Spontaneous spectinomycin resistance mutations detected after biolistic transformation of Daucus carota L.

Spectinomycin resistant mutant carrot (Daucus carota L.) callus lines detected in the experiments on biolistic transformation of plastome were analyzed. It has been found that this antibiotic resistance is determined by point nucleotide substitutions at two distinct sites of the chloroplast gene rrn16, coding for 16S rRNA, namely, G1012T, G1012C, and A1138G. The detected mutations are localized to the 16S rRNA region forming helix h34, which contains spectinomycin binding site, and lead to its destabilization by several kilocalories per mole. Comparative analysis of rrn16 gene sequences has demonstrated conservation of the positions of the nucleotide substitutions determining this antibiotic resistance in carrot (D. carota L.), tobacco (Nicotiana tabacum L.), and bladder pod (Lesquerella fendleri L.), as well as in Escherichia coli.     

Spontaneous spectinomycin resistance mutations of the chloroplast <Emphasis Type="Italic">rrn16</Emphasis> gene in <Emphasis Type="Italic">Daucus carota</Emphasis> callus lines

Bioballistic transformation of carrot (Daucus carota L.) callus cultures with a plasmid containing the aadA (aminoglycoside 3′-adenyltransferase) gene and subsequent selection of transformants on a selective medium containing spectinomycin (100–500 mg/l) yielded ten callus lines resistant to this antibiotic. PCR analysis did not detect exogenous DNA in the genomes of spectinomycin-resistant calluses. Resistance proved to be due to spontaneous mutations that occurred in two different regions of the chloroplast rrn16 gene, which codes for the 16S rRNA. Six lines displayed the G > T or G > C transverions in position 1012 of the rrn16 gene, and three lines had the A > G transition in position 1138 of the gene. Chloroplast mutations arising during passages of callus cultures in the presence of spectinomycin were described in D. carota for the first time. The cause of spectinomycin resistance was not identified in one line. The mutations observed in the D. carota plastid genome occurred in the region that is involved in the formation of a double-stranded region at the 3′ end of the 16S rRNA and coincided in positions with the nucleotide substitutions found in spectinomycin-resistant plants of tobacco Nicotiana tabacum L. and bladderpod Lesquerella fendleri L.     

Chlorsulfuron resistance in Daucus carota cell lines and plants:Involvement of gene amplification

Daucus carota L. cell lines stably resistant to the herbicide chlorsulfuron (CS) have been isolated according to a stepwise selection. Studies carried out during different selection steps show that the specific activity of the target enzyme acetohydroxyacid synthase (AHAS) increases along with CS resistance. Southern hybridization analysis performed with aBrassica napus AHAS probe in a CS highly-resistant cell line reveals the presence of a greatly amplifiedEcoRI fragment of genomic DNA. This indicates that AHAS overproduction induced by stepwise selection is due to gene amplification. Regenerants from some resistant cell lines maintained the CS-resistant trait at the whole plant level.     

Spectinomycin resistance mutations in the rrn16 gene are new plastid markers in Medicago sativa

We report here the isolation of spectinomycin-resistant mutants in cultured cells of Medicago sativa line RegenSY-T2. Spectinomycin induces bleaching of cultured alfalfa cells due to inhibition of protein synthesis on the prokaryotic type 70S plastid ribosomes. Spontaneous mutants resistant to spectinomycin bleaching were identified by their ability to form green shoots on plant regeneration medium containing selective spectinomycin concentrations in the range of 25–50?mg/l. Sequencing of the plastid rrn16 gene revealed that spectinomycin resistance is due to mutations in a conserved stem structure of the 16S rRNA. Resistant plants transferred to the greenhouse developed normally and produced spectinomycin-resistant seed progeny. In light of their absence in soybean, a related leguminous plant, the isolation of spectinomycin-resistant mutants in M. sativa was unexpected. The new mutations are useful for the study of plastid inheritance, as demonstrated by detection of predominantly paternal plastid inheritance in the RegenSY-T2?×?Szapko57 cross, and can be used as selective markers in plastid transformation vectors to obtain cisgenic plants.     

Development of embryoids in carrot root callus culture (Daucus carota L.)

Embryogenesis occurred in carrot root callus (Daucus carota L.) cultivated on simple synthetic medium containing IAA and 2,4-D. Embryoid development continued also during successive years when the tissue was cultivated on the same nutrient medium without those substances. Sometimes production of plants with atypical leaves was also observed. In those plants development of adventive embryoids occurred repeatedly. Result of this work confirmed reports about the organogenic potentiality of this species and about its sensitivity to some chemical substances.     

