Evidence on the clonal versus non-clonal origin of dark green islands in virus infected tobacco leaves

Abstract

The origin of green islands which appear as virus-free areas in a tobacco leaf systemically infected with tobacco mosaic virus, was investigated by means of genetic markers. ‘Twin spots’ are visually identifiable regions of clonally related green homozygous (+/+) and yellow homozygous (Su/Su) tissue in leaves of Nicotiana tabacum var. John William Broadleaf heterozygous (Su/+) for the sulfur mutation. Green island which were totally contained within the genetically green portion of twin spots were excised. The upper epidermis plus the mesophyll layers, and the lower epidermis, were separately cultured, yielding plants of two distinct genetic clones. Green (+/+) plants were regenerated from the mesophyll layers while heterozygous, yellow-green (Su/+) plants were recovered from the lower epidermis. When tissues from the regenerated plants were indexed for virus on leaves of Pinto bean, 38% of the plants derived from both the green (+/+) and the heterozygous (Su/+) tissue of green islands were found to be free of the virus. Based on observations of the extent of green islands, it was calculated that only 0.56% of both plant genotypes would be expected to have been virus free if the event producing a green island involved a clonal origin of cells. Our results support the notion that a diffusible substance may be involved in the origin of green islands.