Chromosome doubling methods in doubled haploid and haploid inducer-mediated genome-editing systems in major crops.

The doubled haploid technique aims to generate pure inbred lines for basic research and as commercial cultivars. The doubled haploid technique first generates haploid plants and is followed by chromosome doubling, which can be separated in time or overlapped, depending the procedure for each species. For a long time, much effort has been focused on haploid production via androgenesis, gynogenesis, or parthenogenesis. The obtention of haploid plants has frequently required more optimization and has lagged behind research and improvements in chromosome doubling methods. Nevertheless, chromosome doubling has recently been of renewed interest to increase the rates and efficiency of doubled haploid plant production through trialing and optimizing of different procedures. New antimitotic compounds and application methods are being studied to ensure the success of chromosome doubling once haploid material has been regenerated. Moreover, a haploid inducer-mediated CRISPR/Cas9 genome-editing system is a breakthrough method in the production of haploid plant material and could be of great importance for species where traditional haploid regeneration methods have not been successful, or for recalcitrant species. In all cases, the new deployment of this system will demand a suitable chromosome doubling protocol. In this review, we explore the existing doubled haploid and chromosome doubling methods to identify opportunities to enhance the breeding process in major crops.

Four years of experimental warming do not modify the interaction between subalpine shrub species.

Climate warming can lead to changes in alpine plant species interactions through modifications in environmental conditions, which may ultimately cause drastic changes in plant communities. We explored the effects of 4 years of experimental warming with open-top chambers (OTC) on Vaccinium myrtillus performance and its interaction with neighbouring shrubs at the Pyrenean treeline ecotone. We examined the effects of warming on height, above-ground (AG) and below-ground (BG) biomass and the C and N concentration and isotope composition of V. myrtillus growing in pure stands or in stands mixed with Vaccinium uliginosum or Rhododendron ferrugineum. We also analysed variations in soil N concentrations, rhizosphere C/N ratios and the functional diversity of the microbial community, and evaluated whether warming altered the biomass, C and N concentration and isotope composition of V. uliginosum in mixed plots. Our results showed that warming induced positive changes in the AG growth of V. myrtillus but not BG, while V. uliginosum did not respond to warming. Vaccinium myrtillus performance did not differ between stand types under increased temperatures, suggesting that warming did not induce shifts in the interaction between V. myrtillus and its neighbouring species. These findings contrast with previous studies in which species interactions changed when temperature was modified. Our results show that species interactions can be less responsive to warming in natural plant communities than in removal experiments, highlighting the need for studies involving the natural assembly of plant species and communities when exploring the effect of environmental changes on plant-plant interactions.

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 has been studied in the past. However, important gaps remain in understanding the effect of shoot cutting on nodule performance and its relevance during regrowth. In this study, isotopic labelling was conducted at root and canopy levels with both (15) N2 and (13) C-depleted CO2 on exclusively nitrogen-fixing alfalfa plants. As expected, our results indicate that the roots were the main sink organs before shoots were removed. Seven days after regrowth the carbon and nitrogen stored in the roots was invested in shoot biomass formation and partitioned to the nodules. The large depletion in nodule carbohydrate availability suggests that root-derived carbon compounds were delivered towards nodules in order to sustain respiratory activity. In addition to the limited carbohydrate availability, the upregulation of nodule peroxidases showed that oxidative stress was also involved during poor nodule performance. Fourteen days after cutting, and as a consequence of the stimulated photosynthetic and N2 -fixing machinery, availability of Cnew and Nnew strongly diminished in the plants due to their replacement by C and N assimilated during the post-labelling period. In summary, our study indicated that during the first week of regrowth, root-derived C and N remobilization did not overcome C- and N-limitation in nodules and leaves. However, 14 days after cutting, leaf and nodule performance were re-established.

Metabolic origin of δ15 N values in nitrogenous compounds from Brassica napus L. leaves.

