Tissue Culture and Somatic Embryogenesis in Warm-Season Grasses-Current Status and Its Applications: A Review.

Warm-season grasses are C4 plants and have a high capacity for biomass productivity. These grasses are utilized in many agricultural production systems with their greatest value as feeds for livestock, bioethanol, and turf. However, many important warm-season perennial grasses multiply either by vegetative propagation or form their seeds by an asexual mode of reproduction called apomixis. Therefore, the improvement of these grasses by conventional breeding is difficult and is dependent on the availability of natural genetic variation and its manipulation through breeding and selection. Recent studies have indicated that plant tissue culture system through somatic embryogenesis complements and could further develop conventional breeding programs by micropropagation, somaclonal variation, somatic hybridization, genetic transformation, and genome editing. This review summarizes the tissue culture and somatic embryogenesis in warm-season grasses and focus on current status and above applications including the author's progress.

Varietal differences in flavonoid and antioxidant activity in Japanese soybean accessions.

Soybean seeds contain many antioxidants, including flavonoids and other phytochemicals. Isoflavone is a phytoestrogen that mimics estrogenic effects on target tissues and also exerts antioxidant activity by sequestering free radicals. Despite many cultivars developed to date, varietal differences in flavonoid content and antioxidant activity in Japanese soybean accessions remain less well characterized. Here, we evaluated the seed content of isoflavones, total flavonoids, and total phenolics in 26 soybean accessions. Next, the antioxidant activity of each accession was examined using antioxidant responsive element (ARE) linked to a luciferase reporter in human HepG2 stable cells. The relative ARE luciferase activity rate of all soybean accessions varied up to 4-fold which ranged from 1.00 to 4.02; and 22 accessions exhibited significant antioxidant activities. Correlation analysis indicated that the level of total isoflavone moderately correlated with antioxidant activity.

Lotus japonicus Genetic, Mutant, and Germplasm Resources.

A quarter of a century has passed since Lotus japonicus was proposed as a model legume because of its suitability for molecular genetic studies. Since then, a comprehensive set of genetic resources and tools has been developed, including recombinant inbred lines, a collection of wild accessions, published mutant lines, a large collection of mutant lines tagged with LORE1 insertions, cDNA clones with expressed sequence tag (EST) information, genomic clones with end-sequence information, and a reference genome sequence. Resource centers in Japan and Denmark ensure easy access to data and materials, and the resources have greatly facilitated L. japonicus research, thereby contributing to the molecular understanding of characteristic legume features such as endosymbiosis. Here, we provide detailed instructions for L. japonicus cultivation and describe how to order materials and access data using the resource center websites. The comprehensive overview presented here will make L. japonicus more easily accessible as a model system, especially for research groups new to L. japonicus research. © 2018 by John Wiley & Sons, Inc.

Field trial of insect-resistant and herbicide-tolerant genetically modified cotton (Gossypium hirsutum L.) for environmental risk assessment in Japan.

Japan imports cottonseed mainly from Australia and the USA where more than 96% of all cotton varieties grown are genetically modified (GM). GM crops undergo an environmental risk assessment (ERA) under the Law Concerning the Conservation and Sustainable Use of Biological Diversity before import into Japan. Potential adverse effects on biodiversity are comprehensively assessed based on competitiveness, production of harmful substances and outcrossing ability. Even though imported cottonseed is intended for food and feed uses and not for cultivation, the potential risks from seed spillage during transport must be evaluated. In most cases, the ERA requires data collected from in-country field trials to demonstrate how the GM crop behaves in Japan's environment. Confined field trials in Japan were conducted for the ERA of Lepidoptera-resistant and glufosinate-tolerant GM cotton (Gossypium hirsutum L.) lines GHB119 and T304-40. These lines were compared with conventional varieties for growth habit, morphological characteristics, seed dormancy, and allelopathic activity associated with competitiveness and production of harmful substances. Outcrossing ability was not a concern due to the absence of sexually compatible wild relatives in Japan. Although slight statistical differences were observed between the GM line and its conventional comparator for some morphological characteristics, transgenes or transformation were not considered to be responsible for these differences. The trial demonstrated that competitiveness and production of harmful substances by these GM cotton lines were equivalent to conventional cotton varieties that have a long history of safe use, and no potential adverse effects to biosafety in Japan were observed.

Plant regeneration from embryogenic callus derived from shoot apices and production of transgenic plants by particle inflow gun in dwarf napier grass (Pennisetum purpureum Schumach.).

Napier grass (Pennisetum purpureum Schumach.) is a highly productive C4 tropical forage grass that has been targeted as a potential bioenergy crop. To further increase the efficiency of bioethanol production by molecular breeding, a reliable protocol for genetically transforming napier grass is essential. In this study, we report the creation of transgenic napier grass plants derived from embryogenic callus cultures of shoot apices. Embryogenic callus was initiated in three accessions of napier grass and a napier grass×pearl millet hybrid using Murashige and Skoog (MS) medium supplemented with 2.0 mg L-1 2,4-dichlorophenoxyacetic acid (2,4-D), 0.5 mg L-1 6-benzylaminopurine (BAP) and 50 µM copper sulfate (CuSO4). Of the accessions tested, a dwarf type with late-heading (DL line) had the best response for embryogenic callus formation. Highly regenerative calli that formed dense polyembryogenic clusters were selected as target tissues for transformation. A plasmid vector, pAHC25, containing an herbicide-resistance gene (bar) and the ß-glucuronidase (GUS) reporter gene was used in particle bombardment experiments. Target tissues treated with 0.6 M osmoticum were bombarded, and transgenic plants were selected under 5.0 mg L-1 bialaphos selection. Although a total of 1400 target tissues yielded nine GUS-positive bialaphos-resistant calli, only one transgenic line that was derived from target tissue with the shortest culture term produced four transgenic plants. Thus, the length of time that the target tissue is in callus culture was one of the most important factors for acquiring transgenic plants in napier grass. This is the first report of successfully producing transgenic napier grass plants.

Sequencing and comparative analyses of the genomes of zoysiagrasses.

Zoysiais a warm-season turfgrass, which comprises 11 allotetraploid species (2n= 4x= 40), each possessing different morphological and physiological traits. To characterize the genetic systems of Zoysia plants and to analyse their structural and functional differences in individual species and accessions, we sequenced the genomes of Zoysia species using HiSeq and MiSeq platforms. As a reference sequence of Zoysia species, we generated a high-quality draft sequence of the genome of Z. japonica accession 'Nagirizaki' (334 Mb) in which 59,271 protein-coding genes were predicted. In parallel, draft genome sequences of Z. matrella 'Wakaba' and Z. pacifica 'Zanpa' were also generated for comparative analyses. To investigate the genetic diversity among the Zoysia species, genome sequence reads of three additional accessions, Z. japonica'Kyoto', Z. japonica'Miyagi' and Z. matrella'Chiba Fair Green', were accumulated, and aligned against the reference genome of 'Nagirizaki' along with those from 'Wakaba' and 'Zanpa'. As a result, we detected 7,424,163 single-nucleotide polymorphisms and 852,488 short indels among these species. The information obtained in this study will be valuable for basic studies on zoysiagrass evolution and genetics as well as for the breeding of zoysiagrasses, and is made available in the 'Zoysia Genome Database' at http://zoysia.kazusa.or.jp.