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

Nitrate- and nitric oxide-induced plant growth in pea seedlings is linked to antioxidative metabolism and the ABA/GA balance

This study looks at the effects of potassium nitrate (KNO3) and sodium nitroprusside (SNP), a nitric oxide (NO)-donor, on the development, antioxidant defences and on the abscisic acid (ABA) and gibberellin (GA) levels inpea seedlings. Results show that 10 mM KNO3and 50μM SNP stimulate seedling fresh weight (FW), althoughthis effect is not reverted by the action of 2-4-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide(cPTIO), a NO-scavenger.The KNO3treatment increased peroxidase (POX) and ascorbate oxidase (AOX) activities. SNP, on the otherhand, reduced monodehydroascorbate reductase (MDHAR) activity and produced a significant increase in su-peroxide dismutase (SOD), POX and AOX activities. The“KNO3plus cPTIO”treatment increased ascorbateperoxidase (APX), MDHAR, glutathione reductase (GR) and SOD activities, but POX activity decreased in re-lation to the KNO3treatment. The“SNP plus cPTIO”treatment increased APX and MDHAR activities, whereas ahuge decrease in POX activity occurred. Both the KNO3and the SNP treatments increased reduced ascorbate(ASC) concentrations, which reached control values in the presence of cPTIO. All treatments increased thedehydroascorbate (DHA) level in pea seedlings, leading to a decrease in the redox state of ascorbate. In the“KNO3plus cPTIO”treatment, an increase in the redox state of ascorbate was observed. Glutathione contents,however, were higher in the presence of SNP than in the presence of KNO3. In addition, KNO3produced anaccumulation of oxidised glutathione (GSSG), especially in the presence of cPTIO, leading to a decrease in theredox state of glutathione. The effect of SNP on reduced glutathione (GSH) levels was reverted by cPTIO, sug-gesting that NO has a direct effect on GSH biosynthesis or turnover.Both the KNO3and SNP treatments produced an increase in GA4 and a decrease in ABA concentrations, andthis effect was reverted in the presence of the NO-scavenger. Globally, the results suggest a relationship betweenantioxidant metabolism and the ABA/GA balance during early seedling growth in pea. The results also suggest arole for KNO3and NO in the modulation of GA4 and ABA levels and antioxidant metabolism in pea seedlings.Furthermore, this effect correlated with an increase in the biomass of the pea seedlingsinfo:eu-repo/semantics/acceptedVersio

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

Metabolomics and biochemical approaches link salicylic acid biosynthesis to cyanogenesis in peach plants

Despite the long-established importance of salicylic acid (SA) in plant stress responses and other biological processes, its biosynthetic pathways have not been fully characterized. The proposed synthesis of SA originates from chorismate by two distinct pathways: the isochorismate and phenylalanine (Phe) ammonia-lyase (PAL) pathways. Cyanogenesis is the process related to the release of hydrogen cyanide from endogenous cyanogenic glycosides (CNglcs), and it has been linked to plant plasticity improvement. To date, however, no relationship has been suggested between the two pathways. In this work, by metabolomics and biochemical approaches (including the use of [C-13]-labeled compounds), we provide strong evidences showing that CNglcs turnover is involved, at least in part, in SA biosynthesis in peach plants under control and stress conditions. The main CNglcs in peach are prunasin and amygdalin, with mandelonitrile (MD), synthesized from phenylalanine, controlling their turnover. In peach plants MD is the intermediary molecule of the suggested new SA biosynthetic pathway and CNglcs turnover, regulating the biosynthesis of both amygdalin and SA. MD-treated peach plants displayed increased SA levels via benzoic acid (one of the SA precursors within the PAL pathway). MD also provided partial protection against Plum pox virus infection in peach seedlings. Thus, we propose a third pathway, an alternative to the PAL pathway, for SA synthesis in peach plantsThis work was supported by the Spanish Ministry of Economy and Competitiveness (Project AGL2014-52563-R). PDV and CP thank CSIC and UPCT, respectively, as well as the Spanish Ministry of Economy and Competitiveness for their ‘Ramon & Cajal’ research contract, co-financed by FEDER funds. We also acknowledge Prof. Manuel Acosta Echeverría for his very useful commentaries and discussion

Elucidating the plant growth-promoting effects of three microorganisms on deciduous fruit tree plants using in vitro culture conditions

