Expression of a receptor kinase in Arabidopsis roots is stimulated by the basidiomycete Piriformospora indica and the protein accumulates in Triton X-100 insoluble plasma membrane microdomains

Piriformospora indica, an endophytic fungus of the Sebacinaceae family, colonises the roots of a wide variety of plant species and promotes their growth, in a manner similar to mycorrhizal fungi. We demonstrate that the fungus also interacts with the non-mycorrhizal host Arabidopsis thaliana. Promotion of root growth was detectable even before noticeable root colonization, and was accompanied by a massive transfer of phosphate from the media to the aerial parts of the seedlings. During the recognition period of both organisms, the message for a receptor kinase with leucine-rich repeats is transiently upregulated. The kinase is located in Triton X-100-insoluble plasma membrane microdomains. Thus, this is one of the earliest events of a plant root in response to a fungus reported to date.     

Adventitious rooting performance in micropropagated Cornus mas

Axillary buds sampled from a mature 27-year-old Cornus mas cv. Macrocarpa were grown in vitro on modified woody plant medium (WPM). Adventitious rooting performance of microshoots was assayed on half-strength WPM supplemented with 1-naphthaleneacetic acid (NAA) or indole-3-butyric acid (IBA) under various pH. NAA induced significantly higher rooting frequencies than IBA. The pH of 6.8 inhibited rooting, and differentiated roots were extremely thick and fragile. The highest rooting frequency was recorded on half-strength WPM supplemented with 5.37 µM NAA at the pH value adjusted to 6.2 (73 % of rooted shoots). In the presence of IBA, the formation of adventitious roots was observed only in the basal part of the microshoot dipped into rooting medium. In the case of NAA, however, adventitious roots arose also from the parts of microshoots that were not in contact with medium. The growth of aerial roots was always positively gravitropic. The nuclear microsatellite Cf-G17 gave a monomorphic fingerprinting pattern across the mother shrub and micropropagated plantlets. Acclimatized plants did not show any visually detectable morphological variation and the aerial adventitious root formation was no longer observed.     

Transformation of Populus tomentosa by Agrobacterium and Regeneration of Transformed Plantlets

The establishment of efficient transformation system of Populus tomentosa by Agrobacterium is reported. The strains of Agrobacterium used in experiments were: 1. A. rhizogenes R1000, which harboured the Ri plasmid pRiA4b. 2. A. rhizogenes R1000 (pTVK85), which carried the plasmids pRiA4b and pTVK85 Containing supervirulent region. 3. A. tumefaciens C58C1 (pBZ693), the plasmid pBZ693 containing genes 1 and 2. After being cocultured with the bacteria on media containing 0.5 ppm kinetin for 2 days, explants of P. tomentosa were transferred to MS medium containing 500 ppm cefotaxime. Roots appeared on the explants in a week. The roots induced by A. tume[aciens were morphologically different from those induced by A. rhizogenes. The frequency of the explants transformed by A. rhizogenes R1000 (pTVK85) was nearly up to 60%. Some Ri plasmid transformed roots could spontaneously produce adventitious shoots or calli. By adding appropriate plant growth regulators in the media, we could have all of the root lines transformed produce adventitious shoots which would develop into intact plantlets on a hormone-free medium. Some phenotypical differences were observed among clones of the transformed plantlets. Some clones had short internodes, large number of leaves, reduced apical dominance, rich root systems with a great quantity of branches and root hairs, whereas in other clones aboveground parts of plantlets were morphologically normal and only their root systems were different from those of untransformed plantlets. None of the plantlets transformed by A. rhizogenes had the phenomenon of wrinkle leaves and shapes these leaves were analogous to normal plantlets. It was often observed that roots were regenerated from stems above the medium surfaces. Southern analysis on three clones of the putative transformed plantlets by A. rhizogenes R1000 (pTVKS5) showed that two of them were hybridized positively with the probe covering the TL-DNA region of the plasmid pRiA4b.     

