On-site analysis and rapid identification of citrus herbs by miniature mass spectrometry and machine learning.

BACKGROUND: Natural medicines present a considerable analytical challenge due to their diverse botanical origins and complex multi-species composition. This inherent complexity complicates their rapid identification and analysis. Tangerine peel, a product of the Citrus species from the Rutaceae family, is widely used both as a culinary ingredient and in traditional Chinese medicine. It is classified into two primary types in China: Citri Reticulatae Pericarpium (CP) and Citri Reticulatae Pericarpium Viride (QP), differentiated by harvest time. A notable price disparity exists between CP and another variety, Citri reticulatae "Chachi" (GCP), with differences being based on the original variety. METHODS: This study introduces an innovative method using portable miniature mass spectrometry for swift on-site analysis of QP, CP, and GCP, requiring less than a minute per sample. And combined with machine learning to differentiate the three types on site, the method was used to try to distinguish GCP from different storage years. RESULTS: This novel method using portable miniature mass spectrometry for swift on-site analysis of tangerine peels enabled the characterization of 22 compounds in less than one minute per sample. The method simplifies sample processing and integrates machine learning to distinguish between the CP, QP, and GCP varieties. Moreover, a multiple-perceptron neural network model is further employed to specifically differentiate between CP and GCP, addressing the significant price gap between them. CONCLUSIONS: The entire analytical time of the method is about 1 minute, and samples can be analyzed on site, greatly reducing the cost of testing. Besides, this approach is versatile, operates independently of location and environmental conditions, and offers a valuable tool for assessing the quality of natural medicines.

Electrical impedance spectroscopy for potassium content analysis and botanical origin identification of honey.

Minerals are reported to dominate the electrical properties of honey and indicate its botanical and geographical origins. In this study, Electrochemical Impedance Spectroscopy (EIS) was used to assess the relation between mineral elements, electrical properties and botanical origin using three honey varieties - Citrus sp., Eucalyptus sp., and Erica sp. These varieties are identified through pollen analysis and market labelling. Flame atomic absorption and emission spectroscopies were used to quantify the concentrations of eight elements (potassium, sodium, calcium, magnesium, manganese, zinc, copper, and iron). Among all the mineral elements, potassium showed a consistent correlation with impedance. The potassium estimation in honey and standard solutions (calibration curve) had similar sensitivities of 153.43 nF/mM and 132.68 nF/mM, respectively. Additionally, the analysis revealed that potassium dominates the mineral composition, with the other species present in minimal quantities. The EIS technique showed high sensitivity to potassium and other ionisable species, making it possible to classify the botanical origin of these three honey types. The EIS technique proved to be both time and cost effective, yielding a classification rate higher than that achieved by analysing mineral composition.

Comparative Evaluation of Secondary Metabolite Chemodiversity of Citrus Genebank Collection in Greece: Can the Peel be More than Waste?

Citrus fruits are among the most economically important crops in the world. In the global market, the Citrus peel is often considered a byproduct but substitutes an important phenotypic characteristic of the fruit and a valuable source of essential oils, flavonoids, carotenoids, and phenolic acids with variable concentrations. The Mediterranean basin is a particularly dense area of autochthonous genotypes of Citrus that are known for being a source of healthy foods, which can be repertoires of valuable genes for molecular breeding with the focus on plant resistance and quality improvement. The scope of this study was to characterize and compare the main phenotypic parameters (i.e., peel thickness, fruit volume, and area) and levels of bioactive compounds in the peel of fruits from the local germplasm of Citrus in Greece, to assess their chemodiversity regarding their polyphenolic, volatile, and carotenoid profiles. A targeted liquid chromatographic approach revealed hesperidin, tangeretin, narirutin, eriocitrin, and quercetin glycosides as the major polyphenolic compounds identified in orange, lemon, and mandarin peels. The content of tangeretin and narirutin followed the tendency mandarin > orange > lemon. Eriocitrin was a predominant metabolite of lemon peel, following its identification in lower amounts in mandarin and at least in the orange peel. For these citrus-specific metabolites, high intra- but also interspecies chemodiversity was monitored. Significant diversity was found in the essential oil content, which varied between 1.2 and 3% in orange, 0.2 and 1.4% in mandarin, and 0.9 and 1.9% in lemon peel. Limonene was the predominant compound in all Citrus species peel essential oils, ranging between 88 and 93% among the orange, 64 and 93% in mandarin, and 55 and 63% in lemon cultivars. Carotenoid analysis revealed different compositions among the Citrus species and accessions studied, with ß-cryptoxanthin being the most predominant metabolite. This large-scale metabolic investigation will enhance the knowledge of Citrus peel secondary metabolite chemodiversity supported by the ample availability of Citrus genetic resources to further expand their exploitation in future breeding programs and potential applications in the global functional food and pharmaceutical industries.

