Application Note

pdfCommercial production of hybrid seed in Zea mays L

Hybrid seed production is predominant in modern agriculture because hybrids can improve the characteristics of the resulting plants: such as better yield, greater uniformity, improved color, and disease resistance. This process is referred to as heterosis or hybrid vigor. Hybrid seed is produced by cross-pollinating plants, and to maintain the high purity of hybrid seeds in successive generations, it is critical to eliminate self-pollination.  In maize, a major cause of contamination to the genetic purity of hybrids is the self-pollination of the female parent.

Several methods have been used to prevent self‐pollination of the female inbred parent during maize hybrid seed production, including manual removal of male parts (detasseling) or the development of male-sterility systems. However, detasseling in maize is labor-intensive, and male sterility systems require maintainer lines for fertility restoration so that the male sterile line can be propagated.

RHS2 – a method of inducible male sterility

Roundup® and other glyphosate-based herbicides target an enzyme required for plant survival. Scientists at Monsanto have developed a method for fine control of the glyphosate effect on maize. Glyphosate tolerance is conferred by inserting a transgene expressing a glyphosate insensitive version of the enzyme and then RNA interference (RNAi) is used to turn off this tolerance gene resulting in glyphosate sensitivity. By placing targets within the tolerance gene that are specific to siRNAs expressed only male tissue, the scientists have developed a method (RHS2) of inducible male sterility by application of glyphosate herbicide at late growth stages. The ability to selectively induce male sterility effectively eliminates the need for maintainer lines and removal of male parts for commercial hybrid seed production.

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Dual-utility. CP4 EPSPS protein (produced by the tolerance transgene) is normally accumulated in vegetative and female tissues, providing excellent protection against glyphosate sprays for weed control in the early season, therefore the inbred can be maintained. By contrast, little CP4 EPSPS protein is present in the male reproductive cell. Upon glyphosate sprays at the stages critical for pollen development, male sterility will be induced and the plants can be used as female parent to produce hybrid seeds.

Identification of mts-siRNAs

To identify male tissue-specific small interfering RNAs (mts-siRNAs), the scientists employed a Seq-Array™ strategy where next-gen sequencing was initially performed on various maize tissues to identify the small RNA sequences. Then a selection of the tassel-enriched (putative mts) siRNAs were chosen for further evaluation across a broad group of WT plants using custom microarray technology. LC Sciences’ µParaflo® microarray technology enables massively parallel oligonucleotide and peptide synthesis on a microchip. This technology offers a fast and cost efficient method for the production of small or large batches of microarrays with probe content based on custom sequences curated from next-gen sequencing data. Microarray data from custom chips designed for detection of the selected mts-siRNAs revealed that more than 500 mts-siRNAs exhibited tassel-specific expression in a panel of organs.

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Tassel specificity of 10 representative mts-siRNAs

Germplasm penetrance

It is crucial that the endogenous mts-siRNAs are present across as broad a range of germplasms as possible to achieve optimal germplasm penetrance. The scientists again used microarray to test for the presence of the mts-siRNAs in the tassels of additional representative maize germplasms commonly cultivated in North America.  These microarray experiments indicated that the majority of the mts-siRNAs were present in tassels from the thirty other inbred germplasms tested.

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Presence of the mts-siRNAs A to J in V8 tassels of thirty additional representative maize germplasms. Graphic representation is based on microarray signal intensities.

Selection of commercial transgenic events

The scientists then selected a cDNA sequence EU974548 containing regions rich in potential mts-siRNA targets with perfect or near perfect sequence matches, that has previously been isolated from a maize hybrid via large-scale sequencing of expressed sequence tags. Using PCR, they determined that the EU974548 sequence is present in diverse maize germplasms cultivated in North America, and bioinformatic searches using multiple sequence databases confirmed that the EU974548 sequence is present in representative germplasms cultivated in global regions across the world, indicating the EU974548 sequence is broadly conserved in maize. The conservation of the mts-siRNAs and the EU974548 sequence across a wide range of maize germplasms makes EU974548 ideal for use in transgene regulation. Finally, field testing, trait efficacy trials, and molecular characterizations were then conducted for multiple years in order to select the commercial event MON 87429 that contains the RHS2 trait.

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