Preface

Citrus is a nutrient-rich fruit crop, predominantly cultivated in tropical and subtropical regions of the world. The *Citrus* genus belongs to the Rutaceae family and consists of a variable number of species due to the admixture of wide morphological diversity, intra- and interspecific sexual compatibility, apomixis, and spontaneous mutations. Citrus fruits are highly nutritious and beneficial for health due to the presence of bioactive compounds that have antioxidant, antitumor, antiinflammatory, and blood clot-inhibiting characteristics. However, citrus production and quality are challenged with several biotic and abiotic problems. Conventional research has played a pivotal role in the improvement of citrus and the introduction of novel biotechnological approaches reduces the time involved in the development of varieties and fixes problems where traditional approaches have failed. Further, transgenic technology and omics approaches have great potential to improve this fruit crop and address biotic as well as abiotic problems.

*Citrus - Research, Development and Biotechnology* consists of fourteen chapters that are divided into three sections. Section I, "Citrus Genealogy, Classification, and Biotechnology", consists of Chapters 1 through 3. In Chapter 1, Dr. Khan highlights citrus genealogy, production, and crop management. Further, it demonstrates the nutritional and nutraceutical importance of citrus and the biotechnology interventions to improve citrus. In Chapter 2, Dr. Sindhu et al. examine the horticultural classification of *Citrus* cultivars and the sexual and asexual means through which these varieties have been evolved. In Chapter 3, Dr. Khan et al. describe the milestones achieved in citrus improvement employing conventional approaches as well as the achievements made through biotechnology interventions.

Section II, "Citrus Biotic and Abiotic Stress Management", consists of Chapters 4 through 8. In Chapter 4, Drs. Daniele and Etienne explain the importance of weather conditions in cropping systems and substantiate how pathogen and disease spreading is managed through the structural features of the cocoa-based agroforestry system (CBAFS) in the humid forest zones of Cameroon. In Chapter 5, Dr. Lahlali et al. describe two prevailing viroids—Citrus Exocortis Viroid (CEVd) and Hop Stunt Viroid (HSVd)—and stress the use of transcriptomic and proteomic approaches to fully analyze and understand the mechanisms of host-pathogen interactions. In Chapter 6, Dr. Iftikhar et al. highlight that the indexing of diseases caused by a virus and virus-like pathogens is essential for producing disease-free citrus nurseries. Further, they briefly describe the commonly used biological, serological, and molecular tests for the detection of citrus viruses and virus-like pathogens. In Chapter 7, Drs. Caicedo and Villamizar review the main structural and functional features of bacterial responsible for spreading disease and causing symptoms in a susceptible host, including bacterial attachment, antagonism, effector production, quorum sensing regulation, and genetic plasticity at phenotypical and genotypical levels. In Chapter 8, Dr. Naqvi et al. explain the effects of changing climate on citrus and highlight how agronomic, breeding, and biotechnological interventions can mitigate climate change effects on citrus.

Section III, "Citrus Nutritional and Nutraceutical Importance", consists of Chapters 9 through 14. In Chapter 9, Drs. Saeid and Ahmed highlight the intervention of novel approaches in converting citrus byproducts into valuable commodities. Further, they explain the primary and secondary research findings of citrus fruits, especially lemon and pomelo, their chemical properties, composition, and use in health and cosmetic needs. In Chapter 10, Dr. Khanikor et al. discuss the potential and effective use of green pesticides, developed from citrus essential oils, for indoor and outdoor insect management. In Chapter 11, Dr. Fontana stresses the recycling processes of a huge amount of citrus waste as fruit pomace and the outstanding chemical value of peel of *Citrus sinensis*, rich in useful chemicals, such as polyphenols, polymethoxylated phenols, glycosylated flavonoids, volatile and non-volatile terpenoids, enzymes, and pectin. In Chapter 12, Dr. Minamisawa highlights the importance of yuzu seed-derived nutrients and experimentally demonstrates that limonoid and spermine improve the proportions of beneficial bacteria and their metabolites in the intestinal flora. In Chapter 13, Cavaco et al. review the application of Vis-NIRS in the assessment of the quality and ripening of citrus fruit. In Chapter 14, Demir et al. highlight the use of electrochemical methods to analyze the antioxidant ingredients in food and fruit samples.

Citrus fruits, diverse in color and size, are highly nutritious and beneficial for health because of bioactive compounds such as carotenoids, flavonoids, and ascorbic acid, which have medicinal properties. Thus, this book serves as a guide for students and professionals of biotechnology, medicinal chemistry, and food technologists.

> **Muhammad Sarwar Khan, Ph.D. and Iqrar Ahmad Khan, Ph.D.** Center Agricultural Biochemistry and Biotechnology (CABB), University of Agriculture, Faisalabad, Pakistan

Section 1

Citrus Genealogy, Classification, and Biotechnology

#### **Chapter 1**

## Introductory Chapter: Citrus for a Healthy Life

*Muhammad Sarwar Khan*

#### **1. An overview**

Citrus is an extensively produced fruit crop and is cultivated predominantly in tropical and subtropical regions of the world. The genus Citrus and related genera (Fortunella, Poncirus, Eremocitrus, and Microcitrus) belong to the family Rutaceae. Of these genera, Citrus is the widely grown genus and is well known for fruits like oranges, mandarins, lemons, limes, and grapefruits [1]. The classification of citrus is complexed however, the genus Citrus consists of more than 100 species. The number of species is variable and this species variation in a single genus is due to the admixture of wide morphological diversity, intra- and interspecific sexual compatibility, apomixis, and spontaneous mutations. However, intergenic hybrids such as citranges, citrumelos, and citrandarins, citremons, citradias, and citrumquats are also reported and are getting increasing importance. Several indigenous varieties are developed and consumed locally in specific regions. The citrus fruits are tangy with pleasant flavor and taste, a combination of sweet and sour flavors. Oranges and mandarins are predominant species of genus Citrus, marketed as fresh or processed juice [2].

The citrus plants in the orchards are confronted worldwide with increasing biotic and abiotic factors due to the changing climate. Amongst abiotic factors, fluctuating temperature and unexpected frosts are the main limiting factors whereas, bacteria, viruses, viroids, nematodes, fungi, and phytoplasmas are major biotic factors. Some factors result in a massive reduction in production and quality while others may destroy altogether the citrus industry. Citrus improvement through conventional approaches is discouraged due to the genetic and reproductive characteristics of the plant. The omics and biotechnology-based interdisciplinary interventions may allow combating such external factors and improving the health, nutritional quality of the fruit. The book, Citrus: Research and Development describe the citrus plant, the biotic as well as abiotic challenges, nutrients, and nutritional value, and nutraceutical applications to improve human health. Citrus production, management, detection, and documentation of citrus pathogens and their management, fruit nutritional quality, and potential use as nutraceutical is an interdisciplinary endeavor; therefore, it is difficult to cover all aspects of this subject in a single book. The editor of the book is conscious of the fact that there is considerable scope for improving citrus production and controlling the diseases and benefitting from the availability of chemicals of nutritional and nutraceutical importance, novel approaches for detection of such chemicals, enriching the genetic information through next-generation sequencing and improving the genome by incorporating new genes through genetic engineering and knocking out genes using CRISPR/Cas technology, and hence the information relevant to the topics is covered in the book.

#### **2. Citrus genealogy**

Citrus domesticated in Southeast Asia started several thousand years ago and was distributed to different regions of the world through ancient land and sea routes. The genealogy of the modern cultivated citrus is controversial because these are either selections from or hybrids of wild progenitors (**Figure 1**). The biological features and cultivation of Citrus have further complicated the lineage of modern species. Citrus is being cultivated either through clonal grafting or by asexual means of propagation to maintain the identified superior traits however, diversity within such populations is due to somatic mutations. Further, spontaneous mutants have been reported and are occasionally selected from limb sports or nucellar seedlings. The genetic diversity in citrus is the major impediment in the classification of the ever-increasing number of varieties. Several methods have been used to classify the citrus varieties however the methods proposed by Swingle [3] and Tanaka [4] are commonly adopted. Swingle's classification is based on native varieties rather than cultivated, and he placed two subgenera Papeda with six species and Citrus with ten species in the genus Citrus [3], and the rest as natural

#### **Figure 1.**

*The genealogy and evolution of citrus fruits. Four ancestral species namely;* Citrus reticulata*,* Citrus maxima*,*  Citrus medica*, and Citrus micrantha, depicted in the central circle, have contributed to the evolution of cultivated species. The figure is derived from Velasco and Licciardello, 2014 [8] and other published articles.*

*Introductory Chapter: Citrus for a Healthy Life DOI: http://dx.doi.org/10.5772/intechopen.99355*

hybrids of native species. Whereas Tanaka [4] classified both indigenous and cultivated varieties as species and placed two subgenera namely; Archicitrus with 111 species and Metacitrus with 48 species, the list of species was swelled to 159 [5]. The intervention of biotechnology approaches like RAPD, RFLP, AFLP, SSR, SRAP, and more recently the next-generation sequencing and pan-genomics has made it easier to determine the genealogy of citrus, extensively reviewed elsewhere [6]. The genomes of clementines, mandarins, pummelos, sweet oranges, and sour oranges have been sequenced using Sanger whole-genome sequencing method [7]. The sequence data together with earlier similar work was a step forward in elucidating the phylogenetic history of citrus domestication and highlights the genetic basis of the diversity in the colors, flavors, sizes, and aromas of citrus fruits, which could be introduced in novel varieties [8]. Another exciting and challenging work has been published, again by Wu et al. [9], where they worked out that distant genera i.e., Fortunella, Eremocitrus, and Microcitrus are a part of the citrus monophyletic group, whereas a related genus Poncirus, originally believed to be a part of Citrus group, is declared as a distinct clade based on whole-genome phylogeny. These findings have challenged the earlier taxonomic and phylogenetic developments and have warranted reformulation of the genus Citrus.

#### **3. Citrus production and management**

Worldwide, citrus is cultivated for consumption as fresh or processed fruit. Major citrus-producing countries are; China, Brazil, USA, Mexico, India, Spain, Iran, Italy, Nigeria, and Turkey. Production and consumption trends are diverse in different regions and countries however, variably 147 million tones citrus is produced, annually [10]. As citrus is grown from subtropical to tropical and the Mediterranean regions of the world, hence, its production is dependent on soil and climate conditions. The global orange production is projected to grow 3.6 million metric tons from the previous year due to favorable weather in Brazil and Mexico. Only a slight increase in production, as well as consumption, is expected for mandarins/tangerines. Unexpectedly, global grapefruit consumption and exports will rise to their highest levels in three years due to favorable weather conditions and expanded areas in China and Mexico. More than 80% of the fruit is being processed for juice production in developed countries, and the juice demand is increasing day by day. The market and consumption trends of major types of citrus fruit as well as of juice are not affected even by the COVID-19 pandemic, this is perhaps due to the perception of the consumers that citrus fruits are immunity boosters being rich in vitamin C.

In addition to climate, plant–soil interaction affects citrus production by affecting the availability of nutrients to the plants [11]. For efficient nutrient availability, the practices of controlled release of fertilizers are preferred [12]. The published data confirm that the nitrogen uptake is greater by using controlled means, compared to conventional approaches, resultantly, the plant growth is improved [13]. Hence, technology-based precision management of orchards including the application of balanced fertilizers, herbicides, and pesticides is required for sustainable citrus production. However, there are several biotic and abiotic factors including diseases that affect the citrus industry. Amongst diseases, bacterial, fungal, and viral are constant threats and cause substantial economic impact in all growing areas around the world. Citrus greening is an extremely dangerous disease, caused by different species of the bacterium Candidatus Liberibacter including Candidatus Liberibacter asiaticus, Candidatus Liberibacter americanus, and Candidatus Liberibacter africanus. Of these three species, the Candidatus Liberibacter asiaticus (CLas) has become a serious threat to the citrus industry. The disease was reported from Brazil in 2004, from Florida, the USA in 2005, in 2007 from Cuba, and molecularl detection of the disease has been reported in 2007 from Pakistan, in 2008 from the Dominican Republic; in 2010 from Mexico [14–18]. However, the disease has been reported and described in China since 2019. The disease is transmitted by the psyllid *Diaphorina citri* Kuwayama (Hemiptera: Psyllidae), commonly called Asian citrus psyllid. As CLas is difficult to culture on artificial media hence its detection is possible through polymerase chain reaction (PCR) and particularly by quantitative real-time polymerase chain reaction (qPCR) targeting the 16S rDNA gene [14]. Recently, chloroplasts provide opportunities for pathogens to directly or indirectly target 'chloroplast immunity' as these organelles are the main sites for the synthesis of precursors of phytohormones, hence are coordinating plant defense responses. As hormonal crosstalk between host and pathogens is well established. The identification of chloroplast genes targeted by the CLas effectors will open the window to control this disease. The pathogen genome has been sequenced from *Diaphorina citri* from America [19] and the genome is composed of 1231639 bp with GC 36.5% contents. The pathogen genome has been sequenced from two strains from Pakistan. The CLas genomes of two strains (PA19 and PA20) were sequenced that is comprised of 1224156 bp and 1226225 bp, respectively with an average GC content of 36.4% [20]. The genome sequence of CLas from Thailand has also been reported with total GC contents of 36.4% and 1230623 bp genome. Several genes from the Candidatus spp. have been identified that interact with the genes of the plant defense system and scientists are working to identify the plant genes regulated by these pathogen effectors. The chloroplast being the main site of phytohormone precursor synthesis provides opportunities for pathogens to target, directly or indirectly, the 'chloroplast immunity'. As hormonal crosstalk between host and pathogens is now well established hence identification and editing of chloroplast genes using CRISPR/Cas technologies hold promise to control the disease. Similarly, the canker susceptibility gene, CsLOB1, of Duncan grapefruit has been knockout. The infection by Xanthomonas citri was significantly reduced with no disease development on plants [21].

