First Report of Agrobacterium rhizogenes-induced Hairy Root Formation in Selaginella bryopteris: a Pteridophyte Recalcitrant to Genetic Transformation

Abstract we report A. rhizogenes-induced hairy root formation in S. bryopteris, a medicinally and commercially important plant. A. rhizogenes strain LBA1334 co-cultivated with explants (root, rhizophore, stem portion near the root, and stem with intact fronds) for 24 and 48 h after transformation for induction of hairy roots. The induction of hairy root was observed after 6 days of infection in case of 48 h co-cultivation only. PCR with rolA and virC gene specific primers confirmed the induced hairy roots were due to Ri T-DNA integration and not due to contaminating A. rhizogenes. The root network as explants showed the maximum transformation efficiency. We tested different media like MS, SHFR (Stage Hog Fern Root) and KNOP's during transformation for hairy root induction. The SHFR based media showed good response in transformation as well as propagation. Further, transformation efficiency was enhanced by addition of TDZ (2 mg/L) and Bevistin (0.1%) in SHFR media. The present work would be helpful in hairy roots-based in vitro production of secondary metabolites and on aspect of functional genomics of S. bryopteris.

[Ex vitro production of transgenic composite plants in Glycyrrhiza glabra via Agrobacterium rhizogenes-mediated transformation as a new method]

Background: Licorice, Glycyrrhiza glabra [belong to Leguminosae family] is one of the most popular medicinal plants in the world and it is widely used in many fields such as medical, pharmaceutical, confectionery and health industries. Different parts of licorice [shoots, leaves and roots] were had various components such as Glycyrrhzin that was used for some proposes

Objective: The current study was done with the aim of gene transfer via Agrobacterium rhizogenes by ex vitro method for hairy root production in licorice

Methods: The experiment was laid out as a completely randomized design [CRD] with five treatments in three replications. At first, root of young plantlets was eliminated and excited plantlets were putted in the glass wool contain suspension of bacteria. After 10 to 14 days of inoculation with Agrobacterium rhizogenes, the roots were appeared. The percentage of root induction by four strains of Agrobacterium [ATCC 15834, GMI 9534, A4 and A13] with check [without bacteria] was investigated

Results: The results of PCR analysis with specific primers for roots of composite plants [putative transgenic] was shown that three strains of bacteria [A4, A13 and GMI 9534] and strain ATCC 15834, were produced 100% and 66.66% transgenic roots respectively

Conclusion: Thus, production of composite licorice plants was remarked due to it has low cost, fast and simple

[ production of hairy roots from [Datura innoxia L.] by Agrobacterium rhizogenes and effects of salicylic acid and methyl jasmonate biological elicitors on atropine and scopolamine content]

Background: Using Agrobacterium rhizogenes due to create hairy roots, is a useful method to increase secondary metabolites many plants

Objective: Purpose of this research is to transgenic hairy roots culture, in order to produce secondary metabolites in Datura innoxia

Methods: Explants leaf and cotyledon of Datura innoxia were inoculated for two months with A7, A4 and 15834 strains of Agrobacterium rhizogenes; Furthermore injection and Immersion methods were used in this scrutiny. The presence of T-DNA in transgenic hairy roots were confirmed by PCR. Transgenic hairy roots in liquid medium of 1/2MS were cultured. In order to induct elicitors, methyl jasmonate in tow densities of 50 micro M and 100micro M, and salicylic acid in three densities of 1mM, 0.1mM and 0.01 mM were used randomly three times. Atropine and scopolamine content of transgenic hairy roots were examined by HPLC

Results: The highest and lowest rate of transgenic hairy roots production was respectively related to the strains of A4 and 15834. Best explants for inoculation, leaf with A4 strain and cotyledon with A7 strain, were reported. With highest production rate of hairy roots, Simple deposit using a syringes method was recognized as the best method of inoculation. The effect of salicylic acid at a density of 0.1 mM increases the content of atropine concentrations. Also the results showed that usage of Methyl jasmonate at higher doses [100 micro M] reduces the content of atropine and scopolamine

Conclusion: A. rhizogenes as an appropriate method to produce hairy roots and elicitors the best treatment for increase alkaloids

Métodos para la detección de Agrobacterium a partir de muestras de material vegetal, suelo y agua / Methods for the detection of Agrobacterium from plant, soil and water samples

