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Rhizophores arise from angle meristems located at shoot branch points in many species of Selaginella and produce roots at their distal ends. We reinvestigated the origin and development of rhizophores and roots in Selaginella martensii using a nondestructive sequential replica technique for scanning electron microscopy and anatomical studies of sectioned material. We followed development in excised branch systems containing undetermined ventral angle meristems Analysis of patterns of division and expansion of cells on the surface of apical meristems and in transverse and longitudinal sections showed that the outgrowth from a ventral angle meristem had three distinct developmental phases: rhizophore, aerial root, and subterranean root, each with a distinctive apical meristem structure and activity. Rhizophore formation involved division of all cells in the angle meristem and establishment of multiple surface initial cells from which rhizophore growth ensued. The aerial root phase began with formation of two internal root meristems, each with a single prominent apical cell, replacing the multiple surface initials. From these meristems, dichotomously branched aerial roots formed, which continued to grow and branch internally. Eventually, subterranean roots formed, bearing root hairs and root caps. Formation of subterranean roots was induced at any time after aerial roots reached 0.5-1 cm in length when the tip of the organ touched a substrate. Our data provided support for the original concept of the rhizophore as a unique root-bearing organ in S. martensii, as well as for a developmental succession of apical meristem structure and activity, specific forms of which characterized the rhizophore and true root stages of angle meristem outgrowth.

The effect of concurrent salinity (0-60 mM NaCl) and rootzone hypoxia (flooding for up to 15 d) on shoot and root growth and shoot ion concentrations of six species of Trifolium (T. subterraneum L., T. fragiferum L., T. michelianum Savi., T. isthmocarpum Bot., T. purpureum Lois., and T. repens L.), was studied in two greenhouse experiments. There was a significant salinity × flooding effect for shoot yield but no significant salinity × flooding × species interaction although individual species differed significantly (P < 0·001) in their growth response to the saline or flooded conditions separately. Concentrations of Na and Cl in the shoots of all species increased with increasing periods of saline flooding and there was a significant salinity × flooding interaction. Sodium and Cl concentrations were significantly higher (P < 0·001) in T. purpureum, the species in which shoot growth was most depressed by saline flooding, than other species. In T. michelianum, T. fragiferum and T. repens, fresh and dry weight of roots increased with flooding under both saline and non-saline conditions while in T. subterraneum root growth decreased. A significant proportion of the increased root growth in the first three species occurred as new adventitious roots. These roots had higher percentages of internal gas spaces within the root tissue even in the presence of salinity compared with roots from non-flooded conditions. There were also significantly more gas spaces in the total root tissue in T. fragiferum and T. repens under saline-flooding than in roots of T. subterraneum. Electron micrographs of the root cross sections illustrated the presence of these gas spaces or aerenchyma. Trifolium fragiferum, T. repens and T. michelianum are more likely to be suited to growth in soils prone to high salinity and to flooding than are T. subterraneum, T. purpureum and T. ishmocarpum.

Background and aims This work aimed to quantify the critical external requirement for phosphorus (P) (i.e. extractable-P concentration required for 90% of maximum yield) for a number of temperate legume species and understand differences in dry matter allocation, P distribution and P acquisition efficiency among these species. Methods Shoot and root growth of five legume and one grass species was assessed in response to six rates of P mixed into the top 45 mm of soil in a pot experiment. Dactylis glomerata and Trifolium subterraneum were used as benchmark species; they are commonly grown together in mixed temperate pastures and have low and high critical external requirements for P, respectively. Growth was compared with four alternative legume species: Ornithopus compressus, Ornithopus sativus, Biserrula pelecinus and Trifolium hirtum, that have root morphologies better suited to soil exploration and nutrient acquisition than that of Trifolium subterraneum. Results Dactylis glomerata, Ornithopus compressus and Ornithopus sativus had maximum yields equal to or greater than Trifolium subterraneum but achieved this at rates of P less than half that of Trifolium subterraneum. Biserrula pelecinus and Trifolium hirtum had critical P requirements between that of Trifolium subterraneum and the Ornithopus species, but also had lower yields. Root dry matter of Dactylis glomerata and the Ornithopus species in the fertilised soil layer was only marginally changed in response to low P supply. In contrast, Trifolium subterraneum, Trifolium hirtum and to a lesser extent Biserrula pelecinus markedly increased root dry matter allocation to this soil layer. Species with lower critical P requirements were able to take up more P per unit root dry mass than those with higher critical P requirements, particularly at lower levels of P addition. Conclusions The high P acquisition efficiencies of the Ornithopus species and Dactylis glomerata were likely to have contributed to their low critical external P requirements. It was surmised that differences in root morphology traits underpin the differences in acclimation to low P stress and P acquisition efficiency among the species.

