The sum of saponins in the hairy root lines (6, 17) of Platycodon grandiforum A.DC. was compared. Hairy root line 6 showed a higher total saponin content (6.92%) than the line 17 (6.01%). According to the Chinese Pharmacopoeia standards the content of saponins in Platycodi Radix should be not less than 2%. Our results seem to indicate that the hairy root culture of Platycodon grandiforum A.DC. is a good source of saponins. The Haemolytic Index of the hairy root line 6 was 1600. Digitonin was used as a reference. Moreover, the haemolytic activity of TLC subfractions of saponins varied.
P. ixocarpa hairy root cultures were obtained after the transformation with A. rhizogenes strain ATCC 15834. The ability of P. ixocarpa hairy roots to biotransform HQ to arbutin was examined. The roots were treated 3 times with the same HQ concentration on 3 consecutive days or every 3 days. Despite these differences the highest arbutin yield and the highest biotransformation ratio were similar in both variants, 13.1 and 14.4 mg·25 cm⁻³ of the cultures and 67.6% and 70.6%, respectively. However, in the case of shorter intervals between treatments the highest levels of these parameters were achieved earlier. Multiple treatment of lower HQ concentration reduced its harmful effects on root biomass growth.
In order to initiate hairy root culture initiation cotyledons and hypocotyls of Calendula officinalis L. were infected with Agrobacterium rhizogenes strain ATCC 15834 or the same strain containing pCAMBIA 1381Z vector with β-glucuronidase reporter gene under control of promoter of NIK (Nematode Induced Kinase) gene. The efficiency of induction of hairy roots reached 33.8% for cotyledons and 66.6% for hypocotyls together for both transformation experiments. Finally, eight control and nine modified lines were established as a long-term culture. The hairy root cultures showed the ability to synthesize oleanolic acid mainly (97%) as glycosides; control lines contained it at the average 8.42 mg·g-1 dry weight in tissue and 0.23 mg·dm-3 in medium; modified lines: 4.59 mg·g-1 for the tissue, and 0.48 mg·dm-3 for the medium. Additionally lines showed high positive correlation between dry/fresh weight and oleanolic acid concentration in tissue. Using the Killiani mixture in acidic hydrolysis of oleanolic acid glycosides released free aglycones that were partially acetylated in such conditions.
In a hairy root culture of Tanacetum parthenium treated with yeast extract (YE), silver nitrate (AgNO3) and microalgal glycoproteins (MGPS), contents of four spiroketal enol ether type diacetylenes were mesured. The elicitors transiently reduced contents of three constitutive spiroketal enol ethers and selectively enhanced accumulation of cis-C13-spiroketal enol ether epoxide ((E)-3,4-epoxy-2-(2,4-hexadiynylidene)-1,6-dioxaspiro[4.4]nonane) in the roots. The most abundant formation of cis-C13-spiroketal enol ether epoxide was observed after 48-96 h of AgNO3 treatment and 96 h of YE treatment (over 3-fold increase compared with the control). The applied elicitors caused enhanced liberation of cis-C13-spiroketal enol ether epoxide to the culture medium. The results show that diacetylene accumulation pattern in the elicited hairy roots is affected in a similar manner, irrespectively of the elicitor applied.
To study the influence of genetic background on the transformation and regeneration of cultivated tomato plants, hairy root lines of tomato (Lycopersicon esculentum) were obtained by inoculating the hypocotyl explants of three tomato cultivars with the Agrobacterium rhizogenes strain DCAR-2, which harbors the pBI-121 binary vector. The Ri-T-DNA transformation into the plant DNA was confirmed by both of mikimopine and GUS assay analyses. The regeneration efficiency from hairy root explants was assessed. The data indicated that white embryonic calli were formed within two weeks in the presence of 2 mgl-1 2, 4-D plus 0.25 mgl-1 kinetin. Adventitious shoots emerged from the embryonic callus in the presence of 1 mgl-1 GA3 along with 0.5 mgl-1 NAA. The regeneration frequency was higher in the cultivar UC-97, followed by Momotaro and then Edkawi. Molecular confirmation of the integration of the GUS gene into the hairy root-derived plants genomes was done via PCR using GUS-specific primers and also using Southern blotting analysis. Our data shows that regeneration is possible from hairy roots of the cultivated tomato and this system could be used to produce transgenic tomato plants expressing the genes present in Agrobacterium rhizogenes binary vectors.
The growth and saponin accumulation were measured in two lines of transgenic hairy roots of Platycodon grandiflorum, Pl 6 and Pl 17, cultured for 8 weeks in 250-ml shake flasks containing 50 ml of hormone-free woody plant medium supplemented with 40 g/l sucrose and in the Pl 17 line cultured for 12 weeks in a 5-l mist bioreactor containing 1.5 l of the same medium. With both methods, the growth of transgenic hairy roots was assessed as both fresh and dry weight and the biomass growth was correlated with the conductivity and sucrose uptake. The accumulation of saponins was measured and compared with that in roots derived from the field cultivation. The saponin concentrations were significantly higher in the two hairy root lines cultured in shake flasks [6.92 g/100 g d.w. (g%) and 5.82 g% in Pl 6 and Pl 17, respectively] and the line cultured in the bioreactor (5.93 g%) than in the roots derived from the field cultivation (4.02 g%). The results suggest that cultures of P. grandiflorum hairy roots may be a valuable source for obtaining saponins.
The study focused on the production of compounds with antioxidant activity in hairy root and shoot cultures of Salvia officinalis grown in laboratory-scale sprinkle nutrient bioreactors. HPLC analysis showed that production of rosmarinic acid in transformed roots (34.65±1.07 mg l-1) was higher that in shoot culture (26.24±0.48 mg l-1). In the latter diterpenoids: carnosic acid (1.74±0.02 mg l-1) and carnosol (1.34±0.01 mg l-1) were also found. Biomass accumulation after a growth period in the bioreactor was also studied. An 18-fold increase in hairy root biomass was recorded after 40 days of culture. In sage shoot culture, biomass increased 43 times after 21 days of bioreactor run. The current operating conditions of the bioreactor were not suitable for the propagation of Salvia officinalis mainly due to the hyperhydricity problem of leaves and stems.
Rehmannia glutinosa hairy roots were used to evaluate the effect of methyl jasmonate (MeJa) and salicylic acid (SA) on increase of root biomass and production of iridoids (catalpol, harpagide) and phenylethanoids (verbascoside and isoverbascoside). The elicitors were added to 23-day-old culture separately at concentrations between 50 and 200 μM or in combinations at concentrations of 50 and 100 μM. Roots were harvested 72 h and 120 h after elicitation. The type of elicitor, its concentration and exposure time were found to strongly affect the content of each analyzed compound. A 72-hour treatment with 200 μM MeJa was the most effective in increase of verbascoside content (60.07 mg·DWˉ¹ equivalent to 845.45 mg·Lˉ¹) and isoverbascoside (1.77 mg·DWˉ¹ equivalent to 24.94 mg·Lˉ¹): these respective amounts were roughly 10- and 6.4-fold higher than the control values (unelicited roots). Exposure to 150 μM MeJa provided optimal harpagide content after 72 hours (0.136 mg·DWˉ¹; 7.5-fold increase compared to the control), and catalpol content after 120 hours (up to 2.145 mg·DWˉ¹). The combination of MeJa and SA also resulted in higher levels of secondary metabolites compared to the control culture, although these levels were lower than those observed for MeJa alone at the optimal concentration and exposure time. SA alone was less efficient in enhancing metabolite production than MeJa.