Optimization of Factors Influencing Microinjection Method for Agrobacterium tumefaciens-Mediated Transformation of Tomato

Abstract

A simple and efficient protocol for Agrobacterium-mediated genetic transformation of tomato was developed using combination of non-tissue culture and micropropagation systems. Initially, ESAM region of 1-day-old germinated tomato seeds were microinjected for one to five times with Agrobacterium inoculums (OD(600) = 0.2-1.0). The germinated seeds were cocultivated in the MS medium fortified with (0-200 mM) acetosyringone and minimal concentrations of (0-20 mg L(-1)) kanamycin, and the antibiotic concentration was doubled during the second round of selection. Bacterial concentration of OD(600) = 0.6 served as an optimal concentration for infection and the transformation efficiency was significantly higher of about 46.28 %. In another set of experiment, an improved and stable regeneration system was adapted for the explants from the selection medium. Four-day-old double cotyledonary nodal explants were excised from the microinjected seedlings and cultured onto the MS medium supplemented with 1.5 mg L(-1) thidiazuron, 1.5 mg L(-1) indole-3-butyric acid, 30 mg L(-1) kanamycin, and 0-1.5 mg L(-1) adenine sulphate. Maximum of 9 out of 13 micropropagated shoots were shown positive to GUS assay. By this technique, the transformation efficiency was increased from 46.28 to 65.90 %. Thus, this paper reports the successful protocol for the mass production of transformants using microinjection and micropropagation techniques.

Agrobacterium-mediated transformation of tomato (Lycopersicon esculentumL. cv. Hezuo 908) with improved efficiency

“…[19] Commonly examined factors include explant type, medium composition and selective agent. Tomato cotyledon and hypocotyl are common explants used in tomato transformation.…”

Piercing and incubation method of in planta transformation producing stable transgenic plants by overexpressing DREB1A gene in tomato (Solanum lycopersicum Mill.)

“…Similarly, acetosyringone (200 lM) have been reported for in planta transformation in lentil (Mahmoudian et al 2002), alfalfa (Weeks et al 2008) and wheat ). Various parameters affecting microinjection in planta transformation in tomato have been optimized and concluded that infection medium fortified with acetosyringone (100 mM) exhibited 28.68 % TE compared to control that gave only 2.6 % TE by microinjecting the 1-day-old germinated seeds (Vinoth et al 2013). Similarly, a recent report has modelled the in planta seed transformation via Agrobacterium in brinjal (Solanum melongena L.) and revealed that infiltration and co-cultivation media fortified with acetosyringone (100 lM) remarkably increased the TE (16.38 %) in brinjal .…”

“…Earlier reports have revealed the significance of soil: vermiculite growth medium for successful seedling establishment in tomato (Khare et al 2010;Vinoth et al 2012;Sinha and Rajam 2013). Vinoth et al (2013) developed an efficient tissue culture independent Agrobacterium-mediated transformation system in tomato and argued that sterilized sand: soil: vermiculite (1:2:1) was proved to be the best growth medium for successful plantlet formation. Similarly, Hazarika and Rajam (2011) raised T 1 transgenic tomato seedlings on soil: vermiculite (1:1) growth medium and reported that soil and vermiculite in equal ratio found efficient in yielding the highest germination frequency ranging from 35 to 88 % in transgenic lines.…”

“…In previous studies, different methods of in planta transformation have been documented in literature such as injection of bacterial culture into meristematic tissues or fruit in tomato (Orzaez et al 2006;Vinoth et al 2013), infection of germinating seeds by Agrobacterium in cotton , dropping of DNA directly onto florets in Arabidopsis thaliana (Tague and Mantis 2006), infection of shoot apical node in alfalfa (Weeks et al 2008) and vaccum infiltration of green leaves (Ben-Amar et al 2013). Among them, infection of germinating seeds have been found as better explants source for efficient in planta transformation strategy in various crops such as Medicago truncatula, radish and brinjal (Trieu et al 2000;Park et al 2005;Subramanyam et al 2013).…”

Generation of Transgenic Maize by Two Germinating Seed Transformation Methods

“…Transformants were obtained by followed herbicide screening and molecular verification of seedlings from the treated seeds. Also transformation has been achieved in rice, tomato and Morinda citrifolia with this and similar methods (Liu et al, 1999;Lee et al, 2013;Vinoth et al, 2013). Electron microscopy analysis of rice embryos has revealed that the internal structure and cell surface of apical growing points go through a series of microscopic and submicroscopic structural changes during soaking; these structural changes were highly favorable for invasion by Agrobacterium (Liu et al, 1999).…”