TY - JOUR
T1 - Stable Agrobacterium-mediated transformation of embryogenic tissues from Pinus pinaster Portuguese genotypes
AU - Tereso, Susana
AU - Miguel, Célia
AU - Zoglauer, Kurt
AU - Valle-Piquera, Carolina
AU - Oliveira, M. Margarida
PY - 2006/9/1
Y1 - 2006/9/1
N2 - Protocols for genetic transformation of maritime pine (Pinus pinaster Sol. ex Aiton) embryogenic tissues were developed using the Agrobacterium C58pMP90/pPCV6NFGUS. This is the first report of Agrobacterium-mediated T-DNA integration in P. pinaster confirmed by Southern blot analysis. The omission of casein hydrolysate from culture medium during cocultivation and subsequent subculture was crucial to control Agrobacterium growth. Two different transformation protocols were compared: (1) bacterial drops were spread over embryogenic clumps; (2) a mixture of bacterial and embryogenic cell suspensions was plated on filter paper. The highest frequency of transformation (22 independent transformed lines per g fresh weight, for embryogenic clone 31/668/00) was obtained with Protocol 2. The same basic procedure allowed transformation of embryogenic cell suspensions, which was dependent on subculture age. From 52 hygromycin-resistant independent lines obtained, 47 showed stable uidA gene expression and were PCR-positive for uidA gene and 42 for hpt gene. No residual Agrobacterium was detected in the transformed lines. Transgene integration was achieved using both protocols, as confirmed by Southern hybridization. From 38 (90%) transformed lines successfully cryopreserved and recovered, 71% regrown replicates have maintained the frequency of cell aggregates and early-formed embryos with uidA expression. Maturation of 44 transformed lines gave rise to 3 mature somatic embryos, each one coming from a different transformed line. Our results show the high potential of Protocol 2 for application to different culture systems.
AB - Protocols for genetic transformation of maritime pine (Pinus pinaster Sol. ex Aiton) embryogenic tissues were developed using the Agrobacterium C58pMP90/pPCV6NFGUS. This is the first report of Agrobacterium-mediated T-DNA integration in P. pinaster confirmed by Southern blot analysis. The omission of casein hydrolysate from culture medium during cocultivation and subsequent subculture was crucial to control Agrobacterium growth. Two different transformation protocols were compared: (1) bacterial drops were spread over embryogenic clumps; (2) a mixture of bacterial and embryogenic cell suspensions was plated on filter paper. The highest frequency of transformation (22 independent transformed lines per g fresh weight, for embryogenic clone 31/668/00) was obtained with Protocol 2. The same basic procedure allowed transformation of embryogenic cell suspensions, which was dependent on subculture age. From 52 hygromycin-resistant independent lines obtained, 47 showed stable uidA gene expression and were PCR-positive for uidA gene and 42 for hpt gene. No residual Agrobacterium was detected in the transformed lines. Transgene integration was achieved using both protocols, as confirmed by Southern hybridization. From 38 (90%) transformed lines successfully cryopreserved and recovered, 71% regrown replicates have maintained the frequency of cell aggregates and early-formed embryos with uidA expression. Maturation of 44 transformed lines gave rise to 3 mature somatic embryos, each one coming from a different transformed line. Our results show the high potential of Protocol 2 for application to different culture systems.
KW - Conifer
KW - Cryopreservation
KW - Embryogenic cell suspensions
KW - Gus expression
KW - Maritime pine
KW - Somatic embryogenesis
UR - http://www.scopus.com/inward/record.url?scp=33751509025&partnerID=8YFLogxK
U2 - 10.1007/s10725-006-9126-2
DO - 10.1007/s10725-006-9126-2
M3 - Article
AN - SCOPUS:33751509025
VL - 50
SP - 57
EP - 68
JO - Plant Growth Regulation
JF - Plant Growth Regulation
SN - 0167-6903
IS - 1
ER -