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Improving the growth of tropical nitrogen-fixing forest trees in the genera acacia and casuarina through tissue culture and genetic transformation

Objective

The goal of the project is to develop genetic transformation strategies for nitrogen-fixing species of tropical trees in the genera Acacia (Acacia mangium, Acacia mearnsii and Acacia crassicarpa) and Casuarina (Caruarina glauca and Allocasuarina verticillata). Gene transfer technologies will be used to introduce agronomically important traits such as insect resistance and to obtain new tree-microorganism symbiosis that are more adapted to the ecological features of the planting sites.
- Techniques for the micropropagation of superior clones of Acacia sp. and Casuarina will be developed;
- Efficient regeneration of whole plants starting from somatic embryogenesis of Acacia and Casuarina will be established;
- Using the beta-glucuronidase gene and a selection marker, transformation of Acacia and Casuarina will be achieved using either the natural vector Agrobacterium or direct gene transfer techniques (high velocity microprojectiles), transgenic plants will be regenerated;
- Using the beta-glucuronidase gene under the control of different known regulatory sequences, constitutive or tissue-specific expression vectors will be identified for Acacia and Casuarina, stability of the transgenes will be studied;
- Using the most appropriate vector, insect resistance genes will be introduced into Acacia and Casuarina and transgenic plants will be regenerated.
Expected Outcome

- Established in vitro tissue culture and regeneration of Acacia species and Casuarina glauca;
- Techniques for the micropropagation of selected mature clones of Acacia and Casuarina developed and tested;
- Most efficient selective marker for the genetic transformation of the tree species identified;
- Meristem-specific promoters used to follow the transfer of the beta-glucuronidase gene into meristematic cells of Acacia and Casuarina;
- Transformed tissues of Acacia and Casuarina obtained using either Agrobacterium or direct gene transfer techniques (electroporation and high velocity microprojectiles);
- Efficiency of transformation improved;
- Transformed plants of Acacia and Casuarina regenerated;
- Level and specificity of expression of different gene constructs in transgenic tissues and in transformed plants analysed and quantified;
- Insect resistance genes introduced into Acacia and Casuarina.

Results

- Identification of superior clones of A. mangium with good organogenic potential.
- Micropropagation of A. mangium and A. mearnsii.
- Micropropagation of C. glauca from using shoots from mature trees.
- Using thidiazuron, differentiation of buds on A. mangium calli derived from hypcotyls.
- Regeneration of rooted plants from cotyledons of A. crassicarpa.
- Identification of selection markers for Acacia and Casuarina.
- Induction of tumors after inoculation of A. mangium, A. mearnsii and A. crassicarpa by wild-type A. tumefaciens strains.
- Transfer of the beta-glucuronidase gene into A. mangium using a wild-type A. tumefaciens strain.
- Transient expression of the uidA gene in Acacia mangium calli following particle bombardment.
- Transgenic buds of Casuarina glauca expressing the beta-glucuronidase and nptII genes were obtained after transformation of epicotyls with the disarmed strain C58C1(GV2260, BIN19GUSint).
- Regeneration of transgenic A. verticillata plants expressing the beta-glucuronidase gene under the control of constitutive or tissue-specific promoters.
- Nodulation by Frankia of transgenic A. verticillata.

Follow-up

- Gene transfer into Acacia using disarmed strains of Agrobacterium tumefaciens and growth of transformed tissues;
- Regeneration and characterisation of transgenic modulated C. glauca trees;
- Stability of expression of the 35S promoter in a tropical nitrogen-fixing tree of the family Casuarinaceae, identification of expression vectors for Casuarinaceae;
- Basic knowledge of the specificity of expression of several promoters in nodulated Casuarinaceae trees;
- Transformation of Casuarinaceae with valuable genes.
- Micropropagation;
- Regeneration procedure;
- Identification of a selection marker for the genetic transformation of Acacia and Casuarina;
- Transformation vectors to follow shoot meristem formation;
- Agrobacterium-mediated DNA transfer;
- Direct DNA transfer by high velocity microprojectiles;
- Analysis of transformed tissues;
- Expression vectors for Acacia and Casuarina;
- Introduction of important trait.

Funding Scheme

CSC - Cost-sharing contracts

Coordinator

Institut Français de Recherche Scientifique pour le Développement en Coopération (ORSTOM)
Address
45 Bis Avenue De La Belle Gabrielle
94736 Nogent-sur-marne
France

Participants (5)

Fundação Bio Rio
Brazil
Address
N,ccs-bloco A 2 Andar-sala 098 Ufrj-rj
21944 Rio De Janeiro
Innoprise Corporation SDN BHD
Malaysia
Address

91017 Tawau
TEAGASC - AGRICULTURE AND FOOD DEVELOPMENT AUTHORITY
Ireland
Address
Malahide Road
17 Dublin
Universiteit Gent
Belgium
Address
35,K.l. Ledeganckstraat 35
9000 Gent
Université Mohammed V
Morocco
Address
Avenue Ibn Batouta
10000 Rabat