Molecular aspects of albinism in anther culture derived barley plants.

Haploid cereal plants can be regenerated from single pollen grains via the process of anther culture. Anther culture of cereals is of potential use in crop improvement programmes. One problem associated with anther culture of cereal plants is a high incidence of albino individuals which cannot be used in crop breeding schemes. Albinos derived from barley anther culture (albino pollen plants) are severely pigment deficient and from electron microscopy studies appear to possess plastids that are developmentally arrested at a stage prior to the differentiation of proplastids to mature chloroplasts. The aim of the project has been to investigate some of the molecular aspects of albinism in these individuals. In vitro propagation experiments were carried out to find the conditions necessary to improve the growth and maintenance of albino pollen plants with the objective of producing a continuous supply of albino tissue for molecular analysis. However, use of various media containing organic and inorganic supplements including a number of plant growth regulators failed to improve the growth of albino plants. Southern analysis revealed that four out of the five albino plants studied exhibit ptDNA restriction patterns that are different to that expected from the wild type map of the barley plastid genome due to the alteration or deletion of specific ptDNA fragments. One plant appears to contain a major form of ptDNA that has undergone a deletion event removing 75% of all sequences. This confirms that the albino pollen plants examined in this study contain forms of the plastid genome that have undergone structural alteration. I have termed these variant plastid genomes ptDNAs. Most of the albino plants studied appear to contain heterogenous populations of ptDNAs. One albino barley pollen plant appears to possess an intact plastid genome. For all the albinos studied the overall levels of ptDNA are reduced 5-15 fold compared to the levels found in normal green tissues. Northern analyses revealed that the transcripts from the ptDNA genes rbcL and psbD-psbC do not accumulate or are present in albino tissues at 5-10% the level found in seed-derived green shoots. Levels of the plastid encoded 16S and 23S rRNAs are similarly reduced in albino tissues. Further Northern analysis revealed that the abundance of transcripts from the nuclear genes rbcS and cab are present in most albino plants at 10% the level found in normal green tissues. Southern analysis indicated that the nuclear DNA restriction fragments encompassing the cab and rbcS genes in two albino plants had not been altered or deleted during the anther culture process. Analysis of green pollen plants indicated that they contain ptDNA of apparently normal structure and abundance and accumulate transcripts from plastid genes and nuclear genes encoding chloroplast polypeptides to the same levels found in the leaves of light grown seedlings. These results represent the first determination of the levels of photosynthetic gene expression in both albino and green pollen plants.