Improved Agrobacterium -mediated transformation of sunflower ( Helianthus annuus L.): assessment of macerating enzymes and sonication

Abstract

Agrobacterium -mediated transformation of shoot apices of sunflower (Helianthus annuus L.) was evaluated following wounding by cell-wall-digesting enzymes and sonication. The frequency of explants with regenerated shoots expressing GUS (beta-glucuronidase) or GFP (green fluorescent protein) increased following treatment with the macerating enzymes cellulase Onozuka R-10 and pectinase Boerozym M5, whereas treatment with macerozyme R-10 had a negative effect. When a combination of cellulase (0.1%) and pectinase (0.05%) was used, the rate of explants with uniformly GUS-positive shoots increased at least twofold. The transient expression of reporter genes was also enhanced using sonication (50 MHz; 2, 4 and 6 s), but stable expression in regenerated shoots following 4 weeks of selection did not increase with this treatment. Enzyme treatment alone (0.1% cellulase and 0.05% pectinase) was superior to a combined treatment of sonication and enzymes with respect to stable transformation. Polymerase chain reaction analyses of shoots recovered by grafting from transformation experiments using GFP as the reporter gene demonstrated the stable integration of the transgene. Regenerated plants were fertile and seeds could be harvested.

Rooting in Km selective media as efficient in vitro selection method for sunflower genetic transformation

“…Thus, the proper selection of a selective gene determines the possibility of success in the production of transgenic plants. Despite several publications in sunflower genetic transformation (Hewezi et al 2002;Weber et al 2003), there is no efficient or reproducible protocol with a low number of escapes, indicating that the selection method using Km is not effective. The aim of this work was to establish the use of the antibiotic Km as an efficient selective agent, trying to relate transgene presence with the differential in vitro behaviour.…”

Suitability of small and branching sunflower varieties for molecular genetic experiments and their transformation by <i>Agrobacterium</i> infection

“…With all of the tested varieties, we were able to obtain small plantlets of normal morphology, including shoots and roots ( Figure 2I-L). While Weber et al (2003) used grafting to obtain seeds from regenerated sunflower plants, no grafting was necessary for the sunflower varieties tested in our study. The regenerated plantlets of Hybrid Sunflower ( Figure 2M), Sonja ( Figure 2N), and Pacino ( Figure 2P) formed normal plants and developed flowers.…”

“…Several attempts have been made to overcome these problems using wounded explants derived from immature seeds (Lucas et al 2000;Hewezi et al 2002Hewezi et al , 2003. Others have attempted to improve shoot regeneration systems from seedling shoot apical meristem explants by using sonication, macerating enzymes and/or wounding methods (Grayburn et al 1995;Scott and Vick 1995;Alibert et al 1999;Weber et al 2003). Weber et al (2000) reported successful shoot regeneration in some interspecific hybrid progenies.…”

Agrobacterium-Mediated Genetic Transformation and Regeneration From Leaf Explants of Solanum Thorvum (Swartz) a Medicinally Important Plant.

“…In Some countries, the highest percentage of the acreage of some economically important crops is transgenic; an increasing number of these transgenic varieties are or will soon be generated by Agrobacterium-mediated transformation (Gelvin, 2003 (Weber et al, 2003). Brassica is also a suitable host for Agrohacterium spp.…”