Rice seed quality development and temperature during late development and maturation

The potential longevity of japonica rice (Oryza sativa L. subsp. japonica) seed is particularly sensitive to high temperature – and thus climate change – during development and maturation. Cultivar Taipei 309 was grown at 28/208C (12 h/12 h) and then from 19 DAA (days after 50% anthesis), when seeds were just over half filled, at 28/208C, 30/228C, 32/248C or 34/268C (12 h/12 h). Whereas ability to germinate ex planta had been achieved in almost all seeds by 24 DAA, only half the population were desiccation tolerant. Desiccation tolerance continued to increase over the subsequent 28 d, similarly at all four temperatures. Subsequent longevity, assessed by p50 (period in days to reduce viability to 50% in hermetic storage at 408C with c. 15% moisture content), increased progressively at 28/208C until 38 DAA, and remained constant until the final harvest (52 DAA). The three warmer temperature regimes provided similar longevity to 28/208C at any one harvest, except at 38 DAA where the warmest (34/268C) was poorer. That temperature regime also provided greater seed-to-seed variability within each survival curve. The results confirm that appreciable improvement in seed quality occurs during seed development and also subsequent maturation in japonica rice, but that increase in temperature from 28/208C to 34/268C during late seed filling onwards has comparatively little effect thereon. Comparison with previous investigations suggests that seed quality development may be less sensitive to high temperatures during late development and maturation than during the early seed development that precedes it.

Farmers' management of rice varietal diversity in the mid-hills of Nepal: implications for on-farm conservation and crop improvement

Season-long monitoring of on-farm rice (Oryza sativa, L.) plots in Nepal explored farmers' decision-making process on the deployment of varieties to agroecosystems, application of production inputs to varieties, agronomic practices and relationship between economic return and area planted per variety. Farmers deploy varieties [landraces (LRs) and modern varieties (MVs)] to agroecosystems based on their understanding of characteristics of varieties and agroecosystems, and the interaction between them. In marginal growing conditions, LRs can compete with MVs. Within an agroecosystem, economic return and area planted to varieties have positive relationship, but this is not so between agroecosystems. LRs are very diverse on agronomic and economic traits; therefore, they cannot be rejected a priori as inferior materials without proper evaluation. LRs have to be evaluated for useful traits and utilized in breeding programmes to generate farmer-preferred materials for marginal environments and for their conservation on-farm.

On-farm management of rice genetic diversity: understanding farmers' knowledge on rice ecosystems and varietal development

The paper highlights the methodological development of identifying and characterizing rice (Oryza sativa L.) ecosystems and the varietal deployment process through participatory approaches. Farmers have intricate knowledge of their rice ecosystems. Evidence from Begnas (mid-hill) and Kachorwa (plain) sites in Nepal suggests that farmers distinguish ecosystems for rice primarily on the basis of moisture and fertility of soils. Farmers also differentiate the number, relative size and specific characteristics of each ecosystem within a given geographic area. They allocate individual varieties to each ecosystem, based on the principle of ‘best fit’ between ecosystem characteristics and varietal traits, indicating that competition between varieties mainly occurs within the ecosystems. Land use and ecosystems determine rice genetic diversity, with marginal land having fewer options for varieties than more productive areas. Modern varieties are mostly confined to productive land, whereas landraces are adapted to marginal ecosystems. Researchers need to understand the ecosystems and varietal distribution within ecosystems better in order to plan and execute programmes on agrobiodiversity conservation on-farm, diversity deployment, repatriation of landraces and monitoring varietal diversity. Simple and practical ways to elicit information on rice ecosystems and associated varieties through farmers’ group discussion at village level are suggested.

Profiling dehydrin gene sequence and physiological parameters in drought tolerant and susceptible spring wheat cultivars.

