Agrobacterium rhizogenes T-DNA genes capable of inducing hairy root phenotype

Summary Segments of the TL-DNA of the agropine type Ri plasmid pRi 1855 encompassing single and groups of open-reading frames were cloned in the Ti plasmid-derived binary vector system Bin 19. Leaf disc infections on Nicotiana tabacum led to transformed plants, some of which showed typical hairy root phenotypes, such as the wrinkled leaf morphology, excessive and partially non geotropic root systems and the ability of leaf explants to differentiate roots in a hormone-free culture medium. Particularly interestingly, most of these traits were shown by plants transformed with a TL-DNA segment encompassing the single ORF 11, corresponding to the rolB locus. Hairy root can be induced by this latter T-DNA segment on wounded stems of tobacco plants; hairy root induction on carrot discs requires, on the contrary, a more complex complement of TL-DNA genes.Abbreviations YMB yeast mannitol broth - MS Murashige and Skoog medium - 6-BAP 6-benzylaminopurine - NAA naphthalene acetic acid - Km kanamycin - Cb carbenicillin     

Regenerants from Ajuga hairy roots with high productivity of 20-hydroxyecdysone

Summary Regenerants derived from hairy roots of Ajuga reptans var. atropurpurea induced by infection with Agrobacterium rhizogenes MAFF03-01724 harboring pRi revealed a dwarfing response, i.e. decrease in leaf size, reduction in internode distance, and increase in leaf number. These morphogenic alterations were accompanied by an increase in root mass and lack of floral differentiation. In the pRi-transformed regenerants, the proportion of root mass to whole plant mass was higher than that of the untransformed ones, although both kinds of regenerants were comparable on a fresh weight basis. High capacity of rooting and 20-hydroxyecdysone production associated with the original hairy root line were stably maintained in clonal regenerants.     

A novel life cycle arising from leaf segments in plants regenerated from horseradish hairy roots

Summary Horseradish (Armoracia rusticana) hairy root clones were established from hairy roots which were transformed with the Ri plasmid in Agrobacterium rhizogenes 15834. The transformed plants, which were regenerated from hairy root clones, had thicker roots with extensive lateral branches and thicker stems, and grew faster compared with non-transformed horseradish plants. Small sections of leaves of the transformed plants generated adventitious roots in phytohormone-free G (modified Gamborg's) medium. Root proliferation was followed by adventitious shoot formation and plant regeneration. Approximately twenty plants were regenerated per square centimeter of leaf. The transformed plants were easily transferable from sterile conditions to soil. When leaf segments of the transformed plants were cultured in a liquid fertilizer under non-sterile conditions, adventitious roots were generated at the cut ends of the leaves. Adventitious shoots were generated at the boundary between the leaf and the adventitious roots and developed into complete plants. This novel life cycle arising from leaf segments is a unique property of the transformed plants derived from hairy root clones.     

Optimization of induction and culture conditions and tropane alkaloid production in hairy roots of <Emphasis Type="Italic">Anisodus acutangulus</Emphasis>

In this study, an efficient transformation system for the medicinal plant Anisodus acutangulus was successfully developed and optimized using Agrobacterium rhizogenes. Three bacterial strains, A4, R1601, and modified C58C1 and three explant types, leaf blade, petiole, and stem, were examined. The highest transformation efficiency of 94.44% was achieved using strain C58C1 with stem explants. Over 20 independent hairy root lines were successfully established with strain C58C1 using stem explants, all of which contained the ro/B and ro/C genes as confirmed by polymerase chain reaction (PCR). Out of four media compositions, the liquid 1/2 MS medium was found the most suitable for hairy root growth. The maximum biomass of one hairy root line increased up to 80 times in liquid 1/2 MS medium after a 30 day culture period. Different hairy root lines displayed a varied capacity for tropane alkaloid production and the best hairy root line (T4) from the C58C1-stem combination produced up to 10.21 mg/g (dw) of hyoscyamine, which was about 1.5-fold higher than in the wild type plants. To our knowledge, this is the first report to demonstrate the production of tropane alkaloids in hairy roots of A. acutangulus.     

