The plastid genome (plastome) has proved a valuable source of data for evaluating evolutionary relationships among angiosperms. Through basic and applied approaches, plastid transformation technology offers the potential to understand and improve plant productivity, providing food, fiber, energy and medicines to meet the needs of a burgeoning global population. The growing genomic resources available to both phylogenetic and biotechnological investigations are allowing novel insights and expanding the scope of plastome research to encompass new species. In this chapter we present an overview of some of the seminal and contemporary research that has contributed to our current understanding of plastome evolution and attempt to highlight the relationship between evolutionary mechanisms and tools of plastid genetic engineering.

The plastid genomes of flowering plants.

In this review we focus on recent progress in protoplast regeneration, symmetric and asymmetric hybridization and novel technology developments. Regeneration of new species and improved culture techniques opened new horizons for practical breeding in a number of crops. The importance of protoplast sources and embedding systems is discussed. The study of reactive oxygen species effects and DNA (de)condensation, along with thorough phytohormone monitoring, are in our opinion the most promising research topics in the further strive for rationalization of protoplast regeneration. Following, fusion and fragmentation progress is summarized. Genomic, transcriptomic and proteomic studies have led to better insights in fundamental processes such as cell wall formation, cell development and chromosome rearrangements in fusion products, whether or not obtained after irradiation. Advanced molecular screening methods of both genome and cytoplasmome facilitate efficient screening of both symmetric and asymmetric fusion products. We expect that emerging technologies as GISH, high resolution melting and next generation sequencing will pay major contributions to our insights of genome creation and stabilization, mainly after asymmetric hybridization. Finally, we demonstrate agricultural valorization of somatic hybridization through enumerating recent introgression of diverse traits in a number of commercial crops.

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Progress in plant protoplast research

Hybridization events are relatively common in vascular plants. However, the frequency of these events is unevenly distributed across the plant phylogeny. Plant families in which individual species are pollinated by specific pollinator species are predicted to be less prone to hybridization than other families. However, exceptions may occur within these families, when pollinators shift host-plant species. Indeed, host shifts are expected to increase the rate of hybridization events. Pollinators of Ficus section Galoglychia are suspected to have changed host repeatedly, based on several cases of incongruence between plant phylogeny and taxonomy, and insect phylogeny and taxonomy. We tracked cyto-nuclear discordance across section Galoglychia as evidence for hybridization. To achieve a proper global view, we first clarified the monophyly of section Galoglychia as it had been questioned by recent phylogenetic studies. Moreover, we investigated if fig size could be a factor facilitating host shifts. Phylogenetic chloroplast and nuclear results demonstrated the monophyly of section Galoglychia. Within section Galoglychia, we detected several cases of statistically significant cyto-nuclear discordance. Discordances concern both terminal nodes of the phylogenetic trees and one deep node defining relationships between subsections. Because nuclear phylogeny is congruent with morphological taxonomy, discordances were caused by the chloroplast phylogeny. Introgressive hybridization was the most likely explanation for these discordances. We also detected that subsections pollinated by several wasp genera had smaller figs and were pollinated by smaller wasps than subsections pollinated by a single wasp genus. As hypothesized, we discovered evidences of past hybridization in Ficus section Galoglychia. Further, introgression was only detected in subsections presenting incongruence between plant and pollinator phylogenies and taxonomy. This supports the hypothesis that host shift is the cause for plant-pollinator incongruence. Moreover, small fig size could facilitate host shifts. Eventually, this study demonstrates that non-coding chloroplast markers are valuable to resolve deep nodes in Ficus phylogeny.

