amma Radiation: Physical Elicitor for In vitro Culture Techniques of Cajanus cajan.

Gamma rays are very important in mutation breeding and in in vitro mutagenesis in order to develop required features of plants and increase the genetic variability. Cajanus cajan when subjected to absorbed doses 30 Gy, 50 Gy, 100 Gy, 150 Gy and 200 Gy showed a direct corelation between callus induction, regeneracy frequency and absorbed doses of gamma radiation as compared to control. Gamma irradiation resulted in the induction of autonomous growth in callus, which led to the formation of callus tumors resembling the shape of crown gall tumors. Gamma irradiation in the present study proved to be an important tool in increasing the breeding efficiency, and regeneration frequency, especially that of the recalcitrant varieties.

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.

Isolation and purification of polysaccharides from transgenic crown gall cultures of Panax quinquefolium / 中草药

Objective To investigate the polysaccharides constituents from the transgenic crown gall cultures of Panax quinquefolium. Methods The crude polysaccharides PPQ50 and PPQ70 were extracted by water-ethanol method from the crown gall of P. quinquefolium. Four polysaccharides, named PPQ50-1, PPQ50-2, PPQ70-1, and PQ70-2, were isolated and purified by DEAE Sepharose and Sephacry S-100 column chromatography. Results The homogeneity of four polysaccharides was examined by gel chromatography and polarimetry. Their molecular weights were determined by gel filtration chromatography. They were 4.7?104, 2.9?105, 7.5?104, and 1.3?105, respectively. The sugar composition was analyzed by HPLC. The polysaccharides are mainly composed of neutral sugar particularly of D-glucose. The structure elucidation of PPQ50-2 was also supported by 1H-NMR and 13C-NMR spectroscopy. Conclusion All of the four polysaccharides are first isolated and purified from the transgenic crown gall cultures of P. quinquefolium.The result shows the PPQ50-2 and PPQ70-2 are both glucans.

Biosynthesis of arbutin from crown gall of transgenic Panax quinquefolium / 中草药

Objective To investigate the biosynthesis of arbutin by suspension culture of crown gall of transgenic Panax quinquefolium. Methods Hydroquinone in methanol(60 mg/mL) was added to the medium of the crown gall of P.quinquefolium after precultured for 20 d,then they were co-cultured for another 60 h.The product was isolated and purified by column chromatography and its structure was identified by physicochemical properties and spectroscopic data.The dynamic curve of biotransformation was investigated by quantative analysis of arbutin with HPLC.Results The product could be obtained from both of the culture and medium,which was isolated and elucidated as 4-hydroxyphenyl-?-D-glucopyranoside(arbutin).After co-cultured for 60 h,the mole conversion ratio of hydroquinone is 88.4%,the product contents in culture and medium are 63.69 mg/flask and 1.86 mg/flask,respectively,and the excretion ratio of arbutin reaches the highest(2.92%).Conclusion It's the first time around the world that the crown gall of transgenic P.quinquefolium is used as a biotransformation system and arbutin which shows varied pharmacological activites have been got successfully.

IDENTIFICATION OF PLANT PATHOGENIC BACTERIUM FROM CROWNGALL OF JAPANESE CHERRY (PRUNUS XYROLOEUSIS) ANDCONTROL TECHNOLOGY / 微生物学通报

11 plant pathogenic strains were isolated from the crown gall of Japanese cherry (Prunus xyroloensis)in Japanese cherry growing areas, such as Chixing. fenhua and Shenzhou counties of Zhejiang province. Ten ofthem were identified as Agrobacterium tumefaciens biotype 1 based on their morphological. physiological andbiochemical characterizations and bacterial protein SDS-PAGE test. The plate antagonistic assay and the potbiocontrol test indicated that the isolated strains of A tumefaciens biotype 1 were sensitive to the well-knownantagonistic bacterium , K84 strain. The K84 strain could inhabit the tumor production by the pathogenic satins.