Agrobacterium rhizogenes-mediated transformation of Picrorhiza kurroa Royle ex Benth.: establishment and selection of superior hairy root clone

A protocol for induction and establishment of Agrobacterium rhizogenes-mediated hairy root cultures of Picrorhiza kurroa was developed through optimization of the explant type and the most suitable bacterial strain. The infection of leaf explants with the LBA9402 strain resulted in the emergence of hairy roots at 66.7% relative transformation frequency. Nine independent, opine and TL-positive hairy root clones were studied for their growth and specific glycoside (i.e., kutkoside and picroside I) productivities at different growth phases. Biosynthetic potentials for the commercially desirable active constituents have been expressed by all the tested hairy root clones, although distinct inter-clonal variations could be noted in terms of their quantity. The yield potentials of the 14-P clone, both in terms of biomass as well as individual glycoside contents (i.e., kutkoside and picroside I), superseded that of all other hairy root clones along with the non-transformed, in vitro-grown control roots of P. kurroa. The present communication reports the first successful establishment, maintenance, growth and selection of superior hairy root clone of Picrorhiza kurroa with desired phyto-molecule production potential, which can serve as an effective substitute to its roots and thereby prevent the indiscriminate up-rooting and exploitation of this commercially important, endangered medicinal plant species. CIMAP Publication No.: 2007-28J     

Displacement of an herbaceous plant species community by mycorrhizal and non-mycorrhizal Gmelina arborea, an exotic tree, grown in a microcosm experiment

Gmelina arborea Roxb. (Gmelina, Yemane) is a fast growing tree, native from India and considered as a potentially invasive woody plant in West Africa. Mycorrhizal inoculation of seedlings with Glomus intraradices was performed to study (1) the effect on the growth of G. arborea, (2) the impact on the catabolic diversity of soil microbial communities and (3) the influence on the structure of herbaceous plant species communities in microcosms. Treatments consisted of control plants, pre-planting fertilizer application and arbuscular mycorrhizal (AM) inoculation. After 4 months’ culture in autoclaved soil, G. arborea seedlings were either harvested for growth measurement or transferred into containers filled with the same soil but not sterilized. Other containers were kept without G. arborea seedlings. After 12 months’ further culture, effects of fertilizer amendment and AM inoculation on the growth of G. arborea seedlings were recorded. AM colonization was significantly and positively correlated with plant diversity. The substrate-induced respiration response to carboxylic acids was significantly higher in the absence of G. arborea and in the presence of G. intraradices as compared to the other treatments. The influence of AM symbiosis on plant coexistence and on allelopathic processes of invasive plants are discussed.     

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.     

Indole alkaloid production by hairy root cultures of Catharanthus roseus

Hairy root cultures of Catharanthus roseus were established by infection with six different Agrobacterium rhizogenes strains. Two plant varieties were used and found to exhibit significantly different responses to infection. Forty-seven hairy root clones derived from normal plants and two derived from the flowerless variety were screened for their growth and indole alkaloid production. The growth rate and morphological appearance showed wide variations between the clones. The alkaloid spectra observed were qualitatively but not quantitatively very similar to that of the corresponding normal plant roots. No vindoline or deacetyltransferase activity could be detected in any of the cultures studied. O-acetylval-lesamine, an alkaloid which has not been previously observed in C. roseus was identified from extracts of hairy root clone No. 8. Two root clones were examined for their growth and alkaloid accumulation during a 26-day culture period. Alkaloid accumulation parallelled growth in both clones with ca. 2 mg ajmalicine and catharanthine per g dry weight being observed.Dedicated to Dr. Friedrich Constabel on the occasion of his 60th birthday     

Influence of arbuscular-mycorrhizal fungi, Rhizobium meliloti strains and PGPR inoculation on the growth of Medicago arborea used as model legume for re-vegetation and biological reactivation in a semi-arid mediterranean area

