Melatonin supplementation enhances browning suppression and improves transformation efficiency and regeneration of transgenic rough lemon plants (Citrus × jambhiri).

Enzymatic browning poses a significant challenge that limits in vitro propagation and genetic transformation of plant tissues. This research focuses on investigating how adding antioxidant substances can suppress browning, leading to improved efficiency in transforming plant tissues using Agrobacterium and subsequent plant regeneration from rough lemon (Citrus × jambhiri). When epicotyl segments of rough lemon were exposed to Agrobacterium, they displayed excessive browning and tissue decay. This was notably different from the 'Hamlin' explants, which did not exhibit the same issue. The regeneration process failed completely in rough lemon explants, and they accumulated high levels of total phenolic compounds (TPC) and polyphenol oxidase (PPO), which contribute to browning. To overcome these challenges, several antioxidant and osmoprotectant compounds, including lipoic acid, melatonin, glycine betaine, and proline were added to the tissue culture medium to reduce the oxidation of phenolic compounds and mitigate browning. Treating epicotyl segments with 100 or 200 µM melatonin led to a significant reduction in browning and phenolic compound accumulation. This resulted in enhanced shoot regeneration, increased transformation efficiency, and reduced tissue decay. Importantly, melatonin supplementation effectively lowered the levels of TPC and PPO in the cultured explants. Molecular and physiological analyses also confirmed the successful overexpression of the CcNHX1 transcription factor, which plays a key role in imparting tolerance to salinity stress. This study emphasizes the noteworthy impact of supplementing antioxidants in achieving successful genetic transformation and plant regeneration in rough lemon. These findings provide valuable insights for developing strategies to address enzymatic browning and enhance the effectiveness of plant tissue culture and genetic engineering methods with potential applications across diverse plant species.

Influence of Anthocyanin Expression on the Performance of Photosynthesis in Sweet Orange, Citrus sinensis (L.) Osbeck.

Anthocyanins are a class of natural pigments that accumulate transiently or permanently in plant tissues, often in response to abiotic and biotic stresses. They play a photoprotective role by attenuating the irradiance incident on the photochemical apparatus and quenching oxyradicals through their powerful anti-oxidative function. The objective of the current study is to understand the impact of introducing Vitis vinifera mybA1 (VvmybA1) in 'Hamlin' sweet orange trees on various aspects, including photosynthetic performance, pigment composition, and gene expression related to photosynthesis and light harvesting. We describe the relationship between anthocyanin accumulation and photosynthetic measurements in genetically modified 'Hamlin' sweet orange trees expressing the grapevine-derived Vitis vinifera mybA1 (VvmybA1). The juvenile leaves of transgenic plants displayed an intense purple color compared to the mature leaves, and microscopic visualization showed anthocyanin accumulation primarily in the leaf epidermal cells. Under optimal growth conditions, there were no significant differences in leaf gas exchange variables, suggesting normal photosynthetic performance. The chlorophyll fluorescence maximum quantum yield of PSII was slightly reduced in VvmybA1 transgenic leaves compared to the performance of the control leaves, while the total performance index per absorbance remained unaffected. Comparison of the chlorophyll and carotenoid pigment contents revealed that chlorophyllide a and carotenoid pigments, including trans-neoxanthin, trans-violaxanthin, cis-violaxanthin, zeaxanthin, antheraxanthin, and total xanthophylls were enhanced in VvmybA1 transgenic leaves. Although there were no significant changes in the rates of the gas exchange parameters, we recorded a high relative expression of the ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (RuBP) and rubisco activase (RCA) in the mature leaves of transgenic plants, indicating activation of Rubisco. Our findings confirm an efficient photoacclimation of the photosynthetic apparatus, allowing the transgenic line to maintain a photosynthetic performance similar to that of the wild type.

Insights into the mechanism of Huanglongbing tolerance in the Australian finger lime (Citrus australasica).

