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New Avenues for Somatic Hybridization in Citrus






Haploid + diploid protoplast fusions and gametosomatic hybridizations

Triploid breeding is an important method in citrus variety improvement, mainly for


small citrus fruit and lemons. For all the strategies involving 2 n gametes, interploid sexual crosses or endosperm culture, both of the parental genomes are submitted to meiotic recombination. Maximum het- erozygosity in triploid progeny will be obtained from interploid crosses involving an allotetraploid parent. Concerning triploids arising in diploid sexual crosses, it appears that only a part of parental het- erozygosity is present in the 2 n gametes producing the spontaneous triploids (Ollitrault et al., 1998). Considering that cultivars are generally highly heterozygous, it is clear that in most cases the selected genetic balances of the diploid parental cul- tivars will be lost in triploid hybrids. So, the effectiveness of selection made at the


 


diploid level for complex characters is low, and it is necessary to make fi nal selections from a large number of triploid hybrids. The production of allotriploids from somatic hybridizations between haploid potato and diploid tomato has been previ- ously described (Schoenmakers et al., 1991), while triploid somatic hybrid plants of Nicotiana and Petunia have been obtained by gametic + somatic (n + 2 n) pro- toplast fusions (Pirrie and Power, 1986; Lee and Power, 1988). These methods allow the synthesis of triploids in one cycle of hybridization, and provide an interesting alternative to diploid ´ tetraploid sexual crosses or spontaneous triploid selection. Indeed it is the only method allowing the addition of a haploid genome to the whole genome of a diploid cultivar without recombination.

Triploid citrus hybrids have been syn- thesized by somatic hybridization between diploid cultivars and haploid lines (Kobayashi et al., 1997; Ollitrault et al., 1997, 1998). Indeed, the regeneration of haploid plants and cell lines by anther cul- ture (Germaná, 1992) or by induced gyno- genesis (Oiyama and Kobayashi, 1993; Ollitrault et al., 1996a) has opened up avenues for this new breeding scheme. Ollitrault et al. (1998, 2000b) have com- bined two haploid cell lines of clementine obtained by induced gynogenesis with 11 diploid cultivars. Triploid and tetraploid hybrids have been obtained for each combi- nation as well as a few pentaploid hybrids. Triploid hybrids should be exploited directly and will soon be evaluated in trop- ical, subtropical and Mediterranean areas. Tetraploid hybrids obtained by this approach will join the pool of allote- traploids for further diploid ´ tetraploid sexual crosses.

Citrus is the fi rst example of triploid hybrids obtained by somatic hybridization in fruit crops. The main limitation of this strategy is the lack of haploid lines. This could be overcome by the applica- tion of gameto-somatic hybridization mentioned by Z. A. Deng et al. (1992) and X.X. Deng et al. (1995), who have


regenerated only chimeric plants with 18 and 19 chromosomes. This technique is currently being developed by CIRAD (France), and will hopefully allow the pro- duction of polymorph triploid progeny recombining only for the haploid source. If successful, this would provide a new avenue for citrus genetics and breeding research.

 

 

Asymmetric nuclear hybridization

For numerous crops, somatic hybridization is a way to by-pass sexual incompatibility between species. In this case, it is of inter- est to transfer only a limited amount of genetic material from a foreign species to a crop rather than to combine two complete genomes (Forsberg et al., 1998b). The diffi - culty in obtaining viable somatic hybrids between Citrus and very distant species suggests that this strategy should be inter- esting for introgression of resistance charac- ters from wild germplasm, such as resistance of Murraya paniculata against Huanglongbing. Moreover the diversifi ca- tion and breeding of species such as sweet orange or grapefruit (that are not amenable to conventional breeding) would probably benefi t greatly from such a strategy, even for partial transfer of the genome of compatible species. Two main methods can be distin- guished for partial nuclear genome transfer leading to the development of asymmetric hybrids: chromosome fragmentation before somatic hybridization and microprotoplast fusion.

