THE UTILISATION OF AN EMBRYO BREEDER TECHNIQUE FOR MATURATION OF SOMATIC EMBRYOS

The formation of embryos from somatic tissue may require several transfers to different media before ripe embryos can be harvested. In a semiopen system, where fresh medium is delivered continuously to the cells through a semipermeable membrane, the problem of repeated transfer can be avoided or the number of transfers reduced. Two types of culture vessels were constructed for studying the growth and differentiation of cells on the membrane. Because cells and embryos are stationary, not moving around as in shaking cultures, it is called an embryo breeder (EB) system. This equipment allows culture periods of unlimited duration without transfer of cultured tissues and any kind of changes in nutrient composition during the culture period. In addition, selection of a membrane with an appropriate molecular weight cut-off (MWCO) enables control of molecule transfer through the membrane. The first prototype was made of Teflon® and the second one of polycarbonate. Both EBs are autoclavable and consist of upper and lower compartments separated by a semipermeable membrane. The cells to be cultured are located in the upper compartment and the nutrients are pumped slowly by a tubing pump through the lower compartment. In preliminary experiments, EBs were loaded with carrot somatic cells and globular embryos. During the culture period of 2 to 4 weeks, differentiation of so-called torpedo-stage embryos with clearly visible shoot and root initials could be observed.