The Australian finger lime (Citrus australasica) is tolerant to Huanglongbing (HLB; Citrus greening). This species can be utilized to develop HLB tolerant citrus cultivars through conventional breeding and biotechnological approaches. In this report, we conducted a comprehensive analysis of transcriptomic data following a non-choice infection assay to understand the CaLas tolerance mechanisms in the finger lime. After filtering 3,768 differentially expressed genes (DEGs), 2,396 were downregulated and 1,372 were upregulated in CaLas-infected finger lime compared to CaLas-infected HLB-susceptible 'Valencia' sweet orange. Comparative analyses revealed several DEGs belonging to cell wall, ß-glucanase, proteolysis, R genes, signaling, redox state, peroxidases, glutathione-S-transferase, secondary metabolites, and pathogenesis-related (PR) proteins categories. Our results indicate that the finger lime has evolved specific redox control systems to mitigate the reactive oxygen species and modulate the plant defense response. We also identified candidate genes responsible for the production of Cys-rich secretory proteins and Pathogenesis-related 1 (PR1-like) proteins that are highly upregulated in infected finger lime relative to noninfected and infected 'Valencia' sweet orange. Additionally, the anatomical analysis of phloem and stem tissues in finger lime and 'Valencia' suggested better regeneration of phloem tissues in finger lime in response to HLB infection. Analysis of callose formation following infection revealed a significant difference in the production of callose plugs between the stem phloem of CaLas+ 'Valencia' sweet orange and finger lime. Understanding the mechanism of resistance will help the scientific community design strategies to protect trees from CaLas infection and assist citrus breeders in developing durable HLB tolerant citrus varieties.

Comparative transcriptome analysis of Citrus macrophylla tree infected with Citrus tristeza virus stem pitting mutants provides new insight into the role of phloem regeneration in stem pitting disease.

Stem pitting is a complex and economically important virus-associated disease of perennial woody plants. Molecular mechanisms and pathways occurring during virus-plant interaction that result in this phenomenon are still obscure. Previous studies indicated that different Citrus tristeza virus (CTV) mutants induce defined stem pitting phenotypes ranging from mild (CTVΔp13) to severe (CTVΔp33) in Citrus macrophylla trees. In this study, we conducted comparative transcriptome analyses of C. macrophylla trees infected with CTV mutants (CTVΔp13 and CTVΔp33) and a full-length virus in comparison to healthy plants as control. The mild CTV stem pitting mutant had very few differentially expressed genes (DEGs) related to plant defense mechanism and plant growth and development. In contrast, substantial gene expression changes were observed in plants infected with the severe mutant and the full-length virus, indicating that both the p13 and p33 proteins of CTV acted as a regulator of symptom production by activating and modulating plant responses, respectively. The analysis of transcriptome data for CTVΔp33 and the full-length virus suggested that xylem specification has been blocked by detecting several genes encoding xylem, cell wall and lignin degradation, and cell wall loosening enzymes. Furthermore, stem pitting was accompanied by downregulation of transcription factors involved in regulation of xylem differentiation and downregulation of some genes involved in lignin biosynthesis, showing that the xylem differentiation and specification program has been shut off. Upregulation of genes encoding transcription factors associated with phloem and cambium development indicated the activation of this program in stem pitting disease. Furthermore, we detected the induction of several DEGs encoding proteins associated with cell cycle re-entry such as chromatin remodeling factors and cyclin, and histone modification. This kind of expression pattern of genes related to xylem differentiation and specification, phloem and cambium development, and cell cycle re-entry is demonstrated during secondary vascular tissue (SVT) regeneration. The microscopy analysis confirmed that the regeneration of new phloem is associated with stem pitting phenotypes. The findings of this study, thus, provide evidence for the association between stem pitting phenotypes and SVT regeneration, suggesting that the expression of these genes might play important roles in development of stem pitting symptoms. Overall, our findings suggest that phloem regeneration contributes to development of stem pitting symptoms.

Overexpression of the salicylic acid binding protein 2 (SABP2) from tobacco enhances tolerance against Huanglongbing in transgenic citrus.