Nitrogen isotope composition (δ(15) N) in plant organic matter is currently used as a natural tracer of nitrogen acquisition efficiency. However, the δ(15) N value of whole leaf material does not properly reflect the way in which N is assimilated because isotope fractionations along metabolic reactions may cause substantial differences among leaf compounds. In other words, any change in metabolic composition or allocation pattern may cause undesirable variability in leaf δ(15) N. Here, we investigated the δ(15) N in different leaf fractions and individual metabolites from rapeseed (Brassica napus) leaves. We show that there were substantial differences in δ(15) N between nitrogenous compounds (up to 30‰) and the content in ((15) N enriched) nitrate had a clear influence on leaf δ(15) N. Using a simple steady-state model of day metabolism, we suggest that the δ(15) N value in major amino acids was mostly explained by isotope fractionation associated with isotope effects on enzyme-catalysed reactions in primary nitrogen metabolism. δ(15) N values were further influenced by light versus dark conditions and the probable occurrence of alternative biosynthetic pathways. We conclude that both biochemical pathways (that fractionate between isotopes) and nitrogen sources (used for amino acid production) should be considered when interpreting the δ(15) N value of leaf nitrogenous compounds.

Concerted changes in N and C primary metabolism in alfalfa (Medicago sativa) under water restriction.

Although the mechanisms of nodule N(2) fixation in legumes are now well documented, some uncertainty remains on the metabolic consequences of water deficit. In most cases, little consideration is given to other organs and, therefore, the coordinated changes in metabolism in leaves, roots, and nodules are not well known. Here, the effect of water restriction on exclusively N(2)-fixing alfalfa (Medicago sativa L.) plants was investigated, and proteomic, metabolomic, and physiological analyses were carried out. It is shown that the inhibition of nitrogenase activity caused by water restriction was accompanied by concerted alterations in metabolic pathways in nodules, leaves, and roots. The data suggest that nodule metabolism and metabolic exchange between plant organs nearly reached homeostasis in asparagine synthesis and partitioning, as well as the N demand from leaves. Typically, there was (i) a stimulation of the anaplerotic pathway to sustain the provision of C skeletons for amino acid (e.g. glutamate and proline) synthesis; (ii) re-allocation of glycolytic products to alanine and serine/glycine; and (iii) subtle changes in redox metabolites suggesting the implication of a slight oxidative stress. Furthermore, water restriction caused little change in both photosynthetic efficiency and respiratory cost of N(2) fixation by nodules. In other words, the results suggest that under water stress, nodule metabolism follows a compromise between physiological imperatives (N demand, oxidative stress) and the lower input to sustain catabolism.

Arundo donax L. growth potential under different abiotic stress.

Arundo donax L. (giant reed) is a fast-growing, vegetatively multiplying, and rhizomatous perennial grass. It is considered a leading crop for biomass production on marginal and degraded lands under different adverse conditions such as drought, salinity, waterlogging, high and low temperatures, and heavy metal stress. The giant reed tolerance to those stresses is reviewed based on its effects on photosynthetic capacity and biomass production. Possible explanations for the giant reed tolerance against each particular stress were elucidated, as well as changes shown by the plant at a biochemical, physiological and morphological level, that may directly affect its biomass production. The use of giant reed in other areas of interest such as bioconstruction, phytoremediation, and bioremediation, is also reviewed. Arundo donax can be key for circular economy and global warming mitigation.

Marker-Assisted Introgression of the Salinity Tolerance Locus Saltol in Temperate Japonica Rice.

BACKGROUND: Rice is one of the most salt sensitive crops at seedling, early vegetative and reproductive stages. Varieties with salinity tolerance at seedling stage promote an efficient growth at early stages in salt affected soils, leading to healthy vegetative growth that protects crop yield. Saltol major QTL confers capacity to young rice plants growing under salt condition by maintaining a low Na+/K+ molar ratio in the shoots. RESULTS: Marker-assisted backcross (MABC) procedure was adopted to transfer Saltol locus conferring salt tolerance at seedling stage from donor indica IR64-Saltol to two temperate japonica varieties, Vialone Nano and Onice. Forward and background selections were accomplished using polymorphic KASP markers and a final evaluation of genetic background recovery of the selected lines was conducted using 15,580 SNP markers obtained from Genotyping by Sequencing. Three MABC generations followed by two selfing, allowed the identification of introgression lines achieving a recovery of the recurrent parent (RP) genome up to 100% (based on KASP markers) or 98.97% (based on GBS). Lines with highest RP genome recovery (RPGR) were evaluated for agronomical-phenological traits in field under non-salinized conditions. VN1, VN4, O1 lines were selected considering the agronomic evaluations and the RPGR% results as the most interesting for commercial exploitation. A physiological characterization was conducted by evaluating salt tolerance under hydroponic conditions. The selected lines showed lower standard evaluation system (SES) scores: 62% of VN4, and 57% of O1 plants reaching SES 3 or SES 5 respectively, while only 40% of Vialone Nano and 25% of Onice plants recorded scores from 3 to 5, respectively. VN1, VN4 and O1 showed a reduced electrolyte leakage values, and limited negative effects on relative water content and shoot/root fresh weight ratio. CONCLUSION: The Saltol locus was successfully transferred to two elite varieties by MABC in a time frame of three years. The application of background selection until BC3F3 allowed the selection of lines with a RPGR up to 98.97%. Physiological evaluations for the selected lines indicate an improved salinity tolerance at seedling stage. The results supported the effectiveness of the Saltol locus in temperate japonica and of the MABC procedure for recovering of the RP favorable traits.