The role of calcium channels in anthocyanin production in callus cultures of Daucus carota

The involvement of Ca²⁺ ATPases in anthocyanin accumulation in callus cultures of Daucus carota was investigated under the influence of calcium and calcium channel modulators. Ionophore (I) treatment enhanced callus growth and anthocyanin accumulation. Increasing the amount of calcium applied to cultures enhanced the anthocyanin level. Ionophore treatment influenced the enhancement of Ca²⁺ATPase and endogenous titres of PAs. Addition of the calcium channel blocker verapamil or the calmodulin antagonist chlorpromazine to the A23187 (ionophore) treated cells caused a reduction in anthocyanin levels. Channel blockers reduced Ca²⁺ATPase activity, which was restored by ionophore treatment, showing the importance of calcium in anthocyanin production. Higher ethylene levels were also found in treatment with ionophore or 2X calcium. Thus the influence of ionophore in anthocyanin production and its inhibition by calcium channel modulators suggests that calcium plays an important role in the production of anthocyanin by carrot callus cultures.     

Synthesis of phenylalanine ammonia-lyase in anthocyanin-containing and anthocyanin-free callus cells of Daucus carota L.

When callus cells of Daucus carota are grown on a medium containing gibberellic acid (GA₃) in a physiological concentration of 3x10^-6 M the cells cease to accumulate anthocyanins. This anthocyanin-free cell line has a very low activity of phenylalanine ammonia-lyase. After density labelling with D₂O an intensive de novo synthesis of the phenylalanine ammonia-lyase (E.C. 4.3.1.5; PAL) in the anthocyanin-containing cells does occur. 58% of the C-bound H-atoms are replaced by deuterium. The anthocyanin-free cells show only a very low enzyme synthesis which is difficult to detect with density labelling experiments. To ascertain that de novo synthesis occurs in the anthocyanin-free cells, the incorporation of ¹⁴C-labelled amino acids into the partially purified enzyme protein was measured after separation of the protein a) in CsCl gradients and b) on polyacrylamide gels. In both cases the enzyme bears ¹⁴C-label. These results suggest that in the anthocyanin-free cells de novo synthesis of PAL is still occuring but the synthesis is reduced in comparison to the anthocyanin-containing cells.Abbreviations GA₃ gibberellic acid - PAL phenylalanine ammonia-lyase (E.C.4.3.1.5) - DCb anthocyanin-containing cells - DCw anthocyanin-free cells     

Historical and contemporary gene dispersal in wild carrot (Daucus carota ssp. carota) populations

Background and Aims

Wild carrot is the ancestor of cultivated carrot and is the most important gene pool for carrot breeding. Transgenic carrot may be released into the environment in the future. The aim of the present study was to determine how far a gene can disperse in wild carrot populations, facilitating risk assessment and management of transgene introgression from cultivated to wild carrots and helping to design sampling strategies for germplasm collections.

Methods

Wild carrots were sampled from Meijendel and Alkmaar in The Netherlands and genotyped with 12 microsatellite markers. Spatial autocorrelation analyses were used to detect spatial genetic structures (SGSs). Historical gene dispersal estimates were based on an isolation by distance model. Mating system and contemporary pollen dispersal were estimated using 437 offspring of 20 mothers with different spatial distances and a correlated paternity analysis in the Meijendel population.

Key Results

Significant SGSs are found in both populations and they are not significantly different from each other. Combined SGS analysis indicated significant positive genetic correlations up to 27 m. Historical gene dispersal σ_g and neighbourhood size N_b were estimated to be 4–12 m [95 % confidence interval (CI): 3–25] and 42–73 plants (95 % CI: 28–322) in Meijendel and 10–31 m (95 % CI: 7–∞) and 57–198 plants (95 % CI: 28–∞) in Alkmaar with longer gene dispersal in lower density populations. Contemporary pollen dispersal follows a fat-tailed exponential-power distribution, implying pollen of wild carrots could be dispersed by insects over long distance. The estimated outcrossing rate was 96 %.

Conclusions

SGSs in wild carrots may be the result of high outcrossing, restricted seed dispersal and long-distance pollen dispersal. High outcrossing and long-distance pollen dispersal suggest high frequency of transgene flow might occur from cultivated to wild carrots and that they could easily spread within and between populations.     

Sequential synthesis of some proteins in cultured carrot explant (Daucus carota) cells during callus induction

Freshly isolated explants of the secondary phloem of carrot roots were exposed to ¹⁴C-leucine for various periods from t₀—to 18 h and the ¹⁴C labelling of protein was studied by 2-dimensional PAGE followed by fluorograph. The labelling pattern of proteins indicated a sequential activation of synthesis of about 130 proteins during the 18 h experimental period prior to the onset of cell division activity.Abbreviations IAA indole acetic acid - 2iP 2-isopentenyladenine - PVP polyvinylpyrrolidone - CBB Coomassie brilliant blue - RuBPCase ribulosebisphosphate carboxylase - LSC liquid scintillation counter - spec.act. specific radioactivity - u.l. uniformly labelled     

Isolation and characterization of Daucus carota L. cell lines resistant to 2-deoxy-D-glucose

Variant carrot (Daucus carota L.) cell lines resistant to the growth inhibitory effects of the glucose analogue 2-deoxy-D-glucose (dGlc) were isolated utilizing a feeder plate technique. Growth of sensitive cells was less than 7.5% of controls on medium supplemented with 3.0 mM dGlc, whereas resistant variants achieved growth ranging from 15% to 70% of that in controls. Increased levels of acid invertase activity in variant cell lines in response to dGlc in the culture medium, together with decreased sensitivity of the acid invertase enzyme (EC 3.2.1.26) to dGlc, is proposed as one of several potential mechanisms contributing to the observed dGlc resistance.     

