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P.F. McCabe, L.J. Dunbar, A. Guri, and K.C. Sink

Sexual hybrid plants of Lycopersicon esculentum × L. pennellii (E × P) have been transformed and the T-DNA inserts genetically mapped. Donor protoplasts of E × P were isolated from leaves, and subsequently irradiated with 0, 5, 10 and 20 krad of a 60Co. They were then fused with suspension-derived protoplasts of S. lycopersicoides using the PEG-CA++-high pH technique. The protoplasts were cultured in medium 8E at 1.5 × 106 protoplasm/ml. Selection of heterokaryon-derived macrocalli was facilitated by the inability of irradiated donor protoplasts to divide and by plating p-calli on regeneration medium containing kanamycin - an antibiotic for which the resistance gene NPTII is part of the T-DNA. Morphological characteristics of the resulting asymmetric somatic hybrid plants indicate that 10 and 20 krad irradiation eliminates a larger port ion of the genome than does 5 krad. This has been confirmed with isozyme analysis and chromosome counts which generally show the 5 krad asymmetric somatic hybrid plants to differ little from symmetric hybrids although they contain some significant exceptions. Isozyme data reveals the 10 and 20 krad plants to have received much less of the donor genome. Exact quantification is continuing using isozyme markers, chromosome counts and cDNA probes.

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Thomas Walters and Elizabeth Earle

Cauliflower protoplasts with male fertile and Ogura male sterile cytoplasm were fused. Organelle reassortment and recombinant mitochondria were found in calli and plants regenerated from the fused protoplasts. Pretreatment (gamma-irradiation or iodoacetate) and protoplasm source (leaf or hypocotyl tissue) were manipulated in a series of fusions to determine their effects on organelle segregation. Some regenerated plants appear to combine Ogura male sterile mitochondria with normal Brassica chloroplasts. Plants with this organelle combination should be free of the cold temperature chlorosis due to incompatibility between the Brassica nucleus and the radish chloroplasts of the Ogura cytoplasm. These plants may have potential for improved cauliflower hybrid production.

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

Protoplasm culture following the chemical fusion of `Valencia' sweet orange [Citrus sinensis (L.) Osb.] protoplasts, isolated from an embryogenic suspension culture, with `Femminello' lemon [Citrus limon (L.) Burro. f.] leaf protoplasts resulted in the regeneration of an interspecific allotetraploid somatic hybrid plant, two autotetraploid lemon plants, and diploid plants from both parents. The regeneration of plants from lemon leaf protoplasts is an example of protoplast-to-plant regeneration from non-nucellus-derived tissue for Citrus. Regenerated plants were classified according to leaf morphology, chromosome number, and analyses of phosphohexose isomerase (PHI), peroxidase (PER), and 6-phosphoglucose dehydrogenase (PGD) zymograms. The somatic hybrid plant was vigorous, with leaves morphologically intermediate to the parents. The tetraploid lemon plants were similar to diploids, although less vigorous and with thicker leaves. The tetraploid lemon and somatic hybrid plants, if fertile, could be used in interploid sexual crosses to breed triploid seedless lemon cultivars with tolerance of mal secco disease from sweet orange. Further investigation of plant regeneration from leaf protoplasts could increase the number of totipotent Citrus clones amenable to somatic hybridization and genetic transformation experiments.

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S. Kobayashi, I. Oiyama, K. Yoshinaga, T. Ohgawara, and S. Ishii

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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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Jude W. Grosser, Frederick G. Gmitter Jr., Franca Sesto, Xiu Xin Deng, and J.L. Chandler

Protoplasm culture following polyethylene glycol (PEG) -induced fusion resulted in the regeneration of somatic hybrid plants from the following six parental combinations: Citrus sinermis (L.) Osbeck cv. Hamlin + Severinia buxifolia (Poir.) Tenore (Chinese box-orange); C. reticulate Blanco cv. Cleopatra + Poncirus trifoliata (L.) Raf. cv. Flying Dragon; C. reticulate cv. Cleopatra + Swingle citrumelo (C. paradisi Macf. × P. trifoliata); C. sinensis cv. Hamlin + C. jambhiri cv. Rough lemon; C. sinensis cv. Valencia + C. jambhiri cv. Rough lemon; and C. paradisi cv. Thompson + `Murcott' tangor (purported hybrid of C. reticulate × C. sinensis). Diploid plants were regenerated from nonfused embryogenic culture-derived protoplasts of `Cleopatra' mandarin and `Hamlin' and `Valencia' sweet orange, and from nonfused leaf-derived protoplasts of Rough lemon and `Mnrcott'. Regenerated plants were classified according to leaf morphology, chromosome number, and isozyme analyses. All of the somatic hybrids reported herein are tetraploid (2n = 4x = 36), with the exception of the `Hamlin' + S. buxifolia hybrid, which was unexpectedly found to have a chromosome number of 2n = 27. These six new somatic hybrids have potential in citrus scion and rootstock improvement for commercial use.

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Cheol Hee Lee, Kee Yoeup Paek, J. Brian Power, and Edward C. Cocking

This study was designed to assess the general limitations of somatic hybridization as one of the key technologies for genetic manipulation in plants. The limits of somatic hybridization against different taxonomic backgrounds, intraspecific to interfamilial, were also assessed. Protoplast culture studies provided essential information relating to the species cultural and morphogenetic capacity. several #elect Ion strategies for the recovery of somatic hybrid colonies/plants were developed and assessed using various combinations of protoplast sources and species in the genera Petunia, Nicotiana, Salpiglossis and Chrysanthemum. Morphological, cytological and biochemical analyses were performed to confirm the hybridity of plants or cell lines recovered following protoplasm fusion (using 4-5 methods) and selection.

The somatic hybrid callus/plants were obtained at intraspecific to interfamilial levels by complementation to chlorophyll proficiency, together with media selection or complementation of nitrate reductase deficient mutants as follows; P. Hybrida var. Monsanto (+) P. hybrida cv. Blue Lace (intraspecific), P. hybrida var. Monsanto (+) P. inflata and P. parviflora (interspecific), P. parviflora (+) N. tabacum (intergeneric), S. sinuata (+) P. hybrida var. Monsanto, P. parodii and N. tabacum (intertribal), and C. morifolium (+) S. sinuata.

From this study, it appeared that there were no taxonomic limits to the production and proliferation of somatic hybrid cell lines. However, obtaining morphologically normal hybrid plants met with limited success as the taxonomic relationships became more distant. The regeneration capacity of somatic hybrids seemed to be controlled by both parental species. Somatic incompatibility mechanism was also shown to operate on chromosome elimination. Such chromosome elimination may well be advantageous in plant improvement.