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.

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.

Alleviation of salt stress in citrus seedlings inoculated with arbuscular mycorrhizal fungi depends on the rootstock salt tolerance.

Seedlings of Cleopatra mandarin (Citrus reshni Hort. ex Tan.) and Alemow (Citrus macrophylla Wester) were inoculated with a mixture of AM fungi (Rhizophagus irregularis and Funneliformis mosseae) (+AM), or left non-inoculated (-AM). From forty-five days after fungal inoculation onwards, half of +AM or -AM plants were irrigated with nutrient solution containing 50 mM NaCl. Three months later, AM significantly increased plant growth in both Cleopatra mandarin and Alemow rootstocks. Plant growth was higher in salinized +AM plants than in non-salinized -AM plants, demonstrating that AM compensates the growth limitations imposed by salinity. Whereas AM-inoculated Cleopatra mandarin seedlings had a very good response under saline treatment, inoculation in Alemow did not alleviate the negative effect of salinity. The beneficial effect of mycorrhization is unrelated with protection against the uptake of Na or Cl and the effect of AM on these ions did not explain the different response of rootstocks. This response was related with the nutritional status since our findings confirm that AM fungi can alter host responses to salinity stress, improving more the P, K, Fe and Cu plant nutrition in Cleopatra mandarin than in Alemow plants. AM inoculation under saline treatments also increased root Mg concentration but it was higher in Cleopatra mandarin than in Alemow. This could explain why AM fungus did not completely recovered chlorophyll concentrations in Alemow and consequently it had lower photosynthesis rate than control plants. AM fungi play an essential role in citrus rootstock growth and biomass production although the intensity of this response depends on the rootstock salinity tolerance.

Improving knowledge of plant tissue culture and media formulation by neurofuzzy logic: a practical case of data mining using apricot databases.

Plant tissue growth can be regulated and controlled via culture media composition. A number of different laborious and time-consuming approaches have been used to attempt development of optimized media for a wide range of species and applications. However, plant tissue culture is a very complex task, and the identification of the influences of process factors such as mineral nutrients or plant growth regulators on a wide spectrum of growth responses cannot always well comprehended. This study employs a new approach, data mining, to uncover and integrate knowledge hidden in multiple data from plant tissue culture media formulations using apricot micropropagation databases as an example. Neurofuzzy logic technology made it possible to identify relationships among several factors (cultivars, mineral nutrients and plant growth regulators) and growth parameters (shoots number, shoots length and productivity), extracting biologically useful information from each database and combining them to create a model. The IF-THEN rule sets generated by neurofuzzy logic were completely in agreement with previous findings based on statistical analysis, but advantageously generated understandable and reusable knowledge that can be applied in future plant tissue culture media optimization.

Physiological and growth changes in micropropagated Citrus macrophylla explants due to salinity.

Salinity is one of the major abiotic stresses affecting arable crops worldwide, and is the most stringent factor limiting plant distribution and productivity. In the present study, the possible use of in vitro culture to evaluate the growth and physiological responses to salt-induced stress in cultivated explants of Citrus macrophylla was analyzed. For this purpose, micropropagated adult explants were grown in proliferation and rooting media supplemented with different concentrations of NaCl. All growth parameters were decreased significantly by these NaCl treatments; this was accompanied by visible symptoms of salt injury in the proliferated shoots from 60mM NaCl and in the rooted shoots from 40mM NaCl. Malondialdehyde (MDA) increased with increasing salinity in proliferated shoots, indicating a rising degree of membrane damage. The concentration of total chlorophyll significantly decreased in the presence of NaCl, and this effect was more pronounced in the rooted explants. The Na(+) and Cl(-) concentrations in the explants increased significantly with the salinity level, but Cl(-) levels were higher in the proliferated explants than in the rooted explants. For osmotic adjustment, high concentrations of compatible solutes (proline and quaternary ammonium compounds-QAC) accumulated in salt-stressed plants in proliferation, but differences were not observed in rooted explants. In proliferation, proline and QAC were highly correlated with the sodium and chloride concentrations in the explants, indicating a possible role of these compounds in osmotic adjustment. The plant concentrations of NO(3)(-), K(+), Mg(2+), Ca(+) and Fe were also affected by the NaCl concentration of the medium. We suggest that the important deleterious effects in the in vitro explants of Citrus macrophylla grown at increasing NaCl concentrations were due mainly to toxic effects of saline ions, particularly Cl(-), at the cellular level.