SuperSour: A New Strategy for Breeding Superior Citrus Rootstocks.
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ABSTRACT: Citrus crops have a long history of cultivation as grafted trees on selected rootstock cultivars, but all current rootstocks have significant limitations and traditional methods of rootstock breeding take at least 2-3 decades to develop and field test new rootstocks. Citrus production in the United States, and other parts of the world, is impaired by a wide range of biotic and abiotic problems, with especially severe damage caused by the disease huanglongbing (HLB) associated with Candidatus Liberibacter asiaticus. All major commercial citrus scion cultivars are damaged by HLB, but tree tolerance is significantly improved by some rootstocks. To overcome these challenges, the USDA citrus breeding program has implemented a multi-pronged strategy for rootstock breeding that expands the diversity of germplasm utilized in rootstock breeding, significantly increases the number of new hybrids evaluated concurrently, and greatly reduces the time from cross to potential cultivar release. We describe the key components and methodologies of this new strategy, termed "SuperSour," along with reference to the historical favorite rootstock sour orange (Citrus aurantium), and previous methods employed in citrus rootstock breeding. Rootstock propagation by cuttings and tissue culture is one key to the new strategy, and by avoiding the need for nucellar seeds, eliminates the 6- to 15-year delay in testing while waiting for new hybrids to fruit. In addition, avoiding selection of parents and progeny based on nucellar polyembryony vastly expands the potential genepool for use in rootstock improvement. Fifteen new field trials with more than 350 new hybrid rootstocks have been established under the SuperSour strategy in the last 8 years. Detailed multi-year performance data from the trials will be used to identify superior rootstocks for commercial release, and to map important traits and develop molecular markers for the next generation of rootstock development. Results from two of these multi-year replicated field trials with sweet orange scion are presented to illustrate performance of 97 new hybrid rootstocks relative to four commercial rootstocks. Through the first 7 years in the field with endemic HLB, many of the new SuperSour hybrid rootstocks exhibit greatly superior fruit yield, yield efficiency, canopy health, and fruit quality, as compared with the standard rootstocks included in the trials.
Project description:Two citrus rootstocks, Rangpur lime (RL) and Sunki Maravilha mandarin (SM), were analyzed either ungrafted or grafted with their reciprocal graft combinations or with shoot scions of two commercial citrus varieties: Valencia orange (VO) and Tahiti acid lime (TAL). All graft combinations were subjected to distinct watering regimes: well-watered, severe drought and rehydration. Growth and water relation parameters, gas exchange as well as sugar and hormone profiles were determined. Data indicated that RL adopted a dehydration avoidance strategy and maintained growth, whereas SM adopted a dehydration tolerance strategy focused on plant survival. Compared with RL, the leaves and roots of SM exhibited higher concentrations of abscisic acid and salicylic acid, which induced drought tolerance, and accumulation of carbohydrates such as trehalose and raffinose, which are important reactive oxygen species scavengers. SM rootstocks were able to transfer their survival strategy to the grafted shoot scions (RL, VO, TAL). Because of their contrasting survival strategies, RL reached the permanent wilting point more quickly than SM whereas SM recovered from prolonged droughts more efficiently than RL. This is one of the most complete studies of drought tolerance mechanisms in citrus crops and is the first to use reciprocal grafting to clarify scion/rootstock interactions.
