Host Root Transformation

Agrobacterium rhizogenes Conn., a gram-negative soil bacterium which induces hairy root disease of dicotyledonous plants, is used to induce hairy roots. In roots transformed with this bacterium, a segment of the bacterial DNA, named the region T (transferred DNA) of the plasmid Ri (root inducing) is incorporated into the host plant cells (Chilton et al. 1982). Integration and expression of this DNA in the plant genome lead to the development of the hairy root phenotype and synthesis of novel low molecular weight compounds called opines (Tepfer and Tempé, 1981). Depending on the strain of A. rhizogenes used for the transformation, different principal opines can be found in the tissues of the hairy root: agropine, mannopine, cucumopine or mikimopine (Dessaux et al. 1992). In addition, depending on the gene incorporated in the plant genome, the root can have a change of geotropism. Some hairy roots show a highly negative geotropic nature, some only have a slightly negative geotropic behaviour whileotherskeeptheir positivegeotropism. Thisisduetoachangeofauxin sensitivity and the redistribution of this hormone after root transformation (Legué et al. 1996). Roots with a negative geotropism should be incubated in inverted position, to make the roots grow inside the medium.

In this section, only the transformation procedure of carrot (Daucus carota L.) with A. rhizogenes A4 is described. For the transformation procedures of other plants used as hosts in monoxenic cultures, we refer to the references given in Table 1 of Chapter 7.

5.2.1 Material

Equipment

- Laminar flow hood

- Cooled heated incubator

- Bunsen burner or bead sterilizer Laboratory material

- Forceps

- Scalpel

- Petri plates filled with 1% water agar

- Petri plates filled with modified White's (Bécard and Fortin 1988) medium supplemented with 500 mg/l carbenicillin

- Small plastic wrap roll

Disinfection solutions

- Distilled water Bacterial strain

- Agrobaterium rhizogenes strain A4

Procedure (Following Becard and Fortin 1988)

1. Thoroughly wash the carrots in water, peel them, dip in 95% (v/v) ethanol for 10 s, and surface sterilize in 1% NaOCl for 15 min.

2. Rinse the carrots in sterile distilled water and cut transversely into 5-mm-thick slices with a sterilized scalpel.

3. Place the slices in Petri plates filled with 1% water agar and inoculate with the A4 A. rhizogenes strain on the distal face of the sections. The bacterial suspension is taken from a 2-day-old culture grown on Difco Nutrient agar.

4. After 3 weeks, some transformed roots proliferate on the inoculated sections. Aseptically excise and transfer the roots to Petri plates containing modified White's medium, supplemented with 500 mg/l car-benicillin. Three successive sub-cultures are necessary to free the transformed roots of bacteria.

5. Finally, excise one root apex from the final subculture (minimum length 3 cm) and place it on fresh modified White's medium to initiate a clonal culture.

6. Seal the Petri plates with plastic wrap and incubate in an inverted position in the dark at 27 °C.

7. Maintain the excised root on MSR medium as described below.

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