 

 

Transfer of fragmented chromosomes

The most common method used for asym- metric hybridization has been to expose the donor protoplasts to g irradiation (Bates et al., 1987) or X irradiation (Dudits et al., 1980). Correlations between irradiation dose and the degree of chromosome elimi- nation have been demonstrated in some studies (Melzer and O’Connell, 1992; Schoenmakers et al., 1994). More recently,


 


 

UV irradiation has also been proven to be effi cient to fragment and eliminate chromo- somes (Jazdzewska et al., 1995; Forsberg et al., 1998a, b). The transfer of fragmented chromosomes demonstrates its effi ciency when it was combined with in vitro selec- tion pressure. For instance, it has allowed transfer of resistances to methotrexate and 5-methyltryptophan from carrot to tobacco (Dudits et al., 1987), resistance to Phoma linguam from Brassica juncea, Brassica car- inata and Brassica nigra to Brassica napus (Sjö din and Glimenius, 1989) or resistance to tobacco mosaic virus from Nicotiana repanda to Nicotiana tabacum (Bates, 1990). More recently, the use of fl uores- cence-activated cell sorting (FACS) has been proven to be effi cient to select asym- metric hybrids just after fusion (Rasmussen et al., 1997). The proportion of foreign genome transfer is usually evaluated with RFLP (Forsberg et al., 1998b), RAPD (Rasmussen et al., 1997) or AFLP (Tian and Rose, 1999).

This technique has not been explored in citrus. It should fi nd application to breed tolerant cultivars when in vitro selection is possible both for pathogens such as Mal Secco by application of toxin in cell cul- tures (Gentile, 1992) and abiotic stresses such as salinity (Spiegel Roy and Ben Hayvin, 1985). This should also be a way to induce genetic diversity in very monomorphic species such as sweet orange and grapefruits. However, the uncontrolled transfer of multiple chromosome fragments would require a very strong effort of selec- tion in the fi eld as well as the development of molecular markers to select specifi c traits in large populations.

 

 

Chromosome transfer by microprotoplast technique

The microprotoplast-mediated chromo- some transfer (MMCT) method was origi- nally developed for mammalian cells. Micronuclear induction in plant by herbi- cides (amiprosphosmethyl, oryzalin) was demonstrated in Solanum tuberosum,


 

Daucus carota, Nicotiana plumbaginifolia and Helianthus sp. (Morejohn et al., 1987; Verhoeven et al., 1990; Ramulu et al., 1994; Binsfeld et al., 2000). Successful induction of micronuclei and microprotoplast isola- tions for asymmetric hybridization have allowed the transfer of a single potato chro- mosome to tomato and tobacco (Ramulu et al., 1996a, b), and addition lines in sun- fl ower (Binsfeld et al., 2000).

For citrus, Louzada et al. (2002) have developed a method to produce citrus microprotoplasts containing a limited number of chromosomes, and used them in protoplast fusions with diploid lines. If suc- cessful, this method should have very inter- esting applications both for genetic study with monosomic addition line and for breeding. Indeed, it is possible that the addition of a single chromosome from wild species may be more efficient to obtain viable and interesting cultivars than symmetric hybridization. Moreover, the preservation of pomological and organoleptic specifi c traits of species such as sweet orange and grapefruit should be more effi cient by adding single chromo- somes of the Citrus gene pool for improving disease tolerance/resistance.

 

 

Somatic cybridization

Production of diploid somatic hybrid plants containing the nuclear genome of one parent and either the cytoplasmic genome of the other parent or a combina- tion of both parents (cybrids) has been a common approach in plant improvement. Characterization of such cybrids can deter- mine cytoplasm inheritance and cytoplasm- coded agronomic traits, and may lead to improved selections (Kumar and Cocking, 1987). Male sterility and tolerance traits with cytoplasmic determinism have been the more common objective of such strate- gies (Pelletier et al., 1983; Barsby et al., 1987; Thomzik and Hain, 1988; Varotto et al., 2001). Classically, cybrid and alloplas- mic plants are obtained by asymmetric pro- toplast fusions between irradiated donor


 


protoplasts (with destroyed nuclei) and recipient protoplasts whose mitochondria and chloroplasts have been inactived by iodoacetate treatments (Sidorov et al., 1981; Galun et al., 1987; Vardi et al., 1987; Li et al., 1993). Cytoplasmic genome recombina- tion should occur and concern principally the mitochondrial genome (Belliard et al., 1979; Rothenberg et al., 1985; D’Hont et al., 1987) while reports of new chloroplast profiles are very rare (Medgyest et al., 1985).

