ABSTRACT

Grapefruit is considered as a minor citrus crop in Pakistan and its annual production is less than 0.5% of total citrus production. Grapefruit industry is depending upon pink flesh cultivar ‘Shamber’ and need varietal diversification. Mutation breeding has played a pivotal role in grapefruit crop improvement and most of the commercial cultivars are bud sports or induced mutants which were selected and later released as new cultivars. Flesh color enhancement, seedlessness and low furanocoumarin level have been main objectives for grapefruit breeders. An overview of potential of mutation breeding in grapefruit and leading mutants produced is presented in the following sections. Though physical mutagens have been more successful, breeders’ interest is rising in more precise and targeted mutagenesis technologies including CRISPR/Cas9 which has enormous potential in genome editing and could shorten breeding and selection cycle. The available leading grapefruit cultivars were screened for horticultural traits and potential candidate varieties has been selected for diversification and selection of better parents for breeding programs. Several putative mutants have been developed using gamma irradiated plant material. The effect of irradiation on plant growth, morphology and biochemical properties has been evaluated and salient findings are discussed. The developed genetically diverse material could be useful for future biotechnology applications.

OVERVIEW OF GLOBAL CITRUS AND GRAPEFRUIT INDUSTRY

Citrus stands amongst the most important tree fruit crops in the world. Citrus and its related genera belong to Rutaceae family, which are widely distributed across monsoon regions. Its geographical origin, timing and dispersal remain unclear across Southeast Asia (Wu et al., 2018). It is gaining popularity due its nutritional importance for human health (Wu et al., 2007). World area under citrus cultivation is 11.14 million ha with annual production of 152.44 million tons while, in Pakistan citrus covers an area of about 0.2 million ha with 2.25 million tons of annual production (FAO, 2018).

The grapefruit (Citrus paradisi Macf.) is an evergreen tree. It is named grapefruit due to its fruit bearing habit in clusters like grapes (Scora et al., 1982). It is a spontaneous hybrid between pummelo (Citrus grandis L.) and sweet orange (Citrus sinensis L.) (Nicolosi et al., 2000; Oueslati et al., 2017). It originated in Barbados in mid-18^th century and was named as “forbidden fruit of Barbados” (Hughes et al., 1750). At present, grapefruit is also considered as one of the “Seven Wonders of Barbados” (Bourne, 1996). Grapefruit is a popular nutraceutical fruit and an excellent source of nutrients and phytochemicals including vitamin C, antioxidants, lycopene and dietary fibers. Due to super nutraceutical properties, it is now widely cultivated and has a great influence in international citrus market. Grapefruit and pomelos are being cultivated on 0.37 million ha area with annual production of 9.37 million tons in the world. Global grapefruit production is less than 5% of the total production of citrus fruits and is mainly confined to China, USA and Mexico (FAO, 2018) as shown in figure 1. In Pakistan, it is being cultivated on an area of about 5,000 ha only which is expected to be enhanced. Grapefruit industry comprises on ‘Shamber’ cultivation which need to be diversified with other potential candidate varieties including ‘Star Ruby’ and ‘Red Blush’ (Fig. 2) (Usman et al., 2020). Staggering trends were observed for citrus production in Pakistan during last few years. The factors affecting citrus industry and causing low production include citrus decline, long juvenility, narrow genetic base, self-incompatibility, sterility and climate change (Nawaz et al., 2019; Iqbal et al., 2020). Fruit fly is another threat that causes severe damage and fruit drop in most of the citrus species including grapefruit (Mangan et al., 2011; Usman et al., 2020). Lack of certified nurseries is major source for dispersal of diseases like citrus greening, canker and tristeza virus which leads to degradation of the citrus industry in Pakistan (Usman and Fatima, 2013; Naqvi et al., 2017).

Figure 1: Leading global grapefruit fresh fruit producers.

Figure 2: Pigmented grapefruit varieties available at institute gardens, University of Agriculture, Faisalabad. Figures include A) Red Blush B) irradiated mutant Star Ruby and C) Shamber.

