Genetic transformation of rice (Oryza sativa L.) with gene encoding Melastoma malabathricum metallothionein type II (MaMt2) using Agrobacterium tumefaciens-mediated transfer.

Abstract

Rice production can be decrease caused by abiotic stresses. These abiotic stresses, cause oxidative stress in plants overproducting radical oxygen species (ROS). ROS could cause damage and death in plants cells. The tolerant varieties adapted to abiotic stress could be created by introducing and expressing the abiotic stress tolerant genes. Metallothionein (MT) has an important role to detoxity some heavy-metal ions, e.g. cadmium and mercury by binding these metal ions. MaMt2 gene encoding for metallothionein type II had been isolated from Melastoma malabathricum. We suppose that plant overexpressing this gene would be tolerant to metal ions, including Al. This research had an objective to introduce this MaMt2 gene under the control of pUbiquitin promoter and NosT terminator into rice (Oryza sativa L.) genom. Two rice cultivars i.e. Kasalath and Nipponbare were transformed with MaMt2, through Agrobacterium-mediated gene transfer method. Kasalath is indica rice and Nipponbare is japonica rice. Genetic transformation of rice was carried out on Kasalath and Nipponbare cultivars by using calli of seeds as explants. The calli were obtained from seeds cultivated in 2N6 medium containing 2 mg/L 2.4 D. These 3-4 week old calli (235 calli of Kasalath and 165 calli of Nipponbare) were co-cultivated with A. tumefaciens LBA4404 carrying pIG6-SMt2 containing MaMt2 gene under the control of Ubiquitin promoter and Nos terminator. Co-cultivation was carried out 3 days in the 2N6 media containing 100 mg/L acetosiringone. After cocultivation, the calli were selected in two steps in 2NBK and nN6C media containing 20 and 30 mg/L hygromycin respectively. The resistant calli were regenerated in 2N6 medium containing 0.5 mg/L kinetin. Based on hygromycin resistant calli on hygromycin selection medium, the transformation efficiency in Kasalath was 14.04%, while in Nipponbare was 19.39%. Based on resistant calli on selective medium, the efficiency of regeneration of transgenic shoots in Kasalath was 6.06%, while in Nipponbare was 28.1%. Molecular analysis by PCR showed that two of eleven putative rice transgenic were confirmed as transgenic ones containing MaMt2 transgene under the control of Ubiquitin promoter and NosT terminator. This transgenic rice can be used as source of MaMt2 that can be transferred to other cultivars of rice by conventional crossing.