A Mini Review of Citrus Rootstocks and Their Role in High-Density Orchards

Dwarfing is an important agricultural trait for intensive cultivation and effective orchard management in modern fruit orchards. Commercial citrus production relies on grafting with rootstocks that reduce tree vigor to control plant height. Citrus growers all over the world have been attracted to dwarfing trees because of their potential for higher planting density, increased productivity, easy harvest, pruning, and efficient spraying. Dwarfing rootstocks can be used to achieve high density. As a result, the use and development of dwarfing rootstocks are important. Breeding programs in several countries have led to the production of citrus dwarf rootstocks. For example, the dwarfing rootstocks ‘Flying Dragon’, ‘FA 517’, ‘HTR-051’, ‘US-897’, and ‘Red tangerine’ cultivated in various regions allow the design of dense orchards. Additionally, dwarf or short-stature trees were obtained using interstocks, citrus dwarfing viroid (CDVd) and various chemical applications. This review summarizes what is known about dwarf citrus rootstocks and the mechanisms underlying rootstock–scion interactions. Despite advances in recent decades, many questions regarding rootstock-induced scion development remain unanswered. Citrus rootstocks with dwarfing potential have been investigated regarding physiological aspects, hormonal communication, mineral uptake capacity, and horticultural performance. This study lays the foundation for future research into the genetic and molecular mechanisms underlying citrus dwarfing.


Introduction
Citrus fruits are one of the most popular tree fruits and are widely grown in tropical and subtropical regions around the world on a commercial scale [1,2]. Citrus fruits belong to the family Rutaceae, which consists of 140 different genera and 1300 different species, including oranges, mandarins, lemons, limes, pummelos, grapefruits, and several others [3]. Citrus fruits are known for their nutritional value, quality, aroma, and attractive flavor. Furthermore, they are an important source of vitamin C, dietary fibers, carbohydrates, and minerals [4][5][6][7][8].
Citrus has been commercially grown for hundreds of years using grafted plants, and rootstocks play a vital role in the growth and development of citrus plants [1]. Rootstocks influence the physiological and biochemical traits of scion cultivars, including plant vigor,

Dwarfing Citrus Rootstocks
Citrus growers worldwide are attracted to dwarfing citrus rootstocks because they are ideal for high-density plantations and are suitable for mechanized farming [25]. Dwarfing citrus rootstocks are well represented in research reports (Table 1). Higher plant densities promote greater productivity; generally, lower densities permit the harvest of larger fruits, which raises the price of fresh fruit on the market [14]. Dwarf trees have several advantages, such as a better yield, high density, and photosynthetic efficiency, which raises potential production. In this system, plants will be trained in the assigned space, facilitating numerous practices such as harvesting, scouting, and spraying [26]. Additionally, high tree densities, in combination with adapted varieties, enable high-efficiency production techniques in many fruits [10,[27][28][29].
It has long been believed that the 'Flying Dragon' trifoliate orange is the only true dwarfing rootstock in the citrus industry. Its commercial feasibility in tropical conditions has been established, particularly for more vigorous scion cultivars such as Persian lime and lemons [21,30]. Mature 'Flying Dragon' trees are typically about 2.5 m tall in most scion varieties. Conversely, this tree grows slowly when grafted to navel oranges, requiring several years to produce a commercial harvest. Hence, employing a dwarfing rootstock that grows faster and produces more fruit than scions grafted to 'Flying Dragon' is needed. However, the extensive use of Flying Dragon with sweet orange scion has not acquired commercial importance in the major producing areas, where farmers generally prefer more vigorous rootstocks [31]. As a result, most citrus breeding programs have developed new, alternative dwarfing rootstocks, and conventional cross-breeding has produced some promising genotypes [32,33] and genetic transformation [34].

Dwarfing by Chemical Treatments
Plant growth inhibitors are substances that slow down plant growth without altering developmental stages [42]. Many species are regularly treated with chemicals to control their height [43]. Plant growth regulators (PGRs), such as gibberellic acid (GA) biosynthesis inhibitors, are often used to limit excessive vegetative growth in various fruit crops, including apples, cashews, pomegranates, and citrus [44][45][46]. In the 19th century, Aron treated 'Minneola' tangelos (Citrus paradisi Macf.) with 1 g·L −1 of paclobutrazol before summer growth; the average shoot length decreased by nearly 50%. According to Garner et al. [47], prohexadione-calcium (P-Ca; 250 mg L −1 ) reduced the shoot growth of different fruit plants. Therefore, the PGRs approach should be further evaluated.

