Summary: We have produced 22 090 primary transgenic rice plants that carry a T‐DNA insertion, which has resulted in 18 358 fertile lines. Genomic DNA gel‐blot and PCR analyses have shown that approximately 65% of the population contains more than one copy of the inserted T‐DNA. Hygromycin resistance tests revealed that transgenic plants contain an average of 1.4 loci of T‐DNA inserts. Therefore, it can be estimated that approximately 25 700 taggings have been generated. The binary vector used in the insertion contained the promoterless β‐glucuronidase (GUS) reporter gene with an intron and multiple splicing donors and acceptors immediately next to the right border. Therefore, this gene trap vector is able to detect a gene fusion between GUS and an endogenous gene, which is tagged by T‐DNA. Histochemical GUS assays were carried out in the leaves and roots from 5353 lines, mature flowers from 7026 lines, and developing seeds from 1948 lines. The data revealed that 1.6–2.1% of tested organs were GUS‐positive in the tested organs, and that their GUS expression patterns were organ‐ or tissue‐specific or ubiquitous in all parts of the plant. The large population of T‐DNA‐tagged lines will be useful for identifying insertional mutants in various genes and for discovering new genes in rice.

In most crop breeding programs, the rate of yield increment is insufficient to cope with the increased food demand caused by a rapidly expanding global population. In plant breeding, the development of improved crop varieties is limited by the very long crop duration. Given the many phases of crossing, selection, and testing involved in the production of new plant varieties, it can take one or two decades to create a new cultivar. One possible way of alleviating food scarcity problems and increasing food security is to develop improved plant varieties rapidly. Traditional farming methods practiced since quite some time have decreased the genetic variability of crops. To improve agronomic traits associated with yield, quality, and resistance to biotic and abiotic stresses in crop plants, several conventional and molecular approaches have been used, including genetic selection, mutagenic breeding, somaclonal variations, whole-genome sequence-based approaches, physical maps, and functional genomic tools. However, recent advances in genome editing technology using programmable nucleases, clustered regularly interspaced short palindromic repeats (CRISPR), and CRISPR-associated (Cas) proteins have opened the door to a new plant breeding era. Therefore, to increase the efficiency of crop breeding, plant breeders and researchers around the world are using novel strategies such as speed breeding, genome editing tools, and high-throughput phenotyping. In this review, we summarize recent findings on several aspects of crop breeding to describe the evolution of plant breeding practices, from traditional to modern speed breeding combined with genome editing tools, which aim to produce crop generations with desired traits annually.

Significance: Contamination of water and foods with arsenic (As) poses a threat to millions of people worldwide. Because the rice grain is the major source of As intake, reducing the transfer of As from soil to the grain is a pressing public health issue. We found that a member of the Oryza sativa C-type ATP-binding cassette transporter (OsABCC) family, OsABCC1, detoxifies As and reduces the amount of As in the rice grain. OsABCC1 in the upper nodes of rice plants restricts the distribution of As to the grain by sequestering it in the vacuoles of the phloem companion cells of diffuse vascular bundles directly connected to the grain. Our work suggests a strategy for limiting As accumulation in rice grains and thereby reducing human As exposure.

The tapetum, the innermost of four sporophytic layers in the anther wall, comes in direct contact with the developing male gametophyte and is thought to play a crucial role in the development and maturation of microspores. Here, we report the identification of rice (Oryza sativa) Undeveloped Tapetum1 (Udt1), which is required for the differentiation of secondary parietal cells to mature tapetal cells. T-DNA or retrotransposon Tos17 insertions in the Udt1 gene caused male sterility. The anther walls and meiocytes of the mutants were normal during the early premeiosis stage, but their tapeta failed to differentiate and became vacuolated during the meiotic stage. In addition, meiocytes did not develop to microspores, and middle layer degeneration was inhibited. Consequently, the anther locules contained no pollen. The UDT1:green fluorescent protein fusion protein was localized to the nucleus. This, together with its homology with other basic helix-loop-helix proteins, suggests that UDT1 is a transcription factor. DNA microarray analysis identified 958 downregulated and 267 upregulated genes in the udt1-1 anthers, suggesting that Udt1 plays a major role in maintaining tapetum development, starting in early meiosis.

We have previously generated a large pool of T-DNA insertional lines in rice. In this study, we screened those T-DNA pools for rice mutants that had defective chlorophylls. Among the 1,995 lines examined in the T2 generation, 189 showed a chlorophyll-deficient phenotype that segregated as a single recessive locus. Among the mutants, 10 lines were β-glucuronidase (GUS)-positive in the leaves. Line 9-07117 has a T-DNA insertion into the gene that is highly homologous to XANTHA-F in barley and CHLH in Arabidopsis. This OsCHLH gene encodes the largest subunit of the rice Mg-chelatase, a key enzyme in the chlorophyll branch of the tetrapyrrole biosynthetic pathway. In the T2 and T3 generations, the chlorina mutant phenotypes are co-segregated with the T-DNA. We have identified two additional chlorina mutants that have a Tos17 insertion in the OsCHLH gene. Those phenotypes were co-segregated with Tos17 in the progeny. GUS assays and RNA blot analysis showed that expression of the OsCHLH gene is light inducible, while TEM analysis revealed that the thylakoid membrane of the mutant chloroplasts is underdeveloped. The chlorophyll content was very low in the OschlH mutants. This is the first report that T-DNA insertional mutagenesis can be used for functional analysis of rice genes.

Abscisic acid (ABA) is a phytohormone that plays important roles in the regulation of seed dormancy and adaptation to abiotic stresses. Previous work identified OsPYL/RCARs as functional ABA receptors regulating ABA-dependent gene expression in Oryza sativa. OsPYL/RCARs thus are considered to be good candidate genes for improvement of abiotic stress tolerance in crops. This work demonstrates that the cytosolic ABA receptor OsPYL/RCAR5 in O. sativa functions as a positive regulator of abiotic stress-responsive gene expression. The constitutive expression of OsPYL/RCAR5 in rice driven by the Zea mays ubiquitin promoter induced the expression of many stress-responsive genes even under normal growth conditions and resulted in improved drought and salt stress tolerance in rice. However, it slightly reduced plant height under paddy field conditions and severely reduced total seed yield. This suggests that, although exogenous expression of OsPYL/RCAR5 is able to improve abiotic stress tolerance in rice, fine regulation of its expression will be required to avoid deleterious effects on agricultural traits.