Anther and Isolated Microspore Culture in Eggplant (Solanum melongena L.).

Eggplant is one of the five important, worldwide-distributed solanaceous crops. The use of anther culture technology to produce pure, 100% homozygous doubled haploid lines for hybrid seed production is possible since 1982, where the first protocol of wide application to different eggplant materials was published. From then on, different improvements and adaptations to different materials have been made. In parallel, protocols to implement isolated microspore culture technology in eggplant have been developed principally in the last decade, which opens the door for a more efficient DH production in this species. In this chapter, two protocols, one for anther and other for isolated microspore culture in eggplant, are described. Some steps and materials are common to both approaches. A detailed description of each step from is provided.

Anther Culture in Eggplant (Solanum melongena L.).

For a long time, conventional breeding methods have been used to obtain pure, 100% homozygous lines for hybrid seed production in crops of agronomic interest. However, by doubled haploid technology, it is possible to produce 100% homozygous plants derived from precursors of male gametophytes (androgenesis), to accelerate the production of pure lines, which implies important time and cost savings. In this chapter, a protocol for anther culture in eggplant is described, from donor plant growth conditions to regeneration and acclimation of doubled haploid plants, as well as a description of how to analyze ploidy levels of regenerated plants.

Embryogenic competence of microspores is associated with their ability to form a callosic, osmoprotective subintinal layer.

Microspore embryogenesis is an experimental morphogenic pathway with important applications in basic research and applied plant breeding, but its genetic, cellular, and molecular bases are poorly understood. We applied a multidisciplinary approach using confocal and electron microscopy, detection of Ca2+, callose, and cellulose, treatments with caffeine, digitonin, and endosidin7, morphometry, qPCR, osmometry, and viability assays in order to study the dynamics of cell wall formation during embryogenesis induction in a high-response rapeseed (Brassica napus) line and two recalcitrant rapeseed and eggplant (Solanum melongena) lines. Formation of a callose-rich subintinal layer (SL) was common to microspore embryogenesis in the different genotypes. However, this process was directly related to embryogenic response, being greater in high-response genotypes. A link could be established between Ca2+ influx, abnormal callose/cellulose deposition, and the genotype-specific embryogenic competence. Callose deposition in inner walls and SLs are independent processes, regulated by different callose synthases. Viability and control of internal osmolality are also related to SL formation. In summary, we identified one of the causes of recalcitrance to embryogenesis induction: a reduced or absent protective SL. In responding genotypes, SLs are markers for changes in cell fate and serve as osmoprotective barriers to increase viability in imbalanced in vitro environments. Genotype-specific differences relate to different responses against abiotic (heat/osmotic) stresses.

BvCOLD1: A novel aquaporin from sugar beet (Beta vulgaris L.) involved in boron homeostasis and abiotic stress.

Beta vulgaris (sugar beet) is one of the most important industrial crops. Screening of a cDNA library for sugar beet genes able to confer cold tolerance upon overexpression in yeast identified a novel aquaporin, which we named BvCOLD1. The amino acid sequence of BvCOLD1 indicated that an acidic protein (pI 5.18) is similar to tonoplast intrinsic protein aquaporins. RNA expression analysis indicated that BvCOLD1 is expressed in all sugar beet organs. Confocal microscopy of a green fluorescent protein-tagged version localized BvCOLD1 in the endoplasmic reticulum in yeast and in plant cells. Experiments in yeast showed that BvCOLD1 has an important role in transporting several molecules, among them is boron, one of the most limiting micronutrients for sugar beet cultivation. Transgenic Arabidopsis thaliana plants overexpressing BvCOLD1 showed enhanced tolerance to cold, to different abiotic stresses, and to boron deficiency at different developmental stages. Searches in databases only retrieved BvCOLD1 orthologues in genomes from the Chenopodioideae, a subfamily of the Amaranthaceae family that includes the closely related crop Spinacea oleracea and halotolerant plants such as Salicornia herbacea or Suaeda glauca. Orthologues share a conserved sequence in the carboxy terminus, not present in other aquaporins, which is required for the functionality of the protein.

Glutathione and transpiration as key factors conditioning oxidative stress in Arabidopsis thaliana exposed to uranium.

Although oxidative stress has been previously described in plants exposed to uranium (U), some uncertainty remains about the role of glutathione and tocopherol availability in the different responsiveness of plants to photo-oxidative damage. Moreover, in most cases, little consideration is given to the role of water transport in shoot heavy metal accumulation. Here, we investigated the effect of uranyl nitrate exposure (50 µM) on PSII and parameters involved in water transport (leaf transpiration and aquaporin gene expression) of Arabidopsis wild type (WT) and mutant plants that are deficient in tocopherol (vte1: null α/γ-tocopherol and vte4: null α-tocopherol) and glutathione biosynthesis (high content: cad1.3 and low content: cad2.1). We show how U exposure induced photosynthetic inhibition that entailed an electron sink/source imbalance that caused PSII photoinhibition in the mutants. The WT was the only line where U did not damage PSII. The increase in energy thermal dissipation observed in all the plants exposed to U did not avoid photo-oxidative damage of mutants. The maintenance of control of glutathione and malondialdehyde contents probed to be target points for the overcoming of photoinhibition in the WT. The relationship between leaf U content and leaf transpiration confirmed the relevance of water transport in heavy metals partitioning and accumulation in leaves, with the consequent implication of susceptibility to oxidative stress.

Identification of a cDNA from the arbuscular mycorrhizal fungus Glomus intraradices that is expressed during mycorrhizal symbiosis and up-regulated by N fertilization.

A cDNA library was constructed with RNA from Glomus intraradices-colonized lettuce roots and used for differential screening. This allowed the identification of a cDNA (Gi-1) that was expressed only in mycorrhizal roots and was of fungal origin. The function of the gene product is unknown, because Gi-1 contained a complete open reading frame that was predicted to encode a protein of 157 amino acids which only showed little homology with glutamine synthetase from Helicobacter pylori. The time-course analysis of gene expression during the fungal life cycle showed that Gi-1 was expressed only during the mycorrhizal symbiosis and was not detected in dormant or germinating spores of G. intraradices. P fertilization did not significantly change the pattern of Gi-1 expression compared with that in the unfertilized treatment, whereas N fertilization (alone or in combination with P) considerably enhanced the Gi-1 transcript accumulation. This increase in gene expression correlated with plant N status and growth under such conditions. The possible role of the Gi-1 gene product in intermediary N metabolism of arbuscular mycorrhizal symbiosis is further discussed.