IMPACT OF MUTATION BREEDING ON FRUIT INDUSTRY

Mutation breeding has been a valuable tool in producing horticultural varieties with improved phenotypic and genotypic traits (Ahloowalia et al., 2004). Cultivars developed through mutation have an advantage over classical breeding as this approach haven’t issues regarding intellectual property rights (IPR) and economics which ultimately leads towards cultivation of transgenic horticultural plants (Alston, 2004; Dobres, 2008). Primary source of genetic variations existing in plants was exploited by mutations (kharkwal, 2012) and induced variations which provided raw material for better selection of traits and acted as a driving force in the process of evolution. About 3222 mutant plant varieties belonging to 170 species have been released in more than sixty countries of the world. These include 20 different fruit species having more than 50 cultivars (Jankowicz-Cieslak et al., 2017). Global leading mutant producers include China (810), Japan (479) and India (341), while Pakistan has released 59 mutants including NIAB Kinnow (Fig. 3). Among citrus, about 15 mutants have been released since 1970 including six mandarin and clementine, six orange, two grapefruit and one lemon cultivar (MVD, 2020).

Figure 3: Overview of leading global mutant variety producers (MVD, 2020).

Spontaneous mutations have been very important in citrus industry; however, frequency of occurrence was rare having random gene alterations which made them difficult to identify and utilize (Lonnig, 2005). In contrast, induced mutations using radiations or chemical compounds can increase the frequency of mutations and enhance genetic variability (Broertjes and van Harten, 1988). Agents responsible for inducing artificial mutations are called as mutagens which are further classified as physical and chemical mutagens. Various types of these mutagens have been used to alter the genetic makeup of different organisms.

INDUCED MUTAGENESIS, TARGET TRAITS AND IMPROVED VARIETIES

Physical mutagens are mostly ionizing agents that have been used extensively for inducing hereditary changes as chromosomal aberrations and enhancing genetic polymorphism. More than 70% of the global mutant cultivars have been developed using physical mutagenesis (Mba et al., 2012). Physical mutagens have been very helpful in induction of seedlessness, dwarfism and early flowering in different fruit crops (Lamo et al., 2017). Use of gamma irradiation is a useful technique to obtain genetically stable and disease-free mutants. Wavelength of gamma rays is shorter therefore these penetrate deeper into tissues than X-rays and neutrons (Amano, 2006). Gamma radiations have been used in many fruit crops successfully. Ion beam, a new mutagen introduced recently gained popularity and is being more widely used than gamma rays, X-rays and neutrons (Watanabe, 2001; Watsumura et al., 2010).

Chemical mutagens are alkylating agents which are very useful for mutation induction. Chemical mutations are more reliable and gene specific than physical mutations. Diethyl sulfate (DES), Ethyl methanesulfonate (EMS), Ethylenimine (EI), Sodium azide and Colchicine are the commonly used chemical mutagens. Colchicine is most widely and commercially used chemical for polyploidization due to its reliability and effectiveness. It is an alkaloid obtained from meadow saffron (Colchicum autumnale L.). Colchicine is used to obtain tetraploids (Predieri, 2001) by restricting the chromosomal segregation during metaphase in meiosis thus doubling the chromosome number and ploidy (Kumar and Rani, 2013; Fatima et al., 2015).

Mutation breeding had been successfully used for introduction of many useful traits including alteration in blooming time and fruit ripening, change in fruit color, inducing self-compatibility, self-thinning and resistance to pathogens, breaking linkages of undesirable traits, inducing variability in already adapted species, restoring fertility in sterile hybrids, increasing ploidy level (autotetraploids), inducing dwarfism, and increasing fruit size with better taste and aroma. Mutation breeding has been exploited in fruit crops like pear, peach, banana, papaya, grapes, almond, plum, sour cherry and sweet cherry (Predieri, 2001), rough lemon (Saini and Gill, 2009), apple (Campeanu et al., 2010) and for chromosome doubling in banana, cherry (James et al., 1987), grapes (Notsuka et al., 2000) and blueberry (Lyrene and Perry, 1982).

