Abstract

A simple efficient protocol for introducing exogenous gene from Agrobacteriumtumfaciens 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 μmol 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.

Key words: Agrobacterium tumefaciens, Physcomitrella patens, transformation, bud.

Abbreviation

PEG, Polyethylene glycol; OD, optical density; CTAB, cetyl trimethyl ammonium bromide; dCTP, deoxycytidine 5’-triphosphate; SDS, sodium dodecyl sulfate; YFP, your favorite protein.