Opportunities and challenges in doubled haploids and haploid inducer-mediated genome-editing systems in cucurbits

Doubled haploids have played a major role in cucurbit breeding for the past four decades. In situ parthenogenesis via irradiated pollen is the preferred technique in order to obtain haploid plantlets further chromosome doubled in Cucurbitaceae like melon, cucumber, pumpkin, squash and winter squash. In contrast to doubled haploid procedures in other species, in situ parthenogenesis in cucurbits present many limiting factors which impede an efficient production of haploids. In addition, is very time-consuming and labor-intense. However, haploid inducer-mediated genome-editing system is a breakthrough technology for doubled haploid production. CRISPR/Cas9 system have been reported several times in cucurbit species and although its application has many bottlenecks the targeting of CENH3 gene for knock-out will allow breeders to obtain haploid inducer lines further used for parthenogenetic embryo obtention. In this review, we have discussed the progress made towards the development of doubled haploids and haploid inducer genotypes by using CRISPR/Cas9 technologies in cucurbit species. The present review provides insights for the application of haploid inducer-mediated genome-editing system in cucurbit species

Carbon and nitrogen allocation and partitioning in traditional and modern wheat genotypes under pre-industrial and future CO2 conditions

The results of a simultaneous 13C and 15N labelling experiment with two different durum wheat cultivars, Blanqueta (a traditional wheat) and Sula (modern), are pre- sented. Plants were grown from the seedling stage in three fully controllable plant growth chambers for one growing season and at three different CO2 levels (i.e. 260, 400 and 700 ppm). Short-term isotopic labelling (ca. 3 days) was performed at the anthesis stage using 13CO2 supplied with the chamber air and 15NH4-15NO3 applied with the nutrient solution, thereby making it possible to track the allocation and par- titioning of 13C and 15N in the different plant organs. We found that photosynthesis was up-regulated at pre-industrial CO2 levels, whereas down-regulation occurred under future CO2 conditions. 13C labelling revealed that at pre-industrial CO2 carbon investment by plants was higher in shoots, whereas at future CO2 levels more C was invested in roots. Furthermore, the modern genotype invested more C in spikes than did the traditional genotype, which in turn invested more in non-reproductive shoot tissue. 15N labelling revealed that the modern genotype was better adapted to assimi- lating N at higher CO2 levels, whereas the traditional genotype was able to assimilate N more efficiently at lower CO2 levels

Parthenogenetic double haploid production in melon 'Piel de Sapo' for breeding purposes

Double haploids in melon breeding are commonly used for hybrid F1 production. Via parthenogenesis, double haploids can be efficiently obtained in melon. Melon fruit traits and pathogen resistances were first appraised to analyse the commercial value of donor genotypes further used in a parthenogenetic process. Parthenogenetic capacity from melon 'Piel de Sapo' germplasm, including six inbred genotypes and Melito cultivar, was evaluated and optimized in this study. Melon fruit set after pollination with irradiated pollen, haploid embryo obtention, in vitro germination and growth of haploid embryos, in vitro and in vivo chromosome doubling with colchicine or oryzalin, and fruit set of double haploid plants were steps deeply assessed in this study. Parthenogenetic efficiencies of 'Piel de Sapo' genotypes showed a high genotypic dependency during the whole process. Pollinated female flowers set fruit after pollination with irradiated pollen (15.78%). Three different methods were assayed for haploid embryo detection of the 178 melon fruits recovered: one-by-one, X-ray and liquid medium. X-ray radiography of seeds was 4-5 times faster than one-by-one method and jeopardized eight times less haploid embryo obtention than liquid medium. One third of those fruits carried haploid embryos, which 50.94% did not developed into plantlets because failed to germinate or plant died at the first stages of development as a consequence of deleterious gene combination in haploid homozygosity. The distribution of the ploidy-level of 26 parthenogenetic plantlets was: 73.08% haploids, 23.08% spontaneous double haploids and 3.84% mixoploids. Two in vitro chromosome doubling methods with colchicine or oryzalin were compared with a third in vivo method. In vivo immersion of apical meristems showed the best results of plant survival, 57.33%, and doubling, 9.30% mixoploids and 20.93% double haploids. Fruit set and seed recovery of double haploids plants was achieved. In this study, double haploid lines were produced from the seven genotypes of melon 'Piel de Sapo', however, further improvements are needed to increase the parthenogenetic efficiency

Limited carbon inputs from plants into soils in arid ecosystems: a study of changes in the d13C in the soil-root interface