Dwarfing by Citrus Dwarfing Viroid (CDVd)
Plant dwarfism has been linked with several viruses and viroids [25,48,49]. The citrus exocortis viroid seems to cause dwarfism in citrus plants by increasing the aboveground hydraulic resistance [50]. Additionally, the rootstock, variety, and species of citrus hosts all affect the symptoms brought on by CDVd. CDVd infection of navel orange trees grafted on 'trifoliate' orange rootstock has revealed that the stunting phenotype caused by CDVd infection decreases canopy volume by about 50% [51][52][53][54]. Field research shows that the CDVd infection approach might be utilized to minimize plant height and maximize plantation density. Substantial findings were made on the possible biological mechanism by which some rootstock scion pairings are affected by CDVd to decrease tree canopy volume. Further information was revealed regarding the putative biological mechanism through which CDVd affects specific scion/rootstock combinations to reduce plant height [55]. According to Lavagi [25], CDVd has been used to produce dwarf citrus trees when grafted on trifoliate rootstock. Further findings demonstrate that CDVd modulates the expression profile of citrus growth and developmental processes, which may be responsible for reduced vegetative growth. However, the molecular mechanisms that restrict the canopy volume of citrus trees in response to CDVd infection are poorly understood. Hence, it seems that latent viruses could potentially contribute to regulating the dwarfing capacity of some rootstocks ( Table 2). The tree canopy was reduced by >20% in CDVd-infected trees. [51] 'Parent Washington' navel orange 'Rich 16-6' trifoliate orange The CDVd-infected trees were planted at a close spacing (3 × 6.7 m), whereas the uninfected trees were planted at a standard spacing (6.1 × 6.7 m).
CDVd modifies the expression profile of citrus growth and developmental processes, which may be related to cellular changes that result in the observed phenotype of reduced vegetative growth and smaller trees. [25] 'Washington navel' 'Carrizo citrange' A graft was infected with viroid isolates.
TsnRNAs (CDVd) can limit tree growth, making citrus grove management and production more flexible and consumer-friendly. [56] 'Grapefruit' 'Troyer citrange' The graft was inoculated with five different kinds of GTDC, together with 225T and 225M.
CVd infection of grafted grapefruit trees decreased the water movement capacity from the roots and within the canopy. [50] 'Valencia orange' 'Poncirus trifoliata' Treatment was performed using citrus viroid (CVd-la, CVd-IIIb and CVd-IIa,).
The canopy volume was reduced while the yield per tree increased. [52]

Dwarfing by Using Interstocks
Interstock grafting is utilized in many fruit trees (Table 3), including citrus trees, as a sustainable approach to controlling plant height, dwarfing traits, and fruit quality [57][58][59]. According to previous studies, interstock and rootstock could be utilized jointly to overcome compatibility issues between the scion cultivar and rootstock [60]. When the 'Flying Dragon' rootstock is used as an interstock, it causes a considerable reduction of scion growth with both 'Troyer' citrange and 'P. trifoliata' as rootstocks. Furthermore, compared to plants without interstock, the average size reduction is approximately 37%. However, using 'Flying Dragon' as a rootstock resulted in a 66% reduction in canopy growth compared with P. trifoliata and 'Troyer citrange' rootstocks [61].
Moreover, similar findings have been documented that lemon trees grafted with interstocks have smaller size, peel, and albedo thicknesses. Furthermore, interstocks affect the growth morphology and photosynthetic characteristics of 'Yuanxiaochun' grafted plants.
In addition, when Kumquat and 'Ponkan' mandarin were employed as interstocks, the 'Yuanxiaochun' scion cultivar displayed greater photosynthetic activities and higher rates of light and CO 2 utilization [62]. Interstocks influence the transport of water, nutrient uptake capacity, hormonal communication, and some other factors, and these interstocks influence overall plant growth, blooming, and fruiting. In addition, methods such as strangling, inarching, girdling, and grafting by budding are frequently employed throughout the interstocked-seedling production stages. Through stomatal and non-stomatal effects (girdling), these techniques can restrict photosynthetic carbon uptake and reduce transpiration [63][64][65].