#### **4. Citrus nutritional and nutraceutical importance**

Citrus fruits, diverse in color and size, are highly nutritious. They are beneficial for health, due to the presence of bioactive compounds such as carotenoids, flavonoids, and ascorbic acid [22]. These compounds have antioxidant, antitumor, antiinflammatory and blood clotting inhibiting characteristics [23, 24]. These fruits are also a rich source of vitamins and minerals like vitamin C, A, and B-complex [25–27], vitamin A benefits skin and vision whereas vitamin B-complex like thiamin, folates, and pyridoxine are required as external sources to replenish. Of minerals, potassium, magnesium, calcium, and sodium are present in citrus from very high to low levels in citrus fruits [28]. However, zinc, iron, and manganese are present in trace amounts (**Figure 2**).

Nutraceutical is a combination of two words; 'nutrition' and 'pharmaceutical', hence the word infers that the nutraceuticals could be regulated as dietary supplements, medicine, and food ingredients. The word, 'nutraceutical' was first coined by Stephen L. DeFelice in 1989 who explained nutraceuticals as; "Food, or parts of food, that provide medical or health benefits, including the prevention and treatment of disease" [29, 30]. However, the government of Japan started approving foods with proven benefits for the general public in the 1980s, reviewed elsewhere [31]. Nutraceuticals protect against diseases developed due to nutrient deficiencies *Introductory Chapter: Citrus for a Healthy Life DOI: http://dx.doi.org/10.5772/intechopen.99355*

#### **Figure 2.**

*The citrus, a source of nutrients and nutraceuticals. Citrus species are a rich source of carotenoids, flavonoids, minerals, and vitamins that function as antioxidants, antitumor, and anti-inflammatory compounds.*

and also have physiological benefits. These are used as dietary supplements and food ingredients. Being dietary supplements, these may contain vitamins, minerals, botanical extracts, essential amino acids, Poly Unsaturated Fatty Acids (PUFA), and enzymes that are probably deficient in most of our diets. Another category of nutraceuticals is nutrient-fortified food. For example, the iron-fortified wheat flour protects the wheat-dependent population from diseases that develop due to iron deficiency. A salient example is iron deficiency anemias. Other examples include purple cauliflower and purple potatoes, having additional anthocyanin content. Golden rice and Golden potatoes as well as Provitamin-A-fortified maize are the rich sources of Provitamin-A and carotenoids. These crops could reduce the disease development in humans caused by Vitamin A deficiency diseases e.g. night blindness, xerophthalmia, prevalent in Africa and Asia. Similarly, Quinoa is of great nutritional value. Laden with fiber, vitamins, and minerals, this plant also is rich in lysine, hence its proteins are nutritionally more complete than many vegetables [32]. Thus, quinoa holds great potential as a Nutraceutical, to curb the malnutrition

rampant in many third-world countries, the likes of Pakistan, India, Nepal, Bangladesh, and several African nations.

Several plants, including food crops, have been reported that contain compounds to prevent diseases. Even in this era of rapid medicine evolution, cancer remains a major threat to the population and a leading cause of mortality in developed nations. Introduction of plants into lifestyle at an early age could reduce cancer risk up to 33%. For instance, blue maize is useful in preventing different types of cancers, such as colon cancer [33]. Several chemotherapeutic agents such as Taxol, Vincristine, and Vinblastine are derived from plants such as *Taxus brevifolia* and alkaloids of Vinca species. Nutraceuticals have also been shown to reduce the toxic effects of chemotherapeutic agents and radiation therapies [34].

Bacterial infections and the growing resistance to synthetic antibiotics is a serious concern. It has been proven experimentally that medicinal plants are effective against bacterial infections. In this era of technology development, the introduction of medicinal and nutritional traits transgenically into food crops is on the top priority of biotechnologists, hence engineering the citrus genome will be a better choice.

#### **5. Citrus improvement through biotech approaches**

Citrus fruits are highly nutritious and are beneficial for health, due to the presence of bioactive compounds such as carotenoids, flavonoids, and ascorbic acid. However, citrus production and quality are challenged with several biotic and abiotic problems. Biotech interdisciplinary interventions including transgenesis, genome editing, and OMICS could offer solutions to the issues of this fruit crop. Genetic transformation has been established in many citrus species thereby transgenic plants have been developed against bacterial, viral, and fungal pathogens. Equally, OMICS approaches; genomics, transcriptomics, proteomics, metabolomics, interactomics, and phenomics are exploited to improve the citrus fruits. Since, first attempt to manipulate the citrus genome remained unsuccessful hence, the protocols for efficient regeneration from explants like seeds, embryogenic cells, epicotyls, callus, nodal stem segments, and protoplasts, followed by transformation and selection have been optimized for different citrus species. Maximum regeneration potential has been observed in explant 'epicotyl' hence, the epicotyl has been used as an explant for the genetic transformation of citrus plants. Transgenically stable plants were recovered from Agrobacterium treated Duncan grapefruit epicotyls. The recovered plants were confirmed for transgene presence using PCR and Southern blotting techniques. Similarly, transgenic plants using epicotyl tissues as explants were developed from sweet orange, and citrange. The transformation efficiency remained as high as 93%. Hence, transgenic technology is proven as one of the most reliable interventions to genetically improve tolerance/resistance to abiotic/biotic factors in citrus [35, 36].

Using the technology, the nutrition and medicine-related traits have been successfully tailored in citrus fruits. For example, the expression of genes that encode enzymes like phytoene synthase, lycopene-β-cyclase, and phytoene desaturase of the carotenoid biosynthesis pathway have been modulated to supplement human nutrition with vitamin A and antioxidants. The Valencia orange is majorly grown for its juice but the quality of the juice is deteriorated due to the degradation of an enzyme, named thermostable pectin methylesterase (TSPME). Hence, the gene (CsPME4) that encodes TSPME was downregulated to improve the juice quality [37]. Further, an environmentally friendly technology named, 'chloroplast transformation' is available to develop transgenic plants [38–46]. This technology offers several superior advantages like overexpression of transgenes up to 70% due to

*Introductory Chapter: Citrus for a Healthy Life DOI: http://dx.doi.org/10.5772/intechopen.99355*

polyploidy at organelle and genome (plastome) levels, accumulation of functional proteins, and natural containment of transgenes since plastids are transmitted to the next generation through ovary, rather than pollens that cause horizontal gene transfer, in most of the cultivated plant species. The chloroplast genome of several citrus species has been sequenced [47–51], thus necessary genetic information of the subcellular organelle is available to develop chloroplast transformation vectors and achieve chloroplast transformation, successfully. Therefore, the development of transgenic plants through chloroplast genome engineering is a promising way forward for cost-effective production of nutraceuticals.

#### **6. Conclusions**

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 enhance cost-effectiveness. Transgenic technology and OMICS have great potential to improve this fruit crop.

#### **Acknowledgements**

The author would like to thank Miss Rimsha Riaz, a Ph.D. scholar for her contribution to the graphical presentations in the chapter.

#### **Author details**

Muhammad Sarwar Khan Center of Agricultural Biochemistry and Biotechnology (CABB), University of Agriculture, Faisalabad, Pakistan

\*Address all correspondence to: sarwarkhan\_40@hotmail.com

© 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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*Introductory Chapter: Citrus for a Healthy Life DOI: http://dx.doi.org/10.5772/intechopen.99355*

'Guanximiyou', Mitochondrial DNA Part B 2020;5(1):482-483 DOI:10.1080/2 3802359.2019.1704661

[50] Bausher MG, Singh ND, Lee SB, Jansen RK, Daniell H. The complete chloroplast genome sequence of Citrus sinensis (L.) Osbeck var 'Ridge Pineapple': Organization and phylogenetic relationships to other angiosperms. BMC Plant Biol. 2006;30(6):21 DOI:10.1186/ 1471-2229-6-21

[51] Lee M, Park J, Lee H. et al. Complete chloroplast genomic sequence of Citrus platymamma determined by combined analysis of sanger and NGS data. Hortic. Environ. Biotechnol. 2015;56:704-711 DOI:10.1007/s13580-015-0061-x

#### **Chapter 2**

## Horticultural Classification of Citrus Cultivars

*Jagveer Singh, Vishal Sharma, Kuldeep Pandey, Shahnawaz Ahmed, Manveen Kaur and Gurupkar Singh Sidhu*

#### **Abstract**

Globally, citrus fruits are grown over an area of 11.42 million ha with 179.0 million tons production. China with 82.7 m tons production is the major producer of citrus fruits followed by Brazil (18.14 m tons) and India (10.53 m tons) (FAOSTAT, 2019). All commercially used scion and rootstock cultivars belong to the genus Citrus, except kumquats, Fortunella spp., and *Poncirus trifoliata*, which are used as rootstock only all over the world. Worldwide citrus cultivars divided into four, reasonably-well-defined horticultural groups: the Sweet oranges, the mandarins, the grapefruits and the pummelos and the common acid members. The true *or 'biological'* citrus, including species of *Citrus (C. reticulata, C. maxima and C. medica)*, share certain characteristics, however, these are clearly differentiated according to the morpho- taxonomic traits. Hundreds of different citrus cultivars are available. Many varieties were chance finds from natural populations, and not the product of intentional breeding efforts. Other varieties in common use have originated from planned citrus hybridization and breeding efforts from worldwide. Most of the readers will be well acquainted with the cultivated types of Citrus scion and rootstocks. This chapter provides ripening season information for worldwide, farmers/gardeners have had success with citrus in many different regions of world where tropical/subtropical climatic conditions occur.

**Keywords:** true citrus species and its relatives, commercial cultivars

#### **1. Introduction**

Generally, there is a strong demand for citrus varieties of superior eating and processing quality. A shortage of supply of consumer-preferred varieties and high prices are the dominant market forces responsible for the revitalisation of the fresh citrus sector. The general demand is for sweet, low acid fruit, with an aromatic flavor. The shortage of supply has meant the acceptance of a range of varieties, some with marginal quality, which it is expected will have a limited commercial potential. Growing citrus in your own backyard and field can be both enjoyable and rewarding! Beautiful green foliage, fragrant blossoms, and delicious, healthful fresh fruit readily available at your doorstep make citrus excellent garden trees. You can choose a citrus variety according to the climate in your area. While this chapter provides ripening season information for worldwide, farmers/gardeners

have had success with citrus in many different regions of world where tropical/ subtropical climatic conditions occur. In general appearance and other respects, the citrus fruits of principal commercial importance fall into four, reasonably-welldefined horticultural groups: the Sweet oranges, the mandarins, the grapefruits and the pummelos and the common acid members. The true *or 'biological'* citrus fruit trees, including species of *Citrus (C. reticulata, C. maxima and C. medica)*, share certain characteristics, however, these are clearly differentiated according to the morpho- taxonomic key of Swingle [1, 2]. Wu et al. [2] found that several named genera (*Fortunella*, *Eremocitrus* and *Microcitrus*) are in fact nested within the citrus clade. These and other distinct clades that they have identified are therefore more appropriately considered species within the genus *Citrus*. The pulp vesicles contain droplets of oil, which are more abundant in Poncirus, Microcitrus, and the papedas. Fruits of the true citrus species are segmented and fruits of the genera other than *Citrus* are smaller than those of Citrus itself.