Medicago arborea can be used for re-vegetationpurposes under semiarid conditions. These woody legumes have the ability toforman association with arbuscular mycorrhizal (AM) fungi and rhizobial bacteria,which can be maximised by microorganisms producing certain stimulatingmetabolites acting as plant growth promoting rhizobacteria (PGPR). The effectsof single and combined inoculations using microorganisms with different andinteractive metabolic capacities, namely three Glomusspecies, two Rhizobium meliloti strains (a wild type, WTand its genetically modified derivative GM) and a plant growth promotingrhizobacterium, (PGPR), were evaluated. All three inoculated AM fungi affectedMedicago growth in different ways. Differences weremaintained when soil was co-inoculated with each of the rhizobial strains (WTorGM) and the PGPR. Mycorrhizal fungi were effective in all cases, but the PGPRonly affected plant growth specific microbial situations. PGPR increased growthof G. mosseae-colonised plants associated withRhizobium WT strain by 36% and those infected byG. deserticola when associated with the rhizobial GMstrainby 40%. The most efficient microbial treatments involved mycorrhizalinoculation, which was an indication of the AM dependency of this plantspecies.Moreover, PGPR inoculation was only effective when associated with specificmycorrhizal endophytes (G. mosseae plus WT andG.deserticola plus GM rhizobial strain). The reduced root/shoot (R/S)ratio resulting from PGPR inoculation, was an indication of more effective rootfunction in treated plants. AM colonisation and nodule formation wereunaffectedby the type of AM fungus or bacteria (rhizobial strain and/or PGPR). AM fromnatural soil were less infective and effective than those from the collection.The results supported the existence of selective microbial interactionsaffecting plant performance. The indigenous AM fungi appeared to be ineffectiveand M. arborea behaved as though it was highly dependentonAM colonisation, which implied that it must have a mycorrhizal association toreach maximum growth in the stressed conditions tested. Optimum growth ofmycorrhizal M. arborea plants was associated with specificmicrobial groups, accounting for a 355% increase in growth overnodulatedcontrol plants. The beneficial effect of PGPR in increasing the growth of awoody legume, such as M. arborea under stress, was onlyobserved with co-inoculation of specific AM endophytes. As a result of theinteraction, only shoot biomass was enhanced, but not as a consequence ofenhancing of the colonising abilities of the endophytes. The growthstimulation,occurring as a consequence of selected microbial groups, may be critical anddecisive for the successful establishment of plants under Mediterraneanclimaticand soil conditions.     

T-DNA analysis of plants regenerated from hairy root tumors

Summary Plants regenerated from hairy root tumors induced on Nicotiana glauca and Nicotiana tabacum by Agrobacterium rhizogenes strain A4 were examined for the presence of T-DNA. Regenerated N. tabacum plants contained intact copies of both TL-DNA and TR-DNA. However, plants regenerated from N. glauca tumors did not contain the TR-DNA region corresponding to the tms (auxin synthesis) genes. Some of the regenerants exhibited an abnormal phenotype which is characterized by severe leaf wrinkling. This phenotype is correlated with the presence of TL-DNA, but not TR-DNA.     

Transgenic Medicago truncatula plants obtained from Agrobacterium tumefaciens -transformed roots and Agrobacterium rhizogenes-transformed hairy roots

Medicago truncatula, barrel medic, is a forage crop that has been developed into a model legume. The development of new transformation methods is important for functional genomic studies in this species. Based on Agrobacterium tumefaciens-mediated transformation of root explants, we developed an effective system for producing M. truncatula (genotype R108) transgenic plants. Among the four A. tumefaciens strains (AGL1, C58C1, EHA105 and LBA4404) tested, EHA105 and AGL1 were most effective in regenerating transgenics. Callus induction frequency from root explants was 69.8%, and plantlet/shoot regeneration frequency was 41.3% when EHA105 was used. Transgenic nature of the regenerated plants was confirmed by PCR and Southern hybridization analyses. Progeny analysis revealed stable Mendelian meiotic transmission of transgenes. Because M. truncatula is particularly useful for the study of root endosymbiotic associations, we further developed a plant regeneration system from A. rhizogenes-transformed hairy roots of M. truncatula. Fertile true transgenic plants were regenerated from the hairy roots, thus allowing the assessment of gene functions at the whole plant level. Segregation analysis revealed that the hairy root genes could be segregated out in the progenies. By coupling A. rhizogenes-mediated hairy root transformation and the regeneration system reported here, once potential genes of interest are identified, the transformed hairy roots carrying such genes could be directly regenerated into plants for more detailed characterization of the genes.     