KEY MESSAGE: Overexpression of the salicylic acid binding protein 2 (SABP2) gene from Tobacco results in enhanced tolerance to Huanglongbing (HLB; citrus greening disease) in transgenic sweet oranges. Huanglongbing (HLB), the most destructive citrus disease, is caused by Candidatus Liberibacter asiaticus (CaLas). Currently, no cure for this disease exists, and all commercially planted cultivars are highly susceptible. Salicylic Acid Binding Protein 2 (SABP2) is a well-characterized protein essential for establishing systemic acquired resistance (SAR) in tobacco. The constitutive over expression of SABP2 from tobacco (NtSABP2) in 'Hamlin' sweet orange resulted in the production of several transgenic lines with variable transcript levels. Transient expression of the NtSABP2-EGFP fusion protein in Nicotiana benthamiana plants demonstrated that NtSABP2 was cytosolic in its subcellular localization. In a long-term field study, we identified a SABP2 transgenic line with significantly reduced HLB symptoms that maintained a consistently low CaLas titer. Transcriptome analysis of this selected transgenic line demonstrated upregulation of several genes related to plant defense and SAR pathways. Genes, such as NPR family genes and those coding for monooxygenases and lipoxygenases, were upregulated in the 35S-NtSABP2 overexpressing line and might be candidates for incorporation into our citrus improvement program.

The Citrus sinensis TILLER ANGLE CONTROL 1 (CsTAC1) gene regulates tree architecture in sweet oranges by modulating the endogenous hormone content.

Citrus is a major fruit crop cultivated on a global scale. Citrus trees are long lived perennials with a large canopy. Understanding the genetic control of tree architecture could provide tools for breeding and selection of citrus cultivars suitable for high density planting with improved light exposure. Tree architecture is modulated by the TILLER ANGLE CONTROL 1 (TAC1) gene which plays an important role in the regulation of the shoot angle. Herein, we used CRISPR/Cas9 technology to knockout the CsTAC1 gene for the biochemical and molecular analysis of its function. Nine transgenic lines were obtained, and five edited plants were confirmed based on T7EI mismatch detection assay and Sanger sequencing. The transgenic citrus lines exhibited pleiotropic phenotypes, including differences in branch angle and stem growth. Additionally, silencing CsTAC1 led to enhanced CsLAZY1 transcript levels in the tested lines. Analysis of the phytohormonal profile revealed that TAC1-edited plants exhibited lower auxin contents and increased cytokinin levels in the leaves compared to the wild-type plants. The GA7 gibberellin level was enhanced in most of the edited lines. Collectively, TAC1 affects branch angle in association with hormone signals in citrus.

A cationic lipid mediated CRISPR/Cas9 technique for the production of stable genome edited citrus plants.

BACKGROUND: The genetic engineering of crops has enhanced productivity in the face of climate change and a growing global population by conferring desirable genetic traits, including the enhancement of biotic and abiotic stress tolerance, to improve agriculture. The clustered regularly interspaced short palindromic repeats (CRISPR/Cas9) system has been found to be a promising technology for genomic editing. Protoplasts are often utilized for the development of genetically modified plants through in vitro integration of a recombinant DNA fragment into the plant genome. We targeted the citrus Nonexpressor of Pathogenesis-Related 3 (CsNPR3) gene, a negative regulator of systemic acquired resistance (SAR) that governs the proteasome-mediated degradation of NPR1 and developed a genome editing technique targeting citrus protoplast DNA to produce stable genome-edited citrus plants. RESULTS: Here, we determined the best cationic lipid nanoparticles to deliver donor DNA and described a protocol using Lipofectamine™ LTX Reagent with PLUS Reagent to mediate DNA delivery into citrus protoplasts. A Cas9 construct containing a gRNA targeting the CsNPR3 gene was transfected into citrus protoplasts using the cationic lipid transfection agent Lipofectamine with or without polyethylene glycol (PEG, MW 6000). The optimal transfection efficiency for the encapsulation was 30% in Lipofectamine, 51% in Lipofectamine with PEG, and 2% with PEG only. Additionally, plasmid encapsulation in Lipofectamine resulted in the highest cell viability percentage (45%) compared with PEG. Nine edited plants were obtained and identified based on the T7EI assay and Sanger sequencing. The developed edited lines exhibited downregulation of CsNPR3 expression and upregulation of CsPR1. CONCLUSIONS: Our results demonstrate that utilization of the cationic lipid-based transfection agent Lipofectamine is a viable option for the successful delivery of donor DNA and subsequent successful genome editing in citrus.