Fortunella and Eremocitrus have ovaries with three to five locules, each of which has only two ovules, whereas Citrus, Microcitrus, and Poncirus have ovaries with six to eight locules, each of which contains many ovules. Members of the true citrus fruit trees are generally cross and graft compatible with other members of the group [3–5]. Fortunella (Kumquat) trees, leaves, flowers, and fruits are generally smaller than those of Citrus. Kumquats are adapted to climates that are marginally cool for most of the other members of the subfamily Aurantioideae, they require less heat to achieve fruit maturity and have a certain level of winter dormancy [1]. Eremocitrus and Microcitrus are both endemic to the Oceania region. Both differ from Citrus in having dimorphic foliage and free stamens; however, Microcitrus has an ovary with four to eight locules, whereas Eremocitrus has an ovary with three to five locules. The cold hardiness of Eremocitrus stated in Swingle [1] and Swingle and Reece [6] is in error; Eremocitrus can probably tolerate temperatures as low as −5.5 °C, consistent with the original description of the genus in 1914 [3, 7, 8]. Microcitrus, on the other hand, is considered semixerophytic and able to withstand prolonged periods of drought [1, 6]. Trifoliate orange was considered as a mono-typic genus for many years, represented by *Poncirus trifoliata* [1], with distinctive trifoliate leaves (unique among the true citrus fruit trees) and deciduous growth habit. This gives to trifoliate oranges the highest degree of cold hardiness among the true citrus fruit trees, surpassing that of kumquats. The adaptation of Poncirus to cold conditions led Swingle [1] to speculate that the remote ancestor of the true citrus fruit trees originated in a tropical or semitropical climate. While the other genera of the true citrus fruit trees remained in these climates, *Poncirus* (or its ancestors) "migrated" to the temperate climate of Northeastern Asia, during which time it developed the adaptations to colder winters mentioned previously. In addition to cold tolerance, Poncirus exhibits many other characteristics that have been and continues to be used in citrus rootstock breeding, notably disease tolerance (including citrus tristeza virus immunity) and dwarfing. For a more complete utilization of Poncirus, the reader is referred to Krueger and Navarro [3, 9, 10]. Relatively recently, a new species, *Poncirus polyandra*, was published [11, 12], which differs from *P. trifoliata* by its larger leaves, some floral differences, and most notably, being evergreen. Perhaps, this latter characteristic is related to its habitat in Yunnan, the southernmost province of China. Clymenia is a very distinctive member of the other true citrus fruit trees. Clymenia was separated from Citrus by Swingle [1] based upon the structure of the pulp vesicles, which are short, plump, blunt, ovoid or sub-globose, sessile or very short stalked, and attached to the side walls of the 14–16 locules. **Table 1** summarizes the correspondence between the proposed classification and the former most important ones of Tanaka [13], Swingle and Reece [6], and Mabberley [22] revised by Zhang and Mabberley [14]. Commercially


#### *Horticultural Classification of Citrus Cultivars DOI: http://dx.doi.org/10.5772/intechopen.96243*


#### *Citrus - Research, Development and Biotechnology*


#### *Horticultural Classification of Citrus Cultivars DOI: http://dx.doi.org/10.5772/intechopen.96243*

**Table 1.**

 *Correspondences between the new phylogenomic classification and the former classifications of Tanaka, Swingle and Reece, and Mabberley revised by Zhang and Mabberley.* grown citrus trees such as the varieties discussed in this publication are not grown from seed but are grafted or budded onto a seedling of a rootstock variety. Varieties that are used as rootstocks provide a number of important qualities to the entire tree such as disease tolerance, cold hardiness, soil adaptation, and, to a certain degree, tree size. In the world, most citrus nurseries do not label or identify the rootstock of a tree, but they do select rootstocks that protect trees from important diseases of commercial citrus and are adapted to a range of regions and soil conditions.

### **2. Varietal groups**

Four main varietal groups are distinguished in the international market:

#### **2.1** *Citrus sinensis* **(L.) Osbeck**

Sweet orange is the main group which is used both for fresh fruit and processing. It probably originated in China but its major center of diversification is the Mediterranean Basin (**Figure 1**). Major cultivars in this group are classified as navel oranges (Washington Navel, Navelina, Navelate, Powell, Rhode Navel, Cara Cara), blonde oranges (Shamouti, Valencia Late, Hamlin, Pineapple, Trovita, Salustiana, Delta Valencia, Pera), and blood oranges (Tarocco, Moro, Sanguinelli, Maltese).

#### *2.1.1 Navel oranges*

**Washington (Riverside, Bahia, Baia or Baiana):** Is considered to be a limb sport of a variety 'Selecta' in Bahia, Brazil. Tree medium in size and vigor, crown round topped, anthers are without pollen. Rind medium thick tender flesh deep orange, firm less juicy, rich in flavor and taste. Processing quality poor, ships and stores well. Seedless.

**Figure 1.** *Sweet orange varieties.*

#### *Horticultural Classification of Citrus Cultivars DOI: http://dx.doi.org/10.5772/intechopen.96243*

**Navelencia:** In growth characters is less vigorous than Washington. Flesh light in color (as rind) firm more juicy, flavor good more than Thompson. Fruit matures earlier than Washington and after Thompson, and hangs well on tree, almost seedless.

**Thompson:** Limb sport of Washington. Tree compact semi dwarf type, less in vigor than Washington. Rind and flesh less colored, rind smooth glossy pitted finely, flesh firm more juicy, taste and flavor good. Ripens early, seedless.

#### *2.1.2 Blonde oranges*

**Shamouti (Palestine Jaffa, Jaffa, Chamouti):** Tree upright moderate in growth and vigor, branches thick and thornless, petioles with narrow wing. Rind thick leathery smooth pitted, oil glands faint, flesh light orange, firm juicy fragrant and sweet in taste, peels easily. Shipping and storage very good. Mid-season cultivar. Seedless or nearly.

**Valencia (Valencia Late, Hart's Tardiff, Hart Late):** Has wide adaptation with alternate bearing, tendency to be heavy cropper. Tree large upright vigorous. Matures late. Storage and transportation qualities are very good and hangs well on trees. Seeds few to none.

**Hamlin (Norris):** Originated as chance seedling and named after the owner of the orchard AG Hamlin. Tolerate cold better than most oranges, productive. Tree medium large with moderate vigor. Matures very early in the season, seeds few to none.

**Pineapple:** Chance seedling very sensitive to frost, very productive and excellent for processing. Tree medium large with moderate vigor. A mid-season ripening cultivar. Has two limb sport which are seedless. Seedless Pineapple and Blaquemines.

#### *2.1.3 Pigmented or blood oranges*

This group of oranges differ from the common sweet orange in that the fruit generally has pink or red coloration on the rind, in the flesh and juice and also has distinct flavor.

**Ruby (Ruby Blood):** This cultivar was introduced from Mediterranean region. Plants are compact, not very large, moderate in vigor and production. Ripens in mid-season. Rich in flavor and few seeds.

**Spanish Sanguinelli (Syn. Sanguinelli, SanguinellaNegra):** Originated as a limb sport of Doblefina. Tree foliage light green, spineless, productive small medium in size. Fruit large very attractive with persistent style, late to mid-season in maturity, shipping and storage qualities very good.

**Torocco (Tarocco dal Muso, Taracco di Francofonte):** An Italian cultivar. Tree medium in size, irregular in bearing but moderate production. Fruits are quite large, variable in shape. Shipping and storage quality good and has mid-season maturity, does not retain quality if left for long on trees. Seeds few to none.

**Doblefina (Oval Sangre, Blood Oval):** Tree small poor in growth, branches spreading, crown open, foliage light green. Precocious and heavy bearer. Shipping, storage quality good but does not hang well on tree, almost seedless.

**Blood Red (Blood Red Malta):** Origin unknown probably came from Mediterranean basin. Grown widely and commercially in India and Pakistan. Good coloration is generally attained in the submountain region.

**Maltaise Sanguine (Portugaise):** Origin of this variety is uncertain but the Egyptian variety Baladi Blood introduced from Malta is said to be Maltaise Sanguine. Likewise Maltase Blood and Bloodred Malta seems to be a clone of this cultivar.Trees are very productive, medium large moderate in vigor. Rind soft easy to peel, taste and flavor excellent, seeds few, storage and shipping quality poor.

#### *2.1.4 Sugar or Acidless oranges*

**Maltaise Meski:** Originated in Tunisia, a non-acid orange cultivar. For all plant, foliage, flower and fruit characters it is like the parent cultivar Maltaise Blonde.

**Shamouti Maski or Shamouti Moghrabi:** It is a Labenese cultivar which is indistinguishable from Shamouti (Palestine Jaffa) baring that it is acid less (insipid in taste) and more seedy in nature.

#### *2.1.5 Other sweet oranges*

**Jaffa (Florida Jaffa):** It is a clone of Palestine beledi seedling group. Flesh pale orange, tender juicy with pleasant flavor, seeds few. Has good shipping quality but does not hang well on tree.

**Joppa:** Resembles Jaffa for number of characters and is different in that it starts bearing early and prolifically, branches are stiff and thornless, branchlets are stout. Flesh light orange, soft juicy with rich aroma. Mid-season cultivar, seeds few.

**Foster:** Tree medium in growth semi spreading foliage dense. Seeds large oval, maturity mid to late season.

**Marrs (Marrs Early):** Precocious and heavy bearer matures very early. Flesh orange well colored juicy, sweet in taste, acid very low. Fruits hang well on tree and maintain the quality. For good quality and high juice picking should be delayed. Seeds moderate in number.

**Parson (Parson Brown):** Originated as a chance seedling. It is an early maturing and relatively more seedy. Trees are large vigorous and productive. Rind pitted and pebbled moderately, flesh orange, firm juicy highly flavored.

#### *2.1.6 Indian continents*

In India, Mosambi and Sathgudi are invariably placed under sweet oranges as the acid content is very low.

**Mosambi (Mosambique):** A very popular variety grown commercially in India, early in maturity. Rind quite thick, stripes faint with longitudinal ridges and grooves. Flesh light yellow juicy, acid low, tastes insipid, seeds few.

**Sathgudi:** Origin is unknown and a popular cultivar grown extensively in India. Tree vigorous produces moderately. Flesh orange, juicy flavor fair sweet in taste, acid very low, mid-season cultivar, moderately seedy.

#### *2.1.7 Sequenced oranges*

**Chinese box orange** (*Severinia buxifolia* (Poir.) Tenore) is native to China and grows as a compact tree or a small shrub. Among the trees related to citrus is the hardiest one. It produces small fruits that have no commercial value and it is used as an ornamental species (IVIA-147).

**Amber sweet orange**, [(An unnamed hybrid of Clementine mandarin x Orlando tangelo) x unnamed midseason sweet orange seedling], is a variety released by the USDA because of its resemblance to sweet orange, early maturity and deep flesh color. It is the only such hybrid ever legally designated as a "sweet orange", so that its juice could be used to blend with true sweet orange juice, according to juice industry regulations in Florida. All other known sweet oranges are derived only by somatic mutation, not by sexual hybridization, so Amber sweet is not a true sweet orange [23, 24]. There is one true sweet orange (*C. sinensis*) from which many somatic mutants are derived, including Washington navel and blood

orange. The Amber sweet orange is a mandarin x sweet orange hybrid, and not a true sweet orange, as noted above.

#### *2.1.7.1. Sour oranges*

Wu et al. [2] reserved the name "sour orange" (*C. aurantium*) to refer to the genome from which cultivar Seville and other somatic mutants are derived. It is the maternal parent of lemon (*C. limon*). The two sour oranges from South China [24] represent two different genomes both unrelated to sour orange (*C. aurantium*).

#### **2.2 Lemon and lime**

Lemon and limes are included in the second group. Two main types of limes are distinguished: the small diploid and seedy lime (Mexican) and the big seedless triploid lime (Tahiti, Bears). Several lemon cultivars having major contribution in the world production include Lisbon, Verna, Eureka, Feminello, Fino and Primofiori.

#### *2.2.1 Lemon (C.* limon*)*

There are some distinct fruits in which lemon characteristics are evident, however the differences are to such a magnitude which warrants their separate characterization and classification. In this group, the most important are the karna, the galgal or hill lemon and jambhiri or rough lemon and all of them are widely grown in India. Meyor lemon and the limettas are also lemon like fruits, described below.

*Hill lemon (Galgal) C. pseudolimon*: An ancient Indian citrus whose origin is unknown and is commonly called as hill or kumaon lemon, grown extensively in the submountain areas along the foothills of Himalayas and in Punjab as a substitute for lime or lemon. It is indigenous to North India grown in sub-Himalayan region. Plant is tall, vigorous, upright and spreading with an irregular and loose crown, fruit ovate oblong, yellow, apex nipped, base rounded or nippled, rind medium, axis hollow, segments 7–11, seed 25–50. Ripening during October–December.

**Karna** (*C. karna* Raf): Karna Khatta, Karna Nimbu, Khatta Nimbu. A very old Indian citrus fruit of unknown origin, moderately polyembryonic. Considered to be a natural hybrid between rough lemon and sour orange, as the characters exhibited resemble the two species. Widely employed as a rootstock in northern India, second only to rough lemon. Flowers and fruits only once a year. Rind quite thick adhere tightly, golden yellow to deep orange, smooth or ribbed. Flesh orange, texture coarse, semidry, acidic in taste, flavor sour orange like. Seedy, cotyledons white.

