Phloem transport of arsenic species from flag leaf to grain during grain filling

Strategies to reduce arsenic (As) in rice grain, below concentrations that represent a serious human health concern, require that the mechanisms of As accumulation within grain be established. Therefore, retranslocation of As species from flag leaves into filling rice grain was investigated.

Arsenic species were delivered through cut flag leaves during grain fill. Spatial unloading within grains was investigated using synchrotron X-ray fluorescence (SXRF) microtomography. Additionally, the effect of germanic acid (a silicic acid analog) on grain As accumulation in arsenite-treated panicles was examined.

Dimethylarsinic acid (DMA) and monomethylarsonic acid (MMA) were extremely efficiently retranslocated from flag leaves to rice grain; arsenate was poorly retranslocated, and was rapidly reduced to arsenite within flag leaves; arsenite displayed no retranslocation. Within grains, DMA rapidly dispersed while MMA and inorganic As remained close to the entry point. Germanic acid addition did not affect grain As in arsenite-treated panicles. Three-dimensional SXRF microtomography gave further information on arsenite localization in the ovular vascular trace (OVT) of rice grains.

These results demonstrate that inorganic As is poorly remobilized, while organic species are readily remobilized, from leaves to grain. Stem translocation of inorganic As may not rely solely on silicic acid transporters.

A temporal limit to the association between flag leaf life extension by fungicides and wheat yields

Three field experiments, each repeated over two or three seasons, on winter wheat investigated a possible limit to the association between grain yield and flag leaf life, as extended by fungicide application. The experiments involved up to six cultivars and different application rates, timings and frequencies of the strobilurin azoxystrobin and the triazole epoxiconazole. In the 2000/01 and 2001/02 seasons, the relationships between the thermal time to 37 % green flag leaf area (m) and yield deviated from linearity. 'Broken stick' models were fitted to cultivar x experiment combinations within each season and the limit to the benefit to yield associated with extending flag leaf life was 700 degrees C days (S.E. = 20.7) and 725 degrees C days (S.E. = 9.33) after anthesis in 2000/01 and 2001/02, respectively. In 2002/03, the relationship between yield and in did not deviate significantly (P > 0.05) from linearity, but in this latter year the fungicide application failed to increase In past 700 degrees C days. (c) 2004 Elsevier B.V. All rights reserved.

The effects of adding picoxystrobin, azoxystrobin and nitrogen to a triazole programme on disease control, flag leaf senescence, yield and grain quality of winter wheat

The effect of adding strobilurins to a triazole (epoxiconazole) fungicide programme on the quality of a range of wheat cultivars was assessed in field experiments in three successive years. Strobilurin was applied at just flag leaf emergence (azoxystrobin) or at the start of stem extension (azoxystrobin or picoxystrobin) and again at flag leaf emergence or at flag leaf emergence and again at ear emergence (azoxystrobin). All strobilurin treatments reduced disease levels, delayed senescence of the flag leaf and consistently increased yields, thousand grain weight and specific weight. Reductions in Hagberg falling number were observed, even by fungicide applications at the start of stem extension, but effects were small compared to the variation among cultivars. Application of fungicide (triazole or strobilurin) before ear emergence increased the amount of blackpoint, but this was partly countered by applying azoxystrobin at ear emergence. The effect of fungicide on protein concentration differed over seasons and cultivar. Where they occurred. small reductions in protein concentration could be compensated for by extra application of nitrogen as foliar urea at anthesis. Foliar urea (40 kg N ha(-1)) applied at anthesis also improved Hagberg failing number and reduced blackpoint in one of the growing seasons. In one season, the effect of foliar urea at anthesis was compared with applications of granular fertiliser at flag leaf emergence. The granular treatment produced grain with more concentrated protein, while the later, foliar application produced higher specific weights. (C) 2003 Elsevier Science Ltd. All rights reserved.

Effect of locally increased zinc contents on zinc transport from the flag leaf lamina to the maturing grains of wheat

From previous experiments, it was evident that the accumulation of zinc in maturing wheat grains is highly regulated, but the regulatory mechanisms involved are not yet identified. In this study, we determined the transfer of radiolabelled zinc (fed directly into a leaf flap) from the flag leaf lamina to the grains. We also determined how this zinc transfer was affected by feeding additional unlabeled zinc (1 μmol per plant) either into the flag leaf sheath or the peduncle. Most of the 65Zn was retained in the feeding flap. A high percentage of the zinc exported from the flap accumulated in the grains with little accumulation of radiolabel in the other parts of the shoot. Unlabeled zinc remained mainly in the feeding flap and in the parts reached by the transpiration stream from the feeding position. The transfer of radiolabelled zinc was essentially not influenced by unlabeled zinc fed into another plant part. Our results suggest that the loading of zinc into the phloem and the mass flow in the sieve tubes might regulate zinc redistribution within the wheat shoot.

