Binary Ti vectors are the plasmid vectors of choice in Agrobacterium-mediated plant transformation protocols. The pGreen series of binary Ti vectors are configured for ease-of-use and to meet the demands of a wide range of transformation procedures for many plant species. This plasmid system allows any arrangement of selectable marker and reporter gene at the right and left T-DNA borders without compromising the choice of restriction sites for cloning, since the pGreen cloning sites are based on the well-known pBluescript general vector plasmids. Its size and copy number in Escherichia coli offers increased efficiencies in routine in vitro recombination procedures. pGreen can replicate in Agrobacterium only if another plasmid, pSoup, is co-resident in the same strain. pSoup provides replication functions in trans for pGreen. The removal of RepA and Mob functions has enabled the size of pGreen to be kept to a minimum. Versions of pGreen have been used to transform several plant species with the same efficiencies as other binary Ti vectors. Information on the pGreen plasmid system is supplemented by an Internet site (http://www.pgreen.ac.uk) through which comprehensive information, protocols, order forms and lists of different pGreen marker gene permutations can be found.

pGreen: a versatile and flexible binary Ti vector for Agrobacterium-mediated plant transformation.

A Robust CRISPR/Cas9 System for Convenient, High-Efficiency Multiplex Genome Editing in Monocot and Dicot Plants

Silicon is beneficial to plant growth and helps plants to overcome abiotic and biotic stresses by preventing lodging (falling over) and increasing resistance to pests and diseases, as well as other stresses. Silicon is essential for high and sustainable production of rice, but the molecular mechanism responsible for the uptake of silicon is unknown. Here we describe the Low silicon rice 1 (Lsi1) gene, which controls silicon accumulation in rice, a typical silicon-accumulating plant. This gene belongs to the aquaporin family and is constitutively expressed in the roots. Lsi1 is localized on the plasma membrane of the distal side of both exodermis and endodermis cells, where casparian strips are located. Suppression of Lsi1 expression resulted in reduced silicon uptake. Furthermore, expression of Lsi1 in Xenopus oocytes showed transport activity for silicon only. The identification of a silicon transporter provides both an insight into the silicon uptake system in plants, and a new strategy for producing crops with high resistance to multiple stresses by genetic modification of the root's silicon uptake capacity.

A silicon transporter in rice

Drought and salinity are major abiotic stresses to crop production. Here, we show that overexpression of stress responsive gene SNAC1 (STRESS-RESPONSIVE NAC 1) significantly enhances drought resistance in transgenic rice (22–34% higher seed setting than control) in the field under severe drought stress conditions at the reproductive stage while showing no phenotypic changes or yield penalty. The transgenic rice also shows significantly improved drought resistance and salt tolerance at the vegetative stage. Compared with WT, the transgenic rice are more sensitive to abscisic acid and lose water more slowly by closing more stomatal pores, yet display no significant difference in the rate of photosynthesis. SNAC1 is induced predominantly in guard cells by drought and encodes a NAM, ATAF, and CUC (NAC) transcription factor with transactivation activity. DNA chip analysis revealed that a large number of stress-related genes were up-regulated in the SNAC1-overexpressing rice plants. Our data suggest that SNAC1 holds promising utility in improving drought and salinity tolerance in rice.

Overexpressing a NAM, ATAF, and CUC (NAC) transcription factor enhances drought resistance and salt tolerance in rice

Grain weight is one of the most important components of grain yield and is controlled by quantitative trait loci (QTLs) derived from natural variations in crops. However, the molecular roles of QTLs in the regulation of grain weight have not been fully elucidated. Here, we report the cloning and characterization of GW2, a new QTL that controls rice grain width and weight. Our data show that GW2 encodes a previously unknown RING-type protein with E3 ubiquitin ligase activity, which is known to function in the degradation by the ubiquitin-proteasome pathway. Loss of GW2 function increased cell numbers, resulting in a larger (wider) spikelet hull, and it accelerated the grain milk filling rate, resulting in enhanced grain width, weight and yield. Our results suggest that GW2 negatively regulates cell division by targeting its substrate(s) to proteasomes for regulated proteolysis. The functional characterization of GW2 provides insight into the mechanism of seed development and is a potential tool for improving grain yield in crops.