Dwarfing by Using Tetraploid Rootstocks
In citrus, tetraploid trees can be used for the diversification of rootstocks because they have more genetic variability because of new recombination possibilities and their capability to serve as dwarf rootstock [70]. Tetraploids (4×), which result from incomplete mitosis of somatic embryos, might occur naturally or artificially in seedlings with diploid (2×) apomictic genotypes. Tetraploid rootstocks are characterized by shorter and thicker roots, which results in slower growth [71,72]. Furthermore, tetraploidy affects phenotypic features such as leaf and root morphology, fruit quality, stomatal size, and density. These alterations may disrupt normal physiological processes [73]. Tetraploid trifoliate orange rootstocks lowered scion canopy development and fruit yield; however, clementine's sugar content, acidity, juiciness, and carotenoid content remained unaffected; hesperidin concentration increased, and this was only true for clementine scions grafted onto tetraploid rootstocks [74]. Allario et al. [75] evaluated diploid and tetraploid plants derived from the same seed ('Rangpur' lime; C. limonia Osbeck), and found that polyploid seedlings were smaller than diploid plants. According to Syvertsen [76], the lowest growth rates reported in citrus seedlings obtained from tetraploid rootstocks are attributed to decreased transpiration rates due to a lower number of stomata. Variation concerning plant height was noticed, and the diploid plants presented higher growth than tetraploid plants. Moreover, tetraploid plants were smaller and grew more slowly [72].

Dwarfing Mechanism of Scion Reduction
Grafting is an ancient horticultural practice that joins the aerial part (scion) with another segment (rootstock) to produce a new plant [10]. Scion cultivars grafted with rootstock are the foundation of modern fruit orchards. Rootstocks influence the morphological, biochemical, and physiological characteristics of the scion portion [77]. Several studies have been conducted to investigate the rootstock-induced dwarfing effect; however, the associated mechanisms in citrus plants have not been fully explained. Scion vigor is known to be influenced by multiple factors, such as the transport of minerals [2], level of hormones [78], hydraulic conductance [79], and anatomical studies [48,80]. Therefore, it can be concluded from the literature that the impact of citrus rootstocks on scion growth and dwarfing mechanisms are mediated by numerous factors (Figure 1 and Table 4), including mineral uptake capacity, hormonal alterations, hydraulic conductance, and anatomical features.
rates reported in citrus seedlings obtained from tetraploid rootstocks are attributed to creased transpiration rates due to a lower number of stomata. Variation concerning p height was noticed, and the diploid plants presented higher growth than tetraploid pla Moreover, tetraploid plants were smaller and grew more slowly [72].

Dwarfing Mechanism of Scion Reduction
Grafting is an ancient horticultural practice that joins the aerial part (scion) with other segment (rootstock) to produce a new plant [10]. Scion cultivars grafted with r stock are the foundation of modern fruit orchards. Rootstocks influence the morphol cal, biochemical, and physiological characteristics of the scion portion [77]. Several stu have been conducted to investigate the rootstock-induced dwarfing effect; however, associated mechanisms in citrus plants have not been fully explained. Scion vigo known to be influenced by multiple factors, such as the transport of minerals [2], leve hormones [78], hydraulic conductance [79], and anatomical studies [48,80]. Therefor can be concluded from the literature that the impact of citrus rootstocks on scion gro and dwarfing mechanisms are mediated by numerous factors (Figure 1 and Table 4) cluding mineral uptake capacity, hormonal alterations, hydraulic conductance, and a tomical features.    Mineral analysis, scion vigor, and photosynthetic processes.
The primary shoot length of the 'Salustiana' scion grafted onto the Rough lemon rootstock was the longest. In addition, the 'Rough lemon' rootstock had a vigorous root system, which improved its ability to absorb minerals and nutrients. Higher mineral uptake was associated with a stronger root system (total root length, number of forks, and root tips), which can directly impact nutrient uptake. clementines.
[85] IAA levels were highest in the fresh shoots of 'Swingle citrumelo' and lowest in the 'Flying Dragon' rootstock. [78]

Type of Dwarf Rootstock
Dwarfing rootstocks produce a mature tree with a height of no more than 2.5 m, in combination with any scion cultivar, regardless of environmental influences [87]. The vigor of citrus trees (Citrus spp.) is affected by the canopy/rootstock combination, soil, and phytosanitary conditions. Bitters [19] proposed a classification in which a tree taller than 6.0 m was used as the standard. Sub-standard, semi-dwarf, and dwarf plants had a reduction of 25%, 50%, and 75%, respectively, regarding the standard.

