In vitro screening of durum wheat against water-stress mediated through polyethylene glycol.

Three durum wheat (Triticum durum Desf.) genotypes with three levels of drought tolerance were screened in order to evaluate their response to water stress at callus induction and plant regeneration levels. Significant differences were observed among the genotypes, and polyethylene glycol (PEG) levels used, and their interactions were however, significant for all the studied characters. Increase in PEG concentration increased the time required for callus initiation and reduced the number of calli frequency of embryogenic structures and number of plants regenerated, showing the adverse effect of PEG on the somatic embryogenesis developmental., under in vitro conditions tested, and Djenah Khetifa was the most tolerant genotype, followed by Oued Zenati and Waha. This pattern was per their drought tolerance behavior under field conditions. Principal component analysis (PCA) showed that 95.56% of the total variation was explained by the first two principal components. Biplot analysis allowed the stress-tolerant genotype to be distinguished from the two less tolerant genotypes. Time required for callus initiation was strongly negatively correlated with all other studied traits. These traits can be recommended as suitable selection criteria for screening drought-tolerant genotypes. The selected cells and plants will provide a tool for determining the mechanisms involved in tolerance to water stress.

Change in ATP-binding cassette B1/19, glutamine synthetase and alcohol dehydrogenase gene expression during root elongation in Betula pendula Roth and Alnus glutinosa L. Gaertn in response to leachate and leonardite humic substances.

Humic substances (HS) are complex and heterogeneous compounds of humified organic matter resulting from the chemical and microbiological decomposition of organic residues. HS have a positive effect on plant growth and development by improving soil structure and fertility. They have long been recognized as plant growth-promoting substances, particularly with regard to influencing nutrient uptake, root growth and architecture. The biochemical and molecular mechanisms through which HS influence plant physiology are not well understood. This study evaluated the bioactivity of landfill leachate and leonardite HS on alder (Alnus glutinosa L. Gaertn) and birch (Betula pendula Roth) during root elongation in vitro. Changes in root development were studied in relation to auxin, carbon and nitrogen metabolisms, as well as to the stress adaptive response. The cDNA fragments of putative genes encoding two ATP-binding cassette (ABC) transporters (ABCB1 and ABCB19) belonging to the B subfamily of plant ABC auxin transporters were cloned and sequenced. Molecular data indicate that HS and their humic acid (HA) fractions induce root growth by influencing polar auxin transport (PAT), as illustrated by the modulation of the ABCB transporter transcript levels (ABCB1 and ABCB19). There were also changes in alcohol dehydrogenase (ADH) and glutamine synthetase (GS) gene transcript levels in response to HS exposure. These findings confirmed that humic matter affects plant growth and development through various metabolic pathways, including hormonal, carbon and nitrogen metabolisms and stress response or signalization.

Morpho-histology and genotype dependence of in vitro morphogenesis in mature embryo cultures of wheat.

Cellular totipotency is one of the basic principles of plant biotechnology. Currently, the success of the procedure used to produce transgenic plants is directly proportional to the successful insertion of foreign DNA into the genome of suitable target tissue/cells that are able to regenerate plants. The mature embryo (ME) is increasingly recognized as a valuable explant for developing regenerable cell lines in wheat biotechnology. We have previously developed a regeneration procedure based on fragmented ME in vitro culture. Before we can use this regeneration system as a model for molecular studies of the morphogenic pathway induced in vitro and investigate the functional links between regenerative capacity and transformation receptiveness, some questions need to be answered. Plant regeneration from cultured tissues is genetically controlled. Factors such as age/degree of differentiation and physiological conditions affect the response of explants to culture conditions. Plant regeneration in culture can be achieved through embryogenesis or organogenesis. In this paper, the suitability of ME tissues for tissue culture and the chronological series of morphological data observed at the macroscopic level are documented. Genetic variability at each step of the regeneration process was evaluated through a varietal comparison of several elite wheat cultivars. A detailed histological analysis of the chronological sequence of morphological events during ontogeny was conducted. Compared with cultures of immature zygotic embryos, we found that the embryogenic pathway occurs slightly earlier and is of a different origin in our model. Cytological, physiological, and some biochemical aspects of somatic embryo formation in wheat ME culture are discussed.

Analysis of expression profiles of selected genes associated with the regenerative property and the receptivity to gene transfer during somatic embryogenesis in Triticum aestivum L.

