Efficiency of Agrobacterium rhizogenes–mediated root transformation of Parasponia and Trema is temperature dependent
Parasponia trees are the only non-legume species that form nitrogen-fixing root nodules with rhizobium. Based on its taxonomic position in relation to legumes (Fabaceae), it is most likely that both lineages have gained this symbiotic capacity independently. Therefore, Parasponia forms a bridging species to understand the evolutionary constraints underlying this symbiosis. However, absence of key technologies to genetically modify Parasponia seriously impeded studies on these species. We employed Agrobacterium rhizogenes to create composite Parasponia andersonii plants that harbour transgenic roots. Here, we provide an optimized protocol to infect P. andersonii as well as its non-symbiotic sister species Trema tomentosa with A. rhizogenes. We show that the transformation efficiency is temperature dependent. Whereas the optimal growth temperature for both these species is 28 °C, the transformation is most efficient when co-cultivation with A. rhizogenes occurs at 21 °C. Using this optimized protocol up to 80 % transformation efficiency can be obtained. These robust transformation platforms will provide a strong tool to unravel the Parasponia–rhizobium symbiosis.
KeywordsParasponia Trema Agrobacterium transformation Symbiosis Transgenic root Composite plant
This work was supported by the Dutch Science Foundation (NWO) (VIDI 864.06.007 to R.G.) and a visitors fellowship of the Dutch Graduate School of Experimental Plant Sciences to Q.C.
- Becking JH (1992) The Rhizobium symbiosis of the nonlegume Parasponia. In: Stacey G, Burris RH, Evans HJ (eds) Biological nitrogen fixation: achievements and objectives. Chapman & Hall, New York, pp 497–559Google Scholar
- Burrill TJ, Hansen R (1917) Is symbiosis possible between legume bacteria and non-legume plants? University of Illinois Agricultural Experiment Station, pp 161–181Google Scholar
- Colpaert N, Tilleman S, Montagu MV, Gheysen G, Terryn N (2008) Composite Phaseolus vulgaris plants with transgenic roots as research tool. Afr J Biotech 7:404–408Google Scholar
- Dillen W, Clercq JD, Kapila J, Zambre M, Van Montagu M, Angenon G (1997) The effect of temperature on Agrobacterium tume- faciens-mediated gene transfer to plants. Plant J 12:1459–1463Google Scholar
- Estrada-Navarrete G, Alvarado-Affantranger X, Olivares JE, Díaz-Camino C, Santana O, Murillo E, Guillén G, Sánchez-Guevara N, Acosta J, Quinto C, Li D, Gresshoff PM, Sánchez F (2006) Agrobacterium rhizogenes transformation of the Phaseolus spp.: a tool for functional genomics. Mol Plant Microb Int 19:1385–1393CrossRefGoogle Scholar
- Kondo T, Hasegawa H, Suzuki M (2000) Transformation and regeneration of garlic (Allium sativum L.) by Agrobacterium-mediated gene transfer. Plant Cell Rep 19:989–993Google Scholar
- Lloyd G, McCown B (1980) Commercially feasible micropropagation of mountain laural (Kalmia latifolia) by use of shoot tip cultures. Comb Proc Intl Soc 30:421–427Google Scholar
- Salas M, Park S, Srivatanakul M, Smith R (2001) Temperature influence on stable T-DNA integration in plant cells. Plant Cell Rep 20:701–705Google Scholar
- Young ND, Debellé F, Oldroyd GED, Geurts R, Cannon SB, Udvardi MK, Benedito VA et al (2011) The Medicago genome provides insight into the evolution of rhizobial symbioses. Nature. doi: 10.1038/nature10625