Short-Term Waterlogging in Citrus Rootstocks.

Changes in climate are provoking flooding events that cause waterlogging in the fields. Citrus are mainly cultivated in areas with a high susceptibility to climate change. Therefore, it is vital to explore their responses to these events to anticipate future challenges by means of genetic improvement of the commercial rootstocks. In this experiment, three popular commercial rootstocks, namely 'Cleopatra' (C. reshni Hort. Ex Tanaka), C. macrophylla, and 'Forner Alcaide no. 5' (Citrus reshni Hort. Ex Tanaka × Poncirus trifoliata), were evaluated after being submitted to short-term waterlogging and a period of recovery of 7 days in each case. Photosynthesis rate and stomatal conductance decreased in 'Cleopatra', while in the other two genotypes they were maintained (C. macrophylla) or restored after recovery ('Forner Alcaide no. 5''). Relative water content and chlorophylls also decreased in 'Cleopatra'. This indicates a deeper effect of flooding in 'Cleopatra', which suffered changes during flooding that were also sustained during the recovery phase. This did not occur in the other two rootstocks, since they showed signs of recovery for those parameters that decreased during waterlogging.

Comparison of Four Systems to Test the Tolerance of 'Fortune' Mandarin Tissue Cultured Plants to Alternaria alternata.

Alternaria brown spot is a severe disease that affects leaves and fruits on susceptible mandarin and mandarin-like cultivars, and is produced by Alternaria alternata. Consequently, there is an urge to obtain new cultivars resistant to A. alternata, and mutation breeding together with tissue culture can help shorten the process. However, a protocol for the in vitro selection of resistant citrus genotypes is lacking. In this study, four methods to evaluate the sensitivity to Alternaria of mandarin 'Fortune' explants in in vitro culture were tested. The four tested systems consisted of: (1) the addition of the mycotoxin, produced by A. alternata in 'Fortune', to the propagation culture media, (2) the addition of the A. alternata culture filtrate to the propagation culture media, (3) the application of the mycotoxin to the intact shoot leaves, and (4) the application of the mycotoxin to the previously excised and wounded leaves. After analyzing the results, only the addition of the A. alternata culture filtrate to the culture media and the application of the mycotoxin to the wounded leaves produced symptoms of infection. However, the addition of the fungus culture filtrate to the culture media produced results, which might indicate that, in addition to the mycotoxin, many other unknown elements that can affect the plant growth and behavior could be found in the fungus culture filtrate. Therefore, the application of the toxin to the excised and wounded leaves seems to be the most reliable method to analyze sensitivity to Alternaria of 'Fortune' explants cultured in vitro.

In Vitro Plant Evaluation Trial: Reliability Test of Salinity Assays in Citrus Plants.

Salinity is one of the major abiotic stresses affecting crops worldwide, and breeders are urged to evaluate new genotypes to know their degree of tolerance to this selective agent. However, obtaining a number of plants high enough to make the evaluation can prove to be a long and laborious process which could be overcome by using tissue culture techniques. In the present study, the reliability of tissue culture evaluations is called into question through two parallel experiments, in vitro and ex vitro, using Citrus macrophylla and four mutants thereof, previously selected by their different behavior to salinity, as a plant material. Plants were subjected to salinity for 8 weeks in both in vitro (80 mM NaCl) and ex vitro (100 mM NaCl) experiments, and differences with plants grown in control conditions without salt were analyzed. After the experiments, length, leaf damage, shoot dry weight, chlorophylls and ions were measured in both conditions and experiments. As a result, it was demonstrated that tissue culture is a reliable tool to determine whether a genotype is tolerant to salinity or not, since plants of the same genotype responded in a similar way to salinity in both experiments. Henceforth, in vitro evaluations can be employed to test genotypes in a very early stage and using very little time and space. However, genotypes that showed the biggest or lowest changes when cultured in salinity were not always the same in both experiments. Thus, only ex vitro experiments can be performed if the goal is to compare genotypes and see which genotype is the most or least resistant to salinity.

Mutants of Citrus macrophylla rootstock obtained by gamma radiation improve salt resistance through toxic ion exclusion.

