Factors Influencing Embryogenesis in Carrot Cultures (Daucus carota L.)

There is no doubt that isolated vacuolated carrot cells cande-differentiate and give rise to embryogenic clusters fromwhich embryoids arise. However a study of the origins of embryogeniccells in culture suggests that the most frequent source is fromgroups of small meristematic cells liberated from the primaryexplant, and maintained as meristematic cells through numeroussubcultures. Groups of vacuolated cells divide to give riseto callus nodules, which can undergo morphogenesis in a varietyof ways. Transitions from one cell type to another are relativelyinfrequent, and cells generally divide to give rise to cellsof similar type to the parent. The occurrence of a low proportionof embryogenic cells in an inoculum is sufficient to resultin large numbers of embryoids when medium conditions are changedto favour their proliferation and development. The various routesby which plants can arise from carrot cultures are discussed.     

Myo-inositol Biosynthesis and Galactose Utilization by Wild Carrot (Daucus carota L. var. carota) Suspension Cultures

Wild carrot (Daucus carota var. carota) cell suspensions (63–120µm in diameter) were grown on a mineral salt medium containingdifferent carbon sources in the presence (10 mM) and absenceof myo-inositol. The data obtained after 14 and 21 days of growthshow that an external supply of myo-inositol is not essentialfor growth and development of wild carrot embryos. A linearrelationship was found between growth (d. wt) and embryo numberin the presence and absence of myo-inositol. Standard stock cell suspensions never exposed to exogenous myo-inositoland grown in the absence of 2, 4-D with glucose or galactoseas the carbon source synthesized radioactive myo-inositol whenexposed to D-[1–¹⁴C]glucose or D-[1–¹⁴C]galactose.Gas chromatographic analyses revealed the presence of myo-inositolin the bulk tissue grown in the presence of 2.25 µM 2,4-D with glucose, galactose, fructose or mannose as the solecarbohydrate. We could not detect any component indicating anisomer or a methylated derivative of an inositol in the tissueextracts. Stock cultures were maintained (with 2, 4-D) successfully forat least three successive sub-cultures on D-galactose as thesole carbohydrate. The growth achieved over this culture periodshowed that wild carrot cells used by us could quickly adaptto grow on D-galactose as rapidly as they grow on sucrose. Daucus carota L., wild carrot, suspension cultures, myo-inositol, galactose     

The Decline of Embryogenic Potential as Callus and Suspension Cultures of Carrot (Daucus carota L.) are Serially Subcultured

Callus and suspension cultures derived from seedling root segmentsof carrot can be assessed for their embryogenic potential (EP)by transfer of a standard culture inoculum to 25 ml culturemedium with 2,4-D omitted and incubation for a fixed period;the EP is expressed as the number of embryoids (0·5–2·5mm in length) developed per culture under these standard conditions.The initial decline in EP is indicative of increasing sensitivity,as culture proceeds, to inhibition by the auxin essential tocontinuing growth of the cultures. However, during culture changesoccur in the nuclear cytology of the cells leading to the appearanceof cells of impaired or nil totipotency and some such cellsare at a selective advantage so that eventually the cultures,as they are serially subcultured, no longer contain any totipotentcells. Normally the cells of such cultures have chromosome numbersin excess of the diploid complement. Evidence for the view thatthe cultures, as they exhibit declining EP, come to containa mixed population of cells comes from microscopic examinationof the cultures and from the isolation, cytological examinationand assessment of the EP of cell lines isolated by plating.Evidence that cells which lack totipotency and which in singleculture have similar growth rates to totipotent cells may neverthelessbe at a strong selective advantage in mixed culture is presentedfrom a study of the growth, and changing cellular compositionand EP during serial subculture of an artificially-preparedmixed culture initially containing equal numbers of diploidtotipotent cells and tetraploid cells lacking totipotency.     