Tissue culture of Alhagi camelorum – a legume of high regenerative capacity

Alhagi camelorum cultures provide a system with a high propensity for plantlet regeneration from root, hypocotyl, stem, and leaf explants. Excluding leaf explants, all explants regenerate to form shoot-buds on a simple basal medium suggesting a differential morphogenic potential of different parts of the same plant. All parts of the plant including leaves, form shoot-buds on cytokinin-containing media which markedly promote shoot-bud differentiation, alone or in combination with indoleacetic acid or naphthaleneacetic acid. Rooting occurs on media containing indoleacetic acid and naphthaleneacetic acid. 2, 4-Dichlorophenoxyacetic acid is inhibitory for differentiation and induces callusing. Callus induced on benzylaminopurine and 2,4-dichlorophenoxyacetic acid differentiates to form shoot-buds on transfer to cytokinin-containing medium. Upon transfer to basal medium shoots produce roots. Plants have been transferred to soil.     

The root endophyte fungus Piriformospora indica leads to early flowering,higher biomass and altered secondary metabolites of the medicinal plant,Coleus forskohlii

This study was undertaken to investigate the influence of plant probiotic fungus Piriformospora indica on the medicinal plant C. forskohlii. Interaction of the C. forskohlii with the root endophyte P. indica under field conditions, results in an overall increase in aerial biomass, chlorophyll contents and phosphorus acquisition. The fungus also promoted inflorescence development, consequently the amount of p-cymene in the inflorescence increased. Growth of the root thickness was reduced in P. indica treated plants as they became fibrous, but developed more lateral roots. Because of the smaller root biomass, the content of forskolin was decreased. The symbiotic interaction of C. forskohlii with P. indica under field conditions promoted biomass production of the aerial parts of the plant including flower development. The plant aerial parts are important source of metabolites for medicinal application. Therefore we suggest that the use of the root endophyte fungus P. indica in sustainable agriculture will enhance the medicinally important chemical production.     

The novel paclitaxel-producing system: establishment of Corylus avellana L. hairy root culture

Hazelnut (Corylus avellana L.), contains a valuable medicinal substance known as Paclitaxel®, which is one of the most effective anticancer drugs. The original plants produce negligible amount of paclitaxel; therefore, tissue culture techniques, especially hairy root culture, could be one of the most practical methods to enhance the amount of paclitaxel. The main goal of this study was to assess the induction of hairy roots in C. avellana. The effects of different strains of Agrobacterium rhizogenes including c58c1pRiA₄, K599, and 15834, and six culture media, MS (Murashige and Skoog), half-strength MS, quarter-strength MS, WPM (woody plant media), half-strength WPM, and quarter-strength WPM, were evaluated. The results showed that the maximum amounts of the rooted explants were obtained with c58c1pRiA4 strain in quarter-strength WPM medium. The investigations of explant type (leafstalk, petiole, lamina, and stem) and different propagation media (quarter-strength WPM, half-strength MS, and half-strength SH ((Schenk and Hildebrandt) medium) showed that the leafstalk was the most optimal explant for hairy root induction, and half-strength SH was the best culture medium for growth of the hairy roots in liquid medium. HPLC analyses confirmed the presence of paclitaxel (3.2 μg g⁻¹ (DW)) in hairy root extracts.

     

Piriformospora indica-a mutualistic basidiomycete with an exceptionally large plant host range

Piriformospora indica is a basidiomycete of the order Sebacinales, representing a model for the study of mutualistic symbiosis and, beyond that, the plant immune system. The fungus colonizes the roots of a wide range of vascular plants, increasing their growth, seed yield and adaptation to abiotic and biotic stresses. The fungal colonization of roots begins with a biotrophic growth phase, in which living cells are colonized, and continues with a cell death-dependent phase, in which root cells are actively killed by the fungus. The complexity of sebacinalean symbiosis is further enhanced by the presence of endocellular bacteria which may represent significant determinants for a successful outcome of the symbioses. Molecular ecological analyses have revealed an exceptional relevance of sebacinoid fungi in natural ecosystems worldwide. This natural competence could be rooted in their phenotypic adaptability, which, for instance, allows P. indica to grow readily on various synthetic media and to colonize distinct hosts. In molecular and genetic studies, P. indica's mutualistic colonization strategy has been partly unravelled, showing that the jasmonate pathway is exploited for immune suppression and successful development in roots. Research on P. indica supports efforts to make the bioprotective potential of the fungus accessible for agricultural plant production. The decoding of P. indica's genome has revealed its potential for application as bioagent and for targeted improvement of crop plants in biotechnology-based approaches.     

