Hairy root culture


Hairy roots result from the transfer and integration of the genes located on the Ri (root-inducing) plasmid of Agrobacterium rhizogenes into the plant genome and their expression therein (White and Nester, 1980). The transferred genes are involved in the synthesis of plant hormones,  expressing in the plant cell and giving rise to rooty tumors at the infection site. These types of roots are characterized by fast growth, frequent branching, plagiotropism, and the ability to synthesize the same compounds as the roots of the intact plant (David, 1984).

The responsible bacteria

Agrobacterium rhizogenes is a gram negative soil bacterium. It incites hairy root disease of many dicotyledonous plants (Brown, 1929; DeCleene and DeLey, 1981; Riker et al.,1930; Siegler, 1928). The ability of A. rhizogenes to incite hairy root disease is determined by a virulence plasmid (Chilton. et al.,1982; Moore et al.,1979; White and Nester, 1980) similar to Ti (tumor inducing) plasmid found in Agrobacterium tumefaciens which causes Crown gall tumors of plants. The virulence plasmid of A. rhizogenes is known as the Ri-plasmid to distinguish it from the tumor-inducing (Ti) plasmid.

Mechanism

The Ri–plasmid contains a distinct segments of DNA which is transferred to plant genome during infection (Chilton et al.,1982; White et al.,1982; Willmitzer et al.,1982). The transfer of the DNA (T-DNA) to the plant genome is mediated by another segment on the plasmid known as the virulence (vir) region. Hairy root induction and morphology are controlled by the rol (A, B, C and D) genes from the A. rhizogenes Ri plasmid (White et al. 1985). The rol genes have also been found to affect secondary metabolite production (Sevón et al., 1997; Bonhomme et al., 2000; Bulgakov et al., 2004). The T-DNA confers on the plant cells the ability to grow in the absence of exogenous plant hormones. The T-DNA also confers on the transformed tissue the ability to produce modified amino acids (opines), which, in turn, are utilized only by the inciting bacteria as the carbon, nitrogen and energy source. The Agrobacterium species thus establish a unique ecological niche by genetically engineering the host plant—a highly sophisticated parasitism! Modern Agrobacterium mediated gene transfer to plants utilizes binary vectors in which the T-DNA and the vir region can reside on separate plasmids (Hellens et al., 2000).


Establishment of transformed root cultures

1. Transformed roots are obtained by infecting surface sterilized leaf and stem segments with Agrobacterium rhizogenes strain A4.


2. The strain A4 harbors the root inducing plasmid pRiA4 and engineered to contain in additional plasmid Bin 19, which harbors a kanamycin-resistant gene as a selectable marker. 

3. An overnight bacterial suspension in yeast mannitol broth (YMB) (Hooykaas 1977) supplemented with 50µM acetosyringone (as virulence inducer) is used for inoculation into freshly wounded explants. 

4. Infected samples are transferred to one tenth Murashige and Skoog (MS) agar solidified media (Murashige & Skoog 1962), kept in the dark for 48 h then incubated under 16 hour photoperiod at light intensity of approximately 1.8 wm-2 at 20 ± 2 °C. 

5. The putative hairy roots, are transferred to hormone-free MS liquid media supplemented with 30 g l-1 sucrose. Ampicillin sodium salt 500 mg l-1 is added until cultures are free from the residual bacteria. 

6. The putative transgenic roots are maintained on the same liquid media (50 ml in 250 ml flasks) on gyratory shakers (90 rpm), at 20 ± 2 °C in the dark or under illumination for 16 h day -1 or under continuous light. 

7. The roots are sub-cultured every two weeks. 

Ref: 
Amani M. Marzouk, Stanley G. Deans, Robert J. Nash and Alexander I. Gray (2011). Transformed Root Cultures of Solanum dulcamara L.: A Model for Studying Production of Secondary Metabolites, Genetic Transformation, Prof. MarÃa Alvarez (Ed.), ISBN: 978-953-307-364-4
Fig.  Agrobacterium rhizogenes induced hairy root culture of Hyoscyamus muticus (Courtesy Annika Wilhelmson)

Fig. Agrobacterium injects SS-DNA into the host cells

Fig. Schematic representation of Agropine type Ri plasmid of A. rhizogenes


Fig. A petri dish culture of Agrobacterium rhizogenes



Fig. Agrobacterium (Courtesy Shirley Owens, Michigan State University)