**Rough Lemon** (*C. jambhiri* Lush.), Jatti Khatti, Lemon, Citronelle [Red rough lemon (*C x jambhiri* (Lush)) Wu et al. [2]. The species is regarded to be native of Himalayan foothills in India, where even today it grows wild. It was thought to be a natural hybrid between citron and lemon. However, Wu et al. [2] was reported that it originated from an F1 cross *C. reticulata* x *C. medica* by whole genome sequence comparison and is not a true lemon. It is presumed that Portuguese while returning home introduced it in the southeast Africa. Later towards the end of the fifteenth or early sixteenth century it was brought to Europe from where it reached new world. Fruits are acidic, medium sized, shape variable, usually oblate to elliptic oblong. Rind lemon yellow to brownish orange in color, medium thick, surface typically deeply pitted, bumpy (sunken oil glands) deeply pitted or ribbed, separates readily. Flesh pale yellow to pale orange, acidic in taste, juice moderate, segments 10, hollow and large. Seeds many, small highly polyembryonic, cotyledons light green in color.

**Eureka** (*C. x limon* L. (Burm. f.))**:** Carvalho et al. [25] were sequenced cultivar Eureka and related somatic mutants. Its seed parent is sour orange and pollen parent is an unknown citron. It is one of the most important commercial varieties around the world [2]. It is seedling selection of Sicilian lemons. Tree is medium, spreading and having few thorns. Its fruit color is lemon yellow, surface rugose, pitted, shape obovate, size medium, apex round, rind medium thin axis small, solid, segments 8–10, juice acidic with excellent flavor and quality. Eureka is heavy yielder and begins bearing at early age. It has tendency of tip bearing. Rind semi thick pitted, oil glands sunken. Fruiting more in winter, spring and early summer. Seeds few to none.

**Lisbon:** Originated in Portugal. Its appearance and yield is superior to Eureka. It is resistant to frost, heat and high wind velocity. Tree is large and vigorous with spreading shoots. It has upright thorny growth, lemon yellow fruit color, smooth surface, medium size, pitted rind, small axis, solid, 6–10 segments with 0–8 seeds. Rind quite thick adherence tight, pitted surface smooth and less ribbed than Eureka.

**Lucknow seedless:** It is hardy, medium, vigorous, spreading drooping, dense foliage, thorny, fruit color yellow, smooth, nippled apex, base round, thin rind, hollow axis, segments 10–12 maturity during November–February.

**Plant lemon:** Fruit size medium, juicy, heavy fruiting, tolerant to pests and diseases.

*Villafranca lemon* **(CRC 280)**: It belongs to Eureka group and was introduced into Florida from Europe in about 1875. Originated in Sicily. Commercially not very important compared to Eureka and Lisbon. Tree characters resembles to that of Lisbon, but the plants are more erect or upright in growth, foliage less and with few thorns. In fruit characters, it is similar to Eureka but fruits more during winter like Lisbon.

*Nepali oblong (Assam, Nimber or Pat Nebu)*: An ever bearing lemon cultivar. Plant medium sized, hardy, spreading drooping with irregular crown, fruit shape oblong, color lemon yellow, segments 10–12, the fruit ripen during December– January. Rind relatively thick, greenish yellow, glossy and smooth. Flesh fine grained, greenish yellow, juicy, not too acidic, seeds none to few.

*Meyer lemon*: Flowers throughout the year, but more so in spring. Tree semi dwarf, thornless, spreading, cold resistant, fruit color light orange, smooth surface, finely pitted, shape obolate or oblong base rounded, rind thin, axis small, 8–10 segments and 8–12 seeds. Rind thin, adhere tightly, surface smooth yellowish orange to orange, flesh light orange yellow, soft, very juicy and typical lemon flavor.

#### *2.2.2 Limes*

Like the citron and lemon, the limes likely have originated in north eastern India. Limes are generally of two forms- Small fruited acid limes (*C. aurantifolia*), West Indian lime and the large fruited acid limes (*C. lantifolia*). In its natural habitat, several forms are recognized which differs markedly in size, form, shape, spine and seediness character etc. The West Indian or Mexican lime is the Kagzinimbu and has number of vernacular names. Australian desert lime (*Eremocitrus glauca* (Lindl.) Swingle, *C. glauca* (Lindl.) Burkill) is native to Australia and produces fruits of sour taste that can be used as condiment. It is drought tolerant and has very few soil requirements (UCR-12B-38-01). Eremorange, Australian desert lime hybrid (*Eremocitrus glauca* x *C. sinensis*) (SRA-871). Australian finger lime (*Microcitru saustralasica* (F. Muell.) Swingle, *C. australasica* F. Muell), native to Australia, develops elongated finger-shaped fruits of different colors. Juice vesicles that can be broken down and separate very easily are of sharp acid flavor. It is used as a food seasoning (UCR-18B-16-04). Australian finger lime is an accession that we find has Australian round lime admixture. BC2 backcross. (SRA-1002). Australian round lime (*Microcitrus australis*, (Swingle), *C. australis* (A. Cunn. ex Mudie)) native to Australia produces rounded green fruit although at full maturity they become yellow. The pulp has low cohesive juice vesicles as the Australian finger lime. It is used as a food seasoning (UCR-18A-32-01).

#### *Horticultural Classification of Citrus Cultivars DOI: http://dx.doi.org/10.5772/intechopen.96243*

#### *Acid lime* **(***C. aurantifolia* (Christm.) Swingle.**)***:*

**West Indian (Mexican sour lime):** It is native of India and widely cultivated in the tropics. Rouiss et al. [26] reported that it is a natural hybrid between micrantha and citron. It is tenderest species among all the citrus species. Tree medium sized, hardy, semi vigorous, upright growth, thorny, fruit round to oblong, yellow apex rounded and slightly nippled, base round, rind thin, papery, 8–10 segments and seeds both. Rind thin, surface fine, smooth, adhere tightly, greenish yellow. Polyembryonic with few seeds, aroma characteristic, flesh greenish yellow, fine, soft juicy, very acidic.

#### *Tahiti lime (Persian lime) C. lantifolia.*

Cold resistant, same as that of lemons. It is large fruited acid lime. Flowers throughout the year more in spring. Purple pigmentation present on shoot and flowers. The plants are large, spreading, cold resistant, thornless, fruit large, seedless, triploid and produce non-viable pollen. It is considered as hybrid between lime and lemon. Fruit color orange yellow, smooth surface and 8–10 segments. It is late variety. Rind thin lemon yellow, adherence tight, flesh greenish yellow, tender juicy acidic. Usually 10 segments.

#### *Rangpur lime (C. limonia* **Osbeck***):*

Rangpur lime (*C. x limonia* (Osbeck)) produces non-commercial small and very acidic fruits of orange color. It is indigenous to India and is commonly used as rootstock. Rangpur lime is mainly grown for home consumption and ornamental purpose [2]. Fruits are used for making limeade. It is also known as Marmalade orange. It has loose rind, easily separable segments and pulp is light orange yellow. It is very cold hardy.

#### *Sweet lime (C. limettioides):*

In north-eastern India to which it is native it is said that sohsynteng of Assam is the acid form of this fruit. Like acid limes, the Indian sweet lime is the mithanimbu and number of its forms differing in fruitfulness, fruit shape, size and with or without nipple etc. are easily recognized. Generally, sweet lime is grown as a rootstock and for its non-acidic fruits.

**Vikram:** It was developed at MAU, Parbhani, fruit medium size, heavy fruiting, fruit color golden.

**Pramilini:** It was also developed at MAU, Parbhani, high yielder, golden fruit color, tolerant to canker.

**Sai Sarbati:** It was developed at MPKV, Rahuri, high yielder, suitable for summer cropping, tolerant to canker and tristeza.

**Jai devi:** It was developed at FRS, Periakulum, high yield, juicy, thin peel and pleasant aroma.

#### **2.3 Mandarins**

The easy peeling mandarins are becoming more important in the fresh fruit market. Principal in importance in the Orient are the Mandarins, a large, distinctive, and highly varied group that includes some of the finest and most highly reputed citrus fruits. These fruits are commonly referred to as loose-skin oranges. Clementines are the most important mandarins in the Mediterranean Basin, while Satsumas predominate in Japan. Other commercial mandarins include intraspecific or interspecific hybrids such as Fortune, Kinnow, Minneola and several chance seedlings such as Ponkan, Ellendale, Ortanique, Murcott, and Nadorcott (**Figure 2**). In the United States, where the name tangerine first came into common usage, mandarin and tangerine are used interchangeably to designate the whole group. Since mandarin is the older and much more widely employed name, its use is clearly preferable. Presumably because of the orange-red color of the Dancy variety, which originated in Florida and was introduced in the markets as the Dancy tangerine, horticulturists have tended to restrict the use of term tangerine to the mandarins of similar deep

color. Tangerine is applied more strictly to those varieties which produce deep orange or scarlet fruits. Mandarin is known as the mikan of Japan, the suntara or sangtra (numerous modifications) of India, mandarino of Italy and Spain and the mandarine of French-speaking countries.

Due to remarkable diversity of mandarins and the writer's lack of firsthand knowledge of many of the Oriential members, considerable difficulty was experienced in developing a satisfactory horticultural classification for this group. Webber (1948) has separated the mandarin oranges into (a) King group (b) Satsuma group (c) Mandarin group (d) Tangerine group (e) Mandarin-Lime group (f) Mitis group. In this treatment, therefore, the mandarins are presented as the following classes.

#### *2.3.1 The Satsuma mandarins*

The Satsuma mandarins (unshiu) mandarin, cv. Owari (UNS) (*C. unshiu* [(Mak.) Marc]; *C. reticulata* (Swingle)) is a commercial midseason, sterile and parthenocarpic, easy peeling mandarin. Satsumas are a group of commercial varieties with relatively high tolerance to low winter temperatures. It is a Japanese variety that was introduced in Florida in 1876. It is also resistant to canker, gummosis, scaly bark and melanose. Plant is thornless having spreading growth habit, orange fruit color, rough surface, oblate to spherical shape, medium size, thin and easily separable rind, flavor rich and seedless. Ripens early than any other oranges as its heat requirement is very low. Number of segments is 10–12, axis hollow and capillary membranes are loose. Fruits should be picked quickly when mature otherwise quality deteriorates and it stores well.

#### *2.3.2 The King mandarins*

The King mandarins (C. nobilis (Lour.), C. reticulata (Swingle)) is thought to be a natural tangor, i.e., a hybrid between mandarin and orange, that originated in Vietnam. However, this conventional wisdom is evidently wrong, as Wu et al. [2] was reported that whole genome sequence analysis shows that sweet orange is not a direct parent of King mandarin. King mandarin was first introduced from Cochin China into California in 1882. The king is a prolific bearer, frost resistant and produces high quality fruit. Fruits have had much commercial interest since they are large, develop good flavor when ripe and are of late harvest.

#### *2.3.3 Tachibana mandarin*

Tachibana mandarin (*C. tachibana* (Mak.) Tanaka, *C. reticulata* (Blanco)) is thought to be native to Japan and surrounding islands. It develops easy peeling, small fruits of pale-yellow-orange color and acid flavor. Although taste is not completely unpleasant the fruit is not palatable.

#### *2.3.4 Sunki mandarin*

Sunki mandarin (sour mandarin, suanju) (C. sunki (Hayata, Hort. ex Tanaka, C. reticulata (Blanco)) produces easy peeling, very acidic small fruits, of an attractive orange color. Its fruits are not palatable and the plants are used as rootstocks.

#### *2.3.5 Cleopatra mandarin*

Cleopatra mandarin (C. reshni (Hort. ex Tanaka), C. reticulata (Blanco)) is native to India. It produces unpalatable, small and very acidic fruits. It is widely used as a salt tolerant rootstock and also as an attractive ornamental because of the deep red color of the peel. Plant is thornless with dense top. Fruits are produced singly or in bunch, fruit color is dark orange red, shape oblate, flattened at both ends, size is small with12–15 segments.

#### *2.3.6 The Mediterranean mandarins*

The Mediterranean mandarins (C. deliciosa Tenore), which is of principal importance in the Mediterranean Basin. It differs from other mandarins as seeds are plumb and spherical, leaves are small narrow lanceolate and aroma of oil glands and juice is very aromatically flavored.

*Willow leaf mandarin (C. deliciosa):* The tree is willowy in growth, almost thornless, fruits usually born singly at the tip of slender branches. Fruit color orange, surface smooth, glossy, frequently slightly lobed, necked base, apex depressed, wrinkled, rind thin with 10–12 segments. It is an early variety. Trees are cold hardy but at the ripening time rind separates rapidly and lacks storage ability.

