Transgenic tea [Camellia sinensis (L.) O. Kuntze cv. Kangra Jat] plants obtained by Agrobacterium-mediated transformation of somatic embryos

Abstract.

A protocol for the production of transgenic tea [Camellia sinensis (L.) O. Kuntze cv. Kangra Jat] was developed via Agrobacterium-mediated genetic transformation of somatic embryos. Two disarmed Agrobacterium tumefaciens strains, EHA 105 and LBA 4404, both carrying the binary plasmid p35SGUSINT with the nptII gene and gus-intron were evaluated as vector systems. A number of parameters were tested with respect to maximizing transformation efficiency. While pre-culture, wounding and acetosyringone treatment were inhibitory, the bacterial growth phase (optical density; OD₆₀₀ = 0.6), cell density (10⁹/ml), co-cultivation period (5 days) and pH of the co-cultivation medium (5.6) had positive effects on transformation. Following co-cultivation, globular somatic embryos were placed on multiplication medium and stressed with kanamycin (50 µg/ml). Further selection occurred in the maturation and germination medium at an elevated kanamycin level (75 µg/ml). An average of 40% transient expression was evident based on the GUS histochemical assay. Kanamycin-resistant, GUS-positive embryos were germinated, and the resulting microshoots were multiplied in vitro. Integration of the transgenes into the tea nuclear genome was confirmed by PCR analysis using nptII- and gus-specific primers and by Southern hybridization using an nptII-specific probe. The transgenic shoots were micrografted onto seed-grown rootstocks of cv. Kangra Jat and eventually hardened in a walk-in polyhouse. This is the first report on the production of transgenic tea.