The effect of different antibiotics on the elimination of Agrobacterium and high frequency Agrobacterium-mediated transformation of indica rice (Oryza sativa L.)

Abstract

Abstract:We report here the suitable explant with high efficiency of transformation and the positive effects of timentin over other antibiotics like carbenicillin and cefotaxime on the elimination of Agrobacterium tumefaciens during the genetic transformation of popular indica rice (Oryza sativa L.). The tissues assayed were embryogenic calli, embryos with endosperm contamination, intact seeds, leaf blades, leaf bases and coleoptiles. The frequency of transient β-glucuronidase (GUS) expression as revealed by histochemical assay was 90% for embryogenic calli, which was the highest among the explants used. On the basis of disc-diffusion assay, the maximum zone of inhibition (29 mm) at 250 mg/l was obtained for timentin. In tissue culture conditions the frequency of Agrobacterium recurrence after 20 days of infection was minimum (2.3%) at 200 mg/l of timentin. At 250 mg/l of timentin there was no Agrobacterium growth, besides, there were no negative effects on the callus growth unlike other antibiotics, hence it was selected as the optimum concentration for high frequency callus proliferation and regeneration. The effect of the parameters evaluated was determined by the callus proliferation during selection, reduction in browning, transient GUS expression and stable transformation efficiency (23.3%). The resultant plants were stable transformants as confirmed by a molecular analysis of the gus and hpt genes. The developed transformation protocol will be very helpful for the information on indica rice cultivars in general and on IR 64 in particular.

Robust and Reproducible Agrobacterium-Mediated Transformation System of the C4 Genetic Model Species Setaria viridis

“…the callus material was evidenced by the high degree of soft, watery and/or necrotic regions that subsequently formed on the callus (Figure 2A) with similar observations previously reported for rice callus when using the same approach (Priya et al, 2012). In contrast to the dry and wet co-cultivation strategies, the callus material resulting from semi-dry cocultivation continued to proliferate normally.…”

Effect of antibiotic ceftriaxone on elimination of ABI and GV3101 strains of Agrobacterium tumefaciens

“…Ceftriaxone in all the studied concentrations was proved to eliminate the bacterium growth. Timentin was also used for bacteria elimination: 250 mg/l [13] or 400 mg/l [15] for EHA105 strain, 250 mg/l for C58 strain [7], 150 mg/l [16] or 400 mg/l [17] for LBA4404 strain, 50 mg/l for KYRT1strain [18]; 400 mg/l for GV3101 strain [12]. Cefotaxime (500 mg/l) is commonly used for elimination of most mentioned strains.…”

“…The ceftriaxone effect on the ABI and GV3101 Agrobacterium tumefaciens strains was evaluated by ISSN 1993- disk diffusion method [13]. Cefotaxime (500 mg/l) was used as a control.…”

“…Ceftriaxone, a third-generation cephalosporin, characterized by a prolonged half-value period, has the broad-spectrum activity. The successful elimination of various Agrobacterium tumefaciens strains (EHA105, LBA4404, AGL_1) by means of ceftriaxone has been earlier reported [9][10][11][12][13]. However, it has not been yet used for the elimination of ABI Agrobacterium strain.…”

Effect of Ceftriaxone and Timentin antibiotics on morphogensis in the in vitro culture of bread wheat Triticum aestivum L.

“…or to eliminate any bacterial contamination (in particular during in vіtro Agrobacterium-mediated transformation -Cefo taxime, Carbenicillin, etc.). Recently new generation antibiotics of the β-lactam group, such as Timentin and Ceftriaxone are used more often [1][2][3][4][5][6][7][8][9][10][11].…”