The 2,4-D-induced wheat para-nodules are modified lateral roots with structure enhanced by rhizobial inoculation

Nodular outgrowths (para-nodules or p-nodules) on the roots of wheat (Triticum aestivum L.) cv. Miskle seedlings were induced by treatment with 0.3 and 0.6 mg L^-1, 2,4-dichlorophenoxyacetate (2,4-D). When co-inoculated with Rhizobium trifolii strain ATCC 14480, more p-nodules were formed at these levels and p-nodulation occured at 0.1 mg L^-1 indicating that inoculation enhances 2,4-D-induced p-nodulation. Similar to lateral roots, the p-nodules arose from the pericycle opposite the phloem tissues and were free from the cortical cells of the parental root at all stages of development. Structurally, the p-nodules exhibited tissue differentiation. They possessed a highly organized central vascular cylinder connected to that of the parent root, an endodermis, a cap, and an apical and lateral meristems. P-nodules formed by 2,4-D treatment alone were irregularly lobed due to uncoordinated activity of the apical meristem, while those in the combined 2,4-D and inoculation treatment were more globose. The results of the present study indicate that the 2,4-D-induced wheat p-nodules are modified lateral roots, the structure of which is enhanced by rhizobial inoculation.     

Colonization of wheat by Azospirillum brasilense Cd is impaired by saline stress

The effect of saline stress on the colonization of wheat was analyzed by using Azospirillum brasilense Cd carrying the fusion of the reporter gene lacZ (β-galactosidase) with the N₂ fixation gene promoter nifA. Colonization was also studied by inducing para-nodules on wheat roots using 2,4-D, establishing that these structures acted as bacterium protected niches. Bacteria grown under standard conditions were distributed along the whole root system, except the elongation zone, and colonized the para-nodules. Bacteria experiencing saline stress were mainly localized at the root tips and the lateral roots. In 2,4-D treated plants, most of the bacteria were present around the basal surface of the modified lateral root structures. Using the MPN method, there were not statistical differences between the numbers of control and stressed bacteria. As this method estimates endophytic colonization in contrast with the one using X-gal, which emphasizes colonization on the root surface, both procedures demonstrated to be necessary, concluding that salt treatment reduced surface colonization (X-gal) but not colonization inside the root. The bacterial counts made on inoculated wheat roots indicated higher numbers of both control and stressed bacteria in roots treated with 2,4-D compared with untreated roots. This revised version was published online in June 2006 with corrections to the Cover Date.     

Nitrogen fixation inpara-nodules of wheat roots by introduced free-living diazotrophs

Nitrogen-fixation (C₂H₂-reduction) was demonstrated in wheat root nodules (p-nodules) induced by 2,4-dichlorophenoxyacetate (2,4-D) and inoculated withA. brasilense. By lowering the O₂ tension it was possible to distinguish the nitrogenase activity of bacteria located within thep-nodule of the wheat root system from that in the rhizosphere. Using cytological evidence, nitrogenase activity was attributed mainly to be coming from the bacteria within thep-nodule. It was also shown that the host plant was able to supply the necessary substrate required for the bacterial N₂-fixation (C₂H₂-reduction) within thep-nodules.     

Effects of plant growth regulators on acetylene-reducing associations between Azospirillum brasilense and wheat

Treatment of wheat seedlings with the synthetic auxin, 2,4-dichlorophenoxyacetic acid (2,4-d), induced nodule-like structures or tumours (termed para-nodules) where lateral roots would normally emerge. The formation of these structures promoted increased rates of acetylene reduction at reduced oxygen pressure (0.02–0.04 atm) in seedling inoculated with Azospirillum brasilense, compared to seedlings inoculated without auxin treatment. Fluorescent microscopy, laser scanning confocal microscopy and direct bacterial counts all showed that the 2,4-d treatment stimulated internal colonization of the root system with azospirilla, particularly in the basal region of the nodular structures. Both colonization with azospirilla and acetylene-reducing activity were further stimulated by simultaneous treatment with another synthetic auxin, naphthaleneacetic acid (NAA) and, less reliably, with indoleacetic acid (IAA) and indolebutyric acid (IBA). These auxins produced shortening of many initiated lateral roots, although 20 times the concentration of NAA was required to achieve rounded structures similar to those obtained with 2,4-d. Treatment with NAA, IAA or IBA alone also stimulated colonization with azospirilla and acetylene reduction rates at 0.02 atm oxygen, but less effectively than by treatment with 2,4-d. Such exogenous treatments of wheat seedlings with synthetic growth regulators provide an effective laboratory model for studies on the development of a N₂-fixing system in cereals.     