El cultiu in vitro de teixits vegetals és una tècnica molt utilitzada per a l’obtenció d’importants quantitats de material genèticament idèntic i lliure de malalties. No obstant això, en alguns cultius de fruiters, aquesta tècnica es pot veure limitada per la baixa capacitat d’arrelament i aclimatació d’alguns genotips, així com per las grans pèrdues ocasionades per la presència de contaminacions endòfites. L’ús de microorganismes que milloren el creixement de plantes pot constituir una alternativa interessant gràcies a la seva capacitat de produir hormones vegetals i controlar el creixement de patògens. A més a més, també és important desenvolupar productes basats en aquests microorganismes per a l’escalat de la seva aplicació a camp. En aquesta tesi, els tres microorganismes Pseudomonas oryzihabitans PGP01, Cladosporium ramotenellum PGP02 i Phoma spp. PGP03 es van aïllar de cultius in vitro contaminats de Prunus i Pyrus que mostraven un millor creixement que els no contaminats. En plantes produïdes a partir d’embrions de nectarina rescatats in vitro (Capítol 1), es va demostrar que P. oryzihabitans PGP01 augmentava el desenvolupament radicular que afavoria la seva aclimatació en hivernacle. En portaempelts comercials micropropagats in vitro (Capítol 2), C. ramotenellum PGP02 i Phoma spp. PGP03 milloraven el percentatge d’arrelament in vitro, d’un 56,3% a un 100%, en explants tractats amb IBA del portaempelt difícil d’arrelar Py12. En el mateix capítol, P. oryzihabitans PGP01 també promogué el desenvolupament de les arrels del porta-empelt de Prunus RP-20. Tots aquests resultats podrien estar relacionats amb la producció de IAA per part dels tres microorganismes. Mitjançant l’ús de mutants d’Arabidopsis thaliana, es va suggerir que les modificacions en les arrels induïdes per P. oryzihabitans PGP01 podrien estar mediades per les estrigolactones (SLs) i el glutatió (GSH) (Capítol 3). En un cultiu en medi líquid (Capítol 4), es va observar que els efectes en les arrels produïts per P. oryzihabitans PGP01 podrien estar mediats pel contingut d’auxines en el medi de cultiu. A més a més, utilitzant aquest mateix sistema, en presencia de C. ramotenellum PGP02, es va suggerir un efecte del baix pH en el medi de cultiu sobre el creixement d’endòfits en plantes de RP-20. Aquesta hipòtesi va quedar demostrada al Capítol 5, verificant que un pH àcid, en absència de microorganismes, reduïa la concentració d’endòfits sense afectar la multiplicació in vitro. Finalment, es van provar tres subproductes de la industria de la patata, tomàquet i cereals per a l’elaboració d’ún medi de cultiu barato per la producció de P. oryzihabitans PGP01. El creixement d’aquest bacteri en un medi basat en subproductes de patata es va obtenir un creixement màxim de 4.4x109 UFC mL-1 sense afectar la seva activitat biològica (Capítol 6). Els resultats obtinguts aporten noves aproximacions sobre l’ús de microorganismes beneficiosos com alternatives més sostenibles per a promoure el creixement de plantes in vitro.El cultivo in vitro de tejidos vegetales es una técnica muy útil para obtener grandes cantidades de material genéticamente idéntico y libre de enfermedades. Sin embargo, esta técnica se ve limitada en algunas plantas frutales por la poca capacidad de enraizamiento y aclimatación de algunos genotipos, o por las pérdidas de material vegetal causadas por la presencia de contaminaciones endófitas. El uso de microorganismos que mejoran el crecimiento de plantas puede ser una alternativa muy interesante debido a su capacidad de producir hormonas vegetales o de controlar el crecimiento de patógenos. Es importante desarrollar productos basados en estos microorganismos para escalar su posible aplicación en campo. Los tres microorganismos Pseudomonas oryzihabitans PGP01, Cladosporium ramotenellum PGP02 y Phoma spp. PGP03 se aislaron de cultivos in vitro contaminados de Prunus y Pyrus que mostraban un mayor crecimiento que los no contaminados. En plántulas obtenidas a partir de embriones de nectarina rescatados in vitro (Capítulo 1), P.oryzihabitans PGP01 indujo mayor desarrollo radicular que favoreció la aclimatación de las plantas en invernadero. En patrones comerciales micropropagados in vitro (Capítulo 2), C. ramotenellum PGP02 y Phoma spp. PGP03 mejoraron el porcentaje de enraizamiento in vitro, de un 56.3 a un 100%, de explantos tratados con IBA del portainjerto Pyrus Py12 difícil de enraizar. En