The Origin and Development of Embryoids in Suspension Cultures of Carrot (Daucus carota)

In suspension cultures of carrot, embryoids are initiated ator near the surface of characteristic cellular aggregates (embryogenicclumps) and are released from these aggregates as free-floatingstructures capable of further development into plantlets. Embryoiddevelopment is promoted by transfer from a medium containing2,4-D to one from which it is omitted. The structure of theembryogenic clumps, in these two media, has been studied bythe thin section technique involving fixation in glutaraldehydeand embedding in glycol methacrylate. The superficial cellsof the clumps are clearly marked off from the central cellsand contain large starch grains, a large central nucleus, abundantcytoplasm and a number of vacuoles. It is from individual superficialcells that the embryoids are seen to arise in the 2,4-D omittedmedium but it has not been possible to distinguish, in advanceof embryogeny, such cells from within the superficial cell layers.The fragmentation of the proliferating clumps in 2,4-D containingmedium and the release of embryoids into the 2,4-D omitted mediumis promoted by some of the superficial cells becoming free ofstarch and undergoing pronounced enlargement. A sequence of segmentations leading from single superficialcells of the clumps to globular embryoids has been traced. Thisdiffers from that reported to occur during early embryogenyfrom the egg cell of carrot. The resemblance to zygote embryogenyis closer from the late globular stage of embryoid development;embryoids then differ from zygotic embryos in their shortersuspensors. Attention is drawn to the initiation of additionalembryoids from the epidermis of some of the embryoids developingin culture.     

Inositol Trisphosphate Metabolism in Carrot (Daucus carota L.) Cells

The metabolism of exogenously added d-myo-[1-³H]inositol 1,4,5-trisphosphate (IP₃) has been examined in microsomal membrane and soluble fractions of carrot (Daucus carota L.) cells grown in suspension culture. When [³H]IP₃ was added to a microsomal membrane fraction, [³H]IP₂ was the primary metabolite consisting of approximately 83% of the total recovered [³H] by paper electrophoresis. [³H]IP was only 6% of the [³H] recovered, and 10% of the [³H]IP₃ was not further metabolized. In contrast, when [³H]IP₃ was added to the soluble fraction, approximately equal amounts of [³H]IP₂ and [³H]IP were recovered. Ca²⁺ (100 micromolar) tended to enhance IP₃ dephosphorylation but inhibited the IP₂ dephosphorylation in the soluble fraction by about 20%. MoO₄²⁻ (1 millimolar) inhibited the dephosphorylation of IP₃ by the microsomal fraction and the dephosphorylation of IP₂ by the soluble fraction. MoO₄²⁻, however, did not inhibit the dephosphorylation of IP₃ by the soluble fraction. Li⁺ (10 and 50 millimolar) had no effect on IP₃ metabolism in either the soluble or membrane fraction; however, Li⁺ (50 millimolar) inhibited IP₂ dephosphorylation in the soluble fraction about 25%.     

Characterization of Inositol Phosphates in Carrot (Daucus carota L.) Cells

We have shown previously that inositol-1,4,5-trisphosphate (IP₃) stimulates an efflux of ⁴⁵Ca²⁺ from fusogenic carrot protoplasts (M Rincón, WF Boss [1987] Plant Physiol 83: 395-398). In light of these results, we suggested that IP₃ might serve as a second messenger for the mobilization of intracellular Ca²⁺ in higher plant cells. To determine whether or not IP₃ and other inositol phosphates were present in the carrot cells, the cells were labeled with myo-[2-³H]inositol for 18 hours and extracted with ice-cold 10% trichloroacetic acid. The inositol metabolites were separated by anion exchange chromatography and by paper electrophoresis. We found that [³H]inositol metabolites coeluted with inositol bisphosphate (IP₂) and IP₃ when separated by anion exchange chromatography. However, we could not detect IP₂ or IP₃ when the inositol metabolites were analyzed by paper electrophoresis even though the polyphosphoinositides, which are the source of IP₂ and IP₃, were present in these cells. Thus, [³H] inositol metabolites other than IP₂ and IP₃ had coeluted on the anion exchange columns. The data indicate that either IP₃ is rapidly metabolized or that it is not present at a detectable level in the carrot cells.     

