Using plant growth‑promoting microorganisms (PGPMs) to improve plant development under in vitro culture conditions

Plant in vitro culture techniques are highly useful to obtain signifcant amounts of true-to-type and disease-free plant materials. One of these techniques is clonal micropropagation which consists on the establishment of shoot tip cultures, shoot multiplication, in vitro rooting and acclimatization to ex vitro conditions. However, in some cases, the existence of recalcitrant genotypes, with a compromised multiplication and rooting ability, or the difculties to overcome the overgrowth of endophytic contaminations might seriously limit its efciency. In this sense, the establishment of benefcial interactions between plants and plant growth-promoting microorganisms (PGPMs) under in vitro culture conditions might represent a valuable approach to efciently solve those restrictions. During the last years, signifcant evidence reporting the use of benefcial microorganisms to improve the yield of in vitro multiplication or rooting as well as their acclimatization to greenhouse or soil conditions have been provided. Most of these positive efects are strongly linked to the ability of these microorganisms to provide in vitro plants with nutrients such as nitrogen or phosphorous, to produce plant growth regulators, to control the growth of pathogens or to mitigate stress conditions. The culture of A. thaliana under aseptic conditions has provided high-quality knowledge on the root development signaling pathways, involving hormones, triggered in the presence of PGPMs. Overall, the present article ofers a brief overview of the use of microorganisms to improve in vitro plant performance during the in vitro micropropagation stages, as well as the main mechanisms of plant growth promotion associated with these microorganismsinfo:eu-repo/semantics/acceptedVersio

Somatic manipulation of Pyrus and Cydonia : characterization and selection for iron efficiency

Appropriate micropropagation regimes were developed for four Pyrus species (P. amygdaliformis Vill., P. betulaefolia Bunge, P. calleryana Dcne., and P. communis L.) and Cydonia oblonga L. Shoot multiplication was optimal at 10 or 20μM N⁶⁻ benzyladenine (BA) and high light intensity (135μm⁻²s⁻1). Root formation of the Pyrus species was stimulated by exposure of shoots to high levels (10 or 32μM) of β- indolebutyric acid (IBA) for 7 days or a dip in 10mM IBA for 15s, followed by a passage on auxin-free medium. a-Naphthaleneacetic acid (NAA) was more effective than IBA in stimulating rooting of C. oblonga. Adventitious shoots of Cydonia oblonga Quince A were induced from leaves cultured on MS-N6 medium containing thidiazuron (TDZ) and NAA. Optimal regeneration (78% of the cultured leaves with 3.2 shoots per leaf) occurred with 32μM TDZ plus 0.3μM NAA on young leaves obtained from micropropagated shoots. Adventitious shoots of Pyrus amygdaliformis and P. communis were obtained, but at much lower frequency. Effects of Fe-limiting conditions in vitro were determined by comparing the responses of shoots and rooted plantlets to media containing FeEDTA or FeSO₄, with or without bicarbonate. Symptoms of Fe deficiency were genotype-dependent and most severe in the presence of FeSO₄ and bicarbonate. Chlorosis was pronounced in Cydonia, absent in P. amygdaliformis and P. communis, and intermediate in P. betulaefolia and P. calleryana, indicating parallel responses between in vitro and field conditions. Similar responses were obtained with rooted and unrooted shoots. Tolerance to Fe-deficiency chlorosis was correlated with maintenance, under Fe-stress, of a high Fe²⁺/total Fe ratio, high Fe³⁺ reducing activity, and medium acidification. This adaptive response was diminished by bicarbonate. Roots of plantlets, shoot bases, root cultures and cell suspension cultures all manifested Fe-stress inducible Fe³⁺ reducing activity. The adventitious shoot regeneration protocol was successfully used to select somaclonal variants of C. oblonga with increased tolerance to Fe-deficiency chlorosis. Two variants, IE1 and IE2, were recovered which displayed higher Fe³⁺ reducing ability and acidification of the medium than the original C. oblonga clone. These variants may be useful as pear rootstocks in regions with calcareous soils

Inoculation of in vitro cultures with rhizosphere microorganisms improve plant development and acclimatization during immature embryo rescue in nectarine and pear breeding programs