Analysis of CcGASA family members in Citrus clementina (Hort. ex Tan.) by a genome-wide approach.

The Gibberellic Acid Stimulated Arabidopsis (GASA) proteins were investigated in the study to help understand their possible roles in fruit trees, particularly in Citrus. A total of 18 CcGASA proteins were identified and characterized in Citrus clementina via a genome-wide approach. It was shown that the CcGASA proteins structurally shared a conserved GASA domain but varied considerably in primary sequences and motif compositions. Thus, they could be classified into three major phylogenetic groups, G1~G3, and two groups, G1 and G3 could be further classified into subgroups. The cis- elements on all CcGASA promoters were identified and categorized, and the associated transcription factors were predicted. In addition, the possible interactions between the CcGASA proteins and other proteins were predicted. All the clues suggested that these genes should be involved in defense against biotic and abiotic stresses and in growth and development. The notion was further supported by gene expression analysis that showed these genes were more or less responsive to the treatments of plant hormones (GA3, SA, ABA and IAA), and infections of citrus canker pathogen Xanthomonas citri. It was noted that both the segmental and the tandem duplications had played a role in the expansion of the CcGASA gene family in Citrus. Our results showed that the members of the CcGASA gene family should have structurally and functionally diverged to different degrees, and hence, the representative group members should be individually investigated to dissect their specific roles.

Antioxidant Metabolites in Primitive, Wild, and Cultivated Citrus and Their Role in Stress Tolerance.

The genus Citrus contains a vast range of antioxidant metabolites, dietary metabolites, and antioxidant polyphenols that protect plants from unfavorable environmental conditions, enhance their tolerance to abiotic and biotic stresses, and possess multiple health-promoting effects in humans. This review summarizes various antioxidant metabolites such as organic acids, amino acids, alkaloids, fatty acids, carotenoids, ascorbic acid, tocopherols, terpenoids, hydroxycinnamic acids, flavonoids, and anthocyanins that are distributed in different citrus species. Among these antioxidant metabolites, flavonoids are abundantly present in primitive, wild, and cultivated citrus species and possess the highest antioxidant activity. We demonstrate that the primitive and wild citrus species (e.g., Atalantia buxifolia and C. latipes) have a high level of antioxidant metabolites and are tolerant to various abiotic and biotic stresses compared with cultivated citrus species (e.g., C. sinensis and C. reticulata). Additionally, we highlight the potential usage of citrus wastes (rag, seeds, fruit peels, etc.) and the health-promoting properties of citrus metabolites. Furthermore, we summarize the genes that are involved in the biosynthesis of antioxidant metabolites in different citrus species. We speculate that the genome-engineering technologies should be used to confirm the functions of candidate genes that are responsible for the accumulation of antioxidant metabolites, which will serve as an alternative tool to breed citrus cultivars with increased antioxidant metabolites.

A Comparative Study of the Chemical Composition by SPME-GC/MS and Antiradical Activity of Less Common Citrus Species.

Citrus secondary metabolites, such as terpene compounds, are very important for human health due to their bioactivity including anti-inflammatory, anti-cancer, and antioxidant effects. In this work, for the first time, the volatile chemical composition of peels and juices from four different Citrus species (C. junos, Citrus × aurantium, C. aurantium 'Bizzarria' and C. medica 'Florentina', commonly known as Yuzu jeune, Oni Yuzu, Bizzarria orange and Florence cedar, respectively) was investigated by Solid-Phase Microextraction-Gas Chromatography/Mass Spectrometry (SPME-GC/MS) technique and the antiradical activity was also examined. The results showed that limonene and γ-terpinene were the main volatile substances detected both in the juices and in the peels, followed by other minority compounds responsible for the phyto-complex of the unique aromas which characterize each individual analyzed Citrus species. Principal component analysis (PCA), performed on volatile compounds, showed both some correlation as well as a clear separation between the juice and the peel of each species. Among them, Oni Yuzu juice was found to be the richest in total polyphenols and flavonoids while its capacity to scavenge ABTS•+ and DPPH• radicals was similar to that of Yuzu Jeune and Bizzarria orange.

