Keywords

These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

9.1 Basic Botany of the Genus

Poncirus trifoliata (trifoliate orange or Japanese bitter orange) belongs to the subtribe Citrinae, tribe Citreaea, subfamily Aurantioideae of the family Rutaceae and is a close relative of citrus. It is thought to be a native of China and Korea. It was brought to North America as an ornamental plant. It is widely found in all citrus-growing regions of the world, as it is commonly used as a rootstock for citrus and it improves frost hardiness and fruit quality. It is usually propagated from seeds. It is a small, fast-growing, deciduous, perennial tree, which grows upto a height of 3–10 ft (2–3 m) and has a green bark with brown streaks. The twigs are covered with sharp spines. It is a very frost hardy tree and can withstand temperatures upto −5°F (−21°C) and lower. The leaves are trifoliate rarely having four or five leaflets, alternate with leaflets being glabrous, elliptic, oblong to obovate, sessile, and 2–6 cm long. The rachis is broadly winged. Flowers are simple, fragrant, sessile, white, borne on axillary inflorescence on previous year’s growth, and have five sepals and petals, eight to ten stamens, which are entirely free, and a six-celled pubescent ovary. Though the flower buds are formed in early summer, they pass the winter in a dormant condition and are protected by bud scale. Fruits are small, pubescent, round to pear-shaped, yellow when ripe, and sour to bitter in taste with numerous seeds. Unlike citrus, the fruits of Poncirus are inedible in USA. However, in China, the dried mature fruits are used medicinally. The peel is candied and is used as a spice while the juice is used as flavoring syrup. It is used as a source of pectin and grown as an ornamental in the European countries. Due to its spines, it is also grown as a hedge plant in Brazil.

Poncirus species are mostly diploid with 2x = 2n = 18 chromosomes. Chromomycin A 3 (CMA) and 4,6-diamidino-2-phenylindole (DAPI) have been used for chromosome banding for characterization of chromosomes of P. trifoliata (Moraes et al. 2008). This has allowed the separation of the nine chromosome pairs into three groups (4B + 8D + 6F) of which only one F chromosome pair could be distinguished (Befu et al. 2000; Brasileiro-Vidal et al. 2007). In situ hybridization demonstrated that two B chromosome pairs were different from those previously found in citrus as they displayed a 45S rDNA (ribosomal DNA) site colocalized with a CMA + proximal band and a 5S rDNA site adjacent to this band while only one pair of the four D chromosome pairs showed adjacent 5S and 45S rDNA sites, with a 45S rDNA site colocalized with a CMA + band (Brasileiro-Vidal et al. 2007). Bacterial artificial chromosome (BAC) in situ hybridization has also been attempted in P. trifoliata and allowed the identification of seven chromosome pairs while the other two were recognized by the presence of 45S rDNA associated with the CMA + band in the first one, and lack of any single copy signal and the presence of a terminal CMA + band in the second one (Moraes et al. 2008). All chromosome pairs were homomorphic, indicating a high level of chromosomal homozygosity. Thus, a combination of chromosome morphology, fluorochrome banding, and fluorescent in situ hybridization (FISH) with rDNA probes distinguished the Poncirus chromosomes successfully (Roose et al. 1998).

9.2 Conservation Initiatives

Though Poncirus is widely used as a rootstock for citrus, very little information is available on the extent and distribution of its genetic diversity. It has traditionally been conserved in very small scale in clonal orchards belonging to botanical gardens or scientific institutions, where citrus is primarily conserved. It has been conserved along with citrus in large ex situ collections in Argentina, Australia, Brazil, France, Morocco, New Zealand, South Africa, Spain, Turkey, and USA (Krueger and Navarro 2007). No information is available on in situ conservation of Poncirus. Shoot tips from juvenile plants of P. trifoliata have been cryopreserved using encapsulation–dehydration method (Gonzalez-Arnao et al. 1988). Attempts have been made to cryopreserve seeds and embryonic axes of P. trifoliata in vitro. The seeds were found to be sensitive to desiccation, whereas the excised embryonic axes could be easily desiccated and successfully preserved in liquid nitrogen (Radhamani and Chandel 1992). Shoot tips excised from axillary buds of Troyer citrange (P. trifoliata × C. sinensis) have been cryopreserved using encapsulation–dehydration and encapsulation–vitrification methods (Wang et al. 2000).