Phloem transport limitation in Huanglongbing-affected sweet orange is dependent on phloem-limited bacteria and callose.

Huanglongbing (HLB), caused by Candidatus `Liberibacter asiaticus' (CLas), is a phloem-limited disease that disrupts citrus production in affected areas. In HLB-affected plants, phloem sieve plate pores accumulate callose, and leaf carbohydrate export is reduced. However, whether HLB causes a reduction in carbohydrate phloem translocation speed and the quantitative relationships among callose, CLas population and phloem translocation are still unknown. In this work, a procedure was developed to concurrently measure sugar transport, callose deposition and relative pathogen population at different locations throughout the stem. Increasing quantities of CLas genetic material were positively correlated with quantity and density of callose deposits and negatively correlated with phloem translocation speed. Callose deposit quantity was position and rootstock dependent and was negatively correlated with phloem translocation speed, suggesting a localized relationship. Remarkably, callose accumulation and phloem translocation disruption in the scion were dependent on rootstock genotype. Regression results suggested that the interaction of Ct values and number of phloem callose depositions, but not their size or density, explained the effects on translocation speed. Sucrose, starch and sink 14C label allocation data support the interpretation of a transport pathway limitation by CLas infection. This work shows that the interaction of local accumulation of callose and CLas affects phloem transport. Furthermore, the extent of this accumulation is attenuated by the rootstock and provides important information about the disease mechanism of phloem-inhabiting bacteria. Together, these results constitute the first example of a demonstrated transport limitation of phloem function by a microbial infection.

Physiological Responses and Gene Expression Patterns in Open-Pollinated Seedlings of a Pummelo-Mandarin Hybrid Rootstock Exposed to Salt Stress and Huanglongbing.

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.

Utilization of somatic fusion techniques for the development of HLB tolerant breeding resources employing the Australian finger lime (Citrus australasica).

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.

Metabolic Profiling of Hybrids Generated from Pummelo and Citrus latipes in Relation to Their Attraction to Diaphorina citri, the Vector of Huanglongbing.

The citrus industry at present is severely affected by huanglongbing disease (HLB). HLB is caused by the supposed bacterial pathogen "Candidatus Liberibacter asiaticus" and is transmitted by the insect vector, the Asian citrus psyllid, Diaphorina citri Kuwayama. Developing new citrus hybrids to improve HLB management is much needed. In this study, we investigated the metabolomic profiles of three new hybrids produced from the cross of C2-5-12 Pummelo (Citrus maxima (L.) Osbeck) × pollen from Citrus latipes. The hybrids were selected based on leaf morphology and seedling vigor. The selected hybrids exhibited compact and upright tree architecture as seen in C. latipes. Hybrids were verified by simple sequence repeat markers, and were subjected to metabolomic analysis using gas chromatography-mass spectrometry. The volatile organic compounds (VOCs) and polar metabolites profiling also showed that the new hybrids were different from their parents. Interestingly, the levels of stored VOCs in hybrid II were higher than those observed in its parents and other hybrids. The level of most VOCs released by hybrid II was also higher than that released from its parents. Additionally, the preference assay showed that hybrid II was more attractive to D. citri than its parents and other hybrids. The leaf morphology, compact and upright architecture of hybrid II, and its attraction to D. citri suggest that it could be used as a windbreak and trap tree for D. citri (double duty), once its tolerance to HLB disease is confirmed. Our results showed that metabolomic analysis could be successfully used to understand the biochemical mechanisms controlling the interaction of D. citri with its host plants.

The vascular targeted citrus FLOWERING LOCUS T3 gene promotes non-inductive early flowering in transgenic Carrizo rootstocks and grafted juvenile scions.