Genetic transformation of oilseed rape (Brassica napus) by the Ri T-DNA of Agrobacterium rhizogenes and analysis of inheritance of the transformed phenotype

Summary Genetically transformed repeseed (Brassica napus) roots were obtained by in vitro inoculation of excised stem segments with Agrobacterium rhizogenes. Axenic root organ clones were established and they exhibited a phenotype characteristic of transformed roots: rapid growth, reduced apical dominance and root plagiotropism. Stem regeneration was induced by exposing root fragments to 2,4-dichloroacetic acid (2,4-D) in liquid medium, followed by transfer to solid regeneration medium. The resulting plants exhibited the transformed phenotype observed in other species where similar experiments have been performed. Direct evidence for genetic transformation was obtained from opine assays and molecular hybridization. Sexual transmission of the transformed phenotype was Mendelian, and a probable case of T-DNA insertion into two independent loci within the same plant was detected. The estimated optimal time necessary to obtain transformed oilseed rape plants using this approach is 2 months.     

The effect of plant growth regulators on growth, morphology and condensed tannin accumulation in transformed root cultures of Lotus corniculatus

This study concerns the effects of four different classes of plant growth regulators on root morphology, patterns of growth and condensed tannin accumulation in transgenic root cultures of Lotus corniculatus L. (Bird's-foot trefoil). Growth of transformed roots in 2,4-dichlorophenoxyacetic acid (2,4-D) resulted in decreased tannin levels relative to controls at concentrations of 10^-6 M and above, while gibberellic acid (GA₃) inhibited tannin accumulation at concentrations of 10^-7 M and above. Benzyladenine (BA) had little effect at low concentrations (10^-7 M and below) but resulted in an increase in tannin levels at 10^-6 M. Abscisic acid had little effect on levels of condensed tannins at any of the concentrations used. Experiments involving growth regulator addition and medium transfer demonstrated that 2,4-D inhibition of tannin accumulation could be reversed by GA₃ and BA, while GA₃ downregulation could only be reversed by the addition of 2,4-D. Although 2,4-D inhibited tannin accumulation, addition of 2,4-D to root cultures grown for 14 or 28 days in the absence of plant growth regulators stimulated both growth and tannin biosynthesis. Characteristic alterations in root morphologies accompanied growth regulator-mediated modulation of tannin biosynthesis. Growth in 2,4-D resulted in partially de-differentiated root cultures while growth in GA₃ produced roots with an elongated phenotype. Restoration of tannin biosynthesis in 2,4-D-treated roots was accompanied by root re-differentiation and the production of new lateral roots.Abbreviations ABA abscisic acid - BA benzyladenine - 2,4-d 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid 3 - FW fresh weight     

Growth and steroidal saponin production in hairy root cultures of Solanum aculeatissimum

Summary Hairy root cultures of Solanum aculeatissimum were established by trans-formation using Agrobacterium rhizogenes strain 15834. Root growth and production of steroidal saponin were investigated under various culture conditions. Transformed roots grew better in Gamborg's B5 medium containing 3 % sucrose under continuous light than in the dark. Also, the roots turned light green when cultured under continuous light. Green hairy roots produced aculeatiside A (6.71mg ·) L^–1 and aculeatiside B (6.39mg · L^–1) after 8 weeks of culture, while no steroidal saponin was detected in hairy roots cultured in the dark. Of the three culture media tested, Gamborg's B5 medium was superior for growth and steroidal saponin production. Growth and steroidal saponin production were enhanced when 100g · L^–1 auxin except for 2,4-D was added to the medium. The addition of 2,4-D inhibited growth. Production of steroidal saponin was highest with NAA. Transformed roots used in this experiment were confirmed that hairy roots examined contain both TL-DNA and TR-DNA region of Ri plasmid by PCR amplification analysis of DNA.Abbreviations MS medium Murashige and Skoog's medium (1962) - B5 medium Gamborg's B5 medium (1968) - LS medium Linsmaier and Skoog's medium(1965) - HPLC High performance liquid chromatography - NAA -Naphthaleneacetic acid - IAA Indole-3-acetic acid - 2,4-D 2,4-Dichlorophenoxyacetic acid - PCR polymerase chain reaction     