este mismo capítulo, P.oryzihabitans PGP01 también promovió el desarrollo de las raíces del patrón de Prunus RP-20. Todos estos resultados podrían estar relacionados con la producción de IAA por parte de los tres microorganismos. Utilizando mutantes de Arabidopsis thaliana, se sugirió que los efectos en las raíces producidos por P. oryzihabitans PGP01 podrían estar mediados por estrigolactonas (SLs) y glutatión (GSH) (Capítulo 3). En cultivo con medio líquido (Capítulo 4), se observó que los efectos en la raíz producidos por P. oryzihabitans PGP01 podrían estar mediados por el contenido de auxinas en el medio de cultivo. En este mismo sistema de crecimiento en líquido, en presencia de C. ramotenellum PGP02, se sugirió el efecto de un pH bajo en el medio de cultivo sobre el crecimiento de microorganismos endófitos en plantas de RP-20. Esta hipótesis fue finalmente confirmada en el Capítulo 5, demostrando que un pH ácido en ausencia de microorganismos reducía la concentración de endófitos sin afectar la micropropagación in vitro. Finalmente, se probaron tres subproductos de la industria de la patata, tomate y cereales para elaborar un medio barato para la producción de P. oryzihabitans PGP01. El crecimiento de esta bacteria en un medio basado en subproductos de patata proporcionó un crecimiento máximo de 4,4x109 UFC mL-1 sin afectar la actividad biológica del mismo (Capítulo 6). Los resultados presentados en esta tesis proporcionan hallazgos muy novedosos acerca del uso de microoganismos beneficiosos como alternativas más sostenibles para promover el crecimiento de plantas in vitro.In vitro tissue culture constitutes a very versatile technique to obtain large amounts of true-to-type and disease-free-plant materials. However, in some fruit tree crops, the poor in vitro rooting or acclimatization of some genotypes, or the high losses of plant material associated to endophytic contaminations may limit the effectiveness of the process. The use of microorganisms with plant-growth promoting ability might represent a sustainable alternative to overcome those limitations, knowing their ability to produce plant hormones or control pathogens growth. On the other hand, for the scale-up of the application to field conditions, it is of crucial importance to develop a product based on microorganism showing potential agronomical interest. The three microorganisms Pseudomonas oryzihabitans PGP01, Cladosporium ramotenellum PGP02 and Phoma spp. PGP03 were isolated from Prunus and Pyrus contaminated in vitro cultures showing a greater growth than those non-contaminated. In seedlings obtained from in vitro nectarine rescued embryos (Chapter 1), P. oryzihabitans PGP01 promoted root development, favouring the acclimatization to greenhouse conditions. In in vitro micropropagated commercial rootstocks (Chapter 2), C. ramotenellum PGP02 and Phoma spp. PGP03 increased the in vitro rooting percentage, from 56.3 to 100%, of the hard-to-root Pyrus rootstock Py12 explants treated with 3-indolebutyric acid. An effect of P. oryzihabitans PGP01 on root development of the Prunus rootstock RP-20 was observed. The in vitro ability of the three microorganisms to produce IAA supported these results. Using Arabidopsis thaliana defective mutants, the role of strigolactones (SLs) and glutathione (GSH) in the root events induced by P. oryzihabitans PGP01 was suggested (Chapter 3). In a liquid culture (Chapter 4), it was established a link between auxin levels in the medium and root development in the presence of P. oryzihabitans PGP01. Furthermore and regarding endophytes growth in the culture medium, the role of acidic pH to control their growth in RP-20 cultures was suggested in the presence of C. ramotenellum PGP02, being this assumption finally confirmed in Chapter 5 in the absence of microorganisms. In this chapter, the micropropagation at low pH reduced endophytes population without affecting in vitro micropropagation. Finally, wastes based on potato, tomato and cereals industries were tested for the development of a cheap culture medium for P. oryzihabitans PGP01. The growth of this bacterium in a potato wastes-based medium provided a maximum of 4.4x109 CFU mL-1 without losing the plant growth-promoting activity (Chapter 6). The results obtained in the present thesis provide novel insights regarding the use of beneficial microorganisms as more sustainable alternatives to promote in vitro plant growth