Association of Piriformospora indica with Arabidopsis thaliana roots represents a novel system to study beneficial plant–microbe interactions and involves early plant protein modifications in the endoplasmic reticulum and at the plasma membrane

Piriformospora indica , an endophytic fungus of the Sebacinaceae family, colonizes the roots of a wide variety of plant species and promotes their growth, in a manner similar to arbuscular mycorrhizal fungi. The results of the present study demonstrate that the fungus interacts also with the non-mycorrhizal host Arabidopsis thaliana and promotes its growth. The interaction is detectable by the appearance of a strong autofluorescence in the roots, followed by the colonization of root cells by fungal hyphae and the generation of chlamydospores. Promotion of root growth was detectable even before noticeable root colonization. Membrane-associated proteins from control roots and roots after cultivation with P. indica were separated by two-dimensional gel-electrophoresis and identified by electrospray ionization mass spectrometry and tandem mass spectrometry. Differences were found in the expression of glucosidase II, beta-glucosidase PYK10, two glutathione- S -transferases and several so-far uncharacterized proteins. Based on conserved domains present in the latter proteins their possible roles in plant–microbe interaction are predicted. Taken together, the present results suggest that the interaction of Arabidopsis thaliana with P. indica is a powerful model system to study beneficial plant–microbe interaction at the molecular level. Furthermore, the successful accommodation of the fungus in the root cells is preceded by protein modifications in the endoplasmatic reticulum as well as at the plasma membrane of the host.     

Establishment of Camptotheca acuminata regeneration from leaf explants

Plantlet regeneration through shoot formation from young leaf explant-derived callus of Camptotheca acuminata is described. Calli were obtained by placing leaf explants on Woody plant medium (WPM) supplemented with various concentrations of 6-benzyladenine (BA) and naphthaleneacetic acid (NAA) or 2,4-dichlorophenoxyacetic acid (2,4-D). Callus induction was observed in all media evaluated. On the shoot induction medium, the callus induced on the WPM medium containing 19.8 μM BA and 5.8 μM NAA was the most effective, providing high shoot regeneration frequency (70.3 %) as well as the highest number of shoots (11.2 shoots explant⁻¹). The good rooting percentage and root quality (98 %, 5.9 roots shoot⁻¹) were achieved on WPM medium supplemented with 9.6 μM indole-3-butyric acid (IBA). 96 % of the in vitro rooted plantlets with well developed shoots and roots survived transfer to soil.     

混栽杨树-刺槐间磷素养分转移途径的研究

应用^32P同位素示踪法,人工接种VA菌根菌(Glomus mosseae),对种植在根箱中的杨树(Populus euramericana cv.‘I-214')和刺槐(Robinia pseudoacia)苗木进行了树种间P素养分转移途径的研究．结果表明,5室根箱中,接种VA菌根菌的杨树一例,菌根侵染率为34％,根箱隔网另一侧的刺槐根系侵染率为26％,而对照的杨树和刺槐根系侵染率均为零．在杨树一例施放射性同位累^32P,施用后第14—27天,刺槐一例的放射性同位素值,处理显著高于对照(P＜0．05)、在根箱中的杨树和刺槐根系间观察到菌丝连接,表明人工接种VA菌根菌能在杨树和刺槐根系间产生菌丝桥,菌丝桥可以在杨树和刺槐根系间传递P素．养分转移定量分析表明,根系接触和根系分泌物是树种间P素转移的主要途径,其转移P素量占转移总量的62％;菌根菌等微生物活动及其与根系接触和根系分泌物两种途径的交互作用占38％;菌丝桥通过隔网发挥的作用仅表现为一种趋势．     

