[Effects of repeated in vitro bud formation on somatic variation ability on a chlorophyll mutant of Nicotiana tabacum var. Samsun].

A homozygous tl/tl chlorophyll mutant of Nicotiana tabacum var. Samsun shows spontaneous somatic variations on its leaves. These variations are either single (dark or light) or twinned (dark and light). In vitro culture of variation-free leaf fragments, on a particular medium, directly yields neoformed buds. These buds are placed on a medium for root formation, so that neoformed plants are obtained from which a new cycle of bud neoformation can be started. In this way, one obtains several sets of plants of the same somatic origin but which differ from each other by one or more neoformation cycles. At each cycle, the somatic variation ability is characterized on the basis of four criteria: (1) numbers and proportions of dark, light and twinned variations, (2) distribution of dark variations according to their sizes, (3) distribution of variations according to the foliar level, and (4) sexual transmission of each character. As early as at the second neoformation cycle, one observes a significant increase of variability among the numbers of variations and shifts in size distributions. The increase of variability is more striking after the third cycle; particularly, plants obtained in this manner have acquired, on the average, the ability to vary earlier in the development of the foliar initium. This property is expressed through the enlargement of the mean size of the variations. In each case, the observed variability is somatically maintained through cuttings. This, together with the fact that the observed shifts in frequency distributions as well as the changes in numbers of variations are sexually transmitted after the third neoformation cycle, definitely means that genetic changes have occurred. Consequently, it seems that from one neoformation cycle to another, in vitro culture of leaf fragments has cumulative effects which, beyond a particular threshold, are sexually transmitted. The only character, among four, not to be affected by in vitro culture is the distribution of somatic variations according to the foliar level. Thus, it seems that this character is strictly controlled by changes in physiological conditions which occur during the plant growth; in particular, the transition of the shoot apex from vegetative to flowering state. The effects of in vitro culture on the ability to somatically vary are discussed on the basis of four hypotheses: (1) modification in number of chromosomes (polyploidy, aneuploidy); (2) modification in chromosome structure (translocation); (3) selection of a particular kind of cells and (4) cumulative alteration of genetic information either by mutation or by disturbance in cell regulation process.