Induction of male sterility in transgenic chrysanthemums (Chrysanthemum morifolium Ramat.) by expression of a mutated ethylene receptor gene, Cm-ETR1/H69A, and the stability of this sterility at varying growth temperatures

Abstract

Chrysanthemum (Chrysanthemum morifolium Ramat.) is one of the most popular ornamental flowers in the world, and many agronomic traits have recently been introduced to chrysanthemum cultivars by gene transformation. Concerns have been raised, however, regarding transgene flow from transgenic plants to wild plants. In early studies, ethylene receptor genes have been used for genetic modification in plants, such as flower longevity and fruit ripening. Recently, overexpression of ethylene receptor genes from melon (CmETR1/H69A) caused delayed tapetum degradation of the anther sac and a reduction in pollen grains. We therefore introduced the ethylene receptor gene into chrysanthemums to induce male sterility and prevent transgene flow via pollen. The chrysanthemum cultivar Yamate shiro was transformed using a disarmed strain of Agrobacterium tumefaciens, EHA105, carrying the binary vector pBIK102H69A, which contains the CmETR1/H69A gene. A total of 335 shoots were regenerated from 1,282 leaf discs on regeneration medium (26.1%). The presence of the Cm-ETR1/H69A gene was confirmed in all of the regenerated plantlets by Southern blot analysis. These genetically modified (GM) plants and their non-GM counterparts were grown in a closed greenhouse and flowered at temperatures between 10 and 35°C. In 15 of the 335 GM chrysanthemum lines, the number of mature pollen grains was significantly reduced, particularly in three of the lines (Nos. 91, 191 and 324). In these three lines, pollen grains were not observed at temperatures between 20 and 35°C but were observed at 10 and 15°C, and mature pollen grains were formed only at 15°C. In northern blot analyses, expression of the CmETR1/H69A gene was suppressed at low temperatures. This phenomenon was observed as a result of both the suppression of CmETR1/H69A expression at low temperatures and the optimal growth temperature of chrysanthemums (15–20°C). Furthermore, the female fertility of these three GM lines was significantly lower than that of the non-GM plants. Thus, the mutated ethylene receptor is able to reduce both male and female fertility significantly in transgenic chrysanthemums, although the stability of male and/or female sterility at varying growth temperatures is a matter of concern for its practical use.