Non-volatile signals and redox mechanisms are required for the responses of Arabidopsis roots to Pseudomonas oryzihabitans

Soil bacteria promote plant growth and protect against environmental stresses, but the mechanisms involved remain poorly characterized, particularly when there is no direct contact between the roots and bacteria. Here, we explored the effects of Pseudomonas oryzihabitans PGP01 on the root system architecture (RSA) in Arabidopsis thaliana seedlings. Significant increases in lateral root (LR) density were observed when seedlings were grown in the presence of P. oryzihabitans, as well as an increased abundance of transcripts associated with altered nutrient transport and phytohormone responses. However, no bacterial transcripts were detected on the root samples by RNAseq analysis, demonstrating that the bacteria do not colonize the roots. Separating the agar containing bacteria from the seedlings prevented the bacteria-induced changes in RSA. Bacteria-induced changes in RSA were absent from mutants defective in ethylene response factor (ERF109), glutathione synthesis (pad2-1, cad2-1, and rax1-1) and in strigolactone synthesis (max3-9 and max4-1) or signalling (max2-3). However, the P. oryzihabitans-induced changes in RSA were similar in the low ascorbate mutants (vtc2-1and vtc2-2) to the wild-type controls. Taken together, these results demonstrate the importance of non-volatile signals and redox mechanisms in the root architecture regulation that occurs following long-distance perception of P. oryzihabitans.info:eu-repo/semantics/acceptedVersio

The salt-stress response of the transgenic plum line J8-1 and its interaction with the salicylic acid biosynthetic pathway from mandelonitrile

Salinity is considered as one of the most important abiotic challenges that affect crop productivity. Plant hormones, including salicylic acid (SA), are key factors in the defence signalling output triggered during plant responses against environmental stresses. We have previously reported in peach a new SA biosynthetic pathway from mandelonitrile (MD), the molecule at the hub of the cyanogenic glucoside turnover in Prunus sp. In this work, we have studied whether this new SA biosynthetic pathway is also present in plum and the possible role this pathway plays in plant plasticity under salinity, focusing on the transgenic plum line J8-1, which displays stress tolerance via an enhanced antioxidant capacity. The SA biosynthesis from MD in non-transgenic and J8-1 micropropagated plum shoots was studied by metabolomics. Then the response of J8-1 to salt stress in presence of MD or Phe (MD precursor) was assayed by measuring: chlorophyll content and fluorescence parameters, stress related hormones, levels of non-enzymatic antioxidants, the expression of two genes coding redox-related proteins, and the content of soluble nutrients. The results from in vitro assays suggest that the SA synthesis from the MD pathway demonstrated in peach is not clearly present in plum, at least under the tested conditions. Nevertheless, in J8-1 NaCl-stressed seedlings, an increase in SA was recorded as a result of the MD treatment, suggesting that MD could be involved in the SA biosynthesis under NaCl stress conditions in plum plants. We have also shown that the plum line J8-1 was tolerant to NaCl under greenhouse conditions, and this response was quite similar in MD-treated plants. Nevertheless, the MD treatment produced an increase in SA, jasmonic acid (JA) and reduced ascorbate (ASC) contents, as well as in the coefficient of non-photochemical quenching (qN) and the gene expression of Non-Expressor of Pathogenesis-Related 1 (NPR1) and thioredoxin H (TrxH) under salinity conditions. This response suggested a crosstalk between different signalling pathways (NPR1/Trx and SA/JA) leading to salinity tolerance in the transgenic plum line J8-1.This work was supported by the Spanish Ministry of Economy and Competitiveness (Projects AGL2014-52563-R and INIA-RTA2013-00026-C03-00). PDV and CP thank CSIC and UPCT, respectively, as well as the Spanish Ministry of Economy and Competitiveness for their ‘Ramon and Cajal’ research contract, co-financed by FEDER funds. This work was supported by the Spanish Ministry of Economy and Competitiveness (Projects AGL2014-52563-R and INIA-RTA2013-00026-C03-00).Peer reviewe

