| The efficiency of soybean transformation is extremely low. And there was a significant genotype specificity in Agrobacterium-mediated soybean transformation. This study utilized ten soybean cultivars for Agrobacterium-mediated transformation using the cotyledonary node as explants. We screened six cultivars including three high-efficiency and better susceptibility cultivars (Williams, Shennong 9 and Bert) and three low-efficiency and weak susceptibity cultivars (General, Liaodoul6 and Kottman). On the base of these screened cultivars, we investigated influences of plant factors on the Agrobacterium-mediated transformation, and intended to provide theoretical reference for improving the soybean transgenic efficiency.There were significant variations of contents in cotyledonary nodes and cotyledons between the cultivars with different transformation efficiency after germination. Results showed that starch and fat all decreased in cotyledonary nodes, but only starch reduced in cotyledons. Moreover, high-efficiency cultivars presented low starch content and high soluble sugar content in cotyledonary nodes. In addition, There were significant differences in mineral elements content between the cultivars with different transformation efficiency. For example, phosphorus content in high-efficiency cultivars were higher, however, it presented higher potassium, calcium and magnesium contents in low-efficiency cultivars.There were significant changes in endogenous hormones of cotyledonary nodes between the cultivars with different transformation efficiency during the co-cultivation period. It showed that high-efficiency cultivars had higher GA (gibberellin) level, and lower ABA (abscisic acid) level at one day of the co-cultivation. ZR (zeatin riboside) content in high-efficiency cultivars increased rapidly after Agrobacterium infection, and was higher than that in low-efficiency cultivars. High-efficiency cultivars showed a relatively lower IAA (indoleacetic acid) level when compared to low-efficiency cultivars before the third day of the co-cultivation. MeJA (methyl jasmonate) content in low-efficiency cultivars all increased more than 2.5-fold, but high-efficiency cultivars only increased up to 1.5-fold. In addition, MeJA content of low-efficiency cultivars was significantly higher than that of high-efficiency cultivars at 1 day of the co-cultivation.There were significant differences in total phenols content, PAL (phenylalanine ammonia-lyase) activity, PPO (polyphenol oxidase) activity and POD (peroxidase) activity between the cultivars with different transformation efficiency during the co-cultivation period. High-efficiency cultivars showed higher total phenol content and PAL activity in cotyledonary nodes at 1 day and 3 day of the co-cultivation, which can facilitate Agrobacterium infection. At 3 day and 5 day of the co-cultivation, low-efficiency cultivars showed higher PPO activity and POD activity, which increased the defense responses to Agrobacterium infection.The microscopic observation indicated that cotyledonary node meristems, cell size and cell population increased in all cultivars, especially in high-efficiency cultivar. Williams meristems showed conspicuous growing and strongest DAPI staining fluorescence intensity during the co-cultivation period. The results of the cell nucleus and DNA content showed that the nucleus got bigger in all cultivars at the middle and later stage of the co-cultivation. In addition, DNA content of cell nucleus in high-efficiency cultivars was greater than that in low-efficiency cultivars.In order to clarify the connection between the cuilivars with different transformation efficiency and the genes that participate in hormones metabolism, phenol metabolism, Agrobacterium T-DNA transport and integration process, and cell cycle regulation, our study measured the expression of these genes. The study showed that the expression of hormones metabolism genes showed significant differences between the cultivars with different transformation efficiency, which were consistent with the variation of endogenous hormones. The expression of GA synthetase gene GA20ox2 and ABA catabolic enzymes gene CYP707A2 reached at the highest levels at 1 day of the co-cultivation, and high-efficiency cultivars had a higher expression than low efficiency cultivars. After Agrobacterium infection, the expression of ZR synthetase gene IPT3 and IPT5 in high-efficiency cultivars was increased and higher than that in low-efficiency cultivars. However, the expression of MeJA synthetase gene OPR3 was low in high-efficiency cultivars. The expression of phenol metabolism gene showed significant differences between high-efficiency cultivars and low-efficiency cultivars. AfterAgrobacterium infection, the expression of PPO1 and PRX71 in high-efficiency cultivars was increased and higher than that in low-efficiency cultivars, which was consistent with the variation of PPO and POD activity. The expression of T-DNA nuclear input related gene IMPal and T-DNA intergate related gene HTA1 and Ku80 in high-efficiency cultivars was increased and higher than that in low-efficiency cultivars at 5 day of the co-cultivation. The expression of G1 phase activating gene CYCD3 and S phase specific gene CYCA3 in high-efficiency cultivars was increased and higher than that in low-efficiency cultivars at 3 day and 5 day of the co-cultivation. |
