Citrus is a valuable fruit crop worldwide. It not only provides essential minerals and vitamins but is also of great commercial importance. Conventional research has contributed a lot to the improvement of this fruit plant. Numerous improved varieties have been developed through conventional breeding, mutational breeding, polyploidization and tissue culture yet pathogens continue to emerge at a consistent pace over a wide range of citrus species. Citriculture is vulnerable to various biotic and abiotic stresses which are quite difficult to be controlled through conventional research. Biotechnological intervention including transgenesis, genome editing, and OMICS offers several innovative options to resolve existing issues in this fruit crop. Genetic transformation has been established in many citrus species and transgenic plants have been developed having the ability to tolerate bacterial, viral, and fungal pathogens. Genome editing has also been worked out to develop disease-resistant plants. Likewise, advancement in OMICS has helped to improve citrus fruit through the knowledge of genomics, transcriptomics, proteomics, metabolomics, interactomics, and phenomics. This chapter highlights not only the milestones achieved through conventional research but also briefs about the achievements attained through advanced molecular biology research.
- conventional research
- genome editing
Citrus is one of the most diverse members of the family
Citrus is being widely cultivated in the sub-tropical, tropical, and temperate regions of the world. Global citrus production is 157 million tons per annum from an area of 15 million hectares. About 50% of the area and production of citrus is being contributed by the northern hemisphere of the world. China (28%) and the Mediterranean regions (25%) are the major contributors to global citrus production followed by Brazil (13%). China is leading in grapefruit and mandarin production. Among Mediterranean countries, Spain is leading in global citrus production (6 million tons) including mandarins, oranges, limes, lemons and exports. Brazil is leading in global fresh sweet orange and its juice production. Mexico and India are major lime producers . Pakistan’s share in global citrus production is quite low (1.6%) which includes mandarin and sweet oranges as major species whereas limes, grapefruit, and lemons have less production and are dealt as minor species. The global citrus industry is facing many biotic (Citrus greening, Citrus tristeza virus, sudden death, citrus canker, and Phytophthora) and abiotic stress (salinity, drought, and temperature fluctuations) which have a direct impact on fruit crop production and yield .
2. Origin and diversification
Among citrus genetic resource centers, major collections are found in the USA, China, Spain, France, Japan, and Brazil where a large number of wild species, their relatives, old and new varieties, and breeding lines are conserved . In Pakistan, citrus genetic resources are conserved mainly in the field as orchards or germplasm units in Sargodha, Faisalabad, and Sahiwal in different academic and research institutes.
3. Conventional approaches for crop improvement
Citrus breeders have been using different approaches for their improvement including conventional breeding, mutation breeding, polyploidization and
3.1 Classical and mutation breeding
Though conventional breeding has limitations in citrus due to its complex reproductive behavior, nucellar embryony, long juvenility, sterility, sexual incompatibility, and endogametic depression [9, 10]. However, still, many hybrids have been developed by conventional breeding and recovered using
Mutation breeding has played a pivotal role in fruit crop improvement including citrus and has developed several mutants with improved phenotypic and genotypic traits . Spontaneous or induced mutants do not have intellectual property rights (IPR) related issues that have to be faced in the case of conventional breeding and transgenics . Both spontaneous and induced mutations have enhanced genetic diversity in existing varieties and have provided the raw material for making selections for the novel horticultural traits . About 3365 mutant varieties belonging to 170 plant species have been released including citrus and 20 other fruit species . Among continents, Asia is leading with 2052 mutants released followed by Europe (960 mutants). Among countries, China (817), Japan (479), India (341) and the USA (139) are leading in mutant development whereas Pakistan has released 59 mutants in different crops . In citrus, a total of 15 mutants have been released since 1970 including mandarins and clementine (6), sweet oranges (6), grapefruit (2), and lemon (1) . Pakistan has registered a single mutant variety in citrus, a Kinnow mandarin induced mutant having less number of seeds and named it as “NIAB Kinnow” in 2017.
