CHARACTERIZATION OF CHITINASES ABLE TO RESCUE SOMATIC EMBRYOS OF THE TEMPERATURE-SENSITIVE CARROT VARIANT TS11

To characterize the acidic endochitinase EP3, able to rescue somatic e mbryos of the carrot cell line ts11, the enzyme was purified from the medium of wild-type suspension cultures. Peptide sequences, deduced am ino acid sequences of corresponding PCR-generated cDNA clones, serolog ical relation and biochemical properties showed that there were at lea st five closely related chitinases, four of which could be identified as class IV EP3 chitinases with an apparent size of 30 kDa. Two other proteins were identified as a serologically related class I acidic chi tinase (DcChitI) of 34 kDa, and a serologically unrelated 29 kDa class II acidic chitinase (DcChitII), respectively. Additional cDNA sequenc es, Western and Southern analysis showed the presence of a least two, but possibly more, highly homologous class IV EP3 genes in the carrot genome. Two class IV EP3 chitinases were tested and found to be able t o increase the number of ts11 globular embryos formed under non-permis sive conditions. One of the class IV EP3 chitinases as well as the cla ss I chitinase DcChitI promoted the transition from globular to heart- stage ts11 embryos. The class II endochitinase and a heterologous clas s IV chitinase from sugar-beet were not active on ts11. This suggests that there are differences in the specificity of chitinases in terms o f their effect on plant somatic embryos.