Estrogen-inducible GFP expression patterns in rice (Oryza sativa L.)

We investigated estrogen-inducible green fluorescent protein (GFP) expression patterns using an estrogen receptor fused chimeric transcription activator, XVE, in the monocotyledonous model plant rice (Oryza sativa L.). This system has been shown to be an effective chemical-inducible gene expression system in Arabidopsis and has been applied to other plants in order to investigate gene functions or produce marker-free transgenic plants. However, limited information is available on the correlation between inducer concentration and the expression level of the gene induced in monocots. Here, we produced a transgenic rice integrated estrogen-inducible GFP expression vector, pLex:GFP, and investigated dose–response and time-course patterns of GFP induction in rice calli and seedlings for the first time. With 17-β-estradiol treatment at >5 μM, GFP signals were detected in the entire surface of calli within 2 days of culture. Highest GFP signals were extended for 8 days with estradiol treatment at 25 μM. In three-leaf-stage seedlings, GFP signals in the leaves of pLex:GFP-integrated transgenic lines were weaker than those in the leaves of p35S:GFP-integrated transgenic lines. However, GFP signals in the roots of pLex:GFP- and p35S:GFP-integrated transgenic lines were similar with estradiol treatment at >10 μM. With regard to controlling appropriate gene expression, these results might provide helpful indications on estradiol treatment conditions to be used for the XVE system in rice and other monocots

Development of transgenic male-sterile rice by using anther-specific promoters identified by comprehensive screening of the gene expression profile database ‘RiceXPro’

Because genomic selection is designed for the population breeding of allogamous species, a successive outcrossing system is required for efficient use of genomic selection in autogamous crops, such as Oryza sativa L. (rice). Transgenic and dominant male-sterility is a suitable tool for efficient outcrossing of autogamous crops. Though there have been some reports of dominant male-sterile rice developed using transgenic technology, the flowering habit was substandard. Here, to isolate promoters that, when linked to a lethal gene, induce dominant male-sterility while retaining a good flowering habit, we identified 38 candidate genes with anther-specific expression by using the ‘RiceXPro’ database. We then evaluated the abilities of the near-upstream regions of these genes to induce male-sterility when linked to the lethal gene barnase and introduced into the rice cultivar ‘Nipponbare’. Seven of the 38 promoters induced clear dominant male-sterility; promoters expressed in the later stage of anther development induced male-sterility while retaining better flowering habits when compared to ones expressed in the early stage. These seven promoters could potentially be used to facilitate development of an efficient outcross-based breeding system in rice

Interfamily Transfer of Dual NB-LRR Genes Confers Resistance to Multiple Pathogens

A major class of disease resistance (R) genes which encode nucleotide binding and leucine rich repeat (NB-LRR) proteins have been used in traditional breeding programs for crop protection. However, it has been difficult to functionally transfer NB-LRR-type R genes in taxonomically distinct families. Here we demonstrate that a pair of Arabidopsis (Brassicaceae) NB-LRR-type R genes, RPS4 and RRS1, properly function in two other Brassicaceae, Brassica rapa and Brassica napus, but also in two Solanaceae, Nicotiana benthamiana and tomato (Solanum lycopersicum). The solanaceous plants transformed with RPS4/RRS1 confer bacterial effector-specific immunity responses. Furthermore, RPS4 and RRS1, which confer resistance to a fungal pathogen Colletotrichum higginsianum in Brassicaceae, also protect against Colletotrichum orbiculare in cucumber (Cucurbitaceae). Importantly, RPS4/RRS1 transgenic plants show no autoimmune phenotypes, indicating that the NB-LRR proteins are tightly regulated. The successful transfer of two R genes at the family level implies that the downstream components of R genes are highly conserved. The functional interfamily transfer of R genes can be a powerful strategy for providing resistance to a broad range of pathogens

Development and characterization of transgenic dominant male sterile rice toward an outcross-based breeding system

Genomic selection is attracting attention in the field of crop breeding. To apply genomic selection effectively for autogamous (self-pollinating) crops, an efficient outcross system is desired. Since dominant male sterility is a powerful tool for easy and successive outcross of autogamous crops, we developed transgenic dominant male sterile rice (Oryza sativa L.) using the barnase gene that is expressed by the tapetum-specific promoter BoA9. Barnase-induced male sterile rice No. 10 (BMS10) was selected for its stable male sterility and normal growth characteristics. The BMS10 flowering habits, including heading date, flowering date, and daily flowering time of BMS10 tended to be delayed compared to wild type. When BMS10 and wild type were placed side-by-side and crossed under an open-pollinating condition, the seed-setting rate was <1.5%. When the clipping method was used to avoid the influence of late flowering habits, the seed-setting rate of BMS10 increased to a maximum of 86.4%. Although flowering synchronicity should be improved to increase the seed-setting rate, our results showed that this system can produce stable transgenic male sterility with normal female fertility in rice. The transgenic male sterile rice would promote a genomic selection-based breeding system in rice

Agrobacterium-mediated transformation of kabocha squash (Cucurbita moschata Duch) induced by wounding with aluminum borate whiskers

An efficient genetic transformation method for kabocha squash (Cucurbita moschata Duch cv. Heiankogiku) was established by wounding cotyledonary node explants with aluminum borate whiskers prior to inoculation with Agrobacterium. Adventitious shoots were induced from only the proximal regions of the cotyledonary nodes and were most efficiently induced on Murashige–Skoog agar medium with 1 mg/L benzyladenine. Vortexing with 1% (w/v) aluminum borate whiskers significantly increased Agrobacterium infection efficiency in the proximal region of the explants. Transgenic plants were screened at the T0 generation by sGFP fluorescence, genomic PCR, and Southern blot analyses. These transgenic plants grew normally and T1 seeds were obtained. We confirmed stable integration of the transgene and its inheritance in T1 generation plants by sGFP fluorescence and genomic PCR analyses. The average transgenic efficiency for producing kabocha squashes with our method was about 2.7%, a value sufficient for practical use

カワラヨモギの「ほふく型」と「直立型」の開花日, 頭花サイズ, 頭花中の利胆成分含量及び分子レベルの相違について

[論文

Relationship between hybridization frequency of Brassica juncea ^|^times; B. napus and distance from pollen source (B. napus) to recipient (B. juncea) under field conditions in Japan

We describe two data structures that preprocess a set S of n points in R(d) (d constant) so that the sum of Euclidean distances of points in S to a query point q can be quickly approximated to within a factor of ε. This preprocessing technique has several applications in clustering and facility location. Using it, we derive an O(nlogn) time deterministic and O(n) time randomized ε-approximation algorithm for the so called Fermat-Weber proble