Tree Size and Vigor
Rootstocks significantly impact the physiological, biochemical, and molecular characteristics of the scion cultivar [16]. The reduction of scion growth due to rootstock is a fascinating phenomenon in studying fruit trees. Previous studies have demonstrated that the Salustiana scion cultivar grafted on 'Rough lemon' rootstock had the most extended primary shoot length, greater scion trunk diameter, and vigorous root morphology compared with less vigorous rootstocks. Additionally, plants grafted onto vigorous rootstocks have better nutritional properties [2]. The 'Shatangju' mandarin scion cultivar grafted onto the 'Fragrant orange' and 'Red tangerine' rootstocks displayed dwarfing traits with the shortest shoot length, lowest trunk diameter, and shortest internodal length [40]. In another study, the root system of 'Rough lemon' rootstock was shown to be vigorous with increased root projected area, root volume, surface area, and the number of forks and points; however, the 'Carrizo' rootstock displayed lower values of root morphological traits [81]. Recent experiments reported that the 'Shatangju' scion cultivar grafted onto the 'Flying Dragon' rootstock encouraged short-stature trees. In contrast, trees grafted with other rootstocks, such as 'Shatang mandarin', 'Goutou sour orange' and 'Sour orange', grew taller and wider and had more vigorous plant growth. According to the research mentioned above, the vegetative growth of scion cultivars is significantly influenced by citrus rootstocks. Additionally, using dwarfing rootstocks permits high-density planting, which boosts yield and leads to optimal use of water and nutrients [1].

Precocity
Prominent features imparted by dwarfing citrus rootstocks are a decrease in tree size and precocity (early flowering and fruiting). Dwarfing rootstocks are typically connected with precocity, while vigorous rootstocks delay fruiting. Conversely, the performance Plants 2022, 11, 2876 9 of 14 of the fruit trees is linked to a proper balance between fruiting and vegetative growth because excessive vegetative growth lowers the total yield and fruiting [88]. Rootstocks that encourage scion precocity are needed for early crop production [9]. For instance, dwarfing citrus rootstocks limit tree size and increase yield production and precocity. 'Mandared' trees grafted onto C22, C57, and C35 rootstocks bear fruit one year earlier than other tested rootstocks. Furthermore, 'Mandared' trees grafted with C22 rootstock demonstrated yield precocity and higher yield efficiency than C22 rootstock [89]. A lowered canopy volume of trees grafted on C22 rootstocks has been shown in previous studies [90,91], and could be an advantage for new plantings with higher densities.

Planting Density for Citrus Rootstocks
A high-density planting system (Table 5) is an innovative agrotechnology that enhances yield by managing more plants in a given area [92]. In addition, the appropriate plant density should be maintained for maximum yield and good-quality fruit [14]. Citrus trees in a grove compete for resources such as water, nutrients, and light. As the distance between trees decreases and resources become more limiting, competition increases, and there are notable tree responses [87]. A distance of 5-7 ft (1.52-2.13 m) is recommended between plants grafted onto Flying Dragon rootstock despite its limited commercial use in Florida [93]. In Southeast Brazil, 4-5 m row spacing and 1.5-2.5 m plant spacing are advised for the Flying Dragon rootstock [94]. In Japan, orchards of Wase satsuma mandarin with a density of up to 10,000 plants ha −1 were evaluated via long-term tests [23]. Recent research conducted in India with Nagpur mandarin on Rangpur lime rootstock determined that a high-density planting was regarded as one that included between 555 and 625 plants ha −1 and that an ultra-high-density planting was considered as one that contained between 1250 and 2500 plants ha −1 [13]. Therefore, long-term experiments will be needed to examine commercial citrus cultivars with dwarfing rootstocks to determine optimal plant density under modern production circumstances. Kinnow mandarin Rough lemon 6.00 m × 6.00 m, 6.00 m × 5.00 m, 6.00 m × 3.00 m Vegetative growth of plants was greater at larger spacing (6.00 m × 6.00 m). Yield ha −1 was the maximum (220.99 t ha −1 ) at the closest spacing density (6.00 × 3.00 m). [98]

Conclusions
Grafting has been employed as a key tool in the propagation of horticultural crops to manage plant specific features such as early fruit production and vigor control. To encourage cultural application in high-density planting, citrus germplasm should be evaluated for its potential to produce plants with dwarf characteristics or short stature. In several countries, breeding programs have been started to develop dwarfing citrus rootstocks to achieve maximum planting density per unit area. For instance, 'Flying Dragon', 'FA 517', 'HTR-051', 'US-897', and 'Red Tangerine' rootstocks produce dwarf statures that allow for the establishment of dense orchards. Furthermore, short-stature trees have been obtained by using different interstocks and citrus dwarfing viroid (CDVd). Mechanisms for rootstock-induced dwarfing effects have also been covered in this article. Moreover, dwarf rootstocks have smaller root systems; thus, the roots absorb less water and nutrients from the soil medium. Furthermore, the influence of citrus rootstocks on scion growth and the dwarfing mechanism is induced by numerous factors, including mineral uptake capacity, hormonal alterations, hydraulic conductance, and anatomical features.