9.3 Molecular Mapping in Poncirus

Poncirus has been extensively used as a parent in intergeneric crosses to facilitate optimum polymorphism to construct a number of molecular genetic linkage maps (Cai et al. 1994; Gmitter et al. 1996; Tozlu et al. 1999a, b; Ling et al. 2000); however, the hybrids obtained have inedible fruits making these mapping populations of limited use for fruit traits (Gmitter et al. 2007). A population obtained from an intergeneric backcross of C. grandis cv. “Thong Dee” and P. trifoliata cv. “Pomeroy,” using the former as the recurrent (female) parent, has been used for performing genetic linkage analysis using restriction fragment length polymorphism (RFLP) and isozyme (Durham et al. 1992) markers. Another study used isozymes and RFLP for the construction of a genetic map based on the segregation of 8 isozyme, 1 protein, and 37 RFLP loci in 60 progeny of a cross of two intergeneric hybrids, “Sacaton” citrumelo (C. paradisi × P. trifoliata) and “Troyer” citrange (C. sinensis × P. trifoliata), often used as rootstocks (Jarrell et al. 1992). Linkage maps have also been constructed using various molecular markers from intergeneric crosses such as C. grandis × (C. grandis × P. trifoliata), C. sunki × P. trifoliata cv. Rubidoux, C. grandis cv. “Thong Dee,” and P. trifoliata cv. “Pomeroy” (Cai et al. 1994; Cristofani et al. 1999; Sankar and Moore 2001; Table 9.1). Five genetic linkage maps have been constructed for the parents of progenies of C. aurantium × P. trifoliata var. Flying Dragon, C. volkameriana × P. trifoliata var. Rubidoux and a self-pollination of P. trifoliata var. Flying Dragon using simple sequence repeats (SSRs) for genome comparison (Ruiz and Asins 2003). Recently, an F1 intergeneric population of C. sinensis × P. trifoliata was used to construct genetic maps in which 11 linkage groups with 113 markers in C. sinensis, nine with 45 markers in P. trifoliata, and 13 with 123 markers in the cross-pollinator consensus of both, were constructed (Chen et al. 2008).

Table 9.1 Poncirus linkage maps
Full size table

Resistance to citrus tristeza virus (CTV) was found to be dominant by performing enzyme-linked immunosorbent assay (ELISA) on several Poncirus-derived populations. Using bulked segregant analysis (BSA) approach (Michelmore et al. 1991) and RAPD markers, a map was developed and the Ctv gene was identified from Poncirus (Gmitter et al. 1996; Fang et al. 1998). Two BAC contigs with integrated fine maps were constructed that resulted in the full-length sequencing of the locus spanning several hundreds of kilobases and identification of the candidate genes (Deng et al. 1997, 2001; Yang et al. 2001, 2003). Prolonged CTV challenge led to the suggestion that more than one gene may be involved in CTV resistance (Mestre et al. 1997). One CTV-resistant gene was later mapped in a different location within linkage group 4 of Poncirus from a population of citradias (derived from the cross between sour orange and Poncirus) suggesting a deviation from the single gene hypothesis, which could be quantitative trait loci (QTLs) (Bernet and Asins 2003; Asins et al. 2004). It was found that a major QTL, designated Tyr1, controls resistance to citrus nematode (Ling et al. 2000) and was adjacent to the Ctv region (Ling et al. 1999). In the C. volkameriana and P. trifoliata progeny, 11 putative QTLs have been detected in P. trifoliata that control the number of fruits per tree (Garcia et al. 2000). A C. grandis × P. trifoliata F1 pseudo-testcross population was used to map QTLs associated with freezing tolerance (Weber et al. 2003). The dominant trifoliate leaf character of Poncirus has also proven to be advantageous in developing mapping populations, as it allows the direct identification of zygotic hybrids from true nucellar seedlings. F1 progeny of C. sunki × P. trifoliata were evaluated for the detection of QTLs linked to citrus Phythophthora gummosis resistance. Two QTLs linked to gummosis resistance were detected in linkage groups 1 and 5 of the P. trifoliata map and one in linkage group 2 of the C. sunki map (Siviero et al. 2006). QTL analysis of morphological traits in an intergeneric BC1 progeny of C. grandis × P. trifoliata under saline and non-saline environments has also been attempted (Tozlu et al. 1999b).