myo-Inositol Trisphosphate Mobilizes Calcium from Fusogenic Carrot (Daucus carota L.) Protoplasts

To determine whether or not inositol trisphosphate (IP₃) mobilizes calcium in higher plant cells, we investigated the effect of IP₃ on Ca²⁺ fluxes in fusogenic carrot (Daucus carota L.) protoplasts. The protoplasts were incubated in ⁴⁵Ca²⁺-containing medium and the ⁴⁵Ca²⁺ associated with the protoplasts was monitored with time. Addition of IP₃ (20 micromolar) caused a 17% net loss of the accumulated ⁴⁵Ca²⁺ within 4 minutes. There was a reuptake of ⁴⁵Ca²⁺ and the protoplasts recovered to their initial value by 10 minutes. Phytic acid (IP₆), also stimulated ⁴⁵Ca²⁺ efflux from the protoplasts. Both the IP_3⁻ and the IP_6⁻induced ⁴⁵Ca²⁺ efflux were inhibited by the calmodulin antagonist, trifluoperazine.     

Cellular Basis of Tissue Toughness in Carrot (Daucus carota L.) Storage Roots

Carrots are brittle, tending to split and break on harvestingas a result of impacts. Since the phloem tissue of carrot storageroots is largely parenchymatous, there is little to resist thepropagation of a fracture once initiated. In carrots, fracturetoughness is inversely related to water potential, whereas tensilestrength is virtually independent of water status. Fracturetoughness tend to reflect changes in root water potential, buttensile strength increases on two separate occasions despitethe fact that root and cell expansion is accompanied by a reductionin apoplast volume fraction. Possible mechanisms to accountfor carrot tissue toughening towards the end of crop growthare discussed.Copyright 1995, 1999 Academic Press Daucus carota, carrot, toughness, strength, harvest damage, water status, apoplast volume fraction     

Organic Acid Metabolism in Aluminum-Phosphate Utilizing Cells of Carrot (Daucus carota L.)

Carbohydrate metabolism in Al-phosphate utilizing cells of carrot[designated as IPG, Koyama et al. (1992) Plant Cell Physiol.33: 171], which grow normally in Al-phosphate medium accompaniedby citrate excretion, was investigated. The excretion of citratewas strongly related to the availability of sucrose in medium,indicating that citrate excretion was severely limited by sucrosein medium. The ratio of the amount of carbon in the excretedcitrate to the consumed sucrose, was significantly higher inIPG cells than in wild-type cells. When 50% of the sucrose inthe medium was consumed, the ratio was 0.6% for the IPG cellsand 0.2% the wild-type cells. Under these conditions, IPG cellsshowed altered citrate synthesis metabolism, which resultedin increased citrate production. Specific activity of mitochondrialcitrate synthase was higher in IPG cells than in wild-type cells,whereas the activity of cytosolic NADP-specific isocitrate dehydrogenasewas lower in IPG cells than in wild-type cells. (Received August 27, 1998; Accepted February 21, 1999)     

Isolation and Properties of Deoxyribonucleic Acid from Protoplasts of Cell Suspension Cultures of Ammi visnaga and Carrot (Daucus carota)

A procedure is described for the isolation of native DNA from protoplasts of ammi (Ammi visnaga) and carrot (Daucus carota) cells. Protoplasts were produced from 40 grams of fresh cells by enzyme hydrolysis and lysed with sodium dodecyl sulfate. The DNA was purified by treatment with pronase and ribonuclease. Final isolation was achieved by sucrose density gradient centrifugation.     

Transformation and regeneration of carrot (Daucus carota L.)

