Luigi Monti

Uses and usefulness of endosperm balance number

Abstract The Endosperm Balance Number (EBN) hypothesis was developed in the early ’80s to explain the basis for normal seed development after intra- and inter-specific crosses, first in the potato and then in several other crop species. According to this hypothesis, each species has a genome-specific effective ploidy, the EBN, which must be in a 2 : 1 maternal to paternal ratio in the hybrid endosperm for normal development of the endosperm itself. This paper reviews how the EBN may act as a powerful isolating mechanism in sexual reproduction, maintaining the genome integrity of the species and playing an important role in the speciation of polyploids from diploids. We also provide further evidence that EBN is more important than chromosome ploidy in determining the success or failure of interspecific crosses. In fact, results from inter-ploidy and inter-EBN crosses to infuse 1EBN Solanum commersonii into 4EBN S. tuberosum demonstrated that the knowledge and manipulation of EBN is a useful tool in designing breeding schemes and in predicting the offspring ploidy and EBN. In this paper we also discuss the exceptions to the 2 : 1 EBN ratio, and report the evidence for endomitosis in the polar nuclei to explain exceptions to the EBN model in the potato.

Organelle DNA and male fertility variation in Solanum spp. and interspecific somatic hybrids

Abstract Novel and potentially useful genetic variation in cytoplasmic genomes can be induced by interspecific somatic hybridization in plants. To evaluate such variability and correlate it with nuclear-cytoplasmic interactions leading to male sterility in Solanum spp., we examined progeny of male-sterile and male-fertile somatic hybrids between Solanum tuberosum (tbr), the common potato, and S. commersonii (cmm), a wild species showing sexual incongruity with tbr, for fertility and organelle DNA composition. Uniform male-fertile and male-sterile progenies were obtained by selfing the male-fertile hybrid and crossing the male-sterile ones, indicating maternal inheritance of the fertility phenotype. The two fusion partners were only slightly differentiated in the plastidial genome. MtDNA polymorphism between the species was greater, although its extent varied with the genomic region investigated. All somatic hybrids had non-parental organelle genomes, with reassorted organelles and/or rearranged mitochondria (i.e., cmm-specific bands for some regions and tbr-specific bands for others). Mitochondria reassorted independently from chloroplasts. Most hybrids showed the cmm cpDNA hybridization pattern, indicating non-random transmission of chloroplasts. Most male-sterile hybrids showed preferential inheritance of tbr mtDNA fragments. The male-fertile somatic hybrid clone had predominantly cmm mtDNA fragments. This result suggests that a tbr-derived region involved in nuclear-cytoplasmic incompatibility and male sterility has been lost by rearrangement; however, no clear correlation between a specific mitochondrial region and male sterility has been found so far.

Identification of Vigna spp. through specific seed storage polypeptides

SummarySeed globulins of ten Vigna species were separated by means of SDS electrophoresis. Both inter and intraspecific variation were observed.Groups of specific bands which do not vary in the species being present in all the accessions of the species were identified. These species-specific bands, allow the identification of the 10 Vigna spp. analyzed.

Solanum sect. Lycopersicon

In this review, we examine the plant group Solanum sect. Lycopersicon – a clade of 13 species, including the domesticated tomato (Solanum lycopersicum L.) and its wild relatives – along with four allied species in the immediate outgroups Solanum sects. Juglandifolia and Lycopersicoides. We summarize the geographic distribution and morphological characters of these plant groups, describing their evolutionary relationships in the context of a new taxonomic revision at the species level of all these groups. We provide an overview of the role that wild tomato species have played in the development of cytogenetic stocks, in classical and molecular genetic studies as well as in crop improvement through traditional and advanced tools. We discuss how the very narrow genetic basis of cultivated tomato germplasm has forced tomato geneticists and breeders to rely on the wealth of genetic variation present in the wild relatives to address the many breeding challenges. The numerous molecular mapping studies conducted using interspecific crosses have clearly demonstrated that the breeding value of exotic (wild) tomato germplasm goes far beyond its phenotype. These studies also show that we are still far from being able to fully exploit the breeding potential of the thousands of accessions stored in seed banks around the world, in addition to those that may still be found in natural habitats. Over the past decades, tomato breeders have been at the forefront of establishing new principles for crop breeding based on the use of wild species to improve modern cultivars. In this respect, among all model systems, the wild and domesticated species of the tomato clade have pioneered development of novel populations such as “exotic libraries.” These genetic resources, combined with the increasing knowledge deriving from the many “omics” tools, including the tomato genome sequence, are expected to further improve the efficiency with which wild tomato relatives will contribute to the improvement of this important crop.