The rate of spontaneous mutations has been much higher in citrus compared with other fruit crops, however, due to random genetic alterations it has been difficult to identify and utilize such mutants [16, 17]. Induced mutations using different irradiation sources including gamma rays (physical mutagens) and various chemical compounds have enhanced the frequency of genetic variability. Physical mutagens or ionizing radiations have been more commonly used for inducing genetic diversity, chromosomal aberrations, and point mutations. About 70% of the mutant varieties have been developed using physical mutagens . In fruit crops, physical mutagens have altered key horticultural traits like seedlessness, precocious bearing, and dwarfism [19, 20, 21]. Other traits include fruit ripening time, fruit skin and flesh color, fruit aroma, self-compatibility, pathogen resistance, and fertility restoration in sterile hybrids. Among physical mutagens, gamma rays have been most used for the development of mutants due to their shorter wavelength and greater penetration , however, the ion beam is getting more popular and is being widely used due to its greater efficiency and precision compared with gamma rays . Among chemical mutagens, ethyl methanesulfonate (EMS), diethyl sulfate (DES), ethylenimine (EI), sodium azide (SA) has been most frequently used for reliable and gene-specific mutations. A comprehensive review of the role of mutation breeding in mandarins and lime crop improvement has been discussed [24, 25]. Irradiation and chemical mutagen treatment of seeds and budwood have been commonly used by breeders for inducing variation followed by selection and clonal propagation. Mutation breeding applications have been reported in different fruit crops including papaya, peach, pear, grapes, sweet and sour cherries, banana, plum, almond , apple , and rough lemon . Natural bud mutants include Washington navel orange, most of the early grapefruit varieties including Marsh, Foster, Shamber, Salustiana sweet orange, and Shamouti orange have originated as bud sports. Now there are several commercial seedless varieties including Daisy SL, Kinnow SL, Fairchild SL, and Tango that have been developed from their seedy parents through mutation breeding and are being commercially cultivated . Other commercial mutants in citrus include sweet orange varieties Jin Cheng , Kozan , and NIAB Kinnow mandarin . In grapefruit, Rio Red and Star Ruby are two induced mutants that have obtained commercial significance due to their better fruit color and seedlessness, respectively . These are leading grapefruit varieties in Texas, USA. Star Ruby is the leading variety in Turkey, South Africa, Australia, and Spain. Rio Red is the main cultivar in China, India, and Argentine . In Pakistan, Shamber is the main grapefruit variety that needs to be replaced with other potential candidate varieties like Star Ruby, Rio Red, and Flame . Conclusively mutation breeding has shown its enormous potential in citrus crop improvement particularly in economically important horticultural traits. However, it is a slow and long-term process and takes more time to detection of desirable phenotypic variability. Utilization of modern breeding tools including molecular markers, advanced methods for phenotypic screening like Targeting Induced Local Lesions IN Genomes (TILLING) , using targeted mutagenesis and genome editing technologies  like Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR/Cas9) could enhance the efficiency and cost-effectiveness of variety development having novel traits in citrus.
3.2 Ploidy manipulations
Polyploid organisms have a greater number of chromosomes compared with their diploid progenitors. Breeders have utilized polyploidization for the investigation of inheritance patterns in genes of interest. Polyploids have shown tremendous success in nature due to higher heterozygosity, less inbreeding depression, and more tolerance to biotic and abiotic stress conditions compared with their diploid progenitors [36, 37, 38]. The duplicated genes may evolve new functionalization during evolution . Polyploids have been reported in many fruit crops including grapes, apples, strawberry, and citrus [40, 41], however, the frequency of spontaneous polyploid events is quite low and breeders prefer to induce hyperploidy using different chemicals.
Among chemical mutagens, colchicine is mostly used for the induction of polyploids due to its more reliability, higher efficacy, and cost-effectiveness. Colchicine is an alkaloid derived from
Members of the subfamily
In vitroculture: somatic hybridization
Plant tissue culture tools offer advantages related to efficient regeneration, propagation, and crop improvement in citrus and other horticultural crops. Endosperm cultures have been used for the development of triploids in citrus . In interploid and wide hybridizations, the progeny may be sterile or have underdeveloped or shriveled seeds with viable embryos. The embryo rescue technique has shortened the breeding cycle and many plants have been recovered from these embryos through
Another highly promising and most widely used approach is somatic hybridization which is utilized to overcome sexual incompatibility and to enhance genetic variability by combining nuclear and organelle (chloroplast and mitochondria) genomes followed by their characterization for hybrid confirmation and variability assessment . The organelle genomes are known to encode genes related to photosynthesis and male sterility and new hybrids could be developed having novel genetic recombinations. Somatic hybrids may be developed through electrofusion of plant embryogenic protoplasts predominantly with mesophyll protoplasts. The plant progeny having nuclear origin could be characterized and separated using flow cytometry and molecular markers .