Mapping quantitative trait loci for flag leave senescence as a yield determinant in winter wheat under optimal and drought stressed environments

The timing of flag leaf senescence (FLS) is an important determinant of yield under stress and optimal environments. A doubled haploid population derived from crossing the photo period-sensitive variety Beaver,with the photo period-insensitive variety Soissons, varied significantly for this trait, measured as the percent green flag leaf area remaining at 14 days and 35 days after anthesis. This trait also showed a significantly positive correlation with yield under variable environmental regimes. QTL analysis based on a genetic map derived from 48 doubled haploid lines using amplified fragment length polymorphism (AFLP) and simple sequence repeat (SSR) markers, revealed the genetic control of this trait. The coincidence of QTL for senescence on chromosomes 2B and 2D under drought-stressed and optimal environments, respectively, indicate a complex genetic mechanism of this trait involving the re-mobilisation of resources from the source to the sink during senescence.

Leaf application of silicic acid to white oat and wheat

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Development of a transformation system for sorghum (Sorghum bicolor L.)

Sorghum (Sorghum bicolor (L.) Moench) is the world’s fifth major cereal crop and holds importance as a construction material, food and fodder source. More recently, the potential of this plant as a biofuel source has been noted. Despite its agronomic importance, the use of sorghum production is being constrained by both biotic and abiotic factors. These challenges could be addressed by the use of genetic engineering strategies to complement conventional breeding techniques. However, sorghum is one of the most recalcitrant crops for genetic modification with the lack of an efficient tissue culture system being amongst the chief reasons. Therefore, the aim of this study was to develop an efficient tissue culture system for establishing regenerable embryogenic cell lines, micropropagation and acclimatisation for Sorghum bicolor and use this to optimise parameters for genetic transformation via Agrobacterium-mediated transformation and microprojectile bombardment. Using five different sorghum cultivars, SA281, 296B, SC49, Wray and Rio, numerous parameters were investigated in an attempt to establish an efficient and reproducible tissue culture and transformation system. Using immature embryos (IEs) as explants, regenerable embryogenic cell lines (ECLs) could only be established from cultivars SA281 and 296B. Large amounts of phenolics were produced from IEs of cultivars, SC49, Wary and Rio, and these compounds severely hindered callus formation and development. Cultivar SA281 also produced phenolics during regeneration. Attempts to suppress the production of these compounds in cultivars SA281 and SC49 using activated charcoal, PVP, ascorbic acid, citric acid and liquid filter paper bridge methods were either ineffective or had a detrimental effect on embryogenic callus formation, development and regeneration. Immature embryos sourced during summer were found to be far more responsive in vitro than those sourced during winter. In an attempt to overcome this problem, IEs were sourced from sorghum grown under summer conditions in either a temperature controlled glasshouse or a growth chamber. However, the performance of these explants was still inferior to that of natural summer-sourced explants. Leaf whorls, mature embryos, shoot tips and leaf primordia were found to be unsuitable as explants for establishing ECLs in sorghum cultivars SA281 and 296B. Using the florets of immature inflorescences (IFs) as explants, however, ECLs were established and regenerated for these cultivars, as well as for cultivar Tx430, using callus induction media, SCIM, and regeneration media, VWRM. The best in vitro responses, from the largest possible sized IFs, were obtained using plants at the FL-2 stage (where the last fully opened leaf was two leaves away from the flag leaf). Immature inflorescences could be stored at 25oC for up to three days without affecting their in vitro responses. Compared to IEs, the IFs were more robust in tissue culture and showed responses which were season and growth condition independent. A micropropagation protocol for sorghum was developed in this study. The optimum plant growth regulator (PGR) combination for the micropropagation of in vitro regenerated plantlets was found to be 1.0 mg/L BAP in combination with 0.5 mg/L NAA. With this protocol, cultivars 296B and SA281 produced an average of 57 and 13 off-shoots per plantlet, respectively. The plantlets were successfully acclimatised and developed into phenotypically normal plants that set seeds. A simplified acclimatisation protocol for in vitro regenerated plantlets was also developed. This protocol involved deflasking in vitro plantlets with at least 2 fully-opened healthy leaves and at least 3 roots longer than 1.5 cm, washing the media from the roots with running tap water, planting in 100 mm pots and placing in plastic trays covered with a clear plastic bag in a plant growth chamber. After seven days, the corners of the plastic cover were opened and the bags were completely removed after 10 days. All plantlets were successfully acclimatised regardless of whether 1:1 perlite:potting mix, potting mix, UC mix or vermiculite were used as potting substrates. Parameters were optimised for Agrobacterium-mediated transformation (AMT) of cultivars SA281, 296B and Tx430. The optimal conditions were the use of Agrobacterium strain LBA4404 at an inoculum density of 0.5 OD600nm, heat shock at 43oC for 3 min, use of the surfactant Pluronic F-68 (0.02% w/v) in the inoculation media with a pH of 5.2 and a 3 day co-cultivation period in dark at 22oC. Using these parameters, high frequencies of transient GFP expression was observed in IEs precultured on callus initiation media for 1-7 days as well as in four weeks old IE- and IF-derived callus. Cultivar SA281 appeared very sensitive to Agrobacterium since all tissue turned necrotic within two weeks post-exposure. For cultivar 296B, GFP expression was observed up to 20 days post co-cultivation but no stably transformed plants were regenerated. Using cultivar Tx430, GFP was expressed for up to 50 days post co-cultivation. Although no stably transformed plants of this cultivar were regenerated, this was most likely due to the use of unsuitable regeneration media. Parameters were optimised for transformation by particle bombardment (PB) of cultivars SA281, 296B and Tx430. The optimal conditions were use of 3-7 days old IEs and 4 weeks old IF callus, 4 hour pre- and post-bombardment osmoticum treatment, use of 0.6 µm gold microparticles, helium pressure of 1500 kPa and target distance of 15 cm. Using these parameters for PB, transient GFP expression was observed for up to 14, 30 and 50 days for cultivars SA281, 296B and Tx430, respectively. Further, the use of PB resulted in less tissue necrosis compared to AMT for the respective cultivars. Despite the presence of transient GFP expression, no stably transformed plants were regenerated. The establishment of regenerable ECLs and the optimization of AMT and PB parameters in this study provides a platform for future efforts to develop an efficient transformation protocol for sorghum. The development of GM sorghum will be an important step towards improving its agronomic properties as well as its exploitation for biofuel production.

