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Milica Ćalović, Chunxian Chen, Qibin Yu, Vladimir Orbović, Frederick G. Gmitter Jr, and Jude W. Grosser

unconventional methods, somatic hybridization via protoplast fusion and interploid sexual hybridization, has become an integral part of mandarin cultivar improvement programs targeting seedlessness worldwide, because they can easily overcome some mentioned

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Mihoko Tamura, Ryutaro Tao, and Akira Sugiura

Interspecific hybrids between Diospyros glandulosa (2n = 2x = 30) and D. kaki cv. Jiro (2n = 6x = 90) were produced by electrofusion of protoplasts. Protoplasts were isolated from calli derived from leaf primordia, fused electrically, and cultured by agarose-bead culture using modified KM8p medium. Relative nuclear DNA contents of calli derived from fusion-treated protoplasts were determined by flow cytometry. One-hundred-forty-nine of 166 calli obtained had the nuclear DNA content of the sum of those of D. glandulosa and D. kaki cv. Jiro. RAPD analysis showed that the 149 callus lines yielded specific bands for both D. glandulosa and D. kaki cv. Jiro and they appeared to be interspecific somatic hybrid calli. Shoots were regenerated from 63 of the 149 interspecific hybrid calli. PCR-RFLP of chloroplast DNA analysis, flow cytometric determination of nuclear DNA content, and RAPD analysis revealed that the 63 interspecific hybrid shoot lines contained nuclear genome from both the parents but only chloroplast genome from D. glandulosa. Microscopic observation of root tip cells confirmed that somatic chromosome numbers of the interspecific hybrids were 2n = 8x = 120.

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Jude W. Grosser, Hyun Joo An, Milica Calovic, Dong H. Lee, Chunxian Chen, Monica Vasconcellos, and Frederick G. Gmitter Jr

Somatic hybridization in citrus using the embryogenic suspension protoplast plus leaf protoplast fusion model can be considered a reliable technique, because hundreds of allotetraploid somatic hybrids have been produced for both scion and

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Aditi D. Satpute, Chunxian Chen, Fredrick G. Gmitter Jr., Peng Ling, Qibin Yu, Melinda R. Grosser, Jude W. Grosser, and Christine D. Chase

The genetic improvement of citrus is a challenge because of long generation times, polyembryony, and sexual incompatibilities ( Grosser and Gmitter, 1990 ). To overcome these barriers, protoplast fusion and plant regeneration techniques have gained

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Samia Lotfy, Francois Luro, Françoise Carreel, Yann Froelicher, Delphine Rist, and Patrick Ollitrault

Somatic hybridization allows the creation of new patterns of nuclear, mitochondrial and chloroplastic association. It is therefore necessary to master cytoplasmic molecular markers to determine the genetic origin of both organelles of plantlets obtained from protoplasts fusion. In the case of Citrus and related genera, only southern blot hybridization and restriction fragment-length polymorphism (RFLP) techniques were used for this task until now. Here, we describe the use in the Aurantioideae subfamily, of a simple and non labeling cleaved amplified polymorphic sequence (CAPS) technique, to determine the cytoplasmic genome origin of intergeneric somatic hybrids. Mitochondrial and chloroplastic universal primers previously selected for population genetic studies in Quercus by Demesure et al. (1995) are used with some modifications. The variability of cytoplasmic genome among somatic fusion partners is detected by coupling amplification and restriction reactions. Digested DNA fragments are analyzed by agarose gel electrophoresis (PCR-RFLP). This technique has been applied for the analysis of the cytoplasmic constitution of somatic hybrids arising from intergeneric, intersubtribal and intertribal combinations. Systematic transmission of the mitochondria from protoplasts isolated from embryogenic callus parents was confirmed.

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Jude W. Grosser, Victor Medina-Urrutia, Govindarajulu Ananthakrishnan, and Patricia Serrano

Sour orange has been a premier citrus rootstock worldwide due to its ability to perform on challenging soils and to produce and hold high-quality fruit. However, increasingly widespread quick-decline isolates of citrus tristeza virus (CTV) have destroyed entire industries on sour orange in some countries, and are in the process of destroying millions of trees on sour orange in Florida. CTV also threatens other citrus locations planted heavy to sour orange, including Texas and Mexico. An acceptable alternative rootstock to replace sour orange is in high demand but has yet to be developed. Molecular analyses have recently determined that sour orange is probably a hybrid of pummelo and mandarin. We report the production of 12 new mandarin + pummelo somatic hybrids produced by protoplast fusion from selected superior mandarin and pummelo parents, in efforts to develop a suitable replacement sour-orange-like rootstock that is resistant to CTV-induced quick decline. Somatic hybrids from all 12 parental combinations were confirmed by a combination of leaf morphology, flow cytometry, and RAPD analyses (for nuclear hybridity). These new mandarin + pummelo somatic hybrids are being propagated by rooted cuttings as necessary to conduct quick-decline resistance assays and to assess horticultural performance in replicated field trials.

