Genetic transformation of hop (Humulus lupulus L. cv. Tettnanger) by particle bombardment and plant regeneration using a temporary immersion system

Abstract

The efficiency of micropropagation of double-node shoots of hop (Humulus lupulus L. cv. Tettnanger) was evaluated using semi-solid and liquid culture medium in RITA® temporary immersion bioreactors. The highest fresh and dry weight of shoots, average number of shoots, and multiplication rate were obtained using the RITA® system, whereas the longest shoots were obtained on semi-solid medium. Moreover, shoot length was affected significantly by the inoculum density of double-node shoots in RITA® vessels. In addition, the RITA® bioreactors were suitable for shoot induction from organogenic calli. The percentage of shoot induction and the shoot fresh and dry weights were significantly higher in the RITA® system than in semi-solid medium. The age of organogenic calli and inoculum density significantly affected the induction of shoots from organogenic calli. The optimum conditions for DNA delivery into hop organogenic calli using the biolistic particle delivery system were also determined. Organogenic calli were bombarded with the plasmid pSR5-2 (gusA and nptII) varying helium pressure (900, 1,100, or 1,350 psi) and target distance (6, 9, or 12 cm). The highest gusA transient activity was obtained using a pressure of 900 psi and a target distance of 6 cm. For stable genetic transformation, 3-wk-old organogenic calli were bombarded with the plasmid pCAMBIA1303 (gusA, mgfp5, and hpt) using these optimum conditions. Stable gusA expression was observed in organogenic calli and shoots after 4 wk of culture on selection medium containing 2.5 mg l−1 hygromycin. The presence of the mgfp5 gene in the hop genome was confirmed by PCR.