Development of transformation system of rice based on binary bacterial artificial chromosome (BIBAC) vector.
An Agrobacterium-mediated transformation protocol using binary bacterial artificial chromosome (BIBAC) vector system in rice (Oryza sativa L.) was developed. Calli derived from mature embryos of japonica rice cv. H1493 were used as target tissues. Various aspects in transformation and regeneration processes including callus induction and culture, Agrobacterium concentration and duration of co-cultivation, bacterial elimination and transformant selection were examined in order to improve the transformation efficiency. An optimized transformation conditions was established including: using an Agrobacterium strain, LBA4404(HP4404), which carries a super-virulent helper plasmid pCH32, for the infection; a modified N6 medium system for callus induction and culture; pH 5.6 for media in pre-cultivation and co-cultivation; Agrobacterium concentration at OD600 = 1.0 for 3 days co-cultivation and 7 days for a resting period of the infected calli. Based on PCR and Southern blot analysis, it was demonstrated that insert DNA and marker genes carried by BIBAC2 were integrated into the rice genome.
Key Laboratory of Ministry of Education for Plant Development Biology, College of Life Sciences, Wuhan University, Wuhan 430072, China.
Rui-Feng
RF
He
Yuan-Yuan
YY
Wang
Bo
B
Du
Ming
M
Tang
Ai-Qing
AQ
You
Li-Li
LL
Zhu
Guang-Cun
GC
He
D022202:Chromosomes, Artificial, Bacterial / Q000235:genetics*; D018744:DNA, Plant / Q000235:genetics; D017343:Genes, Plant / Q000235:genetics; D005822:Genetic Vectors; D012275:Oryza / Q000235:genetics* / Q000254:growth & development; D030821:Plants, Genetically Modified / Q000235:genetics* / Q000254:growth & development; D012231:Rhizobium / Q000235:genetics; D014170:Transformation, Genetic
D018744:DNA, Plant