A transformation procedure for recalcitrant tomato by addressing transgenic plant‐recovery limiting factors

Abstract

Agrobacterium tumefaciens technology is the battle horse for tomato genetic transformation. However, tomato varieties with low regeneration capacity are very difficult to transform. In the past, tomato transformation through Agrobacterium infection was focused on varieties capable of high regeneration yield, while successful transformation of low regenerable cultivars has not been reported. The genotype response to tissue culture conditions is believed to drive the frequency of regeneration of transgenic plant, whereas the capacity for cell proliferation could determine the transformation efficiency through this technology. The Campbell-28 cultivar is an example of constraints arising from a high morphogenetic potential with low conversion compared to normal plants. In the present work the roles that contribute to improved transgenic plant recovery from this recalcitrant variety were explored for factors like Agrobacterium concentration and antibiotics for bacterial removal and transformant selection. Analysis of the efficiency from independent transformation experiments revealed a more than twofold increase of transformant regeneration after selection on ammonium glufosinate compared to kanamycin selection, showing a transformation efficiency of 21.5%.

Tomato (Solanum lycopersicum L.) in the service of biotechnology

“…The process of plant genetic transformation is very complex, with many factors playing an important role including the application of nurse cells, the addition of acetosyringon to the culture or preculture media, bacterial factors (Agrobacterium strain, culture density) and tissuespecific factors (the genotype and the type of the explants) as well as the plasmid vector, the composition of the culture medium (concentration of fitohormones), the type and concentration of antibiotics, the cocultivation time, etc. (Fuentes et al 2008;Jabeen et al 2009;Sharma et al 2009;El-Siddig et al 2011;Wu et al 2011;Guo et al 2012;Chetty et al 2013;Koul et al 2014). Unfortunately Agrobacterium-mediated transformation is still not suitable for tomato varieties with low regeneration capacity.…”

“…However, some attempts to establish an efficient transformation procedure for such cultivars (e.g. Cambell-28) have been made (Fuentes et al 2008). The development of a system for stable genetic transformation of tomato plastids was a milestone in the transformation of the tomato (Ruf et al 2001).…”

“…There are many reports regarding tomato transformation and in vitro plant regeneration from different explants (including seed-cut cotyledons, hypocotyls, leaves, stem sections, pedicels, petioles and inflorescences) via organogenesis (Khoudi et al 2009;Yasmeen 2009;Goel et al 2011;Koleva Gudeva and Dedejski 2012;Rai et al 2013;Namitha and Negi 2013;Sherkar and Chavan 2014;Wayase and Shitole 2014). These reports also describe the recalcitrance of 'non-competent' tomato explants (partial or total inability to respond to in vitro culture) (Fuentes et al 2008;Mamidala and Nanna 2011).…”

Tomato tolerance to abiotic stress: a review of most often engineered target sequences

“…Despite numerous attempts to improve transformation protocols with regards to effectiveness, progress in this area is limited due to genotype specificity. Notwithstanding this fact, some efforts to determine an effective genetic transformation method for such "stubborn" cultivars were made (Fuentes et al 2008). Agroinfection-mediated modifications utilized both Agrobacterium tumefaciens and A. rhizogenes species.…”

Genetic Transformation in Tomato: Novel Tools to Improve Fruit Quality and Pharmaceutical Production

“…Many factors were believed to be crucial for tomato transformation using Agrobacterium tumefaciens, including the application of nurse cells or acetosyringone to the culture or pre-culture media, the type of explants, the Agrobacterium strain used and its concentration, co-cultivation period and the concentration of thiamine, 6-benzylamino purine (BAP), zeatin and indole acetic acid (IAA). Also, new transformation procedures have been developed for tomato varieties with low in vitro regeneration capacity (Fuentes et al, 2008) and alternative transformation methods, such as floral dip, have been tested (Yasmeen et al, 2009). In addition, novel resources for temporal and tissue-specific manipulation of gene expression in tomato plants are now available for the scientific community.…”