Novel alloplasmic Nicotiana plants by “donor-recipient” protoplast fusion: cybrids having N. tabacum or N. sylvestris nuclear genomes and either or both plastomes and chondriomes from alien species

Abstract

The “donor-recipient” fusion method was utilized to investigate interspecific transfer of organelles and organelle-controlled traits from donor to recipient Nicotiana species. To follow organelle transfer and sorting-out under conditions which will be either selective or non-selective for chloroplast transfer we used, respectively, albino (N. tabacum VBW) or normally pigmented (N. sylvestris) plants as protoplast-recipients. N. alata, N. bigelovii and N. undulata served as donors. Organelle composition of the cybrid plants was investigated by the analysis of chloroplast DNA restriction pattern, tentoxin sensitivity, chloroplast pigmentation, mitochondrial DNA restriction pattern and alloplasmic male sterility. In certain cybrids the analysis of restriction patterns of chloroplast and mitochondrial DNA was augmented by Southern hybridization with the respective organelle-DNA probes. A total of 341 fusion-derived plants (from 109 calli) were analysed and 102 of these were found to be cybrids with novel nuclear/organelle compositions. The incidence of chloroplast and mitochondrial transfer was strongly facilitated when iodoacetate-treated protoplasts of N. tabacum VBW (albino) rather than N. sylvestris (normal chloroplasts) protoplasts served as recipient. Mitochondrial DNA restriction patterns in cybrids commonly differed from those of either fusion partner. All fusion combinations resulted in at least some male-sterile cybrids, having either donor or recipient plastomes thus indicating that interspecific “donor-recipient” protoplast fusion is an efficient mean to produce plants with alloplasmic male sterility.