Amélie C.M. Gaudin

Self-excision of the antibiotic resistance gene nptII using a heat inducible Cre-loxP system from transgenic potato

Resistance to antibiotics mediated by selectable marker genes remains a powerful selection tool for transgenic event production. However, regulatory agencies and consumer concerns favor these to be eliminated from food crops. Several excision systems exist but none have been optimized or shown to be functional for clonally propagated crops. The excision of the nptII gene conferring resistance to kanamycin has been achieved here using a gene construct based on a heat-inducible cre gene producing a recombinase that eliminates cre and nptII genes flanked by two loxP sites. First-generation regenerants with the Cre-loxP system were obtained by selection on kanamycin media. Following a heat treatment, second generation regenerants were screened for excision by PCR using nptII, cre, and T-DNA borders primers. Excision efficiency appeared to be at 4.7% depending on the heat treatment. The footprint of the excision was shown by sequencing between T-DNA borders to correspond to a perfect recombination event. Selectable marker-free sprouts were also obtained from tubers of transgenic events when submitted to similar heat treatment at 4% frequency. Spontaneous excision was not observed out of 196 regenerants from untreated transgenic explants. Biosafety concerns are minimized because the expression of cre gene driven by the hsp70 promoter of Drosophilamelanogaster was remarkably low even under heat activation and no functional loxP site were found in published Solanum sequence database. A new plant transformation vector pCIP54/55 was developed including a multiple cloning site and the self-excision system which should be a useful tool not only for marker genes in potato but for any gene or sequence removal in any plant.

Increasing Crop Diversity Mitigates Weather Variations and Improves Yield Stability

Cropping sequence diversification provides a systems approach to reduce yield variations and improve resilience to multiple environmental stresses. Yield advantages of more diverse crop rotations and their synergistic effects with reduced tillage are well documented, but few studies have quantified the impact of these management practices on yields and their stability when soil moisture is limiting or in excess. Using yield and weather data obtained from a 31-year long term rotation and tillage trial in Ontario, we tested whether crop rotation diversity is associated with greater yield stability when abnormal weather conditions occur. We used parametric and non-parametric approaches to quantify the impact of rotation diversity (monocrop, 2-crops, 3-crops without or with one or two legume cover crops) and tillage (conventional or reduced tillage) on yield probabilities and the benefits of crop diversity under different soil moisture and temperature scenarios. Although the magnitude of rotation benefits varied with crops, weather patterns and tillage, yield stability significantly increased when corn and soybean were integrated into more diverse rotations. Introducing small grains into short corn-soybean rotation was enough to provide substantial benefits on long-term soybean yields and their stability while the effects on corn were mostly associated with the temporal niche provided by small grains for underseeded red clover or alfalfa. Crop diversification strategies increased the probability of harnessing favorable growing conditions while decreasing the risk of crop failure. In hot and dry years, diversification of corn-soybean rotations and reduced tillage increased yield by 7% and 22% for corn and soybean respectively. Given the additional advantages associated with cropping system diversification, such a strategy provides a more comprehensive approach to lowering yield variability and improving the resilience of cropping systems to multiple environmental stresses. This could help to sustain future yield levels in challenging production environments.

Novel temporal, fine-scale and growth variation phenotypes in roots of adult-stage maize (Zea mays L.) in response to low nitrogen stress

There is interest in discovering root traits associated with acclimation to nutrient stress. Large root systems, such as in adult maize, have proven difficult to be phenotyped comprehensively and over time, causing target traits to be missed. These challenges were overcome here using aeroponics, a system where roots grow in the air misted with a nutrient solution. Applying an agriculturally relevant degree of low nitrogen (LN) stress, 30-day-old plants responded by increasing lengths of individual crown roots (CRs) by 63%, compensated by a 40% decline in CR number. LN increased the CR elongation rate rather than lengthening the duration of CR growth. Only younger CR were significantly responsive to LN stress, a novel finding. LN shifted the root system architectural balance, increasing the lateral root (LR)-to-CR ratio, adding ∼70 m to LR length. LN caused a dramatic increase in second-order LR density, not previously reported in adult maize. Despite the near-uniform aeroponics environment, LN induced increased variation in the relative lengths of opposing LR pairs. Large-scale analysis of root hairs (RHs) showed that LN decreased RH length and density. Time-course experiments suggested the RH responses may be indirect consequences of decreased biomass/demand under LN. These results identify novel root traits for genetic dissection.

