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The potential of storing ‘Budd Blood’ blood orange fruit at different temperatures (6, 8, 10, and 12 °C) for 60 days after harvest was investigated to enhance anthocyanin, total phenolic content, antioxidant activity, and to understand temperature effects on other physicochemical attributes. Fruit weight and firmness losses increased during storage, with the lowest losses occurring at 6 °C. Titratable acidity (TA) decreased, and the lowest values were recorded at 10 and 12 °C. Total soluble solids (TSS), TSS/TA, and juice pH increased, with the highest values recorded at 10 and 12 °C. The sucrose content exhibited slight changes while glucose and fructose levels increased during storage, with the highest levels of glucose and fructose observed at 12 °C. Total anthocyanin concentration, the levels of cyanidin-3-glucoside and cyanidin 3-(6″-malonylglucoside), total phenolic content, and total antioxidant activity significantly increased during storage and the highest values were observed at 10 and 12 °C. Total anthocyanin content increased 2950 % at 10 °C and 3000 % at 12 °C. Interestingly, higher temperatures (10 and 12 °C) were more effective in enhancing anthocyanin levels, total phenolic content, and antioxidant activity during storage, while lower temperatures (6 and 8 °C) were more effective in preserving the other physicochemical attributes.

Callose is a 1,3-β glucan responsible for several processes in planta such as cell division, ripening of pollen mother cells, maintaining the plasmodesmata opening, and providing structure to the sieve plates. Besides the physiological roles, callose is deposited during pathogen attacks, forming papillae to prevent pathogen entrance into the tissue or plugging the sieve pores to limit the spread of phloem pathogens. Candidatus Liberibacter asiaticus (CLas), the putative causal agent of Huanglongbing (HLB), is a phloem-restricted pathogen whose infection leads to the production of callose in the phloem. Characterizing the dynamics of callose plugging during HLB is important for understanding the disease, but no published protocols are available for the extraction and quantification of the callose in citrus trees, and quantitative data are limited. Detection of callose by microscopy is time-consuming and expensive and does not give information about the distribution of callose in the entire plant. Here, we present a short protocol for the efficient extraction and quantification of the total callose from citrus plants. We compared different tissues from healthy and CLas-infected plants and identified an increase in callose levels in the midribs, the stems, and the fruit peduncles of infected plants. Callose levels were highest in leaves, especially midribs, compared to stems, roots, and fruit peduncles. This method could be applied to other woody plant species.

Mukaku Kishu (‘MK’), a small sized mandarin, is an important source of seedlessness in citrus breeding. Identification and mapping the gene(s) governing ‘MK’ seedlessness will expedite seedless cultivar development. In this study, two ‘MK’-derived mapping populations- LB8-9 Sugar Belle® (‘SB’) × ‘MK’ (N=97) and Daisy (‘D’) × ‘MK’ (N=68) were genotyped using an Axiom_Citrus56 Array encompassing 58,433 SNP probe sets, and population specific male and female parent linkage maps were constructed. The parental maps of each population were integrated to produce sub-composite maps, which were further merged to develop a consensus linkage map. All the parental maps (except ‘MK_D’) had nine major linkage groups, and contained 930 (‘SB’), 810 (‘MK_SB’), 776 (‘D’) and 707 (‘MK_D’) SNPs. The linkage maps displayed 96.9 (‘MK_D’) to 98.5% (‘SB’) chromosomal synteny with the reference Clementine genome. The consensus map was comprised of 2588 markers including a phenotypic seedless (Fs)-locus and spanned a genetic distance of 1406.84 cM, with an average marker distance of 0.54 cM, which is substantially lower than the reference Clementine map. For the phenotypic Fs-locus, the distribution of seedy and seedless progenies in both ‘SB’ × ‘MK’ (55:42, χ2 = 1.74) and ‘D’ × ‘MK’ populations (33:35, χ2 = 0.06) followed a test cross pattern. The Fs-locus mapped on chromosome 5 with SNP marker ‘AX-160417325’ at 7.4 cM in ‘MK_SB’ map and between two SNP markers ‘AX-160536283’ and ‘AX-160906995’ at a distance of 2.4 and 4.9 cM, respectively in ‘MK_D’ map. The SNPs ‘AX-160417325’ and ‘AX-160536283’ correctly predicted seedlessness of 25-91.9% progenies in this study. Based on the alignment of flanking SNP markers to the Clementine reference genome, the candidate gene for seedlessness hovered in a ~ 6.0 Mb region between 3.97 Mb (AX-160906995) to 10.00 Mb (AX-160536283). This region has 131 genes of which 13 genes (belonging to seven gene families) reportedly express in seed coat or developing embryo. The findings of the study will prove helpful in directing future research for fine mapping this region and eventually underpinning the exact causative gene governing seedlessness in ‘MK’.