Development and function ofAzospirillum-inoculated roots

Summary The surface distribution ofAzospirillum on inoculated roots of maize and wheat is generally similar to that of other members of the rhizoplane microflora. During the first three days, colonization takes place mainly on the root elongation zone, on the base of root hairs and, to a lesser extent, on the surface of young root hairs.Azospirillum has been found in cortical tissues, in regions of lateral root emergence, along the inner cortex, inside xylem vessels and between pith cells. Inoculation of several cultivars of wheat, corn, sorghum and setaria with several strains ofAzospirillum caused morphological changes in root starting immediately after germination. Root length and surface area were differentially affected according to bacterial age and inoculum level. During the first three weeks after germination, the number of root hairs, root hair branches and lateral roots was increased by inoculation, but there was no change in root weight. Root biomass increased at later stages. Cross-sections of inoculated corn and wheat root showed an irregular arrangement of cells in the outer layers of the cortex. These effects on plant morphology may be due to the production of plant growth-promoting substances by the colonizing bacteria or by the plant as a reaction to colonization. Pectic enzymes may also be involved. Morphological changes had a physiological effect on inoculated roots. Specific activities of oxidative enzymes, and lipid and suberin content, were lower in extracts of inoculated roots than in uninoculated controls. This suggests that inoculated roots have a larger proportion of younger roots. The rate of NO^– ₃, K⁺ and H₂PO^– ₄ uptake was greater in inoculated seedlinds. In the field, dry matter, N, P and K accumulated at faster rates, and water content was higher inAzospirillum-inoculated corn, sorghum, wheat and setaria. The above improvements in root development and function lead in many cases to higher crop yield.     

Establishment of Orthosiphon stamineus Cell Suspension Culture for Cell Growth

Callus of Orthosiphon stamineus could be induced successfully from petiole, leaf and stem tissues but not roots when cultured on MS medium containing different concentration of NAA (0–4.0 mg l^–1) and 2,4-D (0–2.0 mg l^–1). Highest fresh weight callus production was obtained from leaf explants and those with best friability were obtained on MS medium plus 1.0 mg l^–1 2,4-D plus 1.0 mg l^–1 NAA. Cell suspension cultures were established from these cultures. The appropriate cell inoculum size for the best cell growth was 0.75 g of cells in 20 ml culture medium. Cell suspension culture using MS medium supplemented with 1.0 mg l^–1 2,4-D promoted the best cell growth with maximum biomass of 8.609 g fresh weight and 0.309 g dry weight 24 days after inoculation. Cells that grew in MS medium supplemented with 1.0 mg l^–1 2,4-D reached the stationary growth phase in 15 days as compared to the cells that grew in MS medium supplemented with 1.0 mg l^–1 2,4-D + 1.0 mg l^–1 NAA reached the stationary phase in 24 days. MS medium supplemented with 1.0 mg l^–1 2,4-D was considered as the maintenance medium for maintaining the optimum cell growth of O. stamineus in the cell suspension cultures with 2-week interval subculture.     

植物激素和接种方式对广西金线莲壮苗生根的影响

该文以广西野生金线莲无菌播种离体茎段为材料,采用单因素对比试验,研究了植物激素(NAA、IBA、6-BA、GA_3、KT、ZT、TDZ、2-IP)以及接种方式(竖直接种和水平接种)对壮苗生根培养的影响。结果表明:与对照相比,生长素有利于壮苗生根,NAA的效果优于IBA;细胞分裂素对壮苗生根的效果依次为6-BATDZKT=ZT 2-IPCK,其中6-BA诱导平均株高8.4 cm,3.6条根,茎粗为2.84 mm,植株生长健壮,诱导效果最好;赤霉素GA_3诱导出的植株高且直,但植株细弱,且抑制根系生长,不利于壮苗生根培养;在激素组合6-BA 0.5 mg·L~(-1)、NAA1.0 mg·L~(-1)处理中,组培苗生长健壮且根数量较多,效果最佳;水平接种能诱导出更多的根系,且便于接种操作,可以节省接种时间。因此,确定广西金线莲最适宜的壮苗生根培养基配方为1/2MS+6-BA 0.5 mg·L~(-1)+NAA 1.0 mg·L~(-1)+香蕉汁100 g·L~(-1)+AC(活性炭) 1.0 g·L~(-1)+蔗糖20 g·L~(-1),最佳接种方式为水平接种。     