Dual Δ¹³C/δ¹8O response to water and nitrogen availability and its relationship with yield in field-grown durum wheat.

The combined use of stable carbon and oxygen isotopes in plant matter is a tool of growing interest in cereal crop management and breeding, owing to its relevance for assessing the photosynthetic and transpirative performance under different growing conditions including water and N regimes. However, this method has not been applied to wheat grown under real field conditions. Here, plant growth, grain yield (GY) and the associated agronomic components, carbon isotope discrimination (Δ¹³C) plus oxygen isotope composition (δ¹8O) as well as leaf and canopy gas exchange were measured in field-grown wheat subjected to different water and N availabilities. Water limitation was the main factor affecting yield, leaf and canopy gas exchange and Δ¹³C and δ¹8O, whereas N had a smaller effect on such traits. The combination of Δ¹³C and δ¹8O gave a clear advantage compared with gas exchange measurements, as it provides information on the instantaneous and the long-term plant photosynthetic and transpirative performance and are less labour intensive than gas exchange measurements. In addition, the combination of plant Δ¹³C and δ¹8O predicted differences in GY and related agronomical parameters, providing agronomists and breeders with integrative traits for selecting crop management practices and/or genotypes with better performance under water-limiting and N-limiting conditions.

Plant physiology and proteomics reveals the leaf response to drought in alfalfa (Medicago sativa L.).

Despite its relevance, protein regulation, metabolic adjustment, and the physiological status of plants under drought is not well understood in relation to the role of nitrogen fixation in nodules. In this study, nodulated alfalfa plants were exposed to drought conditions. The study determined the physiological, metabolic, and proteomic processes involved in photosynthetic inhibition in relation to the decrease in nitrogenase (N(ase)) activity. The deleterious effect of drought on alfalfa performance was targeted towards photosynthesis and N(ase) activity. At the leaf level, photosynthetic inhibition was mainly caused by the inhibition of Rubisco. The proteomic profile and physiological measurements revealed that the reduced carboxylation capacity of droughted plants was related to limitations in Rubisco protein content, activation state, and RuBP regeneration. Drought also decreased amino acid content such as asparagine, and glutamic acid, and Rubisco protein content indicating that N availability limitations were caused by N(ase) activity inhibition. In this context, drought induced the decrease in Rubisco binding protein content at the leaf level and proteases were up-regulated so as to degrade Rubisco protein. This degradation enabled the reallocation of the Rubisco-derived N to the synthesis of amino acids with osmoregulant capacity. Rubisco degradation under drought conditions was induced so as to remobilize Rubisco-derived N to compensate for the decrease in N associated with N(ase) inhibition. Metabolic analyses showed that droughted plants increased amino acid (proline, a major compound involved in osmotic regulation) and soluble sugar (D-pinitol) levels to contribute towards the decrease in osmotic potential (Ψ(s)). At the nodule level, drought had an inhibitory effect on N(ase) activity. This decrease in N(ase) activity was not induced by substrate shortage, as reflected by an increase in total soluble sugars (TSS) in the nodules. Proline accumulation in the nodule could also be associated with an osmoregulatory response to drought and might function as a protective agent against ROS. In droughted nodules, the decrease in N(2) fixation was caused by an increase in oxygen resistance that was induced in the nodule. This was a mechanism to avoid oxidative damage associated with reduced respiration activity and the consequent increase in oxygen content. This study highlighted that even though drought had a direct effect on leaves, the deleterious effects of drought on nodules also conditioned leaf responsiveness.