Physiological and yield traits such as stomatal conductance (mmol m-2s-1), Leaf relative water content (RWC %) and grain yield per plant were studied in a separate experiment. Results revealed that five out of sixteen cultivars viz. Anmol, Moomal, Sarsabz, Bhitai and Pavan, appeared to be relatively more drought tolerant. Based on morphophysiological results, studies were continued to look at these cultivars for drought tolerance at molecular level. Initially, four well recognized primers for dehydrin genes (DHNs) responsible for drought induction in T. durum L., T. aestivum L. and O. sativa L. were used for profiling gene sequence of sixteen wheat cultivars. The primers amplified the DHN genes variably like Primer WDHN13 (T. aestivum L.) amplified the DHN gene in only seven cultivars whereas primer TdDHN15 (T. durum L.) amplified all the sixteen cultivars with even different DNA banding patterns some showing second weaker DNA bands. Third primer TdDHN16 (T. durum L.) has shown entirely different PCR amplification prototype, specially showing two strong DNA bands while fourth primer RAB16C (O. sativa L.) failed to amplify DHN gene in any of the cultivars. Examination of DNA sequences revealed several interesting features. First, it identified the two exon/one intron structure of this gene (complete sequences were not shown), a feature not previously described in the two database cDNA sequences available from T. aestivum L. (gi|21850). Secondly, the analysis identified several single nucleotide polymorphisms (SNPs), positions in gene sequence. Although complete gene sequence was not obtained for all the cultivars, yet there were a total of 38 variable positions in exonic (coding region) sequence, from a total gene length of 453 nucleotides. Matrix of SNP shows these 37 positions with individual sequence at positions given for each of the 14 cultivars (sequence of two cultivars was not obtained) included in this analysis. It demonstrated a considerable diversity for this gene with only three cultivars i.e. TJ-83, Marvi and TD-1 being similar to the consensus sequence. All other cultivars showed a unique combination of SNPs. In order to prove a functional link between these polymorphisms and drought tolerance in wheat, it would be necessary to conduct a more detailed study involving directed mutation of this gene and DHN gene expression.

Temporal sensitivity of rice seed development from spikelet fertility to viable mature seed to extreme-temperature

Extreme temperature during reproductive development affects rice (Oryza sativa L.) yield and seed quality. A controlled-environment reciprocal-transfer experiment was designed where plants from two japonica cultivars were grown at 28/24 ⁰C and moved to 18/14 ⁰C and vice versa, or from 28/24 to 38/34 ⁰C and vice versa, for 7-d periods to determine the respective temporal pattern of sensitivity of spikelet fertility, yield, and seed viability to each temperature extreme. Spikelet fertility and seed yield per panicle were severely reduced by extreme temperature in the 14 d period prior to anthesis; and both cultivars were affected at 38/34 ⁰C while only cv. Gleva was affected at 18/14 ºC. The damage was greater the earlier the panicles were stressed within this period. Later-exserted panicles compensated only partly for yield loss. Seed viability was significantly reduced by 7-d exposure to 38/34 ⁰C or 18/14 ⁰C at 1 to 7 and 1 to 14 d after anthesis, respectively, in cv. Gleva. Cultivar Taipei 309 was not affected by 7 d exposure at 18/14 ⁰C; and no consistent temporal pattern of sensitivity was evident at 38/34 ⁰C. Hence, brief exposure to low or high temperature was most damaging to spikelet fertility and yield 14 to 7 d before anthesis, coinciding with microsporogenesis; and it was almost as damaging around anthesis. Seed viability was most vulnerable to low or high temperature in the 7 or 14 d after anthesis, when histodifferentiation occurs.

Resilience of rice (Oryzaspp.) pollen germination and tube growth to temperature stress

