Regulatory approaches for genome edited agricultural plants in select countries and jurisdictions around the world.

Genome editing in agriculture and food is leading to new, improved crops and other products. Depending on the regulatory approach taken in each country or region, commercialization of these crops and products may or may not require approval from the respective regulatory authorities. This paper describes the regulatory landscape governing genome edited agriculture and food products in a selection of countries and regions.

Evolution of GA Metabolic Enzymes in Land Plants.

Gibberellins (GAs) play key roles in various developmental processes in land plants. We studied the evolutionary trends of GA metabolic enzymes through a comprehensive homology search and phylogenetic analyses from bryophytes to angiosperms. Our analyses suggest that, in the process of evolution, plants were able to acquire GA metabolic enzymes in a stepwise manner and that the enzymes had rapidly diversified in angiosperms. As a good example of their rapid diversification, we focused on the GA-deactivating enzyme, GA 2-oxidase (GA2ox). Although the establishment of a GA system first occurred in lycophytes, its inactivation system mediated by GA2oxs was established at a much later time: the rise of gymnosperms and the rise of angiosperms through C19-GA2ox and C20-GA2ox development, respectively, as supported by the results of our direct examination of their enzymatic activities in vitro. Based on these comprehensive studies of GA metabolic enzymes, we discuss here that angiosperms rapidly developed a sophisticated system to delicately control the level of active GAs by increasing their copy numbers for their survival under different challenging environments.

OsIDD2, a zinc finger and INDETERMINATE DOMAIN protein, regulates secondary cell wall formation.

Previously, we found 123 transcription factors (TFs) as candidate regulators of secondary cell wall (SCW) formation in rice by using phylogenetic and co-expression network analyses. Among them, we examined in this work the role of OsIDD2, a zinc finger and indeterminate domain (IDD) family TF. Its overexpressors showed dwarfism, fragile leaves, and decreased lignin content, which are typical phenotypes of plants defective in SCW formation, whereas its knockout plants showed slightly increased lignin content. The RNA-seq and quantitative reverse transcription polymerase chain reaction analyses confirmed that some lignin biosynthetic genes were downregulated in the OsIDD2-overexpressing plants, and revealed the same case for other genes involved in cellulose synthesis and sucrose metabolism. The transient expression assay using rice protoplasts revealed that OsIDD2 negatively regulates the transcription of genes involved in lignin biosynthesis, cinnamyl alcohol dehydrogenase 2 and 3 (CAD2 and 3), and sucrose metabolism, sucrose synthase 5 (SUS5), whereas an AlphaScreen assay, which can detect the interaction between TFs and their target DNA sequences, directly confirmed the interaction between OsIDD2 and the target sequences located in the promoter regions of CAD2 and CAD3. Based on these observations, we conclude that OsIDD2 is negatively involved in SCW formation and other biological events by downregulating its target genes.

Engineering the lodging resistance mechanism of post-Green Revolution rice to meet future demands.

Traditional breeding for high-yielding rice has been dependent on the widespread cultivation of gibberellin (GA)-deficient semi-dwarf varieties. Dwarfism lowers the "center of gravity" of the plant body, which increases resistance against lodging and enables plants to support high grain yield. Although this approach was successful in latter half of the 20th century in rice and wheat breeding, this may no longer be enough to sustain rice with even higher yields. This is because relying solely on the semi-dwarf trait is subject to certain limitations, making it necessary to use other important traits to reinforce it. In this review, we present an alternative approach to increase lodging resistance by improving the quality of the culm by identifying genes related to culm quality and introducing these genes into high-yielding rice cultivars through molecular breeding technique.

Comprehensive gene expression analysis of rice aleurone cells: probing the existence of an alternative gibberellin receptor.

