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
Chrysanthemum (Chrysanthemum morifoliumRamat.) is one of the most popular ornamentalflowers in the world, and many agronomic traits haverecently been introduced to chrysanthemum cultivarsby gene transformation. Concerns have been raised,however, regarding transgene flow from transgenicplants to wild plants. In early studies, ethylene receptorgenes have been used for genetic modification inplants, such as flower longevity and fruit ripening.Recently, overexpression of ethylene receptor genesfrom melon (CmETR1/H69A) caused delayed tapetumdegradation of the anther sac and a reduction in pollengrains. We therefore introduced the ethylene receptorgene into chrysanthemums to induce male sterility andprevent transgene flowvia pollen. The chrysanthemumcultivar Yamate shiro was transformed using adisarmed strain of Agrobacterium tumefaciens,EHA105, carrying the binary vector pBIK102H69A,which contains theCmETR1/H69A gene.Atotal of 335shoots were regenerated from 1,282 leaf discs onregeneration medium (26.1%). The presence of theCm-ETR1/H69A gene was confirmed in all of theregenerated plantlets by Southern blot analysis. Thesegenetically modified (GM) plants and their non-GMcounterparts were grown in a closed greenhouse andflowered at temperatures between 10 and 35 C. In 15of the 335 GM chrysanthemum lines, the number ofmature pollen grains was significantly reduced, particularlyin three of the lines (Nos. 91, 191 and 324). Inthese three lines, pollen grains were not observed attemperatures between 20 and 35 C but were observedat 10 and 15 C, and mature pollen grains were formedonly at 15 C. In northern blot analyses, expression ofthe CmETR1/H69A gene was suppressed at lowtemperatures. This phenomenon was observed as aresult of both the suppression of CmETR1/H69Aexpression at low temperatures and the optimal growthtemperature of chrysanthemums (15–20 C). Furthermore,the female fertility of these three GM lines wassignificantly lower than that of the non-GM plants.Thus, the mutated ethylene receptor is able to reduceboth male and female fertility significantly in transgenic chrysanthemums, although the stability ofmale and/or female sterility at varying growth temperaturesis a matter of concern for its practical use
