SummaryMaize ARGOS8 is a negative regulator of ethylene responses. A previous study has shown that transgenic plants constitutively overexpressing ARGOS8 have reduced ethylene sensitivity and improved grain yield under drought stress conditions. To explore the targeted use of ARGOS8 native expression variation in drought‐tolerant breeding, a diverse set of over 400 maize inbreds was examined for ARGOS8 mRNA expression, but the expression levels in all lines were less than that created in the original ARGOS8 transgenic events. We then employed a CRISPR‐Cas‐enabled advanced breeding technology to generate novel variants of ARGOS8. The native maize GOS2 promoter, which confers a moderate level of constitutive expression, was inserted into the 5′‐untranslated region of the native ARGOS8 gene or was used to replace the native promoter of ARGOS8. Precise genomic DNA modification at the ARGOS8 locus was verified by PCR and sequencing. The ARGOS8 variants had elevated levels of ARGOS8 transcripts relative to the native allele and these transcripts were detectable in all the tissues tested, which was the expected results using the GOS2 promoter. A field study showed that compared to the WT, the ARGOS8 variants increased grain yield by five bushels per acre under flowering stress conditions and had no yield loss under well‐watered conditions. These results demonstrate the utility of the CRISPR‐Cas9 system in generating novel allelic variation for breeding drought‐tolerant crops.

Key messageA simple and versatile ternary vector system that utilizes improved accessory plasmids for rapid maize transformation is described. This system facilitates high-throughput vector construction and plant transformation.AbstractThe super binary plasmid pSB1 is a mainstay of maize transformation. However, the large size of the base vector makes it challenging to clone, the process of co-integration is cumbersome and inefficient, and some Agrobacterium strains are known to give rise to spontaneous mutants resistant to tetracycline. These limitations present substantial barriers to high throughput vector construction. Here we describe a smaller, simpler and versatile ternary vector system for maize transformation that utilizes improved accessory plasmids requiring no co-integration step. In addition, the newly described accessory plasmids have restored virulence genes found to be defective in pSB1, as well as added virulence genes. Testing of different configurations of the accessory plasmids in combination with T-DNA binary vector as ternary vectors nearly doubles both the raw transformation frequency and the number of transformation events of usable quality in difficult-to-transform maize inbreds. The newly described ternary vectors enabled the development of a rapid maize transformation method for elite inbreds. This vector system facilitated screening different origins of replication on the accessory plasmid and T-DNA vector, and four combinations were identified that have high (86–103%) raw transformation frequency in an elite maize inbred.Electronic supplementary materialThe online version of this article (10.1007/s11103-018-0732-y) contains supplementary material, which is available to authorized users.

SummaryA transgenic gene-silencing approach was used to modulate the levels of ethylene biosynthesis in maize (Zea mays L.) and determine its effect on grain yield under drought stress in a comprehensive set of field trials. Commercially relevant transgenic events were created with down-regulated ACC synthases (ACSs), enzymes that catalyse the rate-limiting step in ethylene biosynthesis. These events had ethylene emission levels reduced approximately 50% compared with nontransgenic nulls. Multiple, independent transgenic hybrids and controls were tested in field trials at managed drought-stress and rain-fed locations throughout the US. Analysis of yield data indicated that transgenic events had significantly increased grain yield over the null comparators, with the best event having a 0.58 Mg/ha (9.3 bushel/acre) increase after a flowering period drought stress. A (genotype 9 transgene) 9 environment interaction existed among the events, highlighting the need to better understand the context in which the downregulation of ACSs functions in maize. Analysis of secondary traits showed that there was a consistent decrease in the anthesis-silking interval and a concomitant increase in kernel number/ ear in transgene-positive events versus nulls. Selected events were also field tested under a lownitrogen treatment, and the best event was found to have a significant 0.44 Mg/ha (7.1 bushel/ acre) yield increase. This set of extensive field evaluations demonstrated that down-regulating the ethylene biosynthetic pathway can improve the grain yield of maize under abiotic stress conditions.

Increasing maize grain yield has been a major focus of both plant breeding and genetic engineering to meet the global demand for food, feed, and industrial uses. We report that increasing and extending expression of a maize MADS-box transcription factor gene, zmm28, under the control of a moderate-constitutive maize promoter, results in maize plants with increased plant growth, photosynthesis capacity, and nitrogen utilization. Molecular and biochemical characterization of zmm28 transgenic plants demonstrated that their enhanced agronomic traits are associated with elevated plant carbon assimilation, nitrogen utilization, and plant growth. Overall, these positive attributes are associated with a significant increase in grain yield relative to wild-type controls that is consistent across years, environments, and elite germplasm backgrounds.

Nitrogen (N) application in maize (Zea mays L.) reached a maximum of 145 kg N ha−1 in the US Midwest in 1975. Grain yield has continued to increase at a rate of 111 kg ha−1 yr−1, implying an improvement in N efficiency. Our objective was to measure the rate of genetic gain and the traits that contributed to the observed N efficiency for a set of DuPont Pioneer hybrids released between the era decades (ERA) of 1934 to 2013. These hybrids represent the most widely sold hybrids (by volume) in each ERA. A randomized complete block experiment in a split‐plot arrangement was conducted at Sciota, IL, and Marion, IA, during 2013 and 2014, with plant densities of 39,500 and 79,000 plants ha−1 as the whole plot, respectively, and 47 ERA hybrids as the split plot. This experiment was grown in a low‐N (56 kg N ha−1) block and in a high‐N (>200 kg N ha−1) block at each location. Grain yield increased at an average rate of 109 kg ha−1 yr−1 from 1934 to 2013. Partial factor productivity increased from 13.8 in 1934 to 55 kg grain kg applied N−1 in 2013 under high‐N conditions and 79,000 plants ha−1. Traits associated with yield improvement without increasing N application were (i) greater synchrony in floral development, (ii) reduced concentration of grain N, (iii) increased specific leaf nitrogen, (iv) increased kernel number per ear (KPE), and (v) increased kernel mass. Breeding efforts that select for increased KPE under increased plant density should increase yield, and this yield increase could partially be supported through greater postanthesis N remobilization from vegetative tissue without requiring greater N application.