Resilience of rice cropping systems to potential global climate change will partly depend on temperature tolerance of pollen germination (PG) and tube growth (PTG). Germination of pollen of high temperature susceptible Oryza glaberrima Steud. (cv. CG14) and O. sativa L. ssp. indica (cv. IR64) and high temperature tolerant O. sativa ssp. aus (cv. N22), was assessed on a 5.6-45.4°C temperature gradient system. Mean maximum PG was 85% at 27°C with 1488 μm PTG at 25°C. The hypothesis that in each pollen grain, minimum temperature requirements (Tn) and maximum temperature limits (Tx) for germination operate independently was accepted by comparing multiplicative and subtractive probability models. The maximum temperature limit for PG in 50% of grains (Tx(50)) was lowest (29.8°C) in IR64 compared with CG14 (34.3°C) and N22 (35.6°C). Standard deviation (sx) of Tx was also low in IR64 (2.3°C) suggesting that the mechanism of IR64's susceptibility to high temperatures may relate to PG. Optimum germination temperatures and thermal times for 1mm PTG were not linked to tolerating high temperatures at anthesis. However, the parameters Tx(50) and sx in the germination model define new pragmatic criteria for successful and resilient PG, preferable to the more traditional cardinal (maximum and minimum) temperatures.

Contrasting responses to drought stress in herbaceous perennial legumes

Background and aims Medicago sativa L. is widely grown in southern Australia, but is poorly adapted to dry, hot summers. This study aimed to identify perennial herbaceous legumes with greater resistance to drought stress and explore their adaptive strategies. Methods Ten herbaceous perennial legume species/accessions were grown in deep pots in a sandy, low-phosphorus field soil in a glasshouse. Drought stress was imposed by ceasing to water. A companion M. sativa plant in each pot minimised differences in leaf area and water consumption among species. Plants were harvested when stomatal conductance of stressed plants decreased to around 10% of well watered plants. Results A range of responses to drought stress were identified, including: reduced shoot growth; leaf curling; thicker pubescence on leaves and stems; an increased root:shoot ratio; an increase, decrease or no change in root distribution with depth; reductions in specific leaf area or leaf water potential; and osmotic adjustment. The suite of changes differed substantially among species and, less so, among accessions. Conclusions The inter- and intra-specific variability of responses to drought-stress in the plants examined suggests a wide range of strategies are available in perennial legumes to cope with drying conditions, and these could be harnessed in breeding/selection programs.

Variation in seedling growth of 11 perennial legumes in response to phosphorus supply

Phosphorus (P) deficiency is a major problem for Australian agriculture. Development of new perennial pasture legumes that acquire or use P more efficiently than the current major perennial pasture legume, lucerne (Medicago sativa L.), is urgent. A glasshouse experiment compared the response of ten perennial herbaceous legume species to a series of P supplies ranging from 0 to 384 µg g−1 soil, with lucerne as the control. Under low-P conditions, several legumes produced more biomass than lucerne. Four species (Lotononis bainesii Baker, Kennedia prorepens F.Muell, K. prostrata R.Br, Bituminaria bituminosa (L.) C.H.Stirt) achieved maximum growth at 12 µg P g−1 soil, while other species required 24 µg P g−1. In most tested legumes, biomass production was reduced when P supply was ≥192 µg g−1, due to P toxicity, while L. bainesii and K. prorepens showed reduced biomass when P was ≥24 µg g−1 and K. prostrata at ≥48 µg P g−1 soil. B. bituminosa and Glycine canescens F.J.Herm required less soil P to achieve 0.5 g dry mass than the other species did. Lucerne performed poorly with low P supply and our results suggest that some novel perennial legumes may perform better on low-P soils.

Root distributions of Australian herbaceous perennial legumes in response to phosphorus placement

