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Comparative Study
. 2015 Aug 7;16(8):18522-43.
doi: 10.3390/ijms160818522.

Comparison of Soybean Transformation Efficiency and Plant Factors Affecting Transformation during the Agrobacterium Infection Process

Yuying Jia  1 , Xingdong Yao  2 , Mingzhe Zhao  3 , Qiang Zhao  4 , Yanli Du  5 , Cuimei Yu  6 , Futi Xie  7
Affiliations
Comparative Study

Comparison of Soybean Transformation Efficiency and Plant Factors Affecting Transformation during the Agrobacterium Infection Process

Yuying Jia et al. Int J Mol Sci. .

Abstract

The susceptibility of soybean genotype to Agrobacterium infection is a key factor for the high level of genetic transformation efficiency. The objective of this study is to evaluate the plant factors related to transformation in cotyledonary nodes during the Agrobacterium infection process. This study selected three genotypes (Williams 82, Shennong 9 and Bert) with high transformation efficiency, which presented better susceptibility to Agrobacterium infection, and three low transformation efficiency genotypes (General, Liaodou 16 and Kottman), which showed a relatively weak susceptibility. Gibberellin (GA) levels and soybean GA20ox2 and CYP707A2 transcripts of high-efficiency genotypes increased and were higher than those of low-efficiency genotypes; however, the opposite performance was shown in abscisic acid (ABA). Higher zeatin riboside (ZR) content and DNA quantity, and relatively higher expression of soybean IPT5, CYCD3 and CYCA3 were obtained in high-efficiency genotypes. High-efficiency genotypes had low methyl jasmonate (MeJA) content, polyphenol oxidase (PPO) and peroxidase (POD) activity, and relatively lower expression of soybean OPR3, PPO1 and PRX71. GA and ZR were positive plant factors for Agrobacterium-mediated soybean transformation by facilitating germination and growth, and increasing the number of cells in DNA synthesis cycle, respectively; MeJA, PPO, POD and ABA were negative plant factors by inducing defence reactions and repressing germination and growth, respectively.

Keywords: Agrobacterium infection; cotyledonary node; plant factors; soybean; transformation.

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Figures

Figure 1
Figure 1
Cotyledonary node for determination, LibertyLink strip analysis of T0 plants, phenotype of explants after shoot induction, and GUS staining after co-cultivation for five days. (A) Sampling standard of cotyledonary node, numbers on the ruler represent centimeters; (B) Transgenic soybean plants were tested using LibertyLink strips, +: positive T0 plants, −: negative T0 plants; (C) Explants with phosphinothricin-resistant multiple buds; (D) Explants with no buds; (E) Cotyledonary node after GUS staining dyeing; and (F) Cotyledonary node with no GUS staining.
Figure 2
Figure 2
PCR analysis of BAR gene accumulation from different genotypes during co-cultivation period. Abbreviations represent different phases: 0 HAI (0 h after infection), 1 DAC (one day after co-cultivation), 2 DAC (two days after co-cultivation), 3 DAC (three days after co-cultivation), 4 DAC (four days after co-cultivation), and 5 DAC (five days after co-cultivation). M: DL2000 marker.
Figure 3
Figure 3
Comparisons of gibberellin (GA) and abscisic acid (ABA) levels, and the expression levels of genes ralated to GA and ABA metabolism between different genotypes. The values represent means ± SD based on three biological replications. (A) GA content in cotyledonary nodes after 0 h of infection and after 1 day of co-cultivation; (B) qRT-PCR analysis of GA synthetic gene GmGA20ox2; (C) ABA content; and (D) qRT-PCR analysis of ABA catabolic gene GmCYP707A2.
Figure 4
Figure 4
Comparison of zeatin riboside (ZR) levels and the expression levels of ZR synthetic gene between different genotypes. The values represent means ± SD based on three biological replications. (A) ZR content in cotyledonary nodes after 0 h of infection, and after one day, three days, and five days of co-cultivation; (B) qRT-PCR analysis of ZR synthetic gene GmIPT5.
Figure 5
Figure 5
Comparison of the expression levels of cell cyclin-associated genes between different genotypes. The values represent means ± SD based on three biological replications. (A) qRT-PCR analysis of GmCYCD3 in cotyledonary nodes after 0 h of infection, and after one day, three days, and five days of co-cultivation; (B) qRT-PCR analysis of GmCYCA3.
Figure 6
Figure 6
Comparison of nuclear DNA content in meristem cells of cotyledonary nodes between different genotypes during the co-cultivation period. The data represent the means ± SD of all cell nucleus in the cotyledonary node.
Figure 7
Figure 7
Comparison of methyl jasmonate (MeJA) levels and the expression levels of MeJA synthetic gene between different genotypes. The values represent means ± SD based on three biological replications. (A) MeJA content in cotyledonary nodes after 0 h of infection and after one day of co-cultivation; (B) qRT-PCR analysis of MeJA synthetic gene GmOPR3.
Figure 8
Figure 8
Comparison of polyphenol oxidase (PPO) and peroxidase (POD) activity, and the expression of encoding genes between different genotypes. The values represent means ± SD based on three biological replications. (A) PPO activity in cotyledonary nodes after 0 h of infection, and after one day, three days, and five days of co-cultivation; (B) qRT-PCR analysis of the gene GmPPO1 that encodes PPO; (C) POD activity; and (D) qRT-PCR analysis of the gene GmPRX71 that encodes POD.
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