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J.W. Grosser, J. Jiang, F.A.A. Mourao-Fo, E.S. Louzada, K. Baergen, J.L. Chandler, and F.G. Gmitter Jr.

Seedlessness is an important breeding objective of most citrus scion improvement programs, but production of quality seedless triploid citrus via interploid crosses has historically been limited by the low quality of available tetraploid parents. Production of tetraploid hybrid parents from elite diploid scion cultivars via protoplast fusion is now a practical strategy, and numerous hybrids can be produced on a timely basis from a wide range of parents. Such hybrids can be used as pollen parents in interploid crosses to generate improved seedless triploid fresh fruit cultivars. Herein we report the production of 15 such hybrids from 17 different parents, including sweet orange [Citrus sinensis (L.) Osbeck], mandarin/tangerine (C. reticulata Blanco), grapefruit (C. paradisi Macf.), pummelo [C. grandis (L.) Osbeck], tangor (C. reticulata × C. sinensis), and tangelo (C. reticulata × C. paradisi) germplasm. All hybrids were confirmed by cytological and RAPD analyses, and have been budded to selected rootstocks to expedite flowering.

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N. Tusa, J.W. Grosser, F.G. Gmitter Jr., and E.S. Louzada

Allotetraploid somatic hybrid plants of `Hamlin' sweet orange (Citrus sinensis L. Osbeck) + `Femminello' lemon (C. limon L. Burm. f.), and Milam lemon (purported hybrid of C. jambhiri Lush) + `Femminello' lemon were regenerated via somatic embryogenesis following protoplast fusion. `Hamlin' and Milam protoplasts were isolated from undeveloped ovule-derived embryogenic callus cultures and fused using a polyethylene glycol method with seedling leaf-derived protoplasts of `Femminello' lemon. Somatic hybrids were identified on the basis of leaf morphology, root-tip cell chromosome number, and electrophoretic analyses of phosphoglucose isomerase, phosphoglucose mutase, and 6-phosphogluconate dehydrogenase leaf isozymes. The somatic hybrids will be used in interploid crosses with lemon in an effort to generate seedless triploid lemon types with improved tolerance to mal secco disease.

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J.W. Grosser, J. Jiang, E.S. Louzada, J.L. Chandler, and F.G. Gmitter Jr.

Production of tetraploid somatic hybrids that combine complementary diploid rootstock germplasm via protoplast fusion has become a practical strategy for citrus rootstock improvement, with the overall objective of packaging necessary disease and pest resistance into horticulturally desirable, widely adapted rootstocks. Citrus somatic hybridization techniques have been advanced to the point where numerous somatic hybrid rootstocks can now be produced and propagated for evaluation on a timely basis. Herein we report the production of 11 new somatic hybrid rootstock candidates from 12 different parents, including Milam lemon hybrid (Citrus jambhiri Lush.), Cleopatra mandarin (C. reticulata Blanco), sour orange (C. aurantium L.), `Succari' sweet orange [C. sinensis (L.) Osbeck], `Redblush' grapefruit (C. paradisi Macf.), `Nova' tangelo [C. reticulata × (C. paradisi × C. reticulata)], `Kinkoji' (C. obovoidea Hort. Ex Takahashi), Swingle citrumelo [C. paradisi × Poncirus trifoliata (L.) Raf.], Carrizo citrange (C. sinensis × P. trifoliata), rough lemon 8166 (C. jambhiri), and Palestine sweet lime (C. limettoides Tan.). All hybrids were confirmed by cytological and VNTR-PCR analyses, and have been propagated, budded with a commercial scion, and field-planted for performance evaluation.

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Jude W. Grosser, Frederick G. Gmitter Jr., E.S. Louzada, and J.L. Chandler

Allotetraploid somatic hybrid plants of `Nova' tangelo [a sexual hybrid of `Clementine mandarin (C. reticulata Blanco) × `Orlando' tangelo (C. reticulata × C. paradisi Macf.)] + `Succari' sweet orange (C. sinensis L. Osbeck), and `Hamlin' sweet orange (C. sinensis L. Osbeck) + `Dancy' tangerine (C. reticulata) were regenerated following protoplast fusion. `Nova' and `Hamlin' protoplasts were isolated from ovule-derived embryogenic callus and suspension cultures, respectively, and fused using a polyethylene glycol method with seedling leaf-derived protoplasts of `Succari' and `Dancy', respectively. Plants were regenerated via somatic embryogenesis, and somatic hybrids were identified on the basis of leaf morphology, root-tip cell chromosome number, and electrophoretic analysis of peroxidase and phosphoglucose mutase isozyme banding patterns. Diploid plants were regenerated from unfused protoplasts of `Hamlin', `Nova', and `Succari'. Tetraploid plants of `Hamlin' and `Succari' were also recovered, apparently resulting from homokaryotic fusions. No `Dancy' plants were recovered. The somatic hybrid and autotetraploid plants can be used for interploid hybridization with selected monoembryonic scions to generate improved seedless triploid tangor/tangelo cultivars. The lack of suitable tetraploid breeding parents has previously inhibited the development of quality seedless cultivars by this method.