A QTL for rice grain width and weight encodes a previously unknown RING-type E3 ubiquitin ligase

Summary
A large number of morphologically normal, fertile, transgenic rice plants were obtained by co-cultivation of rice tissues with Agrobacterium tumefaciens The efficiency of transformation was similar to that obtained by the methods used routinely for transformation of dicotyledons with the bacterium Stable integration, expression and inheritance of transgenes were demonstrated by molecular and genetic analysis of transformants in the R0, R1 and R2 generations Sequence analysis revealed that the boundaries of the T-DNA in transgenic rice plants were essentially identical to those in transgenic dicotyledons Calli induced from scutella were very good starting materials A strain of A tumefaciens that carried a so-called ‘super-binary’ vector gave especially high frequencies of transformation of various cultivars of japonica rice that included Koshihikari, which normally shows poor responses in tissue culture

https://onlinelibrary.wiley.com/doi/pdf/10.1046/j.1365-313X.1994.6020271.x

Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA.

Transformants of maize inbred A188 were efficiently produced from immature embryos cocultivated with Agrobacterium tumefaciens that carried "super-binary" vectors. Frequencies of transformation (independent transgenic plants/embryos) were between 5% and 30%. Almost all transformants were normal in morphology, and more than 70% were fertile. Stable integration, expression, and inheritance of the transgenes were confirmed by molecular and genetic analysis. Between one and three copies of the transgenes were integrated with little rearrangement, and the boundaries of T-DNA were similar to those in transgenic dicotyledons and rice. F1 hybrids between A188 and five other inbreds were transformed at low frequencies.

High efficiency transformation of maize (Zea mays L.) mediated by Agrobacterium tumefaciens

Novel 'super-binary' vectors that carried two separate T-DNAs were constructed. One T-DNA contained a drug-resistance, selection-marker gene and the other contained a gene for beta-glucuronidase (GUS). A large number of tobacco (Nicotiana tabacum L.) and rice (Oryza sativa L.) transformants were produced by Agrobacterium tumefaciens LBA4404 that carried the vectors. Frequency of co-transformation with the two T-DNAs was greater than 47% GUS-positive, drug-sensitive progeny were obtained from more than half of the co-transformants. Molecular analyses by Southern hybridization and polymerase chain reactions confirmed integration and segregation of the T-DNAs. Thus, the non-selectable T-DNA that was genetically separable from the selection marker was integrated into more than a quarter of the initial, drug-resistant transformants. Since various DNA fragments may be inserted into the non-selectable T-DNA by a simple procedure, these vectors will likely be very useful for the production of marker-free transformants of diverse plant species. Delivery of two T-DNAs to plants from mixtures of A. tumefaciens was also tested, but frequency of co-transformation was relatively low.

https://onlinelibrary.wiley.com/doi/pdf/10.1046/j.1365-313X.1996.10010165.x

Vectors carrying two separate T-DNAs for co-transformation of higher plants mediated by Agrobacterium tumefaciens and segregation of transformants free from selection markers

Phospholipase D (PLD) was purified to high homogeneity from rice bran (Oryza sativa L.). Two peaks of PLD activity were resolved by Mono Q anion-exchange chromatography. The molecular mass of PLD in both peaks was 82 kDa on SDS-PAGE and 78 kDa in gel filtration. Antibodies raised against the protein in one of the peaks precipitated the enzyme activities in both peaks. Enzymatic characteristics of PLD in the two peaks were identical except for a difference of 0.1 in the isoelectric points. Sequence analysis covering more than 10% of the amino acids of the proteins and peptide mapping did not detect any difference in the primary structure of the proteins. A cDNA for PLD was isolated from rice and it encoded a protein of 812 residues. The N-terminal sequences of purified PLDs matched the deduced amino acid sequence starting from residue 47. A Northern blot showed this gene was expressed in leaves, roots, developing seeds and cultured cells, and a Southern blot detected a single band of rice genomic DNA hybridizing to the cDNA. A cDNA for PLD was also isolated from maize. The similarity of the deduced amino acid sequences of PLD was 90% between rice and maize, 73% between the cereals and castor bean.

Takashi Kumashiro

Papers

Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA.

High efficiency transformation of maize (Zea mays L.) mediated by Agrobacterium tumefaciens

Vectors carrying two separate T-DNAs for co-transformation of higher plants mediated by Agrobacterium tumefaciens and segregation of transformants free from selection markers

Purification and characterization of phospholipase D (PLD) from rice (Oryza sativa L.) and cloning of cDNA for PLD from rice and maize (Zea mays L.).

Advances in cereal gene transfer.