Current gibberellin (GA) research indicates that GA must be perceived in plant nuclei by its cognate receptor, GIBBERELLIN INSENSITIVE DWARF1 (GID1). Recognition of GA by GID1 relieves the repression mediated by the DELLA protein, a model known as the GID1-DELLA GA perception system. There have been reports of potential GA-binding proteins in the plasma membrane that perceive GA and induce α-amylase expression in cereal aleurone cells, which is mechanistically different from the GID1-DELLA system. Therefore, we examined the expression of the rice (Oryza sativa) α-amylase genes in rice mutants impaired in the GA receptor (gid1) and the DELLA repressor (slender rice1; slr1) and confirmed their lack of response to GA in gid1 mutants and constitutive expression in slr1 mutants. We also examined the expression of GA-regulated genes by genome-wide microarray and quantitative reverse transcription-polymerase chain reaction analyses and confirmed that all GA-regulated genes are modulated by the GID1-DELLA system. Furthermore, we studied the regulatory network involved in GA signaling by using a set of mutants defective in genes involved in GA perception and gene expression, namely gid1, slr1, gid2 (a GA-related F-box protein mutant), and gamyb (a GA-related trans-acting factor mutant). Almost all GA up-regulated genes were regulated by the four named GA-signaling components. On the other hand, GA down-regulated genes showed different expression patterns with respect to GID2 and GAMYB (e.g. a considerable number of genes are not controlled by GAMYB or GID2 and GAMYB). Based on these observations, we present a comprehensive discussion of the intricate network of GA-regulated genes in rice aleurone cells.

Genome-wide association study identifies a gene responsible for temperature-dependent rice germination.

Environment is an important determinant of agricultural productivity; therefore, crops have been bred with traits adapted to their environment. It is assumed that the physiology of seed germination is optimised for various climatic conditions. Here, to understand the genetic basis underlying seed germination, we conduct a genome-wide association study considering genotype-by-environment interactions on the germination rate of Japanese rice cultivars under different temperature conditions. We find that a 4 bp InDel in one of the 14-3-3 family genes, GF14h, preferentially changes the germination rate of rice under optimum temperature conditions. The GF14h protein constitutes a transcriptional regulatory module with a bZIP-type transcription factor, OREB1, and a florigen-like protein, MOTHER OF FT AND TFL 2, to control the germination rate by regulating abscisic acid (ABA)-responsive genes. The GF14h loss-of-function allele enhances ABA signalling and reduces the germination rate. This allele is found in rice varieties grown in the northern area and in modern cultivars of Japan and China, suggesting that it contributes to the geographical adaptation of rice. This study demonstrates the complicated molecular system involved in the regulation of seed germination in response to temperature, which has allowed rice to be grown in various geographical locations.

Enabling Trade in Gene-Edited Produce in Asia and Australasia: The Developing Regulatory Landscape and Future Perspectives.

Genome- or gene-editing (abbreviated here as 'GEd') presents great opportunities for crop improvement. This is especially so for the countries in the Asia-Pacific region, which is home to more than half of the world's growing population. A brief description of the science of gene-editing is provided with examples of GEd products. For the benefits of GEd technologies to be realized, international policy and regulatory environments must be clarified, otherwise non-tariff trade barriers will result. The status of regulations that relate to GEd crop products in Asian countries and Australasia are described, together with relevant definitions and responsible regulatory bodies. The regulatory landscape is changing rapidly: in some countries, the regulations are clear, in others they are developing, and some countries have yet to develop appropriate policies. There is clearly a need for the harmonization or alignment of GEd regulations in the region: this will promote the path-to-market and enable the benefits of GEd technologies to reach the end-users.

Development and characterization of GR2E Golden rice introgression lines.

Golden Rice with ß-carotene in the grain helps to address the problem of vitamin A deficiency. Prior to commercialize Golden Rice, several performance and regulatory checkpoints must be achieved. We report results of marker assisted backcross breeding of the GR2E trait into three popular rice varieties followed by a series of confined field tests of event GR2E introgression lines to assess their agronomic performance and carotenoid expression. Results from confined tests in the Philippines and Bangladesh have shown that GR2E introgression lines matched the performance of the recurrent parents for agronomic and yield performance, and the key components of grain quality. Moreover, no differences were observed in terms of pest and disease reaction. The best performing lines identified in each genetic background had significant amounts of carotenoids in the milled grains. These lines can supply 30-50% of the estimated average requirements of vitamin A.

