Hop (Humulus lupulus L.) is a dioecious perennial climbing plant, mainly known for the use in the brewing industry. Among several diseases that damage hop, the most devastating for hop production are severe hop stunt disease caused by citrus bark cracking viroid (CBCVd) and Verticillium wilt. Developing resistant cultivars are therefore the principal goals of hop breeding programmes. Today, almost all plant breeding programs involve use of new techniques based on advances in biotechnology. With that aim we developed a protocol for isolation of mesophyll protoplasts from hop leaves. Protoplasts are cells with removed cell wall, bounded only by the plasma membrane. They are often used for genetic transformations. In our work, we found that MS medium with vitamins and glucose, without the addition of hormones, is the most suitable for hop growth. The highest yield and viabilty of isolated protoplasts were obtained after one hour plasmolysis in 0.4 M mannitol from 0.4 g leaves digested overnight in 8 ml of 2.0 % cellulase Onozuka R-10 and 0.5% macerozyme R-10 enzymatic solution. Protoplasts were isolated from different hop genotypes and different types of plant material. We used leaves of wild hop, hop grown in greenhouse and under in vitro conditions. The highest number of viable protoplasts (85.6 % viability) were obtained from genotype ‘Celeia’. Wild hop, hop plants grown in greenhouse and genotype ‘Styrian Eureka’ grown under in vitro conditions appeared not suitable plant material for protoplast isolation with used procedure.
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