Keunsub Lee, Minjeong Kang, Qing Ji, Sehiza Grosic, Kan Wang; New T-DNA binary vectors with NptII selection and RUBY reporter for efficient maize transformation

Efficient genetic transformation is a prerequisite for rapid gene functional analyses and crop trait improvements. We recently demonstrated that new T-DNA binary vectors with NptII/G418 selection and a compatible helper plasmid can efficiently transform maize inbred B104 using our rapid Agrobacterium-mediated transformation method. In this work, we implemented the non-integrating Wuschel2 (Wus2) T-DNA vector method for Agrobacterium-mediated B104 transformation and tested its potential for recalcitrant inbred B73 transformation and gene editing. The non-integrating Wus2 (NIW) T-DNA vector-assisted transformation method uses two Agrobacterium strains: one carrying a gene-of-interest (GOI) construct and the other providing an NIW construct. To monitor Wus2 co-integration into the maize genome, we combined the maize Wus2 expression cassette driven by a strong constitutive promoter with a new visible marker RUBY, which produces the purple pigment betalain. As a GOI construct, we used a previously tested CRISPR-Cas9 construct pKL2359 for Glossy2 gene mutagenesis. When both GOI and NIW constructs were delivered by LBA4404Thy- strain, B104 transformation frequency was significantly enhanced by about two-fold (10% vs. 18.8%). Importantly, we were able to transform a recalcitrant inbred B73 using the NIW-assisted transformation method and obtained three transgene-free edited plants by omitting the selection agent G418. These results suggest that NIW-assisted transformation can improve maize B104 transformation frequency and provide a novel option for CRISPR technology for transgene-free genome editing.

Maize genetic transformation is a critical tool for functional genomics and crop improvement. Many laboratories, however, continue to face multiple challenges in attempting to achieve routine genetic transformation of maize inbred genotypes. Here, we describe a rapid and robust maize B104 transformation method using immature embryos as explants. This method uses an Agrobacterium ternary vector system, which includes a conventional T-DNA binary vector (pCBL101-RUBY) and a compatible ternary helper plasmid (pKL2299) that carries extra copies of essential virulence genes. The T-DNA binary vector carries the neomycin phosphotransferase II ( NptII ) gene for selection and a betalain biosynthesis marker, RUBY, for visual screening. We provide step-by-step instructions for immature embryo explant preparation, Agrobacterium infection, tissue culture procedures, and greenhouse care for acclimatization of regenerated plantlets.

Conventional maize transformation has largely relied on immature embryos as explants, and is thus often hampered by the limited access to high-quality immature embryos year-round. Here, we present a detailed protocol using seedling leaf whorls as alternative explants for tropical maize inbred transformation. This approach involves the use of a cassette that drives the expression of the morphogenic transcription factors (MTFs) Baby boom ( Bbm ) and Wuschel2 ( Wus2 ), which have been shown to greatly enhance transformation efficiency. We outline here the steps required for the preparation of seedling leaf whorl explants and subsequent Agrobacterium infection, and describe the tissue culture regimen that results in transgenic plant regeneration. Because constitutive expression of Bbm and Wus2 prevents normal plant regeneration and the production of fertile plants, the cassette containing these genes must be excised. As such, we include the steps for the Cre / loxP -mediated excision of the MTF gene cassette. The protocol outlines a year-round, more affordable, and efficient approach for carrying out maize transformation for crop improvement.

The introduction of maize genetic transformation in the 1990s brought forth a powerful tool for crop improvement and a deeper understanding of plant genetics. Despite decades of genetics research, however, and the promise of CRISPR-mediated gene editing, maize transformation currently faces several challenges, such as genotype dependence and limitations in explant availability. Indeed, although the most commonly used method, immature embryo transformation, has been improved through optimization of tissue culture media composition and selection methods, the approach is only applicable to a limited number of public genotypes, including B104 and Hi II. Recently, genotype-flexible methods have been developed using coexpression cassettes of morphogenic transcription factors (MTFs) Baby boom ( Bbm ) and Wushel2 ( Wus2 ), which have enabled the successful transformation of many previously recalcitrant maize lines. This MTF-based transformation method has also allowed for the use of alternate explants, such as seedling leaf whorl, whose production is cost-effective and requires only minimum controlled growth space. In this review, we summarize recent advances in Agrobacterium -mediated maize transformation methods that use immature embryos or seedling leaf whorls as starting material.

Breeding for stress-tolerant hybrids begins with screening germplasm for tolerant genotypes. We employed a non-destructive and objective method to evaluate the low-temperature response of sweet corns at the seedling stage, using a digital image analysis. It was estimated using summed leaf area, a new parameter defined as the sum of the leaf area measured from images taken at different angles. The summed leaf area, SPAD, shoot and root fresh weight, and total root length were significantly different among hybrids. The group mean of sugary endosperm type was significantly higher than that of shrunken type for all traits but SPAD. For the summed leaf area, the top three ranked hybrids were from the sugary type, but the area for the following three hybrids from the shrunken type did not differ from that for the first three hybrids. The summed leaf area was correlated with SPAD (r = 0.49 **), shoot (r = 0.99 **) and root (r = 0.93 **) fresh weight, and total root length (r = 0.76 **). Phytoglycogen in seeds only had a significant correlation (r = 0.46 **) with the area. The summed leaf area of only one hybrid differed between normal- and low-temperature conditions. The low-temperature response based on the summed leaf area was reflected in the field condition, with a few exceptions. The results suggest that the summed leaf area via digital image analysis can be used to evaluate low-temperature response in sweet corns.