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Eustoma grandiflorum is a popular ornamental flower worldwide. The production of transgenic Eustoma is expected to help in the study and modification of its traits, such as growth, flowering time, flower color, and flower shape. Agrobacterium-mediated transformation of leaf disks is a frequently used method of plant transformation. To improve the transformation efficiency of Eustoma, I focused on two steps: the inoculation of Agrobacterium into the leaf disks, and subsequent cocultivation. The effect of detergent in the inoculation buffer was tested. Given its marginal effect on the viability of the disks, Tween 20 was determined to be a better candidate for use in the buffer. Acetosyringone is known to improve transformation efficiency by up-regulating the virulence of Agrobacterium. In previous studies using Eustoma, the effect of this compound has only been tested during inoculation. However, I studied the effect of acetosyringone by adding it to the inoculation as well as the co-cultivation medium. When added to the co-cultivation medium, acetosyringone significantly increased the number of transformed calli harboring a marker transgene. More than 0.7 callus per leaf disk was obtained by adding acetosyringone, whereas less than 0.03 callus per disks were obtained in its absence. Thus, the mediated incorporation of a transgene into the Eustoma genome can be intensified by the addition of acetosyringone to the co-cultivation medium. Discipline: Horticulture Additional key words: Agrobacterium tumefaciens *Corresponding author: e-mail ynakano@affrc.go.jp Received 3 October 2016; accepted 27 February 2017. Introduction Eustoma grandiflorum, a member of the family Gentianaceae, has become a popular ornamental flower worldwide. However, there are some problems, such as heat-induced rosetting (Ohkawa et al. 1991), reduced rate of flowering under low temperature (Nakano et al. 2011), and flower bud necrosis under low light intensity (Ushio and Fukuta 2010), which reduce the productivity of Eustoma. To study the growth and flowering habits of Eustoma by introducing a gene of interest, it is important to develop an efficient transformation method. Transgenic breeding is employed in ornamental flower plants for altering the flower shape, color, and other properties. In a study aimed at improving the disease resistance in Eustoma by the stable integration of a candidate gene, Isuzugawa et al. (2002) evaluated the factors affecting the transformation of Eustoma. Thiruvengadam and Yang (2009) also reported a transformation method for altering the flowering time and flower shape in Eustoma.

Effect of Acetosyringone on <i>Agrobacterium</i>-mediated Transformation of <i>Eustoma grandiflorum</i> Leaf Disks