Somatic hybridization has emerged as a valuable tool for developing novel genetic combinations in citrus breeding programs, including the creation of salt-tolerant rootstocks. In this study, the performance of a tetraploid somatic hybrid, obtained by fusing protoplasts derived from salt-tolerant Cleopatra mandarin (Citrus reshni hort. ex Tanaka) and salt-sensitive Carrizo citrange (Citrus sinensis L. Osbeck × Poncirus trifoliata L. Raf), was assessed under in vitro salt stress. Hybrid plants were characterized by leaf morphology, and ploidy level by flow cytometry and molecular markers. In vitro shoots were generated from the micropropagation of mature stem pieces of the somatic hybrid and its parents, and these were challenged by exposure to NaCl (0, 50, or 100 mM) supplemented to the media for three weeks to induce salt stress. The leaves of the somatic hybrid display intermediate morphology compared to the parental Cleopatra mandarin and Carrizo citrange rootstocks. All molecular markers successfully amplified DNA from the three cultivars; however, only 11 of 14 unequivocally confirmed somatic hybridity. The physiological and biochemical parameters, including chlorophyll content, lipid peroxidation, total phenolic compounds, antioxidants activity and proline content, were measured in the leaves. The somatic hybrid exhibited superior salt stress tolerance compared to the parent varieties, as evidenced by the reduced cellular membrane damage indicated by the lower levels of malondialdehyde and electrolyte leakage, particularly under 100 mM NaCl treatment. The somatic tetraploid hybrid also displayed higher total phenolic content than either parent, while Cleopatra mandarin exhibited the highest proline levels under 50 mm NaCl. These results demonstrate the enhanced salinity stress tolerance of the somatic hybrid compared to its parent lines, highlighting its potential as a valuable candidate for developing salt-tolerant citrus rootstocks.

To explore the nature of wild and cultivated mandarins, the authors carry out genomic analysis of diverse east Asian citrus. The discovery of a wild species Citrus ryukyuensis native to the Ryukyu islands and a new population of wild mainland Asian mandarin explains the origin and diversity of mandarins and their ability to reproduce apomictically.

Citriculture is considered the most important fruit industry and involves the cultivation of several fruit varieties, which are susceptible to many plant pathogens. In this sense, soil-borne pathogenic fungi, such Rosellinia necatrix, threaten citrus fruit production worldwide because they can cause fruit loss. Therefore, we assayed the physiological reaction of novel citrus rootstocks against white root rot disease during long-term management. Data from above-ground symptoms and chlorophyll content were periodically obtained during the experimental process. In addition, plant leaf area and percentage of biomass reduction were determined for each rootstock when the experiment finished. The behavior of the inoculated tolerant rootstocks was as follows: the lowest symptom rate of SAUDPC was achieved by B11R5T25 and N40R3T25; AMB+CZO manifested the highest disease incidence; B11R5T25 and A+VOLK × Orange 19-11-8 displayed the highest and the lowest chlorophyll content, respectively; AMB+CZO and A+VOLK × Orange 19-11-8 showed the highest biomass reduction, and the lowest was detected in B11R5T25 and N40R2T19; concerning the leaf area, N40R1T18, N40R3T25 and N40R2T19 showed the lowest response, and 2247 × 2075-01-2 achieved the highest rate. In summary, B11R5T25 and N40R3T25 displayed the lowest disease rate.

