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Genetic transformation of Physcomitrella patens mediated by Agrobacterium tumefaciens
Abstract
A simple efficient protocol for introducing exogenous gene from Agrobacterium tumfaciens into Physcomitrella patens was established. When the gametophores of gametophytes about 12 leaves were
inoculated into the wells of PP3 medium at 25°C under the continuous light energy of 30 mol m-2 s-1 from cool-white fluorescent lamps, they grew up to form many buds at about 11 days, then A. tumefaciens at OD600 0.3 was added into the whole wells for 4 times at an interval of 12 h for continuous co-cultivation. After about 17 days of co-culture, the new juvenile gametophytes about 12 leaves grew up and were incubated into PP3 medium containing 50 mg L-1 kanamycin for selection. 100% positive
plants could be obtained after 4 generations of selective culture. PCR analysis showed that gene Cameleon YC2.1 in A. tumefaciens was transferred into all detected plants of P. patens and southern blotting analysis confirmed that exogenous gene was integrated into genomic DNA at a single locus. The results facilitated to analyze genes from more plants via genetic transformation by this system.
inoculated into the wells of PP3 medium at 25°C under the continuous light energy of 30 mol m-2 s-1 from cool-white fluorescent lamps, they grew up to form many buds at about 11 days, then A. tumefaciens at OD600 0.3 was added into the whole wells for 4 times at an interval of 12 h for continuous co-cultivation. After about 17 days of co-culture, the new juvenile gametophytes about 12 leaves grew up and were incubated into PP3 medium containing 50 mg L-1 kanamycin for selection. 100% positive
plants could be obtained after 4 generations of selective culture. PCR analysis showed that gene Cameleon YC2.1 in A. tumefaciens was transferred into all detected plants of P. patens and southern blotting analysis confirmed that exogenous gene was integrated into genomic DNA at a single locus. The results facilitated to analyze genes from more plants via genetic transformation by this system.