Does ear C sink strength contribute to overcoming photosynthetic acclimation of wheat plants exposed to elevated CO2?

Wheat plants (Triticum durum Desf., cv. Regallo) were grown in the field to study the effects of contrasting [CO(2)] conditions (700 versus 370 µmol mol(-1)) on growth, photosynthetic performance, and C management during the post-anthesis period. The aim was to test whether a restricted capacity of sink organs to utilize photosynthates drives a loss of photosynthetic capacity in elevated CO(2). The ambient (13)C/(12)C isotopic composition (δ(13)C) of air CO(2) was changed from -10.2‰ in ambient [CO(2)] to -23.6‰ under elevated [CO(2)] between the 7th and the 14th days after anthesis in order to study C assimilation and partitioning between leaves and ears. Elevated [CO(2)] had no significant effect on biomass production and grain filling, and caused an accumulation of C compounds in leaves. This was accompanied by up-regulation of phosphoglycerate mutase and ATP synthase protein content, together with down-regulation of adenosine diphosphate glucose pyrophosphatase protein. Growth in elevated [CO(2)] negatively affected Rubisco and Rubisco activase protein content and induced photosynthetic down-regulation. CO(2) enrichment caused a specific decrease in Rubisco content, together with decreases in the amino acid and total N content of leaves. The C labelling revealed that in flag leaves, part of the C fixed during grain filling was stored as starch and structural C compounds whereas the rest of the labelled C (mainly in the form of soluble sugars) was completely respired 48 h after the end of labelling. Although labelled C was not detected in the δ(13)C of ear total organic matter and respired CO(2), soluble sugar δ(13)C revealed that a small amount of labelled C reached the ear. The (12)CO(2) labelling suggests that during the beginning of post-anthesis the ear did not contribute towards overcoming flag leaf carbohydrate accumulation, and this had a consequent effect on protein expression and photosynthetic acclimation.

Measurement of 13C and 15N isotope labeling by gas chromatography/combustion/isotope ratio mass spectrometry to study amino acid fluxes in a plant-microbe symbiotic association.

We have developed a method based on a double labeling with stable isotopes and gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) analyses to study amino acid exchange in a symbiotic plant-microbe association. Isotopic precision was studied for 21 standards including 15 amino acid derivatives, three N-protected amino acid methyl esters, three amines and one international standard. High correlations were observed between the δ(13)C and δ(15)N values obtained by GC/C/IRMS and those obtained by an elemental analyzer (EA) coupled to an isotope ratio mass spectrometer (R(2) = 0.9868 and 0.9992, respectively). The mean precision measured was 0.04‰ for δ(13)C and 0.28‰ for δ(15)N (n = 15). This method was applied in vivo to the symbiotic relationship between alfalfa (Medicago sativa L.) and N(2)-fixing bacteria. Plants were simultaneously labeled over 10 days with (13)C-depleted CO(2) ((12)CO(2)), which was assimilated through photosynthesis by leaves, and (15)N(2) fixed via nodules. Subsequently, the C and N isotope compositions (i.e. δ(13)C and δ(15)N) of free amino acids were analyzed in leaves and nodules by GC/C/IRMS. The method revealed the pattern of C and N exchange between leaves and nodules, highlighting that γ-aminobutanoic acid and glycine may represent an important form of C transport from leaves to the nodules. The results confirmed the validity, reliability and accuracy of the method for assessing C and N fluxes between plants and symbiotic bacteria and support the use of this technique in a broad range of metabolic and fluxomic studies.

Maintenance of C sinks sustains enhanced C assimilation during long-term exposure to elevated [CO2] in Mojave Desert shrubs.