The Chemical Compositions, and Antibacterial and Antioxidant Activities of Four Types of Citrus Essential Oils.

Nanfeng mandarins (Citrus reticulata Blanco cv. Kinokuni), Xunwu mandarins (Citrus reticulata Blanco), Yangshuo kumquats (Citrus japonica Thunb) and physiologically dropped navel oranges (Citrus sinensis Osbeck cv. Newhall) were used as materials to extract peel essential oils (EOs) via hydrodistillation. The chemical composition, and antibacterial and antioxidant activities of the EOs were investigated. GC-MS analysis showed that monoterpene hydrocarbons were the major components and limonene was the predominate compound for all citrus EOs. The antibacterial testing of EOs against five different bacteria (Bacillus subtilis, Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and Salmonella typhimurium) was carried out using the filter paper method and the broth microdilution method. Kumquat EO had the best inhibitory effect on B. subtilis, E. coli and S. typhimurium with MIC (minimum inhibitory concentration) values of 1.56, 1.56 and 6.25 µL/mL, respectively. All citrus EOs showed the antioxidant activity of scavenging DPPH and ABTS free radicals in a dose-dependent manner. Nanfeng mandarin EO presented the best antioxidant activity, with IC50 values of 15.20 mg/mL for the DPPH assay and 0.80 mg/mL for the ABTS assay. The results also showed that the antibacterial activities of EOs might not be related to their antioxidant activities.

Diversification of mandarin citrus by hybrid speciation and apomixis.

The origin and dispersal of cultivated and wild mandarin and related citrus are poorly understood. Here, comparative genome analysis of 69 new east Asian genomes and other mainland Asian citrus reveals a previously unrecognized wild sexual species native to the Ryukyu Islands: C. ryukyuensis sp. nov. The taxonomic complexity of east Asian mandarins then collapses to a satisfying simplicity, accounting for tachibana, shiikuwasha, and other traditional Ryukyuan mandarin types as homoploid hybrid species formed by combining C. ryukyuensis with various mainland mandarins. These hybrid species reproduce clonally by apomictic seed, a trait shared with oranges, grapefruits, lemons and many cultivated mandarins. We trace the origin of apomixis alleles in citrus to mangshanyeju wild mandarins, which played a central role in citrus domestication via adaptive wild introgression. Our results provide a coherent biogeographic framework for understanding the diversity and domestication of mandarin-type citrus through speciation, admixture, and rapid diffusion of apomictic reproduction.

Volatilomic Profiling of Citrus Juices by Dual-Detection HS-GC-MS-IMS and Machine Learning-An Alternative Authentication Approach.

A prototype dual-detection headspace-gas chromatography-mass spectrometry-ion mobility spectrometry (HS-GC-MS-IMS) system was used for the analysis of the volatile profile of 47 Citrus juices including grapefruit, blood orange, and common sweet orange juices without requiring any sample pretreatment. Next to reduced measurement times, substance identification could be improved substantially in case of co-elution by considering the characteristic drift times and m/z ratios obtained by IMS and MS. To discriminate the volatile profiles of the different juice types, extensive data analysis was performed with both datasets, respectively. By principal component analysis (PCA), a distinct separation between grapefruit and orange juices was observed. While in the IMS data grapefruit juices not from fruit juice concentrate could be separated from grapefruit juices reconstituted from fruit juice concentrate, in the MS data, the blood orange juices could be differentiated from the orange juices. This observation leads to the assumption that the IMS and MS data contain different information about the composition of the volatile profile. Subsequently, linear discriminant analysis (LDA), support vector machines (SVM), and the k-nearest-neighbor (kNN) algorithm were applied to the PCA data as supervised classification methods. Best results were obtained by LDA after repeated cross-validation for both datasets, with an overall classification and prediction ability of 96.9 and 91.5% for the IMS data and 94.5 and 87.9% for the MS data, respectively, which confirms the results obtained by PCA. Additional data fusion could not generally improve the model prediction ability compared to the single data, but rather for certain juice classes. Consequently, depending on the juice class, the most suitable dataset should be considered for the prediction of the class membership. This volatilomic approach based on the dual detection by HS-GC-MS-IMS and machine learning tools represent a simple and promising alternative for future authenticity control of Citrus juices.