Compositional Analysis of Genetically Engineered GR2E "Golden Rice" in Comparison to That of Conventional Rice.

Compositional analyses were performed on samples of rice grain, straw, and derived bran obtained from golden rice event GR2E and near-isogenic control PSBRc82 rice grown at four locations in the Philippines during 2015 and 2016. Grain samples were analyzed for key nutritional components, including proximates, fiber, polysaccharides, fatty acids, amino acids, minerals, vitamins, and antinutrients. Samples of straw and bran were analyzed for proximates and minerals. The only biologically meaningful difference between GR2E and control rice was in levels of ß-carotene and other provitamin A carotenoids in the grain. Except for ß-carotene and related carotenoids, the compositional parameters of GR2E rice were within the range of natural variability of those components in conventional rice varieties with a history of safe consumption. Mean provitamin A concentrations in milled rice of GR2E can contribute up to 89-113% and 57-99% of the estimated average requirement for vitamin A for preschool children in Bangladesh and the Philippines, respectively.

New path towards a better rice architecture.

New plant type (NPT) or ideal plant architecture (IPA) is an attractive way of increasing yield potential by promoting high resource use efficiency combined with better lodging resistance. In a recent paper in Cell Research, Wang et al. describe how a QTL they identified could bring about the desired NPT architecture by elucidating the role of its encoded gene in controlling the stability of IPA1/OsSPL14, a previously reported NPT protein, in the context of ubiquitination.

Molecular Breeding of Sorghum bicolor, A Novel Energy Crop.

Currently, molecular breeding is regarded as an important tool for the improvement of many crop species. However, in sorghum, recently heralded as an important bioenergy crop, progress in this field has been relatively slow and limited. In this review, we present existing efforts targeted at genetic characterization of sorghum mutants. We also comprehensively review the different attempts made toward the isolation of genes involved in agronomically important traits, including the dissection of some sorghum quantitative trait loci (QTLs). We also explore the current status of the use of transgenic techniques in sorghum, which should be crucial for advancing sorghum molecular breeding. Through this report, we provide a useful benchmark to help assess how much more sorghum genomics and molecular breeding could be improved.

Isolation of a novel lodging resistance QTL gene involved in strigolactone signaling and its pyramiding with a QTL gene involved in another mechanism.

Lodging has been a major roadblock to attaining increased crop productivity. In an attempt to understand the mechanism for culm strength in rice, we isolated an effective quantitative trait locus (QTL), STRONG CULM3 (SCM3), the causal gene of which is identical to rice TEOSINTE BRANCHED1 (OsTB1), a gene previously reported to positively control strigolactone (SL) signaling. A near-isogenic line (NIL) carrying SCM3 showed enhanced culm strength and increased spikelet number despite the expected decrease in tiller number, indicating that SL also has a positive role in enhancing culm strength and spikelet number. We produced a pyramiding line carrying SCM3 and SCM2, another QTL encoding APO1 involved in panicle development. The NIL-SCM2+SCM3 showed a much stronger culm than NIL-SCM2 and NIL-SCM3 and an increased spikelet number caused by the additive effect of these QTLs. We discuss the importance of utilizing suitable alleles of these STRONG CULM QTLs without inducing detrimental traits for breeding.

Utilization of stiff culm trait of rice smos1 mutant for increased lodging resistance.