Salinity is one of the biggest challenges that need to be faced in crop production. Citrus is highly sensitive to salt stress and obtaining rootstocks with improved resistance to salinity is key for the citrus growing industry. In this study, five mutants of Citrus macrophylla rootstock, obtained through gamma radiation and in vitro pre-selected for their resistance to salinity, were irrigated with a solution containing 100 mM of NaCl. After 8 weeks of exposure, the mutants were evaluated for their performance (growth, visual leaf damage) and chlorophyll, proline, starch, soluble sugars and ion contents to determine their degree of resistance to this salinity level. In the saline conditions assayed, all the mutants showed better performance and less leaf damage than Citrus macrophylla. Our data suggest that this improved resistance to salinity was based on their capacity to accumulate less Na (MM4B and MMN1) or Cl- (MM1A, MM4A and MM3B). Besides having the lowest Cl- content, the mutants MM1A, MM4A and MM3B, had the highest NO3- concentrations in salinity. Furthermore, mutants did not show chlorophyll degradation and showed less leaf damage and acceptable plant growth. Other parameters including proline and soluble sugars, did not prove decisive in the salinity resistance of these genotypes.

Genetic Transformation in Peach (Prunus persica L.): Challenges and Ways Forward.

Almost 30 years have passed since the first publication reporting regeneration of transformed peach plants. Nevertheless, the general applicability of genetic transformation of this species has not yet been established. Many strategies have been tested in order to obtain an efficient peach transformation system. Despite the amount of time and the efforts invested, the lack of success has significantly limited the utility of peach as a model genetic system for trees, despite its relatively short generation time; small, high-quality genome; and well-studied genetic resources. Additionally, the absence of efficient genetic transformation protocols precludes the application of many biotechnological tools in peach breeding programs. In this review, we provide an overview of research on regeneration and genetic transformation in this species and summarize novel strategies and procedures aimed at producing transgenic peaches. Promising future approaches to develop a robust peach transformation system are discussed, focusing on the main bottlenecks to success including the low efficiency of A. tumefaciens-mediated transformation, the low level of correspondence between cells competent for transformation and those that have regenerative competence, and the high rate of chimerism in the few shoots that are produced following transformation.

Inducing mutations in Citrus spp.: Sensitivity of different sources of plant material to gamma radiation.

Gamma radiation represents an alternative for improving genotypes for which breeding through hybridization involves many difficulties because of their complex reproductive biology, as in the case of citrus. In this study in vitro and ex vitro mutagenesis was induced by gamma radiation in several citrus species ('Alemow' and sour orange as citrus rootstocks, lemon cv. 'Fino 49' and 'Verna 51', mandarin cv. 'Nova' and lime cv. 'Bearss'). Three different sources of materials - seeds, budwoods and nodal segments - from in vitro explants, were tested. Seeds and budwoods were germinated or induced to sprout, and de novo regeneration was obtained from in vitro nodal segments from which preexisting buds were previously removed. Budwoods were tested in summer and winter in order to assess their capacity for mutation and further sprouted in different seasons. Seeds were seen to be more resistant to gamma radiation (LD50 of 127 Gy in 'Alemow and 156 Gy in sour orange) than budwoods (LD50 about 50 Gy for all cultivars) and nodal segments (LD50 about 25 Gy for both lemon cultivars), the last being the most sensitive material tested. Similar LD50 were found for budwoods collected in winter and summer for all the genotypes. All the tested plant material can be considered suitable for gamma irradiation, although budwood is the most widely available and tissue culture material needs the highest degree of expertise.

Are commercial sweet cherry rootstocks adapted to climate change? Short-term waterlogging and CO2 effects on sweet cherry cv. 'Burlat'.

High CO2 is able to ameliorate some negative effects due to climate change and intensify others. This study involves the sweet cherry (Prunus avium) cultivar 'Burlat' grafted on the 'Mariana 2624', 'Adara' and 'LC 52' rootstocks. In a climate chamber at two CO2 concentrations, ambient (400 µmol mol-1 ) and elevated (800 µmol mol-1 ), the plants were submitted to waterlogging for 7 d, followed by 7 d of recovery after drainage. Waterlogging drastically decreased the rate of photosynthesis, significantly endangering plant survival, particularly for the 'LC 52' and 'Adara' rootstocks. 'Mariana 2624' was also clearly affected by waterlogging that increased lipid peroxidation and the Cl- and SO42- concentrations in all the studied plants. Nevertheless, CO2 was able to overcome this reduction in photosynthesis, augmenting growth, increasing soluble sugars and starch, raising turgor and regulating the concentrations of Cl- and SO42- , while lowering the NO3- concentration in leaves of all the studied rootstocks. In concordance with these results, the proline levels indicated a more intense stress at control CO2 than at high CO2 for waterlogged plants. 'Mariana 2624' was more resistant to waterlogging than 'Adara', and both were more resistant than 'LC 52' in control CO2 conditions; this clearly enhanced the chance of survival under hypoxia.

Fruit quality of sweet pepper as affected by foliar Ca applications to mitigate the supply of saline water under a climate change scenario.