Transgenic chloroplasts are efficient sites for high‐yield production of the vaccinia virus envelope protein A27L in plant cells†

Orthopoxviruses (OPVs) have recently received increasing attention because of their potential use in bioterrorism and the occurrence of zoonotic OPV outbreaks, highlighting the need for the development of safe and cost-effective vaccines against smallpox and related viruses. In this respect, the production of subunit protein-based vaccines in transgenic plants is an attractive approach. For this purpose, the A27L immunogenic protein of vaccinia virus was expressed in tobacco using stable transformation of the nuclear or plastid genome. The vaccinia virus protein was expressed in the stroma of transplastomic plants in soluble form and accumulated to about 18% of total soluble protein (equivalent to approximately 1.7 mg/g fresh weight). This level of A27L accumulation was 500-fold higher than that in nuclear transformed plants, and did not decline during leaf development. Transplastomic plants showed a partial reduction in growth and were chlorotic, but reached maturity and set fertile seeds. Analysis by immunofluorescence microscopy indicated altered chlorophyll distribution. Chloroplast-synthesized A27L formed oligomers, suggesting correct folding and quaternary structure, and was recognized by serum from a patient recently infected by a zoonotic OPV. Taken together, these results demonstrate that chloroplasts are an attractive production vehicle for the expression of OPV subunit vaccines.

The expression in tobacco plants of Aedes aegypti Trypsin Modulating Oostatic Factor (Aea-TMOF) alters growth and development of the tobacco budworm, Heliothis virescens

The production and characterisation of transgenic tobacco plantsexpressing a precursor of a regulatory peptide from Aedesaegypti (Trypsin Modulating and Oostatic Factor, Aea-TMOF) whichinterferes with the development of tobacco budworm larvae is described. Tobaccoplants were transformed with a synthetic gene containing 6 TMOF units spaced bydibasic residues, Arg-Arg, as potential post-translational cleavage sites.Peptide extracts from transgenic plants had TMOF activity and inhibitedin vitro the biosynthesis of serine proteases. Thisactivity was consistently present in T1 plants and absent in control plants.Tobacco budworm larvae, fed with transgenic leaves showed a reduced growth ratecompared to those fed with control plants. The low rather than acute toxicityofthis low impact gene is discussed in the context of alternative integrated pestmanagement strategies.

Organelle DNA variation in parental Solanum spp. genotypes and nuclear-cytoplasmic interactions in Solanum tuberosum (+) S. commersonii somatic hybrid-backcross progeny

Nuclear-cytoplasmic interactions can influence fertility and agronomic performance of interspecific hybrids in potato as well as other species. With the aim of assessing the potential value of a novel recombinant cytoplasm derived by interspecific somatic hybridization, backcross progeny were produced by crossing a somatic hybrid between Solanum tuberosum (tbr) and the wild incongruous species S. commersonii (cmm) with various potato clones. BC1 clones were evaluated for male fertility and other agronomic traits. Male fertility clearly depended on the cross direction and the cytoplasm source. Genotypes with cytoplasms sensitive to nuclear genes derived from Solanum commersonii and inducing male sterility showed identical mtDNA composition, as based on mtDNA analyses with various PCR-based and RFLP markers. On the other hand, genotypes with cytoplasms not inducing male sterility in the presence of the cmm nuclear genes showed a different mtDNA organisation. Analysis of cpDNA confirmed similarity of cytoplasmic composition in CMS-inducing genotypes and clear differences with the others. Genotypes with recombinant cytoplasm induced by somatic hybridization generally showed similar agronomic performances in reciprocal hybrids with tbr cytoplasm, suggesting that the novel cytoplasm can be used in potato breeding.

An insect peptide engineered into the tomato prosystemin gene is released in transgenic tobacco plants and exerts biological activity.

Tomato systemin is a signalling peptide produced in response to wounding that locally and systemically activates several defence genes. The peptide is released from the C-terminus of prosystemin, the 200 amino acid precursor, following post-translational modifications involving unknown events and enzymes. In tobacco, two systemin molecules have been recently isolated, neither sharing any sequence homologies with the tomato prosystemin gene/protein, but performing similar functions. We modified the tomato prosystemin gene by replacing the systemin-encoding region with a synthetic sequence encoding TMOF (trypsin-modulating oostatic factor), a 10 amino acid insect peptide hormone toxic to Heliothis virescens larvae, and expressed the chimeric gene in tobacco. The results reported here show that transformed leaves contain the TMOF peptide and exert toxic activity against insect larvae reared on them. In addition, subcellular localization studies showed the cytoplasmic location of the released TMOF, suggesting that in tobacco the enzymes responsible for the post-translational modifications of the tomato precursor protein are present and act in the cytoplasm to recognise the modified prohormone. The molecular engineering of the precursor, beside supplying new clues towards the understanding of prosystemin processing, constitutes an useful tool for plant genetic manipulation, by enabling the delivery of short biological active peptides.

