Agrobacterium-mediated transformation of safflower and the efficient recovery of transgenic plants via grafting

Abstract

BackgroundSafflower (Carthamus tinctorius L.) is a difficult crop to genetically transform being susceptible to hyperhydration and poor in vitro root formation. In addition to traditional uses safflower has recently emerged as a broadacre platform for the production of transgenic products including modified oils and pharmaceutically active proteins. Despite commercial activities based on the genetic modification of safflower, there is no method available in the public domain describing the transformation of safflower that generates transformed T1 progeny.ResultsAn efficient and reproducible protocol has been developed with a transformation efficiency of 4.8% and 3.1% for S-317 (high oleic acid content) and WT (high linoleic acid content) genotypes respectively. An improved safflower transformation T-DNA vector was developed, including a secreted GFP to allow non-destructive assessment of transgenic shoots. Hyperhydration and necrosis of Agrobacterium-infected cotyledons was effectively controlled by using iota-carrageenan, L-cysteine and ascorbic acid. To overcome poor in vitro root formation for the first time a grafting method was developed for safflower in which ~50% of transgenic shoots develop into mature plants bearing viable transgenic T1 seed. The integration and expression of secreted GFP and hygromycin genes were confirmed by PCR, Southern and Western blot analysis. Southern blot analysis in nine independent lines indicated that 1-7 transgenes were inserted per line and T1 progeny displayed Mendelian inheritance.ConclusionsThis protocol demonstrates significant improvements in both the efficiency and ease of use over existing safflower transformation protocols. This is the first complete method of genetic transformation of safflower that generates stably-transformed plants and progeny, allowing this crop to benefit from modern molecular applications.

Optimization of the conditions for Casuarina cunninghamiana Miq. genetic transformation mediated by Agrobacterium tumefaciens

“…The concentration of A. tumefaciens was also found to be a key factor affecting the transformation frequency (Belide et al 2011;Mishra et al 2013). Previous studies on casuarinas reported that Agrobacterium concentration of OD 600 at 0.1 was used in both C. glauca and A. verticillata transformation (Le et al 1996;Franche et al 1997).…”

Salt and drought stresses in safflower: a review

“…However, in safflower, genetic engineering until now is confined to vector-mediated (Agrobacterium-mediated) transformation via callus-mediated regeneration (Orlikowska et al 1995;Sankara Rao and Rohini 1999) or embryo transformation (Rohini and Sankara Rao 2000). Belide et al (2011) developed a new protocol for transformation with significant improvements in both the efficiency (about 5 %) and simplicity of implementation over existing safflower transformation protocols. Certainly, safflower is a complex crop to genetically engineer, and many studies describe a series of limitations (Orlikowska et al 1995;Sankara Rao and Rohini 1999) like the lack of genotype-dependent regeneration system, the low efficiency of transformation, the hyper-hydration and necrosis of Agrobacterium-infected cotyledons, the growth retardation of shoots, and the poor rooting and low survival following acclimatization of selected shoots.…”

Effects of Media Composition and Auxins on Adventitious Rooting of Bienertia sinuspersici Cuttings

“…Moreover, vegetative propagation is efficient because it reduces the amount of time required for seedling establishment and a large number of healthy living stock plants can be produced rapidly from a small amount of available original plant material. Rooted cuttings can also serve as potential rootstocks for grafting experiments for plant species that are recalcitrant to adventitious root induction process or for multiplication of transgenic shoots [8] [9]. In addition, findings on nutrient requirements and effects of auxins on rooting of B. sinuspersici cuttings may provide valuable insight into factors that influence the in vitro regeneration process.…”