Many Australian plant species have specific root adaptations for growth in phosphorus-impoverished soils, and are often sensitive to high external P concentrations. The growth responses of native Australian legumes in agricultural soils with elevated P availability in the surface horizons are unknown. The aim of these experiments was to test the hypothesis that increased P concentration in surface soil would reduce root proliferation at depth in native legumes. The effect of P placement on root distribution was assessed for two Australian legumes, Kennedia prorepens F. Muell. and Lotus australis Andrews, and the exotic Medicago sativa L. Three treatments were established in a low-P loam soil: amendment of 0.15 g mono-calcium phosphate in either (i) the top 50 mm (120 µg P g–1) or (ii) the top 500 mm (12 µg P g–1) of soil, and an unamended control. In the unamended soil M. sativa was shallow rooted, with 58% of the root length of in the top 50 mm. K. prorepens and L. australis had a more even distribution down the pot length, with only 4 and 22% of their roots in the 0–50 mm pot section, respectively. When exposed to amendment of P in the top 50 mm, root length in the top 50 mm increased 4-fold for K. prorepens and 10-fold for M. sativa, although the pattern of root distribution did not change for M. sativa. L. australis was relatively unresponsive to P additions and had an even distribution of roots down the pot. Shoot P concentrations differed according to species but not treatment (K. prorepens 2.1 mg g–1, L. australis 2.4 mg g–1, M. sativa 3.2 mg g–1). Total shoot P content was higher for K. prorepens than for the other species in all treatments. In a second experiment, mono-ester phosphatases were analysed from 1-mm slices of soil collected directly adjacent to the rhizosphere. All species exuded phosphatases into the rhizosphere, but addition of P to soil reduced phosphatase activity only for K. prorepens. Overall, high P concentration in the surface soil altered root distribution, but did not reduce root proliferation at depth. Furthermore, the Australian herbaceous perennial legumes had root distributions that enhanced P acquisition from low-P soils.

‘Symptomless’ infection by Botrytis cinerea

The study was carried out to clarify the nature of symptomless infection by Botrytis cinerea and to what extent it differs from aggressive necrotic infection in Lactuca sativa (lettuce) and Arabidopsis thaliana. Symptomless plants were produced by dry spore inoculation in plants growing in controlled environmental conditions or in glasshouses. Plating out of surface-disinfected and non-surface-disinfected samples of inoculated, apparently healthy, plants on selective medium revealed that the fungus was spreading from the initial inoculation site to newly developing plant organs both internally and externally. Similar findings were obtained in microscope experiments in which host plants were inoculated with GFP labelled B. cinerea and symptomless spreading was monitored under confocal laser scanning microscope. Spore germination on leaf surface was followed by development of sub-cuticular vesicles and plant cell damage in the infected epidermal cell and a few nearby cells. Sparsely branched long hyphae arose from the vesicles and spread on the leaf surface; spread was mostly on the outer surface of the epidermal layer but occasionally below the cuticle or epidermal cells. In the late symptomless phase, mycelium arising from single vesicles formed several mycelial networks on leaves. Experiments were carried out to compare the extent of gene expression in symptomless and necrotic infections, using RT-qPCR. Expression of selected genes was quantified in tissue samples based on the amount of mRNA of the respective genes found. In both host species, the mRNA concentration of signalling genes bcg1, bmp1 and calcineurin, and the pathogenicity genes bcsod1 and bcpg1 were similar to or slightly greater in symptomless samples than in necrotic samples. The mRNA of the signalling gene bac and pathogenicity genes bcbot1 and bcnep1, were not detected or detected in lower abundance than in necrosis. In lettuce, the leaves developing distant from the site of inoculation showed similar results to A. thaliana, but in healthy leaves close to the site of inoculation mRNA concentrations of bac and bcnep1 were similar to necrotic samples. Thus, in both host species, the fungus grew along with the plant and moved to newly growing plant parts without producing symptoms; during this growth some pathogenicity genes were less expressed than in necrotic infection.

The effect of simulated flooding post-anthesis on pre-harvest sprouting and subsequent seed longevity in contrasting rice cultivars

