An approach to standardize transient expression of GUS in sesame (Sesamum indicum L.) using the Sonication-Assisted Agrobacterium tumefaciens-mediated Transformation (SAAT) method

Abstract

Sonication-Assisted Agrobacterium-mediated Transformation (SAAT) dramatically improved Agrobacterium infection efficiency in Sesamum indicum (sesame) cotyledon. Different parameters of this method such as the duration of sonication, Agrobacterium concentration and acetosyringone concentration during co-cultivation was standardized to develop a balanced protocol that gave optimum β-glucuronidase (GUS) expression with minimal tissue damage. Nine seconds was identified as the optimum duration for sonication. Agrobacterium concentration having OD600 nm 1.0 was found not to induce tissue damage. Usage of 100 μM acetosyringone, which facilitates T-DNA transfer by activating the vir gene in Agrobacterium, increased the transient GUS activity. The optimized protocol facilitated 85.56 ± 0.35% transient GUS activity. Mimicking SAAT by increasing the concentration of acetosyringone during the co-cultivation period to 400 μM in the conventional Agrobacterium-mediated transformation gave only 32.22 ± 1.53% GUS activity. The results suggest that not only acetosyringone but also other compounds were released during formation of micro-wound by sonication that increased the infection efficiency.

An efficient protocol for in vitro direct shoot organogenesis of Sesamum indicum L. using cotyledon as explant

“…Sesame is often considered as "The Queen of Oil-Seed" due to its highly nutritious oil for human consumption. Since the yield of sesame is very low compared to the other oil-seed crops of India, a potential increase of productivity demands the prime importance to improve this ancient-most oil-seed crop (Debnath et al 2014). The major constraint to achieve the goal is the inadequate information about sesame regeneration, which is a pre-requisite for this approach.…”

“…Moreover, the regeneration efficacy of sesame is highly genotype dependent (Taskin and Turgut 1997;Yadav et al 2010;Al-Shafeay et al 2011). These factors usually result in failure of obtaining stable transgenic plants using "sonication assisted Agrobacterium-mediated transformation" (SAAT) method in sesame in our previous attempt (Debnath et al 2014). Therefore, it was necessary to standardise a regeneration protocol for the sesame cultivar JK-1 used in this study, using which the improvement strategy of sesame can be designed using the transgenic approach in future research.…”

“…Previously, we have standardised an efficient protocol of gene transfer in sesame using a relatively less known technique named "sonication-assisted Agrobacterium-mediated transformation" (SAAT) (Debnath et al 2014). The optimised protocol facilitated 85.56 ± 0.35% transient GUS activity in sesame cotyledon.…”

Current Research Trends and Prospects for Yield and Quality Improvement in Sesame, an Important Oilseed Crop

“…The genetic transformation (cotyledon explants) method used the intron interrupted Gus gene, with nptII as a selectable marker, with a transformation frequency of 1.01% (Yadav et al, 2010). In another study, sesame (6-day-old cotyledons) was transformed using the sonication-assisted Agrobacterium tumefaciens-mediated method (Debnath et al, 2014). Several authors have produced transgenic sesame with useful traits, such as increased biosynthesis of phenylpropane, improved abiotic stress tolerance, and increased oil content (Chowdhury et al, 2017;Bhunia et al, 2016) (Table 10).…”