An improved procedure for production of white spruce (Picea glauca) transgenic plants using Agrobacterium tumefaciens

Abstract

An efficient and reproducible procedure for the transformation of white spruce (Picea glauca [Moench] Voss) embryogenic tissues was developed using A. tumefaciens-mediated gene transfer. Rapidly dividing white spruce embryogenic tissues were co-cultivated with disarmed A. tumefaciens strains containing additional copies of the virulence regions from plasmid PToK47. The plasmid pBi121, containing the neomycin phosphotransferase II (nptII) gene providing kanamycin resistance as a selectable marker and the beta-glucuronidase (uidA) reporter gene, was used as binary vector. The highest frequency of transformation (15 transformed tissues g(-1) FW of treated embryogenic tissue) was obtained with 5-d-old tissues grown in liquid medium and co-cultivated with Agrobacterium for 2 d in the same medium but containing 50 microM acetosyringone. Recovery of kanamycin-resistant tissues was improved when tissues were first grown for 10 d on a timentin-containing medium (400 mg l(-1)), to prevent bacterial overgrowth, before application of the selection pressure. After 6 weeks on kanamycin-selection medium, resistant tissues were obtained and showed stable uidA expression. The presence of the transgenes was demonstrated by PCR analysis and their integration into the genome was confirmed by Southern hybridization. Transgenic plants were regenerated from transformed tissues within 4 months after co-culture.

Susceptibility of embryogenic and organogenic tissues of maritime pine (Pinus pinaster) to antibiotics used in Agrobacterium-mediated genetic transformation

“…In genetic transformation experiments using the same Agrobacterium strain (C58pMP90), 200 mg l -1 timentin was used for Pinus taeda and Picea abies embryogenic suspensions (Wenck et al 1999), whereas 250 mg l -1 cefotaxime was used for Pinus strobus freshly prepared embryogenic suspensions (Levée et al 1999). Tissue growth from transgenic Cef cefotaxime, Carb carbenicillin, Tim timentin embryogenic suspensions of Picea glauca was not affected by cefotaxime or carbenicillin at 100-500 mg l -1 or by timentin at 100-400 mg l -1 but 400 mg l -1 timentin was more efficient for Agrobacterium elimination (Le et al 2001). For P. pinaster French genotypes, an antibiotic treatment adequate for all embryogenic lines has not been found, with one line persistently showing Agrobacterium contamination (Trontin et al 2002).…”

“…At concentrations of 20-50 mg l -1 of kanamycin, this antibiotic even caused loss of weight from the tissues. In Agrobacterium-mediated transformation of Larix and Picea, 25-50 mg l -1 kanamycin has been used successfully (Levé e et al 1997;Le et al 2001). This suggests that the optimal antibiotic and concentration have to be found for each species, type of explant and contact between tissue and selection medium.…”

“…The most commonly used antibiotics for Agrobacterium elimination include the b-lactam antibiotics cefotaxime and carbenicillin. Cefotaxime is a cephalosporin antibiotic that has been frequently used in genetic transformation of various conifer species (Hood et al 1990;Levé e et al 1997Levé e et al , 1999Ló pez et al 2000;Klimaszewska et al 2001;Le et al 2001). It has high resistance to b-lactamase activity but in conifers it can inhibit adventitious bud regeneration (Holland et al 1997) and embryo development (Tsang et al 1989;Sarma et al 1995).…”

“…Carbenicillin has been used for Pinus radiata (Cerda et al 2002), but in Picea sitchensis it inhibited the development of somatic embryos and tissue growth (Sarma et al 1995). Timentin, which is a combination of ticarcillin with clavulanic acid that is a specific b-lactamase inhibitor (Verbist and Verhaegen 1986), has been selected for transformation studies in some conifers (Wenck et al 1999;Le et al 2001;Charity et al 2002;Grant et al 2004). In Pinus pinaster French genotypes, neither augmentin (amoxicillin with clavulanic acid) nor a mixture of carbenicillin with cefotaxime were effective in Agrobacterium elimination from all the embryogenic lines tested (Trontin et al 2002).…”

Persistence ofAgrobacterium tumefaciensin transformed conifers

“…While A. tumefaciens-mediated transformation methods have been developed for a number of important forest species (e.g. Charity et al, 2005;Klimaszewska et al, 2001;Le et al, 2001;Levée et al, 1999;Trontin et al, 2002), there are currently no published data on the persistence of A. tumefaciens in transgenic trees or any data evaluating the persistence of A. tumefaciens in any soil-grown plants for longer than six months. However, this sort of information is an important pre-requisite for field release of transgenic plants from a regulatory standpoint.…”