A novel method for high-frequency transgenic shoot regeneration viaAgrobacterium tumefaciensin flax (Linum usitatissimumL.)

Abstract

In this study, routinely used transformation method, which includes transferring explants onto co-cultivation medium after inoculating them with bacterial solution for a while, was compared with 3 different inoculation methods. In every 3 methods, hypocotyl explants excised from 7-day-old sterile flax seedlings having cotyledon leaves and no root system dried under air flow in sterile cabin for 35 min were inoculated with different volumes of bacterial solution at different inoculation periods. GV2260 line of Agrobacterium tumefaciens having 'pBIN 19' plasmid containing npt II (neomycin phosphotransferase II) gene and GUS reporter gene was used in transformation studies. After inoculation, hypocotyl segments of seedlings (0.5 cm in length)-were excised and left to co-cultivation for 2 days. Then, explants were transferred to regeneration medium supplemented with different antibiotics. The presence of npt-II and GUS genes in transformants was confirmed by PCR and GUS analysis. The highest results in all characters examined in all cultivars were obtained from the 2 inoculation method in which hypocotyls excised from seedlings inoculated with 500 µl of bacterial solution after drying in sterile cabin for 35 min were used.

Explant position effect on gene transformation to flax (Linum usitatissimum L.) via Agrobacterium tumefaciens

“…In addition to this protocol, different positions of the hypocotyl region from 7-day-old sterile seedlings were used. That was why the transformation efficiency in our study (93.54%) was found to be higher than the 84.19% in the study by Beyaz et al (5).…”

“…In the study conducted by Beyaz et al (5), the mean transformation efficiency was reported to be 30.45% in the routine inoculation method in which hypocotyl explants excised from 7-day-old sterile flax seedlings were directly inoculated with a 500 μL bacterial solution for 20 minutes. In the newly described inoculation method, the highest mean transformation efficiency was found to be 84.19%.…”

“…In the newly described inoculation method, the highest mean transformation efficiency was found to be 84.19%. In our study, we used the same inoculation method developed by Beyaz et al (5). In addition to this protocol, different positions of the hypocotyl region from 7-day-old sterile seedlings were used.…”

“…All bacterial . Seven-day-old sterile seedlings having cotyledon leaves and no root system were dried with an air flow in a sterile cabin for 35 minutes in order to enable the seedlings to rapidly take in the bacterial solution towards the inner cells by employing the increased osmotic pressure of seedlings at the stage of inoculation, and, in this way, increase the number of cells inoculated and consequently increase the transformation efficiency as reported by Beyaz et al (5). Then, the cut edges of the hypocotyl segment of the seedlings were immersed in liquid regeneration medium with 1 mg L −1 of BAP and 0.02 mg L −1 of NAA for 20 minutes with 500 μL of bacterial solution for inoculation.…”

New Approaches to Agrobacterium tumefaciens-Mediated Gene Transfer to Plants

“…In the study conducted by Beyaz et al [58], the aim was to increase transformation efficiency in flax (Linum usitatissimum L.) by increasing osmotic pressure of the tissue as plant material flax cultivars "Madaras," "Clarck," and "1886 Sel." were used in the study.…”