Purple tea plant cultivars, enrich with flavonoids and anthocyanins, are valuable materials for manufacturing tea with unique color and flavor. Researchers found that ‘Zijuan’ leaves changed from purple to green mainly caused by the decreased flavonoids and anthocyanins concentrations. The mechanism of flavonoids and anthocyanin biosynthesis has been studied in many purple tea plant cultivars and the key genes which regulated the biosynthesis of flavonoid and anthocyanins in different purple tea plant cultivars were quite different. Also, the molecular regulation mechanism underlying the flavonoids and anthocyanins biosynthesis during leaves development and color changes is less-thoroughly understood. In this study, an integrative analysis of transcriptome and metabolome was performed on the purple leaves and green leaves of ‘Zijuan’ tea plant to reveal the regulatory networks correlated to flavonoid biosynthesis and to identify key regulatory genes. Our results indicated that the ‘Zijuan’ new shoots leaves were purple might be due to the copigmentation of quercetin and kaempferol derivatives. In ‘Zijuan’ tea plant cultivar, flavonoids metabolites concentrations in purple leaves and green leaves were significantly influenced by the genes involved in flavonoid biosynthesis, transcriptional regulation, transport, and hormone response. Transcription factors including NAC008, MYB23, and bHLH96 and transporters such as ABC transporter I might be responsible for the flavonoid and anthocyanins accumulation in purple leaves. This study provides a new insight into the metabolism and molecular mechanisms underlying flavonoid and anthocyanin biosynthesis in tea plant.

Citrus is among the most economically important fruit crops. Its vast species diversity and global production was observed by N.I. Vavilov during his international plant explorations from the early to mid-1900s. Currently, ex situ citrus collections located around the world conserve and protect citrus genetic resources, as revealed in a survey conducted in 2021. Responses were received from 43 collections in 27 countries, of which 35 provided data regarding collection composition, management practices, and security, as well as other information. The six largest citrus collections have between 1000 and 1735 accessions. The largest accession holdings are mandarins and sweet oranges, although all citrus fruit types are maintained: mandarin, sweet orange, lemon, pummelo, grapefruit, hybrids, lime, sour orange, citron, kumquat, papeda, finger lime, and crop wild relatives. Diseases pose significant threats to collections, though some collections are maintained in a clean-plant state as a result of intensive sanitation efforts. National and regional quarantine regulations often limit the export and import of citrus plants or propagative materials, thus limiting the availability of materials at an international level. Resources, both financial and human, are necessary to ensure the long-term safety and security of citrus collections on a global scale. Future efforts to develop citrus genebanking communities will provide opportunities for improved conservation, as well as collaborations and training.

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.

Citrus are one of the most relevant fruit crops in the Mediterranean basin, which is the second-highest citrus-growing region in the world. However, these crops are mainly grown under Mediterranean semi-arid conditions, characterized by long periods of drought and torrential rain. In this work, we have assessed the response of three promising HLB-tolerant citrus rootstocks (Orange-14, UFR-1, and B11R3T27) to the application of four water stress conditions (Control, Mild water stress, Drought, and Flooding), comparing them with Carrizo citrange. Aerial plant symptoms were recorded during the experimental period, whereas plant water parameters, including stomatal conductance, leaf water potential, and relative water content, were obtained at the end of the assay. For all assessed rootstocks and variables, drought treatment was the most limiting factor, with Carrizo citrange being the most suitable rootstock under this condition. Flooding was the second restrictive treatment, in which UFR-1 was the least affected rootstock. Mid-water stress with 50% water requirements did not differ from the Control treatment, which can help save water resources in semi-arid regions. This information can be helpful for the citrus industry to increase the efficiency of citrus crops subject to water stress in semi-arid regions.

Huanglongbing (HLB) caused by ‘Candidatus Liberibacter asiaticus’ (CLas) is the most costly disease for the global citrus industry. Currently, no effective tools have been found to control HLB. Most commercial citrus varieties are susceptible to HLB, though some citrus hybrid cultivars have reduced sensitivity to the disease. Citrus breeding populations contain a large diversity of germplasm, with thousands of unique genotypes exhibiting a broad range of phenotypes. Understanding phenotypic variation and genetic inheritance in HLB-affected mandarin hybrid populations are crucial for breeding tolerant citrus varieties. In this study, we assessed 448 diverse mandarin hybrids coming from 30 crosses, and 45 additional accessions. For HLB tolerance, we measured HLB severity visual score and CLas titers by qPCR. We also measured seven morphophysiological traits indirectly related to HLB tolerance with leaf area index (LAI), leaf area (LA), leaf mass per area (LMA), photosystem II parameters (Fv/Fo, Fv/Fm), and photochemical performance index (PIabs). By estimating the genetic variation in five half-sib families, we estimated the heritability of phenotypic traits and found a significant genetic effect on HLB visual score and photosynthesis parameters, which indicates opportunities for the genetic improvement of HLB tolerance. In addition, although it is easy to identify infected trees based on HLB symptomatic leaves, visually phenotyping whole trees can be difficult and inconsistent due to the interpersonal subjectivity of characterization. We investigated their relationships and found that LAI was highly correlated with HLB score, with correlation coefficients of r = 0.70 and r = 0.77 for the whole population and five half-sib families, respectively. Photochemical parameters showed significant correlation with HLB severity and responded differentially with the side of the canopy. Our study suggests that LAI and photochemical parameters could be used as a rapid and cost-effective method to evaluate HLB tolerance and inheritance in citrus breeding programs.