Transcriptional Analysis of C-Repeat Binding Factors in Fruit of Citrus Species with Differential Sensitivity to Chilling Injury during Postharvest Storage.

Citrus fruit are sensitive to chilling injury (CI) during cold storage, a peel disorder that causes economic losses. C-repeat binding factors (CBFs) are related to cold acclimation and tolerance in different plants. To explore the role of Citrus CBFs in fruit response to cold, an in silico study was performed, revealing three genes (CBF1, CBF2, and CBF3) whose expression in CI sensitive and tolerant cultivars was followed. Major changes occurred at the early stages of cold exposure (1-5 d). Interestingly, CBF1 was the most stimulated gene in the peel of CI-tolerant cultivars (Lisbon lemon, Star Ruby grapefruit, and Navelina orange), remaining unaltered in sensitive cultivars (Meyer lemon, Marsh grapefruit, and Salustiana orange). Results suggest a positive association of CBF1 expression with cold tolerance in Citrus cultivars (except for mandarins), whereas the expression of CBF2 or CBF3 genes did not reveal a clear relationship with the susceptibility to CI. Light avoidance during fruit growth reduced postharvest CI in most sensitive cultivars, associated with a rapid and transient enhance in the expression of the three CBFs. Results suggest that CBFs-dependent pathways mediate at least part of the cold tolerance responses in sensitive Citrus, indicating that CBF1 participates in the natural tolerance to CI.

Identification of the 'Haryejosaeng' mandarin cultivar by multiplex PCR-based SNP genotyping.

Most satsuma mandarin (Citrus unshiu Marc.) cultivars are difficult to identify in the seedling stage based only on morphological traits. Therefore, simple polymerase chain reaction (PCR)-based single-nucleotide polymorphism (SNP) markers were developed to specifically and rapidly distinguish the 'Haryejosaeng' cultivar, which is generally supplied to breeders of other satsuma mandarin cultivars. SNP markers were verified using high-resolution melt (HRM)-specific primers. PCR was performed to distinguish 'Haryejosaeng' from eight other satsuma mandarin cultivars using six SNP markers (P1-P6) specific for 'Haryejosaeng', with one negative control SNP primer pair. The best results were obtained using three SNP markers (P1, P2, and P5). In the multiplex PCR, markers P1, P2, and P5 yielded 165-, 150-, and 526-base pair amplicons, respectively, in 'Haryejosaeng', distinguishing it from other satsuma mandarin cultivars. The selected SNP markers were validated by HRM with HRM-specific primers. The multiplex PCR with P1/P5 and P2/P5 also identified 'Haryejosaeng' obtained from a farm growing 17 different cultivars of satsuma mandarin. Specific SNP molecular markers were determined for accurately identifying the 'Haryejosaeng' cultivar by multiplex PCR to save the time and costs associated with its supply to breeders of satsuma mandarin.

The application of artificial neural networks in modeling and predicting the effects of melatonin on morphological responses of citrus to drought stress.

Drought stress as one of the most devastating abiotic stresses affects agricultural and horticultural productivity in many parts of the world. The application of melatonin can be considered as a promising approach for alleviating the negative impact of drought stress. Modeling of morphological responses to drought stress can be helpful to predict the optimal condition for improving plant productivity. The objective of the current study is modeling and predicting morphological responses (leaf length, number of leaves/plants, crown diameter, plant height, and internode length) of citrus to drought stress, based on four input variables including melatonin concentrations, days after applying treatments, citrus species, and level of drought stress, using different Artificial Neural Networks (ANNs) including Generalized Regression Neural Network (GRNN), Radial basis function (RBF), and Multilayer Perceptron (MLP). The results indicated a higher accuracy of GRNN as compared to RBF and MLP. The great accordance between the experimental and predicted data of morphological responses for both training and testing processes support the excellent efficiency of developed GRNN models. Also, GRNN was connected to Non-dominated Sorting Genetic Algorithm-II (NSGA-II) to optimize input variables for obtaining the best morphological responses. Generally, the validation experiment showed that ANN-NSGA-II can be considered as a promising and reliable computational tool for studying and predicting plant morphological and physiological responses to drought stress.