Although the introduction of semi-dwarf trait into rice has led to improved lodging resistance making it capable of supporting high grain yield, lodging still remains a concern when attempting to further increase the grain yield of rice. However, improving the lodging resistance in rice by depending on the semi-dwarf trait alone is possible only up to a certain limit, beyond which other traits may be needed for reinforcement. To search for alternative traits relating to high lodging resistance, we identified 9 rice mutant lines possessing improved culm strength. To evaluate whether such lines can be useful for breeding lodging resistant rice, small organ size1 (smos1) mutant having increased lodging resistance but low tiller number and low grain yield, was chosen as a representative for a breeding trial. smos1 was crossed with ST-4 (from the Stock rice collection of Nagoya University Togo field #4), a cultivar with high tiller number and high grain yield, and from their progeny, LRC1 (lodging resistance candidate-1) was selected. Although the low tiller number trait of smos1 was not fully reversed in LRC1, this was compensated by an increase in grain weight per panicle, thereby resulting in high grain yield per plant. This important attribute of LRC1 was further enhanced by the improved lodging resistance trait inherited from smos1. Such improved lodging resistance in LRC1 and smos1 was revealed to be mainly due to increased culm diameter and culm thickness, which led to a high section modulus (SM) value, a parameter defining the physical strength of the culm. Since smos1 possesses high breaking-type lodging resistance which is different from semi-dwarf plants with high bending-type lodging resistance, an alternative approach of using thick culm lines for the creation of rice with increased lodging resistance is hereby proposed.

Antheridiogen determines sex in ferns via a spatiotemporally split gibberellin synthesis pathway.

Some ferns possess the ability to control their sex ratio to maintain genetic variation in their colony with the aid of antheridiogen pheromones, antheridium (male organ)-inducing compounds that are related to gibberellin. We determined that ferns have evolved an antheridiogen-mediated communication system to produce males by modifying the gibberellin biosynthetic pathway, which is split between two individuals of different developmental stages in the colony. Antheridiogen acts as a bridge between them because it is more readily taken up by prothalli than bioactive gibberellin. The pathway initiates in early-maturing prothalli (gametophytes) within a colony, which produce antheridiogens and secrete them into the environment. After the secreted antheridiogen is absorbed by neighboring late-maturing prothalli, it is modified in to bioactive gibberellin to trigger male organ formation.

Gibberellin deficiency pleiotropically induces culm bending in sorghum: an insight into sorghum semi-dwarf breeding.

Regulation of symmetrical cell growth in the culm is important for proper culm development. So far, the involvement of gibberellin (GA) in this process has not yet been demonstrated in sorghum. Here, we show that GA deficiency resulting from any loss-of-function mutation in four genes (SbCPS1, SbKS1, SbKO1, SbKAO1) involved in the early steps of GA biosynthesis, not only results in severe dwarfism but also in abnormal culm bending. Histological analysis of the bent culm revealed that the intrinsic bending was due to an uneven cell proliferation between the lower and upper sides of culm internodes. GA treatment alleviated the bending and dwarfism in mutants, whereas the GA biosynthesis inhibitor, uniconazole, induced such phenotypes in wild-type plants--both in a concentration-dependent manner, indicating an important role of GA in controlling erectness of the sorghum culm. Finally, we propose that because of the tight relationship between GA deficiency-induced dwarfism and culm bending in sorghum, GA-related mutations have unlikely been selected in the history of sorghum breeding, as could be inferred from previous QTL and association studies on sorghum plant height that did not pinpoint GA-related genes.

Isolation of a novel lodging resistance QTL gene involved in strigolactone signaling and its pyramiding with a QTL gene involved in another mechanism.

Lodging has been a major roadblock to attaining increased crop productivity. In an attempt to understand the mechanism for culm strength in rice, we isolated an effective quantitative trait loci (QTL), STRONG CULM3 (SCM3), the causal gene of which is identical to rice TEOSINTE BRANCHED1 (OsTB1), a gene previously reported to positively control strigolactone (SL) signaling. A near-isogenic line (NIL) carrying SCM3 showed enhanced culm strength and increased spikelet number despite the expected decrease in tiller number, indicating that SL also has a positive role in enhancing culm strength and spikelet number. We produced a pyramiding line carrying SCM3 and SCM2, another QTL encoding APO1 involved in panicle development. The NIL-SCM2+SCM3 showed a much stronger culm than NIL-SCM2 and NIL-SCM3 and an increased spikelet number caused by the additive effect of these QTLs. We discuss the importance of utilizing suitable alleles of these STRONG CULM QTLs without inducing detrimental traits for breeding.