Abstract Background Valencene is a major sesquiterpene in citrus oil and biosynthesized by valencene synthase (Cstps1; EC: 4.2.3.73) from the 15-carbon substrate farnesyl diphosphate. It is abundant in juice of some mandarins (e.g. Citrus reticulata Blanco cv. Fortune), however, it is undetectable in others (e.g. C. reticulata Blanco cv. Murcott), We have discovered that the Murcott mandarin Cstps1 gene expression is severely reduced. A previous genetic mapping study using an F1 population of Fortune × Murcott found that the segregation of valencene production in fruit exhibited a Mendelian inheritance ratio of 1:1. There was only one dominant locus associated with valencene content detected on the mandarin genetic map. The goal of this study was to understand the molecular mechanism underlying the valencene deficiency observed in some citrus hybrids. Results There was a clear relationship between presence or absence of the valencene synthase gene (Cstps1) expression, and presence or absence of valencene among randomly selected mandarin hybrids. Cloning the coding regions of Cstps1 from Fortune and Murcott mandarin, and aligning with previous reported Valencia orange Cstps1 sequence, showed that they both exhibited extremely high similarity with the known Cstps1. By further cloning and analyzing the promoter region of Cstps1 from Valencia, Fortune and Murcott, a 12-nucleotide deletion at approximately − 270 bp from the Cstps1 coding region was only found in Murcott. Three binary vectors, designated as p1380-FortP-GUSin, p1380-MurcP-GUSin and p1380-MurcP(+ 12)-GUSin, were developed for promoter activity analysis. Transient over-expression of Fortune Cstps1 promoter in sweet orange showed notable GUS activity, but the Murcott Cstps1 promoter did not. In addition, by re-inserting the 12-nucleotide fragment, the activity of the Murcott Cstps1 promoter was mostly recovered. Conclusion The deficiency of valencene production in some mandarins is probably due to a 12-nucleotide deletion in the promoter region of the Cstps1, which could be a crucial switch of Cstps1 transcription. Our results further enhanced the understanding of valencene biosynthesis in citrus.

Huanglongbing (HLB), which is believed to be caused by the phloem-restricted bacterium Candidatus Liberibacter asiaticus (CLas), has decimated Florida’s citrus production. Grapefruit production has declined 75%, mandarin 78%, and sweet orange 52% due to the high sensitivity of commercial scions and rootstocks to the disease. New combinations of scions and hybrid rootstocks may provide better performance than current commercial selections for Florida’s fresh citrus production, particularly in the Indian River District. The objective of this study was to evaluate and compare University of Florida rootstocks and other recently released rootstocks grafted with grapefruit, navel orange, and mandarin scions by measuring tree growth and HLB tolerance. Three independent large-scale field trials were established in September 2019 in Fort Pierce, FL, USA. Trial 1 (T1) included 36 rootstocks with ‘Ray Ruby’ grapefruit as the scion; Trials 2 and 3 (T2 and T3, respectively) included 30 rootstocks with ‘Glenn’ navel orange F-56-11 and ‘UF-950’ mandarin as the scion, respectively. Tree canopy volume, trunk diameter, CLas titer, HLB severity index, and leaf nutrient concentrations were evaluated during 2020 and 2021. Significant differences among rootstock-scion combinations were found in each trial for most of the assessed traits. In T2, UFR-15 consistently developed the largest ‘Ray Ruby’ grapefruit trees during both years. In T3, ‘Glenn’ navel orange F-56-11 trees were larger on C-22, and US-802. Similarly, US-802 and US-942 generated the largest ‘UF-950’ mandarin trees. Overall, trees had optimum levels of macro- and micronutrients except for calcium. CLas infection and HLB visual index varied among scion-rootstock combinations, especially during the first year of growth when intensive flushing was produced. Generally, trees grew vigorously with WGFT+50-7 and Willits inducing the lowest HLB symptoms in all evaluations. Production and fruit quality need to be evaluated to determine the suitability of potential scion-rootstock combinations that can confer consistent economical and biological advantages under the current HLB scenario in the Indian River District.