Project description:In recent years, the pressure for replanting and resetting huanglongbing (HLB or citrus greening) affected citrus groves has led to an inadequate seed supply for the most popular rootstock cultivars in the State of Florida, United States. Early fruit harvesting of citrus rootstock source trees might reduce fruit losses and enhance seed availability, especially in HLB-endemic and hurricane susceptible areas, if the physiological quality of the seeds is adequate. The effects of fruit maturity on seed quality and seedling performance of US-802, US-897, and US-942 citrus rootstocks were investigated for two consecutive growing seasons. The study included the evaluation of seed germination and nursery performance of the citrus rootstock seedlings. The germination test was performed in vitro, where seeds were hand-peeled, surface-sterilized and placed in culture tubes containing basal Murashige and Skoog medium. For the emergence test, seeds were sown in seedling trays containing sterilized growing substrate in a greenhouse with controlled-environment conditions. Rootstock fruits from all three varieties harvested in August and September had seeds with higher germination potential, as more than 90% of the seeds generated seedlings. US-942 had more % of emergence than US-802 and US-897, resulting in faster seed germination; in contrast, US-802 had the faster shoot growth rate. Assays on fruit abscission response showed that by August, fruit from all three varieties were responsive to ethylene and abscised, although response varied and was higher in US-942, suggesting the seeds were mature enough. Taken together, our findings indicate that fruits these three rootstocks can be harvested as early as August in contrast to the current procedures without losing germination potential. This will result in an increase in available seeds for nurseries in Florida.
Project description:Citrus is one of the most important fruit crops in Mediterranean countries such as Spain, which is one of the main citrus-producing countries worldwide. Soil-borne pathogens, such as Rosellinia necatrix, are relevant limiting biotic factors in fruit trees, due to their tricky management. This fungus is a polyphagous plant pathogen with worldwide distribution, causing white root rot in woody crops, including citrus trees in Spain. The objective of this study was to evaluate the tolerance of new plant material against R. necatrix infection. Therefore, plants of 12 different citrus rootstocks were inoculated with one R. necatrix isolate. During the assay, and periodically, above-ground symptoms and chlorophyll content were evaluated. At the end of the experiment, leaf area and plant biomass measures were obtained. Rootstocks B11R5T64 and B11R5T60 achieved the lowest disease incidence of symptoms and reduction of biomass, and were similar to their respective controls in chlorophyll content and leaf area. Carrizo citrange, CL-5146 and UFR-5 were the most affected rootstocks in symptoms and biomass reduction. This work provides information about R. necatrix-tolerant citrus rootstocks, which can constitute a new integrated, sustainable and effective long-term strategy to avoid white root rot.
Project description:Iron is an essential micronutrient for citrus, playing an important role in photosynthesis and yield. The aim of this paper was to evaluate the tolerance to Fe deficiency of five citrus rootstocks: sour orange (S), Carrizo citrange (C), Citrus macrophylla (M), Troyer citrange (T), and Volkamer lemon (V). Plants were grown for 5 weeks in nutrient solution that contained the following Fe concentrations (in µM): 0, 5, 10, 15, and 20. At the end of the experiment, biomass (dry weight-DW), leaf area, total leaf chlorophyll (CHL), and the activity of root chelate reductase (FCR) were recorded. Additionally, the mineral composition of roots (R) and shoots (S) was evaluated. Principal component analysis was used to study the relationships between all parameters and, subsequently, the relations between rootstocks. In the first component, N-S, P-S, Ca-S, Cu-S, Zn-S, Mn-S, Zn-R, and Mn-R concentrations were related to leaf CHL and FCR. Increases in leaf CHL, Mg-R, and DW (shoots and roots) were inversely related to Cu-R, which was shown in the second component. The values obtained were consistent for V10, C15, and C20, but in contrast for S0 and S5. In conclusion, micronutrient homeostasis in roots and shoots of all rootstocks were affected by Fe stress conditions. The Fe/Cu ratio was significantly related to CHL, which may be used to assist rootstock performance.