During the first few years of elevated atmospheric [CO(2)] treatment at the Nevada Desert FACE Facility, photosynthetic downregulation was observed in desert shrubs grown under elevated [CO(2)], especially under relatively wet environmental conditions. Nonetheless, those plants maintained increased A (sat) (photosynthetic performance at saturating light and treatment [CO(2)]) under wet conditions, but to a much lesser extent under dry conditions. To determine if plants continued to downregulate during long-term exposure to elevated [CO(2)], responses of photosynthesis to elevated [CO(2)] were examined in two dominant Mojave Desert shrubs, the evergreen Larrea tridentata and the drought-deciduous Ambrosia dumosa, during the eighth full growing season of elevated [CO(2)] treatment at the NDFF. A comprehensive suite of physiological processes were collected. Furthermore, we used C labeling of air to assess carbon allocation and partitioning as measures of C sink activity. Results show that elevated [CO(2)] enhanced photosynthetic performance and plant water status in Larrea, especially during periods of environmental stress, but not in Ambrosia. δ(13)C analyses indicate that Larrea under elevated [CO(2)] allocated a greater proportion of newly assimilated C to C sinks than Ambrosia. Maintenance by Larrea of C sinks during the dry season partially explained the reduced [CO(2)] effect on leaf carbohydrate content during summer, which in turn lessened carbohydrate build-up and feedback inhibition of photosynthesis. δ(13)C results also showed that in a year when plant growth reached the highest rates in 5 years, 4% (Larrea) and 7% (Ambrosia) of C in newly emerging organs were remobilized from C that was assimilated and stored for at least 2 years prior to the current study. Thus, after 8 years of continuous exposure to elevated [CO(2)], both desert perennials maintained their photosynthetic capacities under elevated [CO(2)]. We conclude that C storage, remobilization, and partitioning influence the responsiveness of these desert shrubs during long-term exposure to elevated [CO(2)].

Anther Culture and Chromosome Doubling in Mediterranean Japonica Rice.

Anther culture is the most used technique to produce doubled haploid lines in rice. This technique is well developed in a wide range of indica rice genotypes. However, in japonica type, and more specifically, the Mediterranean japonica, the protocols are yet to be optimized. Japonica and indica have different androgenic response, as well as different induction and regeneration rates, albinism ratios and chromosome doubling competence. The step-by-step anther culture protocol presented in this chapter allows to regenerate doubled haploid rice plantlets from anther microspores in 8 months. We also include an in vitro chromosome doubling protocol to induce doubled haploids from haploid plantlets by immersion in a colchicine solution. This chromosome doubling protocol complements the anther culture by taking advantage of the regenerated haploid plantlets.

Doubled Haploid Parthenogenetic Production of Melon 'Piel de Sapo'.

Parthenogenesis is the main technique to produce doubled haploid lines in melon. Although parthenogenesis is a genotype-dependent process and melon has a huge genotypic diversity, we developed a successful protocol for haploid embryo production via pollination with irradiated pollen and a protocol for chromosome doubling of haploid plants of 'Piel de Sapo' genotypes. 'Piel de sapo' genotype has lower efficiencies during the process in comparison with other genotypes, for instance, of the agrestis subspecies. Nevertheless, the doubled haploid lines produced have a great potential as pure parentals for hybrid seed production.

Integrative Approach for Precise Genotyping and Transcriptomics of Salt Tolerant Introgression Rice Lines.

Rice is the most salt sensitive cereal crop and its cultivation is particularly threatened by salt stress, which is currently worsened due to climate change. This study reports the development of salt tolerant introgression lines (ILs) derived from crosses between the salt tolerant indica rice variety FL478, which harbors the Saltol quantitative trait loci (QTL), and the salt-sensitive japonica elite cultivar OLESA. Genotyping-by-sequencing (GBS) and Kompetitive allele specific PCR (KASPar) genotyping, in combination with step-wise phenotypic selection in hydroponic culture, were used for the identification of salt-tolerant ILs. Transcriptome-based genotyping allowed the fine mapping of indica genetic introgressions in the best performing IL (IL22). A total of 1,595 genes were identified in indica regions of IL22, which mainly located in large introgressions at Chromosomes 1 and 3. In addition to OsHKT1;5, an important number of genes were identified in the introgressed indica segments of IL22 whose expression was confirmed [e.g., genes involved in ion transport, callose synthesis, transcriptional regulation of gene expression, hormone signaling and reactive oxygen species (ROS) accumulation]. These genes might well contribute to salt stress tolerance in IL22 plants. Furthermore, comparative transcript profiling revealed that indica introgressions caused important alterations in the background gene expression of IL22 plants (japonica cultivar) compared with its salt-sensitive parent, both under non-stress and salt-stress conditions. In response to salt treatment, only 8.6% of the salt-responsive genes were found to be commonly up- or down-regulated in IL22 and OLESA plants, supporting massive transcriptional reprogramming of gene expression caused by indica introgressions into the recipient genome. Interactions among indica and japonica genes might provide novel regulatory networks contributing to salt stress tolerance in introgression rice lines. Collectively, this study illustrates the usefulness of transcriptomics in the characterization of new rice lines obtained in breeding programs in rice.

