Pretreatment and posttreatment in the biolistic transformation of tea plant (Camellia sinensis) somatic embryos

Abstract

The tea plant (Camellia sinensis) contains various metabolic substances, including catechins and caffeine, for which genetic transformation techniques are essential for investigating the associated metabolic pathways. In this study, we sought to optimize the conditions and culture process for particle bombardment-mediated transformation of tea plant somatic embryos. We describe somatic embryo pretreatment for effective transient transformation in biolistic bombardment and the posttreatment conditions of somatic embryos for accelerating differentiation after bombardment. For the purpose of transformation, we used the somatic embryos of C. sinensis var. assamica ‘Tingamira normal,’ which were cultured in Murashige and Skoog (MS) medium containing 2 mg l⁻¹ indole-3-butyric acid (IBA) and 4 mg l⁻¹ 6-benzyladenine (BA) at 25°C ±2°C under a 16-h photoperiod. With respect to the optimization of particle bombardment conditions for tea somatic embryos, we examined the effects of different Au colloid particle diameters and bombardment pressures, and accordingly established bombardment with 1.0-µm-diameter DNA-coated Au colloid at 1,100 psi as optimal conditions for introducing DNA for the transient expression of GUS. Additionally, we found that transplantation of tea somatic embryos from IBA/BA medium to a hormone-free medium prior to bombardment and incubation in the dark post-bombardment increased the frequency of secondary embryo production. Furthermore, osmotic treatment by culturing the somatic embryos in medium supplemented with 0.4 M mannitol improved transient transformation efficiency. After transformation, the culture of somatic embryos on filter papers or Kimwipes soaked in MS medium facilitated rapid and effective development of the somatic embryos.