Genetic transformation of maize (Zea mays L.) using scutellar tissue of immature embryo mediated by Agrobacterium tumefaciens
DOI:
https://doi.org/10.14719/pst.4192Keywords:
Agrobacterium, immature embryo, GUS expressionAbstract
Agrobacterium-mediated transformation emerges as a more promising avenue than the biolistic approach for genetically transforming maize plants. Within our transformation endeavours, we harnessed Agrobacterium tumefaciens strain EHA 105, equipped with the p CAMBIA 1305.1 plant transformation vector. This dynamic combination served as the driving force behind the alteration of both mature seed-derived calli and immature embryo explants. When considering the performance of these explants, immature embryos exhibited superior characteristics and were therefore favoured as the primary target material for transformation. This preference was underscored by their ability to yield plantlets with a remarkable transformation frequency of 24-30 % within the COH(M) 5 maize variety. Critical parameters were uncovered to heighten the success of the transformation process. Notably, collecting immature embryos at the 10-12 days after pollination (DAP) stage, boasting a size of 0.8 - 1.5 mm and subsequently inoculating them after 2 days of Agrobacterium tumefaciens cultivation (with an optical density of 1.0 at 600 nm) emerged as pivotal factors that significantly elevated the transient GUS expression rate. The art of cocultivation was found to be optimally executed through the immersion of explants within the bacterial suspension, while the subsequent washing step utilizing sterile distilled water infused with 150 mg L-1 of cefotaxime and 250 mg L-1 of carbenicillin yielded the most favourable outcomes in terms of transient GUS expression for the COH(M) 5 maize variety, surpassing alternative methods. Moreover, the application of cefotaxime up to 150 mg L-1 and carbenicillin up to 250 mg L-1 proved instrumental in securing the highest frequency of regeneration, underscoring their optimal range. Similarly, when considering the selection agent hygromycin, concentrations of up to 30 mg L-1 surfaced as the ideal range, fostering not only maximum regeneration but also a higher count of shoot formations.
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