Expression of Foreign Genes Demonstrates the Effectiveness of Pollen-Mediated Transformation in Zea mays

Abstract

Plant genetic transformation has arguably been the core of plant improvement in recent decades. Efforts have been made to develop in planta transformation systems due to the limitations present in the tissue-culture-based methods. Herein, we report an improved in planta transformation system, and provide the evidence of reporter gene expression in pollen tube, embryos and stable transgenicity of the plants following pollen-mediated plant transformation with optimized sonication treatment of pollen. The results showed that the aeration at 4°C treatment of pollen grains in sucrose prior to sonication significantly improved the pollen viability leading to improved kernel set and transformation efficiency. Scanning electron microscopy observation revealed that the removal of operculum covering pollen pore by ultrasonication might be one of the reasons for the pollen grains to become competent for transformation. Evidences have shown that the eGfp gene was expressed in the pollen tube and embryos, and the Cry1Ac gene was detected in the subsequent T1 and T2 progenies, suggesting the successful transfer of the foreign genes to the recipient plants. The Southern blot analysis of Cry1Ac gene in T2 progenies and PCR-identified Apr gene segregation in T2 seedlings confirmed the stable inheritance of the transgene. The outcome illustrated that the pollen-mediated genetic transformation system can be widely applied in the plant improvement programs with apparent advantages over tissue-culture-based transformation methods.

The Tale of Cotton Plant: From Wild Type to Domestication, Leading to Its Improvement by Genetic Transformation

“…For foreign DNA incorporation, vacuum infiltration or gene gun can be employed [132]. Targeted pollen having genes of interest are transferred to recipient plant at embryo forming stage through the process of pollination [133]. Secondary approach is the process of transformation in recipient plant; the stigma is removed shortly after pollination from style.…”

Opportunities for Innovation in Genetic Transformation of Forest Trees

“…An alternative method is to target pollen grains using methods such as sonication to penetrate pollen apertures in a DNA-containing sucrose solution. Southern analyses demonstrated the successful transfer of transgenes to progenies by pollination in sorghum ( Wang et al, 2007 ), Brassica juncea ( Wang et al, 2008 ), and maize ( Yang et al, 2017 ). Pre-treatment of pollen grains by aeration (e.g., 20 min at 4°C) increased pollen viability, mitigating a common adverse effect of sonication ( Yang et al, 2017 ).…”

“…Southern analyses demonstrated the successful transfer of transgenes to progenies by pollination in sorghum ( Wang et al, 2007 ), Brassica juncea ( Wang et al, 2008 ), and maize ( Yang et al, 2017 ). Pre-treatment of pollen grains by aeration (e.g., 20 min at 4°C) increased pollen viability, mitigating a common adverse effect of sonication ( Yang et al, 2017 ). Zhao et al (2017) have recently investigated an innovative method by which DNA was delivered to pollen via magnetic nanoparticles; stable transformants in cotton, pepper, and pumpkin were generated.…”

Maize Transformation: From Plant Material to the Release of Genetically Modified and Edited Varieties

“…However, the methods remain controversial due to their low frequency or lack of reproducibility. In the case of maize, the aeration of pollen grains at 4°C in sucrose before sonication results in improved transformation ( Yang et al, 2017 ). However, no successful transformation using this method has been published thus far.…”