In the absence of visual signals, subterranean arthropods rely on olfactory and tactile cues to navigate toward resources. Here, in a series of pairwise dual-choice bioassays, we investigated the Limonius californicus (Col., Elateridae) larva response to wheat, pea, and bean seedlings in sand-filled olfactometers. We then quantified volatile organic compounds (VOC) emitted from roots. Wireworm preference for beans compared to wheat was attributed to the higher CO 2 emission. Wireworm preference for peas compared to wheat was attributed to the higher amounts (µg/hr) of hexanal emitted from pea roots. Wireworms preferred synthetic hexanal over clean air control and the higher amount of hexanal (200 µg) over the lower amount of 20 µg. In the presence of CO 2 at both ends of the olfactometer, wireworms did not respond preferentially toward hexanal. 2-Hexenal was also attractive to wireworms relative to the control, but wireworms did not discriminate between hexanal and 2-hexenal. While our results confirmed wireworms’ positive response to the presence of CO 2 and some VOCs in isolation, their host choice appears to be driven by the combination and the concentrations of the present cues, allowing the insect to distinguish among host plants.

Background and aims The growth and root morphology responses to soil phosphorus (P) fertility by five cultivars of Trifolium subterraneum (a temperate annual pasture legume) were examined to assess whether differences in root morphology and/or root acclimation to P stress influenced P-acquisition by the clover varieties, or their critical P requirements (i.e. the rate of P supply needed for maximum shoot yield). Methods The clovers were grown as microswards in soil with P stratified in a topsoil layer to mimic growth conditions and soil P availability in a pasture. Yield and P content of shoots, and roots from the topsoil and subsoil layers was determined after 5 weeks growth in a controlled-environment cabinet. The lengths, diameters, and root hair lengths of nutrient foraging roots from the topsoil layer were quantified. Results The shoot yield of the cultivars was similar when grown with high soil P fertility. However, the cultivars varied up to 1.5-fold in their ability to yield at low levels of soil P supply, and by 1.6-fold in their critical P requirements. All cultivars acclimated to low P soil by increasing root length density in the topsoil but those that yielded relatively well did so by maintaining a large root hair cylinder volume (i.e. they explored more soil) under low soil P conditions. This was associated with maintenance of dry matter allocations to topsoil roots and higher specific root lengths. Both factors assisted development of high root length density for nutrient foraging. Root acclimation responses to P were compared among the cultivars at equivalent relative shoot yields to assess the influence of plant P stress as a trigger for nutrient foraging. The least P-efficient cultivars slowed their allocation of dry matter to foraging roots at lower levels of P stress. Conclusions The results suggest variation within T. subterraneum for root proliferation and specific root length could be targeted in breeding for improved P-acquisition efficiency.

Subterranean clover (Trifolium subterraneum) is the most widely grown annual pasture legume in southern Australia. With the advent of advanced sequencing and genome editing technologies, a simple and efficient gene transfer protocol mediated by Agrobacterium tumefaciens was developed to overcome the hurdle of genetic manipulation in subterranean clover. In vitro tissue culture and Agrobacterium transformation play a central role in testing the link between specific genes and agronomic traits. In this paper, we investigate a variety of factors affecting the transformation in subterranean clover to increase the transformation efficiency. In vitro culture was optimised by including cefotaxime during seed sterilisation and testing the best antibiotic concentration to select recombinant explants. The concentrations for the combination of antibiotics obtained were as follows: 40 mg L−1 hygromycin, 100 mg L−1 kanamycin and 200 mg L−1 cefotaxime. Additionally, 200 mg L−1 cefotaxime increased shoot regeneration by two-fold. Different plant hormone combinations were tested to analyse the best rooting media. Roots were obtained in a medium supplemented with 1.2 µM IAA. Plasmid pH35 containing a hygromycin-resistant gene and GUS gene was inoculated into the explants with Agrobacterium tumefaciens strain AGL0 for transformation. Overall, the transformation efficiency was improved from the 1% previously reported to 5.2%, tested at explant level with Cefotaxime showing a positive effect on shooting regeneration. Other variables in addition to antibiotic and hormone combinations such as bacterial OD, time of infection and incubation temperature may be further tested to enhance the transformation even more. This improved transformation study presents an opportunity to increase the feeding value, persistence, and nutritive value of the key Australian pasture.