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Cyto-nuclear discordance in the phylogeny of Ficus section Galoglychia and host shifts in plant-pollinator associations

Salinity is one of the major stresses threatening potato plants (Solanum tuberosum L.) by affecting their growth and yield. It leads to oxidative stress by the production of reactive oxygen species responsible for alteration of macromolecules. To improve the tolerance of potato to salt stress, we have used somatic hybridization to produce interspecific potato hybrids by protoplast fusion between the BF15 variety and the wild Solanum berthaultii species. These hybrids showed an improved tolerance to salt stress when cultivated in vitro. The present work aims to analyze the response of the hybrids to salt stress in greenhouse conditions. Thus, the development of plants and their antioxidant capacity in response to salt stress were followed. All hybrids showed better growth and stable chlorophyll content compared to those of the BF15 parent plant. Membrane lipid peroxidation, evaluated by measuring the malondialdehyde accumulation (MDA) in plant organs, showed low levels in the hybrids. Higher antioxidant enzyme activities were measured in the roots of the hybrids when compared to those of the BF15 parent. These hybrids also showed an improved control of Na+ accumulation and a stable K+/Na+ ratio. These results therefore confirm the better tolerance of these hybrids to salt stress when compared to their BF15 parent.

Investigation of the response to salinity and to oxidative stress of interspecific potato somatic hybrids grown in a greenhouse

Publisher Summary Potato breeders are challenged by the tetraploid nature of the potato, limited variability for economically important traits in adapted breeding clones, and a complex set of requirements necessary for the successful adoption of new cultivars. An understanding of the genetic basis of the photoperiod response for tuberization would help geneticists and breeders to maximize their use of wild species germplasm. Genetic variance for yield in potato is mainly nonadditive, involving intralocus and interlocus interactions. There is good reason for optimism about the prospects for continued genetic improvements in potato. The germplasm resources are abundant in genetic diversity, and breeders have access to a well-stocked toolbox for the utilization of those resources.

Breeding, Genetics, and Cultivar Development

In this study three somatic hybrid lines originating from protoplast fusion between Solanum tuberosum cv. BF15 and Solanum berthaultii were subjected to a detailed molecular analysis using the I-SSR-PCR technique based on 5′-anchored microsatellite primers. The data obtained revealed a polymorphism between the different lines, suggesting that they correspond to symmetric hybrids. The analysis of chloroplast genome of these hybrids showed that they are resulting from a recombination between parental plastomes. When transferred to a greenhouse, these hybrid lines displayed an improved vigour compared to the cultivated potato BF15 parent. Indeed, an important growth rate and high tuber yield and weight were obtained for these hybrids compared to the parent. Some of these hybrids showed also an improved ion homeostasis control and they seem to display a better tolerance to salt stress compared to the potato BF15 parent.

Interspecific potato somatic hybrids between Solanum berthaultii and Solanum tuberosum L. showed recombinant plastome and improved tolerance to salinity

Production of potato intraspecific somatic hybrids with improved tolerance to PVY and Pythium aphanidermatum

In the present survey, interspecific somatic protoplast fusion between a dihaploid cultivated potato Solanum L. tuberosum (BF15) and a wild species Solanum vernei (V3) has been performed using the PEG method. Five putative hybrids were first selected. Among them, only two were retained. DNA analysis, using flow cytometry, showed that the first hybrid (VB2) was dihaploid (2n=2x=24 chromosomes), whereas the second (VB1) was hexaploid (2n=6x=72 chromosomes). In the greenhouse, these putative hybrids showed that they were able of raising and producing large tubers but with a modified shape compared to parental ones. Furthermore, they harbored an intermediate leaf morphology compared to their parents. The hybrid nature of these plants was first confirmed according to their esterase and peroxidase isoenzyme patterns. The RAPD analysis of genomic DNA and microsatellite based amplification (I-SSR) showed that both clones VB1 and VB2 are asymmetric somatic hybrids. They seem to have eliminated the major part of V3 parental nucleus but not the totality. The analysis of chloroplast DNA suggests that both hybrid plastomes were the result of a recombination between parental ones.

Lilia Lakhoua

Papers

Interspecific potato somatic hybrids between Solanum berthaultii and Solanum tuberosum L. showed recombinant plastome and improved tolerance to salinity

Production of potato intraspecific somatic hybrids with improved tolerance to PVY and Pythium aphanidermatum

Somatic hybrids between potato Solanum tuberosum and wild species Solanum verneï exhibit a recombination in the plastome