Induction and growth properties of carrot roots with different complements of Agrobacterium rhizogenes T-DNA

Single and multiple infections of carrot discs were carried out with Agrobacterium strains harbouring different segments of pRi1855 TL-DNA cloned in the binary vector Bin 19 and with a strain carrying the TR-DNA from the same Ri plasmid. Roots induced by the various co-inoculations were cultured and their growth patterns were followed. Abundant roots could be induced by TL-DNA rol genes A, B and C as a single insert (rolA+B+C) and by rolB alone provided an extended segment beyond its 5 noncoding region was included in the construction. A depression of rooting capability was caused by the inclusion of rolC together with rolB (rolB+C). In all cases co-inoculation with the Agrobacterium carrying TR-DNA-borne auxin genes was necessary for root induction since none of the rol constructions was in itself capable of eliciting any response; an exceeding majority of these roots were however shown to contain rol genes but no TR-DNA. Rooting was also elicited if rol constructions were co-inoculated with a strain carrying TL-DNA genes 13 and 14 (ORF13+14) instead of the TR-DNA strain. These roots were shown to contain both rol genes and ORF13+14. Striking differences in growth properties were shown by roots containing different complements of TL-DNA genes. Typical hairy root traits, high growth rate, branching and, most noticeably, absence of geotropism, were shown by roots containing rolB alone, while roots with rolA+B+C were geotropic as normal carrot roots. Hairy root traits were conferred to rolA+B+C roots by the concomitant presence of ORF13+14 and by the addition of auxin to the culture medium. A model is presented which attempts to rationalize the growth patterns by assigning interplaying roles to the various TL-DNA genes involved.     

The influence of mycorrhizal inoculation and paclobutrazol on water and nutritional status of Arbutus unedo L.

The purpose of this study was to analyze morphological and physiological aspects of Arbutus unedo L. plants treated with paclobutrazol (PAC), compounds characterized by their double activity as plant growth regulators and fungicides, and the ectomycorrhizal fungus Pisolithus tinctorius (Pers.) Coker and Couch, which forms a special type of mycorrhizal colonization called arbutoid mycorrhiza. Native A. unedo L. seedlings were grown in a greenhouse and subjected to four treatments for 4 months: 0 or 60 mg of PAC and inoculated or not with P. tinctorius (Pers.). The arbutoid mycorrhizal inoculation increased in plants treated with PAC. Paclobutrazol reduced shoot and root biomass, plant height, internode length, stem diameter, leaf area, total root length and number of tips. P. tinctorius increased plant height and had a beneficial effect on the root system (increasing root diameter and the number of tips). PAC treatment led to an increase in ion levels in the leaf tissue, while mycorrhizal inoculation induced lower K and higher P contents in the roots. Leaf water potentials (at predawn and at midday) increased with the combined treatment. The absence of water deficit conditions meant there was no osmotic adjustment. Higher photosynthesis (P_n) values were associated with higher stomatal conductance (g_s) values in the mycorrhizal plants, which influenced water uptake from the roots. However, g_s decreased in the PAC-treated plants, reducing photosynthesis and, as a consequence, growth. The higher hydraulic conductivity (L_p) in the plants treated with PAC may have induced a better water energy status and good water transport. The combined treatment produced beneficial effects in the plants, improving their water and nutritional status.     