Shortening the juvenile stage in citrus and inducing early flowering has been the focus of several citrus genetic improvement programs. FLOWERING LOCUS T (FT) is a small phloem-translocated protein that regulates precocious flowering. In this study, two populations of transgenic Carrizo citrange rootstocks expressing either Citrus clementina FT1 or FT3 genes under the control of the Arabidopsis thaliana phloem specific SUCROSE SYNTHASE 2 (AtSUC2) promoter were developed. The transgenic plants were morphologically similar to the non-transgenic controls (non-transgenic Carrizo citrange), however, only AtSUC2-CcFT3 was capable of inducing precocious flowers. The transgenic lines produced flowers 16 months after transformation and flower buds appeared 30-40 days on juvenile immature scions grafted onto transgenic rootstock. Gene expression analysis revealed that the expression of SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1) and APETALA1 (AP1) were enhanced in the transgenics. Transcriptome profiling of a selected transgenic line showed the induction of genes in different groups including: genes from the flowering induction pathway, APETALA2/ETHYLENE RESPONSE FACTOR (AP2/ERF) family genes, and jasmonic acid (JA) pathway genes. Altogether, our results suggested that ectopic expression of CcFT3 in phloem tissues of Carrizo citrange triggered the expression of several genes to mediate early flowering.

Efficient CRISPR/Cas9 genome editing with Citrus embryogenic cell cultures.

BACKGROUND: Development of precise genome editing strategies is a prerequisite for producing edited plants that can aid in the study of gene function and help understand the genetic traits in a cultivar. Citrus embryogenic cell cultures can be used to rapidly produce a large population of genome edited transformed citrus lines. The ability to introduce specific mutations in the genome of these cells using two constructs (pC-PDS1 and pC-PDS2) was evaluated in this study. RESULTS: Citrus sinensis 'EV2' embryogenic cell cultures are amenable to Agrobacterium-mediated CRISPR/Cas9-based genome editing. Guide RNAs (gRNAs) targeting two locations in the phytoene desaturase (PDS) gene were either driven by the Arabidopsis U6-26 promoter (pC-PDS1) or assembled as a Csy4 array under the control of the CmYLCV promoter (pC-PDS2). All transgenic embryos were completely albino and no variegated phenotype was observed. We evaluated 12 lines from each construct in this study and the majority contain either insertion (1-2 bp), substitution (1 bp), or deletion (1-3 bp) mutations that occurred close to the protospacer adjacent motif. CONCLUSIONS: Both the pC-PDS1 and pC-PDS2 could successfully edit the citrus embryogenic cell cultures. However, the editing efficiency was dependent on the gRNA, confirming that the selection of a proper gRNA is essential for successful genome editing using the CRISPR/Cas9 technique. Also, utilization of embryogenic cell cultures offers another option for successful genome editing in citrus.

Potential Mechanisms of AtNPR1 Mediated Resistance against Huanglongbing (HLB) in Citrus.

Huanglongbing (HLB), a bacterial disease caused by Candidatus Liberibacter asiaticus (CLas), is a major threat to the citrus industry. In a previous study conducted by our laboratory, several citrus transgenic trees expressing the Arabidopsis thaliana NPR1 (AtNPR1) gene remained HLB-free when grown in a field site under high HLB disease pressure. To determine the molecular mechanisms behind AtNPR1-mediated tolerance to HLB, a transcriptome analysis was performed using AtNPR1 overexpressing transgenic trees and non-transgenic trees as control, from which we identified 57 differentially expressed genes (DEGs). Data mining revealed the enhanced transcription of genes encoding pathogen-associated molecular patterns (PAMPs), transcription factors, leucine-rich repeat receptor kinases (LRR-RKs), and putative ankyrin repeat-containing proteins. These proteins were highly upregulated in the AtNPR1 transgenic line compared to the control plant. Furthermore, analysis of protein-protein interactions indicated that AtNPR1 interacts with CsNPR3 and CsTGA5 in the nucleus. Our results suggest that AtNPR1 positively regulates the innate defense mechanisms in citrus thereby boosting resistance and effectively protecting the plant against HLB.

Dynamics of Candidatus Liberibacter asiaticus Movement and Sieve-Pore Plugging in Citrus Sink Cells.

