Transformation and regeneration of loblolly pine:: shoot apex inoculation with Agrobacterium

Loblolly pine (Pinus taeda L.) is the most important tree species in US commerce and has much to gain through genetic engineering. This species can be transformed using particle bombardment and Agrobacterium; however, the regeneration of plants from transgenic tissues has been difficult and the recovery of transgenic plants has been rare. A shoot-based and genotype-independent transformation method employing Agrobacterium tumefaciens was used to facilitate recovery of plants and permit the transformation of elite germplasm. Shoots from 4-6 week old seedlings and adventitious shoots from culture were inoculated with A. tumefaciens EHA101 (pGUS3), or EHA105 (pSSLa.3), subjected to selection and regenerated. Shoots that survived exhibited expression of the uidA gene (GUS) in a pattern characteristic of the either the CaMV35S promoter (pGUS3), or the larch RbcS promoter (pSSLa.3) transferred. Recovered plants were screened using PCR amplification. Southern DNA analyses and amplification of the T-DNA border junction confirmed genomic integration of both transferred uidA and nptII genes. In this proof of concept study, the overall recovery of P. taeda shoots was fair (10-20%), while recovery of intact rooted plants was poor (>1%) due to difficulty in rooting. Recovery of intact rooted plants from inoculated shoots of P. eldarica and P. radiata was more efficient (10-30%). The addition of a shoot multiplication step and effective rooting protocols will improve the efficiency of this genotype independent transformation method in P. taeda, and in other Pinus spp.