Optimization of regeneration and Agrobacterium-mediated transformation of Stevia (Stevia rebaudiana Bertoni): a commercially important natural sweetener plant

Abstract

Stevia rebaudiana Bertoni is a commercially important zero calorie natural-sweetener herb which produce sweet compounds known as steviol glycosides. Rising demands of steviol glycosides by food and beverage industries has led to an increase in its cultivation in various countries. Unfortunately, stevia cultivation faces 2–25% yield penalty due to weeds which further adds to its cultivation cost. To resolve this major challenge, Agrobacterium-mediated genetic transformation of in vitro derived stevia-nodal explants using herbicide resistance gene (bar) has been optimized, for the production of stable transgenic stevia plants. Several parameters including explant type, pre-incubation duration, acetosyringone (As) concentration, Agrobacterium cell density, Agro-inoculation duration, co-cultivation duration, selection regime and plant growth regulators (PGRs) combination and concentration, have been successfully optimized. Among the two types of explants used, nodal explants showed a higher regeneration response of 82.85%, with an average of 25 shoots/explant. The best PGRs combination and concentration for shoot-induction, shoot-elongation and root-induction was found to be 6-benzyladenine (1.0 mg l−1) + naphthalene acetic acid (0.5 mg l−1), gibberellic acid (1.0 mg l−1), and half-strength MS medium, respectively. The two-step selection (phosphinothricin) regime resulted in an average transformation efficiency of 40.48% with nodal explants. Molecular characterization of putative transformants through PCR, RT-PCR, qRT-PCR and Southern-blot hybridization confirmed the presence, stability, expression as well as copy number of bar gene respectively. Compared to the non-transgenic plants, the T0 transgenic plants successfully tolerated 8 mg l−1 glufosinate ammonium sprays. Thus, the optimized protocol can be useful for the introduction of other genes (inter-kingdom transfer) into stevia genome.

Critical parameters for robust Agrobacterium‐mediated transient transformation and quantitative promoter assays in Catharanthus roseus seedlings

“…We showed that seedling incubation in the dark, both pre‐ and post‐infiltration, were critical for successful transformation, increasing transformation rates by five‐ to 13‐fold. Most Agrobacterium ‐infiltration methods do not include a pre‐infiltration dark incubation (Acanda et al, 2021 ; Cao et al, 2017 ; Lu et al, 2017 ; Salazar‐González et al, 2023 ; Sparkes et al, 2006 ; Taak et al, 2020 ; Zhang et al, 2020 ). Our previous Agro infiltration method included a pre‐infiltration dark incubation based on the Lee and Yang protocol (Lee & Yang, 2006 ).…”

“…To the best of our knowledge, we are the first to experimentally show that an overnight pre-infiltration dark incubation of seedlings significantly enhanced transformation efficiency by seven-to 13-fold. In contrast to pre-infiltration dark incubation, post-infiltration dark incubations followed by light treatment are common but not standardized in Agroinfiltration protocols (Lee & Yang, 2006;Salazar-González et al, 2023;Taak et al, 2020;Zhang et al, 2020); many protocols contain post-infiltration light treatments (Acanda et al, 2021;Cao al., 2017;Lu et al, 2017). The dark encourages Agrobacterium growth (Oberpichler et al, 2008)…”

Identification and Expression Analysis of EPSPS and BAR Families in Cotton

“…At present, the BAR gene was isolated from Streptomyces hygroscopicus in existing transgenic crops resistant to phosphine glufosinate, and it was transferred into the crops to make them resistant to phosphine glufosinate. At present, researchers have studied different crops with BAR gene transfer [30,[57][58][59][60]. BAR gene not only can improve resistance to the herbicide glufosinate but also play a role in the salt stress and flowering of plants.…”

In vitro regeneration and Agrobacterium-mediated genetic transformation of Dragon’s Head plant (Lallemantia iberica)

“…Several studies have demonstrated that cotyledonary node was the most appropriate explant for regeneration 21 – 23 and it performed best for in vitro regeneration or Agrobacterium -mediated transformation in several plant species such as Lupinus albus 24 , Passiflora caerulea L . 25 , Cicer arietinum L. 26 , Lallemantia iberica 6 , Glycine max 27 and Stevia rebaudiana Bertoni 28 .…”