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

E Eissa

doi:10.21608/ejgc.2013.10454

Abstract

Common bean is one of the most recalcitrant species for in vitro manipulation and transformation. In this study efficient genetic transformation methods were developed and performed in commercialized bean varieties calli by utilized microparticle bombardment (direct biolistic gun) and indirect A. tumefaciens-based technique. Hypocotyl segments cultured on Gamborgs medium supplemented with 1 mg l-l kinetin and 2 mg l-l 2,4-dichlorophenoxy acetic acid proved the best for callus induction and maintenance. Co-transformed calli were double selected on selective medium using 150 mg l-l kanamycin and 0.8 mol l-l mannitol. Transient genes expressions were obtained in calli cells and the results showed that the cells shot by biolistic DNA delivery method can be transformed to get kanamycin resistant and mannitol tolerant. The presence and integration of the neomycin phosphotransferase II (npt II) and osmosis protector mannitol-1-phosphate dehydrogenase (mtlD) genes into P. vulgaris L. genome were confirmed and verified by double selection tests. Transgenic calli were selected after co-cultivated with Agrobacterium method on selective medium containing 5 mg l-l phosphinotricin (PPT). Transient genes expressions were obtained in the calli and the results showed that the common beans calli co-cultivated with Agrobacterium can be transformed to get phosphinotricin resistant and β-glucuronidase reporter positive tissues. The presence and integration of the phosphinotricin acetyl transferase and β-glucuronidase genes into P. vulgaris L. genome were confirmed and verified by marker selection and histochemical assay tests. These results suggest an efficient biolistic gun, and Agrobacterium-mediated transformation methods for stable integration of economically important genes into common beans calli, and that these transformations systems could be useful for future studies on transferring economically important genes into common bean plants.

Highlights

MATERIALS AND METHODSTwo commercial common bean varieties Fönix and Maxidor (Phaseolus vulgaris) were used as source of explants
B5 medium supplemented with 1 mg l-l kinetin and 2 mg l-l 2,4-D proved to be optimal in callus induction from hypocotyl and epicotyl in P. vulgaris L. (Gémesné Juhász et al, 1995)
Selection tests and assay analysis would confirm the transgenic nature of common beans calli

Summary

MATERIALS AND METHODS

Two commercial common bean varieties Fönix and Maxidor (Phaseolus vulgaris) were used as source of explants. The seeds are kindly supplied by the Genetics and Horticultural Plant Breeding Department, Faculty of Horticultural Sciences, Corvinus University, Budapest, Hungary, to whom my thanks are due. Sterilized seeds were germinated on MS basal salts medium (Murashige and Skoog, 1962) pH 5.7 without plant growth regulators for 7-10 days before being used for callus induction and transformation experiments. This study was carried out at the Tissue Culture Laboratory and Genetic Modified Organisms Laboratory, Genetics and Horticultural Plant Breeding Department, Faculty of Horticultural Sciences, Corvinus University, Budapest, Hungary. Hypocotyls segments from 7-10 days old aseptically grown seedlings of common beans were dissected and incubated on B5 medium (Gamborg et al, 1968) supplemented with 1 mg l-l kinetin and 2 mg l-l 2,4dichlorophenoxy acetic acid (2,4-D) as the auxin source. The experiment was repeated three times, and the procedure and conditions of callus induction were followed as described by Eissa et al (2001)

Transformation methods

RESULTS AND DISCUSSION

Concluding remarks

SUMMARY