Optimization of a food industry-waste-based medium for the production of the plant growth promoting microorganism Pseudomonas oryzihabitans PGP01 based on agro-food industries by-products

In this study, three wastes based on potato peels and pulps, tomato seeds and wheat bran were used as basis for the preparation of a cheap medium to produce the bacterium P. oryzihabitans PGP01. In flasks experiments, P. oryzihabitans PGP01 growth at 25 °C in a medium based on frozen potato peels and pulp (FPP) with tryptone as a nitrogen source resulted in the maximum production compared to the commercial TSB medium. In the scale-up to 2 L bioreactors, FPP supplemented with tryptone, molasses, NaCl and K2HPO4 allowed to reach similar biomass production than in the TSB medium. A maximum growth of 4.4 × 109 CFU mL−1 after setting the agitation and the air flux conditions at 400 rpm and 0.75 vvm. Finally, P. oryzihabitans PGP01 growing in this optimized medium conserved its biological activity showing the expected effect in root development previously reported for this microorganism.info:eu-repo/semantics/publishedVersio

Acidification of the culture medium as a strategy to control endophytic contaminations in Prunus spp. rootstocks cultured in GreenTray TIS bioreactor

Overgrowth of endophytes in some in vitro cultures may disrupt the normal shoot tip growth and proliferation, being necessary to obtain endophytes-free cultures to achieve a normal plant micropropagation process. To remove these contaminations from the culture medium, antibiotics are commonly added to the culture medium. However, its use in plant production must be urgently reduced because of the current restrictions imposed by the European Union. For that purpose, the effect of acidic low (pH 3) and neutral (pH 7) pH was tested in the GreenTray® TIS bioreactor as an alternative to control endophytes growth without affecting the micropropagation of the Prunus rootstock RP-20 explants. The results demonstrated that culture at pH 3 did not affect the number of shoots, shoot FW, shoot length and the amount of chlorophyll pigments, but significantly reduced endophytes population. The identification also revealed that Roseomonas mucosa, Microbacterium oxydans, Bacillus subtilis and Luteibacter yeojuensis were the bacterial isolates responsible of those contaminations. These results might suppose a real breakthrough in the in vitro tissue culture field, although more research is required to meet the pH requirements for the different plant species and other endophytic.info:eu-repo/semantics/acceptedVersio

Rhizosphere microorganisms enhance in vitro root and plantlet development of Pyrus and Prunus rootstocks

The rooting of fruit tree rootstocks is the most challenging step of the in vitro propagation process. The use of rhizosphere microorganisms to promote in vitro rooting and plant growth as an alternative to the addition of chemical hormones to culture media is proposed in the present study. Explants from two Pyrus (Py170 and Py12) rootstocks and the Prunus RP-20 rootstock were inoculated with Pseudomonas oryzihabitans PGP01, Cladosporium ramotenellum PGP02 and Phoma sp. PGP03 following two different methods to determine their effects on in vitro rooting and plantlet growth. The effects of the microorganisms on the growth of fully developed Py170 and RP-20 plantlets were also studied in vitro. All experiments were conducted using vermiculite to simulate a soil system in vitro. When applied to Py12 shoots, which is a hard-to-root plant material, both C. ramotenellum PGP02 and Phoma sp. PGP03 fungi were able to increase the rooting percentage from 56.25% to 100% following auxin indole-3-butyric acid (IBA) treatment. Thus, the presence of these microorganisms clearly improved root development, inducing a higher number of roots and causing shorter roots. Better overall growth and improved stem growth of treated plants was observed when auxin treatment was replaced by co-culture with microorganisms. A root growth-promoting effect was observed on RP-20 plantlets after inoculation with C. ramotenellum PGP02, while P. oryzihabitans PGP01 increased root numbers for both Py170 and RP-20 and increased root growth over stem growth for RP-20. It was also shown that the three microorganisms P. oryzihabitans PGP01, C. ramotenellum PGP02 and Phoma sp. PGP03 were able to naturally produce auxin, including indole-3-acetic acid (IAA), at different levels. Overall, our results demonstrate that the microorganisms P. oryzihabitans PGP01 and C. ramotenellum PGP02 had beneficial effects on in vitro rooting and plantlet growth and could be applied to in vitro tissue culture as a substitute for IBA.info:eu-repo/semantics/acceptedVersio