Efficient selection of somatic hybrids in Nicotiana tabacum L. using a combination of drug-resistance markers introduced by transformation

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Abstract
Kanamycin resistance and chloramphenicol resistance were introduced separately to two different tobacco plants (Nicotiana tabacum L.) by Agrobacterium-mediated transformation. Leaf mesophyll protoplasts were prepared from the progeny of these transformants and were subjected to electrofusion. On the 10th day and the 20th day after the fusion treatment, respectively, kanamycin (100 mg/l) and chloramphenicol (30 mg/l) were added to the suspension of protoplasts. The parental protoplasts and an unfused mixture of these protoplasts failed to form colonies when this selection procedure was employed. However, three independent fusion experiments yielded more than 7000 doubleresistant colonies with a frequency between 0.30% and 0.54%. All of the surviving colonies showed continuous growth in the presence of the two antibiotics. The majority of regenerated plants resembled, morphologically, a tetraploid tobacco plant, and their somatic chromosome numbers were 2n=96. The leaf segments from these putative somatic hybrids formed calli and proliferated vigorously on a medium that contained both antibiotics. Southern hybridization permitted the detection of the DNA fragments that conferred kanamycin and chloramphenicol resistance on these somatic hybrid plants. The system described here can be considered to be a universal system for the selection of somatic hybrids and is applicable to various combinations of protoplasts in which pre-selected genetic markers are absent.