There have been efforts to develop citrus cultivars that are tolerant of Huanglongbing (HLB), a catastrophic phloem-limited disease. Previous studies demonstrated that continuous plant growth with phloem regeneration is one of the major characteristics of HLB tolerance. In this study, the metabolic mechanisms of HLB tolerance in citrus were elucidated using a multiple pathway-targeted metabolomic approach. Comparative analysis of healthy and infected HLB-tolerant and HLB-sensitive mandarin cultivars (Citrus reticulata) revealed differentially expressed metabolic responses among different groups. Pathway enrichment analysis indicated aspartate and glutamate metabolism, purine metabolism, and biosynthesis of plant hormones were upregulated in the tolerant group, except salicylic acid signaling. Catabolic pathways linked to energy-yielding metabolism were also upregulated in the tolerant group. These metabolisms and pathways were interconnected with each other, unveiling a pivotal metabolic network associated with HLB tolerance. In the network, auxins and cytokinins, the plant hormones responsible for plant growth and phloem regeneration, were accumulated. In addition, purine metabolites serving as energy carriers and nitrogen sources of plants were increased. Only salicylic acid-related metabolites for plant defense responses were decreased in the tolerant group. Our findings may evidence the strategy of HLB-tolerant cultivars that sustain plant growth and phloem formation rather than displaying direct plant defense to overcome the disease.

Several citrus varieties show gametophytic self-incompatibility (GSI), which can contribute to seedless fruit production in several cultivars. This study investigated the genes regulating this trait through RNA-seq performed using styles collected from the flowers of Japanese citrus cultivars ‘Hyuganatsu,' ‘Tosabuntan,' ‘Hassaku,' ‘Banpeiyu,' and ‘Sweet Spring'. We screened the transcripts of putative T2 RNases, i.e., the protein family including all S-RNases from S-RNase-based GSI plants, and constructed a phylogenetic tree using the screened T2 RNases and S-RNases retrieved from citrus genome databases and a public database. Three major clusters (class I–III) were formed, among which, the class III cluster contained family specific subclusters formed by S-RNase and a citrus-specific cluster monophyletic to the S-RNase clusters. From the citrus class III cluster, six transcripts were consistent with the S haplotypes previously determined in Japanese citrus accessions, sharing characteristics such as isoelectric point, extracellular localization, molecular weight, intron number and position, and tissue-specific expression with S-RNases. One T2 RNase gene in self-incompatible Hyuganatsu was significantly down-regulated in the styles of a self-compatible mutant of Hyuganatsu in RNA-seq and qPCR analyses. In addition, the inheritance pattern of some T2 RNase genes was consistent with the pattern of the S haplotype in the progeny population of Hyuganatsu and Tosabuntan. As all results supported citrus self-incompatibility being based on S-RNase, we believe that six T2 RNase genes were S-RNases. The homology comparison between the six T2 RNases and S-RNases recently reported in Chinese citrus revealed that three out of six T2 RNases were identical to S-RNases from Chinese citrus. Thus, the other three T2 RNases were finally concluded to be novel citrus S-RNases involved in self-incompatibility.

The Australian finger lime is a unique citrus species that has gained importance due to its unique fruit characteristics and perceived tolerance to Huanglongbing (HLB), an often-fatal disease of citrus trees. In this study, we developed allotetraploid finger lime hybrids and cybrids by utilizing somatic cell fusion techniques to fuse diploid ‘OLL8’ sweet orange or ‘Page’ tangelo callus-derived protoplasts with finger lime (FL) mesophyll-derived protoplasts. Six somatic fusions were regenerated from the ‘OLL8’ + FL fusion, while three putative cybrids were regenerated from the ‘Page’ + FL fusion. Ploidy levels and nuclear-expressed sequence tag derived simple sequence repeat (EST-SSR) markers confirmed the somatic hybrid production, and mitochondrial DNA primer sets confirmed the cybrid nature. Several trees produced by the somatic fusion remained HLB negative even after 6 years of growth in an HLB-endemic environment. Pathogenesis related (PR) and other genes that are often upregulated in HLB-tolerant trees were also upregulated in our somatic fusions. These newly developed somatic fusions and cybrids could potentially be used as breeding parents to develop the next generation of improved HLB-tolerant rootstocks and scions.