Elucidation of meiotic nuclear restitution mechanisms in potato through analysis of microtubular cytoskeleton

Organization and rearrangements of microtubular cytoskeleton (MTs) during microsporogenesis are determined in ‘historical’ potato genotypes producing 2n pollen following different meiotic nuclear restitution mechanisms (‘parallel spindles’, ‘fused spindles’, ‘premature cytokinesis’). The scope of the investigation is to assess the abnormalities in MT cytoskeleton and their relationships to 2n pollen formation. The genotypes of Solanum used for MT immunofluorescence in this study are diploid interspecific hybrids (2n=2x=24) coming from Wisconsin (USA)and Wageningen (NL) Universities. Based on MT analysis, a reinterpretation of the restitution mechanisms following spindle abnormalities was performed in this study. The fusion of the nuclei at prophase II occurs through the perinuclear MTs in the genotype with ‘fused spindles’. In genotypes with misoriented spindles, the ‘non-spindle’ cytoskeletal arrays,occurring at telophase II, are clue in determining the meiotic products such as dyads and triads. A particular MT array could be crucially involved in meiotic nuclear restitutions both at telophase I and II: the interzonal MTs. It is discussed which is the relationship between this array and the spindle orientation at meta-anaphase II. It is hypothesized that the fusion of non-sister nuclei at each pole depends on the lack of secondary interzonal MTs and on the short distance between nuclei. In the genotype with premature cytokinesis, effectively, a premature phragmoplast occurs at the end of meiosis I. Furthermore, two genotypes are desynaptic with a high degree of male fertility restored by the nuclear restitutions. However, no clear relationship was found between univalents and restitution mechanisms.

Tuber quality and soft rot resistance of hybrids betweenSolarium tuberosum and the incongruent wild relativeS. commersonii

Tetraploid BC3 hybrids between sexually incompatible speciesSolanum commersonii andS. tuberosum were characterized for tuber quality (stolon length, tuber eye depth, chipping color from 3-month storage at 5 C, and specific gravity) and soft rot resistance traits. For all analysed morphological traits, except stolon length, genotypes resembled theS. tuberosum phenotype. On average, the specific gravity of selected genotypes was quite high (1.087), and 12 of them had a value >1.090. As for chipprocessing ability, 4.8% of genotypes chipped well directly from cold storage, and 14.3% after reconditioning. All but one of these genotypes also possessed a high specific gravity of tubers. Variability was found for resistance toErwinia carotovora subsp.carotovora. In particular, 24% of hybrids had a rotted area diameter ≤ 4 mm and were classified as resistant. The average percentage ofcommersonii-specific markers still present in the BC3 hybrids was 22%, ranging from 9% to 37%. Genotypes combining good qualitative tuber traits with a wild genome content lower than the average value of the population were selected for further breeding efforts. The importance of molecular-assisted selection is discussed.ResumenLos híbridos tetraploides BC3 entre especies sexualmente incompatibles deSolanum commersonii y S. Tuberosum fueron caracterizados por la calidad del tubérculo (longitud de los estolones, profundidad de los ojos del tubérculo, color de las hojuelas después de 3 meses de almacenamiento a 5°C y gravedad específica) y características de su resistencia a la podredumbre blanda. Para todas las características analizadas, excepto tamaño de los estolones, los genotipos se parecían al fenotipoS. tuberosum. En promedio, la gravedad específica de los genotipos seleccionados fue absolutamente más alta (1.087), y 12 de ellos tenían un valor de >1.090. Respecto a su habilidad para el procesamiento en hojuelas, el 4.8% de los genotipos eran de buen color cuando se extrajeron directamente del almacenamiento en frio y 14.3% cuando se extrajeron después del reacondicionamiento. Pero uno de esos genotipos poseía también una alta gravedad específica de tubérculos. Se encontró resistencia por variabilidad aErwinia carotovora subsp.carotovora. En particular, 24% de híbridos tuvieron un diámetro de área de descomposición de ≤ 4 mm y fueron clasificados como resistentes. El porcentaje promedio de los marcadores específicoscommersonii- todavía presente en los híbridos BC3 fue 22%, con una amplitud de 9% a 37%. Los genotipos combinaban buena calidad de los tubérculos con un contenido de genoma silvestre menor que el valor porcentual de la población seleccionada para futuros esfuerzos de mejoramiento. La importancia de la selección molecular asistida está en discusión.