In the present study, two fungi Cladosporium ramotenellum strain PGP02 and Phoma spp. strain PGP03 and the bacterium Pseudomonas oryzihabitans PGP01 were isolated from Pyrus and Prunus in vitro rescued embryos, whose plantlets showed a better growth than non-contaminated cultures. Upon identification, concentrated solutions of the three microorganisms were applied to pear (Pyrus communis L.) in vitro rooted plantlets, increasing in different ways biometric parameters such as plant fresh weight (FW), stem length and root length. Then, these microorganisms were tested in embryos derived from three directed crosses between early ripening nectarine varieties (Prunus persica cv. Nectarina). In a first cross, in vitro cultured embryos were inoculated with both fungi, C. ramotenellum PGP02 and Phoma spp. PGP03, at 2 × 107 esp mL−1, and the bacterium P. oryzihabitans PGP01, at 2 × 108 CFU mL−1. In the following crosses, only the bacterium P. oryzihabitans PGP01, at 2 × 108 CFU mL−1, was employed. The effects on number of germinated embryos, development of the subsequent plants, after 24 weeks of in vitro culture, as well as their ex vitro acclimatization performance were analysed. These microorganisms had no effect on the germination efficiency of nectarine embryos. However, the presence of the bacterium P. oryzihabitans PGP01 modified root system architecture in the three crosses, increasing root volume and thickness, which in consequence enhanced the acclimatization efficiency to soil in those crosses with poor acclimation efficiencies. These results enforce a breakthrough in the use of microorganisms along the in vitro embryo rescue used in early ripening peaches and nectarines breeding programmes, and the production of plants more resistant to the stressful conditions imposed by the acclimatization to soil.info:eu-repo/semantics/acceptedVersio

Differential response to calcium-labelled (44Ca) uptake and allocation in two peach rootstocks in relation to transpiration under in vitro conditions

Calcium-labelled (44Ca) uptake and transport under in vitro GreenTray® temporary immersion bioreactor conditions have been studied related to aeration conditions. For this aim, Rootpac®-20 (RP-20) and Garnem® (G × N) were selected as two main rootstocks used in peach production. Two transpiration conditions, aerated and unaerated, were established for each plant material. 44Ca location, plant development and foliar stomata surface were measured after the in vitro culture period. The results showed that aeration improved Ca transport within the shoot, but it did not enhance Ca uptake by the roots. Regarding plant material, G × N presented a better Ca uptake capacity and concentration. The findings suggest that Ca uptake in the roots is a precise process that is influenced by transpiration. However, it was observed that transpiration and thus the water flux is not the only force promoting Ca uptake by roots. Furthermore, the transport of Ca to the shoot was primarily determined by transpiration, indicating that water flux plays a crucial role in the aboveground movement of Ca. The study also revealed distinct behaviors in Ca uptake and allocation between the different peach rootstocks, emphasizing the importance of considering these factors in the selection process of rootstocks. These findings contribute to our understanding of the mechanisms involved in Ca uptake and transport in peach rootstocks under in vitro conditions. They provide valuable insights for rootstock selection processes and highlight the need for further research in this area

El progreso de la mejora genética en frutales: ¿Cómo serán las variedades y patrones en el horizonte 2040?

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Rhizosphere Acidification as the Main Trait Characterizing the Differential In Vitro Tolerance to Iron Chlorosis in Interspecific Pyrus Hybrids

