Genetic instability of multiple buds of Pinuscoulteri regenerated from tissue culture

Abstract

Embryo expiants ofPinuscoulteri D. Don. were cultured on agar media (modified from Campbell and Durzan 1975) only supplemented with 2.25 mg•L−1 of benzyl amino purine (BAP). Nuclear DNA contents were measured in cells of the embryo explants, callus, and buds regenerated from these explants, and sand-germinated seedlings. Measurements were made at weekly intervals during 6 weeks of culture. Absolute amounts of cellular DNA were determined with a microspectrophotometer using chicken erythrocytes as an internal standard. Initially, the nuclei had a DNA level between 2C and 4C with a mean of 3C. The sand-grown seedlings remained within this range, with an increased frequency of 4C nuclei in the 6-week seedlings. However, cells from callus and regenerated buds showed a progressive increase in DNA level over time. After 42 days in culture, the buds contained large populations of cells at the 8C level and a considerable number of cells with DNA levels above 8C. In addition, the nuclei showed a considerable increase in chromosomal aberrations including bridges, micronuclei, lagging chromosomes, and fragmentation. Nuclear volume also increased over time in the regenerated buds. It is therefore concluded that culture conditions, perhaps cytokinin content, increased the levels of nuclear DNA. These increased levels could be the result of polyploidy, polyteny, hyperaneuploidy, or a combination of these types of nuclear organization.