Transfer of resistance traits from carrot into tobacco by asymmetric somatic hybridization: Regeneration of fertile plants

Abstract

Transfer of methotrexate and 5-methyltryptophan resistance from carrot (Daucus carota) to tobacco (Nicotiana tabacum) was achieved by fusion between leaf mesophyll protoplasts of tobacco and irradiated cell culture protoplasts of carrot. Some of the regenerated somatic hybrids exhibited normal tobacco morphology with coexpression and independent segregation of the transferred resistance markers. Chromosomal instability resulted in aneuploid somatic hybrids with significantly lower chromosome number than predicted by simple addition of parental chromosome number. The methotrexate resistance phenotype was correlated with the expression of carrot-specific dihydrofolate reductase as judged by isozyme and immunological characteristics of the enzyme. The genomic construct of these somatic hybrids made the transmission of the resistance character into the next sexual generation possible.protoplasts (11). In some of the resulting nitrate reductasepositive tobacco plants, barley enzyme was detected with immunological methods. Because of leakiness of selection techniques, based on a single recessive and unstable mutation, the authors did not conclude that cell fusion-mediated gene transfer had occurred. At present, available experimental data are insufficient to explore the potential of asymmetric cell fusion for widening genetic variability.In this paper we describe a fusion system that allowed the monitoring of two dominantly acting selectable markers in tobacco + carrot fusion products. Molecular evidence confirming the expression of carrot-specific dihydrofolate reductase (DHFR; EC 1.5.1.3) in regenerated hybrid plants with normal tobacco morphology is presented. Furthermore, sexual progenies are analyzed to test the inheritance of transferred genetic markers.Gene flow between incompatible species is strongly restricted by evolutionary boundaries in sexual crossings. The use of somatic cells as targets in genetic manipulation experiments, based on DNA transformation or cell hybridization, has opened new horizons for combining diverse genes from unrelated species. If the gene of interest has been cloned, advanced transformation systems may provide the most efficient way of introducing it into a foreign host genome (1, 2). When isolated genes are lacking, asymmetric hybridization mediated by protoplast fusion offers an alternative approach for alien gene transfer. The asymmetric nature of nuclear genomes in cell hybrids originates from spontaneous or induced genome instability, with preferential loss of chromosomes belonging to one of the parental species. Enforced chromosome elimination can be a prerequisite of hybrid plant production in several wide fusion combinations in which somatic incompatibility prohibits hybrid development (3-5). The transient coexistence of two diverse genomes in hybrid cells may provide the opportunity for recombination of chromosomes or a few genes from the eliminated partner. To test this hypothesis, experimental methods are needed to control chromosome elimination from fusion produ...

Asymmetric somatic hybridization between wheat (Triticum aestivum L.) and Agropyron elongatum (Host) Nevishi

“…Production of highly asymmetric somatic hybrids with a few chromosomes, or chromosomal fragments of the donor species, simultaneously overcomes both hybrid sterility (Dudits et al 1987;Hinnisdales et al 1991) and the introgression of too much exotic genetic material into the crop species. Beside the ionizing radiation, UV has also been used to induce production of asymmetric somatic hybrids.…”

Protoplast-Fusion Derived Cybrids in Solanaceae

“…If the respective fusion product is cultured in a kanamycin containing medium --only those plantlets that contain the nuclear components of the donor, with the kanamycin resistance, will survive selection; especially during rooting on kanamycin. A selectable nuclear-coded resistance to methatrexate was used in a similar manner (Dudits et al, 1987). Selection of nuclear components can be performed also by using a recipient with nuclear-coded chlorophyll deficiency (Gupta et al, 1984).…”

The potential of somatic hybridization in crop breeding

“…To date, asymmetric hybridization has met with mixed results . In general, irradiation reduced the amount of transferred donor DNA but the fraction may vary from a few traits (Dudits et al ., 1987), one or a few chromosomes (Gupta et al ., 1984 ;Bates et al ., 1987a;Melzer & O'Connell, 1992) to numerous chromosomes (Gleba et al ., 1988 ;Famelaer et al ., 1989;Sacristan et al ., 1989 ;Sjodin & Glimelius, 1989b ;Yamashita et al ., 1989 ;Wijbrandi et al ., 1990a, b, c, Wolters et al ., 1991Derks et al ., 1992) the latter result being obtained in the majority of cases . Furthermore, chromosome elimination is random and it is not possible to predict which of the chromosomes will be lost .…”