Physiological responses of different interspecific Pyrus hybrids and an open pollinated Pyrus communis ‘Williams’ (Pcw) grown under in vitro culture conditions simulating lime induced chlorosis were studied. The hybrids were derived from crosses between the ‘Pyriam’ pear rootstock and four Pyrus species of the Mediterranean region, namely P. amygdaliformis Vill. (Pa), P. amygdaliformis persica Bornme. (Pap), P. communis cordata (Desv.) Hook. (Pcc), and P. elaeagrifolia Pall (Pe), all known for their higher field tolerance to iron-chlorosis than P. communis. Twenty hybrids and one open pollinated Pcw were micropropagated, and plantlets were in vitro characterized for their physiological responses to iron-deficiency conditions. Rooted plantlets were transferred to a culture medium with 2 µM Fe3+ DTPA and 10 or 20 mM NaHCO3. These physiological responses were scored at 1, 3, 7, and 28 days from the start of the in vitro assay. Leaf total chlorophyll content, the capacity of roots to acidify the medium, reduced iron, and exudates of phenolic acids and organic acids were analyzed in each media and time sample. Leaf chlorophyll levels for the clones derived from Pcc were the highest, especially under the highest bicarbonate concentration, followed by those derived from Pap and Pa. The higher chlorophyll content of Pcc clones were related with their higher capacity to acidify the media but not with their iron reduction capacity at the root level. On the other hand, hybrid clones derived from Pe showed a higher Fe3+ reduction ability than clones from all the other species during the whole assay but only when the bicarbonate concentration was lower. The exudation of phenolic acids by the roots was higher in Pcw than in the other species, and this response might explain why the total chlorophyll levels in Pcw clones are similar to those of Pe and Pa ones. These results with Pyrus spp. bring more evidence in support of the idea that iron reduction capacity at the root level is not directly related with a higher tolerance to iron deficiency caused by the high pH of calcareous soils. Instead, the ability to acidify the rhizosphere is the trait of choice for the selection of the pear hybrid clones better adapted to lime induced chlorosis. In addition, the in vitro assay to select the Pyrus clones for tolerance to iron chlorosis could be shortened to one week of culture in 10 mM NaHCO3, measuring the leaf chlorophyll level, acidification of the culture medium, and exudation of phenolic acids as the physiological responses to predict tolerance to lime-induced chlorosisinfo:eu-repo/semantics/publishedVersio

Analysis of expressed sequence tags generated from full-length enriched cDNA libraries of melon

Abstract Background Melon (Cucumis melo), an economically important vegetable crop, belongs to the Cucurbitaceae family which includes several other important crops such as watermelon, cucumber, and pumpkin. It has served as a model system for sex determination and vascular biology studies. However, genomic resources currently available for melon are limited. Result We constructed eleven full-length enriched and four standard cDNA libraries from fruits, flowers, leaves, roots, cotyledons, and calluses of four different melon genotypes, and generated 71,577 and 22,179 ESTs from full-length enriched and standard cDNA libraries, respectively. These ESTs, together with ~35,000 ESTs available in public domains, were assembled into 24,444 unigenes, which were extensively annotated by comparing their sequences to different protein and functional domain databases, assigning them Gene Ontology (GO) terms, and mapping them onto metabolic pathways. Comparative analysis of melon unigenes and other plant genomes revealed that 75% to 85% of melon unigenes had homologs in other dicot plants, while approximately 70% had homologs in monocot plants. The analysis also identified 6,972 gene families that were conserved across dicot and monocot plants, and 181, 1,192, and 220 gene families specific to fleshy fruit-bearing plants, the Cucurbitaceae family, and melon, respectively. Digital expression analysis identified a total of 175 tissue-specific genes, which provides a valuable gene sequence resource for future genomics and functional studies. Furthermore, we identified 4,068 simple sequence repeats (SSRs) and 3,073 single nucleotide polymorphisms (SNPs) in the melon EST collection. Finally, we obtained a total of 1,382 melon full-length transcripts through the analysis of full-length enriched cDNA clones that were sequenced from both ends. Analysis of these full-length transcripts indicated that sizes of melon 5' and 3' UTRs were similar to those of tomato, but longer than many other dicot plants. Codon usages of melon full-length transcripts were largely similar to those of Arabidopsis coding sequences. Conclusion The collection of melon ESTs generated from full-length enriched and standard cDNA libraries is expected to play significant roles in annotating the melon genome. The ESTs and associated analysis results will be useful resources for gene discovery, functional analysis, marker-assisted breeding of melon and closely related species, comparative genomic studies and for gaining insights into gene expression patterns.This work was supported by Research Grant Award No. IS-4223-09C from BARD, the United States-Israel Binational Agricultural Research and Development Fund, and by SNC Laboratoire ASL, de Ruiter Seeds B.V., Enza Zaden B.V., Gautier Semences S.A., Nunhems B.V., Rijk Zwaan B.V., Sakata Seed Inc, Semillas Fitó S.A., Seminis Vegetable Seeds Inc, Syngenta Seeds B.V., Takii and Company Ltd, Vilmorin and Cie S.A. and Zeraim Gedera Ltd (all of them as part of the support to ICuGI). CC was supported by CNRS ERL 8196.Peer Reviewe