Highly efficient Agrobacterium-based transformation system for callus cells of the C3 halophyte Suaeda salsa

Abstract

Genetic manipulation technologies have been limited in the halophyte Suaeda salsa L. due to the lack of an efficient transformation system. Here, we examined factors affecting transformation and developed an efficient transformation system at the cell level using S. salsa hypocotyl as starting material. S. salsa hypocotyl explants from 10-day-old seedlings were precultured for 2 days on a hygromycin (hyg)-free callus induction medium (CIM) and then inoculated with Agrobacterium tumefaciens suspension at a concentration of 0.5 at OD 600 for 5-10 min. After cocultivation with A. tumefaciens for 4 days in the dark, followed by selection on carbenicillin (carb) for 3 days, explants were placed on CIM containing 10 mg l -1 hyg and 500 mg l -1 carb with three to four consecutive subcultures for up to 45 days. b-Glucuronidase assays showed an average transformation frequency of 62.89%. Gene integration was confirmed by polymerase chain reaction analysis and Northern blot analysis. To our knowledge, this is the first study to show Agrobacterium-mediated transformation in the C 3 halophyte S. salsa.

Establishment of a Rapid and Stable Transgenic System by Agrobacterium-Mediated Transformation of the Germination Seeds in Diploid Strawberry.

“…We also con rm that 24-h infection time has maximum transformation e ciency, in consistent with the previous reports in seed transformation [31,32]. Generally, in the conventional leaf disk method, Agrobacterium with concentration at 0.3-0.5 of OD600 has high vigor and is used as an optimal concentration for infection [39], in that a higher Agrobacterium concentration causes harm to explant differentiation including browning even death, and a lower concentration is not enough Agrobacterium for T-DNA integration of explants with lower transformation e ciency. In the present study, we nd that the germination seeds were more tolerant to Agrobacterium tumefaciens, and OD600 of 1.5 is an optimal concentration for highest transformation e ciency (Fig.…”

Establishment of A Rapid and Stable Infected System by Agrobacterium-Mediated Transformation of Germination Seeds in Diploid Strawberry.

“…Second, Agrobacterium concentrations and infection time are also critical to infection efficiency [38], and thereby we further confirm them, finding that the 24-h infection time has maximum infection efficiency, in consistent with the previous reports in seed infection [31,32]. Generally, in the conventional leaf disk method, Agrobacterium with concentration at 0.3-0.5 of OD 600 has high vigor and is used as an optimal concentration for infection [39], in that a higher Agrobacterium concentration causes harm to explant differentiation including browning even death, and a lower concentration is not enough Agrobacterium for T-DNA integration of explants with lower infection efficiency. In the present study, we find that the germination seeds were more tolerant to Agrobacterium tumefaciens, and OD 600 of 1.5 is an optimal concentration for highest infection efficiency (Fig.…”