In folio isotopic tracing demonstrates that nitrogen assimilation into glutamate is mostly independent from current CO2 assimilation in illuminated leaves of Brassica napus.

*Nitrogen assimilation in leaves requires primary NH(2) acceptors that, in turn, originate from primary carbon metabolism. Respiratory metabolism is believed to provide such acceptors (such as 2-oxoglutarate), so that day respiration is commonly seen as a cornerstone for nitrogen assimilation into glutamate in illuminated leaves. However, both glycolysis and day respiratory CO(2) evolution are known to be inhibited by light, thereby compromising the input of recent photosynthetic carbon for glutamate production. *In this study, we carried out isotopic labelling experiments with (13)CO(2) and (15)N-ammonium nitrate on detached leaves of rapeseed (Brassica napus), and performed (13)C- and (15)N-nuclear magnetic resonance analyses. *Our results indicated that the production of (13)C-glutamate and (13)C-glutamine under a (13)CO(2) atmosphere was very weak, whereas (13)C-glutamate and (13)C-glutamine appeared in both the subsequent dark period and the next light period under a (12)CO(2) atmosphere. Consistently, the analysis of heteronuclear ((13)C-(15)N) interactions within molecules indicated that most (15)N-glutamate and (15)N-glutamine molecules were not (13)C labelled after (13)C/(15)N double labelling. That is, recent carbon atoms (i.e. (13)C) were hardly incorporated into glutamate, but new glutamate molecules were synthesized, as evidenced by (15)N incorporation. *We conclude that the remobilization of night-stored molecules plays a significant role in providing 2-oxoglutarate for glutamate synthesis in illuminated rapeseed leaves, and therefore the natural day : night cycle seems critical for nitrogen assimilation.

On the 13C/12C isotopic signal of day and night respiration at the mesocosm level.

While there is currently intense effort to examine the (13)C signal of CO(2) evolved in the dark, less is known on the isotope composition of day-respired CO(2). This lack of knowledge stems from technical difficulties to measure the pure respiratory isotopic signal: day respiration is mixed up with photorespiration, and there is no obvious way to separate photosynthetic fractionation (pure c(i)/c(a) effect) from respiratory effect (production of CO(2) with a different delta(13)C value from that of net-fixed CO(2)) at the ecosystem level. Here, we took advantage of new simple equations, and applied them to sunflower canopies grown under low and high [CO(2)]. We show that whole mesocosm-respired CO(2) is slightly (13)C depleted in the light at the mesocosm level (by 0.2-0.8 per thousand), while it is slightly (13)C enriched in darkness (by 1.5-3.2 per thousand). The turnover of the respiratory carbon pool after labelling appears similar in the light and in the dark, and accordingly, a hierarchical clustering analysis shows a close correlation between the (13)C abundance in day- and night-evolved CO(2). We conclude that the carbon source for respiration is similar in the dark and in the light, but the metabolic pathways associated with CO(2) production may change, thereby explaining the different (12)C/(13)C respiratory fractionations in the light and in the dark.

In situ Parthenogenetic Doubled Haploid Production in Melon "Piel de Sapo" for Breeding Purposes.

Doubled haploids in cucurbit species are produced through in situ parthenogenesis via pollination with irradiated pollen for further use as parental lines for hybrid F1 production. In this study, seven genotypes of melon "Piel de Sapo" were appraised for agronomic traits and pathogen resistances to evaluate its commercial value and used as donor plant material for the parthenogenetic process. Then, in situ parthenogenetic capacity of melon "Piel de Sapo" germplasm was evaluated and optimized. Several steps of the parthenogenetic process were assessed in this study such as melon fruit set after pollination with irradiated pollen, haploid embryo obtention, in vitro germination and growth of parthenogenetic embryos and plantlets, in vitro and in vivo chromosome doubling with colchicine or oryzalin and fruit set of doubled haploid lines. Parthenogenetic efficiencies of "Piel de Sapo" genotypes showed a high genotypic dependency during the whole process. Three different methods were assayed for parthenogenetic embryo detection: one-by-one, X-ray and liquid medium. X-ray radiography of seeds was four times faster than one-by-one method and jeopardized eight times less parthenogenetic embryo obtention than liquid medium. One third of melon fruits set after pollination with irradiated pollen contained at least one parthenogenetic embryo. The 50.94% of the embryos rescued did not develop into plantlets because failed to germinate or plantlet died at the first stages of development because of deleterious gene combination in haploid homozygosity. The distribution of the ploidy-level of the 26 parthenogenetic plantlets obtained was: 73.08% haploid, 23.08% spontaneous doubled haploid and 3.84% mixoploid. Two in vitro chromosome doubling methods, with colchicine or oryzalin, were compared with a third in vivo colchicine method. In vivo immersion of apical meristems in a colchicine solution for 2 h showed the highest results of plant survival, 57.33%, and chromosome doubling, 9.30% mixoploids and 20.93% doubled haploids. Fruit set and seed recovery of doubled haploids lines was achieved. In this study, doubled haploid lines were produced from seven donor genotypes of melon "Piel de Sapo," however, further improvements are need in order to increase the parthenogenetic efficiency.

