Abstract
An effective transformation protocol for Dunaliella, a β-carotene producer, was developed using the synergistic mechanism of D-glucose and Acetosyringone on three different Agrobacterium strains (EHA105, GV3101 and LBA4404). In the present study, we investigated the pre-induction of Agrobacterium strains harboring pMDC45 binary vector in TAP media at varying concentrations of D-glucose (5 mM, 10 mM, and 15mM) and 100 μM of Acetosyringone for co-cultivation. Induction of Agrobacterium strains with 10 mM D-glucose and 100 μM Acetosyringone showed higher rates of efficiency compared to other treatments. The presence of GFP and HPT transgenes as a measure of transformation efficiency from the transgenic lines were determined using fluorescent microscopy, PCR, and southern blot analyzes. Highest transformation rate was obtained with the Agrobacterium strain LBA4404 (181 ± 3.78 cfu per 106 cells) followed by GV3101 (128 ± 5.29 cfu per 106 cells) and EHA105 (61 ± 5.03 cfu per 106 cells). However, the Agrobacterium strain GV3101 exhibited more efficient single copy transgene (HPT) transfer into the genome of D. salina than LBA4404. Therefore, future studies dealing with genetic modifications in D. salina can utilize GV3101 as an optimal Agrobacterium strain for gene transfer.
Highlights
Dunaliella salina is a well-established natural producer of β-carotene in which synthesis of the pigment is enhanced under extreme environmental conditions, such as illumination intensity, temperature fluctuations and nutrition
From the understanding of these reports, we developed an efficient transformation protocol for Dunaliella by pre-induction of Agrobacterium strains (EHA105, GV3101and LBA4404) with D-glucose in the presence of AS
The binary vector was successfully transformed into Agrobacterium strains EHA105 (Lane 2 and 3), GV3101 (Lane 4 and 5) and LBA4404 (Lane 6 and 7) using the freeze-thaw method
Summary
Dunaliella salina is a well-established natural producer of β-carotene in which synthesis of the pigment is enhanced under extreme environmental conditions, such as illumination intensity, temperature fluctuations and nutrition (depletion or limitation). Pre-Induced Agrobacterium and Dunaliella Transformation into the algal host. The efficiency of the transformation is found to be low due to two potential reasons: poor induction of Vir genes for T-DNA transfer and species specificity of the Agrobacterium strains. Inducers are compounds which play a crucial role in the activation of the Vir gene which prompts genome-integration resulting in an overall increase in the rate of transformation efficiency [10]. Few investigations have reported Agrobacterium-mediated transformation in microalgae in the presence or absence of phenolic compounds acting as inducers [11, 12] and some studies have investigated pre-induction of Agrobacterium with Acetosyringone (AS) and plant-derived compounds for efficient transformation in microalgae under low pH [9, 13, 14, 8]
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