X-ray irradiation promoted asymmetric somatic hybridisation and molecular analysis of the products

Abstract

Complementation of two metabolic deficiences - nitrate reductase and tryptophan synthase - was used to select for somatic fusion hybrids between tobacco (Nicotiana tabacum) and henbane (Hyoscyamus muticus) with prior X-irradiation of one partner. Using species specific, radioactively labelled DNA probes it could be shown that a) irradiation significantly reduced the amount of chromosomal DNA of the irradiated fusion partner in the somatic hybrid, b) irradiation with doses which completely inhibit protoplast division did not pevent transfer of substantial amounts of chromosomal DNA into the fusion hybrids (so called 'cybrids') and c) this method transfers functional nuclear genes together with the partial genome from the irradiated partner.

Protoplast fusion technology

“…One of the most promising techniques is asymmetric fusion (1,5,11,24). By the use of chromatin-damaging treatments such as irradiation, it has been possible to transfer only limited genetic information from one species into another species.…”

“…Recently, chromosome-damaging treatments such as irradiation have been used (1,5,11,24) for specific gene transfer by protoplast fusion. This approach has the added benefit of transferring limited genetic information which can be selected for in vitro or in vivo and does not require the recombination of two entire genomes.…”

Cauliflower and Broccoli

“…Effective probes of the artificial hybridization of the different genomes are based on isoenzyme are RFLP analysis and lastly by repetitive species-specific sequences which estimate also the proportion of parental genomic material in asymmetric hydrids (Sundberg and Glimelius, 1986;Imamura et al, 1987;Piastuch and Bates, 1990;Fahleson et al, 1994;Fahleson et al, 1997).…”

The potential of somatic hybridization in crop breeding

“…Preliminary evidence of hybridity can be obtained by scoring the morphology which is often intermediate between the parents . More direct proof can be obtained by biochemical analysis using the isoenzyme pattern (Scandalios & Sorenson, 1977 ;Gleba & Sytnik, 1984) or various types of molecular probes such as species-specific repetitive DNA sequences (Saul & Potrykus, 1984;Imamura et al ., 1987 ;Schweizer et al ., 1988), rDNA (Gleba et al ., 1988) and other RFLP markers Melzer & O'Connell, 1990) . Recently, the use of PCR technology has also been utilized for hybrid identification (Baird et al ., 1992 ;Xu et al ., 1993b) .…”

“…This can be achieved by spontaneous chromosome elimination (Pijnackeret al ., 1987 ;Fahleson et al, 1988 ;Pijnacker et al ., 1989 ;Sundberg et al ., 1991b;Babiychuk et al ., 1992) or by irradiation prior to fusion (Dudits, 1980 ;Gupta et al ., 1984 ;Bates et al ., 1987a ;Imamura et al ., 1987) . Alternative methods limiting the amount of DNA transferred, such as micronucleation (Ramulu et al ., 1992) and UV irradiation (Hall et al ., 1992), are currently also being investigated.…”