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, 36 (9), 1477-1491

Selectable Marker Independent Transformation of Recalcitrant Maize Inbred B73 and Sorghum P898012 Mediated by Morphogenic Regulators BABY BOOM and WUSCHEL2

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Selectable Marker Independent Transformation of Recalcitrant Maize Inbred B73 and Sorghum P898012 Mediated by Morphogenic Regulators BABY BOOM and WUSCHEL2

Muruganantham Mookkan et al. Plant Cell Rep.

Abstract

Discriminatory co-expression of maize BBM and WUS transcriptional factor genes promoted somatic embryogenesis and efficient Agrobacterium -mediated transformation of recalcitrant maize inbred B73 and sorghum P898012 genotypes without use of a selectable marker gene. 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 RAB17M 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.

Keywords: BABY BOOM; Maize; Morphogenic regulators; Sorghum; WUSCHEL2.

Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Fig. 1
Fig. 1
PHP78891 vector. PHP78891 vector comprises four expression cassettes: (1) a RAB17M: CRE; (2) a NOS At:WUS2; (3) UBI M:BBM; and (4) UBI M: GFP. The CRE:WUS2:BBM cassette is bracketed by lox P sites. One lox P site is flanked by Agrobacterium T-DNA right border (RB) and the other flanks the UBI M: GFP cassette within Agrobacterium T-DNA left border (LB)
Fig. 2
Fig. 2
Transient expression of GFP in maize B73 using PHP78891. a AGL1/empty vector control brightfield image and b GFP fluorescence image (negative control). c AGL1/PHP78891 brightfield image and d GFP fluorescence image showing adaxial surface of the scutellum. e AGL1/PHP78891-SBV containing the super-binary vector brightfield image and f AGL1/PHP78891-SBV showing GFP foci on the surface of the scutellum cells. Micrographs taken 3 days post-inoculation
Fig. 3
Fig. 3
Expression of PHP78891 in T0 transgenics of maize B73. Maize B73 AGL1 PHP78891 calli after 3-day desiccation stress calli; comparable a (Brightfield image) and b (GFP image), AGL1 PHP78891-SBV after 3-day desiccation shows higher GFP sector expression frequencies; c (brightfield image) and d (GFP image), homogeneously GFP expressing shoot in regeneration after 3 days desiccation; e (Brightfield image) and f (GFP image), GFP positive roots (right) and wild type (left); g (Brightfield image) and h (GFP image), tassel from PHP7889-SBV event after anthesis with GFP positive anthers; i (Brightfield image) and j (GFP image), isolated pollen showing 1:1 segregation for GFP; k (Brightfield image) and l (GFP image), silk from regenerated plant; m (Brightfield image) and n (GFP image), kernels from regenerated plant; o (Brightfield image) and p (GFP image), kernels from regenerated plant
Fig. 4
Fig. 4
Transient and stable sorghum P898012 Transformation using PHP78891. a Transient expression EHA 101 PHP78891 3-day post-inoculation (GFP image); b Early stage somatic embryos (arrows) 13-day post-inoculation (GFP image). EHA 101 PHP78891 derived events after desiccation shows heterogeneous callus. c Brightfield and GFP image shows GFP positive embryogenic cluster within GFP negative organized callus. d Homogeneous GFP expressing embryogenic callus before desiccation (brightfield image); f corresponding GFP image showing heterogeneous callus after desiccation; g Heterogenous GFP expression in callus during plant regeneration (brightfield image); h corresponding GFP image
Fig. 5
Fig. 5
PCR amplification of ZsGreen and CRE fragments from maize B73 events. Lanes 1, 300 ng NE Biolabs PCR Marker; lane 2, positive control, amplification of ZsGreen (upper band, expected size 594 bp) and Mo CRE (lower band, expected size 227 bp; lane 3, B73 wild-type negative control; lanes 4–8, transformed plants, M-1-1, M-1-2, M-1-5, M-1-6, and M-1-8
Fig. 6
Fig. 6
Southern blot of PHP78891 transformed maize B73 events using a DIG-labeled probe. a EcoRV restriction digest probed for GFP as shown in Fig. 1. b BamHI restriction digest probed for GFP, as shown in Fig. 1. c Stripped BamHI restriction digest shown in b, re-probed for CRE. Lanes 1, DIG-labeled molecular weight ladder III (Roche Diagnostics Corporation, IN, USA); lane 2, nontransformed maize B73; lanes 3–8, PHP78891-transformed B73 events, i.e., M-1-6, M-1-8, MM-2-9, MM-4-1b, MM-4-1F, and MM-5-0, respectively
Fig. 7
Fig. 7
PCR of inheritance and segregation of the GFP transgene in T1 plants generated from self pollinations. Shown are PCR analysis of 14 T1 plants from the MM-4-1F line. Lane 1, molecular weight markers; lane 2, control reaction for GFP marker; lane 3, wild-type nontransformed control; lanes 4–17, T1 plants testing positive for the transgene in lanes 4, 5, 6, 7, 8, 10, 11, 13, 14, and 17; and, negative in lanes 9, 12, 15, and 16
Fig. 8
Fig. 8
Inheritance and segregation of the functional GFP transgene in T1 plants generated from self pollinations. Wild-type control a is negative for GFP expression. Roots from T1 plants from the MM-4-1F line bo are shown in paired brightfield and GFP micrographs corresponding to the PCR results. The T1 plants in bo correlate directly with lanes 3–17 in Fig. 7

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