Effect of Agrobacterium co-cultivation stage on explant response for subsequent genetic transformation in Soybean (Glycine max (L.) Merr.)

Abstract

The establishment of an efficient in vitro genetic transformation protocol in soybean depends upon an effective interaction between the explants and Agrobacterium tumefaciens. Therefore, a study was conducted at the University of Limpopo, South Africa, between September 2019 and May 2020 to evaluate explant amenability and effects of Agrobacterium co-cultivation stage on the induction of oxidative stress. This stress potentially causes lipid peroxidation, reduction of phytochemicals and chlorophyll pigments on explant tissue targeted for genetic transformation. This study, used double cotyledonary node explants infected and co-cultured with A. tumefaciens to evaluate total phenolics, antioxidant activity, lipid peroxidation and oxidative stress-induced tissue senescence during the co-cultivation stage. The results, showed that, explant co-cultivation with Agrobacterium for 2, 4 and 6 days caused reductions in the amounts of phenolic compounds, chlorophylls and antioxidant activity due to tissue senescence, high oxidative stress and malondialdehyde contents. Percentage phenolic content of all bacteria infected explants ranged between 10.3?10.6 compared to 20.9% in the control. Chlorophyll content of about 1.49?4.00 mg/ml and malondialdehyde content ranging between 1.0?5.7 mM-1g-1 were also recorded. Overall, findings suggest that the infection of explants with A. tumefaciens can induce oxidative stress and tissue senescence depending on the period of co-cultivation. However, reduced oxidative stress and senescence of explant tissues may potentially improve soybean shoot regeneration and transformation efficiency.

Chlorophyll and Protein Changes Observed Post Co-Cultivation of Explants with Agrobacterium Tumefaciens in Soybean (Glycine max L. Merr.)

“…1A). Our previous studies indicated that healthy seedlings, typically with smooth and enlarged surfaces suitable for incisions were obtained using MS basal culture medium containing 1.0-4.0 mgL -1 6-BA (Mangena et al, 2017;Mangena, 2021). Furthermore, the use of a double cotyledonary node and inclusion of cytokinins in the medium showed a high regeneration frequency of multiple shoots, even without co-cultivation of explants with Agrobacterium (Dan and Reichert, 1998;Franklin et al, 2004;Kendir et al, 2008).…”

“…As such, further physiological and biochemical insights emanating from the interaction of A. tumefaciens with cotyledonary explants are still required to guide and improve the optimization of in vitro and in vivo protocols for soybean transformation. Previous studies showed that the infection of cotyledonary explants with Agrobacterium induced oxidative stress causing physiological changes and metabolic imbalances involving the production of ROS and reduced antioxidant activity, both leading to intense damage of plant cellular structures (Mangena et al, 2017;Mangena, 2021). Further insights are thus, required to ameliorate adverse changes in the metabolic profile and ROS accumulations affecting the proliferation of transformed cells by altering the plant's physiological and defense response mechanisms during soybean transformation (Pitzschke, 2013).…”