. The -helical proportions of nonreduced -lactoglobulin A and B were not affected below 2 M guanidine or below 3 M urea, but those of the reduced proteins began to decrease in much lower concentrations of these denaturants. By contrast, the -helical proportions of the nonreduced and reduced proteins increased to 40–44% in SDS. The -sheet proportions of both nonreduced and reduced proteins, which remained unaffected even in 6 M guanidine and 9 M urea, decreased to 24–25% in SDS.     

A simple and efficient gene transfer system of trifoliate orange (Poncirus trifoliata Raf.)

Summary A simple and efficient gene transfer system of trifoliate orange (Poncirus trifoliata Raf.) was developed using epicotyl segments. The segments were infected with Agrobacterium harboring the binary vector pBI121 or pBI101-O12-p1. Both vectors contained the neomycin phosphotransferase II (NPTII) and the -glucuronidase (GUS) genes. In the plasmid pBI101-O12-p1, the GUS gene was directed to the promoter region of ORF12 (rolC) of the Ri plasmid. On a selection medium containing 100 or 200 g/ml kanamycin, adventitious shoots were formed from 21.7–44.6% of the segments. Histochemical GUS assay showed that 55.4–87.7% of the shoots expressed the GUS gene. The stable integration of this gene was also confirmed by polymerase chain reaction (PCR) analysis and by Southern blot analysis. When the pBI101-O12-p1 plasmid was used, the GUS activity was found to be located in phloem cells of leaf, stem and root. More than 100 transformed plants were obtained using this method within 2–3 months.     

Effects of antibiotics on the elimination of <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis> from loblolly pine (<Emphasis Type="Italic">Pinus taeda</Emphasis>) zygotic embryo explants and on transgenic plant regeneration

Three antibiotics were evaluated for their effects on the elimination of Agrobacterium tumefaciens during the genetic transformation of loblolly pine ( Pinus taeda L.) using mature zygotic embryos as targets. Agrobacterium tumefaciens strains, EHA105, GV3101, and LBA 4404, all harbouring the plasmid pCAMBIA1301, which carries the selectable marker gene, hygromycin phosphotransferase ( hpt) controlled by the cauliflower mosaic virus 35S promoter and terminator, and the uidA reporter gene (GUS) driven by the cauliflower mosaic virus 35S promoter and the terminator of nopaline synthase gene, were used in this study. Exposure to 350 mg l^-1 carbenicillin, claforan, and timentin respectively for up to 6 weeks did not eliminate the Agrobacterium, while antibiotics at 500 mg l^-1 eradicated them from the co-cultivated zygotic embryos. All three antibiotics increased callus growth and shoot regeneration at 350 and 500 mg l^-1 each, but reduced callus growth and shoot regeneration at 650 mg l^-1 when compared with controls. Putative transgenic calli were selected for continued proliferation and differentiation on 4.5 mg l^-1 hygromycin-containing medium. Transformed calli and transgenic plants produced on a selection medium containing 4.5 mg l^-1 hygromycin were confirmed by GUS histochemical assays, by polymerase chain reaction (PCR), and by Southern blot analysis. These results are useful for future studies on optimizing genetic transformation procedures in loblolly pine.     

Promoter sequences from two different Brassica napus tapetal oleosin-like genes direct tapetal expression of β-glucuronidase in transgenic Brassica plants

To investigate the sequences responsible for the regulated expression of tapetal-specific oleosin-like genes, ca. 2 kb of the 5-upstream regions from two divergent genes, OlnB;4 and OlnB;13, were isolated, sequenced and fused to the reporter gene -glucuronidase for study in transgenic Brassica napus plants. Although the proteins encoded by these two genes are highly divergent, except for the conserved oleosin-like domain, the first 250 bp of their 5-upstream regions was 86% identical, including a region of 150 bp upstream from the TATA box. Analysis of 42 independent transformants by histochemical and fluorometric methods showed that both promoters directed tapetal-specific expression that peaked at the 4 mm flower bud stage.     

Selective impairment of protein kinase C isotypes in murine macrophage by Leismania donovani

Leishmania donovani, an obligate intracellular parasite resides and multiplies within macrophage of the reticuloendothelial system. The intracellular signalling mechanism involved in the impaired oxidative response in leishmaniasis has not yet been clearly established. Generation of superoxide anion (O₂ ^–) is supposed to be the first line of host defence during microbial invasion. We found a substantial inhibition of superoxide anion generation in parasitized macrophages, which was just the reverse in case of macrophages challenged with Lipophosphoglycan (LPG) deficient attenuated leishmanial parasite UR-6. The generation of O₂ ^– essentially needs the prior activation of protein kinase C (PKC) mediated phosphorylation events. Our study proposed that phosphorylation of 67, 54, 47 and 36 kDa proteins was attenuated during infection. This was supported by PKC activity study, where Ca-dependent PKC activity was inhibited but, Ca-independent PKC activity was enhanced. This result was further confirmed by using isotype specific pseudosubstrate inhibitors of Ca-dependent PKC and Ca-independent PKC . Application of -pseudosubstrate could not alter the Ca-dependent PKC activity but -pseudosubstrate inhibited the Ca-independent PKC activity in infected macrophages. Our immunoblot analysis with specific antibody against PKC and PKC isotypes showed down regulation of PKC -II expression with concomitant induction of PKC . Such inhibition of Ca-dependent PKC was reversed in macrophages treated with UR-6. Taken together, our observations revealed that infection with L. donovani selectively attenuates both the expression and activity of Ca-dependent PKC .