Somatic hybridization experiments in Citrus that involve the fusion of protoplasts of one parent isolated from either nucellus-derived embryogenic callus or suspension cultures with leaf-derived protoplasts of a second parent, often result in the regeneration of diploid plants that phenotypically resemble the leaf parent. In this study, plants of this type regenerated following somatic fusions of the following three parental combinations were analyzed to determine their genetic origin (nuclear and organelle): (embryogenic parent listed first, leaf parent second) (1) calamondin (C. microcarpa Bunge) + 'Keen' sour orange (C. aurantium L.), (2) Cleopatra mandarin (C. reticulata Blanco) + sour orange, and (3) 'Valencia' sweet orange (C. sinensis (L.) Osbeck) + 'Femminello' lemon (C. limon (L.) Burm. f.). Isozyme analyses of PGI, PGM, GOT, and IDH zymograms of putative cybrid plants, along with RFLP analyses using a nuclear genome-specific probe showed that these plants contained the nucleus of the leaf parent. RFLP analyses using mtDNA-specific probes showed that these plants contained the mitochondrial genome of the embryogenic callus donor, thereby confirming cybridization. RFLP analyses using cpDNA-specific probes revealed that the cybrid plants contained the chloroplast genome of either one or the other parent. These results support previous reports indicating that acquisition of the mitochondria of embryogenic protoplasts by leaf protoplasts is a prerequisite for recovering plants with the leaf parent phenotype via somatic embryogenesis following somatic fusion.

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.

Microsporogenesis was analysed in a tetraploid somatic hybrid (SH) (2n 4x 36) of Citrus and its diploid fusion parents (2n 2x 18), Valencia sweet orange (C. sinensis L. Osbeck) and Femminello lemon (C. limon L. Burm. f.). Intergenomic pairing between lemon and orange occurred in the somatic hybrid which showed multivalent chromosome associations in diakinesis, although one quadrivalent was de®nitely because of a reciprocal translocation present in Valencia. The behaviour of univalents was variable in the somatic hybrid and its parents. In the somatic hybrid and Valencia, the univalents preferentially formed micronuclei and polyads whereas, in Femminello, they were generally enclosed in a nucleus although distributed randomly. The somatic hybrid showed a rate of pollen stainability of 64% and germinability of 41%. 1The chromosomally unbalanced pollen from the tetraploid SH was presumed viable and able to fertilize because di erent nuclear DNA contents were found in the back-cross progeny. Moreover, meiotic nuclear restitution mechanisms, which could be mainly dependent on the abnormal orientation of the spindles in meiosis II, are described.

We report the accurate determination of the allelic configurations of a total of eight new citrus tetraploid hybrids by means of SSR analysis, coupled with capillary electrophoresis, and PCR based dosage effects. Tetraploid hybrids were spontaneously obtained from different interploid crosses (2x 9 4x) between diploid 'Femminello' lemon and the allotetraploid somatic hybrid (2n = 4x = 36) 'Key' lime ? 'Valencia' orange, and between diploid 'Wilking' and 'Fortune' mandarins and an autotetraploid 'Dancy' mandarin (2n = 4x = 36). To understand the opportunity to employ them in further backcross programs, the cytological mechanisms underlying their ploidy level were unambiguously determined using six SSR primers. PCR conditions were optimized and skewness in template/product ratios were verified. Tetraploid allelic configurations were determined from PCR based dosage effects using electropherogram peak heights to estimate the copy number per allele. In all the tetraploid hybrids we found out that diploginy (2n eggs) has occurred, contributing the extra haploid genome in the tetraploids. According to the marker genotypes, it was further inferred that the 2n eggs in 'Femminello' lemon resulted from first division restitution (FDR), while in 'Wilking' and 'Fortune' mandarins 2n eggs occurred in second division restitution (SDR). These new genotypes, with their improved genetic female background, can be therefore considered very valuable in our citrus genetic improvement program as pollen donors in backcrosses suitable to eliminate negative traits.

Protoplast culture following polyethylene glycol-induced fusion resulted in the regeneration of vigorous tetraploid somatic hybrid plants from eight complementary parental rootstock combinations: Citrus reticulata Blanco (Cleopatra mandarin) + C. aurantium L. (sour orange), C. reticulata (Cleopatra mandarin) + C. jambhiri Lush (rough lemon), C. reticulata (Cleopatra mandarin) + C. volkameriana Ten. & Pasq. (Volkamer lemon), C. reticulata (Cleopatra mandarin) + C. limonia Osb. (Rang-pur), C. sinensis (L.) Osb. (Hamlin sweet orange) + C. limonia (Rangpur), C. aurantium (sour orange) + C. volkameriana (Volkamer lemon) zygotic seedling, C. auruntium hybrid (Smooth Flat Seville) + C. jambhiri (rough lemon), and C. sinensis (Valencia sweet orange) + Carrizo citrange [C. paradisi Macf. × Poncirus trifoliata (L.) Raf.]. Diploid plants were regenerated from nonfused callus-derived protoplasts of Valencia sweet orange and Smooth Flat Seville and from nonfused leaf protoplasts of sour orange, Rangpur, rough lemon, and Volkamer lemon. Regenerated plants were classified according to leaf morphology, chromosome number, and leaf isozyme profiles. All somatic hybrid plants were tetraploid (2n = 4× = 36). One autotetraploid plant of the Volkamer lemon zygotic was recovered, apparently resulting from a homokaryotic fusion. These eight new citrus somatic hybrids have been propagated and entered into field trials.