Pathogenic profiling: why fruit from bacteria-infected trees tastes bad The bacteria Candidatus Liberibacter asiaticus (CaLas) causes orange trees to produce poor-tasting fruit thanks to the decreased production of flavour-enhancing proteins, sugars, and metabolites. The University of Florida’s Frederick Gmitter and his team of US and Chinese scientists profiled the proteins and metabolites of healthy Valencia sweet orange trees infected with CaLas, a bacterial pathogen that causes the citrus disease Huanglongbing and reduces the quantity and quality of fruit and juice. The researchers found 123 differentially-expressed proteins and decreased numbers of taste-enhancing constituents, including those mediated by key energy-producing processes. This degradation involved a class of chemicals called terpenoids, which the authors link to poor quality fruit. These results provide insights into the pathogenesis of CaLas infection and could empower future studies to prevent the impact of the bacteria and Huanglongbing infection.

The causal agent of mal secco disease is the fungus Plenodomus tracheiphilus, mainly affecting lemon tree survival in the Mediterranean area. Using a fully compatible host-pathogen interaction, the aim of our work was to retrieve the fungus transcriptome by an RNA seq approach during infection of rough lemon (Citrus jambhiri Lush.) to identify crucial transcripts for pathogenesis establishment and progression. A total of 2438 clusters belonging to P. tracheiphilus were retrieved and classified into the GO and KEGG categories. Transcripts were categorized mainly within the “membrane”, “catalytic activity”, and “primary metabolic process” GO terms. Moreover, most of the transcripts are included in the “ribosome”, “carbon metabolism”, and “oxidative phosphorylation” KEGG categories. By focusing our attention on transcripts with FPKM values higher than the median, we were able to identify four main transcript groups functioning in (a) fungus cell wall remodeling and protection, (b) destroying plant defensive secondary metabolites, (c) optimizing fungus development and pathogenesis, and (d) toxin biosynthesis, thus indicating that a multifaceted strategy to subdue the host was executed.

Crop breeding: A new technique for using CRISPR/Cas9 to create non-transgenic mutant crop plants A group led by Dr. Yi Li of University of Connecticut, USA has developed a technique to reliably create desirable mutations in crop plants without introducing any foreign DNA, thus generating non-transgenic mutant plants. Their technique will be particularly important for application of genome editing technologies in perennial crop plants. Genome editing technologies have been used to introduce desired mutations into plants but the approach normally incorporates foreign genes such as Cas9 into the plant’s genome. To avoid this, Dr. Li’s team has used Agrobacterium to transiently express the CRISPR/Cas9 components, a bacterium commonly used to genetically engineer plants. With their method, CRISPR/Cas9 from the bacteria edit the plant genome without introducing any foreign DNA, and then a two-step screening process is used to identify non-transgenic mutant plants. This new method provides a reliable and efficient alternative for producing non-transgenic genetically engineered crops.

Boron (B) toxicity is a common limiting factor both in arid and semiarid regions, such as the Mediterranean Basin. Citrus trees are sensitive to B-toxicity, which generates a negative impact in citrus orchards. In this work, two promising citrus rootstocks (UFR-6 and 2247 x 6070-02-2) have been assessed against B-toxicity and compared with Carrizo citrange, a common commercial citrus rootstock in Mediterranean Basin. Three B concentration treatments (Control, 1 and 2.5 mM H3BO3) were established, irrigating the plants three times per week for 21 days under greenhouse conditions. During the assay, above-ground symptoms, and chlorophyll index (SPAD) were recorded. At the end of the experiment, stomatal conductance, relative water content, and B concentration in leaves and roots were determined. The increasing B concentration in plants generates visual damage in leaves for all citrus rootstocks assayed. Carrizo citrange displayed the greatest visual symptoms, decreased its chlorophyll index (SPAD), and stomatal conductance throughout the B-treatment. However, UFR-6 and 2247 x 6070-02-2 displayed less symptoms than Carrizo citrange and only reduced its parameters under the 2.5 mM H3BO3 treatment. These results can aid citrus grower rootstock planting decisions with under B-toxicity conditions.

