Abstract: One of the tasks in contemporary biotechnology, pharmacology and other fields of human activities is to obtain biologically active substances from plants. They are very essential in the treatment of many diseases due to their actually high therapeutic value without visible side effects. However, sometimes the possibility of obtaining the metabolites is limited due to the reduction of wild-growing plants. That is why the plant cell cultures are of great interest as alternative sources of biologically active substances. Besides, during the monitored cultivation, it is possible to obtain substances that are not synthesized by plants in nature. Isolated culture of Ajuga genevensis with high growth activity and ability of regeneration was obtained using MS nutrient medium. The agar-diffusion method showed that aqueous extracts of callus culture revealed high antimicrobial activity towards various gram-positive (Bacillus subtilis A1WT; B. mesentericus WDCM 1873; Staphylococcus aureus WDCM 5233; Staph. citreus WT) and gram-negative (Escherichia coli WKPM M-17; Salmonella typhimurium TA 100) microorganisms. The broth dilution method revealed that the minimal and half maximal inhibitory concentration values against E. coli corresponded to the 70 μg/mL and 140 μg/mL concentration of the extract respectively. According to the photochemiluminescent analysis, callus tissue extracts of leaf and root origin showed higher antioxidant activity than the same quantity of A. genevensis intact plant extract. A. genevensis intact plant and callus culture extracts showed no cytotoxic effect on K-562 suspension cell line of human chronic myeloid leukemia. The GC-MS analysis showed deep differences between the qualitative and quantitative composition of callus culture and intact plant extracts. Hexacosane (11.17%); n-hexadecanoic acid (9.33%); and 2-methoxy-4-vinylphenol (4.28%) were the main components of intact plant extracts. 10-Methylnonadecane (57.0%); methoxyacetic acid, 2-tetradecyl ester (17.75%) and 1-Bromopentadecane (14.55%) were the main components of A. genevensis callus culture extracts. Obtained data indicate that callus culture of A. genevensis can be used as an alternative source of biologically active substances.
Abstract: The in vitro culture procedure of purple nutsedge
(Cyperus rotundus L.) for multiple shoot induction and tuber
formation was established. Multiple shoots were significantly
induced from a single shoot of about 0.5 – 0.8 cm long, on Murashige
and Skoog (MS) medium supplemented with 4.44 μM 6-
benzyladinine (BA) alone or in combination with 2.85 μM 1-
indoleacetic acid (IAA), providing 17.6 and 15.3 shoots per explant
with 31.2 and 27.5 leaves per explant, respectively, within 6 weeks of
culturing. Moreover, MS medium supplemented with 4.44 μM BA
and 2.85 μM IAA was suitable for tuber induction, obtaining 5.9
tubers with 3.4 rhizomes per explant. In combination with ancymidol
and higher concentration of sucrose, 11.1 μM BA and 60 g/L sucrose
or 11.1 μM BA, 7.8 μM ancymidol and 60 g/L sucrose induced 3.5
tubers with 1.6 rhizomes or 3.5 tubers without rhizome, respectively.
However, MS medium containing 3.9 or 7.8 μM ancymidol in
combination with either 60 or 80 g/L sucrose enchanced significant
root formation at 20.9 – 23.6 roots per explant.
Abstract: The alternative technique for sterilization of culture
medium to replace autoclaving was carried out. For sterilization of
culture medium without autoclaving, some commercial pure essential
oils, bergamot oil, betel oil, cinnamon oil, lavender oil and turmeric
oil, were tested alone or in combinations with some disinfectants,
10% povidone-iodine and 2% iodine + 2.4% potassium iodide. Each
essential oil or combination was added to 25-mL Murashige and
Skoog (MS) medium before medium was solidified in a 120-mL
container, kept for 2 weeks before evaluating sterile conditions.
Treated media, supplemented with essential oils, were compared to
control medium, autoclaved at 121 degree Celsius for 15 min. In
vitro sterile conditions were found 20 – 100% from these treated
media compared to 100% sterile condition from autoclaved medium.
Treated media obtained 100% sterile conditions were chosen for
culturing chrysanthemum shoots. It was found that 10% povidoneiodine
in combination with cinnamon oil (3:1) and 2% iodine + 2.4%
potassium iodide in combination with lavender oil (1:3) at the
concentration of 36 3L/25 mL medium provided the promising
growth of shoot explants.