Protoplast fusion of distantly related citrus species bypasses the biological barriers and develops allopolyploids that could not be obtained through classical breeding. Somatic hybridization is an important tool and has been widely used in citrus scion and rootstock breeding. The first intergeneric allotetraploid somatic hybrid of Trovita sweet orange and
Polyploids developed through somatic hybridization have also shown enhanced tolerance to abiotic and biotic stress conditions. Allotetraploids of cv. FlhorAG1 (FL-4x) developed by somatic hybridization of diploid
4. Innovative approaches/technologies
Since the advent of recombinant DNA technology, transgenesis has proved its significance, and 190.4 million hectares of transgenic crops were grown in more than 29 countries in 2019. They have significantly contributed to food security, climate change mitigation, sustainability thus uplifted the lives of 17 million biotech farmers worldwide. The first transgenic plant was developed in the 1980s and was available as commercial food in the 1990s. More than 400 transformation events have been approved so far wherein 356 events have been approved for crop plants, 23 for ornamentals, 22 for fruit plants, and 2 for trees. Hence a wide range of plant species (maize, cotton, canola, papaya, rice, tomato, sweet pepper, squash, popular, petunia, sugarcane, alfalfa, and citrus) have been engineered for various valuable traits i.e. insect resistance, herbicide tolerance , abiotic stress tolerance, improved nutritional value, and disease resistance. In addition to nuclear transformation plastid genome has also been targeted and has proved to be of more value as multiple genes can be introduced at a specific target site, the transgene is contained owing to maternal inheritance, and hyperexpression of the transgene, etc. [68, 69].
Citrus is an economically important fruit crop worldwide. It not only provides essential minerals and vitamins but is also of great commercial importance. Conventional research has contributed a lot to the improvement of this fruit yet serious problems are evolving which are difficult to tackle with these conventional approaches . Juvenility, sexual incompatibility, high heterozygosity, apomixes, large plant size, and nucellar polyembryony, and certain other biological limitations hinder the improvement of these plant species through conventional breeding. Genetic manipulation through advanced innovative techniques is a potential approach to improve crop plants as well as fruit species. Though citrus species are recalcitrant to transformation and subsequent rooting, yet consistent efforts by the researchers have resolved these bottlenecks and proficient protocols have been established. Likewise, various transformation methods i.e. Agrobacterium-mediated transformation , biolistic transformation , and chemically assisted uptake of recombinant DNA by protoplasts  have been attempted to introduce genes of agronomic value as well as to strengthen it against bacterial, viral, and fungal pathogens (Figure 1).
Genetic manipulation of vegetatively propagated crops like citrus is very tricky as the expression of transgenes over a long period during numerous cycles of graft propagation should be stable.