The Agrobacterium rhizogenes pRi TL-DNA segment as a gene vector system for transformation of plants

Summary A plant gene transfer system was developed from the Agrobacterium rhizogenes pRi15834 TL-DNA region. Intermediate integration vectors constructed from ColE1-derived plasmids served as cloning vectors in Escherichia coli and formed cointegrates into the TL-DNA after transfer to A. rhizogenes. An A. rhizogenes strain with pBR322 plasmid sequences replacing part of the TL-DNA was also constructed. Plasmids unable to replicate in Agrobacterium can integrate into this TL-DNA by homologous recombination through pBR322 sequences. No loss of pathogenicity was observed with the strains formed after integration of intermediate vectors or strains carrying pBR322 in the TL-DNA segment. Up to 15 kb of DNA have been transferred to plant cells with these systems. The T-DNA from a binary vector was cotransformed into hairy roots which developed after transfer of the wild-type pRi T-DNA. Tested on Lotus corniculatus the TL-derived vector system transformed 90% of the developed roots and the T-DNA from the binary vector was cotransformed into 60% of the roots. Minimum copy numbers of one to five were found. Both constitutive and organ-specific plant genes were faithfully expressed after transfer to the legume L. corniculatus.     

Rose-scented geranium (<Emphasis Type="Italic">Pelargonium</Emphasis> sp.) generated by <Emphasis Type="Italic">Agrobacterium rhizogenes</Emphasis> mediated Ri-insertion for improved essential oil quality

Transgenic plants of rose-scented geranium (Pelargonium graveolens cv. Hemanti) have been produced from Agrobacterium rhizogenes (strains A₄ and LBA9402) mediated hairy root cultures. Amongst the explants tested, leaves were most responsive followed by the petioles and internodal segments, respectively. The A₄ strain performed better for all the three explants both in terms of frequency of response and time requirement for hairy root induction. Transgenic shoots could be obtained by spontaneous regeneration without intervening callus phase amongst 16% and 12% root lines of A₄ and LBA 9402 origin, respectively, or they were induced in 29% and 22% hairy root lines of A₄ and LBA9402 origin, respectively, with different hormonal supplementation. These transgenic plants showed 30% survival as against 90% of their control under the confined environment of glasshouse. The transgenic plants were of similar morphotype having increased branching, higher number of leaves with increased dentations, short and round stature, highly branched root system and absence of leaf wrinkling. These transgenic plants showed opine positive results even after 5 months of their transfer to the glasshouse. The essential oil compositions of 81% of these transgenics were qualitatively similar to that of the wild type parent. However, two transgenic plants (LZ-3 and 14TG) showed increase in concentrations of geraniol and geranyl esters signifying improved oil quality with respect to the citronellol:geraniol ratio. These two oils having better olfactory value represent an improvement over that of the wild type parent from the commercial point of view.     

Relationship between expression of the PM H<Superscript>+</Superscript>-ATPase,growth and ion partitioning in the leaves of salt-treated <Emphasis Type="Italic">Medicago</Emphasis> species

The role of the plasma membrane (PM) H⁺-ATPase (E.C. 3.6.1.3) in the plants response to salt stress was studied in the perennial leguminosae forage Medicago arborea L. and its close relative Medicago citrina (Font-Quer) Greuter, a species exposed to saline conditions in its original habitat. Plants were solution cultured for 8 days in 1 or 100 mM NaCl. Leaf growth and CO₂ assimilation were more inhibited by salt in M. arborea than in M. citrina. Both species were able to osmoregulate, and salt-treated plants maintained turgor potentials, with no differences between species. Contrasting ion distribution patterns showed that M. citrina was able to exclude Na⁺ from the leaves more selectively, while M. arborea had a greater buildup of leaf blade Na⁺. Isolation of purified PM and quantification of H⁺-ATPase protein by Western blot analysis against the 46E5B11D5 or AHA3 antibodies showed an increase in response to salt stress in the expanding (92%) and expanded leaves (87%) of M. citrina, while no differences were found in the corresponding leaves of M. arborea. The assay of H⁺-ATPase specific activity of the two leaf types in salinized M. citrina confirmed this increase, as activities increased with 55% and 104% for the expanded and expanding leaves, respectively, while no significant differences were found for either leaf type of salinized M. arborea. A possible role of the increased expression of the PM H⁺-ATPase for leaf expansion and ion exclusion in salt-stressed plants is discussed.     

