Genetic transformation of the actinorhizal tree Allocasuarina verticillata by Agrobacterium tumefaciens

C. Franche1,*, D. Diouf1, Q.V. Le1, D. Bogusz1, A. This capacity for rapid growth comes from a symbiotic association with an actinomycete of the genus Frankia, N’Diaye1, H. Gherbi1, C. Gobé1 and E. Duhoux1,2 1Physiologie Cellulaire et Moléculaire des Arbres which allows A. verticillata to fix dinitrogen in root nodules, so-called actinorhizae (Benson and Silvester, 1993). (ORSTOM GeneTrop), 911 avenue Agropolis, BP 5045, 34032 Montpellier cédex 1, France Actinorhizae are found in at least 20 other angiosperm genera that belong to eight different families. Little is 2Université Paris VII-Denis Diderot, 2 Place Jussieu, 75005 Paris, France known about the biochemistry and molecular biology of the infection process and nodule formation in actinorhizal plants. Evidence for nodule-specific gene expression in Summary two actinorhizal plants, Alnus glutinosa and Casuarina glauca, has been recently reported (Gherbi et al., 1996; We have developed an efficient transformation system for the tropical actinorhizal tree Allocasuarina verticillata Goetting-Minesky and Mullin, 1994; Jacobsen-Lyon et al., 1995; Ribeiro et al., 1995). Studying the similarities and using Agrobacterium tumefaciens mediated gene transfer. Mature zygotic embryos were inoculated with the disdifferences between these actinorhizal symbioses and the Rhizobium–legume symbiosis is of great interest since it armed strain C58C1 carrying, in the binary vector BIN19, the nptII gene, providing kanamycin resistance as a could lead to a better understanding of the evolution of symbiotic associations in general. selectable marker, and the reporter gene β-glucuronidase containing an intron. The transformed embryos were Transgenic plants are valuable tools in exploring the regulation of plant symbiotic gene expression (de Bruijn cultivated on nutrient medium supplemented with 0.5 μM NAA, 2.5 μM BA, 100 mg l–1 kanamycin and 250 mg l–1 et al., 1990). The capacity of actinorhizal trees for transformation has been reviewed by Sederoff (1995). Tumours cefotaxime. After 2 months, a 21% transformation frequency was obtained. Within 6–9 months, transgenic were obtained after genetic transformation of Alnus incana and Alnus glutinosa by wild-type A. tumefaciens strains plants were recovered from 70% of the transformed calli. The presence of the transgenes was demonstrated by PCR (Mackay et al., 1988). Transient expression of the β-glucuronidase gene under the control of the 35S promoter was analysis and by the expression of the β-glucuronidase; integration of the T-DNA was confirmed by Southern observed after electroporation of A. incana protoplasts (Seguin and Lalonde, 1988). Up to now the only transgenic hybridization. More than 100 transgenic plants from a total of 23 independent transformation events have been actinorhizal plant reported in the literature was obtained when A. verticillata was transformed by A. rhizogenes successfully established in soil. The possibility to obtain nitrogen-fixing nodules after inoculation of transgenic (Phelep et al., 1991). This paper reports that Agrobacterium tumefaciens can A. verticillata plants by the actinomycetal strain of Frankia Allo2 was established. be used successfully to transfer foreign genes from a binary vector into Allocasuarina verticillata. Transgenic plants were recovered, and we obtained nitrogen-fixing Introduction actinorhizae expressing the β-glucuronidase gene under the control of the 35S promoter. Allocasuarina verticillata Lam. belongs to the family Casuarinaceae, which consists of about 96 species of trees and shrubs native to Malaysia and the Australo-Pacific Results region. Casuarinaceae are multipurpose pioneer tree species and have a wide range of uses, from environmental Tolerance of Allocasuarina verticillata to selective agents protection to fuelwood production (Dawson, 1990; National Allocasuarina verticillata sensitivity to kanamycin (KM) and Research Council, 1984). A. verticillata has the ability to cefotaxime (CFX) was established prior to transformation grow vigorously on poor coastal sands and saline soils. experiments in order to determine the effective concentration for selecting transformed cells and eliminating Received 20 May 1996; revised 11 December 1996; accepted 7 January 1997. Agrobacterium. In the absence of antibiotics, green com*For correspondence (fax 133 4 67 63 82 65; e-mail [email protected]). pact calli developed after 1 week on all the wounded