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Ying Jia, Dianren Xia, and E.S. Louzada

A cDNA coding for a putative terpene synthase (Grtps) was isolated from `Rio Red' grapefruit (Citrus paradisi Macf.) mature fruit by differential display RT-PCR and the corresponding full-length cDNA and genomic clone were subsequently obtained. The isolated cDNA clone was 1644 bp in length encoding a protein of 548 amino acids with a predicted molecular mass of 64 kDa and of pI 5.38. The genomic clone was 3203 bp in length with 6 introns and 7 exons. This Grtps appears to be a sesquiterpene synthase based on molecular weight, genomic organization, and similarity with the other terpene synthases. Both RT-PCR and Northern blot expression analysis indicated that Grtps is not expressed in immature fruits, roots, or leaves, but only in mature fruits. Southern blot analysis of genomic DNA demonstrated that Grtps is one of the members in the family of terpene synthases.

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E.S. Louzada, H.S. del Rio, D. Xia, and J.M. Moran-Mirabal

Large-scale production of microprotoplasts from `Ruby Red' grapefruit (Citrus paradisi Macf.) and from the Citrus L. sp. relative Swinglea glutinosa (Blanco) Merr., was performed after treatment of suspension cells with APM. An average of 75.2% of the microprotoplasts contained a single chromosome, followed by 17.1% with two, 4.6% with three, and 2.0% with four. Only 1.1% had more than five chromosomes. Maximum chromosome number observed was eight and the average yield was 2 × 106 of total microprotoplasts per gram of suspension cells. Flow cytometry analysis confirmed low DNA content. The polyethylene glycol fusion method was used to fuse microprotoplasts from `Ruby Red' grapefruit with protoplasts of `Succari' sweet orange [Citrus sinensis (L.) Osbeck], and microprotoplasts from S. glutinosa with protoplasts from sour orange (C. aurantium L.). Embryos or suspension cells from the recipient species with a few additional chromosomes were obtained; however, embryogenesis of the fusion products was reduced or inhibited. Chemical name used: amiprophos-methyl (APM).

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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.

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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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Thomas M. Gradziel, Robert J. Knight Jr., William Reid, Chad E. Finn, John R. Clark, Eliezer S. Louzada, and Kim E. Hummer

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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.