No preferential carbon-allocation to storage over growth in clipped birch and oak saplings.

Herbivory is one of the most globally distributed disturbances affecting carbon (C)-cycling in trees, yet our understanding of how it alters tree C-allocation to different functions such as storage, growth or rhizodeposition is still limited. Prioritized C-allocation to storage replenishment vs growth could explain the fast recovery of C-storage pools frequently observed in growth-reduced defoliated trees. We performed continuous 13C-labeling coupled to clipping to quantify the effects of simulated browsing on the growth, leaf morphology and relative allocation of stored vs recently assimilated C to the growth (bulk biomass) and non-structural carbohydrate (NSC) stores (soluble sugars and starch) of the different organs of two tree species: diffuse-porous (Betula pubescens Ehrh.) and ring-porous (Quercus petraea [Matt.] Liebl.). Carbon-transfers from plants to bulk and rhizosphere soil were also evaluated. Clipped birch and oak trees shifted their C-allocation patterns above-ground as a means to recover from defoliation. However, such increased allocation to current-year stems and leaves did not entail reductions in the allocation to the rhizosphere, which remained unchanged between clipped and control trees of both species. Betula pubescens and Q. petraea showed differences in their vulnerability and recovery strategies to clipping, the ring-porous species being less affected in terms of growth and architecture by clipping than the diffuse-porous. These contrasting patterns could be partly explained by differences in their C cycling after clipping. Defoliated oaks showed a faster recovery of their canopy biomass, which was supported by increased allocation of new C, but associated with large decreases in their fine root biomass. Following clipping, both species recovered NSC pools to a larger extent than growth, but the allocation of 13C-labeled photo-assimilates into storage compounds was not increased as compared with controls. Despite their different response to clipping, our results indicate no preventative allocation into storage occurred during the first year after clipping in either of the species.

Water and nitrogen conditions affect the relationships of Delta13C and Delta18O to gas exchange and growth in durum wheat.

Whereas the effects of water and nitrogen (N) on plant Delta(13)C have been reported previously, these factors have scarcely been studied for Delta(18)O. Here the combined effect of different water and N regimes on Delta(13)C, Delta(18)O, gas exchange, water-use efficiency (WUE), and growth of four genotypes of durum wheat [Triticum turgidum L. ssp. durum (Desf.) Husn.] cultured in pots was studied. Water and N supply significantly increased plant growth. However, a reduction in water supply did not lead to a significant decrease in gas exchange parameters, and consequently Delta(13)C was only slightly modified by water input. Conversely, N fertilizer significantly decreased Delta(13)C. On the other hand, water supply decreased Delta(18)O values, whereas N did not affect this parameter. Delta(18)O variation was mainly determined by the amount of transpired water throughout plant growth (T(cum)), whereas Delta(13)C variation was explained in part by a combination of leaf N and stomatal conductance (g(s)). Even though the four genotypes showed significant differences in cumulative transpiration rates and biomass, this was not translated into significant differences in Delta(18)O(s). However, genotypic differences in Delta(13)C were observed. Moreover, approximately 80% of the variation in biomass across growing conditions and genotypes was explained by a combination of both isotopes, with Delta(18)O alone accounting for approximately 50%. This illustrates the usefulness of combining Delta(18)O and Delta(13)C in order to assess differences in plant growth and total transpiration, and also to provide a time-integrated record of the photosynthetic and evaporative performance of the plant during the course of crop growth.