三种除草剂对五爪金龙的防除作用及2,4-D丁酯对环境的影响

用3种除草剂(2,4-D丁酯、麦草畏和塔隆)对五爪金龙(Ipomoea cairica L.Sweet)进行化学防除试验。结果表明:1.00 mL L-1的2,4-D丁酯可以彻底杀灭五爪金龙。喷施1.00 mL L-12,4-D丁酯20 d后,五爪金龙茎叶枯死率接近100%;60 d后五爪金龙的总生物量显著低于其它处理及对照;90 d后未出现生长恢复,最终盖度防效为99.8%。而喷施1.00 mL L-1的麦草畏40 d后,五爪金龙的茎叶枯死率为99.0%,但仍有少量存活的根,90 d后再次萌生率为10.0%;喷施1.00 mL L-1塔隆40 d后,五爪金龙的茎叶枯死率为100%,90 d后再次萌生率为100%。土壤残留分析表明:在有机质含量较高[(10.14±1.01)g kg-1]的土壤中2,4-D丁酯降解速率较快,半衰期为14 d,施药后80 d的土壤中已检测不到2,4-D丁酯。此外,在野外喷洒1.0 mL L-1的2,4-D丁酯对其它植物是安全的,施药1年后,样地内的植物均能恢复生长。因此,实践中可用1.00 mL L-1的2,4-D丁酯来防除五爪金龙。     

Repetitive somatic embryogenesis and plant recovery in white clover

Summary Breeding and selection was used to generate a population of white clover (Trifolium repens L.) from cultivar Osceola with a high embryogenic capacity. Somatic embryos were obtained from immature cotyledons of white clover placed onto EC6 basal medium containing 40 mg L^–1 of 2,4-D and 6% sucrose. The effects of 2,4-D at 20 and 40 mg L^–1 and of the carbohydrates, sucrose and maltose, were evaluated for their influence in the establishment of repetitive somatic embryogenesis. To determine the optimal protocol for plant recovery from somatic embryos, the effects of MS vs. EC6 basal salts, sucrose vs. maltose, B5 vitamins vs. yeast extract, and inclusion or exclusion of activated charcoal were evaluated. Repeated subculture of white clover somatic embryos on EC6 basal medium containing 6% sucrose with 2,4-D at 20 or 40 mg L^–1 effectively maintains repetitive embryogenesis. Medium containing MS salts with 6% maltose as the carbohydrate source was the most efficient for plant recovery.     

Accumulation of 14C from exogenous labelled auxin in lateral root primordia of intact pea seedlings (Pisum sativum L.)

When [¹⁴C]indol-3yl-acetic acid was applied to the apical bud of 5-day old dwarf pea seedlings which possessed unbranched primary roots, a small amount of ¹⁴C was transported into the root system at a velocity of 11–14 mm h^-1. Most of the ¹⁴C which entered the primary root accumulated in the young lateral root primordia, including the smallest detectable (20–30 mm from the primary root tip). In older (8-d old) seedlings in which the primary root bore well-developed lateral roots, ¹⁴C also accumulated in the tertiary root primordia. In contrast, little ¹⁴C was detected in the apical region of the primary root or, in older plants, in the apices of the lateral roots.Abbreviations IAA indol-3yl-acetic acid     

The integration of whole-root and cellular hydraulic conductivities in cereal roots

