Kenjirou Ozawa

Japanese cedar (JC) pollinosis is a serious type I allergic disease in Japan. Although subcutaneous immunotherapy and sublingual immunotherapy have been applied to treat JC pollinosis, high doses of allergens may cause IgE-mediated allergic reactions. The transgenic rice seeds that contain genetically modified Cry j 1 and Cry j 2, the two major allergens of JC pollen, have been developed as candidates for oral immunotherapy. Although the antigens in the transgenic rice seeds (Tg-rice seeds) were engineered such that they decrease binding ability with IgE and they are of insufficient length to cross-link IgE on the surface of mast cells or basophils, the safety of Tg-rice seeds for patients with JC pollinosis was unclear. To verify the safety of Tg-rice seeds in terms of allergies, we investigated the percentage of activated basophils induced by Tg-rice seed extract in the basophil activation test. Blood samples from 29 patients with JC pollinosis were collected. Tg-rice seed extract, non-transgenic wild-type rice seed extract, and Cry j 1 and Cry j 2 were mixed with the blood with reagents. The percentage of activated basophils was assessed by CD203c expression, a basophil activation marker. The percentage of activated basophils after the stimulation with Tg-rice seed extract was 4.5 ± 1.6% (mean ± SD) compared with 62.9 ± 20.2% after Cry j 1- and Cry j 2-stimulation (difference 58.4%, P < 0.001, 95% confidence interval 51.0–65.9%). The results will contribute to the safety of Tg-rice seeds in terms of allergies.

We have developed a high-frequency method for Agrobacterium-mediated gene targeting by combining an efficient transformation system using rice suspension-cultured calli and a positive/negative selection system. Compared with the conventional transformation system using calli on solid medium, transformation using suspension-cultured calli resulted in a 5- to 10-fold increase in the number of resistant calli per weight of starting material after positive/negative selection. Homologous recombination occurred in about 1.5% of the positive/negative selected calli. To evaluate the efficacy of our method, we show in this report that knockout rice plants containing either a disrupted Waxy (granule-bound starch synthase) or a disrupted Xyl (β1,2-xylosyltransferase) gene can be easily obtained by homologous recombination. Study of gene function using homologous recombination in higher plants can now be considered routine work as a direct result of this technical advance.

Agrobacterium-mediated transformation of rice has been routinely performed according to the protocol reported by Hiei et al. (Plant J. 6:271–282, 1994). However, several elite japonica and many indica varieties cannot be efficiently transformed by Agrobacterium system. Also a large number of transformants are required to generate T-DNA insertion and FOX libraries as well as gene-targeting studies. To overcome these challenges, we established a high-efficiency transformation system in rice by cocultivating rice calli with Agrobacterium on filter papers moistened with enriched liquid media instead of using solid media (Ozawa, Plant Sci. 176:522–527, 2009; Ozawa and Takaiwa, Plant Sci. 179:333–337, 2010). In this system, the transformation efficiency of the calli is almost 100% in many varieties.