Besides the success achieved in sym- metric citrus somatic hybridization world- wide, only a few reports can be found pursuing the production of citrus cybrid plants by asymmetric somatic hybridization (Vardi et al., 1987, 1989; Li and Deng, 1997; Liu and Deng, 2002). The challenging methodology involved with the original donor–recipient method and the lack of information concerning cytoplasmic traits initially slowed the evolution of cybridiza- tion in citrus. However, chance and nature have played an important role in this fi eld, allowing the regeneration of several citrus alloplasmic plants as a by-product from the application of standard somatic hybridiza- tion procedures (Kobayashi et al., 1988; Ohgawara et al., 1989, 1991; Tusa et al., 1990; Saito et al., 1993, 1994; Yamamoto and Kobayashi, 1995; Grosser et al., 1996a; Moreira et al., 2000a; Moriguchi et al., 1996, 1997; Ollitrault et al., 1996b, 2000b; Cabasson et al., 2001).

Potential seedlessness via cybridization

As mentioned, seedlessness is a prerequi- site for new fresh market citrus cultivars. Seedlessness in diploid citrus generally relates to male and/or female sterility. The seedless satsuma mandarin is typically male sterile, and its male sterility has been identifi ed to be a cytoplasmic male-sterile (CMS) type (Yamamoto et al., 1997). CMS in higher plants is known to be controlled by mitochondrial DNA (mtDNA). Navel orange is also male sterile, accompanied by partial ovule sterility, and it has not been


determined whether its sterility is the CMS type. Due to complex citrus biology, it is not easy to transfer the sterility character from satsuma mandarin to other seedy citrus cultivars by conventional breeding. However, it may be possible to transfer the CMS trait of satsuma (and possibly navel orange) into commercially important diploid cultivars via cybridization, and efforts are underway to achieve this.

For citrus somatic hybridization, the fusion model of ‘diploid embryogenic pro- toplasts + diploid leaf-derived protoplasts’ has been used extensively. Normally, unfused leaf protoplasts do not divide and regenerate into plants. However, diploid plants resembling the leaf parent morpho- logically have been recovered unexpectedly from more than 30 symmetrical fusion com- binations (Deng et al., 2000; Grosser et al., 2000). In all such cases examined, RFLP analysis indicated that these plants were not directly regenerated from unfused leaf protoplasts, but were cybrids with the nuclear DNA from the leaf parent and the mtDNA from the corresponding embryo- genic parent (Saito et al., 1993; Yamamoto and Kobayashi, 1995; Grosser et al., 1996a; Moriguchi et al., 1996; Moreira et al., 2000a; Cabasson et al., 2001; Guo et al., 2002, Wuhan, PR China, unpublished data). Chloroplast DNA was randomly inherited in these plants. Moreira et al. (2000a) theo- rized that leaf protoplasts do not have an adequate quantity of mitochondria as needed to undergo somatic embryogenesis, and that cybridization fulfi ls this need. These results suggest that it should be pos- sible to transfer the mtDNA from male-ster- ile cultivars to seedy diploid fresh fruit cultivars by simply conducting symmetric fusion experiments, and the research groups directed by X.X. Deng and J.W. Grosser are collaborating to achieve this. Using an embryogenic suspension culture of ‘Guoqing No. 1’ satsuma mandarin pro- vided by X.X. Deng, the Grosser laboratory has produced diploid cybrid plants of ‘Hirado Buntan Pink’ pummelo, ‘Sunburst’ mandarin and an unnamed ‘Clementine’ ´ ‘Murcott’ hybrid (Guo et al., 2004a).