Drought and flooding conditions are increasingly common abiotic factors that affect citrus crops in both the Mediterranean Basin and Florida. Furthermore, emerging diseases, such as Huanglongbing (HLB), are a potential risk for these crops in those producing areas. This study aimed to evaluate the behavior under water-stress treatments of three new citrus rootstocks (UFR-6, B11R5T60, and 2247 x 6070-02-2) with reported tolerance of HLB, comparing them with a common commercial citrus rootstock (Carrizo citrange). Four water conditions were established: Control, Medium Water Stress (MWS), Drought, and Flooding. Chlorophyll index (SPAD), growth in height, relative growth rate, biomass (fresh and dry weight) and plant water status were evaluated. Citru rootstock response were different for each genotype; Carrizo citrange was negatively affected by all water treatments in the chlorophyll index (SPAD) and biomass production. By contrast, UFR-6 showed a positive response in SPAD and growth under MWS and Drought, B11R5T60 displayed similar behavior to Control under all water stresses, and the response of 2247 x 6070-02-2 under MWS treatment was adequate but was not under Drought or Flooding conditions. Our study describes the behavior of these promising new citrus rootstocks against water stress; B11R5T60 exhibiting the best performance. These results can be useful for the citrus industry to address water-stress problems in these crops.

Huanglongbing (HLB), caused by the phloem-limited bacterium Candidatus Liberibacter asiaticus (CaLas), is the primary biotic stress causing significant economic damage to the global citrus industry. Among the abiotic stresses, salinity affects citrus production worldwide, especially in arid and coastal regions. In this study, we evaluated open-pollinated seedlings of the S10 (a diploid rootstock produced from a cross between two siblings of the Hirado Buntan Pink pummelo (Citrus maxima (Burm.) Merr.) with the Shekwasha mandarin (Citrus reticulata Blanco)) for their ability to tolerate HLB and salinity stresses. In a greenhouse study, ‘Valencia’ sweet orange (either HLB-positive or negative) was grafted onto six clonally propagated lines generated from the screened seedlings in the greenhouse and the trees were irrigated with 150 mM NaCl after eight months of successful grafting and detection of CaLas in the leaf petioles. Cleopatra mandarin was used as a salt-tolerant and HLB-sensitive rootstock control. CaLas infection was monitored using a quantitative polymerase chain reaction before and after NaCl treatments. Following three months of NaCl treatment, ‘Valencia’ leaves on the S10 rootstock seedlings recorded lower levels of chlorophyll content compared to Cleopatra under similar conditions. Malondialdehyde content was higher in HLB-infected ‘Valencia’ grafted onto Cleopatra than in the S10 lines. Several plant defense-related genes were significantly upregulated in the S10 lines. Antioxidant and Na+ co-transporter genes were differentially regulated in these lines. Based on our results, selected S10 lines have potential as salt-tolerant rootstocks of ‘Valencia’ sweet orange under endemic HLB conditions. However, it is necessary to propagate selected lines through tissue culture or cuttings because of the high percentage of zygotic seedlings derived from S10.

The prevalence of rough lemon rootstock in India is presently threatened by the spread of Phytophthora root rot. Developing an alternative Phytophthora-resistant rootstock that also withstands other abiotic constraints is a priority for a sustainable citrus industry in India. Complementary progenitors can be found in citrus germplasm to combine the desired traits, particularly between Citrus and Poncirus, a closely related genus. The production of hybrids accumulating the desired dominant traits, irrespective of the heterozygosity level of the parents, is an effective way to develop new candidate rootstocks. Rough lemon was hybridized with Swingle citrumelo, X-639 citrandarin, and GouTou Cheng. A reliable method was developed for greater recovery of hybrid seedlings from these crosses on the basis of morphology and simple sequence repeat (SSR) markers. The greatest proportion of hybrid seedlings resulted from embryo rescue at 95 days after pollination (DAP) (48.5% in rough lemon × X-639 citrandarin and 56.7% in rough lemon × Swingle citrumelo) on the basis of multifoliate leaves. The proportion of hybrid seedlings decreased with increase in embryo development stage after 95 DAP until the last stage of observation (125 DAP). Significantly higher proportion of multifoliate seedlings was recovered by in vitro culture compared with in vivo germination from the mature seeds (26.9% vs. 5.2%, respectively). Swingle and X-639 citrandarin are not homozygous for the multifoliate allele from Poncirus, and GouTou Cheng lacks the multifoliate allele; thus, unifoliate hybrids were selected from all crosses on the basis of SSR marker genotypes.