BACKGROUND: Sweet pepper fruit quality disorders have been related mainly to an unbalanced nutrient supply and non-optimal growth conditions. Increases in the atmospheric CO2 concentration ([CO2 ]) have been associated with a reduction of transpiration, which can affect calcium (Ca) uptake as it is linked closely to water uptake. We investigated whether foliar application of Ca can counterbalance the effects of saline water and elevated [CO2 ]. RESULTS: High CO2 favoured generative growth instead of vegetative growth. Foliar Ca supply did not affect the marketable yield, but reduced the total yield when combined with salinity and 400 µmol mol-1 CO2 . Salinity affected negatively the total yield but this was overcome when CO2 was applied. The B and K concentrations were reduced by foliar Ca application, while Ca and Mn were increased at 400 µmol mol-1 CO2 . Salinity increased the Mn, Cl, and Na concentrations, regardless of the [CO2 ], and decreased K at 800 µmol mol-1 CO2 . The total protein was affected negatively only by elevated [CO2 ], and the total free amino acid concentration was reduced by all treatments. CONCLUSION: The effect of Ca application differed according to the other treatments applied. This procedure should be optimised to overcome future climate impacts on fruit quality. © 2017 Society of Chemical Industry.

Two minuses can make a plus: waterlogging and elevated CO2 interactions in sweet cherry (Prunus avium) cultivars.

The increase in the ambient concentration of CO2 and other greenhouse gases is producing climate events that can compromise crop survival. However, high CO2 concentrations are sometimes able to mitigate certain stresses such as salinity or drought. In this experiment, the effects of waterlogging and CO2 are studied in combination to elucidate the eventual response in sweet cherry trees. For this purpose, four sweet cherry cultivars ('Burlat', 'Cashmere', 'Lapins and 'New Star') were grafted on a typically hypoxia-tolerant rootstock (Mariana 2624) and submitted to waterlogging for 7 days at either ambient CO2 concentration (400 µmol mol-1 ) or at elevated CO2 (800 µmol mol-1 ). Waterlogging affected plants drastically, by decreasing photosynthesis, stomatal conductance, transpiration, chlorophyll fluorescence and growth. It also brought about the accumulation of proline, chloride and sulfate. Nonetheless, raising the CO2 supply not only mitigated all these effects but also induced the accumulation of soluble sugars and starch in the leaf. Therefore, sweet cherry plants submitted to waterlogging were able to overcome this stress when grown in a CO2 -enriched environment.

CO2 effects on the waterlogging response of 'Gisela 5' and 'Gisela 6' (Prunus cerasusxPrunus canescens) sweet cherry (Prunus avium) rootstocks.

Climate change is submitting countries of the Mediterranean arc to periods of drought alternating with heavy rain and waterlogging. Eventual floods along with the rising CO2 in the atmosphere present an unpredictable scenario that affects crop survival. The effect of both stresses combined has been studied in sweet cherry plants. 'Gisela 5' and 'Gisela 6' were evaluated as rootstocks of the sweet cherry cultivar 'Burlat'. Plants were placed in a controlled-climate chamber for 7days, then they were submitted to waterlogging for another 7days and the response to this stress and the subsequent recovery were studied (7 more days). The experiment was carried out at 400µmolmol-1 CO2 (ambient CO2) and 800µmolmol-1 CO2, at 26°C, and plant water status and growth, net CO2 assimilation, transpiration, stomatal conductance, water potential, chlorophyll fluorescence, relative water content, anions content, proline, lipid peroxidation, soluble sugars, and starch were measured. Differences in the response and in its intensity were detected in both rootstocks. Some parameters - such as photosynthesis, soluble sugars, starch, TBARS, and NO3- - varied depending on the CO2 conditions and the waterlogging effect. Elevated CO2 was able to increase photosynthesis and thereby help plants to overcome waterlogging.

Amelioration of boron toxicity in sweet pepper as affected by calcium management under an elevated CO2 concentration.

We investigated B tolerance in sweet pepper plants (Capsicum annuun L.) under an elevated CO2 concentration, combined with the application of calcium as a nutrient management amelioration technique. The data show that high B affected the roots more than the aerial parts, since there was an increase in the shoot/root ratio, when plants were grown with high B levels; however, the impact was lessened when the plants were grown at elevated CO2, since the root FW reduction caused by excess B was less marked at the high CO2 concentration (30.9% less). Additionally, the high B concentration affected the membrane permeability of roots, which increased from 39 to 54% at ambient CO2 concentration, and from 38 to 51% at elevated CO2 concentration, producing a cation imbalance in plants, which was differentially affected by the CO2 supply. The Ca surplus in the nutrient solution reduced the nutritional imbalance in sweet pepper plants produced by the high B concentration, at both CO2 concentrations. The medium B concentration treatment (toxic according to the literature) did not result in any toxic effect. Hence, there is a need to review the literature on critical and toxic B levels taking into account increases in atmospheric CO2.

