- Poster presentation
- Open Access
Effect of inorganic nitrogen concentration in co-culture and regeneration media on Agrobacterium tumefaciens growth and on the regenerative capacity of transformed Pinus radiataembryonal mass
BMC Proceedingsvolume 5, Article number: P141 (2011)
The Genetic Engineering program at Genomica Forestal SA, Chile (GFSA) has a goal of generating stably transformed radiata pine for in planta evaluation of candidate genes. Regeneration of transgenic plants depends mainly on two factors: regeneration ability of transformed cells and stable transgene integration and expression.
In several conifer species, including radiata pine, transgenics have been regenerated through cocultivation of Agrobacterium tumefaciens with embryogenic cells [1–3]. However, in our first experiments using MSG  culture medium we found that radiata pine embryonal masses did not recover easily after co cultivation and that there was an excessive overgrowth of bacterial cells in spite of using bacteriostatics in the medium. This impediment prompted our study on testing other culture medium formulations, routinely used in conifer somatic embryogenesis, on the growth of A. tumefaciens GV3101. The tested media were: MSG, DCR , and modified Litvay  MLV. Of the three media MSG supported significantly higher bacterial growth than the other two media. One of the major differences in the composition of these media is inorganic nitrogen concentration (NH4NO3 and KNO3). Compared with MLV and DCR, MSG has the lowest concentration of inorganic nitrogen (100 mgl-1 compared with 340 in DCR and 950 mgl-l in MLV) provided in the sole form of KNO3. Based on our results and the work of others, we concluded that low nitrate concentration in MSG medium promoted A. tumefaciens growth and this had a deleterious influence on the viability of radiata pine cells during co cultivation, and also rendered eradication of bacterial cells difficult. Comparison of growth of radiata pine embryonal mass on the three media did not show statistically significant differences. A strategy for producing transgenic radiata pine for in planta transgene expression and stability study will be presented.
Levée V, Lelu M, Jouanin L, Cornu D, Pilate G: Agrobacterium tumefaciens-mediated transformation of hybrid larch (Larix kaempferi x L. deciduas) and transgenic plant regeneration. Plant Cell Reports. 1997, 16: 680-685. 10.1007/s002990050301.
Klimaszewska K, Park Y, Overton C, MacEacheron I, Bonga J: Optimized somatic embryogenesis in Pinus strobes L. In vitro Cell Dev. Biol. 2001, 37: 392-399. 10.1007/s11627-001-0069-z.
Grace L, Charity J, Gresham B, Kay N, Walter C: Insect-resistant transgenic Pinus radiata. Plant Cell Reports. 2005, 24: 103-111. 10.1007/s00299-004-0912-x.
Beckwar M, Nagmani R, Wann S: Initiation of embryogenic cultures and somatic embryo development in loblolly pine (Pinus taeda). Can. J. For. Res. 1990, 20: 810-817. 10.1139/x90-107.
Gupta P, Durzan D: Shoot multiplication for mature trees of Douglas fir (Pseudotsuga menziesii) and sugar pine (Pinus lambertiana). Plant Cel. Rep. 1985, 4: 177-179. 10.1007/BF00269282.
Litvay J, Verma D, Johnson M: Influence of a loblolly pine (Pinus taeda L.) culture medium and its components on growth and somatic embryogenesis of the wild carrot (Daucus carrot L:). Plant Cell. Rep. 1985, 4: 325-328. 10.1007/BF00269890.