Application of antibiotics in tomato oral vaccine against tuberculosis

Objective To seek a protocol for application of kanamycin (Km), carbenicillin (Carb) and cefotaxine (Cef) in tomato vaccine against tuberculosis. Methods The sensitivity of untransformed tomato cotyledons to kanamycin and carbencillin was tested, the sensitivity of untransformed shoots to carbencillin and cefotaxine was tested. Transformed cotyledons were screened by 10.0mg/L kanamycin for regenerated shoots resistant to kanamycin, the roots was again screened by kanamycin. Results Regeneration of cotyledons were inhibited by kanamycin. The number of days of recovery of cotyledons and budding were increased, while the number of shoots reduced along with the increase of concentration of kanamycin. Regeneration of cotyledons were inhibited completely when the concentration of kanamycin reached 10.0mg/L. 30% cotyledons regenerated callis, and 13% regenerated shoots if 10.0mg/L kanamycin was used to screen transformed cotyledons. A few shoots grew roots when the shoots were screened by 10.0mg/L kanamycin and the days of growing roots were increased and the number of roots reduced. Two months were needed by two steps of the screening. The concentrations of 0～500.0mg/L carbenicillin did not affect the regeneration of cotyledons. 500.0mg/L carbenicillin promoted the generation of roots, but the roots were short and thick. While roots were long and thin under the concentration of 500.0mg/L cefotaxine. Conclusion The appropriated screening concentration of kanamycin is 10.0mg/L. 500.0mg/L of carbenicillin be used during bud differentiating stage, while 500.0mg/L cefotaxin during root-inducing stage. The method of two-step screening of daughter leaf (calli) and roots were sensitive and rapid, which reduced screening time.