#### *2.3.7 The common mandarins*

The common mandarins (*C. reticulata* Blanco), which have worldwide importance and are represented by numerous varieties.

#### *2.3.8 The small-fruited mandarins*

The small-fruited mandarins, which are of considerable importance in the Orient and consist of many varieties.

#### *2.3.9* Clementine (Algerian Tangerine)

*Clementine (Algerian Tangerine) C. clementina Hort.ex Tanaka:* It is a tangerine and is probably an accidental hybrid of the mandarin and sour orange which originated in Algeria. Fruit color is deep orange, shape globose to elliptical, size medium with depressed apex, rind thick and segments are 8–12, adhered slightly. It is an early variety. Cotyledons are green in color and seeds are monoembryonic.

#### *2.3.10 Kishu mandarin*

Kishu mandarin (Kinokuni mandarin) (*C. kinokuni* Hort. Ex Tanaka). The seeded form of this small tangerine grows in southern China and also in Japan, where it was introduced. We sequenced the seedless mutant known in Japan as Mukakukishu; sweet, juicy, and easy to peel, it is appreciated because of its pleasant taste and wonderful aroma. Whole genome sequence comparison shows that it has the same base genotype (i.e., is a somatic mutant of) Huanglingmiao1 mandarin.

#### *2.3.11 Dancy mandarin*

Dancy mandarin, Dancy tangerine (*C. tangerine* (Tanaka), *C. reticulata* (Swingle)) is an easy peeling commercial late harvesting variety of excellent color and good size and perdurability on the tree. Originated in 1867 from a chance seedling. In USA, dancy is the best known and highly prized of all the mandarin oranges. Tree is large, nearly thornless and has upright growth. It has tendency towards alternate bearing and seeds are polyembryonic.

**Fallglo** [Hybrid of Bower mandarin (Clementine mandarin x Orlando tangelo) x Temple tangor, a presumed mandarin-sweet orange hybrid of unknown parentage], a seeded, early maturing and large fruited mandarin hybrid, developed by the USDA and produced primarily in Florida, USA [24].

#### *2.3.12* Calamondin (C. madurensis *(Lour.))*

*Calamondin (C. madurensis* (Lour.))**:** Tanaka has recognized it as a loose skinned orange group. It has less value as a fruit but hangs well on tree and is widely used as an ornamental fruit. It is very cold resistant true citrus fruit and as hardy as Satsuma. Fruit color is orange to deep orange, smooth and glossy surface, pitted shape oblate, size small with flattened base having 7–10 segments.

**Sun Chu Sha Kat mandarin** (*C. reticulata* (Blanco), *C. reticulata* var. austere (Swingle), *C. erythrosa* (Tanaka)) is characterized by small flowers, small but narrow leaves and small fruits. These are broader than long, peel color may change from yellow to deep red and taste is acidic or acidic-sweet. It is used as rootstock.

**Changsha mandarin** (*C. reticulata* (Blanco)) produces small, juicy, puffy, brilliant orange-red and seedy fruit. The taste is sweet or acidic-sweet. The tree is rather tolerant to frost and yields heavy crops. It is also grown as an ornamental (UCR-12B-23-07).

**Wilking** [It is a sister hybrid of Kinnow mandarin both having King mandarin x Willowleaf mandarin parentage], developed by the University of California, Riverside in 1915. Fruit are small, quite fragrant and richly aromatic. Because it produces monoembryonic (zygotic) seeds, it has been used in breeding programs, but not grown commercially to any great extent [24].

**Feutrell's early:** It is an old variety of New South Wales. Its parents are unknown. The fruit characteristics indicate that it may be a natural tangore and those of the three suggest the possibility that medaterian or Willow leaf might have been the mandarin parent.

**Coorg orange:** It is an important variety of South India particularly in Coorg and Wynad tracts. Fruits are medium to large, bright orange color, oblate to globose in shape, finally papillate and winkled, glossy with 9–11 segments.

**Deshi mandarin (Pathankot):** This variety is mainly grown in Punjab hills. The tree is large with semi-upright growth habit with compact foliage and spineless.

#### *Horticultural Classification of Citrus Cultivars DOI: http://dx.doi.org/10.5772/intechopen.96243*

Fruit is ovoid to sub globose, color uniformly cadmium, surface pitted, semi glossy and finally wrinkled, rind medium, adherence slight with 7–10 segments.

**Khasi mandarin:** It is commercial variety of Assam. Fruit is depressed, globose to oblate, orange yellow to bright orange, surface smooth, glossy, base even or obtuse, rind thin, soft and 8–10 segments.

**Nagpur santra (Ponkan, Warnucro):** This variety occupies premier position in Indian market and is one of the finest mandarins grown in the world. It is also known as Ponkan. Tree is large, vigorous, spineless with compact foliage. Fruit size is medium, cadmium color, smooth surface, glossy, rind thin, soft, and slightly adhered with 10–12 segments.

**Kinnow mandarin:** It is first generation hybrid between the King and Willow leaf mandarin that was developed by H.B. Frost at the California Citrus Experiment Station in 1915. It was introduced into Indian Punjab from USA. Tree is vigorous, large, top erect, dense symmetrical with few scattered thorns. Fruit color resembles of King, deep yellowish orange, surface smooth, glossy, very shallow pitted, shape slightly oblate, size medium with flattened base, rind thin, peel tough and leathery, 9–10 segment easily separable and 12–24 seeds. It is a late variety, cold resistant, has strong alternate bearing tendency and seeds are polyembryonic.

**PAU Kinnow-1:** It is bud mutant of Kinnow mandarin and is low seeded compared to Kinnow.

**Tengors:** The mandarin-like fruits include the synthetic tangors; the so-called natural tangor, Temple. Kiyomi [Hybrid of Miyagawa-wase satsuma mandarin x Trovita sweet orange], developed by the Okitsu Branch Fruit Research Station, now known as the Okitsu Citrus Research Station, National Institute of Fruit Tree Science. This is a large fruited juicy tangor, with aroma closely resembling sweet orange, and is seedless in the absence of cross pollination. It produces abundant monoembryonic (zygotic) seeds when cross pollinated and has been used as a scion breeding parent in Japan and elsewhere [24].

**Temple mandarin:** It is a hybrid between Tangerine and sweet orange. Temple mandarin is most beautiful and highly flavored fruit of the citrus group. Tree is medium, thorny, spreading with deep orange to reddish fruit color, rugose glossy surface, medium to large, depressed or nearly flat apex, loose rind, solid axis with 10–12 segments and orange pulp. It is late in maturity.

**Dweet:** Evolved from a cross between Mediterranean Sweet Orange and Dancy Tangerine. Late in maturity. Tree character intermediate of the two parents. Fruits do not hang well on tree. Fruit reddish-orange in color, globose-oblate, neck distinct, medium to large, surface pebbled. Rind adherence tight and peels very poorly, also puffs, flesh orange, firm very juicy, flavor rich, seeds numerous.

**Mency:** Originated from a reciprocal cross of Dweet. Fruits ripen early, do not hang well on tree, susceptible to sunburn, small, reddish orange, somewhat oblate and necked, surface pebbled due to oil glands. Rind adherence is not tight and peels easily. Flesh is orange colored and juicy with acidic flavor. Seeds are many.

**Tangelos:** The mandarin-like fruits include the synthetic tangelos; the so-called natural tangelo, Ugli. Hybrids of mandarin, grapefruit and pummelo are designated as tangelos. They exhibit characters of both the parents.

**Clement:** It is a hybrid between Duncan grapefruit and Clementine mandarin. Tree and foliage characters are intermediate and productive. Fruit is subglobose to oblong, light orange yellow in color and medium large. Rind is quite thick, pebbled and peels easily. Flesh is dull yellow soft and moderately sweet. Fruit is early to medium in ripening.

**Minneola:** It is hybrid of Duncan grapefruit and Dancy tangerine. Less cold hardy than Orlando, requires cross pollination for regular and higher yields. Maturity midseason. Rind deep reddish orange, smooth, medium thick, adherence moderate, surface pitted.

**Orlando:** A hybrid of Duncan grapefruit x Dancy tangerine. Early in maturity. Fruit broad oblate to subglobose without neck, medium large. Rind, thin orange in color, adherence quite tight, does not peel easily, pebbled. Flesh orange, very juicy tender, somewhat sweet seedy.

**Seminole:** Parentage is same as that of Orlando and Minneola. More fruitful. Late in maturity, trees are productive, medium large, leaves small to medium, rounded and cupped. Fruit broad oblate deep reddish orange in color. Rind adhere moderately, thin, pebbled, peelable, axis usually hollow. Flesh dark orange, juicy soft, somewhat acidic in taste, seedy.

**Page:** An early ripening cultivar obtained from a cross between Minneola tangelo x Clementine mandarin.

#### **2.4 Grapefruit and Pummelo**

The last group is grapefruit which is divided into the yellow flesh cultivars (Marsh, Duncan) and the red flesh cultivars ('Hudson', 'Star Ruby', 'Ray Ruby' 'Rio Red'). In the Southeast Asia and the Pacific, pummelo (*C. maxima*) and many traditional local mandarin cultivars are still important in the domestic market.

#### *2.4.1* Citrus paradisi *Macf. Grapefruit*

It is closely related to pummelo, originated in Barbados (West Indies), as old records refer to 'forbidden fruit' (**Figure 3**). It has become popular as a breakfast fruit because of its typical flavor and mild bitterness of the juice. Grapefruit is regarded to be either an interspecific hybrid of pummelo and sweet orange or a hybrid or a mutant of pummel Wu et al. [2]. Seeds are polyembryonic. The fruit is also called as small shaddock, obtained from pummelo, and the name has been derived from the fact that it bears in clusters, and flower resemble that of grape. Like pummelo, grapefruit is also of two types- the common and pigmented grapefruits. The varieties obviously differ in a number of characters i.e., maturity time, seedless or seedy and flavor etc. Important cultivars grown worldwide are described briefly. It is a hybrid between a pummelo and sweet orange. The Cocktail grapefruit is not a true grapefruit.

#### **Common Grapefruit Varieties:**

**Marsh:** (Syn. White Marsh, Marsh seedless, (*C. x paradisi* (Macfadyen)). It is most extended varieties of grapefruit, originated as a chance seedling around 1860

**Figure 3.** *Grapefruit varieties.*

#### *Horticultural Classification of Citrus Cultivars DOI: http://dx.doi.org/10.5772/intechopen.96243*

in Lakeland, Florida. It is a late-ripening, self-incompatible variety that shows long tree storage capability and very good behavior during postharvest. It comes under pallid pulp group of grapefruit. It is bud sport from cultivar Marsh. Fruit color light yellow, surface smooth, shape oblate to globose, size medium to large, basal area small, rind thin, segments 12–14 and 2–5 seeds. It is a late cultivar. Cultivars more significant value is its seedlessness character and its late ripening. Fruit medium sized, spherical or oblate, light yellow, areole ring almost absent. Rind surface even, smooth semi thick, flesh light yellow, soft very juicy, with pleasant flavor, but less than that in seedy cultivars. Has very good storage quality and fruit hangs for long on the tree. Seeds few to none.

**Duncan:** It was developed as chance seedling in Florida. It is the hardiest variety for cold, fruit color yellow, surface smooth, shape oblate to globose, size large, basal area depressed, apex round, rind medium thick, firm, axis medium in size segments 12–14 and 25–50 seeds. Plants are large, very vigorous and productive early to medium in ripening and also among the few which are cold hardy. Rind surface even/ smooth, glossy, medium in thickness. Flesh light yellow/ buff, soft very juicy, flavor very characteristics strong and pleasant.

#### **Pigmented Grapefruit Cultivars**

**Foster (Foster Pink):** It belongs to pink or red pulp group and originated as bud sport of Walters grapefruit by R.B. Foster in 1906–1907. Fruit color is light yellow, surface smooth, oblate or globose shape, size medium large, base rounded, apex round, rind medium thick with 12–15 segments and 50 or more seeds. It matures during November–December. Cultivar is said to be the first pigmented grapefruit variety that ripens in mid-season, originated as a limb sport of cv. Walters. Fruit medium large in size, round flat to spherical, light yellow overlaid by pink blush, which is also seen in the albedo (mesocarp), areole inconspicuous, furrows at base are small and diverging. Rind smooth, medium thick, flesh light yellow, pink under favorable conditions, soft, flavor good.

**Red blush (Syn. Ruby, Red Marsh, Red Seedless):** It belongs to pink or red pulp group. It originated as bud sport from Thompson. Deep red color which uniformly distributed throughout pulp. Fruit resembles Thomson for most of the characters except intense pigmentation of the flesh, albedo/ mesocarp also pink with bright red blush on the fruit surface. Hangs well on the tree as the parent cultivar.

