Most agriculturally important traits are regulated by genes known as quantitative trait loci (QTLs) derived from natural allelic variations. We here show that a QTL that increases grain productivity in rice, Gn1a, is a gene for cytokinin oxidase/dehydrogenase (OsCKX2), an enzyme that degrades the phytohormone cytokinin. Reduced expression of OsCKX2 causes cytokinin accumulation in inflorescence meristems and increases the number of reproductive organs, resulting in enhanced grain yield. QTL pyramiding to combine loci for grain number and plant height in the same genetic background generated lines exhibiting both beneficial traits. These results provide a strategy for tailormade crop improvement.
The chronic food shortage that was feared after the rapid expansion of the world population in the 1960s was averted largely by the development of a high-yielding semi-dwarf variety of rice known as IR8, the so-called rice 'green revolution'. The short stature of IR8 is due to a mutation in the plant's sd1 gene, and here we identify this gene as encoding an oxidase enzyme involved in the biosynthesis of gibberellin, a plant growth hormone. Gibberellin is also implicated in green-revolution varieties of wheat, but the reduced height of those crops is conferred by defects in the hormone's signalling pathway.
Agrobacterium-mediated transformation of rice is an important method that has been widely adopted by many laboratories. However, because current approaches rely on culture systems, routine protocols have been established only in japonica rice, especially those varieties with higher regeneration potential. Some very efficient methods have been developed for japonica varieties that enable high-throughput functional analysis in rice; however, many elite japonica, and most indica, varieties are difficult to regenerate, leading to low transformation efficiencies. Much effort has been devoted to improving transformation efficiency for all rice genotypes. Here, we describe an Agrobacterium-mediated rice transformation method that is applicable to easily cultured varieties in addition to elite japonica varieties that are more difficult to culture. Using this method, transgenic rice plants can be obtained in about 2-3 months with a transformation frequency of 30-50%, both in easily cultured varieties and recalcitrant elite japonica rice.
A rice semi-dwarf variety, IR8, known as "miracle rice" enabled dramatic increases rice production and its widespread adoption averted predicted food shortages in Asia during the 1960s to 1990s. This remarkable achievement was referred to as "green revolution". The short stature of IR8 was derived from the semi-dwarf gene, sd1, and the sd1 gene contributed significantly to the rice "green revolution". In this paper, we described the physiological, molecular genetic and biochemical characterization of the sd1 gene. The sd1 mutant contained lower gibberellin (GA) levels than wild-type plants but responded sensitively to exogenous GA. Cloning and sequence analyses revealed that the SD1 gene encoded a GA biosynthetic enzyme, GA20 oxidase. In all of the sd1 mutants tested, nucleotide deletions or substitutions were observed in the GA20 oxidase gene (GA20ox-2), which induced an internal stop codon or single amino acid substitutions, respectively. The sd1 plants, which the wild-type GA20ox-2 gene was introduced showed the normal height. A recombinant GA20ox-2 protein produced from the cDNA clone in E. coli catalyzed the conversion of GA 53 to GA 20 . These results confirmed that SD1 encodes an active GA20 oxidase. The expression of GA20ox-2 was down-regulated by GA in a similar manner to that of some GA20oxs in other plants. The rice genome carried at least two GA 20-oxidase genes (GA20ox-1 and GA20ox-2) and SD1 corresponded to GA20ox-2, which is highly expressed in the leaves and flowers, whereas GA20ox-1 is preferentially expressed in the flowers. The reduced plant height associated with the sd1 alleles was due to the low amount of active GA in leaves, which was caused by a mutation of the GA20ox-2 gene. On the basis of these results, we discussed the importance of GA in the regulation of plant height in crop breeding.
