[Genetic transformation of OSISAP1 gene to onion (Allium cepa L.) mediated by amicroprojectile bombardment].

Abstract

Microprojectile bombardment-mediated transformation method has been developed for onion (Allium cepa L.) using embryogenic calli, induced from stem discs, as target tissue. Zinc-finger protein gene OSISAP1 (Oryza sative subspecies indica stress-associated protein gene) was introduced into the open-pollinated onion cultivar (subs.) 'HG400B'. Bombardment parameters were optimized as: the pressure is 1,100 psi, the distance is 6 cm, two times, the ratio of mass between plasmid DNA and golden particles is 1:320. An efficient microprojectile bombardment-mediated transformation system of onion (Allium cepa L.) callus has been established. The binary vector used carried the nptII gene for kanamycin resistance and the GUS reporter gene. Transgenic cultures were screened for their ability to express the GUS reporter gene and to grow in the presence of kanamycin (150 mg/L). Transient expression of GUS reporter gene was observed through histochemical staining of embryogenic callus transformed by microprojectile bombardment. The putative transgenic plants were analysed at the molecular level using PCR, southern hybridization, and RT-PCR. The results confirmed that the OSISAP1 gene was integrated as one copy into the genome of onion and expression. Transgenic plants were produced efficiently with a transformation frequency of about 10%. Test of salinity-alkali stress showed that sodium chloride and sodium bicarbonate at 200 mmol/L effectively killed non-transgenic plants within 1 week of irrigation, while the transgenic plants were completely unaffected by salinity of 400 mmol/L. So transformation with the OSISAP1 gene raised the salinity-alkali-tolerance of the transgenic plants to a high level.