Efficient production of transgenic Alstroemeria plants by using Agrobacterium tumefaciens

Abstract

A highly efficient and reproducible protocol was developed to obtain transgenic Alstroemeria plants by combining Agrobacterium tumefaciens with friable embryogenic callus (FEC). To develop this transformation method, factors such as infection time, cocultivation period, effect of acetosyringone (AS), different dilution concentrations of the bacterium and temperature during cocultivation were evaluated. A protocol was developed in which transient GUS expression activity was observed ranging from 25% to 55% out of the cocultivated FEC cultures, when FEC cultures were infected for 30 min with 50 lM AS, 1:10 dilution of bacteria, and then cocultivated at 24°C in the dark for 7 days with Agrobacterium strain LBA4404 (pTOK233) that carried gus, nptII and hpt genes. Seven independent experiments produced a total of 1300 transformed somatic embryos with shoots from 3.5 g of FEC. Of these germinated embryos, 50% developed into plants in vitro. Thus, on average, 500 mg of FEC infected with A. tumefaciens produced approximately 80-100 transgenic plants within 6-8 months via a selection process with 2.5-20 mg L 21 hygromycin. Additionally, transformation was also performed with Agrobacterium strain AGL1 (containing the uidA and ppt genes), and this showed that luciferase-based selection was less detrimental to the transgenic lines than was herbicide-based selection. The transformation efficiency was 18.6% for the luciferase-based selection and 7.6% for the PPT-based selection, although with luciferase-based selection, more false positives were obtained (about a quarter of the lines were escapes). The nptII and uidA genes were detected by polymerase chain reaction analysis in nine of the 19 tested lines. The results indicate that the system developed here can be used as an alternative to particle bombardment of Alstroemeria.

Characterization of CYCLOIDEA-like genes in controlling floral zygomorphy in the monocotyledon Alstroemeria

“…Furthermore, molecular mechanisms controlling flower development in this plant species have been studied by analysing class B genes (Hirai et al, 2007) and LEAFY-like gene (Hirai et al, 2012). Additionally, genetic transformation of Alstroemeria has been established by using Agrobacterium tumefaciens (Kim et al, 2007;Hoshino et al, 2008). These research findings will be useful for further analyses of flower development in this plant species.…”

Early GFP gene assessments influencing Agrobacterium tumefaciens-mediated transformation system in Phalaenopsis violacea orchid

“…Low temperature of 24°C for co-cultivation of Agrobacterium has been optimized for other monocots such as Alstromeia plants (Kim et al, 2007) and dicots such as tomato (Ahsan et al, 2007). Low temperatures were suggested to improve pili number on cell surface for better attachment and better functioning of the virB-virD4 (Fullner et al, 1996), hence, the enhancement of transformation.…”

Review on the development of virus resistant plants in Alstroemeria

“…Particle bombardment has been applied in Alstroemeria (Lin et al 2000b), Gladiolus (Kamo et al 1995(Kamo et al , 2000(Kamo et al , 2005, Dendrobium (Kuehnle and Sugii 1992), Lily (Watad et al 1998), and Tulip (Wilmink et al 1995). Agrobacterium-mediated transformation has been applied in Alstroemeria (Akutsu et al 2004a(Akutsu et al , 2004bKim et al 2007;Hoshino et al 2008b), Anthurium (Chen and Kuehele 1996), Cymbidium (Yang et al 1999), Iris (Jeknic et al 1999), and Phalaenopsis (Chai et al 2002;Belarmino and Mii 2000). However, in spite of several successful reports, Agrobacterium-mediated transformation is still cumbersome for quite a number of monocot ornamentals.…”