Changes in the salinity tolerance of sweet pepper plants as affected by nitrogen form and high CO2 concentration.

The assimilation and availability of nitrogen in its different forms can significantly affect the response of primary productivity under the current atmospheric alteration and soil degradation. An elevated CO2 concentration (e[CO2]) triggers changes in the efficiency and efficacy of photosynthetic processes, water use and product yield, the plant response to stress being altered with respect to ambient CO2 conditions (a[CO2]). Additionally, NH4(+) has been related to improved plant responses to stress, considering both energy efficiency in N-assimilation and the overcoming of the inhibition of photorespiration at e[CO2]. Therefore, the aim of this work was to determine the response of sweet pepper plants (Capsicum annuum L.) receiving an additional supply of NH4(+) (90/10 NO3(-)/NH4(+)) to salinity stress (60mM NaCl) under a[CO2] (400µmolmol(-1)) or e[CO2] (800µmolmol(-1)). Salt-stressed plants grown at e[CO2] showed DW accumulation similar to that of the non-stressed plants at a[CO2]. The supply of NH4(+) reduced growth at e[CO2] when salinity was imposed. Moreover, NH4(+) differentially affected the stomatal conductance and water use efficiency and the leaf Cl(-), K(+), and Na(+) concentrations, but the extent of the effects was influenced by the [CO2]. An antioxidant-related response was prompted by salinity, the total phenolics and proline concentrations being reduced by NH4(+) at e[CO2]. Our results show that the effect of NH4(+) on plant salinity tolerance should be globally re-evaluated as e[CO2] can significantly alter the response, when compared with previous studies at a[CO2].

Endogenous hormones response to cytokinins with regard to organogenesis in explants of peach (Prunus persica L. Batsch) cultivars and rootstocks (P. persica × Prunus dulcis).

Organogenesis in peach (Prunus persica L. Batsch) and peach rootstocks (P. persica × Prunus dulcis) has been achieved and the action of the regeneration medium on 7 phytohormones, zeatin (Z), zeatin riboside (ZR), indole-3-acetic acid (IAA), abscisic acid (ABA), ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC), salicylic acid (SA), and jasmonic acid (JA), has been studied using High performance liquid chromatography - mass spectrometry (HPLC-MS/MS). Three scion peach cultivars, 'UFO-3', 'Flariba' and 'Alice Bigi', and the peach × almond rootstocks 'Garnem' and 'GF677' were cultured in two different media, Murashige and Skoog supplemented with plant growth regulators (PGRs) (regeneration medium) and without PGRs (control medium), in order to study the effects of the media and/or genotypes in the endogenous hormones content and their role in organogenesis. The highest regeneration rate was obtained with the peach × almond rootstocks and showed a lower content of Z, IAA, ABA, ACC and JA. Only Z, ZR and IAA were affected by the action of the culture media. This study shows which hormones are external PGRs-dependent and what is the weight of the genotype and hormones in peach organogenesis that provide an avenue to manipulate in vitro organogenesis in peach.

Relationship between endogenous hormonal content and somatic organogenesis in callus of peach (Prunus persica L. Batsch) cultivars and Prunus persica×Prunus dulcis rootstocks.

The relationship between endogenous hormones content and the induction of somatic peach plant was studied. To induce multiple shoots from callus derived from the base of stem explants of the scion cultivars 'UFO-3', 'Flariba' and 'Alice Bigi', and the peach×almond rootstocks 'Garnem' and 'GF677', propagated plants were cultured on Murashige and Skoog salts augmented with 0.1mgL(-1) of indolebutyric acid, 1mgL(-1) of 6-benzylaminopurine and 3% sucrose. The highest regeneration rate was obtained with the peach×almond rootstocks. Endogenous levels of abscisic acid (ABA), indole-3-acetic acid (IAA), zeatin (Z), zeatin riboside (ZR), ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC), salicylic acid (SA), and jasmonic acid (JA) were analyzed in the organogenic callus. Lower levels of several hormones, namely Z, ZR, ABA, and ACC were found in the peach×almond rootstock compared to peach cultivars, while IAA and SA presented inconclusive returns. These results suggest that the difference in somatic organogenesis capacity observed in peach and peach×almond hybrids is markedly affected by the endogenous hormonal content of the studied genotypes.