Agrobacterium rhizogenes-mediated transformation of scented geranium

A method is described for producing genetically transformed plants from explants of three scentedPelargonium spp. Transgenic hairy root lines were developed fromPelargonium spp leaf explants and microcuttings after inoculation withAgrobacterium rhizogenes strains derived from the agropine A4 strain. Hairy root lines grew prolifically on growth regulator-free medium. Transgenic shoots were regenerated from hairy roots and the plants have been successfully transferred to soil. The phenotype of regenerated plants has been characterized as having abundant root development, more leaves and internodes than the controls, short internodes and highly branched roots and aerial parts. Southern blot analyses have confirmed the transgenic nature of these plants.     

Agrobacterium rhizogenes-mediated transformation of scented geranium

A method is described for producing genetically transformed plants from explants of three scentedPelargonium spp. Transgenic hairy root lines were developed fromPelargonium spp leaf explants and microcuttings after inoculation withAgrobacterium rhizogenes strains derived from the agropine A4 strain. Hairy root lines grew prolifically on growth regulator-free medium. Transgenic shoots were regenerated from hairy roots and the plants have been successfully transferred to soil. The phenotype of regenerated plants has been characterized as having abundant root development, more leaves and internodes than the controls, short internodes and highly branched roots and aerial parts. Southern blot analyses have confirmed the transgenic nature of these plants.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of <Emphasis Type="Italic">Rhododendron wattii</Emphasis> Cowan—a critically endangered and endemic plant from India

Rhododendron wattii Cowan is a rare and endangered plant found in northeast India. In an effort to boost specimen numbers, experiments of in vitro seed germination, shoot induction on different media supplemented with the cytokinin isopentenyladenine (2iP), and root induction with auxins α-naphthaleneacetic acid (NAA), indole-3-butyric acid (IBA) and indole-3-acetic acid (IAA) in woody plant medium (WPM) were carried out. A maximum mean shoot number of 7.72 per explant were obtained from nodal explants cultured on WPM and 39.36 μM 2iP with a maximum mean shoot length of 2.30 cm per explant. Among the auxins investigated for root induction, IBA at 2.45 μM was found to produce the most and the longest roots, when compared to other treatments. However, WPM supplemented with 0.2% (w/v) activated charcoal also showed 100% root formation with shoots having broader leaves compared to auxin treatments. About 60% of in vitro rooted plantlets transferred from lab to greenhouse conditions survived. Sixty acclimatized plants were reintroduced in the vicinity of their natural habitat at Naga Heritage Village, Kisama, Nagaland, in May 2016 for ex situ conservation. Survival of the reintroduced plants was confirmed during the field visit conducted in November 2016.     

Growth promotion of Chinese cabbage and Arabidopsis by Piriformospora indica is not stimulated by mycelium-synthesized auxin

Piriformospora indica, an endophytic fungus of the order Sebacinales, interacts with the roots of a large variety of plant species. We compared the interaction of this fungus with Chinese cabbage (Brassica campestris subsp. chinensis) and Arabidopsis seedlings. The development of shoots and roots of Chinese cabbage seedlings was strongly promoted by P. indica and the fresh weight of the seedlings increased approximately twofold. The strong stimulation of root hair development resulted in a bushy root phenotype. The auxin level in the infected Chinese cabbage roots was twofold higher compared with the uncolonized controls. Three classes of auxin-related genes, which were upregulated by P. indica in Chinese cabbage roots, were isolated from a double-subtractive expressed sequence tag library: genes for proteins related to cell wall acidification, intercellular auxin transport carrier proteins such as AUX1, and auxin signal proteins. Overexpression of B. campestris BcAUX1 in Arabidopsis strongly promoted growth and biomass production of Arabidopsis seedlings and plants; the roots were highly branched but not bushy when compared with colonized Chinese cabbage roots. This suggests that BcAUX1 is a target of P. indica in Chinese cabbage. P. indica also promoted growth of Arabidopsis seedlings but the auxin levels were not higher and auxin genes were not upregulated, implying that auxin signaling is a more important target of P. indica in Chinese cabbage than in Arabidopsis. The fungus also stimulated growth of Arabidopsis aux1 and aux1/axr4 and rhd6 seedlings. Furthermore, a component in an exudate fraction from P. indica but not auxin stimulated growth of Chinese cabbage and Arabidopsis seedlings. We propose that activation of auxin biosynthesis and signaling in the roots might be the cause for the P. indica-mediated growth phenotype in Chinese cabbage.     

