SummaryRapeseed plants have been regenerated after fusion between protoplasts bearing cytoplasms of different genera. Cybrids combine, in a first experiment Brassica napus chloroplasts and a cytoplasmic male sterility (cms) trait coming from Raphanus sativus, in a second experiment chloroplasts of a triazine resistant Brassica campestris and cms trait from Raphanus sativus. Transfer of chloroplasts has been confirmed by restriction cp DNA analysis and two dimensional thylakoïd protein electrophoresis. These plants may be very useful for Brassica hybrid seed production.

Intergeneric cytoplasmic hybridization in cruciferae by protoplast fusion

The domestication of plants, as a bio-cultural process, is a continuous phenomenon intrinsically associated with the use of plants. Traditional and scientifi c knowledge constitute the basis of the various uses of plants from in situ harvesting to complete domestication of crops. One of the most important challenges of our time is to achieve the conservation and sustainable use of plant genetic resources of landraces, species in the process of domestication and species used in situ. The in situ conservation of agricultural biodiversity is a basic element for the development of more sustainable agroecosystems, the adaptation to climate change, the conservation of ecosystem services and to ensure local food security—a conception that is strongly linked to the local development and the protection of cultural and biological diversity. Through case studies from the Pampa Biome we will discuss the valorization of plant genetic resources through new domestication, the promotion of the use of scientifi cally developed best management practices for in situ conservation, the widening of the germplasm base for breeding programs, plant breeding for stress tolerance, the development of participatory plant breeding programs and the development of high quality products.

Advances in plant breeding strategies: Breeding, biotechnology and molecular tools

In recent years, the rapid development of somatic cell genetics has made possible the transfer of alien genes over wide taxonomic distances by somatic hybridization. In this review, the potential of somatic hybridization in the breeding of crops within the Brassicaceae, Fabaceae, Poaceae and Solanaceae is discussed. It is evident from these studies that many hybrids, either symmetric or asymmetric, which are fertile have the potential to be used as a bridge between the alien species and the crop. Progeny analysis of some hybrid combinations also reveals intergenomic translocations which may lead to the introgression of the alien genes. Furthermore, fusion techniques enable the resynthesis of allopolyploid crops to increase their genetic variability and to restore ploidy level and heterozygosity after breeding at reduced ploidy level in polyploid crops.

The potential of somatic hybridization in crop breeding

https://www.cell.com/trends/biotechnology/pdf/0167-7799(91)90008-6.pdf

Gene transfer via somatic hybridization in plants

Cold tolerant cytoplasmic male-sterile (CMS) cabbage (Brassica oleracea var. capitata) was produced by the fusion of leaf protoplasts from fertile cabbage and cold-tolerant Ogura CMS broccoli lines. The cabbage lines tested showed great variation in plant regeneration from unfused protoplasts; three with high regenerability were selected as the fusion partners. Several procedures for eliminating the nuclear DNA of the broccoli fusion partner were tested. Diploid cabbage plants were identified by flow cytometry and morphological characters. Gamma-irradiation (30 krad) was the most successful procedure; isolation of cytoplasts from broccoli leaf protoplasts, followed by gamma-irradiation of the cytoplast fraction, also produced diploids. UV-irradiation of the broccoli protoplasts was less effective. PCR using primers for an Ogura CMS-specific mitochondrial DNA sequence permitted the identification of cybrids likely to be CMS. Over 200 diploid plants with the CMS-specific sequence were obtained from 66 independent fusion products and three cabbage lines. Plants were ready for transfer into soil within 8 months after fusion. The plants identified as CMS by PCR produced male-sterile flowers. Our procedures permit the transfer of a desirable male-sterile cytoplasm into cabbage much more rapidly than conventional backcrossing procedures.

/pdf/direct-transfer-of-a-cold-tolerant-ogura-male-sterile-254pjfzwbz.pdf

Direct transfer of a cold-tolerant Ogura male-sterile cytoplasm into cabbage (Brassica oleracea ssp. capitata) via protoplast fusion

An efficient procedure for obtaining somatic hybrids between B. oleracea and B. campestris has been developed. Hypocotyl protoplasts of B. oleracea were fused with mesophyll protoplasts from three different varieties of B. campestris by the polyethylene glycoldimethylsulfoxide method. The selection of somatic hybrids utilized the inactivation of B. oleracea protoplasts by iodoacetamide (IOA) and the low regeneration ability of B. campestris. The efficiency of recovery of somatic hybrids depended upon the IOA concentration, and when 15 mM IOA was used, 90% of the regenerated plants were found to be hybrid. The somatic hybrids were examined for i) leaf morphology, ii) leucine aminopeptidase (LAP) isozyme and iii) chromosome number. All the hybrids had intermediate leaf morphology and possessed LAP isozymes of both parental species. The chromosome analysis revealed a considerable variation in chromosome number of somatic hybrids, showing the occurrence of multiple fusion and chromosome loss during the culture. Some of the hybrids flowered and set seeds.

Somatic hybrids between Brassica oleracea and B. campestris: selection by the use of iodoacetamide inactivation and regeneration ability

To examine the possibility of producing asymmetric somatic hybrids of Brassica having a complete genome of one species and a part of the other, we fused inactivated B. oleracea protoplasts with X-irradiated B. campestris protoplasts. The plants obtained were studied with regard to their morphology, isozymes and chromosomes. The morphology of the hybrids was similar to B. oleracea in 9 out of 22 hybrids studied and the rest showed the intermediate phenotype of the parents. Analysis of three isozymes, leucine aminopeptidase, acid phosphatase and esterase indicated that ten hybrids lost B. campestris-specific bands in one or more of the three isozymes examined. The chromosome analysis showed that 90% of the hybrids were aneuploids. In addition, abnormal chromosomes were often found in root tip cells. These results suggested that the hybrids obtained were asymmetric in nature and resulted from elimination of B. campestris chromosomes by X-ray irradiation.

Asymmetric somatic hybrids of Brassica: partial transfer of B. campestris genome into B. oleracea by cell fusion.

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Somatic hybrids between Brassica oleracea and B. campestris: selection by the use of iodoacetamide inactivation and regeneration ability

Asymmetric somatic hybrids of Brassica: partial transfer of B. campestris genome into B. oleracea by cell fusion.