9.4 Role in Crop Improvement Through Traditional and Advanced Tools

9.4.1 Traditional Breeding Efforts

Poncirus is commonly used as a rootstock for most citrus species and is also the most valuable genetic resource for the genetic improvement of citrus (Gmitter et al. 2007). It produces fertile hybrids with citrus and is an important source of useful genes for citrus rootstocks (Roose et al. 1998). It is resistant or tolerant to CTV, Phytophthora root rot, citrus nematode, cold accumulation, and other environmental stresses and has been explored for use in citrus scion and rootstock genetic improvement programs via conventional breeding and molecular approaches (Cai et al. 1994; Gmitter et al. 1996; Tozlu et al. 1999a, b; Ling et al. 2000). Sexual hybridization using P. trifoliata as one of the parents has been used to produce genetically improved combinations of rootstocks for use in citrus propagation. Carrizo and Troyer citranges (C. sinensis × P. trifoliata) and Swingle citrumelo (C. paradisi × P. trifoliata) rootstocks were selected from intergeneric hybrid progeny and were found to have Phythophthora, virus, and nematode tolerance inherited from P. trifoliata. “US-852,” a hybrid obtained from sexual hybridization of C. reticulata × P. trifoliata, exhibited outstanding effects on sweet orange fruit yield, producing fruit with high soluble solids on medium size trees (Bowman et al. 1999). Four new rootstocks, two (Forner Alcaide 5 and Forner Alcaide 13) obtained by sexual hybridization between Cleopatra mandarin × P. trifoliata, one (Forner Alcaide 418) of Troyer citrange × P. trifoliata, and one (Forner Alcaide 517) of King mandarin × P. trifoliata, resistant or tolerant to CTV and salinity have been released (Nicotra 2001; Forner et al. 2003). “X639”, a hybrid between “Cleopatra” mandarin × P. trifoliata, though susceptible to nematodes and root pathogens, proved to be an excellent rootstock for lemons and mandarins (Miller et al. 2003).

9.4.2 Ploidy Manipulation

Anther culture has been used to recover haploid plantlets from P. trifoliata (Hidaka et al. 1979). Deng et al. (1992a) were able to obtain only heterozygous plantlets from anther culture of P. trifoliata. In P. trifoliata, pollen culture has also been attempted; however, plantlets were not obtained (Germana et al. 1996). The pollen developmental stages, genotype used as well as the tissue culture parameters affect the success of anther culture. The effect of different developmental stages of P. trifoliata pollen grains on the formation of embryoids, pseudobulbils, and calli has been studied (Hidaka et al. 1979). For embryoid production, the early uninucleate stage was the most suitable while anthers at other developmental stages from pollen mother cell to bicellular stage produced only calli (Germana 2007). Hidaka (1984) studied the effects of sucrose concentration (1, 3, 5, 7, and 9%) on embryoid and callus formation and found that 3% sucrose was ideal for embryoid formation in P. trifoliata. Medium supplemented with 0.2 mg/l of both indole-3-acetic acid (IAA) and kinetin (Kn) was found to be efficient for embryoid formation while the addition of 2,4-dicholorphenoxy acetic acid (2,4-d) induced callus formation in P. trifoliata. Deng et al. (1992b) found that the addition of α-naphthaleneacetic acid (NAA) and activated charcoal in the medium induced embryoid formation in P. trifoliata.

