1994

Puupponen-Pimiä, Riitta

Differentiation in plant cell cultures was studied by tissue culture and genetic approaches. The influence of various environmental factors on differentiation was elucidated using Digitalis lanata protoplasts in the first part of the study. The molecular genetics of differentiation were studied in embryogenic birch (Betula pendula Roth.) cell cultures in the second part. Isolation and culture methods were developed for Digitalis lanata seedling protoplasts. The physiological state and the age of the explant proved to be critical for protoplast yield and viability. Suitable osmotic pressure, high protoplast density in the beginning of culture, gentle enzyme treatment and efficient purification procedures were essential for protoplast regeneration. In addition, the use of agarose as a gelling agent of the medium was an essential factor for the initiation and maintenance of cell division. In optimized conditions the first cell division occurred after three weeks and callus colonies were formed in two months. In order to achieve embryogenesis or organogenesis, the hormone contents of the culture medium were changed. However, abundant root formation was observed in all cultures regardless of the hormones used, whereas structures resembling embryos or small shoots were observed in only a few cases. This result suggests that differentiation occurs much earlier than previously believed, already in the callus induction medium. Thus, early markers are needed in order to identify competent cells for differentiation. The BP8 gene was isolated from a birch genomic library by heterologous hybridization using a carrot embryospecific cDNA as a probe. The BP8 gene was sequenced and compared with the homologous carrot gene, DC8. On the basis of DNA sequence analysis the BP8 gene codes for a protein of molecular weight 53 kDa and it exhibits 52% identity with the DC8 sequence at the amino acid level. On the basis of its structure and homology the BP8 protein belongs to the group of LEA (late embryogenesis abundant) proteins. The promoter regions of the BP8 and DC8 genes contained several homologous sequences. Furthermore, similar regulatory elements were found in both genes. The expression of the BP8 gene was studied in cell cultures of birch representing different developmental stages of somatic embryogenesis. The BP8 gene was shown to be expressed at a low level in proembryogenic cell aggregates. The low expression level of the BP8 gene does not encourage the use of this gene as a molecular marker for embryogenesis in birch cell cultures. The homology between birch and carrot genes suggests, however, that the methods described here can also be used more generally in order to identify early genetic markers for differentiation in other recalcitrant species. A genomic library and six cDNA libraries of different developmental stages of birch somatic embryogenesis were constructed and stored for future investigation of differentiation and development in plant cells.