The physiological, biochemical and molecular mechanisms regulating the initiation of a regenerative pathway remain partially unknown. Efforts to identify the biological features that confer transformation ability, or the tendency of some cells to induce transgene silencing, would help to improve plant genetic engineering. The objective of our study was to monitor the evolution of plant cell competencies in relation to both in vitro tissue culture regeneration and the genetic transformation properties. We used a simple wheat regeneration procedure as an experimental model for studying the regenerative capacity of plant cells and their receptivity to direct gene transfer over the successive steps of the regenerative pathway. Target gene profiling studies and biochemical assays were conducted to follow some of the mechanisms triggered during the somatic-to-embryogenic transition (i.e. dedifferentiation, cell division activation, redifferentiation) and affecting the accessibility of plant cells to receive and stably express the exogenous DNA introduced by bombardment. Our results seem to indicate that the control of cell-cycle (S-phase) and host defense strategies can be crucial determinants of genetic transformation efficiency. The results from studies conducted at macro-, micro- and molecular scales are then integrated into a holistic approach that addresses the question of tissue culture and transgenesis competencies more broadly. Through this multilevel analysis we try to establish functional links between both regenerative capacity and transformation receptiveness, and thereby to provide a more global and integrated vision of both processes, at the core of defense/adaptive mechanisms and survival, between undifferentiated cell proliferation and organization.

Expression of PiABP19, Picdc2 and PiSERK3 during induction of somatic embryogenesis in leaflets of Prunus incisa (Thunb.).

Somatic embryogenesis is a useful tool of plant breeding. In this context, a procedure for inducing somatic embryogenesis in Prunus incisa leaf explants had been previously developed. The original in vitro protocol relies on picloram treatments and exposure to darkness as inductive conditions, the best frequency of embryogenesis being obtained on the second leaf (F(2)) exposed to 4 µM picloram during 30 days. The morphological and biochemical changes observed during somatic embryogenesis occur in response to alterations in gene expression regulation patterns. A molecular study was conducted in order to provide deeper insight into the fundamental biological factors involved in the induction of this process using a gene candidate strategy and semi-quantitative reverse transcription polymerase chain reaction analysis. So far, no sequence data related to somatic embryogenesis has been available in cherry. In the present study, we cloned and sequenced cDNA fragments of putative genes encoding auxin-binding protein, cell cycle regulator and somatic embryogenesis receptor kinase. Time-course differential transcript accumulations were observed for all investigated genes in leaves or derived callus tissues during the observation period (first month of culture). Their possible involvement in the sequential steps of the embryogenic pathway (dedifferentiation, cell proliferation, differentiation through somatic embryogenesis) is presented and discussed.

Insights into the regenerative property of plant cells and their receptivity to transgenesis: wheat as a research case study.

From a holistic perspective, the discovery of cellular plasticity, a very interesting property of totipotency, underlies many topical issues in biology with important medical applications, while transgenesis is a core research tool in biology. Partially known, some basic mechanisms involved in the regenerative property of cells and in their receptivity to transgenesis are common to plant and animal cells and highlight the principle of the unity of life. Transgenesis provides an important investigative instrument in plant physiology and is regarded as a valuable tool for crop improvement. The economic, social, cultural and scientific importance of cereals has led to a rich stream of research into their genetics, biology and evolution. Sustained efforts to achieve the results obtained in the fields of genetic engineering and applied biotechnology reflect this deep interest. Difficulties encountered in creating genetically modified cereals, especially wheat, highlighted the central notions of tissue culture regeneration and transformation competencies. From the perspective of combining or encountering these competencies in the same cell lineage, this reputedly recalcitrant species provides a stimulating biological system in which to explore the physiological and genetic complexity of both competencies. The former involves two phases, dedifferentiation and redifferentiation. Cells undergo development switches regulated by extrinsic and intrinsic factors. The re-entry into the cell division cycle progressively culminates in the development of organized structures. This is achieved by global chromatin reorganization associated with the reprogramming of the gene expression pattern. The latter is linked with surveillance mechanisms and DNA repair, aimed at maintaining genome integrity before cells move into mitosis, and with those mechanisms aimed at genome expression control and regulation. In order to clarify the biological basis of these two physiological properties and their interconnectedness, we look at both competencies at the core of defense/adaptive mechanisms and survival, between undifferentiated cell proliferation and organization, constituting a transition phase between two different dynamic regimes, a typical feature of critical dynamic systems. Opting for a candidate-gene strategy, several gene families could be proposed as relevant targets for investigating this hypothesis at the molecular level.