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The objective of this study was to produce citrus somatic asymmetric hybrids by fusing gamma-irradiated protoplasts with iodoacetamide-treated protoplasts. Protoplasts were isolated from embryogenic suspension cells of grapefruit (Citrus paradisi Macfad.) cultivars Ruby Red and Flame, sweet oranges (C. sinensis Osbeck) 'Itaboraí', 'Natal', Valencia', and 'Succari', from 'Satsuma' (C. unshiu Marcow.) and 'Changsha' mandarin (C. reticulata Blanco) and 'Murcott' tangor (C. reticulata x C. sinensis). Donor protoplasts were exposed to gamma rays and receptor protoplasts were treated with 3 mmol L-1 iodoacetamide (IOA), and then they were fused for asymmetric hybridization. Asymmetric embryos were germinated, and the resulting shoots were either grafted onto sour orange, rough lemon or 'Swingle' (C. paradisi x Poncirus trifoliata) x 'Sunki' mandarin rootstock seedlings, or rooted after dipping their bases in indol-butyric acid (IBA) solution. The products were later acclimatized to greenhouse conditions. Ploidy was analyzed by flow cytometry, and hybridity was confirmed by amplified fragment length polymorphism (AFLP) analysis of plantlet DNAsamples. The best treatment was the donor-recipient fusion combination of 80 Gy-irradiated 'Ruby Red' protoplasts with 20 min IOA-treated 'Succari' protoplasts. Tetraploid and aneuploid plants were produced. Rooting recalcitrance was solved by dipping shoots' stems in 3,000 mg L-1 IBA solution for 10 min. O objetivo deste trabalho foi produzir híbridos somáticos assimétricos de citros pela fusão de protoplastos irradiados com raios gama e protoplastos tratados com iodoacetamida. Protoplastos foram isolados de suspensões celulares embriogênicas de pomelo (Citrus paradisi Macfad.), cultivares Ruby Red e Flame, de laranja doce (C. sinensis Osbeck) 'Itaboraí', 'Natal', Valencia' e 'Succari', de tangerinas 'Satsuma' (C. unshiu Marcow.) e 'Changsha' (C. reticulata Blanco) e de tangor 'Murcott' (C. reticulata x C. sinensis). Protoplastos doadores foram expostos a raios gama e protoplastos receptores foram tratados com 3 mmol L-1 de iodoacetamida (IOA) e fusionados para hibridação assimétrica. Embriões assimétricos foram germinados e as brotações produzidas foram enxertadas sobre porta-enxertos de sementes de 'Laranja azeda', 'Limão rugoso' ou citrumelo 'Swingle' (C. paradisi x Poncirus trifoliata) x 'Sunki' ou enraizadas após embebição em solução de ácido indol-3-butírico (IBA) e aclimatadas em casa de vegetação. A análise de ploidia foi realizada por meio de citometria de fluxo e a hibridicidade foi confirmada por análise de polimorfismo de comprimento de fragmentos amplificados (AFLP) de amostras de DNA das plântulas. A melhor combinação doador-recipiente foi de protoplastos de 'Ruby Red' irradiados com 80 Gy, fusionados com protoplastos de 'Succari' tratados por 20 min com IOA. Plantas tetraploides e aneuploides foram produzidas e o problema de recalcitrância ao enraizamento foi resolvido pela introdução das bases das brotações em 3.000 mg L-1 de IBA por 10 min.

Agrobacterium tumefaciens is established as a vector for gene transfer in many dicotyledonous plants but is not accepted as a vector in monocotyledonous plants, especially in the important Gramineae. The use of Agrobacterium to transfer genes into monocot species could simplify the transformation and improvement of important crop plants. In this report we describe the use of Agrobacterium to transfer a gene into corn, the regeneration of plants, and detection of the transferred genes in the F(1) progeny. Shoot apices of Zea mays L. variety Funk's G90 were cocultivated with A. tumefaciens EHA 1, which harbored the plasmid pGUS3 containing genes for kanamycin resistance (NPT II) and beta-glucuronidase (GUS). Plants developed from these explants within 4 to 6 weeks. Fluorometric GUS assays of leaves and immature seeds from the plants exhibited low GUS activity. Both NOS and GUS gene fragments were amplified by polymerase chain reaction in the DNA isolated from the F(1) generations of one of the original transformed plants. Southern analysis showed both GUS and NPT probes hybridized to DNA in several of the F(1) progeny, demonstrating the incorporation of GUS and NPT II genes into high molecular weight DNA. These data establish successful gene transfer and sexual inheritance of the genes.