Citrus species show spontaneous polyploidization such as triploids (3n), tetraploids (4n) and pentaploids (5n). The recovered spontaneous polyploids were originated from nucellar embryos and had somatic origin (Barrett and Hutchison, 1978). There are several examples of spontaneous polyploid species including tetraploid Triphasia desert lime (Esen and Soost, 1972), Clausena excavata (Froelicher and Ollitrault, 2000), tetraploid Hong Kong wild kumquat (Longley, 1925), triploid Tahiti lime (Bachi, 1940), tetraploids in Feutrell’s Early and Kinnow mandarins and Mosambi and Succari sweet oranges (Usman et al., 2006; (Fatima, 2015).

Citrus improvement programs are using mutagenesis for the development of better cultivars with novel traits including lemon, oranges and mandarins having improved fruit color, seedlessness and fruit setting percentage (Mlauszynski et al., 2000), and mandarins for fruit size and taste (Agisimanto et al., 2016). A detailed overview of mandarins and lime species improved through mutation and other breeding tools has already been presented (Usman and Fatima, 2018; Usman et al., 2019b). Other commercial mutants include Minneola tangelo, Kinnow low seeded (Altaf and Khan, 2007; Altaf et al., 2014) and Jin Cheng sweet orange (Zhang et al., 2014). Spontaneous tetraploids were also observed in oranges (Shafieizargar et al., 2013), Citrumelo and Troyer citrange having large sized leaf, stem and fruit. Fepagro C13 and C37 are hybrid rootstocks with larger seeds and roots, being widely used in Brazil (Guerra et al., 2014). Colchiploids were produced in Mandarin cv. Baladi having larger inflorescence and variable flowering period (Elyazid and El-Shereif, 2014) and in Rangpur lime for drought tolerance, cold tolerance and pest resistance (Allario et al., 2013).

ORIGIN OF GRAPEFRUIT FAMILY

Most of the present-day grapefruit cultivars have developed spontaneously as bud sports which were identified, characterized, multiplied and released as new varieties. Duncan was the first white flesh cultivar that originated spontaneously in seedlings of wild grapefruit in 1830s. White flesh cultivars Marsh (1850) and Walters (1887) were also discovered as spontaneous mutants in Duncan seedlings. Foster (1907) was developed as bud sport of Walters. Pink fleshed Thompson (1913) and Shamber (1936) also originated as bud sport of Marsh. Ruby Red (1929), Red Blush (1931) and Burgandy (1943) developed as bud sport of Thompson. Ray Ruby (1970), Roug La Toma ARG (1970) and Henderson (1973) were found as bud sport of Ruby Red. Seedling selections of these bud sports were discovered as Flame (1983), Nel Ruby (1987) and Oran Red RG (1989). Rio Red was developed as bud sport from A & I 1-48-Tx irradiated bud wood in 1976. Hudson (1930) and Ruben Pink (1963) bud sports were developed from Foster (Da Graca et al., 2004). Only two induced mutants are reported in grapefruit which got commercial importance i.e. Rio Red (better fruit color) and Star Ruby (seedless) (Maluszynski et al., 2000). Among these cultivars, Star Ruby is leading red flesh cultivar and is being widely cultivated in Spain, Turkey, Australia and South Africa. Star Ruby and Rio Red are main lycopene rich cultivars in Texas. Ruby Red has been main cultivar in India, China and Argentina (Singh et al., 2002). Flame is also being cultivated on large acreage in Australia, South Africa and has replaced Ruby Red in Argentina (Da Graca et al., 2004). Many of the above-mentioned bud sports and irradiation-induced mutant varieties have been available in Pakistan for a long time; however, no significant work was reported about their characterization, bearing behavior and performance under local climatic conditions for making future selections.