Citrus greening or Huanglongbing (HLB) is caused by the phloem-limited intracellular Gram-negative bacterium Candidatus Liberibacter asiaticus (CLas). HLB-infected citrus phloem cells undergo structural modifications that include cell wall thickening, callose and phloem protein induction, and cellular plugging. However, very little is known about the intracellular mechanisms that take place during CLas cell-to-cell movement. Here, we show that CLas movement through phloem pores of sweet orange (Citrus sinensis) and grapefruit (Citrus paradisi) is carried out by the elongated form of the bacteria. The round form of CLas is too large to move, but can change its morphology to enable its movement. CLas cells adhere to the plasma membrane of the phloem cells specifically adjacent to the sieve pores. Remarkably, CLas was present in both mature sieve element cells and nucleated nonsieve element cells. The sieve plate plugging structures of host plants were shown to have different composition in different citrus tissues. Callose deposition was the main plugging mechanism in the HLB-infected flush, where it reduced the open space of the pores. In the roots, pores were surrounded by dark extracellular material, with very little accumulation of callose. The expression of CALLOSE SYNTHASE7 and PHLOEM PROTEIN2 genes was upregulated in the shoots, but downregulated in root tissues. In seed coats, no phloem occlusion was observed, and CLas accumulated to high levels. Our results provide insight into the cellular mechanisms of Gram-negative bacterial cell-to-cell movement in plant phloem.

Development of Improved Fruit, Vegetable, and Ornamental Crops Using the CRISPR/Cas9 Genome Editing Technique.

Horticultural crops, including fruit, vegetable, and ornamental plants are an important component of the agriculture production systems and play an important role in sustaining human life. With a steady growth in the world's population and the consequent need for more food, sustainable and increased fruit and vegetable crop production is a major challenge to guarantee future food security. Although conventional breeding techniques have significantly contributed to the development of important varieties, new approaches are required to further improve horticultural crop production. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) has emerged as a valuable genome-editing tool able to change DNA sequences at precisely chosen loci. The CRISPR/Cas9 system was developed based on the bacterial adaptive immune system and comprises of an endonuclease guided by one or more single-guide RNAs to generate double-strand breaks. These breaks can then be repaired by the natural cellular repair mechanisms, during which genetic mutations are introduced. In a short time, the CRISPR/Cas9 system has become a popular genome-editing technique, with numerous examples of gene mutation and transcriptional regulation control in both model and crop plants. In this review, various aspects of the CRISPR/Cas9 system are explored, including a general presentation of the function of the CRISPR/Cas9 system in bacteria and its practical application as a biotechnological tool for editing plant genomes, particularly in horticultural crops.

Overexpression of the CsFUS3 gene encoding a B3 transcription factor promotes somatic embryogenesis in Citrus.

In citrus, genetic improvement via biotechnology is challenging due to insufficient understanding of molecular barriers that prevent regeneration by somatic embryogenesis (SE). Our previous study indicated that LEC genes were involved in SE in citrus, but their regulatory roles remain to be elucidated. Here, we cloned one of the LEC genes, CsFUS3, and show that it is preferentially expressed during SE and in the embryogenic callus (EC) derived from citrus varieties with strong embryogenic competence. The overexpression of CsFUS3 in recalcitrant citrus callus restored embryogenic competence. Complementation of the loss-of-function Arabidopsis fus3 mutant with the CsFUS3 gene restored normal late embryogenesis, which is consistent with the CsFUS3 and AtFUS3 proteins contributing to the same regulatory network in Arabidopsis. Transcriptome profiling revealed that the expression of particular TFs that promote SE was up-regulated in the citrus overexpression (OE) line. The 104 differentially expressed genes associated with hormone biosynthesis, catabolism, and signaling are particularly noteworthy. The dynamic change in the ratio of ABA to GA during SE in wild-type callus mirrored the expression pattern of CsFUS3. In contrast, in the OE line, the ratio of ABA to GA was higher and the capacity for SE was greater when the OE line was separately treated with ABA and GA biosynthesis inhibitors. Taken together, our results demonstrate that the overexpression of CsFUS3 appears to establish a cellular environment favorable to SE, at least in part by promoting a high ABA to GA ratio and by regulating the expression of TFs that promote SE.