Project description:This research utilized the E. coli manA gene encoding phosphomannose isomerase (PMI) selection on sucrose/mannose medium to increase transformation efficiencies after biolistic transformation of two immature citrus rootstock cultivars. Plasmid DNA, containing the manA gene and the enhanced green fluorescent protein (egfp) reporter gene, was bombarded into epicotyl explants of immature Carrizo citrange and Swingle citrumelo. GFP positive shoots were micro-grafted onto in vitro grown immature Carrizo rootstocks. Nineteen transgenic Carrizo shoots were obtained from ten paired shots, and eight Swingle shoots from five paired shots. The mean transformation efficiency of Carrizo was 1.9 transgenics/paired shot while the transformation efficiency of Swingle was comparable at 1.6 transgenics/paired shot. The transformants were analyzed by PCR for the presence of transgenes. Southern blot analysis of eight representative Carrizo transgenic events and four Swingle transgenic events showed that all transgenics had one to three copies of the manA gene. The PMI enzyme activity in the transgenic lines was confirmed using the chlorophenol red assay.
Project description:Soil and irrigation water salinity are major limiting factor to citrus industry in arid environments. The objective of this study was to assess the effects of different salt stress levels on growth and ion uptake of three-month-old citrus rootstocks; sour orange (Citrus aurantium) and Volkamer lemon (Citrus volkameriana). Six levels of NaCl-salinity were used, 0.7 (control), 2, 4, 8, 12 and 15 dS m-1. Salinity increment from 2.0 to 15.0 dS m-1 significantly reduced seedlings height, stem diameter, leaf area, root dry weight, leaf relative water content, chlorophyll content index and chlorophyll fluorescence by one to three folds. In addition, leaf and root N concentration reduced by 10%-50%, P 6%-50%, K 8%-47%, Ca+2 7%-51% and Mg+2 7%-50% when salt stress in the irrigation water increased from 2.0 to 15.0 dS m-1. Conversely, salt stress increment (2.0-15.0 dS m-1) increased leaf stomatal resistance (5 folds), proline concentration (1 fold), Na+ and Cl- in the leaf (10 fold) and root (4 fold) when compared to control (0.7 dS m-1). In term of rootstock, Volkamer had higher seedling height, stem diameter, and root constituents (length, fresh and dry weight) than sour orange. While sour orange had higher leaf Cl-, Ca+2 and Mg+2, Volkamer lemon had higher N, Na+, K+, and P. However, root nutrient (N, Na+, Cl-, P and Mg+2) from Volkamer had consistently higher concentration compared to sour orange at 4.0, 8.0, 12.0 and 15.0 dS m-1. Therefore, we believe that the Volkamer rootstock is more tolerant to salt stress than sour orange.
Project description:BackgroundWhile the rootstock genotype (belowground part of a plant) can impact rhizosphere microbial communities, few studies have examined the relationships between rootstock genotype-based recruitment of active rhizosphere bacterial communities and the availability of root nutrients for plant uptake. Rootstocks are developed to provide resistance to disease or tolerance of abiotic stresses, and compost application is a common practice to also control biotic and abiotic stresses in crops. In this field study, we examined: (i) the effect of four citrus rootstocks and/or compost application on the abundance, diversity, composition, and predicted functionality of active rhizosphere bacterial communities, and (ii) the relationships between active rhizosphere bacterial communities and root nutrient concentrations, with identification of bacterial taxa significantly correlated with changes in root nutrients in the rhizosphere.ResultsThe rootstock genotype determined differences in the diversity of active rhizosphere bacterial communities and also impacted how compost altered the abundance, diversity, composition, and predicted functions of these active communities. Variations in the active bacterial rhizobiome were strongly linked to root nutrient cycling, and these interactions were root-nutrient- and rootstock-specific. Direct positive relationships between enriched taxa in treated soils and specific root nutrients were detected, and potentially important taxa for root nutrient uptake were identified. Significant differences in specific predicted functions were related to soil nutrient cycling (carbon, nitrogen, and tryptophan metabolisms) in the active bacterial rhizobiome among rootstocks, particularly in soils treated with compost.ConclusionsThis study illustrates that interactions between citrus rootstocks and compost can influence active rhizosphere bacterial communities, which impact root nutrient concentrations. In particular, the response of the rhizobiome bacterial abundance, diversity, and community composition to compost was determined by the rootstock. Specific bacterial taxa therefore appear to be driving changes in root nutrient concentrations in the active rhizobiome of different citrus rootstocks. Several potential functions of active bacterial rhizobiomes recruited by different citrus rootstocks did not appear to be redundant but rather rootstock-specific. Together, these findings have important agronomic implications as they indicate the potential for agricultural production systems to maximize benefits from rhizobiomes through the choice of selected rootstocks and the application of compost. Video Abstract.