INDUCED MUTAGENESIS IN GRAPEFRUIT VARIETIES

Keeping in view the current scenario and potential of mutation breeding in citrus, a research project was initiated for grapefruit germplasm collection, characterization and development of mutants using both physical and chemical mutagens. Fresh bud wood and mature fruits of grapefruit (Citrus paradisi Macf.) cultivars were collected from Citrus Research Institute, Sargodha and Institute of Horticultural Sciences, UAF. Fruits of known elite cultivars were characterized for morphological, physical and biochemical diversity. Fruit weight and size were more in Flame (455 g) and Ray Ruby. Fruit peel was thinner in Star Ruby and Pink Ruby cultivars and mature seeds were minimum (2.2) in Pink Ruby. Total soluble solids (TSS) were higher in Star Ruby (8.51 °Brix) and Pink Ruby, while TSS:TA (6.0-7.8) and ascorbic acid content were higher in Red Blush and Pink Ruby. Anthocyanins were more (0.82 mg/100 g) in Red Blush and Pink Ruby. Total sugars were much higher (5.3%-5.6%) in Flame, Pink Ruby and Star Ruby compared with Shamber. Hence, there is good potential in Pink Ruby, Red Blush and Star Ruby cultivars to replace Shamber which is not a commercial cultivar in most of the leading grapefruit producing countries in the world (Usman et al., 2020). Bud wood of grapefruit cultivars was exposed to gamma rays at different doses at Nuclear Institute of Agriculture and Biology, Faisalabad and grafted on rough lemon plants. Fruits were characterized for morphological variation using standard protocols (IPGRI, 1999). Fruit weight was higher in cultivar Flame compared with Shamber, while TTS were higher in cultivar Pink Ruby. Irradiation affected plant growth and arrested bud sprouting percentage which decreased with increasing irradiation dose (140 Gy). Leaf length was higher in non-irradiated control in Ray Ruby, Red Blush and Pink Ruby, while it was lower in Star Ruby and Flame. Leaf width was more in Flame and Ray Ruby compared with other cultivars. In irradiated germplasm, increase in the irradiation dose from 40 Gy to 140 Gy, markedly reduced leaf size in all the cultivars. Leaf thickness was not much variable in grapefruit cultivars under control conditions; however, thickness increased with irradiation dose and was much higher at higher doses in Ray Ruby and Red Blush. Increase in shoot length, internodal distance and number of leaves per branch were observed in Ray Ruby at different irradiation doses compared with control, while other cultivars showed variable responses at different doses. The variable growth behavior indicated enhanced phenotypic variability induced in response to irradiation in different cultivars (Usman et al., 2019a). The genetically diverse plant material is being screened for genetic polymorphism and promising material could be useful for future horticultural and biotechnology applications.

TARGETED MUTAGENESIS

Conventional mutagenesis has been handicapped by lack of designing and production of new alleles and takes more time to detect any phenotypic variation. Hence, the non-targeted mutagenesis has been coupled with methods of phenotypic screening including Targeting Induced Local Lesions IN Genomes (TILLING) for demonstration of gene functions and gene isolation (Parry et al., 2009). Transgenics have played a major role in crop improvement; however, this technology is limited by random gene insertion and requires efficient plant regeneration particularly in woody crops. Hence, targeted mutagenesis, including genome editing techniques, is getting popular. Clustered Regularly Interspaced Short Palindromic Repeats CRISPR/Cas9 is the most recent gene editing technology which facilitates targeted DNA insertion by non-homologous recombination leading to targeted mutations and gene removal (Luo et al., 2016). In sweet orange (C. sinenesis L.) phytoene desaturase (PDS) gene was knocked out using CRISPR/Cas9 modifications (Jia and Wang, 2014). Higher canker resistance has also been achieved in grapefruit and sweet orange by knockout of CsLOB1 gene using CRISPR/Cas9 system (Jia et al., 2017; Peng et al., 2017). These reports indicate enormous potential of CRISPR/Cas9 based genome editing in citrus for targeted mutagenesis for future crop improvement programs.

Conclusively, mutation breeding has played a key role in grapefruit crop improvement particularly in the development of pink and red flesh seedless cultivars from white flesh and seedy varieties. Application of direct mutagenesis tools including CRISPR/Cas9 will further enhance the progress towards development of improved varieties having economically important novel traits.

ACKNOWLEDGEMENTS

Authors are thankful to Higher Education Commission, Pakistan (HEC) for funding this research under the Project No. 4705.

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