Stable Agrobacterium-mediated transformation of embryogenic tissues from Pinus pinaster Portuguese genotypes

Abstract

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.

Gene Amplification from CryopreservedArabidopsis thalianaShoot Tips

“…They reported functional GUS gene expression in transgenic pears even after cryopreserved for 1 year. Similar types of observations were reported by Tereso et al (2006). They also reported that transgenic maritime pine (Pinus pinaster Sol.…”

Analysis of genetic stability at SSR loci during somatic embryogenesis in maritime pine (Pinus pinaster)

“…Somatic embryo maturation was performed as described by Tereso et al (2006), except that plant growth regulatorfree DCR medium supplemented with 3% of sucrose and 1% activated charcoal, was used for resuspension of embryogenic tissues. Embryogenic suspensions were then spread over filter papers and placed on maturation medium.…”

“…Conversion of somatic embryos to plants growing in the greenhouse (emblings) was obtained following the protocol described by Tereso et al (2006). Emblings with a welldeveloped epicotyle were transferred to a mixture of peat:perlite:pine tree bark (1:1:1) and acclimatized in a growth chamber over a 10-day period (Miguel et al 2004).…”

Norway spruce (Picea abies) genetic transformation with modified Cry3A gene of Bacillus thuringiensis.

“…The whole process from ET to transgenic plantlet(s) takes significantly less time than organogenesis from other explants. In addition, transformed ETs can be easily cryopreserved without compromising re-growth and transgene expression (Tereso et al, 2006). This is an important advantage because some P. abies ET cultures progressively lose their maturation potency already after 6 months (Högberg et al, 1998).…”