Characterization of Volatile Organic Compounds of Healthy and Huanglongbing-Infected Navel Orange and Pomelo Leaves by HS-GC-IMS.

The Asian citrus psyllid (ACP), Diaphorina citri Kuwayama, is the only natural vector of bacteria responsible for Huanglongbing (HLB), a worldwide destructive disease of citrus. ACP reproduces and develops only on the young leaves of its rutaceous host plants. Olfactory stimuli emitted by young leaves may play an important role in ACP control and HLB detection. In this study, volatile organic compounds (VOCs) from healthy and HLB-infected young leaves of navel orange and pomelo were analyzed by headspace-gas chromatography-ion mobility spectrometry (HS-GC-IMS). A total of 36 compounds (including dimers or polymers) were identified and quantified from orange and 10 from pomelo leaves. Some compounds showed significant differences in signal intensity between healthy and HLB-infected leaves and may constitute possible indicators for HLB infection. Principal component analysis (PCA) clearly discriminated healthy and HLB-infected leaves in both orange and pomelo. HS-GC-IMS was an effective method to identify VOCs from leaves. This study may help develop new methods for detection of HLB or find new attractants or repellents of ACP for prevention of HLB.

Target-Genes Reveal Species and Genotypic Specificity of Anthocyanin Pigmentation in Citrus and Related Genera.

BACKGROUND: Anthocyanin pigmentation characterizes a number of tissues of Citrus and its relatives. The gain and loss of pigmentation is intriguing and is inherited variously among species. METHODS: Citrus germplasm was used to investigate the anthocyanin pigmentation of tissues never before considered, including stamen, style and stigma, and of young leaves, petals, rind and flesh of 28 genotypes belonging to 14 species. Citrus genotypes encompassed citron, lemon, sweet orange, lime, and Citrus relatives included Microcitrus, Murraya, and Severinia. A relative qRT-PCR analysis was carried out on the structural and regulatory genes: phenylalanine ammonia-lyase (PAL), chalcone synthase (CHS), chalcone isomerase (CHI), flavanone 3'-hydroxylase (F3H), dihydroflavonol 4-reductase (DFR), anthocyanidin synthase (ANS), uridine diphosphate glucose flavonoid glucosyl-transferase (UFGT), glutathione S-transferase (GST), Ruby and Noemi. Image analysis and a genomic approach were employed to evaluate how the red pigmentation is inherited among tissues and species. RESULTS: Pigmentation of young leaves and petals is specific to citron and its hybrids. Ruby controls the pigmentation of petals, but not of leaves. The red color of the rind and flesh is a trait that particularly characterizes a diversity of sweet oranges, citron hybrids and Citrus relatives. Color expression depends on external factors and also on developmental stage. The coloration of stamen and style is citron-specific, while a red stigma is exclusive to Moro orange and its hybrids. CONCLUSION: It is hypothesized that there is a relationship among Citrus species and genes controlling anthocyanin pigmentation.

A novel fluorescence sensing strategy based on nanoparticles combined with spectral splicing and chemometrics for the recognition of Citrus reticulata 'Chachi' and its storage year.

