Production and genetic characterization of somatic hybrids between leaf mustard (Brassica juncea) and broccoli (Brassica oleracea)

Abstract

Inter-specific somatic hybrids of leaf mustard (Brassica juncea) and broccoli (Brassica oleracea) were established to introduce cytoplasmic male sterility (CMS) and Verticillium dahliae Kleb. resistance from broccoli to leaf mustard. Protoplasts isolated from hypocotyls and cotyledons of inbred lines of leaf mustard and broccoli were fused using 40% (w/v) polyethelene glycol and then cultured in modified k8p medium supplemented with 0.2 mg/l 2,4-dichlorophenoxi-acetic acid, 0.5 mg/l 6-benzyladenine (BA), 0.1 mg/l naphthaleneacetic acid (NAA), and 0.1 mg/l kinetin (Kin), and 0.4 M mannitol as osmoticum. At the eight-to ten-cell stage, divided cells were transferred to Kao's basal medium supplemented with 0.3 M sucrose as carbon source and 0.1% agarose, 2 mg/ l BA, 2 mg/l zeatin (ZEA), 1 mg/l NAA, and 0.5 mg/l Kin for callus proliferation. After 35 d, when small calli reached 2-3 mm in diameter, calli were transferred to regeneration medium containing 5 mg/l ZEA and 2 mg/l indole-3-acetic acid. The chromosome numbers of putative somatic hybrids varied from 46 to 54. A total of ten plants showed a 0.5-kb, CMS-specific band, while two regenerated plants showed a missing band similar to that of leaf mustard by polymerase chain reaction amplification using Ogura CMS-specific primers. Hybrid state was confirmed by random amplified polymorphic DNA analysis. Regenerated plants had normal petals and stamens, but only two plants produced pollen and set seed.

Detection of the Diversity of Cytoplasmic Male Sterility Sources in Broccoli (Brassica Oleracea var. Italica) Using Mitochondrial Markers

“…italica (Jing et al, 2015 ), and B. rapa (Zhang et al, 2007 ; Shi et al, 2012 ; Heng et al, 2015 ). The markers have also been used to analyze genetic diversity in varieties of B. napus (Handa, 2007 ; Zhao et al, 2010 ) and B. rapa (Zhang et al, 2013 ), and to differentiate between somatic hybrids of B. juncea and B. oleracea (Lian et al, 2011 ). Thus, far, mitochondrial markers have not been successfully used to analyze the different types of CMS and genetic diversity of original sources of CMS in broccoli.…”

Interspecific Hybridization for Brassica Crop Improvement

“…Protoplast fusion has been used to transfer Ogu cytoplasmic male sterility factor from Brassica napus to Brassica juncea and for the improvement of male sterile lines in hybrid breeding systems [81]. Somatic hybridization between B. juncea and B. oleracea has also been used to transfer cytoplasmic male sterility and resistance to Turnip mosaic virus from B. oleracea to B. juncea [82,83]. Prakash et al [84] successfully obtained both stable CMS B. juncea and an introgression line carrying the restorer gene via somatic hybridization between M. arvensis and B. juncea followed by backcrossing with B. juncea.…”

Tri-parental protoplast fusion ofBrassicaspecies to produce somatic hybrids with high genetic and phenotypic variability

“…In brief, 3 types of male sterile types were observed in the present study: one type had stamens that were significantly shorter than the stigma length, the second type had flowers with normal stamens and empty pollen, and the third type had normal flowers with stamen carpeloid. In contrast, somatic hybrids derived from 2 parental cell fusions had empty anthers (Lian et al 2011a) or low fertility (Lian et al 2011b). These results indicate that tri-parental somatic distant hybridization can not only introduce new characteristics from donors into phylogenetically close plants such as the CMS trait with wider range and greater ease, but can also create new variant types beyond the variation present in the bi-parental fusion.…”