Project description:Polyploidy is one of the main forces that drives the evolution of plants and provides great advantages for breeding. Somatic hybridization by protoplast fusion is used in citrus breeding programs. This method allows combining the whole parental genomes in a single genotype, adding complementary dominant characters, regardless of parental heterozygosity. It also contributes to surpass limitations imposed by reproductive biology and quickly generates progenies that combine the required traits. Two allotetraploid somatic hybrids recovered from the citrus rootstocks-Citrus macrophylla (CM) and Carrizo citrange (CC)-were characterized for morphology, genome composition using molecular markers (SNP, SSR, and InDel), and their tolerance to iron chlorosis, salinity, and Citrus tristeza virus (CTV). Both hybrids combine the whole parental genomes even though the loss of parental alleles was detected in most linkage groups. Mitochondrial genome was inherited from CM in both the hybrids, whereas recombination was observed for chloroplastic genome. Thus, somatic hybrids differ from each other in their genome composition, indicating that losses and rearrangements occurred during the fusion process. Both inherited the tolerance to stem pitting caused by CTV from CC, are tolerant to iron chlorosis such as CM, and have a higher tolerance to salinity than the sensitive CC. These hybrids have potential as improved rootstocks to grow citrus in areas with calcareous and saline soils where CTV is present, such as the Mediterranean region. The provided knowledge on the effects of somatic hybridization on the genome composition, anatomy, and physiology of citrus rootstocks will be key for breeding programs that aim to address current and future needs of the citrus industry.
Project description:Iron chlorosis is one of the major abiotic stresses affecting fruit trees and other crops in calcareous soils. The most evident symptoms are connected to a reduction in growth and yield and in the interveinal chlorosis of leaves. A custom CombiMatrix 90K microarray was used to identify candidate genes involved in the citrus response to iron deficiency stress, comparing Tarocco Scirè orange [Citrus sinensis (L.) Osbeck] grafted on two different rootstocks, Swingle citrumelo (C. paradisi × Poncirus trifoliata), high sensitive, and Carrizo citrange (C. sinensis × P. trifoliata), tolerant. RNA was extracted from roots of plants grown in two different soils, one volcanic (0% of active lime) used as control, and the other calcareous (10% of active lime).
Project description:New plant breeding techniques (NPBTs) aim to overcome traditional breeding limits for fruit tree species, in order to obtain new varieties with improved organoleptic traits and resistance to biotic and abiotic stress, and to maintain fruit quality achieved over centuries by (clonal) selection. Knowledge on the gene(s) controlling a specific trait is essential for the use of NPBTs, such as genome editing and cisgenesis. In the framework of the international scientific community working on fruit tree species, including citrus, NPBTs have mainly been applied to address pathogen threats. Citrus could take advantage of NPBTs because of its complex species biology (seedlessness, apomixis, high heterozygosity, and long juvenility phase) and aptitude for in vitro manipulation. To our knowledge, genome editing in citrus via transgenesis has successful for induced resistance to Citrus bacterial canker in sweet orange and grapefruit using the resistance gene CsLOB1. In the future, NPBTs will also be used to improve fruit traits, making them healthier. The regeneration of plants following the application of NPBTs is a bottleneck, making it necessary to optimize the efficiency of current protocols. The strengths and weaknesses of using explants from young in vitro plantlets, and from mature plants, will be discussed. Other major issues addressed in this review are related to the requirement for marker-free systems and shortening the long juvenility phase. This review aims to summarize methods and approaches available in the literature that are suitable to citrus, focusing on the principles observed before the use of NPBTs.