Development of an efficient Agrobacterium-mediated genetic transformation for an important ornamental plant, Phlox drummondii Hook

In ornamental plants, continuous attempts are being made to introduce new quality traits that have market value. Of various modes of plant propagation that have been followed for production and propagation new traits, the genetic transformation technology has reportedly played an important role in creating novel varieties of ornamentals of rose, dahlia, carnation, lily, tulips, gerbera, etc. Many of transformed varieties of these ornamentals reportedly produce plants that have improved aesthetic traits like flower colour, fragrance, variant floral architecture, and vase life. Phlox drummondii Hook., also known as “Drummond” Phlox, is a prized ornamental among 67 species of the genus Phlox . In the present investigation, an efficient Agrobacterium -mediated genetic transformation protocol was developed to study its impact on floral morphology, an important commercial trait, of P. drummondii . About 3-4-week-old embryogenic calli derived from zygotic embryo on callus regeneration and multiplication medium (CRMM) comprising MS basal medium + 3% Sucrose + BAP (5 µM) + NAA (10 µM) were pre-cultured on shoot regeneration medium (SRM) comprising MS medium with 3% sucrose + BAP (15 µM) + IAA (2.5 mµ M) for 2–3 days. A. tumefaciens strain GV2260 carrying the GUS gene construct (PPZP 200) was grown overnight in YEM medium and calli were infected with bacteria (A ₆₀₀ 0.3–0.5) for 10 min. Infected calli were subsequently co-cultured on the medium SRM + acetosyringone (100 µM) for 2- days. Selection of transformed calli was achieved by transfer of these co-cultured calli on the SRM I (SRM supplemented with kanamycin (30 mg/l) and augmentin (300 mg/l), which allowed the production of 76% transformed shoots. For shoot elongation SRM II MS basal salts + 3%sucrose + BAP 10 µM + augmentin 300 mg + kanamycin (30 mg/l) was used. Rooting of transformed shoots occurred on MS + IAA (7.5 µM). Transformation status of putative transgenics was confirmed by PCR using primers(Supplementary Table No 5) specific for GUS and NPT-II genes, and by Southern hybridization using NPT-II gene as probe. Genetic transformation protocol has been standardized in this ornamental species for the first time and transformants were observed having unique changes in floral architecture hitherto unknown.