Hybrid embryo rescue has also been exploited for the genetic improvement of Poncirus. P. trifoliata is cold hardy and resistant to root-rot, CTV, and citrus-browning nematode. However, it is susceptible to citrus exocortis viriod (CVC). Controlled crosses, followed by triploid hybrid embryo rescue, were carried out between Red tangerine (which is resistant to CVC) × P. trifoliata as well as Satsuma mandarin (which is citrus canker tolerant) × P. trifoliata to introduce these new characters into P. trifoliata (Tan et al. 2007). To produce triploid intergeneric hybrids, gametosomatic fusion between P. trifoliata tetrads and somatic protoplasts of C. sinensis has been reported (Deng et al. 1992b).

Somatic hybridization allows the production of somatic hybrids that incorporate genomes of the two parents without recombination, thus avoiding the problem of the high heterozygosity (Navarro et al. 2004). Production of tetraploid somatic hybrids that combine complementary diploid rootstock germplasm via protoplast fusion has become a practical strategy with the overall objective of packaging necessary disease and pest resistance into horticulturally desirable, widely adapted rootstock such as Poncirus. In Poncirus, the first somatic hybrid was obtained between C. sinensis and P. trifoliata (Ohgawara et al. 1985). These results allowed the establishment of rootstock breeding programs in several countries. A number of somatic hybrids with Poncirus as one of the parents have been generated (Table 9.2) and are at different stages of field trial (Grosser et al. 2000). Somatic hybrid rootstocks are showing good potential to reduce tree size, as needed, for more efficient high-density plantings with good yields of high quality (Grosser 2003). Seed trees of most of these somatic hybrid rootstocks are also producing adequate nucellar seeds for standard propagation (Grosser et al. 2000).

Table 9.2 Somatic hybrids regenerated using Poncirus as one of the parents
Full size table

9.4.3 Genetic Engineering

Genetic transformation of Poncirus and its hybrids has been achieved (Table 9.3). The first efficient protocol for transformation of seedling explants of P. trifoliata was established by Kaneyoshi et al. (1994), which was subsequently used by many groups (Kobayashi et al. 1996; Kaneyoshi and Kobayashi 1999; Wong et al. 2001; Iwanami et al. 2004; Endo et al. 2005). A similar protocol was used to transform Carrizo citrange (C. sinensis × P. trifoliata) that did not respond as well as P. trifoliata (Pena et al. 1995a). Hence, several factors affecting transformation and regeneration were critically studied (Cervera et al. 1998a). Cocultivation of epicotyl or internodal stem segments with Agrobacterium tumefaciens has been the most commonly used systems to efficiently produce transgenic plants of P. trifoliata (Kaneyoshi et al. 1994) and citrange (C. sinensis × P. trifoliata, Pena et al. 1995b; Gutierrez et al. 1997; Cervera et al. 1998b). To enhance both regeneration and transformation frequency, Yu et al. (2002) proposed cutting longitudinally the epicotyl segments of Carrizo citrange in two halves. Thin layers of about 1–2 mm cut transversally from etiolated epicotyls were found to be highly organogenic in P. trifoliata, Swingle citrumelo and Carrizo citrange transformation (Le et al. 1999; Molinari et al. 2004a, b).

