An efficient Biolistic^® transformation protocol for Picea abies embryogenic tissue and regeneration of transgenic plants

Abstract

An efficient Biolistic® transformation technology was developed to stably transform Picea abies (L.) Karst. Several embryogenic tissue lines were tested for proliferation on standard embryogenesis media. Transient transformation studies with these lines were performed to optimize the parameters for genetic transformation. Selection conditions for transgenic tissue based on the nptII resistance gene in combination with the antibiotic geneticin were defined such that only transgenic P. abies lines were able to develop. Nontransgenic tissue was completely inhibited under these conditions. Stable integration of a uidA reporter gene and a nptII resistance gene into the genome of P. abies was achieved and more than 200 mature embryos were regenerated for every transformation event. Histochemical and fluorometric analysis indicated strong expression of the uidA gene in transgenic material. ELISA studies to detect and quantify the nptII gene product as well as polymerase chain reaction and Southern blotting confirmed the presence and integration of uidA and nptII genes into the P. abies genome. Transgenic P. abies plants from nine independent transformation events were recovered and are currently growing in a greenhouse for genetically modified organisms, awaiting field release.

Towards genetic engineering of maritime pine (Pinus pinaster Ait.)

“…the biolistic and Agrobacterium-mediated procedures. To date, only Picea abies was reported to be genetically modified using both methods [48,49]. In the family Pinaceae, the main group of gymnosperms subjected to transformation attempt [2], pine seemed generally more difficult to transform than spruce [16,43,48] or larch (reviewed in [35]).…”

“…To date, only Picea abies was reported to be genetically modified using both methods [48,49]. In the family Pinaceae, the main group of gymnosperms subjected to transformation attempt [2], pine seemed generally more difficult to transform than spruce [16,43,48] or larch (reviewed in [35]). Transient expression could be obtained in P. taeda, P. banksiana, P. contorta, P. sylvestris and P. palustris [12,30,49], but only two recent studies reported on stable transformation of ESM with regeneration of transgenic plants, i.e.…”

“…Such a promising tissue culture system undeniably provides a good target for stable genetic transformation of conifers such as larch [23,29], spruce [10,48,49], and pine [6,30,47] and offers new prospects for rapid and efficient introduction of desirable traits, mostly unknown (e.g. pests and herbicide tolerance) or with low heritability (e.g.…”

Agrobacterium-mediated transformation and assessment of factors influencing transgene expression in loblolly pine (Pinus taeda L.)

“…Transient expression was observed in electroporated protoplasts of loblolly pine (Pinus taeda L.) [3], as well as black spruce (Picea mariana) [4] tissues transformed by particle bombardment. Transgenic plantlets have been achieved in Norway spruce (Picea abies) [5], white spruce (Picea glauca) [6], black spruce (Picea mariana) [1], and radiata pine (Pinus radiata) [7] via microprojectile bombardment.…”

A Gene Encoding Scots Pine Antimicrobial Protein Sp-AMP2 (PR-19) Confers Increased Tolerance against Botrytis cinerea in Transgenic Tobacco

“…plants at an age of 8 to 10 months over-expressing the SPI1 spruce defensin protein displayed reduced fungal growth in the sapwood after inoculation with H. annosum [44]. Also, SPI1 spruce defensin that was expressed in tobacco permitted less growth of the bacterial pathogen Erwinia carotovora (Jones) Bergey et al [45], suggesting that SPI1 increases resistance in both homologous and heterologous systems. Furthermore, poplar trees transformed with a gene encoding synthetic peptide D4E1 were found to be resistant to bacterial pathogens Agrobacterium tumefaciens and Xanthomonas populi (Ridé) van den Mooter & Swings but not to the fungal pathogen Hypoxylon mammatum (Wahlenb.)…”