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.
Highlights
Genetic transformation has rapidly advanced in the last three decades, and it entails a wide range of regeneration protocols that are intended to broadly enhance plant growth and productivity of valuable legume crops [1]
A total of 0.4 g of homogenised cotyledonary nodes were mixed with 2 mL of 0.1% trichloroacetic acid (TCA) and centrifuged at 14000 g (28 °C) for 15 min
Data obtained in this study suggested that total chlorophyll content, phenolic content and free radical scavenging activity of cotyledonary nodes were significantly reduced by explants co-cultivation with A. tumefaciens
Summary
Genetic transformation has rapidly advanced in the last three decades, and it entails a wide range of regeneration protocols that are intended to broadly enhance plant growth and productivity of valuable legume crops [1]. Problems caused during explant-Agrobacterium interactions, that includes contamination or bacterial overgrowth, explant browning and senescence, as well as oxidative stress still pose many challenges. These factors are additional to constraints such as inefficient regeneration, genotype specificity and the lack of reproducibility of already tested tissue culture-based genetic transformation protocols as reported [2, 3]. Explant tissue senescence observed as a result of Agrobacterium infection was reported as a serious problem because it causes cell death in tissue culture, lowers shoot regeneration rates and negatively impact on soybean transformation frequency [5]. Further insights on the cause and mechanism of this phenomenon in plant tissue culture and genetic transformation of plants would potentially assist in the optimisation of processes involved during genetic transformation in soybean and other valuable grain or forage crops
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