Table 9.3 Summary of Agrobacterium-mediated transformation of Poncirus trifoliata and its hybrids
Full size table

Several authors have proposed the use of rol genes from the Ri plasmid as transgenes to produce dwarf P. trifoliata and citrange rootstocks (Gentile et al. 1998; Kaneyoshi and Kobayashi 1999). rolC gene from A. rhizogenes has been successfully incorporated into P. trifoliata (Kaneyoshi and Kobayashi 1999). Human epidermal growth factor (hEGF) has also been incorporated into P. trifoliata (Kobayashi et al. 1996). A citrus gibberellin (GA) 20-oxidase cDNA (CcGA20ox1) gene that controls the plant architecture and a halotolerance gene HAL2 have been introduced into Carrizo citrange (Cervera et al. 2000a; Fagoaga et al. 2007). Arabidopsis genes such as LEAFY or APETALA1 that alter the growth habit, reduce juvenility, and regulate vegetative and other behavior have been introduced into juvenile Carrizo citrange seedling explants (Pena et al. 2001). Regenerants of Carrizo citrange obtained under selective conditions after Agrobacterium-mediated transformation have been used for the characterization of these regenerants into silenced and/or chimeric plants (Dominguez et al. 2004). Gene constructs have been created for various types of CTV-derived genes and have been introduced into Carrizo citrange in efforts to induce resistance to the CTV virus (Gutierrez et al. 1997). A citrus blight-associated gene has also been introduced into Carrizo citrange (Kayim et al. 2004). Coat protein gene from citrus mosaic virus (CiMV) has been incorporated into P. trifoliata (Iwanami et al. 2004). Another gene FLOWERING LOCUS T that reduces the time of flowering has been incorporated into P. trifoliata and the transformed regenerants flowered in less than 8 months with four out of six transgenic lines developed normal fruits with intact seeds (Endo et al. 2005). In Spain, 16 transgenic plants of Carrizo citrange, with two plants each from eight independent transgenic lines, have been released under an experimentally controlled field for further evaluation (Pena et al. 2008).

9.5 Genomics Resources Developed

Poncirus and its hybrids have been extensively used in EST-sequencing efforts (Talon and Gmitter Jr 2008). They have also been used to generate ESTs from several libraries under biotic (Xylella fastidiosa, CTV, citrus leprosis virus, Phythophthora, mite) and abiotic (drought) stresses, and during fruit development (Machado et al. 2007). Around 62,344 ESTs have been generated from P. trifoliata, and 9,791 from its hybrids with citrus (http://www.int-citrusgenome.org/usa/) using various tissues as seed, leaf, bark, greenhouse and field-grown plants, etc. An Affymetrix citrus GeneChip has been developed, which contains probe sets for detection of several pathogens and commonly used transgenes, and a representation of the region of the P. trifoliata genome containing Ctv, the CTV resistance allele (Close et al. 2006; Talon and Gmitter Jr 2008).

9.6 Conclusion

Poncirus is found in all citrus-growing regions of the world. It is known to have originated in China, where it is also used for its medicinal properties. In certain parts of Europe, it is grown as an ornamental plant. It is propagated by seeds and is a useful rootstock for citrus. Its genetic signatures of resistance to major diseases (such as CTV, nematode, etc.) and environmental stresses (such as salinity, temperature, etc.) have been exploited by citrus breeders in their various rootstock improvement programs. It is also highly amenable to plant tissue culture techniques. Haploid plants have been produced via anther culture, hybrid embryo rescue has been utilized to produce triploids, and somatic hybridization has given rise to tetraploid plants in Poncirus. These haploids, triploids, and tetraploids are highly beneficial for citrus scion and rootstock breeding programs. It has also been genetically transformed to establish and standardize the protocol for use in citrus improvement.

Poncirus has been extensively used as a parent in intergeneric crosses with citrus. This has given rise to optimum polymorphism, which has facilitated the construction of a number of molecular genetic linkage maps. The identification, tagging, and cloning of the economically important genes will provide new information and gene targets for genetic manipulation, and hence will be of great use in citrus genetic improvement. The mapping populations of the intergeneric crosses between citrus and Poncirus have also been used to construct BAC libraries, develop ESTs, used in microarrays, and to develop sequence-based maps. Their gene sequence divergence, synteny, orientation, and possible probable functions have been annotated and compared. These genomic resources will have a great impact on the whole-genome sequencing of citrus. As the whole citrus genome is sequenced, subsequent exploration, comparison, and utilization of that data would be beneficial for the genetic improvement of Poncirus.