Background and aims. The tracing of C assimilation and the subsequent partitioning among plant organs has been a central focus of studies utilising Free Air CO2 Enrichment (FACE) facilities. The approach makes use of the fossil origin of this carbon, which is depleted in 13C. However, there is little data for desert environments. The Nevada Desert FACE Facility (NDFF), located in the Mojave Desert, has been one of the main facilities for the study of C dynamics in arid ecosystems and how they respond to rising atmospheric CO2 concentrations. In this experiment, we studied the incorporation of fixed CO2 during the previous two years (detectable by its lower 13C) in the soil fraction surrounding roots. Methods. The soil was collected monthly in direct vicinity to the roots during a complete growth season, at two depths (5 and 15 cm). Soil samples were dried and fractionated by size (> 50 μm and 50 μm), 13C values ranged between -1 and -2¿ for carbonates and between -23 and -25¿ for soil organic matter. These values did not significantly change throughout the experiment and were not affected by depth (5 or 15 cm). In contrast, 13C values for both organic and inorganic carbon in the fine fraction ( 50 μm). The 13C values for organic C ranged mostly between -20¿ and -27¿, and were roughly maintained throughout the sampling period. For inorganic C, the 13C values were mostly between 0¿ and -15¿, and tended to become less negative during the course of the sampling period. Overall the effect of [CO2] on 13C values of either organic or inorganic carbon was not significant for any experimental condition (plant species, depth, fraction). Conclusion. Little or no signs of recently fixed CO2 (13C-depleted) were detected in the soils close to the roots, in the coarse fraction (> 50 μm), the fine fraction (< 50 μm), the organic matter, or in carbonates. This indicates a slow C turnover 45 in the studied soils, which can result from a highly conservative use of photoassimilates by plants, including a very low release of organic matter into the soil in the form of dead roots or root exudates, and from a conservative use of available C reserves

Characterization of different Arundo donax L. Clones from the Mediterranean region

The present study assessed the behaviour of a tour ecotypes of Arundo donax L. (giant reed) as a perennial rhizomatous grass with an increasing interest due to its high biomass production and great s adaptability to stress conditions. In this study we perform a molecular, physiological and biomass characterization in greenhouse conditions on four mediterranean ecotypes. Not large significant differences were found in physiological and biomass parameters. However, it was possible to observe large differences in the chromosome count for the four ecotypes. In this way, we detected different number of chromosomes for each ecotype (98 to 122), but surprisingly, no correlation was observad between their chromosomes number and their physiological and biomass responses

Non-chemical weed management for sustainable rice production in the Ebro Delta

Weed control is one of the major challenges in rice cultivation, and the use of agrochemicals in this crop is severely restricted under the new European agricultural policy. Therefore, new effective non-chemical weed control agents are the key to sustain European rice production. We investigated four non-chemical weed management strategies in the Ebro Delta in north-eastern Spain, two in dry-seeded rice fields and two in water-seeded rice fields. In addition, two controls per sowing conditions were included; a positive control consisting in chemical herbicides treatment and a negative control consisting in no weeding and no seeding. In all tests but negative controls, the rice variety Argila was employed. "Simple dry seeding" was the best treatment for dry seeding, while "false seeding" (stale seed bed) and planting was the best performing under water seeding conditions. Both mentioned treatments were as effective as chemical control in reducing the density of weeds and the weeding time for those species more abundant in Ebro Delta rice fields (i.e. Echinochloa oryzoides, Echinochloa crus-galli, Scirpus maritimus and Heteranthera reniformis). Our results indicated that some of the non-chemical weed control methods can reduce weed pressure to levels similar to standard chemical herbicide treatments under certain seeding conditions

Morpho-physiological responses of alamo switchgrass during germination and early seedling stage under salinity or water stress conditions

Switchgrass (Panicum virgatum L.) is a warm perennial grass with valuable characteristics as a biofuel crop. To avoid competition with food crops, biofuel crops will be likely relegated to less productive soils such as marginal lands. Consequently, the salinity and water scarcity problems that commonly affect marginal lands compromise biofuel crop germination, emergence, and seedling establishment. The aims of this study were to study the germination and seedling growth of switchgrass under salinity and water stress and to describe the morpho-anatomical responses of the roots and leaves in the seedlings to these stress conditions. The effect of salt and water stress was assessed using sodium chloride (NaCl) and polyethylene glycol (PEG) 8000 at the same water potentials of − 0.8, − 1.0, and − 1.2 MPa. Seeds were moist prechilled for 7 days at 5 °C and germinated at 30/15 °C (8 h light/16 h dark). NaCl treatments (− 0.8 and − 1.0 MPa) delayed germination rates but did not reduce the final germination percentage, whereas at a lower potential (− 1.2 MPa), the final germination percentage was diminished. The effects of PEG (− 1.0 and − 1.2 MPa) on the germination rate and final percentage were more detrimental than those induced by isosmotic concentrations of NaCl. PEG and NaCl reduced significantly the vigor index of − 0.8 to − 1.2 MPa. The morphoanatomical changes such as the reduction in the root cross-sectional area and the thickening of the endodermis walls for both stress conditions and aerenchyma formation in the cortex under salinity could significantly contribute in the survival and tolerance during the early seedling stages

