PEG介导立碗藓转化过程.docVIP

PEG-mediated DNA transformation into protoplasts of Physcomitrella patens Stable transformation using protoplasts and PEG Foreign DNA is introduced into protoplasts prepared from propagated protonemata.the isolation efficiency of protoplasts depends on the age (days from subculture) of the protonemata. We obtained the best results using protonemata grown for 4 to 6 days. When older protonemata were used it was difficult to digest the cell walls using the enzyme driselase. For optimum results ,vigorous, light green protonemata should be used. Transformation schedule We routinely transformed protonemata according to the schedule below. (day 1) Subculture the propagated protonemata at 25℃ for approximately 4-6 days. (day 5 at the earliest ) DNA transfer takes 2 days. After the transfer of DNA, incubate for 5 days. (day 12) Transfer cellophane with top agar containing regenerating protoplasts onto a selection plate supplemented with adequate antibiotics. Cultivate for 5 days on the selection medium. (day 17 ) Transfer cellophane with moss colonies onto a non-selection plate (no antibiotics). Cultivate for 5-7 days on the non-selection medium. (day 24) Transfer cellophane with moss colonies onto a selection plate supplemented with adequate antibiotics. Cultivate for 5-7 days on the selection medium. (day 24) Transplant a selection of the peripheral section of the colonies (100-200 lines)onto a non-selection plate. Cultivate for 7-10 days on the non-selection medium. (day 41 ) Transplant a selection of the peripheral section of the colonies onto a selection plate. Cultivate for 7-10 days on the selection medium. (day 51) Transplant colonies surviving on the selection medium as stable transformants onto non-selection medium. Propagation of protonemata (1)Growth medium Stock medium Stock B(×100)： MgSO4.7H2O 2.5g Fill up to 100ml with H20 （autoclave） Stock C(×100)： KH2PO4 2.5g Fill up to 100ml with H20 （autoclave） Stock D(×100)： KNO3