Abstract
Several approaches have recently been adopted to improve Agrobacterium-mediated transformation of maize; however, about eight months of in vitro culture are still required to isolate transgenic plants. Furthermore, genetic transformation of maize depends on immature embryos, which greatly increases costs. Here, we report a method that ensures the competency of an embryogenic callus secondary culture under laboratory conditions for Agrobacterium-mediated transformation. Moreover, pretreatment of the cell wall with a mixed lytic enzyme solution prior to Agrobacterium infection, significantly improved transformation efficiency and stability. Average stable transformation efficiency was approximately 30.39%, with peaks of 94.46%. Expression and phenotypic analysis of the Rsc reporter gene were tested in the T0 generation of transgenic plants. Using this system, we successfully regenerated transgenic maize plantlets within three months of the emergence of the embryogenic callus. Additionally, we reduced somaclonal variation accompanying prolonged culture of maize cells in the dedifferentiated state, thus facilitating the molecular breeding of maize.
Highlights
Genetic transformation has been extensively used for the preparation of molecular research tools and improvements in breeding
Transgenic maize plants were first obtained from protoplasts using an electroporation method [14]; different types of explants were used for transformation, such as protoplast [15,16,17], immature embryos [18,19,20,21,22,23], shoot apices [24,25,26,27], and immature embryo-derived calli [4,28,29]
To determine the transformation level for different stages of maize immature embryos and calli that are produced from such embryos, we first evaluated instantaneous conversion efficiency and conversion rate using different receptor materials and a green fluorescence reporter
Summary
Genetic transformation has been extensively used for the preparation of molecular research tools and improvements in breeding. Stable genetic transformation of major cereal crops, such as specific varieties of maize, rice, wheat, and barley, have changed from being solely laboratory vehicles for basic research to providing new varieties grown in large areas throughout the world [1,2,3,4,5,6,7,8,9,10,11]. They can be used to alter the expression patterns of individual genes in a more precise and predictable manner than conventional breeding. Agrobacterium-mediated transformation is a simple and cheap technology that inserts a lower copy number of transgenes, which helps to minimize gene silencing
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