The hydraulic conductivities of excised whole root systems of wheat (Triticum aestivum L. cv. Atou) and of single excised roots of wheat and maize (Zea mays L. cv. Passat) were measured using an osmotically induced back-flow technique. Ninety minutes after excision the values for single excised roots ranged from 1.6·10^-8 to 5.5·10^-8 m·s^-1·MPa^-1 in wheat and from 0.9·10^-8 to 4.8·10^-8 m·s^-1·MPa^-1 in maize. The main source of variation was a decrease in the value as root length increased. The hydraulic conductivities of whole root systems, but not of single excised roots, were smaller 15 h after excision. This was not caused by occlusion of the xylem at the cut end of the coleoptile. The hydraulic conductivities of epidermal, cortical and endodermal cells were measured using a pressure probe. Epidermal and cortical cells of both wheat and maize roots gave mean values of 1.2·10^-7 m·s^-1·MPa^-1 but in endodermal cells (measured only in wheat) the mean value was 0.5·10^-7 m·s^-1·MPa^-1. The cellular hydraulic conductivities were used to calculate the root hydraulic conductivities expected if water flow across the root was via transcellular (vacuole-to-vacuole), apoplasmic or symplasmic pathways. The results indicate that, in freshly excised roots, the bulk of water flow is unlikely to be via the transcellular pathway. This is in contrast to our previous conclusion (H. Jones, A.D. Tomos, R.A. Leigh and R.G. Wyn Jones 1983, Planta 158, 230–236) which was based on results obtained with whole root systems of wheat measured 14–15 h after excision and which probably gave artefactually low values for root hydraulic conductivity. It is now concluded that, near the root tip, water flow could be through a symplasmic pathway in which the only substantial resistances to water flow are provided by the outer epidermal and the inner endodermal plasma membranes. Further from the tip, the measured hydraulic conductivities of the roots are consistent with flow either through the symplasmic or apoplasmic pathways.Symbols L _{p, cell} cell hydraulic conductivity - L _{p, root} root hydraulic conductivity - L _{p, root} calculated root hydraulic conductivity - root reflection coefficient     

Valepotriates accumulation in callus, suspended cells and untransformed root cultures of Valeriana glechomifolia

Summary Valeriana glechomifolia is an endemic species of southern Brazil, capable of accumulating, in all of its organs, the terpene derivatives known as valepotriates, the presumed sedative components of the roots of pharmaceutically used species of Valeriana. In vitro cultures of the plant were established and the accumulation of acevaltrate, didrovaltrate, and valtrate in callus, cell suspension, and untransformed root cultures was studied. Leaves of in natura plants and roots of micropropagated plantlets were used as the explants for callus induction and root culture establishment, respectively, on Gamborg B5 basal medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) alone or with kinetin (KIN). Culture growth and secondary metabolite yields were enhanced with 2,4-D (4.52μM) and KIN (0.93μM). Maximum valepotriate contents, quantified by HPLC, of acevaltrate (ACE) 2.6mg g⁻¹ DW, valtrate (VAL) 10.2mgg⁻¹ DW, and didrovaltrate (DID) 2.9mg g⁻¹ DW were observed in root cultures after 7–8wk of culture.     

Recovery of phenotypically normal transgenic plants of Brassica oleracea upon Agrobacterium rhizogenes-mediated co-transformation and selection of transformed hairy roots by GUS assay

In this paper we describe the production of transgenic broccoli and cauliflower with normal phenotype using an Agrobacterium rhizogenes-mediated transformation system with efficient selection for transgenic hairy-roots. Hypocotyls were inoculated with Agrobacterium strain A4T harbouring the bacterial plasmid pRiA4 and a binary vector pMaspro::GUS whose T-DNA region carried the gus reporter gene. pRiA4 transfers T_L sequences carrying the rol genes that induce hairy root formation. Transgenic hairy-root production was increased in a difficult-to-transform cultivar by inclusion of 2,4-D in the medium used to resuspend the Agrobacterium prior to inoculation. Transgenic hairy roots could be selected from inoculated explants by screening root sections for GUS activity; this method eliminated the use of antibiotic resistance marker genes for selection. Transgenic hairy roots were produced from two cauliflower and four broccoli culivars. Shoots were regenerated from transgenic hairy root cultures of all four cultivars tested and successfully acclimatized to glasshouse conditions, although some plants had higher than diploid ploidy levels. Southern analysis confirmed the transgenic nature of these plants. T₀ plants from seven transgenic lines were crossed or selfed to produce viable seed. Genetic analysis of T₁ progeny confirmed the transmission of traits and revealed both independent and co-segregation of Ri T_L-DNA and vector T-DNA. GUS-positive phenotypically normal progeny free of T_L-DNA were identified in three transgenic lines out of the six tested representing all the cultivars regenerated including both cauliflower and broccoli.     

