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Chunxian Chen, Jude W. Grosser, Milica Ćalović, Patricia Serrano, Gemma Pasquali, Julie Gmitter, and Fred G. Gmitter Jr

followed by the processes of validation. Somatic hybrids have been identified first by the complementary and/or intermediate morphology of regenerants followed by confirmation of ploidy level, and finally molecular markers are used to confirm the presence

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

-quality ‘Navel’ sweet orange ( C. sinensis )-like hybrid from a cross of ‘LB8-9’ SugarBelle ® [ C. clementina × ( C. reticulata × C. paradisi )] with autotetraploid ‘Hamlin’ sweet orange. By using somatic hybridization, it is possible to produce novel

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Jane Kahia, Margaret Kirika, Hudson Lubabali, and Sinclair Mantell

weaning. Effect of cytokinin type and concentration on induction of somatic embryos. The presence of cytokinins strongly enhanced the induction and regeneration of somatic embryos in the new hybrid. Initiation of somatic embryos was first observed after 2

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

Protoplasm culture following polyethylene glycol-induced fusion resulted in the regeneration of tetraploid somatic hybrid plants from the following attempted parental combinations: Cleopatra mandarin (Citrus reticulata Blanco) + Argentine trifoliate orange [Poncirus trifoliata (L.) Raf.]; `Succari' sweet orange [C. sinensis (L.) Osb.] + Argentine trifoliate orange; sour orange (C. aurantium L.) + Flying Dragon trifoliate orange (P. trifolita); sour orange + Rangpur (C. limonia Osb.); and Milam lemon (purported sexual hybrid of C. jambhiri Lush × C. sinensis) + Sun Chu Sha mandarin (C. reticulate Blanco). Protoplasm isolation, fusion, and culture were conducted according to previously published methods. Regenerated plants were classified according to leaf morphology, chromosome number, and peroxidase, phosphoglucomutase, and phosphoglucose isomerase leaf isozyme profiles. All of the somatic hybrid plants were tetraploid, as expected (2n = 4x = 36), and all five selections have been propagated and entered into commercial citrus rootstock trials.

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T.J. Montagno, P.S. Jourdan, and S. Z. Berry

Unilateral incompatibility has limited the direction of crossing between L. esculentum and L. hirsutum; the latter can only serve as the pollen parent. In an attempt to introduce the L. hirsutum cytoplasm into L. esculentum, thirty-three somatic hybrid plants have been regenerated following four separate fusions between leaf protoplasts of L. hirsutum PI 126445 and etiolated hypocotyl protoplasts of L. esculentum (`OH7870', `OH832', and `OH8245'). A 33% PEG solution supplemented with 10% DMSO was used as the fusogen. Selection of fusion products was based on treatment of L. hirsutum protoplasts with 1 mM iodoacetic acid and non-regenerability of the L. esculentum genotypes. Hybridity was initially confirmed by intermediate morphology, including leaf shape, type of trichomes, flower shape, stigma placement, and fruit size and color. Isozyme analysis for GOT, PGM, and 6-PDH verified hybridity. Six of the hybrids produced viable seed upon selfing. At least some of the hybrids contained chloroplast DNA from L. hirsutum, indicating that the wild species cytoplasm may be present in these plants.

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Victor Medina-Urrutia, Karla Fabiola, Lopez Madera, Patricia Serrano, G. Ananthakrishnan, Jude W. Grosser, and Wenwu Guo

No presently available rootstock combines all the available rootstock attributes necessary for efficient long-term citriculture (production and harvesting) of Mexican limes and other commercially important scions. In the present study, somatic hybridization techniques were used to combine the widely adapted Amblycarpa mandarin (also known as Nasnaran mandarin) with six different trifoliate/trifoliate hybrid selections: Benton, Carrizo, and C-35 citranges; Flying Dragon and Rubidoux trifoliate oranges; and a somatic hybrid of sour orange + Flying Dragon. The ultimate goal of this research is to generate polyploid somatic hybrids that express the complementary horticultural and disease resistance attributes of the corresponding parents, and have direct potential as improved tree-size controlling rootstocks. Somatic hybrids from all six parental combinations were confirmed by a combination of leaf morphology, flow cytometry, and randomly amplified polymorphic DNA (RAPD) (for nuclear hybridity) and cleaved amplified polymorphic sequence (CAPS) analyses (for mtDNA and cpDNA). This is the first report of citrus somatic hybridization using Amblycarpa mandarin. Unexpected hexaploid somatic hybrid plants were recovered from the fusion of Amblycarpa mandarin + C-35 citrange. Hexaploid hybrids should be very dwarfing and may have potential for producing potted ornamental citrus. Resulting somatic hybrid plants from all six combinations have been propagated by tissue culture and/or rooted cuttings and are being prepared for commercial field evaluation for their potential as improved rootstocks for Mexican lime and other important scions.