Regeneration of plant organs is often the essential step in genetic transformation; however, the regeneration ability of a plant varies depending on the genetic background. By conventional crosses of low-regeneration rice strain Koshihikari with high-regeneration rice strain Kasalath, we identified some quantitative trait loci, which control the regeneration ability in rice. Using a map-based cloning strategy, we isolated a main quantitative trait loci gene encoding ferredoxin-nitrite reductase (NiR) that determines regeneration ability in rice. Molecular analyses revealed that the poor regeneration ability of Koshihikari is caused by lower expression than in Kasalath and the specific activity of NiR. Using the NiR gene as a selection marker, we succeeded in selectively transforming a foreign gene into rice without exogenous marker genes. Our results demonstrate that nitrate assimilation is an important process in rice regeneration and also provide an additional selectable marker for rice transformation.regeneration ability ͉ ferredoxin-nitrate reductase ͉ selectable marker R egeneration of plants from cell culture is a critical step in the production of novel varieties of plants. Generally, it is not easy to culture and regenerate monocot plants, including agronomically important crops such as rice, wheat, and maize. In rice, an efficient culture system using mature seeds has been established based on research with model varieties such as Nipponbare (Japonica) and Kasalath (Indica). However, many leading varieties used for food production, such as Koshihikari in Japan and IR64 in tropical countries, have low regeneration ability in the mature seed culture system, resulting in a serious obstacle to efficient production of transgenic plants. It has been indicated that regeneration ability depends mainly on a few key genes (1-7), but no gene has yet been identified in any plant species. To understand the regeneration process and resolve the low regeneration ability of a leading Japanese variety, Koshihikari, we have attempted to isolate major quantitative trait loci (QTL), which would increase the regeneration ability of Koshihikari. Materials and MethodsCulture Conditions and Regeneration Test. Mature seeds were dehusked and surface-sterilized in 70% ethanol for 30 s, vigorously shaken in 1.5% sodium hypochlorite for 30 min, and rinsed five times in sterilized water. For the induction of calli, sterilized seeds were placed on the surface of an agar medium containing CHU (N 6 ) basal salt mixture (Sigma), 2 mg͞liter glycine, 0.5 mg͞liter nicotinic acid, 0.5 mg͞liter pyridoxine-HCl, 1 mg͞liter thiamine-HCl, 0.1 g͞liter myo-inositol, 0.3 g͞liter casamino acid, 2.878 g͞liter proline, 2 mg͞liter 2,4-dichlorophenoxyacetic acid, 30 g͞liter sucrose, and 3 g͞liter gelrite. The medium was adjusted to pH 5.8. Seeds were incubated in the medium at 29.5°C. Four weeks after inoculation calli formed from seeds were transferred onto regeneration medium containing MS plant salt mixture (Wako), 5 mg͞liter nicotinic acid, 10 mg͞lit...
SummaryIn early plant embryogenesis, the determination of cell fate in the protodermal cell layer is considered to be the earliest event in radial pattern formation. To elucidate the mechanisms of epidermal cell fate determination and radial pattern formation in early rice embryogenesis, we have isolated a GL2-type homeobox gene Roc1 (Rice outermost cell-speci®c gene1), which is speci®cally expressed in the protoderm (epidermis). In early rice embryogenesis, cell division occurs randomly and the morphologically distinct layer structure of the protoderm cannot be observed until the embryo reaches more than 100 mm in length. Nonetheless, in situ hybridization analyses revealed that speci®c expression of Roc1 in the outermost cells is established shortly after fertilization, much earlier than protoderm differentiation. In the regeneration process from callus, the Roc1 gene is also expressed in the outermost cells of callus in advance of tissue and organ differentiation, and occurs independently of whether the cells will differentiate into epidermis in the future or not. Furthermore, this cell-speci®c Roc1 expression could be induced¯exibly in the newly produced outermost cells when we cut the callus. These ®ndings suggest that the expression of Roc1 in the outermost cells may be dependent on the positional information of cells in the embryo or callus prior to the cell fate determination of the protoderm (epidermis). Furthermore, the Roc1 expression is downregulated in the inner cells of ligule, which have previously been determined as protodermal cells, also suggesting that the Roc1 expression is position dependent and that this position dependent Roc1 expression is important also in post-embryonic protoderm (epidermis) differentiation.
SummaryThe Arabidopsis PINHEAD/ZWILLE (PNH/ZLL) gene is thought to play an important role in the formation of the shoot apical meristem (SAM) and in leaf adaxial cell speci®cation. To investigate the molecular mechanisms of rice development, we have isolated a rice homologue of PNH/ZLL, called OsPNH1. Around the SAM, OsPNH1 was strongly expressed in developing leaf primordia, speci®cally in the presumptive vascular domains, developing vascular tissues, a few cell-layers of the adaxial region, and future bundle sheath extension cells. In the SAM, only weak expression was observed in the central region, whereas strong expression was detected in the mid-vein region of leaf founder cells in the peripheral SAM domain. We produced transgenic rice plants containing the antisense OsPNH1 strand. The antisense OsPNH1 plants developed malformed leaves with an altered vascular arrangement and abnormal internal structure. These plants also formed an aberrant SAM with reduced KNOX gene expression. We examined the subcellular localization of the OsPNH1-GFP fusion protein and found that it was localized in the cytoplasm. On the basis of these observations, we propose that OsPNH1 functions not only in SAM maintenance as previously thought, but also in leaf formation through vascular development.
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