The Influence of Meristematic Tissue and Injuries on the Transport of Tobacco Mosaic Virus in Nicotiana tabacum L. cultivar. Samsun

I. The influence of meristematic tissue on virus-transportation in a plant was studied in Nicotiana tabacum L. cultivar. Samsun, inoculated with tobacco mosaic virus (TMV). The rate of transport of the virus in the plant was estimated by determining the rapidity with which the virus appeared in uninoculated leaves. For this purpose the leaves of a plant were cut at different times after inoculation of a leaf and placed in water. Under this condition, virus-material present in the leaves at the moment of cutting was allowed to multiply for three days. After this period each leaf was pressed out and tested for the presence of virus by means of the local lesion test on leaves of Nicotiana glutinosa L. 2. From experiments in which either the lowest, the middle or the topmost leaf was inoculated, it became evident that inoculation of the middle leaf caused the most rapid virus-transportation. 3. The influence of meristematic tissue on virus-transportation was studied in different ways. One of the most obvious methods is to eliminate the meristematic tissue by removing it. In our experiments removal of the stemtip did not influence either the direction or the rate of virus-transportation out of the inoculated leaf. Removal of the axillary buds or both axillary buds and stemtip caused an increased rate of spreading of the virus. 4. When the stem was slightly wounded without removal of plant-parts the same result was obtained and appearently it was the injury that influenced virus-transportation. 5. Microscopical examination of the wound-reactions in Nicotiana tabacum revealed a suberin-coating of cells during the first 48 hours after wounding. 6. It became evident that wounding of the stem of a plant exercised an influence on the rate of virus-transport only when the wound was inflicted 24 hours before, simultaneously with or up to 48 hours after inoculation of a middle leaf. When the injury was made more than 24 hours before or more than 48 hours after inoculation, no influence was perceptible. Probably in the former case wound-reactions had already stopped and, in the latter, the virus-material had already extended too far. From our experiments it became apparent that 48 hour-incubation of virusmaterial within a leaf is required before it comes out of the leaf. Then it may be concluded that wounded cells attract virus-material coming out of the inoculated leaf during a period lasting from the moment of wounding up to 24 + 48 i.e. 72 hours after that moment. If the virus-material reached the attraction-sphere of the wound 96 hours after injuring, no influence was perceptible. Optimal attraction by the wounded cells occurred from 24 to 48 hours after wounding. 7. The influence of wounding was also studied in leaves. One part of a leaf was inoculated, another part was wounded slightly at different times in relation to the moment of inoculation. The leaves were cut into sections 20, 22 or 24 hours after inoculation and these strips were kept on a fluid medium for four days in order to allow the virus-material present at the moment of cutting to multiply. Virus-material was transported quickly to the wounded region only when the injuries were made during a period from 48 hours before to simultaneously with the moment of inoculation. As soon as 18 hours after inoculation virus-material begins to move out of the inoculated region of the leaf. So virus-material seemed to be attracted by wounds 48 + 18, 24 + 18 and 18 hours after wounding, i.e. during a period from 18 to 66 hours after injuring. The attraction was optimal 42 hours after wounding. All effects of wounding of leaves seemed to support the conclusion drawn from the results of experiments with wounded stems of plants. 8. According to Braun wound-activity occurs in a period from 24 to 96 hours with a maximum within 48 to 72 hours after wounding. This period is called the “conditioning phase”. His results were obtained from experiments with Agrobacterium tumefaciens in wounded tomato plants. The duration of the “conditioning phase” found in these trials agrees with that found in our experiments, in which the effect of wound-activity on the rate of virus-transportation was determined. 9. Another method to investigate the influence of meristems on the rate of virus-transport was elimination of meristem-activity without inflicting a wound. The stemtips and the axillary buds of a tobacco plant were powdered with fusarex (2, 3, 5, 6-tetra-chloro-nitrobenzene), whereupon the plants remained in the original stage of development until ten days after treatment. The action of the meristems was inhibited. When the middle leaf of such a plant was inoculated with TMV a very limited virus-multiplication took place but no virus-transport out of that leaf occurred. Inhibition of meristem-activity and cell-growth diminished virus-multiplication to a great extent and, consequently, the spread of virus through the plant. After ten days the inhibiting action of fusarex was terminated and the plants started growing again. After five weeks, treated plants showed virus-symptoms, many leaves were somewhat deformed or discolored. Probably not only the meristemactivity was inhibited by the fusarex-treatment but also the metabolism of the plant was changed and virus-multiplication seemed to be impossible. 10. A third series of experiments was performed with tobacco callus-tissue into which a prepared meristem or a stemtip was introduced without provoking injuries. The advantages of this method were: a) virus-transport could be studied in homogeneous callus-tissue after lateral inoculation with TMV, b) only one meristem could influence the rate of virus-transport in the callus, which is impossible in a plant in possession of many axillary buds, c) no injuries were inflicted upon the callus-tissue by introduction of a meristem or a stemtip. Though the meristems and stemtips developed into small stems with leaves, no histological connection was found between the callus-tissue and the “grafts”. Thirty days after “grafting” the callus-tissue had formed rootlets and the rate of virus-transport was suddenly increased in comparison with the controls without meristem or stemtip. 11. Anatomical observations demonstrated that the stemtip had induced a differentiation in the callus-tissue, consisting of formation of vascular elements. The callus-tissue was no longer homogeneous. Microscopical observations of homogeneous tissue without stemtip revealed thin places in the cell-walls. Probably these places are responsible for the virus-transport from cell to cell in homogeneous tissue in which virus spreads slowly, about 1 mm a week. Apparently an introduced meristem did not influence the rate of transport in callus-tissue directly, but indirectly by inducing a differentiation of the tissue. The homogeneous callus-mass was changed into a potential plant with a stemtip, vascular tissue and roots allowing transport of material, including virus, in a way, comparable with that of a normal plant. 12. It could not be demonstrated by our experiments that primary meristematic tissue itself attracts virus-material. The directing action mentioned in the literature that has been attributed to topmeristems apparently does not derive from the meristems themselves but from the active young tissues formed by the primary meristematic tissues, such as leaf-primordia, which attract virus. Also mature cells reversed into a juvenile state by wounding exercise an attractive action on virusmaterial reaching the attraction-sphere of the wound during the “conditioning phase”, i.e. during the first 72 hours after injuring.