The effects of excess boron with niacin on Daucus carota L. (carrot) root callus

Niacin (Nicotinic acid, B3 vitamin) may be involved in reduction of toxic effects of boron by regulating growth metabolism. This study was designed to examine whether external niacin treatment would improve the boron mobility in carrot callus cells or not. The results showed that excess boron caused tracheary inversions in meristematic root tissue, and also a shortage was seen in tracheary lengths with boric acid treatment. Boron excess induced the plant tolerance to water stress inverting the tracheary cells. This shortage converted nearly to normal size with niacin and boron treatment together. The results showed that boron mobility induced by niacin could reduce significantly the fresh and dry weight of carrot root cells, protein and ABA content was reduced also, in contrary, external boron and boron with niacin treatment considerable increased the two factors after one month stress. Fresh weight reduction and ABA content reduction indicated that niacin treatment caused water stress on the root cells of carrot, but boron treatment and boron with niacin treatment increased drought tolerance in carrot cells by increasing the both factors. In addition, turning the conversion of the length of the trachearies to their normal size proved that niacin treatment ended the polarizing effects of boron on cell walls.     

Evidence for maternal inheritance of the chloroplast genome in cultivated carrot (Daucus carota L. ssp. sativus)

 The incidence and inheritance of a chloroplast DNA (cpDNA) mutation/marker, BP10U, was studied in crosses among cultivated carrots (Daucus carota ssp. sativus). BP10U is about 400 bp larger than the more common BP10L allele. The occurrence of BP10U among carrot inbreds was widespread. Individual plants exhibited only one form of BP10, and cpDNA inheritance was strictly maternal. BP10U only occurred in male-fertile plants. Some male-fertile inbreds and all cytoplasmically male-sterile (petaloid) carrots had the BP10L allele. Alloplasmic cpDNA variation has been reported previously in Daucus, but this is the first report of variation and inheritance of cpDNA within cultivated carrot. Received: 11 August 1998 / Accepted: 8 September 1998     

Proliferation-dependent pattern of expression of a dihydrofolate reductase-thymidylate synthase gene from Daucus carota

The pattern of expression of a carrot dhfr-ts gene was evaluated in different plant organs, in somatic embryos, and in hypocotyl explants induced to dedifferentiate in vitro by the addition of the synthetic auxin 2,4 dichorophenoxyacetic acid. The promoter of this gene was also placed upstream of a uidA (GUS) reporter gene and, using biolistic and protoplasts transient expression assays, was shown to drive a particularly high level of expression in actively growing suspension cells. The results from these expression analyses combined with the presence of putative cell cycle-related cis-acting elements in the dhfr-ts promoter, strongly point to a cell division-dependent expression of this gene.     

Mitochondrial DNA diversity and male sterility in natural populations of Daucus carota ssp carota

Mitochondrial variability was investigated in natural populations of wild carrot (Daucus carota ssp carota) in different regions: South of France, Greece, and various sites in the Mediterranean Basin and Asia. Total DNA was digested with two restriction endonucleases (EcoRV and HindIII) and probed with three mitochondrial DMA-specific genes (coxI, atp6, and coxII). Twenty-five different mitochondrial types were found in 80 analyzed individuals. Thirteen mitotypes were found among the 7 French populations studied. On average, 4.4 different mitotypes were observed per population, and these mitotypes were well-distributed among the populations. All of the mitochondrial types were specific to a single region. However, the proportion of shared restriction fragments between 2 mitotypes from different regions was not particularly lower than that which occurred among mitotypes from a single region. On the basis of the sexual phenotype [male-sterile (MS) or hermaphrodite] of the plants studied in situ and that of their progeny, 2 mitotypes were found to be highly associated with male sterility. Eighty percent of the plants bearing these mitotypes were MS in situ, and all of these plants produced more than 30% MS plants in their progeny. This association with male sterility was consistent in several populations, suggesting an association with a cytoplasmic male-sterility system. Moreover, these two mitotypes had very similar mitochondrial DNA restriction patterns and were well-differentiated from the other mitotypes observed in wild plants and also from those observed in the two CMS types already known in the cultivated carrot. This suggests that they correspond to a third cytoplasmic sterility.     

The greening of chromoplasts in Daucus carota L.

Summary Evidence was obtained for the transformation of chromoplasts to chloroplasts in the cortex parenchyma of carrot during exposure to light. Typical chromoplasts containing carotene crystals but no lamellar system were observed at the onset of illumination. The ensuing synthesis of chlorophyll and a lamellar system was accompanied by disappearance of the carotene crystals. Only chloroplasts were present after 48 hr in the light. The different stages of plastid development were observed using the electron microscope.