A protocol is presented for the efficient transformation of carrot (Daucus carota L. cv. Nantaise) by Agrobacterium tumefaciens. The binary vector contained the marker gene -glucuronidase (GUS), driven by the 35S promoter of cauliflower mosaic virus, and the nptII gene, which confers kanamycin resistance. Highest T-DNA transfer rates were obtained by co-cultivating bacteria with hypocotyl segments of dark-grown seedlings on solidified B5 medium containing naphthaleneacetic acid and 6-benzylaminopurine. After 2 days, bacterial growth was stopped with antibiotics. Two weeks later, the explants were placed on agar containing the kanamycin derivate geneticin; antibiotic-resistant calli developed during the following 4 weeks. Suspension cultures were obtained from resistant calli and plants regenerated via somatic embryogenesis in liquid culture. The majority of plants were phenotypically normal and, depending on the Agrobacterium strain used, harbored single or multiple copies of the T-DNA. About equal levels of GUS activity were found in different organs of young plants up to 6 weeks after embryogenesis. In leaves of older plants, GUS activity was markedly reduced, whereas the activities in phloem and xylem parenchyma cells of developing tap roots were still high and fairly uniform. Thus, the 35S promoter may be a useful tool to drive the expression of transgenes in developing carrot storage roots.     

Purification and Interconversion of Homoserine Dehydrogenase from Daucus carota Cell Suspension Cultures

Homoserine dehydrogenase from cell suspension cultures of carrot (Daucus carota L.) has been purified to apparent homogeneity by a combination of selective heat denaturation, ion exchange and gel filtration chromatographies, and preparative gel electrophoresis. Carrot homoserine dehydrogenase is composed of subunits of equal molecular weight (85,000 ± 5,000). During purification, the enzyme exists predominantly in two molecular weight forms, 180,000 and 240,000. The enzyme can be reversibly converted from one form to the other, and each has different regulatory properties. When the enzyme is dialyzed in the presence of 5 millimolar threonine, the purified enzyme is converted into its trimeric form (240,000), which is completely inhibited by 5 millimolar threonine and is stimulated 2.6-fold by K⁺. When the enzyme is dialyzed in the presence of K⁺ and absence of threonine, the purified enzyme is converted into a dimer (180,000), which is not inhibited by threonine and is only stimulated 1.5-fold by K⁺. The enzyme also can polymerize under certain conditions to form higher molecular weight aggregates ranging in size up to 720,000, which also are catalytically active. This interconversion of homoserine dehydrogenase conformations may reflect the daily stream of events occurring in vivo. When light stimulates protein synthesis, the threonine pool decreases in the chloroplast, while K⁺ concentrations increase. The change in threonine and K⁺ concentrations shift the homoserine dehydrogenase from the threonine-sensitive to the threonine-insensitive conformation resulting in increased production of threonine, which would meet the demands of protein synthesis. The reverse process would occur in the dark.     

Root Development and Source-Sink Relations in Carrot, Daucus carota L.

Growth analysis and ¹⁴CO₂ feeding experiments have shown thatthe developing storage organ became an increasingly importantsink for assimilates, accumulating 40% of the dry matter producedby the carrot plant within 9 weeks of sowing. The relative importanceof each leaf in fixing and exporting ¹⁴C was assessed at twostages of development. Morphogenetic responses indicated thatan absence of thickening in the lateral roots was associatedwith continued meristematic activity in the tap root, in theform of an elongating apex or a vascular cambium. Source-sink relations were examined by observing plant growthfollowing the removal of part of the tap root and/or lateralroots. Pruning the roots at 35 d reduced the subsequent growthof the plant by reducing the AGR of the remaining root systemand the shoot. The reduction in leaf growth was associated witha loss of fibrous roots, removal of part of the tap root havingvery little additional effect on shoot growth although the AGRof the root system was reduced by a further 78%. Increased fibrousroot RGRs following pruning soon re-established the normal fibrousroot/shoot ratio. These experiments demonstrated the importanceof the root system in controlling dry matter production in thecarrot plant, but suggested that the sink activity of the developingstorage organ was less significant than other root functions.     

The Germination of Carrot (Daucus carota L.) Seed Harvested on Two Dates: A Physiological and Biochemical Study

The germination of two batches of carrot seed, harvested 44and 104 d after anthesis, was compared at 10 °C. Proteinand nucleic acid contents of the seeds were measured at intervals,together with fresh weight and respiration rate. The matureseed germinated 3.7 d earlier than the immature seed, with nodifference in percentage germination. The dry mature seed containedmore protein and nucleic acid per unit dry matter than did theimmature seed, and proportions of nucleic acid present as rRNAand poly(A)RNA were greater in the mature seed. The sequenceof metabolic reactivation was the same in both batches of seed,as were the relative rates of increase of each nucleic acidcomponent, fresh weight, and respiration rate. Differences inthe composition of the dry seed appear to be responsible forthe observed difference in rate of germination between the twoseed batches.     