 


 

Confi rmation of the cybridity of the latter two combinations has been diffi cult due to the close relatedness of the parental mito- chondrial genomes. These plants will be fruited and fl owered as soon as possible to determine if the substitution of satsuma mtDNA can result in a new mitochon- dria–nucleus interaction that could result in making these cultivars seedless without otherwise altering their cultivar integrity. If successful, this strategy could be applied to remove seed from many superior diploid cultivars. With the same objective, Xu et al. (2006) have developed a very promising new technique of cytoplast isolation and fusion to generate citrus hybrids.

 

 

Breeding agronomic traits via cybridization

With the exception of potential seedless- ness, the agronomic value of citrus cybrids is currently unknown. The evaluation of citrus cybrids in the fi eld will allow charac- terization of agronomic traits encoded by the cytoplasmic genome. Tusa et al. (2000) suggested from cybrid evaluation that spe- cifi c mechanisms of resistance against Mal secco could be activated in these genotypes. Mandarin and sweet orange cybrids in the fi eld at the CREC (Florida) are showing sig- nifi cant variation in agronomically impor- tant traits including fruit maturity date and seed content. In the same way, Fanciullino et al. (2005) found signifi cant quantitative variation of aromatic compounds in cybrids indicating that cybridization is a potential source of genetic variation for citrus culti- var improvement. As an additional interest, the development of citrus cybrid callus should increase the range of somatic hybridization parents (Saito et al., 1994; Grosser et al., 1996a).

 

 

Scion improvement: potential tetraploid somatic hybrid cultivars

Several allotetraploid somatic citrus hybrids produced for the purpose of serving as tetraploid breeding parents in interploid


 

crosses to generate seedless triploids have fl owered and produced fruit. Although still emerging through juvenility, a few somatic hybrids are producing fruit with cultivar potential. A somatic hybrid of ‘Succari’ sweet orange + ‘Page’ tangelo produces seedless fruit with a shape similar to that of sweet orange. Fruits of this hybrid are very early maturing and have had sugar/acid ratios over 15 by the beginning of October of the past two seasons. These fruits also have adequate juice content and excellent fl avour. This hybrid therefore has potential as an early season fresh fruit cultivar. Another somatic hybrid of ‘Valencia’ sweet orange + ‘Murcott’ tangor produces nearly seedless fruits (about one seed every three fruit). Fruits of this hybrid are very much intermediate to that of the parents, possess- ing excellent fl avour, and are peelable. This hybrid has potential as a mid–late season fresh fruit cultivar. It is possible that some somatic hybrids are seedless because of mutations that accumulate in the embryo- genic callus lines used as parents. These results suggest potential for designing fusion experiments to generate hybrids with cultivar potential at the tetraploid level, and CREC have conducted numerous fusion experiments to achieve this. CREC has focused on using parents that have been successful in previous work, or comple- mentary parents that provide low acidity, high juice content and/or excellent fruit quality (i.e. colour and fl avour). Of course any resulting hybrids would also have great potential as tetraploid breeding parents. Recent successes of CREC in the endeavour are summarized in Table 10.1. (Guo et al., 2004b). As with many other recent somatic fusion experiments, fl ow cytometry (using a Partec table-top model) was used to identify tetraploid embryos early on in the plant regeneration process to save time and money. These hybrids will all be top- worked to mature fi eld trees to expedite fl owering and fruiting as needed to acceler- ate the evaluation process.


 

Table 10.1. New allotetraploid citrus somatic hybrids with direct cultivar potential produced at the CREC.

 

Embryogenic callus parent + Leaf parent
‘Guoquing’ Satsuma No. 1 + ‘Murcott’ tangor
‘Page’ tangelo + LB8-9 (Clementine ´ Minneola)
‘Page’ tangelo + Lee hybrid (Clementine ´ Murcott)
‘Page’ tangelo + unnamed (Clementine ´ Satsuma)
‘Page’ tangelo + ‘Ortanique’ tangor
‘Page’ tangelo + ‘Murcott’ tangor
‘Murcott’ tangor + ‘Dancy’ tangerine

 

 