Molecular and physiological adaptation to prolonged drought stress in the leaves of two Andean potato genotypes

Responses to prolonged drought and recovery from drought of two South American potato (Solanum tuberosum L. ssp. andigena (Juz & Buk) Hawkes) landraces, Sullu and Ccompis were compared under field conditions. Physiological and biomass measurements, yield analysis, the results of hybridisation to a potato microarray platform (44 000 probes) and metabolite profiling were used to characterise responses to water deficit. Drought affected shoot and root biomass negatively in Ccompis but not in Sullu, whereas both genotypes maintained tuber yield under water stress. Ccompis showed stronger reduction in maximum quantum yield under stress than Sullu, and less decrease in stomatal resistance. Genes associated with PSII functions were activated during recovery in Sullu only. Evidence for sucrose accumulation in Sullu only during maximum stress and recovery was observed, in addition to increases in cell wall biosynthesis. A depression in the abundance of plastid superoxide dismutase transcripts was observed under maximum stress in Ccompis. Both sucrose and the regulatory molecule trehalose accumulated in the leaves of Sullu only. In contrast, in Ccompis, the raffinose oligosaccharide family pathway was activated, whereas low levels of sucrose and minor stress-mediated changes in trehalose were observed. Proline, and expression of the associated genes, rose in both genotypes under drought, with a 3-fold higher increase in Sullu than in Ccompis. The results demonstrate the presence of distinct molecular and biochemical drought responses in the two potato landraces leading to yield maintenance but differential biomass accumulation in vegetative tissues.

Taking transgenic rice drought screening to the field

Numerous transgenes have been reported to increase rice drought resistance, mostly in small-scale experiments under vegetative-stage drought stress, but few studies have included grain yield or field evaluations. Different definitions of drought resistance are currently in use for field-based and laboratory evaluations of transgenics, the former emphasizing plant responses that may not be linked to yield under drought. Although those fundamental studies use efficient protocols to uncover and validate gene functions, screening conditions differ greatly from field drought environments where the onset of drought stress symptoms is slow (2-3 weeks). Simplified screening methods, including severely stressed survival studies, are therefore not likely to identify transgenic events with better yield performance under drought in the target environment. As biosafety regulations are becoming established to allow field trials in some rice-producing countries, there is a need to develop relevant screening procedures that scale from preliminary event selection to greenhouse and field trials. Multilocation testing in a range of drought environments may reveal that different transgenes are necessary for different types of drought-prone field conditions. We describe here a pipeline to improve the selection efficiency and reproducibility of results across drought treatments and test the potential of transgenic rice for the development of drought-resistant material for agricultural purposes.

Using Ancient Traits to Convert Soil Health into Crop Yield: Impact of Selection on Maize Root and Rhizosphere Function

The effect of domestication and modern breeding on aboveground traits in maize (Zea mays) has been well-characterized, but the impact on root systems and the rhizosphere remain unclear. The transition from wild ecosystems to modern agriculture has focused on selecting traits that yielded the largest aboveground production with increasing levels of crop management and nutrient inputs. Root morphology, anatomy, and ecophysiological processes may have been affected by the substantial environmental and genetic shifts associated with this transition. As a result, root and rhizosphere traits that allow more efficient foraging and uptake in lower synthetic input environments might have been lost. The development of modern maize has led to a shift in microbiome community composition, but questions remain as to the dynamics and drivers of this change during maize evolution and its implications for resource acquisition and agroecosystem functioning under different management practices. Better understanding of how domestication and breeding affected root and rhizosphere microbial traits could inform breeding strategies, facilitate the sourcing of favorable alleles, and open new frontiers to improve resource use efficiency through greater integration of root development and ecophysiology with agroecosystem functioning.