**Thompson (Pink Marsh):** It originated from bud sport to marsh. Fruit color light yellow, surface smooth, 10–12 segments with 2–5 seeds. Plants are very productive large with vigorous growth. Fruits medium sized, spherical-oblate, areole indistinct light yellow, rind relatively thick, smooth and tough. Has excellent storage and shipping quality, fruits hang well for long period of time. Seeds few to none.

**Saharanpur Special:** Fruit round to oblate, empire yellow, surface small, segments 10–15 and 50–100 seeds. Fruits ripen in November to February.

#### *2.4.2* Citrus grandis *(L.) Osbeck. Pummelo, shaddock*

Seeds are large yellowish and ridged, monoembryonic. It is native of Polinasia and Malaysia and commonly grown in South China. Fruit is pyriform, largest fruit size among citrus fruits, rind thick, juice is acid bitter, juice sacs easily separable. Seeds are monoembryonic. Fruits are of two types (a) elongated pear shaped with neck (b) Oblate or globose, flattened and neckless. In India there is no improved cultivar except Nagpur chakotra.

In all likelihood is indigenous to the Malayan and East Indian archipelago. In respect of fruit characters, the pummelos fall into two major groups the pigmented and the non-pigmented types. The pigmentation in the former types is caused by carotenoid lycopene and varies from pink to deep red some of which are very

attractive with very high in flavor. The non-pigmented (common pummelo) are very variable in number of characters, have moderate to high acid contents and mostly seedy. Description of some known pummelo cultivars is described briefly.

#### **Non-Pigmented Varieties**

**Banpeiyu:** The variety is considered to have originated in Malayan region and is very popular in Japan and Taiwan. Rind thick tightly adhering to vesicles, surface smooth. Flesh pale yellow, soft and juicy, segments many (15–18) central axis solid and large, carpellary membranes thin and tough. Mid-season ripening cultivar, flavor excellent and pleasant taste of acid and sugar, storage quality very good, seedy.

**Hirado (Hirado Buntan):** Originated as a chance seedling at Nagasaki Prefecture, Japan and rated very highly resistant to cold. Fruit large bright yellow, glossy, oblate both ends slightly depressed, surface smooth, rind medium thick, flesh light green, yellow soft semi dry, flavor pleasant with balanced blend of acid and sugar with some bitterness, segments many, carpellary membranes thin and tough. Seedy.

**Kao Phuang:** One of the most famous cultivars grown in Thailand. Mid to late in ripening, fruits hang well on tree for long. Tree upright very vigorous. Rind semi thick adherence medium with the vesicles which are firm large and separates easily, juicy, flavor good, capillary membranes semi thick, axis small and compact. Seedy.

#### **Pigmented Varieties**

In its place of origin-Orient a number of pink and red fleshed types are present some of which are pomologically described below.

**Chandler:** Is a hybrid cultivar obtained from a cross between Siamese Sweet x Siamese Pink. Flesh in pink and in other characters it is intermediate between the parents. Rind smooth medium thick, vesicles moderate in juice flesh firm later turns soft, flavor pleasant sub acidic in taste, seeds many.

**Ogami (Syn. Egami):** This cultivar has performed very well in Florida and other parts of the world. Fruit quite large broadly round flat (oblate), rind smooth shiny and moderate in thickness, pink to deep pink in color, flesh deep pink, pigmentation extends far into the albedo (mesocarp), seedy.

**Siamese Pink (Siam):** Considered to be one of the finest cultivars in respect of its strong flavor, matures late. Rind adheres tightly medium thick, smooth and glossy. Segments many, carpellary membranes quite thick and tough but open at axis when mature. Almost seedless.

**Siamese Sweet:** Commonly called as sweet pummelo practically non-acid. Plants are dwarf type, branches drooping, leaves distinctly round pointed. Vesicles separate easily, are large crisp, lack in juice, sweet in taste.

#### **3. Some wild and semi wild species**

As north eastern region of India has been recognized as one of the major centre of citrus origin, a number of species are said to be native to this place **Table 1**.

*C. indica* (Indian wild orange): Specie is found growing in many parts of Assam, Nagaland, Meghalaya and other north eastern parts of India in wild form. Fruit small broad obovoid or subpyriform, appear singly on terminal twigs, about 2 cm in diameter, pedicel very small. Rind very thin, red in color, segments few, orange red in color, inedible, vesicles spindle shaped, very soft, pulp slimy, acidic in taste with unpleasant flavor. Seeds are very large, smooth, monoembryonic and occupy major portion of fruit.

*C. assamensis* (Adajamir): A very distinct specie known for its peculiar aroma that resembles to ginger or eucalyptus smell that emits from the crushed leaf or from the fruit. A new specie which was indigenous to Assam region. Fruit medium

#### *Horticultural Classification of Citrus Cultivars DOI: http://dx.doi.org/10.5772/intechopen.96243*

small, spherical to round in shape, surface smooth, very acidic juice hence has very limited use as fresh fruit.

The other species *C. latipes*, *C. ichangensis* and *C. macroptera* are usually placed under subgenus Papeda which has number of true wild species of citrus.

*C. macroptera* (Malanesian Papeda): Almost all the species of Papeda group, this species is most promising as a rootstock. Widely distributed in Indo-China, Thailand, Philippines, New Guinea, New Caledonia and Polynesia. All plants parts are with amber colored glands, spine axillary straight. Flowers are like that of orange. Fruit globose, pale yellow, small (2.5 in dia), smooth, glands small, segments 10–12 with 1–2 seed/vesicle. Juice scanty, highly acidic.

*C. latipes* (Khasi Papeda): Cold hardy. Native of northeastern Khasi hills (India), and Northern Burma. A small spiny shrub or a small tree (10–15 feet), twigs angular with sharp stout spines (1.2–2.5 cm), reduced or absent on flowering twigs. Fruit medium sized, globular, rind somewhat thick, leathery, segments 9, quite large pulp, vesicles few, spindle shaped, well developed. Seeds many 5–7 in each vesicle, round in shape.

*C. ichangensis* (Ichang Papeda): Native of south western and west central China. This specie is the most cold hardy of all evergreen types. Differs from other Papeda in that it has large flowers, stamens are connate. Small shrub or a tree (12–15 feet), twigs angular, spines stout. Fruit small (3–4 cm dia) glossy, peel rough, bumpy, medium thick, segments 7–9, each with many seeds large and thick, blunt at both ends, monoembryonic.

**Micrantha, Biasong** (*C. micrantha* (Wester)) it is thought to be native of the Southeast of the Philippines. It produces small, bitter and inedible fruit with a skin comparatively thick and broadly winged leaves (SRA-1114).

**Citron** (*C. medica* L).

Citron is considered as native of India having probable region of origin in South Western Asia. Citron is often ranked as the first specie to be cultivated in the western world and in China.

Major citron cultivars are divided into two groups:


This citrus fruit is most likely to have originated in the north eastern India and the areas nearby. As the fruit is inedible and tree lacks ornamental value, one ponders why the Romans and Greeks liked it so much. This was the first citrus fruit which was known to the Romans, further the fruit fragrance is soft, penetrating and lasting. Features of some citron cultivars grown extensively are described.

**Diamante (Cedro Liscio):** A very important and commercial cultivar of Italy, origin is unknown. New growth, buds and flowers are distinctly purple pigmented. Fruit is usually large, long oval to ellipsoid, basal cavity furrowed, apex pointednipple like and lemon yellow in color. Rind is very thick, fleshy surface lobed or ribbed and smooth. Flesh is crisp, dry, acidic in taste and seeds are many.

**Etrog (Atrog, Ethrog):** New growth, flowers and flower buds all are purple pigmented. Trees are small, productive and moderate in growth. Fruit is small to medium, ellipsoid, neck distinct, nipple at apex sharp, style persistent and lemon yellow. Rind is fleshy, thick, surface bumpy/ribbed, flesh firm and crisp, acidic in

taste and low in juice content. Citrus is a highly heterogeneous group and its various species hybridize freely with each other in nature.

**Humpang citron** (*C. medica* L.) fruit is large, oblong or oval, of green color when growing but generally yellow when ripe. The surface usually is smooth, the rind and the albedo are very thick and the segments are filled with acidic pale greenish pulp-vesicles. Citrons were the first citrus fruit to reach the Mediterranean region and are cold sensitive, monoembryonic, unpalatable and very fragrant.

**Mac Veu citron** (*C. medica* L., *C. lumia* Risso & Poit). Similar to Humpang citron.

**Corsican citron** (*C. medica* L.) is an acidless citron of unknown origin.

**Buddha's hand citron var. Sarcodactylus** (*C. medica* L. (Noot.) Swingle) produces a very characteristic fruit usually without pulp and split into a number of finger-like sections. This fingered citron is well-regarded because of its fragrance for perfuming rooms and clothing. It is also grown as a dwarf plant for ornamental purposes.

#### *Fortunella margarita* **Fortunella/Kumquat.**

Kumquats are part of genus *Fortunella*, and in Chinese language the word kumquat means 'Gold orange'. This distinct group of citrus has been named after Robert Fortune, the well-known English horticulture worker.

Other species of *Fortunella* are:


Pomological description of important Kumquats is briefly summarized below:

**Meiwa- Large round Kumquat** (*F. crassifolia* Swing.): It is less cold hardy compared to Nagami but considered to be the best as fresh fruit. This specie is the Ninpo, Neiha or Meiwa Kinkan of Japan. A natural hybrid of oval and round Kumquats.

**Nagami or Oval Kumquat** (*F. margarita* [Lour] Swing): Popularly called as Naga or Nagami Kinkan of Japan. The characteristic features are that fruits are oval, oblong or obovate in shape having fewer number of segments usually 4–5, rind deep orange, both flesh and rind are richly flavored. Tree and leaves are larger in size. Since trees are small and show slow, cold-tolerant growth it is also used as an ornamental. It produces fertile hybrids when crossed with species of the genus *Citrus*.

**Marumi or Round Kumquat** (*F. japonica* [Thumb.] Swing): Maru or Marumi Kinkan of Japan. Tree is low in vigor with some thorns. Leaves are small and apex is not sharp pointed. Fruit is small round to slightly oblate-obovate. Rind is thin and sweet in taste, segments vary from 4–7.

#### *Poncirus* **and hybrids.**

Trifoliate orange, Poncirus Pomeroy (*Poncirus trifoliata* (L.) Raf.) shows trifoliate leaves and deciduous behavior, two dominant characters that are not present in citrus. The tree also has high resistance to cold. Its fruit has no commercial value and the plant is commonly used as rootstock like its hybrids, especially the citranges, Carrizo and Troyer.

**Citranges:** It is hybrid between *Poncirus trifoliata* and *C. sinensis* and is hardy than sweet orange. It is nearly deciduous, fruit color varies from yellow to deep orange, surface rough, wrinkled, ribbed or smooth, rind thin, juicy pulp, highly

*Horticultural Classification of Citrus Cultivars DOI: http://dx.doi.org/10.5772/intechopen.96243*

acidic. It is used as dwarfing rootstock for grapefruit, Satsuma, sweet orange and lemon.

Varieties: Coleman, Etonia, Mortan, Rusk, Cunnigham, Rustic, Sanford, Savage, Troyer.

**Citrangequats:** It is trigeneric hybrid of Citrange (Sweet orange x Trifoliate orange) and Kumquat. Citranges are subjected to spring frost injury due to its tenderness while Kumquat is tolerant to the spring frost because of their winter dormancy.

#### **4. Sequenced genome of** *Citrus* **spp. and their varieties**

Xu et al. [27] reported Valencia sweet orange (*C. sinensis* cv. Valencia): A model of the sweet orange origin. With pummelo as the female parent crossed with mandarin, the interspecific hybrid was backcrossed with mandarin and produced the ancient sweet orange. Haploid Clementine, Clementine mandarin, Ponkan mandarin, Willowleaf mandarin, W. Murcott mandarin, Chandler pummel, Low-acid pummel, Sweet orange, Seville sour orange [18] **Table 1**.

#### **5. Conclusion**

Classification of the Citrus varieties has long been debated by taxonomists and botanists. The reticulate evolution combined with partial apomixis has led to very different classification systems. Earlier classification was very difficult due to lacking of genomic study but recently, phylogenomic data revealed the origins and admixtures of modern cultivars and wild types. Coupled with reproductive biology, phylogenomy supports the inclusion of all true citrus of the Swingle system plus Oxanthera in the genus Citrus. The variety rank is defined by the old independent reticulation events from which groups of cultivars were differentiated by asexual mechanisms. It provides an unambiguous conceptual framework for Citrus classification based on the phylogenomic and genetic data. However, today, the available genomic data remain available for further study and further WGS studies are needed to establish a definitive classification of the Citrus varieties. Commercially grown citrus varieties discussed in this text, are not grown from seed but are grafted and budded onto a seedling of a rootstock.

