Rhizogenes Research Articles

Transformation ofKalanchoëand oilseed rape with ORFs 11-15 fromAgrobacterium rhizogenes

Transformation of<i>Kalanchoë</i>and oilseed rape with ORFs 11-15 from<i>Agrobacterium rhizogenes</i>

Hairy roots production of transgenic Catharanthus roseus L. plants with Agrobacterium rhizogenes under in vitro conditions.

Hypocotyl explants of Catharanthus roseus L. gave rise to callus at a frequency of 90% when cultured on Murashige and Skoog (MS) basal medium supplemented BA (31.1 µM) and NAA (5.4 µM) on dark conditions. The calli (8 weeks old) were placed onto half strength liquid MS medium that had been polluted by bacterial clones of Agrobacterium rhizogenes. Transgenic of calli regenerated hairy roots after 10 days of inoculation and they have grown after 8 weeks. Hairy roots of in vitro C. roseus L. plant which produces several important terpenoid indole alkaloid by infection with A. rhizogenesis. Transformed hairy root increased and produced alkaloids, including vinblastin and vincristine, which are extracted and analyzed by high performance liquid chromatography (HPLC) and used for anticancer and antimicrobial researches. Key words: Catharanthus roseus, Agrobacterium rhizogenes, genetic transformation, hairy roots.

Rhizogenic Induction in Adult Juglans regia L. cv. Serr Tissue Induced by Indole Butyric Acid and Agrobacterium rhizogenes

La introduccion in vitro de tejido adulto de nogal (Juglans regia L.) representa una oportunidad de clonacion de genotipos elite, cuya seleccion ocurre en estados ontogenicos avanzados. Asi, con el objeto de desarrollar un protocolo que permita la propagacion masiva de genotipos valiosos a partir de material adulto, se compararon dos sistemas de induccion rizogenica de microtallos de nogal provenientes del cuarto subcultivo de un programa de introduccion in vitro de tejido adulto de nogal, previamente revigorizado mediante injerto tradicional. Se utilizo la induccion rizogenica por acido indol-3-butirico (AIB) y Agrobacterium rhizogenes. El proceso rizogenico se analizo tanto para induccion auxinica (T1: 3 mg L-1 AIB; T2: 5 mg L-1 AIB), como para induccion por A. rhizogenes (T3: A-477; T4: A-478), en dos fases. Una primera fase de induccion radicular, con una duracion de 3 dias en oscuridad; y una segunda fase de 27 dias, denominada de manifestacion radicular. En todos los tratamientos se evaluo porcentaje de enraizamiento y se caracterizaron los sistemas radiculares inducidos (numero, largo, diametro y zona de insercion de raices). Los mejores resultados de enraizamiento se obtuvieron en T2; sin embargo, la respuesta obtenida con A. rhizogenes no difiere de aquella lograda en T1, por lo que pareciera ser una metodologia de creciente interes para la rizogenesis adventicia en esta especie.

Isolation of anticancer compound of Artemisia cina hairy root and its inhibition activity on cervix cancer cells.

The research of isolation of anticancer agent of A.cina hairy roots and its inhibition activity on cervix cancer cells have been conducted. Hairy roots transformed by Agrobacterium rhizogenes strains A4 and ATCC-15834 were then extracted by n-hexane and separated by column chromatography with variation of n-hexane/ethyl acetate as eluent. All samples include hexane extract and result of column chromatography tested to cervix HeLa Ohio cells with concentration of 50 μg/ml for hexane extract and 10 μg/ml for column chromatography respectively. The most active fraction was then tested by the concentration of 1 to 5 μg/ml. Confirmation of transformed root of A.cina was conducted by PCR analysis. The result of experiment shown that hexane extract of hairy root, normal root ( in vitro ), leaves of plant from green house as a control gave the inhibition value were about 84 %. The most active fraction from column chromatography was fraction E with IC50 at the concentration of 1 μg/ml and inhibition value was 95 %, the identification compound of this fraction was terpenoid group. The confirmation result showed that TL-DNA was transferred by 780 kb. Key words : anticancer, hairy root, Artemisia cina.

Detection of Tumorigenic Rhizobia in Asymptomatic Peach Plants by PCR.

At present the only method for the detection of Rhizobium radiobacter and R. rhizogenes (ex Agrobacterium tumefaciens) in plants is by isolating the bacteria on selective medium and testing them in vivo for pathogenicity. This procedure is time-consuming and not appropriate for detecting low concentrations of these agrobacteria. In this study a protocol was developed for detecting tumorigenic agrobacteria in the stem and root tissues of artificially inoculated peach plants. DNA was extracted from the plant tissues by a rapid procedure and then a 246 bp sequence of the vir region of the pTi was amplified by PCR. The target sequence was found in all stem and root samples of asymptomatic peach plants, and was evidenced in all the samples analyzed showing the effectiveness and reliability of the method.

Peroxidase production in vitro by Armoracia lapathifolia (horseradish)-transformed root cultures: effect of elicitation on level and profile of isoenzymes.

