Genetic transformation of green bean callus via Agrobacterium mediated DNA transfer

Abstract

Kanamycin resistant callus was produced from leaf disc or hypocotyl expiants of green bean (Phaseolus vulgaris L.) when cultured on a defined medium containing 50 mg/l kanamycin after 4 days of co-cultivation with Agrobacterium tumefaciens strain EHA101 containing the binary vector pKYLX71GUS. The presence of neomycin phosphotransferase II (NPT-II) in crude cellular extracts from the kanamycin resistant callus was confirmed by ELISA. The expression of the ß-glucuronidase (GUS) reporter gene was confirmed by histochemical and fluorimetric analyses. Southern blot border analysis confirmed the integration of the foreign DNA. In addition to the evidence obtained from Southern analysis, the absence of Agrobacterium in the transformed callus cultures was confirmed by microscopic observation and through test cultures. Using the above protocol, bean callus cultures were also transformed with a bean chalcone synthase promoter-GUS fusion. These cultures, when treated with the elicitor glutathione, showed higher levels of GUS expression than the unelicited callus clumps.

Transformation of Medicago truncatula via infiltration of seedlings or flowering plants with Agrobacterium

“…1992 ) or pSKI006 ( Kardailsky et al . 2000 ) (www.salk.edu/LABS/pbio‐w/); A. tumefaciens strain EHA105, carrying pBI121‐ bar or PKYLX7‐Gus ( Franklin et al . 1993 ), or pBIN mgfp ‐ ER ‐ bar , or pGA482‐ bar; A. tumefaciens strain Gv3101 carrying pSKI015 ( Kardailsky et al .…”

GENETIC TRANSFORMATION AND REGENERATION OF COMMON BEAN (Phaseolus vulgaris L.) USING Agrobacterium SYSTEM

“…Early efforts to produce transgenic bean plants failed due to poor DNA delivery and regeneration systems. Franklin et al (1993) produced transformed gusA positive callus from a kidney bean cultivar inoculated with Agrobacterium strain EHA101. So far, transformation of P. vulgaris has been achieved through direct gene transfer into the apical meristem region of seedlings of one cultivar (Russell et al, 1993).…”

GENETIC TRANSFORMATION OF COMMON BEANS (Phaseolus vulgaris L.) CALLI BY BIOLISTIC GUN AND VIA Agrobacterium tumefaciens

“…After direct gene transfer Köhler et al (1987) The kanamycin was increased to 50 mg l -l over a two month period (McClean et al, 1988). 50 mg l -l kanamycin was chosen by Franklin et al (1993) (Kwapata et al, 2012). According to the present results, it is possible to introduce osmosis protective genes into common beans calli by a special biolistic gun mediated DNA transfer.…”