Intergeneric somatic hybrid plants of Citrus sinensis cv. Hamlin and Poncirus trifoliata cv. Flying Dragon

Abstract

Intergeneric somatic hybrid plants between 'Hamlin' sweet orange [Citrus sinensis (L.) Osbeck] and 'Flying Dragon' trifoliate orange (Poncirus trifoliata Raf.) were regenerated following protoplast fusion. 'Hamlin' protoplasts, isolated from an habituated embryogenic suspension culture, were fused chemically with 'Flying Dragon' protoplasts isolated from juvenile leaf tissue. The hybrid selection scheme was based on complementation of the regenerative ability of the 'Hamlin' protoplasts with the subsequent expression of the trifoliate leaf character of 'Flying Dragon.' Hybrid plants were regenerated via somatic embryogenesis and multiplied organogenically. Hybrid morphology was intermediate to that of the parents. Chromosome counts indicated that the hybrids were allotetraploids (2n=4x=36). Malate dehydrogenase (MDH) isozyme patterns confirmed the hybrid nature of the regenerated plants. These genetically unique somatic hybrid plants will be evaluated for citrus rootstock potential. The cell fusion, selection, and regeneration scheme developed herein should provide a general means to expand the germplasm base of cultivated Citrus by intergeneric hybridization with related sexually incompatible genera.

Caipira sweet orange + Rangpur lime: a somatic hybrid with potential for use as rootstock in the Brazilian citrus industry

“…Chromosome counts confirmed the tetraploid nature of the regenerated plants (Figure 3). Somatic hybridization can be further confirmed by the analysis of molecular markers such as isozymes (Grosser et al, 1988;Ling and Iwamassa, 1994), rDNA restriction endonuclease digestion patterns (Ohgawara et al, 1985;Miranda et al, 1997), and RAPD-PCR Mourão Filho et al, 1996). RAPD analysis confirmed the somatic hybridization and revealed complementary banding paterns in the somatic hybrid Caipira sweet orange + Rangpur lime, indicating the presence of DNA from each parent in the corresponding hybrid (Figure 4).…”

Citrus biotechnology: Achievements, limitations and future directions

“…Therefore, it has become an integral part of citrus variety improvement worldwide (Khan, 2007). Various limitations presented by complicated reproductive biology of citrus can be successfully overcome through somatic hybridization by generating inter-specific and inter-generic allotetraploid somatic hybrids of desired cultivars for scion as well as rootstock development (Grosser et al, 1988;Ohgawara et al, 1994;Grosser et al, 1996) that can be utilized in breeding programs. It is also possible to generate somatic hybrids between sexually incompatible species, but has little scope for their incorporation in breeding programs.…”

Stomatal analysis of citrus somatic hybrids obtained by protoplast fusion

“…However, sexual hybridization between individuals of different species generally does not produce viable hybrids due to sexual barriers (Grosser & Gmitter Junior, 1990). In a breeding program, somatic hybridization can overcome sexual incompatibility through somatic cell fusion.…”

“…Observation and confirmation of somatic hybrids can be done through analysis of leaf morphology, molecular markers, such as PCR-RAPD, and determination of chromosome number (Grosser & Gmitter Junior, 1990). Associations of different techniques are common, since some of them may not definitely confirm the hybrid nature of the plantlet obtained from protoplast fusion experiments.…”

“…Variable leaf morphology such as trifoliate leaves (Grosser et al, 1988) or petiole wings (Ballve et al, 1997) are commonly used as morphological markers to identify potential hybrids. When both parentals have specific characteristics in their leaf morphology, such as the trifoliate leaf and petiole wings, and the potential hybrid has leaves with both characteristics, the hybrid character of the plant can be inferred.…”