Changes in Levels and Integrity of Ribosomal RNA During Seed Maturation and Germination in Carrot (Daucus carota L.)

Thompson, S., Bryant, J. A. and Brocklehurst, P. A. 1987. Changesin levels and integrity of ribosomal RNA during seed maturationand germination in carrot (Daucus carota L.).—J. exp.Bot. 38: 1343–1350. Amounts and integrity (percentage of complete un-nicked molecules)of ribosomal RNA (rRNA) during germination in carrot seed lotsof differing vigour and viability were examined using aqueousand formamide gel electrophoresis. In unaged seed, amounts ofrRNA per seed in dry seed are not related to maturity, vigouror viability. However, rRNA in immature seed is more vulnerableto damage by ageing than rRNA in mature seed; thus, amountsof rRNA per seed in aged seed are indicators of vigour and viability.On imbibition of aged or unaged seed, more rRNA fragmentationoccurs in immature seed than in mature seed, and the time atwhich the effects of synthesis outweigh the effects of degradationis earlier during germination in mature than in immature seed. Key words: rRNA, seed maturation, seed germination, Daucus carota L.     

Surgical Experiments on the Differentiation of Vascular Tissue in the Shoot Apex of Carrot (Daucus carota L.)

Surgical techniques were applied to the shoot apex of carrot(Daucus carota L.) to test the interpretation that provasculartissue is the initial stage of vascular differentiation andto localize the sources of the influences that control its differentiation.If the apex is isolated laterally by vertical incisions leavingit at the summit of a plug of pith tissue, vascular differentiationproceeds normally and an independent vascular system is formedin the pith plug. If all leaf primordia are systematically suppressed,provascular tissue continues to differentiate as an acropetalextension of the pre-existing vascular system but no furtherdifferentiation occurs. When the apex is isolated laterallyand all leaf primordia are suppressed, provascular tissue continuesto be formed acropetally and is extended basipetally into thepith plug by redifferentiation of pith cells, but no furtherdifferentiation occurs. This tissue reacts positively to histochemicaltests for esterase indicating its vascular nature. If only oneleaf primordium is allowed to develop on an isolated shoot apex,its vascular system develops normally and extends basipetallyinto the pith plug, but there is no extension of provasculartissue into the pith plug. These results support the interpretationthat the initial stage of vascular differentiation is controlledby the apical meristem but that further maturation of vasculartissue depends upon influences from developing leaf primordia.Copyright 2000 Annals of Botany Company Provascular tissue, differentiation, carrot (Daucus carota L.), shoot apex, surgical techniques, leaf primordia     

Methylation of mitochondrial DNA in carrot (Daucus carota L.)

The methylation status of carrot (Daucus carota L.) mitochondrial DNA (mtDNA) was studied using isoschizomeric restriction enzymes MspI/HpaII (CCGG) and MvaI/EcoRII [CC(A/T)GG]. Southern hybridisations with probes for mitochondrial genes coxII and atpA were performed. MtDNAs isolated from non-embryogenic cell suspensions and roots were analysed. No differences were found using MspI/HpaII but after digesting the mtDNA with MvaI and EcoRII, some qualitative and quantitative differences between the restriction patterns appeared. Distinction was also revealed after Southern hybridisation with the coxII probe. These data indicate that the mtDNA of carrot is methylated in CNG trinucleotides and unmethylated in CG dinucleotides in CCGG sequences. The results were reproducible for cell suspensions of various genotypes and even cultivars but the extent of methylation was different in the root. The possible role of methylation in the mitochondrial genome of higher plants is discussed. Received: 16 April 1997 / Revision received: 4 July 1997 / Accepted: 30 July 1997