Transformed roots of Armoracia lapathifolia (horseradish) were established by infection with Agrobacterium rhizogenes LBA 9402. They were used as a culture system in vitro for peroxidase production in vitro, to avoid many of the problems that affect the traditional production from field-grown species of Armoracia sp. The time course of growth of these cultures showed that total peroxidase attained maximum levels at the end of the exponential growth phase. At this stage of culture, elicitation assays were performed with AgNO3 and CuSO4 as abiotic elicitors and with fungal extracts of Verticillum sp., Monodyctis cataneae and Aspergillus niger as biotic elicitors. The best results were obtained with Verticillum sp., 24 h after elicitation, with an increase of approx. 100% in peroxidase activity. The isoenzyme pattern analysed by isoelectric focusing revealed predominantly basic and acidic isoenzymes in both plant roots and transformed root cultures. Elicited samples showed a similar isoenzyme pattern with a slight increase in basic isoenzymes.

High-frequency transformation of cottonwoods (genus Populus) by Agrobacterium rhizogenes

Many species of Populus, particularly cottonwoods of sections Aigeiros and Tacamahaca, remain recalcitrant to genetic transformation. We demonstrate that transgenic trees can be readily produced in several poplar genotypes using wild-type Agrobacterium rhizogenes. Hairy roots were produced in a variety of clones that included Populus trichocarpa Torr. & A. Gray, Populus deltoides Bartr. ex Marsh., P. trichocarpa × P. deltoides hybrids, and Populus alba L. × Populus grandidentata Michx., some of which were otherwise recalcitrant to regeneration or transformation. The frequency of hairy roots ranged from 17 to 92%; nearly all hairy roots could be regenerated into transgenic plants. Among 18 transgenic lines that were confirmed by Southern analysis and grown into small trees, 5 showed severe dwarfism and other phenotypic abnormalities, while the majority grew normally. Agrobacterium rhizogenes appears to be an effective means for transformation of diverse poplar genotypes and may be useful in genotypes where disarmed strains of Agrobacterium or regeneration protocols are inadequate to produce transgenic plants. Resume : Plusieurs espces du genre Populus, et plus particulirement celles des sections Aigeiros et Tacamahaca, demeurent rOfractaires ‡ la transformation gOnOtique. Les auteurs dOmontrent que des arbres transgOniques peuvent Œtre obtenus facilement pour plusieurs gOnotypes de peuplier ‡ liaide diune souche sauvage de Agrobacterium rhizogenes. Des racines avec poils absorbants ont OtO obtenues pour un nombre important de clones reprOsentatifs des espces Populus trichocarpa Torr. & A. Gray et Populus deltoides Bartr. ex Marsh., ainsi que des hybrides de P. trichocarpa × P. deltoides et de Populus alba L. × Populus grandidentata Michx., dont certains Otaient rOfractaires ‡ la rOgOnOration ou ‡ la transformation. La frOquence ‡ laquelle des racines avec poils absorbants furent obtenues variait de 17 ‡ 92% et presque toutes les lignOes de racines avec poils absorbants pouvaient Œtre rOgOnOrOes en plantes transgOniques. Parmi les 18 lignOes transgOniques qui furent confirmOes par analyse de type Southern et qui se sont dOveloppOes jusquiau stade de petit arbre, 5 ont affichO un nanisme sOvre ainsi que diautres anormalitOs phOnotypiques, alors que la majoritO des lignOes se sont dOveloppOes normalement. Liutilisation de A. rhizogenes apparaOt donc Œtre un moyen efficace pour transformer divers gOnotypes de peuplier. Cette stratOgie pourrait Œtre utile pour la production de plantes transgOniques dans le cas des gOnotypes pour lesquels la transformation par des souches dOsarmOes de Agrobacterium ou les protocoles de rOgOnOration siavrent inadOquats. (Traduit par la ROdaction)

Establishment of Grapevine Axenic Root Lines by Inoculation with "Agrobacterium rhizogenes"

Establishment of Grapevine Axenic Root Lines by Inoculation with "Agrobacterium rhizogenes"

&lt;I&gt;Agrobacterium rhizogenes&lt;/I&gt; A4株によるカキ (&lt;I&gt;Diospyros kaki&lt;/I&gt; L.) の形質転換

&lt;I&gt;Agrobacterium rhizogenes&lt;/I&gt; A4株によるカキ (&lt;I&gt;Diospyros kaki&lt;/I&gt; L.) の形質転換

THE GENETIC DETERMINATION OF HOST RANGE IN THE RHIZOBIACEAE

The family Rhizobiaceae includes bacteria which form nitrogen-fixing root nodules on the roots of leguminous plants (the genus Rhizobium ) and bacteria which form tumours on dicotyledenous plants (the genus Agrobacterium). For both genera classification at the species level is determined by plant interactions: strains of Agrobacterium which induce root proliferation (hairy root disease) are included in the species A. rhizogenes , those which form tumours are classified as A. tumefaciens and those which elicit no plant response as A. radiobacter. For Rhizobium , speciation is determined primarily by the range of legume hosts which are nodulated. Strains of Rhizobium and Agrobacterium contain large amounts of extrachromosomal DNA (plasmids). Host recognition and tumour-forming properties are determined by these plasmids, which can be transferred between different species and also between Agrobacterium and Rhizobium. Such transfer experiments have shown that for Agrobacterium and some fast-growing species of Rhizobium the plant-interaction characteristics of strains and hence the criterion for their taxonomie status is plasmid mediated. Whether or not host range properties are readily exchanged between these bacteria in nature needs to be determined.