Rootstock improvement: building a better sour orange

Sour orange was formerly the most impor- tant rootstock worldwide due to its wide adaptation, tolerance to citrus blight and ability to produce good yields of high qual- ity fruit. However, due to its susceptibility to citrus tristeza virus (CTV)-induced quick decline disease, it can no longer be used in most situations. A suitable replacement rootstock has yet to be developed. Molecular marker analyses indicate that sour orange is a hybrid of pummelo and mandarin (Nicolosi et al., 2000), but it is unlikely that it was produced from the best pummelo or mandarin. This report also demonstrates that the vigorous rootstocks including rough lemon, rangpur, Palestine sweet lime and Volkamer lemon have a sig- nifi cant genetic contribution from citron. Such vigorous rootstocks are also highly susceptible to citrus blight, and generally produce fruit of poor quality. Perhaps avoiding citron-based material in building new rootstocks could minimize these prob- lems. CREC therefore use somatic hybridization to combine widely adapted mandarins (Amblycarpa and Shekwasha mandarins) with tristeza-resistant pumme- los or superior pummelo seedlings selected following germination of seed in fl ats of winder soil (calcareous soil, pH = 5.8) inoc- ulated with both Phytophthora nicotianae and P. palmivora. (Note: a successful replacement for sour orange must also be able to handle challenging soils and


Phytophthora.) To date, ten such mandarin

+ pummelo somatic hybrids have been pro- duced, and several are showing excellent nursery vigour (Table 10.2) (Grosser et al., 2004). These promising hybrids and others produced subsequently will be immedi- ately tested for resistance to tristeza- induced quick decline disease and entered into fi eld trials. It should be possible to develop a quick decline-resistant replace- ment for sour orange that also provides some level of tree size control.

 

 

Rootstock breeding at the tetraploid level: production of ‘tetrazygs’

The breeding of somatic hybrids at the tetraploid level provides an opportunity to mix the genes from three to four proven rootstocks (or other valuable contributing parents) simultaneously, thereby maximiz- ing genetic diversity in progeny. CREC has used two somatic hybrids, Nova mandarin

+ Hirado buntan pummelo (zygotic) and sour orange + rangpur, that are performing well in fi eld trials and produce high per- centages of zygotic seed, as females in such crosses at the tetraploid level. Selected high performance somatic hybrids including sour orange + Carrizo, Cleopatra + trifoliate orange and sour orange + Palestine sweet lime are being used as pollen parents. Grosser et al. (2003) have coined the term ‘tetrazygs’ to identify tetraploid progeny from such crosses. They routinely screen progeny from such crosses by germinating


 

 

Table 10.2. New mandarin + pummelo somatic hybrids produced recently at the CREC – efforts to rebuild a better ‘sour orange’ rootstock.

 

Embryogenic parent + Leaf parent
‘Murcott’ tangor + Citrus grandis ‘Hirado Buntan Pink’ (HBP)a
‘Murcott’ + HBP sdl-JL1a
Amblycarpa mandarin (C. amblycarpa) + HBPa
Amblycarpa + C. grandis ‘Chandler’a
Amblycarpa + HBP sdl-JL1
Amblycarpa + HBP sdl-5-1-99-1Ba
Amblycarpa + HBP sdl-JL2Ba
Amblycarpa + C. grandis ‘LingPingYau’ sdl-8-1-99-4Aa
Amblycarpa + HBP sdl-JL4
Amblycarpa + Chandler sdl-A1-11
Shekwasha mandarin + HBPa
Shekwasha + ‘Chandler’a
Amblycarpa + HBP sdl-JL12
aHybrid confi rmed by RAPD analyses    

 


seed directly in a high pH, calcareous ‘winder’ soil inoculated with Phyto- phthora. Superior ‘tetrazyg’ hybrids are then grafted with sweet orange infected with a quick decline isolate of CTV to deter- mine their resistance to CTV-induced quick decline disease. Simultaneously, the selected hybrids can be propagated by top- working and/or rooted cuttings to provide clonal material for further evaluation. This approach is expected to shorten the time required to develop a new rootstock by 8–10 years. This programme began at the CREC in 1999, and so far more than 300 genetically diverse ‘tetrazyg’ hybrids have been selected for further evaluation. ‘Tetrazygs’ are also being screened for toler- ance to salinity and to the Diaprepes root weevil– Phytophthora complex (Grosser et al., 2003).

 

 


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