Takahiro Gondo

Quantitative trait locus analysis of multiple agronomic traits in the model legume Lotus japonicus

The first quantitative trait locus (QTL) analysis of multiple agronomic traits in the model legume Lotus japonicus was performed with a population of recombinant inbred lines derived from Miyakojima MG-20 x Gifu B-129. Thirteen agronomic traits were evaluated in 2004 and 2005: traits of vegetative parts (plant height, stem thickness, leaf length, leaf width, plant regrowth, plant shape, and stem color), flowering traits (flowering time and degree), and pod and seed traits (pod length, pod width, seeds per pod, and seed mass). A total of 40 QTLs were detected that explained 5%-69% of total variation. The QTL that explained the most variation was that for stem color, which was detected in the same region of chromosome 2 in both years. Some QTLs were colocated, especially those for pod and seed traits. Seed mass QTLs were located at 5 locations that mapped to the corresponding genomic positions of equivalent QTLs in soybean, pea, chickpea, and mung bean. This study provides fundamental information for breeding of agronomically important legume crops.

Arabidopsis galactinol synthase AtGolS2 improves drought tolerance in the monocot model Brachypodium distachyon

Brachypodium distachyon (purple false brome) is a herbaceous species belonging to the grass subfamily Pooideae, which also includes major crops like wheat, barley, oat and rye. The species has been established as experimental model organism for understanding and improving cereal crops and temperate grasses. The complete genome of Bd21, the community standard line of B. distachyon, has been sequenced and protocols for Agrobacterium-mediated transformation have been published. Further improvements to the experimental platform including better evaluation systems for transgenic plants are still needed. Here we describe the growth conditions for Bd21 plants yielding highly responsive immature embryos that can generate embryogenic calli for transformation. A prolonged 20-h photoperiod produced seeds with superior immature embryos. In addition, osmotic treatment of embryogenic calli enhanced the efficiency of transfection by particle bombardment. We generated transgenic plants expressing Arabidopsis thaliana galactinol synthase 2 (AtGolS2) in these experiments. AtGolS2-expressing transgenics displayed significantly improved drought tolerance, increasing with increased expression of AtGolS2. These results demonstrate that AtGolS2 can confer drought tolerance to monocots and confirm that Brachypodium is a useful model to further explore ways to understand and improve major monocot crop species.

Sequencing and comparative analyses of the genomes of zoysiagrasses

Zoysia is 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.

Particle inflow gun-mediated transformation of multiple-shoot clumps in rhodes grass (Chloris gayana).

Rhodes grass (Chloris gayana) is one of the most important warm-season forage grasses. It is cultivated in tropical and subtropical parts of the world and is mostly used for grazing and hay production. We have established a particle-bombardment transformation protocol for rhodes grass using multiple-shoot clumps (MSCs) as the target tissue. A vector pAHC25 containing a herbicide-resistance gene (bar) together with the beta-glucuronidase (GUS) gene was used in transformation experiments. The most efficient recovery of bialaphos-resistant tissue was achieved when the bombarded MSCs were first cultured for 15 d on bialaphos-free medium before being subjected to selection pressure. The resistant tissues regenerated transgenic plants that displayed GUS gene expression. Under optimized conditions, 251 target pieces yielded 46 transgenic plants from 4 independent transgenic lines.

Fertile transgenic Brachiaria ruziziensis (ruzigrass) plants by particle bombardment of tetraploidized callus.

We have produced transgenic plants of the tropical forage crop Brachiaria ruziziensis (ruzigrass) by particle bombardment-mediated transformation of multiple-shoot clumps and embryogenic calli. Cultures of multiple-shoot clumps and embryogenic calli were induced on solidified MS medium supplemented with 0.5mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) and 2mg/L 6-benzylaminopurine (BAP) or 4mg/L 2,4-D and 0.2mg/L BAP, respectively. Both cultures were bombarded with a vector containing an herbicide resistance gene (bar) as a selectable marker and the β-glucuronidase (GUS) reporter gene. Sixteen hours after bombardment, embryogenic calli showed a significantly higher number of transient GUS expression spots per plate and callus than multiple-shoot clumps, suggesting that embryogenic callus is the more suitable target tissue. Following bombardment and selection with 10mg/L bialaphos, herbicide-resistant embryogenic calli regenerated shoots and roots in vitro, and mature transgenic plants have been raised in the greenhouse. Polymerase chain reaction (PCR) and DNA gel blot analysis verified that the GUS gene was integrated into the genome of the two regenerated lines. In SacI digests, the two transgenic lines showed two or five copies of GUS gene fragments, respectively, and integration at different sites. Histochemical analysis revealed stable expression in roots, shoots and inflorescences. Transgenic plants derived from diploid target callus turned out to be sterile, while transgenics from colchicine-tetraploidized callus were fertile.

Assays of the production of harmful substances by genetically modified oilseed rape (Brassica napus L.) plants in accordance with regulations for evaluating the impact on biodiversity in Japan

Environmental risk assessment of transgenic crops is implemented under the Cartagena Protocol domestic law in accordance with guidelines for implementing the assessment established by the Ministry of Agriculture, Forestry and Fisheries (MAFF) and the Ministry of Environment (MOE) in Japan. Environmental risk assessments of transgenic crops are implemented based on the concept of ‘substantial equivalence’ to conventional crops. A unique requirement in Japan to monitor the production of harmful substances, or allelochemicals, is unparalleled in other countries. The potential for allelochemicals to be secreted from the roots of transgenic crops to affect other plants or soil microflora or for substances in the plant body to affect other plants after dying out must be evaluated. We evaluated the allelopathic potential of seven transgenic oilseed rape (Brassica napus L.) lines that express glufosinate tolerance in terms of substantial equivalence to conventional oilseed rape lines, and established evaluation methods. Our results indicate no potential production of allelochemicals for any of the seven transgenic oilseed rape lines compared with conventional oilseed rape lines.

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 Japans 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.