The first attempt to produce transgenic citrus was made in the 1980s wherein protoplast transformation was attempted but it was not successful. The first authentic report was published by Kaneyoshi et al.  who reported transforming NPT II and GUS genes into trifoliate orange through Agrobacterium. Epicotyls of the aforementioned citrus species were used to transform with the selectable marker gene as well as reporter gene and more than 25% transformation efficiency was achieved. Likewise, Yao et al.  reported the first successful transformation through gene gun. They transformed tangelo (
Since genetic transformation has successfully been performed in different species and hybrids including Carrizo citrange, Washington naval orange,
The biolistic transformation has also been performed successfully in tangelo (
Since transgenic technology is the most reliable intervention having the massive potential to improve the citrus crop. The introduction of alien genes is only possible through this technology so any of the desired traits can be engineered. Recent research indicates that citrus growing farmers are facing severe problems due to biotic and abiotic stresses i.e. salinity, cold, drought, and diseases. Hence, the development of improved citrus varieties is direly needed to get a quality crop. Various citrus species have been engineered with alien genes to combat abiotic stresses including salinity and drought. Expression of HLA2 gene, isolated from yeast imparted salinity tolerance and resultant transgenic plants were able to tolerate a higher level of the salts as compared with non-transformants . PsCBL and PsCIPK derived from
Citriculture is prone to be infected by a wide range of diseases that are controlled by chemicals, a drastic non-environmentally friendly strategy. Different types of viral, bacterial, and fungal pathogens infect these plants resulting in drastic losses to crop production and quality of the produce. A range of transgenic citrus lines have been developed varying from fully resistant to susceptible to the diseases. Coat protein (p25) from the CTV (Citrus Tristeza Virus) was expressed in Mexican lime and 33% of the transgenic plants were found to be resistant, neither symptoms appeared nor the viral load was detected. Accumulation of siRNA (small interfering RNA) in the transgenic lines resulted in resistant phenotype and plants were able to withstand viral infection . Expression of viral coat protein (part of
Phytophthora is a noxious fungal pathogen that has been reported to infect a wide range of citrus species. Among these
Transgenic technology has also played a pivotal role to tackle another noxious disease in citrus i.e. Huanglongbing (HLB) which is supposed to be caused by phloem-restricted Gram-negative bacteria;
Another economically important disease, the citrus canker has also been addressed through transgenesis resulting in enhanced tolerance against
4.2 Genome editing
Genome editing through CRISPR-Cas9 has emerged as a breakthrough technology for the precise modification and manipulation of targeted genomic DNA. It has extensively been exploited by several research groups  and certain successes have been achieved. Three major sequence-specific engineered nucleases that have so far been used for genome editing are CRISPR-Cas (clustered regularly interspaced short palindromic repeats), TALENs (transcription activators such as effector nucleases), and ZFNs (zinc finger nucleases). Among these, the CRISPR-Cas9 editing system has been established in many plant species through gene activation, repression, mutation, and epigenome editing in wheat, rice, maize, tomato, potato, carrot, apple, grape, and citrus. Even a few of the genome-edited crops have been approved for commercial cultivation. Through this technology, field crops as well fruit crops can not only be manipulated for improved agronomic traits but can also be manipulated for improved nutritional value .
For citrus, genome editing research is at infancy, yet few successes have been achieved by editing its genome for enhanced resistance against diseases. The CRISPR-Cas9 system was firstly used to target the
Most of the research studies were carried out to target the
A marker gene for pathogen triggered immunity (CsWRKY22) was knocked out in Wanjincheng orange and the resultant mutant plant showed a decreased level of susceptibility to the canker disease . In addition to the CRISPR-Cas9 system, another improved genome editing system (CRISPR/Cas12a (Cpf1) has also been used to edit the
5. Multi-Omics technology: An integrated approach and useful strategy for the improvement of the citrus crop
MultiOMICS including genomics, transcriptomics, proteomics, metabolomics, interactomics, and phenomics approaches have massive potential for citrus improvement just like other crops and fruits. In all disciplines of OMICS, various techniques can be utilized for genome analyses, transcripts, proteins, metabolites, and interactions between different molecules to indicate the molecules which may result in crop improvement.
The field of genomics is a highly applicable part of Omics technologies. It is based on sequencing technologies and the analysis of subsequent genome sequences. Many advanced techniques in genomics for example sequence determination DNA, marker-assisted selection, and transition from marker-assisted selection to genomic selection assist in quick varietal development. Genome sequencing technologies have brought about a revolution in the field of biology. It has also transformed the citrus breeding that helps to understand a relationship between the genetic makeup and response towards various abiotic and biotic stresses like Alternaria brown spot .
A specific pathotype of
Usually, the female parent transmits the 2
In citrus plant, molecular markers are linked to some agronomic traits, e.g. SSR markers are linked to Citrus tristeza virus resistance from
About 40 mandarin genotypes (susceptible and resistant) were tested by the SNP08 marker and were used as breeding parents. An ultimate association was observed between response to
5.2 Proteomics and metabolomics approaches
Proteomics is the comprehensive analysis of all the proteins found in a cell. It includes the identification of proteins, their location in the cell, their interactions with other proteins and other biological components in the cell, and most importantly post-translational modifications that a protein undergoes in the cell . Metabolites are referred to as the last product of any biological activity in a cell and are found in very small quantities . Metabolites are small molecules including intermediates of various metabolic reactions, signaling molecules, hormones, and other regulatory products found in a cell. Hence, metabolomics is defined as the study of metabolites of a cell [117, 118]. It is estimated that around five thousand metabolites are found in any cell depending upon the physical and chemical complexity of that cell .
