Medium hairy root

Fig. 7. Yield of alkaloids in the adventitious roots (left) and hairy roots (center and right) of H. albus cultured in phytohormone-free MS liquid medium (adventitious roots) or in phytohormone-free WP liquid medium (hairy roots).

Bohm, H. and Mack, G., Betaxanthin formation and free amino acids in hairy roots of Beta vulgaris var. lutea depending on nutrient medium and glutamate or glutamine feeding. Phytochemistry 65, 1361, 2004. 

The presence of foreign protein in the medium of plant cultures does not necessarily mean that all or even most of the product can be recovered from the medium. In many expression systems where an appropriate signal sequence has been used, considerable amounts of foreign protein remain within the plant cells and/or tissues. For example, in a comparison of IgG antibody production in tobacco cell suspension and hairy root cultures, a maximum of 72% of the total antibody was found in the medium of the suspension cultures whereas only 26% was found in the medium of the hairy root cultures [17]. This result could indicate that secretion and/or transport across the cell wall was slower in the hairy roots alternatively, it could indicate poorer stability of the secreted protein in the hairy root medium. If foreign proteins are to be purified from the medium, improved secretion and extracellular product stability are desirable. 

Increasing concentrations of PVP 360,000 up to 1.0 g L 1 improved antibody accumulation in hairy root culture medium however, above this concentration there was no further increase in antibody levels [19]. Addition of PVP after extracellular foreign protein levels had decreased during plant suspension culture did not result in a recovery of the protein [66]. 

Supplementation of Gamborg s B5 medium with 0.1% KN03 (in addition to the 0.25% KN03 usually present in B5 medium) resulted in a significant increase in antibody levels in tobacco hairy root cultures [19]. The antibody concentration in the medium increased 2.8-fold and the total antibody accumulation increased by 90% relative to cultures without added nitrate. 

Solution pH has a strong influence on the structure, conformation and solubility of globular proteins. However, when IgO, antibody was added to fresh, sterile B5 medium adjusted to pH values between 4.0 and 8.0, there was no significant difference in the rate at which the antibody disappeared from the different solutions [63]. In other work, antibody-expressing tobacco hairy roots were cultured in B5 medium with initial pH between 3.0 and 11.0 [69]. Root growth was affected severely at the lowest and highest pH values and total antibody levels declined as the initial pH was increased above 5.0-6.0. 

Rhizosecretion is easy to scale up and very cost effective with respect to isolation and purification. However, the bioreactor systems used for hairy root cultures differ from those used for plant cell suspensions. Traditional bioreactor systems have recently been adapted for root culture, and this technology is now being taken to commercial scales. The most traditional system is the airlift bioreactor used for microorganisms or plant cells. This system is adapted for the culturing of roots in liquid medium. Mist culture systems have also been developed. For this technology, the volume of the culture medium is reduced and the concentration of the secreted therapeutic protein is increased. If the protein to be produced is known to be quite stable, then a less expensive hydroponic culture can be designed in a manner suitable for scale-up. 

The hairy roots of P. ginseng C.A. Meyer were initiated and maintained as described previously (18). In all experiments, the hairy roots were cultivated in liquid hormone-free 1/2 MS medium (18) containing 30 g/L of sucrose. The pH of the medium was adjusted to 5.8 with 2 N NaOH, and... 

We reported that an airlift column reactor was superior for the cultivation of hairy roots, in which reticulate polyurethane foam was used as an appropriate support for the even growth and distribution of the hairy roots [44]. A turbine-blade reactor was developed by Nagai et al. [45], in which the cultivation space is separated from the agitation space by a cylindrical stainless-steel mesh and a stainless-steel plate with a slit so that hairy roots do not come into contact with the impeller. The impeller, with 8 turbine blades, is fitted in the agitation space at the bottom of the reactor. The medium flows upwards along the vessel wall, passes through the cylindrical stainless-steel mesh in the center of the re-... 

Through the experiments for characterization of photomixotrophic hairy roots, cultures were carried out at 25 °C using 125-cm3 conical glass flasks containing 50 cm3 MS liquid medium (pH 5.7) with 20 kg m 3 fructose and no phytohormone. The hairy roots were grown with an inoculum of about 0.2 g fresh weight, FW, in flasks shaken at 100 rpm on a rotary shaker located beneath the lamps. 

The inocula for these experiments were prepared by preculturing green hairy roots in MS liquid medium for 14 days at incident light intensity, I, of 11.1 W m 2 under the same conditions as mentioned above. 

The photoautotrophic cultures were carried out at 25 °C using 250-cm3 flat, oblong glass flasks containing 50 cm3 of MS liquid medium. The hairy roots... 

In experiments to evaluate the elongation rate of growing points at root tip meristems, the hairy roots were cultivated under the same conditions with Petri dishes (9 cm in diameter) containing solidified MS medium. 

To determine the resumption potential of the hairy roots, three pieces of the main roots, which were randomly picked up from the liquid cultures at prescribed time, were transferred on the MS solid medium with 20 kg m-3 sucrose... 

The hairy roots, which had various lengths from root tips of L = 5.0 X 10 3 m to 9.0 x 10 2 m, were prepared and placed on MS solid medium after their lateral roots were cut off. The dishes were incubated in a chamber at 5.0% C02 concentration and incident light intensities were kept at 11 W m-2 and 20 W m 2 on the level of the medium surface. 

The Chi in the hairy roots and parent plant organs was extracted with a mixture of 80% acetone and 20% phosphate buffer (2.5 mol m 3,pH 7.8), and analyzed according to the method described by Porra et al. [17]. The activities of SOD and POD in the hairy roots and parent plant organs were spectrophoto-metrically determined using ferricytochrome C [18] and o-aminophenol [19], respectively. Fructose in the medium was analyzed by the Somogyi-Nelson method [20] or was determined using an HPLC with a refractive index detector. Light intensity was measured with a thermopile on the outer walls of flasks at the level of the medium surface or at the top of the Petri dish, and expressed as the mean of values determined at several positions. 

To investigate the influence of a photosynthesis inhibitor on the growth of the hairy roots, 0-10 mmol m 3 of 3-(3,4-dichlorophenyl)-l, 1-dimethylurea, DCMU, was supplemented in the medium according to the procedure described by Horn et al. [21]. The dishes were incubated in a chamber under the varied C02 concentrations of 0.03-8.5%. 

Hairy roots Medium and light conditions Chi content Product tested Ref. 

In the case of culture under plain air, as shown in Fig. 3A, both the Chi content, Cchl, and RubisCO activity, AR, were kept at appreciable levels as long as sucrose was included in the medium (1st to 3rd stages). While being kept in sucrose-free medium (4th stage), however, the hairy roots turned brown and the values of Cchl and AR were ultimately extinguished. 

On the other hand, in the cultures under 3.0% C02 atmosphere, the values of Cqjj and Ar in the hairy roots increased at every culture stage accompanying the decrease in the initial sucrose concentration in the medium as shown in Fig. 3B. At the end of the 2nd and 3rd stages, the sugars (sucrose, glucose, and fructose) were completely consumed in the medium (data not shown). It was thus considered that the hairy roots were acclimating to the photoautotrophic condition... 

---