Photosynthesis, resource acquisition and growth responses of two biomass crops subjected to water stress

This study compares photosynthesis, growth, 13C and 15N labelling patterns of two biomass crops (Arundo donax L. and Panicum virgatum L.) grown under water stress in greenhouse conditions. Plants were exposed to three water stress levels: control (C, 100% Pot Capacity), mild stress (MS, 50% PC) and severe stress (SS, 25% PC). Photosynthesis, fluorescence parameters and relative water content were measured at the beginning (Ti) and the end of the experiment (Tf). Biomass parameters were measured at Tf. Short-term double labelling with 13C and 15N stable isotopes was performed in both species. Isotopic analyses of total organic matter, total soluble sugars and the CO2 respired were undertaken at T0 (prelabelling), T1 (24h after labelling) and T2 (7 days after labelling). Immediately after the 13C and 15N labelling, stems and rhizomes seemed to be the main sinks for labelled carbon and nitrogen in both species. Moreover, not all of the labelled carbon and nitrogen substrate was used by plant metabolism after seven days. Decreases in photosynthesis parameters were observed as a consequence of the increase in water stress (WS) in both species, with a greater magnitude decline in giant reed than in switchgrass. A decrease in height, number of green leaves and total dry weight due to WS was observed in both species. Both species were more 13C-enriched and more 15N-depleted during the increases in WS due to lower stomatal conductance and transpiration. In general, WS accelerated plant phenology and, consequently, the accumulation of storage compounds in the rhizome occurred in response to stress. This effect was more clearly visible in switchgrass than in giant reed

Drought Impact on the Morpho‐Physiological Parameters of Perennial Rhizomatous Grasses in the Mediterranean Environment

The selection of non‐food crops for bioenergy production in limiting environments is a priority for energy security and climate change mitigation. Therefore, more studies are needed on the interactions between species and environmental factors in specific sites which allows their selection for biomass production. The objective of this work is to study the impact of drought on the morpho‐physiological parameters of perennial rhizomatous grasses Panicum virgatum L., Miscanthus × giganteus, and Arundo donax L. in the Mediterranean environment. Plants were grown on field and trials were carried out under support‐irrigation and rainfed conditions during two consecutive years. Morpho‐physiological parameters were measured in May, June and August, and dry biomass at the end of the experiment. Under rainfed conditions, A. donax presented the highest photosynthesis rate (25, 15 and 10 CO2 m−2 s−1), relative water content (85-90%), and dry biomass (~4500 g plant−1) compared with P. virgatum (20, 5 and 5 CO2 m−2 s−1, 65-85% RWC and ~1400 g plant−1) and Miscanthus (18, 4 and 0 CO2 m−2 s−1, 80-10% RWC and ~260 g plant−1). It is concluded that A. donax would be the best perennial rhizomatous grass to be used as bioenergy crop under Mediterranean conditions

Effect of shoot removal on remobilization of carbon and nitrogen during regrowth of nitrogen-fixing alfalfa

The contribution of carbon and nitrogen reserves to regrowth following shoot removal and the processes involved in the reduction of nodule functioning were studied in alfalfa plants (Medicago sativa L.). To do so, isotopic labelling was conducted at root and canopy level with both 15N2 and 13C-depleted CO2 on exclusively nitrogen fixing alfalfa plants. Our results indicate that the roots were the main sink organs before shoot removal as expected. Seven days after regrowth the carbon and nitrogen stored in the roots was invested in shoot biomass formation and partitioned to the nodules in order to sustain respiratory activity. However, this carbon and nitrogen derived from the root did not overcome carbon and nitrogen limitation in nodules and leaves. Together with the limited carbohydrate availability, the up-regulation of nodule peroxidases indicates the involvement of oxidative stress in a worse nodule performance. Fourteen days after shoot removal, leaf and nodule performance were completely reestablished