Standardized Genetic Transformation Protocol for Chrysanthemum cv. ‘Jinba’ with TERMINAL FLOWER 1 Homolog CmTFL1a

Abstract

Chrysanthemum (Chrysanthemum x morifolium Ramat.) cultivar Jinba is a distinctive short-day chrysanthemum that can be exploited as a model organism for studying the molecular mechanism of flowering. The commercial value of Jinba can be increased in global flower markets by developing its proper regeneration and genetic transformation system. By addressing typical problems associated with Agrobacterium-mediated transformation in chrysanthemum, that is, low transformation efficiency and high cultivar specificity, we designed an efficient, stable transformation system. Here, we identify the features that significantly affect the genetic transformation of Jinba and standardize its transformation protocol by using CmTFL1a as a transgene. The appropriate concentrations of various antibiotics (kanamycin, meropenem and carbenicillin) and growth regulators (6-BA, 2,4-D and NAA) for the genetic transformation were determined to check their effects on in vitro plant regeneration from leaf segments of Jinba; thus, the transformation protocol was standardized through Agrobacterium tumefaciens (EHA105). In addition, the presence of the transgene and its stable expression in CmTFL1a transgenic plants were confirmed by polymerase chain reaction (PCR) analysis. The CmTFL1a transgene constitutively expressed in the transgenic plants was highly expressed in shoot apices as compared to stem and leaves. Overexpression of CmTFL1a led to a delay in transition to the reproductive phase and significantly affected plant morphology. This study will help to understand the biological phenomenon of TFL1 homolog in chrysanthemum. Moreover, our findings can explore innovative possibilities for genetic engineering and breeding of other chrysanthemum cultivars.