BACKGROUND: The fluorescence sensing method has been increasingly applied in food quality control because it is fast and sensitive. However, its application in quality evaluation is challenging. Using Citri Reticulatae Pericarpium (CRP; dried mandarin orange peel) as an example, we developed a simple and low-cost fluorescence sensing strategy based on nanoparticles combined with spectral splicing and chemometrics for quality evaluation. This method can recognize Citrus reticulata 'Chachi' (CRC) from other CRP cultivars and further identify the storage year. RESULTS: Nanogold particles and cadmium telluride quantum dots were selected as nanosensors and mixed with aqueous extracts of CRP separately to produce fluorescence quenching spectra. Then, a simple spectral splicing procedure was applied to obtain spliced spectra comprising different combinations of the self-fluorescence and fluorescence quenching spectra of CRP samples. With the aid of partial least-squares discriminant analysis, the new strategy achieved recognition rates of 100% in distinguishing CRC samples from other CRP samples, as well as recognition rates of 100% for the training set and 98.04% for the prediction set in the discrimination of the storage year of CRC. The recognition mechanism is dominated by interactions between the nanoparticles and the fluorescent components in the CRP samples, but other components also have concurrent effects. CONCLUSIONS: This novel fluorescence sensing strategy not only provides a new tool for the quality evaluation of CRC but also has good prospects for the authentication and traceability of other foods and herbs. Crucially, the developed method is convenient, simple and effective. © 2020 Society of Chemical Industry.

Genome-wide analysis of short interspersed nuclear elements provides insight into gene and genome evolution in citrus.

Short interspersed nuclear elements (SINEs) are non-autonomous retrotransposons that are highly abundant, but not well annotated, in plant genomes. In this study, we identified 41,573 copies of SINEs in seven citrus genomes, including 11,275 full-length copies. The citrus SINEs were distributed among 12 families, with an average full-length rate of 0.27, and were dispersed throughout the chromosomes, preferentially in AT-rich areas. Approximately 18.4% of citrus SINEs were found in close proximity (≤1 kb upstream) to genes, indicating a significant enrichment of SINEs in promoter regions. Citrus SINEs promote gene and genome evolution by offering exons as well as splice sites and start and stop codons, creating novel genes and forming tandem and dispersed repeat structures. Comparative analysis of unique homologous SINE-containing loci (HSCLs) revealed chromosome rearrangements in sweet orange, pummelo, and mandarin, suggesting that unique HSCLs might be valuable for understanding chromosomal abnormalities. This study of SINEs provides us with new perspectives and new avenues by which to understand the evolution of citrus genes and genomes.

Effect of fruit maturity on volatiles and sensory descriptors of four mandarin hybrids.

Mandarins (or tangerines) are mainly consumed as fresh fruits due to the ease of peeling and desirable flavor. Sweetness, acidity, and flavor of mandarin are the most important criteria for consumer preference. The objective of this study was to evaluate the effects of harvest date on sensory and chemical components of four mandarin cultivars (Murcott, 411, Temple, and 'LB8-9' Sugar Belle®). Volatiles were extracted from the headspace of juice samples with solid phase microextraction (SPME) and analyzed using gas chromatography-mass spectrometry (GC-MS). The optimum harvest window for eating quality of 411 was late January to mid-February (soluble solids content [SSC]/titratable acidity [TA]: 11.3 to 14.0), Sugar Belle® fruits were best tasted when harvested from mid- to end of January (SSC/TA: 14.1 to 16.1), and February was the best month for harvesting Murcott (SSC/TA: 13.10 to 18.0) and Temple (SSC/TA:10.3 to 12.50). Sensory perception of sweetness, ripeness, and juiciness increased as SSC/TA increased while sourness and bitterness decreased. Pumpkin flavor, an indicator of overripe fruit, was mainly noticed late in the season. Tangerine flavor tended to decrease, whereas fruity-noncitrus flavor tended to increase with fruit maturity. Monoterpenes were the most abundant volatiles and tended to decrease with fruit maturity, whereas alcohols, esters, and aldehydes increase. Aldehydes, esters, and alcohols were positively correlated with sweetness, ripeness, juiciness, and fruity characteristics, and negatively with sourness and bitterness. On the other hand, monoterpenes were positively correlated with bitterness and tangerine flavor, and negatively correlated with sweetness and fruity-noncitrus flavor. The highest number of esters was found in Temple, whereas Murcott and 411 were high in aldehydes.

Storage Behavior and Cryopreservation of Citrus cavaleriei, an Endangered, Cold-resistant Species of Northeast India with Exceptionally Large Seeds.