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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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Jae-Dong Chung, Hong-Yul Kim, Jung-Hae Suh, Oh-Chang Kwon, and Chang-kil Kim

Somatic embryo formation was observed on thin-sectioned leaf explants within 3 weeks of culture from two Phalaenopsis hybrids—Phalaenopsis Hwafeng Redjewell `Ching Ruey' Phalaenopsis Chingruey's Giant Ching Ruey' (R×R), and Phalaenopsis Formosa Best Girl Ching Ruey' Depts. Lih Jiang Beauty `S 566' (WR×WR). Frequency of somatic embryo formation was higher in hybrid WRxWR than R×R and optimal concentration of TDZ for the induction of somatic embryos was 9.08 μM. In (WR×WR) embryo proliferation was simultaneously observed after transferring the explants with somatic embryo clumps onto PGR-free half-strength MS medium. Six months after initiation, the culture plantlets were produced. This is the first report on somatic embryogenesis induced directly from the leaf explants using TDZ in Phalaenopsis.

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Jude Grosser, Milicia Calovic, Patricia Serrano, Fred Gmitter Jr., and J. L. Chandler

The international fresh citrus market now demands high-quality, seedless fruit that must also be easy to peel for consumer convenience, especially when considering new mandarin varieties. High quality varieties that historically perform well in Florida are generally seedy. Florida is therefore losing market-share to `Clementine' and other seedless varieties produced in Mediterranean climates, including Spain, Morocco, and California. In our ongoing program, somatic hybridization and cybridization via protoplast fusion are now playing a key role in strategies to develop competitive seedless mandarin hybrids adapted to Florida. Somatic hybridization is being used to combine elite diploid parents to produce high quality allotetraploid breeding parents that can be used in interploid crosses to generate seedless triploids. Several thousand triploid mandarin hybrids have been produced under the direction of F.G. Gmitter, Jr. Some of our allotetraploid somatic hybrids are producing fruit with direct cultivar potential, i.e., 'Valencia' sweet orange + `Murcott' tangor. New somatic hybrids produced in our program will be discussed, including `Page' tangor + `Dancy' mandarin, `Page' tangor + `Kinnow' mandarin, and `Hamlin' sweet orange + LB8-9 tangelo. Somatic cybridization is being used to transfer CMS (cytoplasmic male sterility) from the seedless `Satsuma' mandarin to other seedy varieties via mtDNA transfer, in efforts to make them seedless. New somatic cybrids produced in our program that contain the `Satsuma' CMS include `Murcott' tangor and `Kinnow' mandarin. Details of these results and other progress will be discussed.

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Francisco A. A. Mourão Fo, Jude W. Grosser, and Frederick G. Gmitter Jr.

Protoplast culture following polyethylene glycol (PEG)-induced fusion resulted in the regeneration of somatic hybrid plants from the following combinations: `Succari' sweet orange (C. sinensis L. Osbeck) + Severinia disticha; `Hamlin' sweet orange (C. sinensisj + S. disticha: `Valencia' sweet orange (C. sinesis) + S. disticha; `Nova' tangelo (C. reticulata hybrid) + S. disticha; `Succari' sweet orange + S. buxifolia; `Nova' tangelo + Citropsis gilletiana; and `Succari' sweet orange + Atlantia ceylanica. `Succari', `Hamlin', `Valencia', and `Nova' protoplasts were Isolated from ovule-derived embryogenic callus and/or suspension cultures whereas protoplasts of S. disticha, S. buxifolia, C. gilletiana, and A. ceylanica were isolated from leaves of potted trees in a greenhouse. Plants were regenerated via somatic embryogenesis and somatic hybrids were identified on the basis of leaf morphology. Electrophoretic analysis of isozyme banding patterns and root tip chromosome counts are being performed. Somatic hybrids with S. disticha are apparently weak whereas the other somatic hybrid plants with S. buxifolia, C. gilletiana, and A. ceylanica exhibit adequate vigor. These are more examples that the the techique of protoplast fusion can be an important tool in overcoming barriers to hybridization of sexually incompatible species.