Huanglongbing (HLB) is considered one of the most devastating citrus diseases that affect not only the production but also the quality of citrus fruit and its juice. Using a combination of proteomics and metabolomics approaches it was found that in symptomatic fruit, the expression of proteins found in the cytoplasm for glycolysis, in mitochondria involved in the tricarboxylic acid (TCA) cycle, and in chloroplasts for the synthesis of amino-acids was downregulated. Similar downregulation was observed for genes involved in terpenoid metabolism for example valencene, limonene, 3-carene, linalool, myrcene, and aterpineol in fruit found on infected trees. Similar phenomena were observed for sucrose and glucose. Hence, the off-flavor found in symptomatic fruits was linked to the downregulation of the above genes and a decrease in the levels of the abovementioned secondary metabolites .
In another study, comparative iTRAQ proteomic profiling was carried out using the fruits of sweet orange which was grafted on sensitive and tolerant rootstocks infected by CaLas. The results showed that symptomatic fruit on sensitive rootstock exhibited a greater number of differentially expressed (DE) proteins as compared to the healthy fruit on a similar rootstock. It was also found that the expression level of various defense-related proteins was reduced in symptomatic fruit on sensitive rootstock, particularly the proteins related to the jasmonate biosynthesis, is signaling, protein hydrolysis, and vesicle trafficking. Hence, it was concluded that the down-regulation of these proteins is likely to be linked with the sensitivity of citrus to the CaLas pathogen .
5.3 Interactomics and metabolomics and phenomics
Interactomics bears a broad scope as it may cover a complete set of interactions in a cell . It covers every type of interaction among interacting molecules including proteins and other molecules. It is a well-known fact that the Protein–protein interactions are major of all cellular processes .
To designate the complete phenotype of a plant, the term phenome is used. Similarly, a phenotype encloses a group of traits that are liable to be distinguished either by utilizing modern science analytical techniques or by a naked eye evaluation. These traits can also be attributed to being an interaction between external factors (environment) and Genotype. David Houle also termed phenomics as the collection of data from varying backgrounds and dimensions in a single entity . Phenomics involves both “extreme phenotyping,” referring to a comprehensive selection of a wide range of valid and correct phenotypes, and “phenome analysis” indicating towards an analysis of specimen and correlation between syndication of genotype and phenotype.
Plant phenomics utilizes screening of large populations to analyze genetic mutations found in the population for a specific trait (drought, salinity, or high-temperature stress tolerance). Various types of imaging techniques are employed in the phenotyping of plants for various growth and developmental processes. The techniques include visible-light imaging , Thermographic imaging , Hyperspectral imaging, Chlorophyll fluorescence, X-ray, MRI, PET .
Using the phenomics approach and tools, we can study the traits regarding plant growth, leaf growth, root growth, and architecture of soil/root interaction, etc. This extensive use of phenomics and its integration into OMICS is the need of the hour to combat food security issues and overcome adverse effects of climate change on crop production.
Conventional research has played a pivotal role in the improvement of citrus. Enhanced heterozygosity has helped in the development of genetically diverse germplasm in most of the citrus species and numerous varieties have been released for commercial cultivation. However, with the advent of modern biotechnological tools, the period involved in crop improvement through indirect mutagenesis and polyploidization could be further reduced and enhancing cost-effectiveness. Transgenic technology and OMICS have great potential to improve this fruit crop. MultiOMICS, integrative-OMICS, or panOMICS technologies may result in better crops having better agronomic traits, enhanced yield potential, and less prone to insect pests. It will ultimately lead towards food security and poverty alleviation. Various OMICS technologies have been used for crop improvement, yet their integrated use will further strengthen the application of this robust technology. Still, there are many challenges associated with tolerant varieties which need to be fine-tuned. Moreover, three thousand reports of enhanced drought and salinity tolerance in wheat, sorghum, canola and rice are present but none of them is in use by farmers. A fundamental reason for this is that salinity and drought are complex multigenic traits. So, to induce tolerance in plants every gene needs to be fine-tuned precisely. However, their evaluation in the field is a long way, and distribution at the commercial level is also a hurdle in their production.