AIMS: The legumes most often used in temperate pastures such as Trifolium subterraneum have relatively high external P requirements for maximum growth. We investigated root traits associated with P acquisition in current and novel pasture legumes, as well as temperate grasses which have lower P requirements. METHODS: Thirteen legume species, two pasture grasses, and three high carboxylate-exuding crop species (Lupinus albus, L. angustifolius, Cicer arietinum) were grown in a glasshouse for six weeks. Rhizosphere carboxylates and root morphological traits were measured. RESULTS: Ornithopus spp. had rhizosphere carboxylates in similar quantities to the Lupinus spp. (> 40 μmol g⁻¹ root dry mass). Trifolium subterraneum lines had relatively large average root diameter, reduced specific root length and very short average root hair length resulting in specific root hair cylinder volumes (RHCVs) only 14–20 % of the grasses. However, O. sativus, O. compressus and Biserrula pelecinus had specific RHCVs more comparable to the grasses. CONCLUSIONS: Novel pasture legume species with root morphology more comparable to that of grasses than T. subterraneum were identified. Of these, Ornithopus spp. were notable as they also had high rhizosphere carboxylates relative to root dry mass.

Background and aims Biochar can be produced from a wide range of organic sources with varying nutrient and metal concentrations. Before making irreversible applications of biochar to soil, a preliminary ecotoxicological assessment is desirable. Methods First, we determined the effect of biochar type and rate on early growth of wheat in a soil-less Petri dish bioassay. Second, we investigated the effect of the same biochars on seed germination and early growth of wheat in ten soils with varying texture using a glasshouse bioassay. Finally, we investigated whether these biochars had similar effects on three plant species when grown in one soil. Results Biochar type and application rate influenced wheat seed germination and seedling growth in a similar manner in both the soil-less Petri dish and soil-based bioassay. Germination and early root growth of mung bean and subterranean clover differed from that of wheat in response to the five biochars. Conclusions We recommend use of the soil-less Petri dish bioassay as a rapid and simple preliminary test to identify potential toxicity of biochars on seed germination and early plant growth prior to biochar application to soil.

Aims Phosphorus (P) is usually stratified in the topsoil layer under pasture, due to the broadcast application of fertiliser, excreta and leaf-litter deposition on the soil surface, and minimal soil disturbance. The objective of this study was to investigate root proliferation and P acquisition in response to P stratification by comparing two Trifolium subterraneum cultivars with contrasting root morphologies. Methods Clover micro-swards were grown with deficient, constrained and sufficient P supplied in a topsoil layer overlying a P-deficient subsoil that mimicked the stratification of P that occurs under pasture. Phosphorus labelled with 33 P- and 32 P-radioisotope tracer was mixed throughout the topsoil and subsoil layers, respectively. Results The shoot yield and total plant P uptake of the cultivars increased in response to increased topsoil P supply. The length of roots produced by the cultivars was equivalent in each of the P treatments, although the specific root length achieved by the cultivars was substantially different. In the P-constrained and P-sufficient treatments, ~91% and ~ 99% of total plant P was acquired by topsoil roots, respectively. In contrast, subsoil roots acquired 60–74% of total plant P in the P-deficient treatment. Conclusions Topsoil roots were most important for P acquisition when P was highly stratified, whereas subsoil roots contributed to P acquisition when P was uniformly distributed throughout the P-deficient soil profile. Selection for prolific nutrient-foraging roots, in conjunction with plasticity for subsoil exploration, may improve the P-acquisition efficiency of T. subterraneum genotypes and confer adaptability across a range of soil-P environments.

Aims: Trifolium subterraneum L. is the predominant annual pasture legume in southern Australia. Cultivars with improved phosphorus (P) foraging ability would improve the P-use efficiency of agricultural systems. We therefore investigated variation in root traits related to Puptake among six cultivars. Methods: Micro-swards were grown at six levels of P in field soil with indigenous arbuscular mycorrhizal (AM) fimgi for six weeks. Dry matter yield, tissue P concentration, rhizosphere carboxylates, AM fungal colonisation and root morphological traits were measured. Results: The cultivars showed similar shoot and root yield responses to P supply. Average root diameter did not change, specific root length (SRL) increased and root tissue density (RTD) decreased with increased P supply. Amounts of total rhizosphere carboxylates were low (<1.2 nmol cm⁻¹ root). The percentage of root length colonised by AM fungi was greatest (29-43 %) at an intermediate level (8 mg kg⁻¹ dry soil) of P supply. Conclusions: Most differences among cultivars were reasonably consistent across P supply levels, indicating greater numbers of lines could be screened reliably at a single P level. Low colonisation by AM fungi at low P supply deserves consideration when selecting soils for cultivar comparisons. Increased SRL and decreased RTD at high P supply likely result from self-shading within the micro-swards and warrant further investigation.