El género Agrobacterium incluye especies ftopatógenas que inducen la formación de agallas en el cuello o la proliferación de raíces en cabellera en más de 600 especies de dicotiledóneas, y especies no patógenas cuyo hábitat natural es el suelo. Como no es posible erradicar a las especies patógenas y habida cuenta de que más del 80 % de las infecciones puede provenir de viveros, es importante evitar la diseminación de la enfermedad. Por ello, el objetivo de este trabajo ha sido desarrollar técnicas sensibles y precisas que, aisladamente o combinadas, permitan detectar la presencia de especies y biovares de Agrobacterium a partir de muestras de material vegetal, suelo y agua. Se comprobó que con la estrategia combinada de realizar aislamientos en los medios semiselectivos D1, D1-M y YEM-RCT; PCR multiplex sobre el gen 23S ADNr; PCR específca sobre los genes virC1 y virC2 y bioensayos en plántulas de pimiento cv. California Wonder y en hojas cortadas de kalanchoe, se reduce la posibilidad de obtener falsos negativos y/o falsos positivos. Por lo expuesto, esta combinación de técnicas constituye una herramienta adecuada para el diagnóstico de cepas patógenas de Agrobacterium a partir de distintos tipos de muestras.

The genus Agrobacterium includes phytopathogenic bacteria that induce the development of root crown galls and/or aerial galls at the base of the stem or hairy roots on more than 600 species of plants belonging to 90 dicotyledonous families and non-pathogenic species. These bacteria being natural soil inhabitants are particularly diffcult to eradicate, which is a problem in nurseries where more than 80% of infections occur. Since early detection is crucial to avoid the inadvertent spread of the disease, the aim of this work was to develop sensitive and precise identifcation techniques by using a set of semi-selective and differential culture media in combination with a specifc PCR to amplify a partial sequence derived from the virC operon, as well as a multiplex PCR on the basis of 23SrDNA sequences, and biological assays to identify and differentiate species and biovars of Agrobacterium obtained either from soil, water or plant samples. The combination of the different assays allowed us to reduce the number of false positive and negative results from bacteria isolated from any of the three types of samples. Therefore, the combination of multiplex PCR, specifc PCR, isolations in semi-selective D1, D1-M and YEM-RCT media combined with bioassays on cut leaves of Kalanchoe and seedlings of California Wonder pepper cultivar constitute an accurate tool to detect species and biovars of Agrobacterium for diagnostic purposes.

On serch of bacterial species definition

The bacterial species concept was examined within the framework of plant and animal associated alpha-2 proteobacteria, taking into consideration the phylogenetic, taxonomic and biological approaches as well as the microbiologists' perception. The virtue of the phylogenetic approach is that it gives an evolutionary perspective of the bacterial lineage; however the methods used possess low resolution for defining species located at the terminal branches of the phylogenetic trees. The merit of the taxonomic approach is that species are defined on the basis of multiple characteristics allowing high resolution at the terminal branches of dendograms; its disadvantage is the inaccuracy in the earlier nodes. On an individual level, the qualitative biological characteristics used for the definition of species frequently reveal shortcomings because many of these properties are the result of coevolution, parallel evolution or the horizontal transfer of genes. Nevertheless, when considered together with the phylogenetic and taxonomic approaches, important uncertainties are discovered: these must be weighed if a practical definition of bacterial species is conceived. The microbiologists' perception is the criterion expressed by a group of sponsors who, based on scientific and practical grounds, propose a new bacterial species. The success of this new proposal is measured by its widespread acceptance and its permanence. A difficult problem concerned with defining bacterial species is how to distinguish if they are independent evolutionary units or if they are reticulate evolutionary units. In the first case the inherence is vertically transmitted as a result of binary fission and clonal expansion. This may be the case of some animal cell associated bacteria in which recombination appears to be precluded or exceptional. In the second case adaptive changes occurring within an individual can be horizontally transferred to many or all group members. This seems to be the condition of many intestinal and plant associated bacteria. Genetic drift and specialization in clonal bacteria will depend almost exclusively on mutation and internal genetic rearrangement processes, whereas specialization in reticulate bacteria will depend not only on these processes but in their genetic interactions with other bacterial strains. This uncertainty, which corresponds to the evolutionary process, is at the same time one of the key factors in defining a bacterial species