Abstract
High transformation efficiency is a prerequisite for study of gene function and molecular breeding. Agrobacterium tumefaciens-mediated transformation is a preferred method in many plants. However, the transformation efficiency in soybean is still low. The objective of this study is to optimize Agrobacterium-mediated transformation in soybean by improving the infection efficiency of Agrobacterium and regeneration efficiency of explants. Firstly, four factors affecting Agrobacterium infection efficiency were investigated by estimation of the rate of GUS transient expression in soybean cotyledonary explants, including Agrobacterium concentrations, soybean explants, Agrobacterium suspension medium, and co-cultivation time. The results showed that an infection efficiency of over 96% was achieved by collecting the Agrobacterium at a concentration of OD650 = 0.6, then using an Agrobacterium suspension medium containing 154.2 mg/L dithiothreitol to infect the half-seed cotyledonary explants (from mature seeds imbibed for 1 day), and co-cultured them for 5 days. The Agrobacterium infection efficiencies for soybean varieties Jack Purple and Tianlong 1 were higher than the other six varieties. Secondly, the rates of shoot elongation were compared among six different concentration combinations of gibberellic acid (GA3) and indole-3-acetic acid (IAA). The shoot elongation rate of 34 and 26% was achieved when using the combination of 1.0 mg/L GA3 and 0.1 mg/L IAA for Jack Purple and Tianlong 1, respectively. This rate was higher than the other five concentration combinations of GA3 and IAA, with an 18 and 11% increase over the original laboratory protocol (a combination of 0.5 mg/L GA3 and 0.1 mg/L IAA), respectively. The transformation efficiency was 7 and 10% for Jack Purple and Tianlong 1 at this optimized hormone concentration combination, respectively, which was 2 and 6% higher than the original protocol, respectively. Finally, GUS histochemical staining, PCR, herbicide (glufosinate) painting, and QuickStix Kit for Liberty Link (bar) were used to verify the positive transgenic plants, and absolute quantification PCR confirmed the exogenous gene existed as one to three copies in the soybean genome. This study provides an improved protocol for Agrobacterium-mediated transformation in soybean and a useful reference to improve the transformation efficiency in other plant species.
Highlights
Soybean [Glycine max (L.)] is one of the most important oil crops and a significant source of protein for food and feed in the world
As the overall transformation efficiency is largely determined by the efficiencies of Agrobacterium infection and explant regeneration, we investigated the key factors affecting Agrobacterium infection efficiency including Agrobacterium concentrations, explants, Agrobacterium suspension medium, co-cultivation time, and soybean varieties (Figure 1, textboxes with red borders), and the major factors affecting the explant regeneration efficiency including different concentration combinations of GA3 and indole-3-acetic acid (IAA) in shoot elongation medium (SEM) as well as adding AgNO3 or Vc in shoot induction medium (SIM) and SEM (Figure 1, textboxes with blue borders)
The half-seed explants from 1-d imbibition were infected by the Agrobacterium suspension medium with or without DTT, and subjected to GUS staining after 5 days of co-cultivation
Summary
Soybean [Glycine max (L.)] is one of the most important oil crops and a significant source of protein for food and feed in the world. Soybean seeds are rich in essential amino acids, and rich in dietary minerals, vitamins, unsaturated fatty acids, and isoflavones, which are implicated as beneficial food for human health (Han et al, 2003). Studies have shown that the new GM crop varieties with important application values must be selected from hundreds, thousands, or even tens of thousands transformation events (Wang et al, 2006). The soybean genome sequence has been released (Schmutz et al, 2010), the functions of most soybean genes are unknown. An efficient and stable genetic transformation method is an important prerequisite to study gene functions in soybean and develop new soybean varieties by molecular breeding
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