Plant Cell Reports (1995) 14:593-597 PlantCell Reports 9 Springer-Verlag 1995 Diplotaxis catholica + Brassica juncea somatic hybrids: molecular and cytogenetic characterization P.B. Kirti, T. Mohapatra, H. Khanna, S. Prakash, and V.L. Chopra Biotechnology Centre, Indian Agricultural Research Institute, New Delhi 110012, India Received 27 June 1994/Revised version received 23 November 1994 - Communicated by C. E Quiros Summary Intergeneric somatic hybrids Diplotaxis catholica (2n=18) + Brassica juncea (2n=36) were produced by fusing mesophyll protoplasts of the former and hypocotyl protoplasts of the latter using polyethylene glycol. Out of 52 somatic embryos, 24 produced plants of intermediate morphology. Cytological analysis of 16 plants indicated that 15 were symmetric hybrids carrying 54 chromosomes, the sum of the parental chromosome numbers. One hybrid was asymmetric with 45 chromosomes. Nuclear hybridity of five putative hybrids was confirmed by the Southern hybridization pattern of full length 18s-25s wheat nuclear rDNA probe which revealed the presence of Hind III fragments characteristic of both the parental species. The hybridization pattern of mitochondria specific gene probe cox I indicated that three of the hybrids carried B. juncea mitochondria and one carried mitochondria of D. catholica. Presence of novel 3.5 kb Hind III and 4.8 kb Bgl II fragments suggested the occurrence of mtDNA recombination in one of the hybrids. The hybrids were pollen sterile. However, seeds were obtained from most of the hybrids by back crossing with B. juncea. Key words : Brassica juncea, Diplotaxis catholica, Somatic hybrids, Organelle constitution, Mitochondrial DNA recombination Introduction Crop Brassicas belong to the sub-tribe Brassicinae, which also includes the wild and weedy species that are a source of agronomically useful genes. These Correspondence to: S. Prakash genes are largely inaccessible to breeders because of barriers to sexual crossability operating at different stages (Shivanna 1991). Somatic hybridization bypasses these barriers and may allow gene introgression from the wild to the cultivated through suitable chromosome manipulation strategies. It also gives an opportunity for creating novel combinations of genes contained in cytoplasmic organelles and producing alloplasmics expressing male sterility. We have attempted to exploit these possibilities by fusing protoplasts of B. juncea (2n=36) and D. catholica (2n=18). The latter is a wild relative from the sandy West Mediterranean and is resistant at the field level to the black spot or alternaria blight disease of oilseed Brassicas. This paper describes results of these fusion experiments. Materials and Methods Brassica juncea cv. RLM 198 (2n=36, AABB) and D. catho/ica (2n=18, DCD c) were used in the protoplast hybridization experiments.Seedsof D. catho/ica were kindly suppliedby Prof. K. Hinata of Tohoku University, Sendai,Japan. Protoplast culture and fusion Protoplasts from hypocotyls of B. juncea, which could be easily regenerated to plants in 8-10 weeks, were fused with leaf protoplasts of D. catho/ica using polyethylene glycol. Pretoplasts were isolated as per Kirti eta/. (1991). Fusion solution was a modification of Menczel and Wolfe (1984) and contained 20% polyethylene glycol (MW 8000, Sigma), 10% dimethylsulfoxide, 3.6% glucose, 0.17% calcium chloride, 0.0095% potassium dihydrogen phosphate at pH 5.8. Fusion solution was filter sterilized. Hypocotyl and leaf protoplastswere mixed in a ratio of 1:1, washedand resuspended in Ws solution (Menczel eta/. 1981) at a density of 2 x 106/m1. Three drops, 50 Id each, of the protoplast mixture were placed in a 5 cm Petri dish and to these was added an equal volume of fusion solution. Five minutes later, the PEG solutionwas replaced by an equal volume of W5 solution. This was followed by the addition of 2 ml Ws solution to the Petri dish in five minutes. After further thirty minutes, 2 ml culture medium was added to each Petri dish after removing the Ws