No abstract

A successful exploitation of in vitro tools for breeding and for the understanding of gene functioning requires the regeneration of true-to-type plants and experiments were therefore performed with several genotypes of Medicago truncatula (J5, TRV25, TR122) to characterize mother plants and regenerating tissues. Each sample was assessed by flow cytometry and, whatever the genotype and regeneration pathway, a divergent phenotype was systematically linked to an abnormal nuclear DNA content. All samples assessed were classed according to their flow cytometry profiles into normal (true-to-type) material, aneuploids, endoreduplicated tissues, tetraploids, mixoploids and senescent tissues. Deviating calluses failed to regenerate or gave rise to infertile, non-viable plants. In turn, all tissues with non true-to-type flow cytometry profiles were examined in terms of isoenzyme banding patterns compared to the mother plants. Esterases, Peroxidases and Leucine aminopeptidase appeared to be the best isozyme systems to show differences between the original genotypes but also between diverging materials and the mother plants. Interestingly, such differences were more often qualitative (presence or absence of bands) than quantitative (i.e. differences in colour intensity of bands) thereby making easier an accurate distinction between genotypes. Peroxidases were prone to variation with culture medium and tissue age. The results stressed the importance of using more than one approach when undertaking the characterisation of materials as, for some of the genotypes analysed, differences compared to the respective mother plants could be shown with flow cytometry that were not reflected in a different banding pattern with isoenzymes.

Polyethylene glycol (PEG) can be used to mimic osmotic stress in plant tissue cultures to study mechanisms of tolerance. The aim of this experiment was to investigate the effects of PEG (M.W. 6000) on embryogenic callus of Medicago truncatula. Leaf explants were cultured on MS medium with 2 mg L-1 NAA and 0.5 mg L-1 BAP for 5 months. Then, calli were transferred to the same medium further supplemented with 10% (w/v) 6000 PEG for 6 months in order to study physiological and putative molecular markers of water stress. There were no significant differences in growth rate of callus or mitotic index ± PEG although embryogenic potential of PEG treated callus was morphologically enhanced. Cells were rounder on PEG medium and cell size, nuclear size and endoreduplication increased in response to the PEG treatment. Significant increases in soluble sugar and proline accumulation occurred under PEG treatment compared with the control. Significantly, high MtWEE1 and MtCCS52 expression resulted from 6 months of PEG treatment with no significant differences in MtSERK1 or MtP5CS expression but down regulation of MtSOS expression. The results are consistent in showing elevated expression of a cell cycle checkpoint gene, WEE1. It is likely that the cell cycle checkpoint surveillance machinery, that would include WEE1 expression, is ameliorating the effects of the stress imposed by PEG.

Changes in global climate and the nonstop increase in demographic pressure have provoked a stronger demand for agronomic resources at a time where land suitable for agriculture is becoming a rare commodity. They have also generated a number of abiotic stresses which exacerbate effects of diseases and pests and result in physiological and metabolic disorders that ultimately impact on yield when and where it is most needed. Therefore, a major scientific and agronomic challenge today is that of understanding and countering the impact of stress on yield. In this respect, in vitro biotechnology would be an efficient and feasible breeding alternative, particularly now that the genetic and genomic tools needed to unravel the mechanisms underlying the acquisition of tolerance to stress have become available. Legumes in general play a central role in a sustainable agriculture due to their capacity to symbiotically fix the atmospheric nitrogen, thereby reducing the need for fertilizers. They also produce grains that are rich in protein and thus are important as food and feed. However, they also suffer from abiotic stresses in general and osmotic stress and salinity in particular. This chapter provides a detailed overview of the methods employed for in vitro selection in the model legume Medicago truncatula for the generation of novel germplasm capable of resisting NaCl- and PEG-induced osmotic stress. We also address the understanding of the genetic determinism in the acquisition of stress resistance, which differs between NaCl and PEG. Thus, the expression of genes linked to growth (WEE1), in vitro embryogenesis (SERK), salt tolerance (SOS1) proline synthesis (P5CS), and ploidy level and cell cycle (CCS52 and WEE1) was upregulated under NaCl stress, while under PEG treatment the expression of MtWEE1 and MtCCS52 was significantly increased, but no significant differences were observed in the expression of genes MtSERK1 and MtP5CS, and MtSOS1 was downregulated. A number of morphological and physiological traits relevant to the acquisition of stress resistance were also assessed, and methods used to do so are also detailed.

In order to understand the mechanisms underlying acquisition of tolerance to salinity, we recently produced callus tissues of tobacco and Medicago truncatula resistant to NaCl-induced salt stress following application of a step-up recurrent selection method. The effects of salinity on cell size are known, but those on cell morphometry including cell and nuclear surface area and position of nuclei within salt stress resistant cells were never studied before. This work fills that gap, using suspension cultured cells of M. truncatula A17 initiated from callus, and Nicotiana tabacum BY-2 cell line resistant to increasing NaCl concentrations up to 150 mM NaCl. The surface area of salinity resistant cells of M. truncatula A17 and N. tabacum BY2 and their nuclei, produced by step-up recurrent selection, were reduced, and cells elongated as NaCl increased, but these parameters proved to be unreliable in explaining cell survival and growth at high NaCl. Conversely, nuclei of resistant cells migrated from the center to the periphery of the cytoplasm close to the walls. Nuclear marginalization was for the first time observed as a result of salt stress in plant cells, and could be a novel helpful morphological marker of acquisition of salinity tolerance.