#### **Acknowledgements**

The author wishes to thank Professor Muhammad Sarwan Khan for critical reading of the manuscript.

#### **Conflict of interest**

The authors declare no conflict of interest.

#### **Author details**

Jagveer Singh1 , Vishal Sharma1 , Kuldeep Pandey2 , Shahnawaz Ahmed3 , Manveen Kaur4 and Gurupkar Singh Sidhu1 \*

1 School of Agricultural Biotechnology, Punjab Agricultural University, Ludhiana, India

2 Indian Agricultural Research Institute-ICAR, New Delhi, India

3 Sri Guru Granth Sahib World University, Punjab, India

4 Dr. J.C. Bakhshi Regional Research Station, Punjab Agricultural University, Abohar, India

\*Address all correspondence to: gurupkar-soab@pau.edu

© 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*Horticultural Classification of Citrus Cultivars DOI: http://dx.doi.org/10.5772/intechopen.96243*

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[15] Curk F, Ancillo G, Ollitrault F, Perrier X, Jacquemoud-Collet JP, Garcia-Lor A, et al. Nuclear species-diagnostic SNP markers mined from 454 amplicon sequencing reveal admixture genomic structure of modern citrus varieties. PLoS One. 2015; 10 (5):e0125628.

[16] Oueslati A, Salhi-Hannachi A, Luro F, Vignes H, Mournet P, Ollitrault P. Genotyping by sequencing reveals the interspecific *C. maxima*/*C. reticulata* admixture along the

genomes of modern citrus varieties of mandarins, tangors, tangelos, orangelos and grapefruits. PLoS One. 2017; 12 (10):e0185618.

[17] Penjor T, Mimura T, Kotoda N, Matsumoto R, Nagano AJ, Honjo MN, et al. RAD-Seq analysis of typical and minor Citrus accessions, including Bhutanese varieties. Breed. Sci. 2016; 66 (5):797-807.

[18] Wu GA, Prochnik S, Jenkins J, Salse J, Hellsten U, Murat F, et al. Sequencing of diverse mandarin, pummelo and orange genomes reveals complex history of admixture during citrus domestication. Nat. Biotechnol. 2014; 32 (7):656-662.

[19] Curk F, Ollitrault F, Garcia-Lor A, Luro F, Navarro L and Ollitrault P. Phylogenetic origin of limes and lemons revealed by cytoplasmic and nuclear markers. Annals in Botany. 2016; 117:565-583. DOI: 10.1093/aob/mcw005.

[20] Ollitrault P, Curk F, and Krueger R. Citrus taxonomy. In: Talon M, Caruso M, Gmitter Jr FG, editors. The Genus Citrus. 1st ed. Elsevier**;** 2020. p. 57-81 DOI: 10.1016/B978-0-12-812163- 4.00004-8. Ch4

[21] Shimizu T, Kitajima A, Nonaka K, Yoshioka T, Ohta S, Goto S, Toyoda A, Fujiyama A, Mochizuki T, Nagasaki H, Kaminuma E, Nakamura Y. Hybrid origins of citrus varieties inferred from DNA marker analysis of nuclear and organelle genomes. PLoS One. 2016; 11 (11): e0166969. DOI: 10.1371/journal. pone.0166969.

[22] Mabberley DJ. Citrus (Rutaceae): a review of recent advances in etymology, systematics and medical applications. Blumea. 2004; 49 (2/3): 481-498.

[23] Bayer RJ, Mabberley DJ, Morton C, Miller CH, Sharma IK, Pfeil BE, Rich S, Hitchcock R, Sykes S. A molecular phylogeny of the orange subfamily (Rutaceae: Aurantioideae) using nine

cpDNA sequences. American Journal of Botany. 2009; 96:668-685. DOI: 10.3732/ ajb.0800341.

[24] Wang X, Xu Y, Zhang S, Cao L, Huang Y, Cheng J, Wu G, Tian S, Chen C, Liu Y, Yu H, Yang X, Lan H, Wang N, Wang L, Xu J, Jiang X, Xie Z, Tan M, Larkin RM, Chen LL, Ma BG, Ruan Y, Deng X, Xu Q. Genomic analyses of primitive, wild and cultivated citrus provide insights into asexual reproduction. Nature Genetics. 2017; 49 (5):765-772. DOI: 10.1038/ ng.3839.

[25] Carvalho R, Soares Filho WS, Brasileiro-Vidal AC, Guerra M. The relationships among lemons, limes and citron: a chromosomal comparison. Cytogenetic and Genome Research. 2005; 109 (1/3):276-282. DOI: 10.1159/000082410.

[26] Rouiss H, Bakry F, Froelicher Y, Navarro L, Aleza P, Ollitrault P. Origin of C. latifolia and C. aurantiifolia triploid limes: the preferential disomic inheritance of doubled-diploid 'Mexican' lime is consistent with an interploid hybridization hypothesis. Annals of Botany. 2018; 121 (3):571-585. DOI: 10.1093/aob/mcx179.molecular.

[27] Xu Q, Chen LL, Ruan X, Chen D, Zhu A, Chen C, et al. The draft genome of sweet orange (*Citrus sinensis*). Nat. Genet. 2013; 45 (1):59-66.

#### **Chapter 3**

## Citrus Biotechnology: Current Innovations and Future Prospects

*Ghulam Mustafa, Muhammad Usman, Faiz Ahmad Joyia and Muhammad Sarwar Khan*

#### **Abstract**

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.

**Keywords:** citriculture, conventional research, transgenesis, genome editing, multi-OMICS

#### **1. Introduction**

Citrus is one of the most diverse members of the family *Rutaceae* and is the leading tree fruit crop in the world. Citrus comprises different species of edible fruits like mandarins (*Citrus reticulata* Blanco), sweet oranges (*C. sinensis* Osbeck), grapefruit (*C. paradisi* Macf.), acid limes (*C. aurantifolia* Swingle), and sweet limes (*C. limettioides*), lemons (*C. limon* Burmf.) and their hybrids including tangerines, tangelos, tangors, etc. [1].

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 [2]. 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 [3].

#### **2. Origin and diversification**

*Citrus* and other genera including *Poncirus*, *Clymenia*, *Fortunella*, *Eremocitrus*, and *Microcitrus* belong to tribe Citreae and sub-tribe Citrineae and are considered as true citrus [4]. Classification in citrus has been controversial since ancient times due to vast morphological diversity, interspecific and intergeneric sexual compatibility. However, molecular biology tools have revealed four species including mandarins, citron (*C. medica*), pummelos (*C. grandis* Linn.), and wild cultivar of papeda (*C. micrantha* Wester) as the true parental species that have contributed to the development of other species during the process of evolution [5, 6]. Based on phylogenetic and genomic studies it is revealed that mandarin originated in China, Vietnam, and Japan whereas citron was originated in northeast India and China. Pummelo originated in Indonesia and Malay whereas *C. micrantha* was originated in the Philippines [6]. Other citrus species including sweet oranges, grapefruit, lime, lemon, sour oranges, and hybrids (tangelos and tangors) have developed from these ancestral species through random hybridization and natural mutation events [7].

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 [8]. 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 *in vitro* culture tools particularly somatic hybridization which has played an essential role in developing new somatic hybrids. These techniques have contributed towards the selection and development of new potential cultivars and are still being used as important fundamental tools for the development of genetically diverse germplasm which could be further screened and characterized using modern breeding technologies.

#### **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 *in vitro* tools.

#### *Citrus Biotechnology: Current Innovations and Future Prospects DOI: http://dx.doi.org/10.5772/intechopen.100258*

Mutation breeding has played a pivotal role in fruit crop improvement including citrus and has developed several mutants with improved phenotypic and genotypic traits [11]. 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 [12]. 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 [13]. About 3365 mutant varieties belonging to 170 plant species have been released including citrus and 20 other fruit species [14]. 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 [15]. 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) [10]. 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 [18]. In fruit crops, physical mutagens have altered key horticultural traits like seedlessness, precocious bearing, and dwarfism [19–21]. 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 [22], however, the ion beam is getting more popular and is being widely used due to its greater efficiency and precision compared with gamma rays [23]. 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 [26], apple [27], and rough lemon [28]. 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 [29]. Other commercial mutants in citrus include sweet orange varieties Jin Cheng [30], Kozan [21], and NIAB Kinnow mandarin [31]. 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 [32]. 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 [33]. 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 [10].

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) [34], using targeted mutagenesis and genome editing technologies [35] 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 [36–38]. The duplicated genes may evolve new functionalization during evolution [39]. 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 *Colchicum autumnale* (meadow saffron). It is used for inducing chromosome doubling or developing tetraploids by restricting the chromosomal segregation at metaphase in meiosis [42, 43]. Other methods of polyploid induction include interploid hybridization [44], unreduced gamete formation [45, 46], and endosperm culture [47, 48].

Members of the subfamily *Aurantioidae* including *Citrus*, *Fortunella*, and *Poncirus* are mainly diploid having chromosome number 2n = 18 [49]. The occurrence of spontaneous polyploids in citrus is known since the 1940s [50]. Important spontaneous polyploids include triploid Tahiti lime [51], Triphasia desert lime [52], *Clausena excavata* [53], tetraploid mandarins [54], sweet oranges [41, 42] and grapefruit [43]. In spontaneous polyploids, triploids and tetraploids are believed to be formed by doubling of chromosomes in nucellar cells and fertilization of the unreduced gametophytes [55, 56]. Polyploids have been induced using colchicine in several citrus species and tetraploids produced have been used for interploid crossing to develop triploid progenies that are usually seedless due to irregular distribution of the chromosomes during cell division particularly gamete formation and formation of unreduced gametes. In interploid crossing, the formation of tetraploids in addition to triploids indicates the predominant formation of the unreduced (2n) gametes which may be formed by the first division restitution (FDR) or second division restitution (SDR) during meiosis. Production of 2n gametes was predominantly via SDR in lemon [44, 45] and monoembryonic Orah mandarin [57]. Higher tetraploid: triploid ratio in the progeny of the interploid hybridization indicates greater production of the 2n megagemtophytes in that cultivar which is promising to produce a greater number of polyploids.

#### **3.3** *In vitro* **culture: somatic hybridization**

Plant tissue culture tools offer advantages related to efficient regeneration, propagation, and crop improvement in citrus and other horticultural crops.

#### *Citrus Biotechnology: Current Innovations and Future Prospects DOI: http://dx.doi.org/10.5772/intechopen.100258*

Endosperm cultures have been used for the development of triploids in citrus [48]. 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 *in vitro* culture in different citrus species [45]. Similarly, micrografting is another tool in which a miniature bud is grafted under aseptic conditions on *in vitro* raised rootstocks and micrografted plants have been reported in many citrus varieties [58]. Micrografting is also useful for the production of virus-free citrus plants.

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 [59]. 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 [60].

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 *Poncirus trifoliata* was reported by Ohgawara et al. [61] followed by several interspecific and intergeneric hybrids in citrus from the USA [62], Japan [63], and other citrus-producing countries. Triploids were also reported from interspecific and intergeneric somatic hybridization of Citrus species, kumquats (*Fortunella japonica*), and *Poncirus trifoliata* by protoplast fusion [64]. Fusion of protoplasts from the haploid lines and diploid cultivars may also yield triploids and hundreds of triploids and tetraploids were developed and planted for field evaluations [65]. Polyethylene glycol may also be used to induce regeneration in the fused protoplast cultures as reported in Willow leaf mandarin (embryogenic parent) and Duncan grapefruit and sweet orange (mesophyll parents). The regenerated plants were identified as alloplasmic cybrids [66].

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 *Poncirus* and *Citrus* showed greater tolerance to cold and higher light conditions compared with parents (diploid) and their tetraploids [36]. Kumquats (*Fortunella* species) chloroplast have demonstrated higher resistance to canker in diploid kumquats and their tetraploid somatic hybrids developed with other citrus species including grapefruit [37].

#### **4. Innovative approaches/technologies**

#### **4.1 Transgenesis**

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 [67], 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 [70]. 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. Agrobacteriummediated transformation [71], biolistic transformation [72], and chemically assisted uptake of recombinant DNA by protoplasts [73] 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. [74] 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. [72] reported the first successful transformation through gene gun. They transformed tangelo (*C. reticulata* × *C. paradisi*) embryogenic cells.