Huanglongbing (HLB), also known as citrus greening, is the most destructive disease of citrus worldwide. In the United States, this disease is associated with a phloem-restricted bacterium, Candidatus Liberibacter asiaticus. Commercial citrus cultivars are susceptible to HLB, but Poncirus trifoliata, a close relative of Citrus, is highly tolerant of HLB. Isolating P. trifoliata gene(s) controlling its HLB tolerance followed by expressing the gene(s) in citrus is considered a potential cisgenic approach to engineering citrus for tolerance to HLB. Previous gene expression studies indicated that the constitutive disease resistance (CDR) genes in P. trifoliata (PtCDRs) may play a vital role in its HLB tolerance. This study was designed to use Arabidopsis mutants as a model system to confirm the function of PtCDRs in plant disease resistance. PtCDR2 and PtCDR8 were amplified from P. trifoliata cDNA and transferred into the Arabidopsis cdr1 mutant, whose resident CDR1 gene was disrupted by T-DNA insertion. The PtCDR2 and PtCDR8 transgenic Arabidopsis cdr1 mutant restored its hypersensitive response to the bacterial pathogen Pseudomonas syringae pv. tomato strain DC3000 (Pst DC3000) expressing avrRpt2. The defense marker gene PATHOGENESIS RELATED 1 (PR1) expressed at much higher levels in the PtCDR2 or PtCDR8 transgenic cdr1 mutant than in the non-transgenic cdr1 mutant with or without pathogen infection. Multiplication of Pst DC3000 bacteria in Arabidopsis was inhibited by the expression of PtCDR2 and PtCDR8. Our results showed that PtCDR2 and PtCDR8 were functional in Arabidopsis and played a positive role in disease resistance and demonstrated that Arabidopsis mutants can be a useful alternate system for screening Poncirus genes before making the time-consuming effort to transfer them into citrus, a perennial woody plant that is highly recalcitrant for Agrobacterium or biolistic-mediated transformation.

Huanglongbing (HLB), or citrus greening, is the most devastating disease in citrus worldwide. Commercial citrus varieties including sweet orange (Citrus sinensis) are highly susceptible to HLB, and trifoliate orange (Poncirus trifoliata, a close Citrus relative) is widely considered resistant or highly tolerant to HLB. In this study, an intergeneric F1 population of sweet orange and trifoliate orange was genotyped by Genotyping-by-Sequencing, and high-density SNP-based genetic maps were constructed separately for trifoliate orange and sweet orange. The two genetic maps exhibited high synteny and high coverage of the citrus genome. Progenies of the F1 population and their parents were planted in a replicated field trial, exposed to intense HLB pressure for 3 years, and then evaluated for susceptibility to HLB over 2 years. The F1 population exhibited a wide range in severity of HLB foliar symptom and canopy damage. Genome-wide QTL analysis based on the phenotypic data of foliar symptom and canopy damage in 2 years identified three clusters of repeatable QTLs in trifoliate orange linkage groups LG-t6, LG-t8 and LG-t9. Co-localization of QTLs for two traits was observed within all three regions. Additionally, one cluster of QTLs in sweet orange (linkage group LG-s7) was also detected. The majority of the identified QTLs each explained 18–30% of the phenotypic variation, indicating their major role in determining HLB responses. These results show, for the first time, a quantitative genetic nature yet the presence of major loci for the HLB tolerance in trifoliate orange. The results suggest that sweet orange also contains useful genetic factor(s) for improving HLB tolerance in commercial citrus varieties. Findings from this study should be very valuable and timely to researchers worldwide as they are hastily searching for genetic solutions to the devastating HLB crisis through breeding, genetic engineering, or genome editing.

Although there are no known sources of genetic resistance, some Citrus spp. are reportedly tolerant to huanglongbing (HLB), presumably caused by ‘Candidatus Liberibacter asiaticus’. Time-course transcriptional analysis of tolerant rough lemon (Citrus jambhiri) and susceptible sweet orange (C. sinensis) in response to ‘Ca. L. asiaticus’ infection showed more genes differentially expressed in HLB-affected rough lemon than sweet orange at early stages but substantially fewer at late time points, possibly a critical factor underlying differences in sensitivity to ‘Ca. L. asiaticus’. Pathway analysis revealed that stress responses were distinctively modulated in rough lemon and sweet orange. Although microscopic changes (e.g., callose deposition in sieve elements and phloem cell collapse) were found in both infected species, remarkably, phloem transport activity in midribs of source leaves in rough lemon was much less affected by HLB than in sweet orange. The difference in phloem cell transport activities is also implicated in the differential sensitivity to HLB between the two species. The results potentially lead to identification of key genes and the genetic mechanism in rough lemon to restrain disease development and maintain (or recover) phloem transport activity. These potential candidate genes may be used for improving citrus tolerance (or even resistance) to HLB by genetic engineering.