Variation in structure and plant regeneration of Agrobacterium rhizogenes transformed and control roots of the potato cv. Bintje

Agrobacterium rhizogenes transformed and control roots of the tetraploid potato cv. Bintje were compared. Transformed roots were obtained after infection by A. rhizogenes 15834 or 1855. Both in leaf and stem segments, more roots were formed at the basal side of the segments, indicative for a polarity in root formation. As compared to control roots the transformed roots are characterized by smaller and more densely stained cells, a zone of cell division, and smaller statoliths. These characteristics are correlated with vigorous growth, high branching incidence and diminished geotropism. The plant regeneration procedure according to Ooms et al. [1] was modified. The transformed roots required less 2,4-D than control roots for the induction of shoot-competent calli. The callus and shoot induction phases were reduced from 8 and 6 weeks to 3 and 3 weeks, respectively. Upon induction, 25%, 58% and 61% of the root clones originating from tuber, stem and leaf, respectively, produced shoots, whereas all of the control roots produced shoots. Shoot outgrowth occurred on liquid MS medium in the absence of hormones.Abbreviations Ri-root Agrobacterium rhizogenes transformed root - BAP benzylaminopurine - IAA indoleacetic acid - GA₃ gibberellic acid - NAA naphthaleneacetic acid - 2,4-D 2,4 dichlorophenoxyacetic acid     

Genotypic variability for callus formation and plant regeneration in rice (Oryza sativa L.)

Summary Sixty rice varieties (Oryza sativa L.), belonging to three subspecies, japonica, indica and javanica (some japonicaXindica hybrids were included), were compared for their capacity for callus growth and plant regeneration. Tissue cultures initiated from mature seeds on Murashige and Skoog's (1962) medium with 2 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) were transferred to a medium containing 0.02 mg/l 2,4-D and 10 mg/l kinetin, from which plantlets were regenerated. Large variabilities in callus growth and plant regeneration potentials were revealed among the varieties tested. Most japonica varieties formed a callus that weighed more than 100 mg per seed 30 days after inoculation, and showed a relatively high regenerative potential, whereas indica varieties, japonica-indica hybrids and javanica varieties showed poor callus growth and plant regeneration, although considerable varietal variation was observed in each subspecies. The callus growth potential was not correlated with the plant regeneration potential. Histological observations revealed that the epithelium cells of the scutellum mainly proliferated to form a callus, from which shoot and root primordia were differentiated independently from each other. The shoot primordia developed into plantlets when roots were formed adventitiously. In a few cases, shoots and roots were bilaterally initiated from a single primordium.     

Auxin-induced rapid changes in translatable mRNAs in tobacco cell suspension

When 2,4-dichlorophenoxyacetic acid (2,4-D)-dependent tobacco cell suspensions, one normal and one transformed by Agrobacterium tumefaciens, were subcultured on hormone-lacking medium the stationary phase of the cell cycle was reached earlier than on medium containing 2,4-D. Addition of the auxin 2,4-D could restore cell division activity within 10–12 h for the most rapidly reacting cell line. The cell-division response was characterized as being auxin-specific and optimal with 2,4-D at 2.2 10^-6 M. Although the cell lines used showed different characteristics, both reacted with a rapid increase in at least three mRNA species within 1 or 2 h after 2,4-D application. Two, 2,4-D-induced protein spots, seen after in-vitro translation, had the same characteristics (MWs 35 kilodaltons (kDa) and 25 kDa with isoelectric points of 7.1 and 6.3, respectively) in both cell lines. Water-treated controls did not show alterations in the translatable mRNA populations. This indicates that the accumulation of the corresponding mRNAs is an early hormone-induced event. Since cell division is the only measurable reaction found after auxin application, cell systems as described here offer excellent possibilities for studying early auxin-induced changes at the molecular level preceding mitosis.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - kDa kilodalton     

The contribution of different regions of the seminal roots of wheat to uptake of nitrate from soil

A technique was developed to determine the physiological activity of defined sections of seminal roots of wheat grown in sand. Wheat plants were grown for 2 weeks in narrow columns of N-deficient sand to which all other nutrients had been added. The columns were split longitudinally and ¹⁵N-labelled nitrate, in an agar medium, supplied to 2 cm sections of root. Shoots and roots were analysed after 24 h to determine the uptake of ¹⁵N. Three sections were examined on either the secondary or tertiary seminal root: 1 cm from the seed (basal segment), 35 cm from the seed (middle segment) and 4 cm from the root apex (apical segment). Total uptake was greatest from the basal and middle segments, declining by 50% from the apical segment. However, uptake per unit root length, including exposed sections of lateral roots, was not significantly different along the root.