Effect of plant growth regulators on plant regeneration of Dioscorea remotiflora (Kunth) through nodal explants

Dioscorea remotiflora (Kunth) is an important wild plant that produces tuberous roots used as a source of food in the Western part of Mexico. Lack of planting material and inefficiency of traditional methods of propagation are the main constraints for implementing large-scale cultivation. In contrast, tissue culture techniques allow increasing multiplication and rapid production of plant material. In this regard, leaves or nodal segments were incubated on MS, B5 and WPM culture media with different PGRs in order to obtain an efficient micropropagation protocol. Leaves explants were unable to inducing shoots or callus. However, nodal segments produced axillary shoots and/or callus in all culture media. MS containing 2.33???M KIN was the most suitable to inducing shoots; an average of 6.6 shoots per segment for 100?% explants was obtained, which displayed also the greater number of nodes (5.0) and leaves (7.9) per segment. A decrease on shoot proliferation was observed combining BA or KIN with 2,4-D or NAA. However, small brownish callus were induced on 100?% of segments using 2.33???M KIN with 5.37???M 2,4-D or 9.30???M KIN plus 2.69???M NAA. In contrast, by adding 2.69???M NAA, 66.4?% of the nodal segments formed shoots and produced also yellowish friable callus on the base of the shoots. Shoots were easily rooted with 8.28???M IBA (96.9?%), displaying the greatest root and shoot biomass, but maximum number of tuberous roots, and root or tuberous root biomass was produced increasing IBA (20.7???M).     

High Frequency Plant Regeneration from Nodal Explants of Paulownia elongata

Abstract: High frequency of plant regeneration from Paulownia elongata was obtained on Murashige and Skoog (MS) medium and Woody Plant Medium (WPM), with appropriate supplements of growth regulators. Leaves, leaves with petioles, internodes and nodes excised from 3-month-old non-aseptically grown P. elongata were used as explants. The highest shoot regeneration efficiency (93.7 %) was obtained from the nodes of P. elongata on MS medium supplemented with 0.1 mg/ml naphthaleneacetic acid (NAA) and 1 mg/ml 6-benzylaminopurine (BAP). The highest root formation efficiency (100 %) from the regenerated shoots was obtained on WPM supplemented with 1 mg/ml indolebutyric acid (IBA). Rooted plantlets were transplanted to soil with a survival efficiency of almost 100 %. The regeneration system reported here could be useful for rapid multiplication of elite genotypes of P. elongata in a short period of time.     

Effect of nutrient media on axillary shoot proliferation and preconditioning for adventitious shoot regeneration of pears

The influence of the nutrient composition of plant tissue culture media on axillary shoot proliferation and their preconditioning effect on subsequent adventitious shoot regeneration from pear leaves was investigated. The goal was to improve both micropropagation and regeneration of ‘Bartlett’ and ‘Beurre Bosc’ pear cultivars. Driver–Kuniyuki walnut (DKW) and Quoirin and Lepoivre (QL) nutrient media were found to be superior to Murashige and Skoog (MS) and Woody Plant Medium (WPM) for axillary shoot proliferation. Shoots on WPM exhibited some chlorosis. Axillary shoot culture on DKW would be preferred to that on QL due to the production of excessively short thin shoots on the latter medium. DKW also was superior to QL and MS for production of young expanding leaves for use as explants in adventitious regeneration. Leaf explants derived from shoot proliferation cultures grown on DKW or QL media produced more adventitious shoots than leaf explants from MS.