Unpredictable flooding is a major constraint to rice production. It can occur at any growth stage. The effect of simulated flooding post-anthesis on yield and subsequent seed quality of pot-grown rice (Oryza sativa L.) plants was investigated in glasshouses and controlled-environment growth cabinets. Submergence post-anthesis (9-40 DAA) for 3 or 5 days reduced seed weight of japonica rice cv. Gleva, with considerable pre-harvest sprouting (up to 53%). The latter was greater the later in seed development and maturation that flooding occurred. Sprouted seed had poor ability to survive desiccation or germinate normally upon rehydration, whereas the effects of flooding on the subsequent air-dry seed storage longevity (p50) of the non-sprouted seed fraction was negligible. The indica rice cvs IR64 and IR64Sub1 (introgression of submergence tolerance gene Submergence1A-1) were both far more tolerant to flooding post-anthesis than cv. Gleva: four days’ submergence of these two near-isogenic cultivars at 10-40 DAA resulted less than 1% sprouted seeds. The presence of the Sub1A-1 allele in cv. IR64Sub1 was verified by gel electrophoresis and DNA sequencing. It had no harmful effect on loss in seed viability during storage compared with IR64 in both control and flooded environments. Moreover, the germinability and changes in dormancy during seed development and maturation were very similar to IR64. The efficiency of using chemical spray to increase seed dormancy was investigated in the pre-harvest sprouting susceptible rice cv. Gleva. Foliar application of molybdenum at 100 mg L-1 reduced sprouted seeds by 15-21% following 4 days’ submergence at 20-30 DAA. Analyses confirmed that the treatment did result in molybdenum uptake by the plants, and also tended to increase seed abscisic acid concentration. The latter was reduced by submergence and declined exponentially during grain ripening. The selection of submergence-tolerant varieties was more successful than application of molybdenum in reducing pre-harvest sprouting.

Glucosinolates, myrosinase hydrolysis products, and flavonols found in rocket (Eruca sativa and Diplotaxis tenuifolia)

Rocket species have been shown to have very high concentrations of glucosinolates and flavonols, which have numerous positive health benefits with regular consumption. In this review we highlight how breeders and processors of rocket species can utilize genomic and phytochemical research to improve varieties and enhance the nutritive benefits to consumers. Plant breeders are increasingly looking to new technologies such as HPLC, UPLC, LC-MS and GC-MS to screen populations for their phytochemical content to inform plant selections. Here we collate the research that has been conducted to-date in rocket, and summarise all glucosinolate and flavonol compounds identified in the species. We emphasize the importance of the broad screening of populations for phytochemicals and myrosinase degradation products, as well as unique traits that may be found in underutilized gene bank resources. We also stress that collaboration with industrial partners is becoming essential for long-term plant breeding goals through research.

Identification and quantification of glucosinolate and flavonol compounds in rocket salad (Eruca sativa, Eruca vesicaria and Diplotaxis tenuifolia) by LC–MS: Highlighting the potential for improving nutritional value of rocket crops

Liquid Chromatography Mass Spectrometry (LC-MS) was used to obtain glucosinolate and flavonol content for 35 rocket accessions and commercial varieties. 13 glucosinolates and 11 flavonol compounds were identified. Semi-quantitative methods were used to estimate concentrations of both groups of compounds. Minor glucosinolate composition was found to be different between accessions; concentrations varied significantly. Flavonols showed differentiation between genera, with Diplotaxis accumulating quercetin glucosides and Eruca accumulating kaempferol glucosides. Several compounds were detected in each genus that have only previously been reported in the other. We highlight how knowledge of phytochemical content and concentration can be used to breed new, nutritionally superior varieties. We also demonstrate the effects of controlled environment conditions on the accumulations of glucosinolates and flavonols and explore the reasons for differences with previous studies. We stress the importance of consistent experimental design between research groups to effectively compare and contrast results.

Use of TD-GC-TOF-MS to assess volatile composition during post harvest storage in seven accessions of rocket salad (Eruca sativa)

An important step in breeding for nutritionally enhanced varieties is determining the effects of the post-harvest supply chain on phytochemicals and the changes in VOCs produced over time. TD- GC-TOF-MS was used and a technique for the extraction of VOCs from the headspace using portable tubes is described. Forty-two compounds were detected; 39 were identified by comparison to NIST libraries. Thirty-five compounds had not been previously reported in Eruca sativa. Seven accessions were assessed for changes in headspace VOCs over 7 days. Relative amounts of VOCs across 3 time points were significantly different - isothiocyanate-containing molecules being abundant on 'Day 0'. Each accession showed differences in proportions/types of volatiles produced on each day. PCA revealed a separation of VOC profiles according to the day of sampling. Changes in VOC profiles over time could provide a tool for assessment of shelf-life.