Somatic hybridization for citrus rootstock breeding: An effective tool to solve some important issues of the Mediterranean citrus industry

UPR 75, Département BIOS, CIRAD, Av. Agropolis, TA A-75/02, 34398, Montpellier Cedex 5, France.
Plant Cell Reports (Impact Factor: 3.07). 05/2011; 30(5):883-900. DOI: 10.1007/s00299-010-1000-z
Source: PubMed


The prevalence of sour orange rootstock in the southern and eastern part of the Mediterranean Basin is presently threatened by the spread of Citrus Tristeza Virus (CTV) and its main vector Toxoptera citricida, combined with abiotic constraints such as drought, salinity and alkalinity. The search for alternative CTV-resistant rootstocks that also withstand the other constraints is now considered an urgent priority for a sustainable citrus industry in the area. Complementary progenitors can be found in citrus germplasm to combine the desired traits, particularly between Poncirus and Citrus genera. The production of somatic hybrids allows cumulating all dominant traits irrespective of their heterozygosity level, and would appear to be an effective way to solve the rootstock challenge facing the Mediterranean citrus industry. This paper presents the results obtained during a regional collaborative effort between five countries, to develop new rootstocks by somatic hybridization. New embryogenic callus lines to be used for somatic hybridization have been created. Protoplast fusions have been performed at CIRAD and IVIA laboratories, focusing on intergeneric combinations. Analysis of ploidy level by flow cytometry and molecular markers confirmed the acquisition of new interesting tetraploid somatic hybrids for six combinations. Diploid cybrids with intergeneric (Citrus × Poncirus) nucleus and C. reticulata or C.
aurantifolia mitochondria were also identified for four combinations. The agronomical performance of a pre-existing somatic hybrid between Poncirus trifoliata and Citrus reticulata was validated in calcareous soils in Morocco. Somatic hybridization is now integrated into the breeding programs of the five Mediterranean countries.

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Available from: Luis Navarro, Dec 10, 2014
    • "The development of somatic hybridization technology by protoplast fusion in citrus has helped approaching goals that seemed unattainable using conventional breeding technologies, trying to combine the characteristics of the best genotypes, and overcoming barriers of sexual incompatibility (Grosser et al., 2000; Dambier et al., 2011). New rootstock hybrids recover either by conventional breeding or somatic hybridization have juvenile characters and takes many years to produce fruits and seeds to start conventional propagation for evaluation (Ollitrault and Navarro, 2012). "

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    • "embryogenic suspension derived protoplasts with leafderived protoplasts, somatic hybrid plants have been created from nearly 500 different parental combinations to date (Grosser and Gmitter 2011). Somatic hybridization played an important role in citrus seedless breeding because allotetraploid somatic hybrids are superior breeding parents in interploid crosses for seedless triploid breeding (Dambier et al. 2011; Grosser and Gmitter 2011). Meanwhile, citrus somatic cybrids were regenerated from over 40 parental combinations by standard symmetric fusion (reviewed by Guo et al. 2013). "
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    ABSTRACT: Seedlessness, an important economic trait for fresh fruit, is among the prior goal for all citrus breeding programs. Symmetric somatic hybridization provides a new strategy for citrus seedless breeding by creating cybrids transferring mitochondrial DNA (mtDNA) controlled cytoplasmic male sterility (CMS) from the callus parent Satsuma mandarin (C. unshiu Marc.) to seedy cultivars. In this study, protoplast fusion was adopted to transfer CMS from C. unshiu Marc. cv. Guoqing No. 1 (G1) to three seedy sweet oranges (C. sinensis L. Osb.), i.e. ‘Early gold’, ‘Taoye’ and ‘Hongjiang’. Flow cytometry analysis showed that 12 of 13 regenerated plants from G1 + ‘Early gold’, 9 of 12 from G1 + ‘Taoye’ and both two plants from G1 + ‘Hongjiang’ were diploids, while the remaining regenerated plants were tetraploids. Molecular analysis using 23 simple sequence repeat (SSR) markers previously proven to map to the citrus genome showed that the nuclear DNA from all recovered diploid and tetraploid plants derived from their corresponding leaf parent, while cleaved amplified polymorphic sequence analysis showed that the mtDNA of all regenerated plants derived from the callus parent, indicating that the regenerated 2X and 4X plants from all these three combinations are authentic cybrids. Furthermore, the Chloroplast SSR analysis revealed that somatic cybrid plants from the three combinations possessed either of their parental chloroplast type in most cases. These results demonstrated that mtDNA of G1 Satsuma mandarin was successfully introduced into the three seedy sweet orange cultivars for potential seedlessness via symmetric fusion.
    Full-text · Article · Jan 2014 · Plant Cell Tissue and Organ Culture
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    • "Citrus is mainly diploid. However, many modern breeding projects for the production of seedless mandarins based on the production of triploid hybrids (Ollitrault et al., 2008; Aleza et al., 2010, 2012a, b) and tetraploid rootstocks are promising (Saleh et al., 2008; Dambier et al., 2011; Grosser and Gmitter, 2011). Triploid populations in citrus can arise from unreduced gametes in crosses between diploid parents or from interploid (diploid × tetraploid or tetraploid × diploid) crosses. "
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    ABSTRACT: Background Polyploidy is a major component of eukaryote evolution. Estimation of allele copy numbers for molecular markers has long been considered a challenge for polyploid species, while this process is essential for most genetic research. With the increasing availability and whole-genome coverage of single nucleotide polymorphism (SNP) markers, it is essential to implement a versatile SNP genotyping method to assign allelic configuration efficiently in polyploids.ScopeThis work evaluates the usefulness of the KASPar method, based on competitive allele-specific PCR, for the assignment of SNP allelic configuration. Citrus was chosen as a model because of its economic importance, the ongoing worldwide polyploidy manipulation projects for cultivar and rootstock breeding, and the increasing availability of SNP markers.Conclusions Fifteen SNP markers were successfully designed that produced clear allele signals that were in agreement with previous genotyping results at the diploid level. The analysis of DNA mixes between two haploid lines (Clementine and pummelo) at 13 different ratios revealed a very high correlation (average = 0·9796; s.d. = 0·0094) between the allele ratio and two parameters [θ angle = tan(-1) (y/x) and y' = y/(x + y)] derived from the two normalized allele signals (x and y) provided by KASPar. Separated cluster analysis and analysis of variance (ANOVA) from mixed DNA simulating triploid and tetraploid hybrids provided 99·71 % correct allelic configuration. Moreover, triploid populations arising from 2n gametes and interploid crosses were easily genotyped and provided useful genetic information. This work demonstrates that the KASPar SNP genotyping technique is an efficient way to assign heterozygous allelic configurations within polyploid populations. This method is accurate, simple and cost-effective. Moreover, it may be useful for quantitative studies, such as relative allele-specific expression analysis and bulk segregant analysis.
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