Traits of transgenic Atropa belladonna doubly transformed with different Agrobacterium rhizogenes strains

Hairy root cultures of Atropa belladonna L. were established by infection either with Agrobacterium rhizogenes ATCC 15834 or MAFF 03-01724, and transgenic plants were obtained from both hairy root cultures. Doubly transformed roots were induced by re-infection of the leaf segments of transgenic Atropa belladonna plants (A. rhizogenes 15834) with MAFF 03-01724. Shoots and viviparous leaves were regenerated from the doubly transformed roots. The genetic transformation was determined by the opine assay (agropine, mannopine and/or mikimopine) and polymerase chain reaction. Physiological changes and tropane alkaloid biosynthesis in the hairy roots (singly and doubly transformed) were investigated. The alkaloid content in the doubly transformed root strain was intermediate as compared to the root strains which were singly transformed. On the other hand endogenous IAA levels in doubly transformed roots were significantly decreased compared to both singly transformed roots.Abbreviations BA benzyladenine - IAA indoleacetic acid - NAA naphthaleneacetic acid - PCR polymerase chain reaction - t-ZR trans-zeatin     

Hairy root culture of <Emphasis Type="Italic">Plumbago indica</Emphasis> as a potential source for plumbagin

Hairy roots of Plumbago indica were established at high frequency (90 %) by infecting leaf explants with Agrobacterium rhizogenes strain ATCC 15834. The axenic root cultures were established under darkness in hormone-free liquid Murashige and Skoog medium containing 3 % sucrose. The highest plumbagin content was found to accumulate in roots at their exponential phase of growth. A low pH (4.6) and a low concentration of sucrose (1 %) were beneficial for root growth in darkness, while pH 5.6 and 3 % sucrose under continuous irradiance enhanced plumbagin accumulation in roots up to 7.8 mg g⁻¹(d.m.). Direct shoot regeneration from hairy root culture was also achieved under continuous irradiance, thus indicated an easy way of obtaining transformed P. indica plants.     

Morphogenesis and Auxin Sensitivity of Transgenic Tobacco with Different Complements of Ri T-DNA

Leaf explants of hairy root tobacco (Nicotiana tabacum) regenerants characteristically differentiate roots from the wound margins on hormonefree medium. The same response can be elicited on normal tobacco by culturing the explants in the presence of auxin. We show here that the spontaneous rooting of transformed plants is neither due to the activity of right T-DNA-borne auxin genes nor to a substantially altered balance of endogenous hormones. Rather, an increased sensitivity to auxin is conferred to transformed cells by the left T-DNA (TL-DNA). Analysis of the morphogenetic behavior of transgenic tobacco plants obtained by transferring segments of TL-DNA cloned in a binary vector system allowed us to pinpoint TL-DNA genes responsible for this increased auxin sensitivity of hairy root tissues. Three genes (open reading frames 10, 11, 12) are responsible for the spontaneous rooting of leaf explants and confer to transgenic plants an exaggerated response to auxin.     

Interaction of Glomus mosseae and Paecilomyces lilacinus on Meloidogyne javanica of tomato

The effects of Glomus mosseae and Paecilomyces lilacinus on Meloidogyne javanica of tomato were tested in a greenhouse experiment. Chicken layer manure was used as a carrier substrate for the inoculum of P. lilacinus. The following parameters were used: gall index, average number of galls per root system, plant height, shoot and root weights. Inoculation of tomato plants with G. mosseae did not markedly increase the growth of infected plants with M. javanica. Inoculation of plants with G. mosseae and P. lilacinus together or separately resulted in similar shoots and plant heights. The highest root development was achieved when mycorrhizal plants were inoculated with P. lilacinus to control root-knot nematode. Inoculation of tomato plants with G. mosseae suppressed gall index and the average number of galls per root system by 52% and 66%, respectively, compared with seedlings inoculated with M. javanica alone. Biological control with both G. mosseae and P. lilacinus together or separately in the presence of layer manure completely inhibited root infection with M. javanica. Mycorrhizal colonization was not affected by the layer manure treatment or by root inoculation with P. lilacinus. Addition of layer manure had a beneficial effect on plant growth and reduced M. javanica infection.