One-step generation of cytoplasmic male sterility by fusion of mitochondrial-inactivated tomato protoplasts with nuclear-inactivated Solanum protoplasts.

Abstract

Mesophyll protoplasts of Lycopersicon esculentum were treated with iodoacetamide to inactivate mitochondria, and protoplasts of Solanum acauk and Solanum tuberosum were irradiated with v-or x-rays to inactivate nuclei. Mixtures of protoplasts thus modified were treated with Ca2+ and polyethylene glycol to obtain heterologous fusion products. Among the fusion products were some tomato plants that were indistinguishabl from the original cultivars with respect to morphology, physiology, and chromosome number (2N = 24) but exhibited various degrees of male sterility (MS): complete lack or malformation of anthers, shrunken pollen, and normal-looking stainable pollen that could not germinate. The MS thus induced in five cultivars ofdifferent growth types, including one of subspecies L. escukntum cerasforme, was inherited maternally over several generations and is, therefore, cytoplasmically determined MS (CMS). Analysis of mitochondrial DNA revealed that the mitochondrial genome of the CMS hybrids does not contain all elements ofthe mitochondrial DNA of either parent but includes sequences of a recombinational nature not present in either parent. The CMS hybrids, therefore, possess a true hybrid mitochondrial genome. The same procedure applied to fusion of tomato with Solaxum lycopersicoides and Nicoiana Atbacum cells did not produce CMS phenotypes. The advantages of this method &ver others for generating MS are as follows: (i) only one step is required; (i) the nuclear genotype of the cultivar is unaffected; (ija the prospect that cytoplasmic determination allows generation of 100% CMS progenies. The normal-appearing but nonfunctional pollen ofcertain CMS types might render them attractive to pollinating bumblebees that thus would facilitate production of hybrid seed.Male sterility (MS) can be artificially introduced into flowering plants by chemical treatment, by delivery of nuclear and/or organellar genes (1), or by transformation with a chimeric gene composed of an anther-specific promoter and a ribonuclease gene (2). In the production of hybrid seed, the need for MS plants is increasing to avoid manual emasculation offemale parents, as =40%o of the total labor is expended for this task, and to avert contamination by self-pollination. In conventional breeding, however, the use of genic MS requires many generations of backcrossing and selection from original sources. A method to produce MS in one step within a desired cultivar would, therefore, be extremely useful.Symmetric hybrids between tomato and potato, produced by protoplast fusion (3-5), showed an intermediate cold tolerance (6). Substitution of Solanum acaule genome into Solanum tuberosum could result in an appreciable increase of frost resistance (7,8). Subsequently, experiments were initiated to compare different fusion methods in this system. For this purpose tomato protoplasts were inactivated with iodoacetamide (IQA), which damages mitochondria and seems not to affect nuclear genomes, and the fusion partners of S. acaule or S. tuberosum were tr...

Transgenic induction of mitochondrial rearrangements for cytoplasmic male sterility in crop plants

“…Sources studied in tobacco have resulted from interspecific hybridization (19) and from in vitro culture (20). In tomato, CMS was also observed as a consequence of interspecific hybridization (21). Both cell culture and alloplasmy routinely give rise to mitochondrial DNA rearrangement and SSS activity and may involve reduced Msh1 expression.…”

Pigment Deficiency in Nightshade/Tobacco Cybrids Is Caused by the Failure to Edit the Plastid ATPase α-Subunit mRNA

“…Nuclear-organellar incompatibilities in higher plants are known to include cytoplasmic male sterility, which is an agriculturally important trait associated with aberrant mitochondria (Melchers et al, 1992;Conley and Hanson, 1995;Kofer et al, 1999). Cytoplasmic male sterility attributable to nuclear-mitochondrial incompatibility is thought to occur as a result of rearrangements of the mitochondrial genome presumably caused by illegitimate recombination (Belliard et al, 1979;Hanson and Bentolila, 2004).…”

Molecular and Ultrastructural Analysis of a Nonchromosomal Variegated Mutant (Tomato Mitochondrial Mutants That Cause Abnormal Leaf Development)

“…109, 1995 alterations in the mitochondrial genome. In one case, CMS tomatoes have been produced following the regeneration of tomato cybrids with portions of the mitochondrial genome of Solanum acaule (Melchers et al, 1992). We have constructed tomato cybrids that have tomato nuclear and chloroplast genomes but mitochondrial genomes that are recombinant between tomato (Lycopersicon esculentum) and L. pennellii (Bonnema et al, 1991).…”