Elimination of mycoplasma in tobacco callus tissues (Nicotiana glauca Grah.) culturedin vitro in the presence of 2,4-D in nutrient medium

Callus tissue cultures were established from stems of tobacco plants ( N. glauca Grah. ) both healthy and mycoplasma (potato witches' broom disease) infected on a modified nutrient medium (with a lower content of mineral salts) according to Murashige and Skoog (1962) in the presence of 2,4-D (1 mg l −1 ) as a growth regulator. No differences were observed in the growth and development of both tissues. Organogenesis appeared on a nutrient medium ( Petrů et al. 1972) supplemented with kinetin (0.64 mg or 2.56 mg l −1 ) and IAA (2 or 4 mg l −1 ). Callus derived from mycoplasma diseased plants started to form numerous buds after three months whereas organogenesis in callus from healthy controls appeared only after six months. We suppose that the reason of this difference is the fact that an expressively higher content of 2,4-D was found in the calli from healthy plants in comparison with the corresponding tissue from mycoplasma diseased ones. Reconstituted plants were isolated, rooted and transferred in the soil. The infectivity of these plants was assayed by grafting their stem tips on tomato plants which indicate very reliably and sensitively this mycoplasma disease. 31 reconstituted plants were obtained in the whole from calli isolated from mycoplasma infected plants and all of them were healthy. It was established that mycoplasma failed in the presence of 2,4-D in vitro . Stem pieces from diseased plants in which mycoplasma presence was proved, lose their infectivity after 4 weeks of cultivation on nutrient medium with this growth regulator. On the contrary 2,4-D which spreads and acts especially through phloem ( Smith et al. 1947) does not kill mycoplasma in vivo even in doses evoking strong symptoms of 2,4-D effect on experimental plants.