Screening Chinese soybean genotypes for Agrobacterium-mediated genetic transformation suitability

Abstract

The Agrobacterium-mediated transformation system is the most commonly used method in soybean transformation. Screening of soybean genotypes favorable for Agrobacterium-infection and tissue regeneration is the most important step to establish an efficient genetic transformation system. In this study, twenty soybean genotypes that originated from different soybean production regions in China were screened for transient infection, regeneration capacity, and stable transgenic efficiency. Three genotypes, Yuechun 04-5, Yuechun 03-3, and Tianlong 1, showed comparable stable transgenic efficiencies with that of the previously reported American genotypes Williams 82 and Jack in our experimental system. For the Tianlong 1, the average stable transformation efficiency is 4.59%, higher than that of control genotypes (Jack and Williams 82), which is enough for further genomic research and genetic engineering. While polymerase chain reaction (PCR), LibertyLink strips, and β-glucuronidase (GUS) staining assays were used to detect the insertion and expression of the transgene, leaves painted with 135 mg/L Basta could efficiently identify the transformants.

Fig. 1

Map of vector pTF102 P35S/T35S, CaMV 35S promoter/terminator; GUS INT, an intron-containing β-glucuronidase gene; bar, encoding phosphinothricin acetyl transferase; Tvsp, soybean vegetative storage protein terminator; aadA gene, a spectinomycin-resistant marker gene; pBR322, origin of replication for Escherichia coli; pVS1, origin of replication for Agrobacterium; LB, left border; RB, right border; MCS, multiple cloning site. HindIII is one of the unique restriction enzyme sites

Fig. 2

Classification standards of GUS staining (a) Strong, with deep dyeing in the cotyledon node; (b) Weak, with light dyeing in the cotyledon node or cotyledon; (c) None, with no dyeing

Fig. 3

Phenotype of explants after shoot induction (SI) for four weeks (a) Subculture explants with a great of herbicide-resistant multiple buds; (b) Discarded explants with no or very little shoots

Fig. 4

Procedure for confirmation of T₀ transgenic plants (a) PCR analysis of genome DNA of putative transgenic soybean plants using bar gene primers. The length of PCR production was 459 bp. M: DL2000 marker DNA; Ctr−: non-transformed soybean; Ctr+: plasmid DNA; (b, c) Events of the PCR positive lines were randomly selected and tested using LibertyLink strips (b) or GUS staining (c). +: positive transgenic soybean plant; −: negative transgenic soybean plant

Fig. 5

Identification of the herbicide resistance of the leaves of transgenic soybean plants using 135 mg/L Basta Half of a leaf with the black mark is not painted with herbicide, and the other side is painted with herbicide. (a) Non-transgenic plant; (b, c) Positive transgenic plants; (d) Negative transgenic plant

Fig. 6

Agrobacterium-mediated soybean transformation using the cotyledonary node as explants (a) Surface sterilization of soybean seeds; (b) A longitudinal cut between the cotyledons and through the hypocotyl to generate two identical explants; (c) Inoculation of explants with Agrobacterium; (d) Two weeks after selection on shoot induction (SI) medium containing 5 mg/L glufosinate; (e) Four weeks after selection on SI medium containing 5 mg/L glufosinate; (f) Elongated shoot in shoot elongation (SE) medium; (g) Rooting of the resistant shoot; (h) A putative transgenic plantlet was transplanted to pots in the greenhouse

Fig. 7

Southern blot analysis of transgenic soybeans Soybean genomic DNA was sampled from five independent events in the T₁ generation (L1, L2, L3, L4, and L5) and digested with the restriction enzyme HindIII, and hybridized with the bar probe labeled with DIG. A DIG-labeled DNA marker (M) was shown