Maize BIG GRAIN1 homolog overexpression increases maize grain yield.

The Zea Mays BIG GRAIN 1 HOMOLOG 1 (ZM-BG1H1) was ectopically expressed in maize. Elite commercial hybrid germplasm was yield tested in diverse field environment locations representing commercial models. Yield was measured in 101 tests across all 4 events, 26 locations over 2 years, for an average yield gain of 355 kg/ha (5.65 bu/ac) above control, with 83% tests broadly showing yield gains (range +2272 kg/ha to -1240 kg/ha), with seven tests gaining more than one metric ton per hectare. Plant and ear height were slightly elevated, and ear and tassel flowering time were delayed one day, but ASI was unchanged, and these traits did not correlate to yield gain. ZM-BG1H1 overexpression is associated with increased ear kernel row number and total ear kernel number and mass, but individual kernels trended slightly smaller and less dense. The ZM-BG1H1 protein is detected in the plasma membrane like rice OS-BG1. Five predominant native ZM-BG1H1 alleles exhibit little structural and expression variation compared to the large increased expression conferred by these ectopic alleles.

Protoplast Isolation and Transfection in Maize.

Protoplast-based transient gene expression platforms can be used to study a range of questions concerning gene regulation. Crucial to the success of these studies is the isolation of large quantities of healthy protoplasts from the tissue of interest. Herein, we describe protocols for isolating and transfecting maize mesophyll protoplasts for gene expression studies. The isolation protocol yields approximately 1.8-1.9 × 107 protoplasts with 80-90% viability from 6 g of etiolated leaf tissue, and the polyethylene glycol-mediated transfection protocol results in 55-58% transfection efficiency. The transfection protocol describes the use of a dual-expression vector that carries the coding sequence for two fluorescent proteins (FPs), one driven by a constitutive promoter for normalization for transfection efficiency and the other driven by the construct of interest. The use of a dual-FP expression vector eliminates the need for co-transfection and separate steps for enzymatic/substrate processing as required for luciferase-based assays. These protocols have been tested on leaf tissue from the maize genotypes B73 and PHR03 and, as written, can be completed in 24 h.