Post-head-emergence frost in wheat and barley: defining the problem, assessing the damage, and identifying resistance

Radiant frost is a significant production constraint to wheat (Triticum aestivum) and barley (Hordeum vulgare), particularly in regions where spring-habit cereals are grown through winter, maturing in spring. However, damage to winter-habit cereals in reproductive stages is also reported. Crops are particularly susceptible to frost once awns or spikes emerge from the protection of the flag leaf sheath. Post-head-emergence frost (PHEF) is a problem distinct from other cold-mediated production constraints. To date, useful increased PHEF resistance in cereals has not been identified. Given the renewed interest in reproductive frost damage in cereals, it is timely to review the problem. Here we update the extent and impacts of PHEF and document current management options to combat this challenge. We clarify terminology useful for discussing PHEF in relation to chilling and other freezing stresses. We discuss problems characterizing radiant frost, the environmental conditions leading to PHEF damage, and the effects of frost at different growth stages. PHEF resistant cultivars would be highly desirable, to both reduce the incidence of direct frost damage and to allow the timing of crop maturity to be managed to maximize yield potential. A framework of potential adaptation mechanisms is outlined. Clarification of these critical issues will sharpen research focus, improving opportunities to identify genetic sources for improved PHEF resistance.

Post-head-emergence frost in wheat and barley: defining the problem, assessing the damage, and identifying resistance

Radiant frost is a significant production constraint to wheat (Triticum aestivum) and barley (Hordeum vulgare), particularly in regions where spring-habit cereals are grown through winter, maturing in spring. However, damage to winter-habit cereals in reproductive stages is also reported. Crops are particularly susceptible to frost once awns or spikes emerge from the protection of the flag leaf sheath. Post-head-emergence frost (PHEF) is a problem distinct from other cold-mediated production constraints. To date, useful increased PHEF resistance in cereals has not been identified. Given the renewed interest in reproductive frost damage in cereals, it is timely to review the problem. Here we update the extent and impacts of PHEF and document current management options to combat this challenge. We clarify terminology useful for discussing PHEF in relation to chilling and other freezing stresses. We discuss problems characterizing radiant frost, the environmental conditions leading to PHEF damage, and the effects of frost at different growth stages. PHEF resistant cultivars would be highly desirable, to both reduce the incidence of direct frost damage and to allow the timing of crop maturity to be managed to maximize yield potential. A framework of potential adaptation mechanisms is outlined. Clarification of these critical issues will sharpen research focus, improving opportunities to identify genetic sources for improved PHEF resistance.