Identification of sieve elements and companion cell protoplasts by a combination of brightfield and fluorescence microscopy.

PREMISE OF THE STUDY: Phloem-limited diseases are becoming increasingly pervasive, threatening the existence of crops worldwide. Studies of phloem diseases are complicated by the inaccessibility of the phloem tissue. Phloem cells are located deep inside the plant body, are interspersed with other cell types, are among the smallest cells in the plant kingdom, and make up a small percentage of the total cell population in a plant. These properties make phloem cells difficult to investigate. METHODS: We used leaf midrib protoplasts and a combination of organelle-specific dyes including Neutral Red (acidic compartments), MitoTracker Green (mitochondria), Hoechst 3342 (nucleus), and chloroplast autofluorescence. We examined the protoplasts under light and fluorescence microscopy. RESULTS: When observed using brightfield and fluorescence microscopy, sieve element protoplasts were distinguished by the lack of vacuole and a nucleus, and by containing various mitochondria. Companion cells showed a dense cytoplasm and various small vacuoles. They also revealed their characteristic large nucleus and abundant mitochondria. DISCUSSION: We present evidence that a combination of organelle-specific dyes and autofluorescence allows for the identification of sieve elements and companion cell protoplasts from citrus leaf tissue.

All roads lead to Rome: Towards understanding different avenues of tolerance to huanglongbing in citrus cultivars.

Citrus tolerance to huanglongbing could result from tolerance to the pathogen Candidatus Liberibacter asiaticus (CLas) and/or to its vector Diaphorina citri. Field observations and greenhouse-controlled studies showed that some citrus cultivars were more tolerant than others. However, the mechanism(s) behind the tolerance has not been determined yet. Using GC-MS, we investigated the volatile organic compounds (VOCs) and the non-volatile metabolite profiles of two tolerant citrus cultivars- Australian finger lime, 'LB8-9' Sugar Belle® mandarin hybrid, and a recently released mandarin hybrid 'Bingo'. The three were grafted onto the rootstock, Carrizo citrange. Our findings showed that the metabolomic profiles of Australian finger lime were different from that of 'LB8-9'. Finger lime was high in many amino acids and tricarboxylic acid intermediates, whereas 'LB8-9' was high in several amino acids, sugars, and sugar alcohols. 'LB8-9' was high in thymol, which is known for its strong antimicrobial activity against a panel of pathogenic bacteria. The metabolomic profiles of 'Bingo' were intensely different from the other mandarin hybrid, 'LB8-9', including a reduced thymol biosynthetic pathway and low amounts of most of the amino acids and sugar alcohols. Remarkably, 1,8-cineole (eucalyptol) was only detected in 'Bingo', indicating that eucalyptol could have feeding and ovipositional repellency against D. citri. The metabolite profiles generated for HLB-tolerant citrus species will improve the ability of citrus breeders and will allow them to take more informed decisions. Metabolomic profiling of HLB-tolerant citrus species could identify tolerance specific markers that can be introduced to other commercial citrus cultivars to improve their tolerance to HLB disease.

Embryo-specific expression of a visual reporter gene as a selection system for citrus transformation.

The embryo-specific Dc3 gene promoter driving the VvMybA1 anthocyanin regulatory gene was used to develop a visual selection system for the genetic transformation of citrus. Agrobacterium-mediated transformation of cell suspension cultures resulted in the production of purple transgenic somatic embryos that could be easily separated from the green non-transgenic embryos. The somatic embryos produced phenotypically normal plants devoid of any visual purple coloration. These results were also confirmed using protoplast transformation. There was minimal gene expression in unstressed one-year-old transgenic lines. Cold and drought stress did not have any effect on gene expression, while exogenous ABA and NaCl application resulted in a minor change in gene expression in several transgenic lines. When gas exchange was measured in intact leaves, the transgenic lines were similar to controls under the same environment. Our results provide conclusive evidence for the utilization of a plant-derived, embryo-specific visual reporter system for the genetic transformation of citrus. Such a system could aid in the development of an all-plant, consumer-friendly GM citrus tree.