Selectable marker independent transformation of recalcitrant maize inbred B73 and sorghum P898012 mediated by morphogenic regulators BABY BOOM and WUSCHEL2

Authors

Muruganatham Mookkan
Kimberly Nelson-Vasilchik, University of Rhode Island
Joel Hague, University of Rhode Island
Zhanyuan J. Zhang
Albert P. Kausch, University of Rhode IslandFollow

Document Type

Article

Date of Original Version

2017

Department

Cell & Molecular Biology

Abstract

The use of morphogenic regulators to overcome barriers in plant transformation is a revolutionary breakthrough for basic plant science and crop applications. Current standard plant transformation systems are bottlenecks for genetic, genomic, and crop improvement studies. We investigated the differential use of co-expression of maize transcription factors BABY BOOM and WUSCHEL2 coupled with a desiccation inducible CRE/lox excision system to enable regeneration of stable transgenic recalcitrant maize inbred B73 and sorghum P898012 without a chemical selectable marker. The PHP78891 expression cassette contains CRE driven by the drought inducible maize RAB17_M promoter with lox P sites which bracket the CRE, WUS, and BBM genes. A constitutive maize UBI_M promoter directs a ZsGreen GFP expression cassette as a reporter outside of the excision sites and provides transient, transgenic, and developmental analysis. This was coupled with evidence for molecular integration and analysis of stable integration and desiccation inducible CRE-mediated excision. Agrobacterium-mediated transgenic introduction of this vector showed transient expression of GFP and induced somatic embryogenesis in maize B73 and sorghum P898012 explants. Subjection to desiccation stress in tissue culture enabled the excision of CRE, WUS, and BBM, leaving the UBI_M::GFP cassette and allowing subsequent plant regeneration and GFP expression analysis. Stable GFP expression was observed in the early and late somatic embryos, young shoots, vegetative plant organs, and pollen. Transgene integration and expression of GFP positive T₀ plants were also analyzed using PCR and Southern blots. Progeny segregation analysis of primary events confirmed correlation between functional GFP expression and presence of the GFP transgene in T₁ plants generated from self pollinations, indicating good transgene inheritance. This study confirms and extends the use of morphogenic regulators to overcome transformation barriers.

Citation/Publisher Attribution

Mookkan, M., Nelson-Vasilchik, K., Hague, J. et al. Selectable marker independent transformation of recalcitrant maize inbred B73 and sorghum P898012 mediated by morphogenic regulators BABY BOOM and WUSCHEL2. Plant Cell Rep, 36: 1477.

Available at: http://dx.doi.org/10.1007/s00299-017-2169-1

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