BACKGROUND: Ichang Papeda (Citrus cavaleriei H.Lév. ex Cavalerie) is a wild and endangered species of NE India that requires urgent preservation of its genetic resources. OBJECTIVE: To characterize the storage physiology of the seeds and to cryopreserve the embryo and embryonic axis (EA). MATERIALS AND METHODS: The morphology and storage physiology of the seeds were determined, and the cryopreservation of embryos and EA attempted using various techniques (viz., air desiccation-freezing, vitrification and encapsulation-dehydration). RESULTS: Weighing up to 4 g, seeds of Citrus cavaleriei are the largest known in the genus. Based on estimates using the seed coat ratio - seed mass (SCR-SM) model there was a very high probability of seed desiccation intolerance, which was validated physiologically; seeds lost vigour on drying below 30% moisture content (MC) and no seeds germinating after drying to <12% MC. Embryos and EAs could be air dried to 25-30% MC and cryopreserved with c. 50% survival. In contrast, EA optimally exposed to PVS2 (20 min) or encapsulated, sucrose pretreated (0.5 M, 24 h) and dehydrated (6 h) had c. 40% survival after cryopreservation. CONCLUSION: Citrus cavaleriei produces large, recalcitrant seeds that can, nevertheless, be cryopreserved as embryos or isolated EA after air drying to c. 25-30% MC; encapsulation-dehydration and vitrification provide alternative options for the cryopreservation of EA.

Simultaneous quantitative analysis of nine constituents in six Chinese medicinal materials from Citrus genus by high-performance liquid chromatography and high-resolution mass spectrometry combined with chemometric methods.

In this study, we aim to determine the chemical constituents of six Chinese medicinal materials from the Citrus genus using high-performance liquid chromatography and high-resolution mass spectrometry. Eight flavonoids and one coumarin were identified and further quantified as marker substances by high-performance liquid chromatography method. The separation was performed on an Agilent TC-C18 column with 0.1% formic acid and acetonitrile as the mobile phase under gradient elution. The analytical method was fully validated in terms of linearity, sensitivity, intra- and inter-day precision and repeatability, limit of detection, limit of quantitation, and recovery. It was subsequently applied to evaluate the quality of 103 batches of the Chinese medicinal materials from the Citrus genus. In addition, the principal constituent analysis was used to compare the samples of different species from the Citrus genus leading to successful classification of the samples in accordance with their origins. It was found that the contents of nine constituents varied greatly in different ripening stages and varieties of the samples from the Citrus genus. In addition, neoeriocitrin and 5,7-dimethoxycoumarin were determined as two unique constituents of 'Zhiqiao' and 'Foshou', respectively. In conclusion, this study provides a chemical basis for quality control of Chinese medicinal materials from the Citrus genus.

Mineral Content of the Pulp and Peel of Various Citrus Fruit Cultivars.

The aim of the study was to compare the mineral content between the peel and the pulp of citrus fruits and to determine which citrus fruit, among orange (Citrus sinensis), pomelo (Citrus maxima), mandarin (Citrus reticulata Blanco), lemon (Citrus limon), key lime (Citrus aurantifolia), and red, yellow, or green grapefruit (Citrus paradisi), is the richest in minerals. The research material consisted of fresh citrus fruits belonging to the genus Citrus L in the family Rutaceae. The fruits were purchased at a supermarket at one time. To prepare laboratory samples, each fruit was cut in half, and one half was homogenized, treating the sample as a whole (peel + flesh), while the other half was peeled and the pulp (F) and peel (P) were homogenized separately. To determine the content of minerals (Na+, K+, Ca+2, Mg+2, Fe+2, Zn+2, Cu+2, Mn+2, and Se+2), the samples were mineralized and analyzed using an Analytik Jena PlasmaQuant PQ 9000 inductively coupled plasma optical emission spectrometer. The content of macro- and micronutrients in the peel of most of the fruits far exceeded their quantity in the pulp. Oranges and pomelos are the fruits richest in iron and copper, so they could be recommended in cases such as hemoglobin production disorders resulting from a deficiency of these elements. Oranges can additionally enrich the body with potassium, phosphorus, and manganese, while lime can be a source of calcium, zinc, sodium, and especially potassium. It should also be noted that all citrus fruits are a very valuable source of potassium, which is needed to ensure the water and electrolyte balance.