Since genetic transformation has successfully been performed in different species and hybrids including Carrizo citrange, Washington naval orange, *Poncirus trifoliata*, Sour orange, Mexican lime, sweet orange, *Citrus reticulata* [75], and a valuable rootstock, swingle citrumelo. Similarly, protocols have been optimized for the genetic transformation of different citrus species by using different explant tissues including seeds, embryogenic cells, epicotyls, embryogenic cells, callus, nodal stem segments, and protoplasts. The most responsive explant tissue has been epicotyl from the *in vitro* germinated seedlings and is preferably used for genetic transformation research. Duncan grapefruit was successfully transformed through *Agrobacterium* for the first time using epicotyl and confirmation of the transgene (NPTII and GUS) integration was carried in the resultant 25 transgenic plants by histochemical staining, PCR, and Southern blot hybridization. Transgenic grapefruit, sweet orange, and citrange plants were developed using epicotyls as target explant whereas selection was carried out on kanamycin [76]. Epicotyl has also been used for Agrobacterium-mediated transformation of citrange and sweet orange [77]. In addition, callus, as well as suspension cultures derived from different parts of flower and seed, have also been attempted to transform. The transformation

*Citrus Biotechnology: Current Innovations and Future Prospects DOI: http://dx.doi.org/10.5772/intechopen.100258*

#### **Figure 1.**

*Schematic sketch showing the importance of conventional and advanced innovative approaches for the improvement of different citrus species.*

efficiency attained, in this case, was lower than 0.5%. Genetic transformation has also been optimized in pomelo (*Citrus grandis*) and sour orange wherein internodal stem segments were used as explants and a promising transformation efficiency was achieved (91%) [78].

The biolistic transformation has also been performed successfully in tangelo (*C. paradisi* Macf. x *C. reticulata* Blanco) using nucellar embryogenic cells raised from the suspension culture and more than 15 stable whereas 600 transient transgenic lines were attained per bombardment. The transformed calli cells showed proficient growth on kanamycin selection medium and positive GUS activity but were not able to regenerate into plants. Calli treatment with 0.3 M osmoticum sorbitol and 0.3 M mannitol appeared to have positive effects for enhanced transformation efficiency for stable and transient transformation. Thin epicotyl segments of germinated seedlings were also targeted through the biolistic gun and more than 93% transformation efficiency was attained for transient transgene integration in *C. citrange*. The incubation of explant on culture medium before bombardment appeared to have profound effects on transformation efficiency which was further improved [79].

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 [80]. PsCBL and PsCIPK derived from *Pisum sativum* were transformed into *Citrus sinensis* and *Citrus reticulata* by targeting calli derived from mature seed. Bacterial strain LBA4404 was used to induce infection in the target calli cells. The putative transformants showed better performance as compared with control plants for salinity and drought tolerance when tested under *in vitro* conditions [81].

*Citrus paradisi* was transformed to improve carotenoid content by manipulating the genes involved in carotenoid biosynthesis i.e. phytoene synthase, lycopeneβ-cyclase, and phytoene desaturase. The multigene transgenic citrus plants were aimed to supplement human nutrition with vitamin A along with antioxidants. Similarly, fruit juice quality has been attempted to improve in Valencia orange, a valuable variety that is majorly grown for its juice. Degradation of TSPME (thermostable pectin methylesterase) deteriorates the quality of the juice. This TSPME is encoded by the *CsPME4* gene. The protoplasts were isolated from embryogenic suspension cultures and transgene was introduced through the PEG mediated transformation method [82] aiming at down-regulation of the *CsPME4* resulting in the citrus with improved juice quality.

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 [83]. Expression of viral coat protein (part of *p23* gene and the 3UTR), in the sense and antisense orientation also delayed viral infection in grapefruit [84].

Phytophthora is a noxious fungal pathogen that has been reported to infect a wide range of citrus species. Among these *Phytophthora parasitica* and *Phytophthora citrophthora* cause more severe damage in the citrus orchards and nurseries all over the world [85]. Expression of *bo* gene (bacterio-opsin) in Rangpur lime rootstock showed an elevated level of tolerance against *Phytophthora parasitica* infection. It was observed that expression of the aforementioned gene led to induce expression of defense-related proteins; chitinase, salicylic acid, and glucanase. Hence plants with an elevated level of transgene expression showed greater resistance against the oomycetic fungi including *Phytophthora parasitica*. Transgenic citrus plants expressing the tomato *PR5* gene showed an enhanced survival rate in the presence of pathogen (*P. citrophthora*). Transgenic grapefruit plants were able to better withstand citrus scab infection when transformed with the *attE* gene encoding for antimicrobial peptide [86].

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; *Candidatus Liberibacter* americanus, *Candidatus Liberibacter* asiaticus, and *Candidatus Liberibacter* africanus [87]. Various genetic strategies have been tested to develop HLB-resistant citrus lines

#### *Citrus Biotechnology: Current Innovations and Future Prospects DOI: http://dx.doi.org/10.5772/intechopen.100258*

with decreased susceptibility to the pathogen. These include the expression of anti-microbial peptides from a bacterial, fungal, plant, or animal origin and engineering host-pathogen interaction pathways. The expression of antimicrobial proteins under phloem-specific promoters has been an effective strategy to control this phloem-resident pathogen. Overexpression of synthetic cecropin B gene under phloem specific promoter resulted in reduced bacterial population after one year of inoculation and no disease symptoms appeared even after two years of inoculation [88]. Overexpression of modified methionine under double 35S promoter also appeared to have an inhibitory effect on bacterial growth and lowered down the CLas (*Candidatus Liberibacter* asiaticus) titer in the roots of transgenic Carrizo citrange (rootstock). Further, newly emerging leaves from the rough lemon, grafted on this transgenic rootstock, also had a non-detectable bacterial titer. Expression of AtNPR1 and chimeric proteins (ThioninLBP and Thionin1-D4E1) demonstrated elimination of CLas providing tolerance against HLB infection [89].

Another economically important disease, the citrus canker has also been addressed through transgenesis resulting in enhanced tolerance against *Xanthomonas citri*. Engineering sweet orange genome with *Xa21* gene showed a significant reduction in disease severity upon inoculation in three lines Hamlin, Pera, and Natal. Expression of the *Xa21* gene under its promoter appeared to be more effective in disease resistance when expressed in highly susceptible Anliucheng sweet orange [90]. Transgenic Carrizo citrange and sweet orange plants were developed by introducing RpfF from *X. fastidiosa* which encodes for a quorum-sensing factor and can disrupt bacterial communication by reducing activation of virulence factors, thus enhancing the ability to tolerate pathogen infection. Similarly, the expression of AMP sarcotoxin from flesh fly also enhanced tolerance against *X. citri* [91].

#### **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 [92] 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 [93].

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 *CsPDS* gene in Duncan grapefruit and sweet orange. The target gene was successfully modified through a transient expression method, Xcc-facilitated agroinfiltration [94]. The modified *CsPDS* sequence was not detectable in the leaves of sweet orange indicating that CRISPR/Cas9 has induced the desired mutation successfully.

Most of the research studies were carried out to target the *LOB1* (LATERAL ORGAN BOUNDARIES 1) gene which has been characterized as a citrus susceptibility gene for *Xanthomonas citri*. The said gene has been explored to be upregulated by TAL (transcription activator-like) effector PthA4 which binds to the EBE (effector

binding elements) in the promoter region of LOB1 thus activates expression of this canker-susceptibility gene [95]. Mutation in the single allele of effector binding elements of the *LOB1* gene resulted in minor alleviation of canker symptoms in Duncan grapefruit. Anyhow, a mutation in effector binding elements of both of the alleles of LOB1 promoters alleviated canker symptoms to great extent thus showing a high degree of resistance in Wanjincheng orange [96]. Another research group explored that editing the coding region of LOB1 in Duncan grapefruit, through the CRISPR-Cas9 system also provides resistance against *X. citri* infection.

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 [97]. In addition to the CRISPR-Cas9 system, another improved genome editing system (CRISPR/Cas12a (Cpf1) has also been used to edit the *CsPDS* gene in the Duncan grapefruit gene. It appeared to be a more efficient editing system with lower off-target effects thus will prove a great milestone in citrus genome editing [98]. These studies indicate that CRISPR-mediated genome editing can be a promising pathway to generate disease-resistant citrus cultivars [99].

#### **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.

#### **5.1 Genomics**

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 [100].

A specific pathotype of *Alternaria alternata* (Fr.) Keisel is a disease with heavy losses [101]. It causes necrosis and resultant lesions on the surface of fruits and young leaves. It leads towards defoliation and fruit drop [102]. Thus, exploitation of innate genetic resistance appears to be the most applicable and effective strategy of disease control. Currently, the control is primarily based on the application of 4–10 sprays of toxic and environmentally injurious fungicide per year [103]. Such limitations are compelling farmers to find alternative cultivars with resistant ones [104].

Usually, the female parent transmits the 2*n* gamete in 2x × 2x citrus crosses [105–108]. Cuenca et al. [109] recognized ABS resistance locus containing genomic region by using BSA-genome scan combined with HTA based. Trait segregation in crosses between two heterozygous ABS-susceptible or between heterozygous ABSsusceptible parents and resistance was used to confirm the recessive inheritance of the ABS resistance in triploid populations. ABSr locus was first located at 10 cM from a

#### *Citrus Biotechnology: Current Innovations and Future Prospects DOI: http://dx.doi.org/10.5772/intechopen.100258*

centromere based on segregation of 368 SDR 2*n* gametes. A genomic region containing several markers with a high probability (> 99%) of association with phenotype variation was identified on chromosome III by performing BSA over 93 triploid hybrids from a Fortune × Willow leaf population. This identified region contains 25 significant SNP markers within an interval of 13.1 cM. The size of the genomic region among these two markers is 15 Mb. Linkage genetic mapping was performed on identified genomic regions by developing new SNP and SSR markers. A 268-diploid mapping population was performed by Cuenca et al. [110] from a heterozygous-susceptible × resistant hybridization. Fine mapping was performed to confirm the location of *ABSr* locus in a region of 1.1 cM between the markers SNP05/SNP06/SNP07/AT21 (at 0.7 cM) and SNP08 (at 0.4 cM). Another region containing eight genes with NBS-LRR repeats was identified by the SNP08 marker and considered ABS resistance genes.

In citrus plant, molecular markers are linked to some agronomic traits, e.g. SSR markers are linked to Citrus tristeza virus resistance from *Poncirus trifoliate*, PCR assay for the anthocyanin content of pulp [111], AFLP markers are associated with polyembryony [112] and RAPD markers are associated to dwarfism and fruit acidity [113]. Some other characteristics such as salinity tolerance and nematode resistance are linked to QTLs [114]. The selection of resistant genotype at the early growth stage was improved by the newly developed SNP08 marker. This marker was mapped at 0.4 cM from the ABD resistance gene and it has role in avoiding the selection of susceptible varieties. On the other side of the gene, some new markers were also identified at 0.7 cM from the *ABSr* locus. Combining these new markers with SNP08, the probability of selection of resistant genotype was increased by 0.0028%. This marker appeared to be very helpful in the selection of resistant and susceptible genotypes and for analyzing the resistant germplasm to configure the ABS genes. So, it is a very valuable tool for the selection of susceptible heterozygous cultivars which may be used as breeding parents allowing manipulation of genetic diversity in citrus and prevents susceptible homozygous genotypes.

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 *Alternaria* infections and SNP08 marker. Recently SNP08 is used in breeding programs of citrus performed at CIRAD and IVIA for the selection of ABS-resistant citrus genotypes. About 2187 resistant hybrids were selected from 4517 total hybrids rising from 10 different parental combinations by using the SNP08 marker since its development. This analysis was very helpful to prevent the growth of more than 2000 susceptible lines which were removed at the early growth stage after selection so, a lot of time, cost, personnel, and resources were saved.

#### **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 [115]. Metabolites are referred to as the last product of any biological activity in a cell and are found in very small quantities [116]. 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 [119].

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 [120].

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 [121].

#### **5.3 Interactomics and metabolomics and phenomics**

Interactomics bears a broad scope as it may cover a complete set of interactions in a cell [122]. 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 [123].

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 [124]. 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 hightemperature 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 [125], Thermographic imaging [126], Hyperspectral imaging, Chlorophyll fluorescence, X-ray, MRI, PET [127].

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.

#### **6. Conclusions**

Conventional research has played a pivotal role in the improvement of citrus. Enhanced heterozygosity has helped in the development of genetically diverse

#### *Citrus Biotechnology: Current Innovations and Future Prospects DOI: http://dx.doi.org/10.5772/intechopen.100258*

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.

### **Author details**

Ghulam Mustafa1 , Muhammad Usman2 , Faiz Ahmad Joyia1 and Muhammad Sarwar Khan1 \*

1 Centre of Agricultural Biochemistry and Biotechnology (CABB), University of Agriculture, Faisalabad, Pakistan

2 Institute of Horticultural Sciences, University of Agriculture, Faisalabad, Pakistan

\*Address all correspondence to: sarwarkhan\_40@hotmail.com

© 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Section 2
