Agrobacterium-mediated silencing of caffeine synthesis through root transformation in Camellia sinensis L

Tea [Camellia sinensis (L.) O. Kuntze] is a perennial and most popular non-alcoholic caffeine-containing beverage crop. Tea has several constraints for its genetic improvement such as its high polyphenolic content and woody perennial nature. The development of transgenic tea is very difficult, laborious, and time taking process. In tea, regeneration requires minimum 8-12 months. In view of this, attempt has been made in this article to develop a rapid, efficient, and quite economical Agrobacterium-mediated root transformation system for tea. The feasibility of the developed protocol has been documented through silencing caffeine biosynthesis. For this, one-month-old tea seedlings were exposed to fresh wounding at the elongation zone of roots and were inoculated with Agrobacterium tumefaciens cultures carrying a RNAi construct (pFGC1008-CS). The pFGC1008-CS contained 376 bp of caffeine synthase (CS) cDNA fragment in sense and antisense direction with an intron in between. This has made the RNAi construct to produce a hairpin RNA (ihpRNA). The suppressed expression of CS gene and a marked reduction in caffeine and theobromine contents in young shoots of tea seedlings were obtained after root transformation through Agrobacterium infiltration. Such transformation system could be useful for functional analysis of genes in tea like woody and perennial plants.