The effect of altered dosage of a mutant allele of Teosinte branched 1 ( tb1-ref ) on the root system of modern maize

BackgroundThere was ancient human selection on the wild progenitor of modern maize, Balsas teosinte, for decreased shoot branching (tillering), in order to allow more nutrients to be diverted to grain. Mechanistically, the decline in shoot tillering has been associated with selection for increased expression of the major domestication gene Teosinte Branched 1 (Tb1) in shoot primordia. Therefore, TB1 has been defined as a repressor of shoot branching. It is known that plants respond to changes in shoot size by compensatory changes in root growth and architecture. However, it has not been reported whether altered TB1 expression affects any plant traits below ground. Previously, changes in dosage of a well-studied mutant allele of Tb1 in modern maize, called tb1-ref, from one to two copies, was shown to increase tillering. As a result, plants with two copies of the tb1-ref allele have a larger shoot biomass than heterozygotes. Here we used aeroponics to phenotype the effects of tb1-ref copy number on maize roots at macro-, meso- and micro scales of development.ResultsAn increase in the tb1-ref copy number from one to two copies resulted in: (1) an increase in crown root number due to the cumulative initiation of crown roots from successive tillers; (2) higher density of first and second order lateral roots; and (3) reduced average lateral root length. The resulting increase in root system biomass in homozygous tb1-ref mutants balanced the increase in shoot biomass caused by enhanced tillering. These changes caused homozygous tb1-ref mutants of modern maize to more closely resemble its ancestor Balsas teosinte below ground.ConclusionWe conclude that a decrease in TB1 function in maize results in a larger root system, due to an increase in the number of crown roots and lateral roots. Given that decreased TB1 expression results in a more highly branched and larger shoot, the impact of TB1 below ground may be direct or indirect. We discuss the potential implications of these findings for whole plant coordination of biomass accumulation and maize domestication.

Addressing agricultural nitrogen losses in a changing climate

Losses of nitrogen from agriculture are a major threat to environmental and human health at local, regional and global scales. Emerging evidence shows that climate change and intensive agricultural management will interact to increase the harmful effects and undermine current mitigation efforts. Identifying effective mitigation strategies and supporting policies requires an integrated understanding of the processes underlying potential agricultural nitrogen responses to climate change. In this Review, we describe these processes, propose a set of multi-scale principles to guide research and policy for decreasing nitrogen losses in the future, and describe the economic factors that could constrain or enable their implementation.Climate change and intensive agricultural management will interact to increase nitrogen (N) losses from agriculture. This Review analyses the processes underlying potential agricultural N responses to climate change, proposes a set of principles to help decrease N losses in the future and describes the economic factors that could affect their implementation.

An Optimized Rhizobox Protocol to Visualize Root Growth and Responsiveness to Localized Nutrients

Roots are notoriously difficult to study. Soil is both a visual and mechanical barrier, making it difficult to track roots in situ without destructive harvest or expensive equipment. We present a customizable and affordable rhizobox method that allows the non-destructive visualization of root growth over time and is particularly well-suited to studying root plasticity in response to localized resource patches. The method was validated by assessing maize genotypic variation in plasticity responses to patches containing 15N-labeled legume residue. Methods are described to obtain representative developmental measurements over time, measure root length density in resource-containing and control patches, calculate root growth rates, and determine 15N recovery by plant roots and shoots. Advantages, caveats, and potential future applications of the method are also discussed. Although care must be taken to ensure that experimental conditions do not bias root growth data, the rhizobox protocol presented here yields reliable results if carried out with sufficient attention to detail.

What is the agronomic potential of biofertilizers for maize? A meta-analysis

ABSTRACT Biofertilizers are promoted as a strategy for sustainable intensification of agriculture, but their efficacy varies widely among published studies and it is unclear whether they deliver the promised benefits. Studies are commonly conducted under controlled conditions prior to deployment in the field, yet the predictive value of such studies for field‐scale productivity has not been critically examined. A meta‐analysis was conducted using a novel host crop‐specific approach to evaluate the agronomic potential of bacterial biofertilizers for maize. Yield increases tended to be slightly higher and more variable in greenhouse studies using field soil than in the field, and greenhouse studies poorly predicted the influence of moderating climate, soil and taxonomic variables. We found greater efficacy of Azospirillum spp. and lower efficacy of Bacillus spp. and Enterobacter spp. under field conditions. Surprisingly, biofertilizer strains with confirmed plant‐growth‐promoting traits such as phosphorus solubilization, nitrogen fixation and phytohormone production in vitro were associated with lower yields in the field than strains not confirmed to possess these traits; only 1‐aminocyclopropane‐1‐carboxylate deaminase synthesis increased yields. These results indicate the need for a novel biofertilizer development framework that integrates information from native soil microbial communities and prioritizes field validation of results.