Abstract: Plant tissue culture is an important in vitro technology applied for agricultural and industrial production. A sterile condition of culture medium is one of the main aspects. The alternative technique for medium sterilization to replace autoclaving was carried out. For sterilization of plant tissue culture medium without autoclaving, ten commercial pure essential oils and 5 disinfectants were tested. Each essential oil or disinfectant was added to a 20-mL Murashige and Skoog (MS) medium before medium was solidified in a 120-mL container, kept for 2 weeks before evaluating sterile conditions. Treated media, supplemented with essential oils or disinfectants, were compared to control medium, autoclaved at 121 degree Celsius for 15 min. Sterile conditions of MS medium were found 100% from betel oil or clove oil (18 mL/20 mL medium), cinnamon oil (36 mL/20 mL medium), lavender oil or holy basil oil (108 mL/20 mL medium), and lemon oil or tea tree oil or turmeric oil (252 mL/20 mL medium), compared to 100% sterile condition from autoclaved medium. For disinfectants, 2% iodine + 2.4% potassium iodide, 2% merbromine solution, 10% povidone-iodine, 6% sodium hypochlorite or 0.1% thimerosal at 36 mL/20 mL medium provided 100% sterile conditions. Furthermore, growth of new shoots from chrysanthemum node explants on treated media (fresh weight, shoot length, root length and number of node) were also reported and discussed in the comparison of those on autoclaved medium.
Abstract: Our results showed that for the growth of qualitative
seedling and vegetative raw material of ðó. marschallianus Willd. and
T. serphyllum L. it is more profitable to use the in vitro and
hydroponics combined method. In in vitro culture it is possible to do
micro-propagation whole year with 98-99% rhizogenesis. 30000
micro-plants were obtained from one explant during 9 months.
Hydroponic conditions provide the necessary microclimate for
microplants where the survival rate without acclimatization was
93.3%. The essential oil content in hydroponic dry herb of both
species in vegetative and blossom phase was 1.3% whereas in wild
plants it was 1.2%, the content of extractive substances and vitamin
C also exceeded wild plants. Our biochemical and radiochemical
investigations indicated that the medicinal raw materials obtained
from hydroponic and wild plants of Thymus species correspond to
the demands of SPh XI, and the content of artificial radionuclides
does not exceed the MACL.
Abstract: The extract of milk thistle contains a mix of flavonolignans termed silymarine.. In order to analysis influence of growth regulators, genotype, explant and subculture on the accumulation of flavonolignans, a study was carried out by using two genotype (Budakalszi and Noor abad moghan cultivars), cotyledon and hypocotyle explants, solid media of MS supplemented by different combinations of two growth regulators; Kinetin (0.1, 1 mg/l) and 2,4-D (1, 2 mg/l). Seeds of the plant were germinated in MS media whitout growth regulators in growth chamber at 26°C and darkness condition. In order to callus induction, the culture media was supplemented whit different concentrations of 2,4-D and kinetin. Calli obtained from explants were sub-cultured four times into the fresh media of the first experiment. flavonoides was extracted from calli in four subcultures. The flavonoid components were determined by high- performance liquid choromatography (HPLC) and separated into Taxifolin, Silydianin+Silychristin, Silybin A+B and Isosilybin A+B. Results showed that with increasing callus age, increased accumulation of silybin A+B, but reduced Isosilybin A+B content. Highest accumulation of Taxifolin was observed at first calli. Calli produced from cotyledon explant of Budakalszi cultivar were superior for Silybin A+B, where calli from hypocotyl explant produced higher amount of Taxifolin and Silydianin+Silychristin. The best cultivar for Silymarin production in this study was Budakalszi cultivar. High amount of SBN A+B and TXF were obtained from hypocotil explant.
Abstract: The effects of chitosan, a biodegradable polymer,
were studied in Grammatophyllum speciosum protocorm-like bodies
(PLBs) in vitro culture. The chitosan concentration of 0, 5, 10, 15,
20, 25, 50 or 100 mg/l were supplemented in half-strength Murashige
and Skoog (1/2 MS) liquid or on agar media containing 2% (w/v)
sucrose. The results showed that liquid medium supplemented with
15 mg/l chitosan showed the highest relative growth rate (7-fold
increase) of PLBs. On 1/2 MS agar medium supplemented with 25
mg/l chitosan gave the highest relative growth rate (4-fold increase).
The relative growth rate of G. speciosum PLBs on agar medium was
significantly lower than that in liquid medium. Moreover, chitosan,
supplemented to agar medium promoted shoot formation but not
rooting. However, supplementation at too high a level, such as 100
mg/l can inhibit growth and kill PLBs.
Abstract: Shoots, with three leaves, of Paphiopedilum 'Delrosi'
were used as explants for multiple shoot induction. Modified
Hyponex medium was supplemented with thidiazuron (TDZ), N6-
benzyladenine (BA) or kinetin (Kn) alone and in combinations with
2,4-dichlorophenoxyacetic acid (2,4-D). All explants were cultured
for 15 weeks. It was found that TDZ alone at the concentration of
0.45μM or in combination with 4.52μM 2,4-D and 8.88μM BA in
combination with 13.56μM 2,4-D promoted multiple shoots. The
highest shoot sprouting efficiencies (80.0, 90.0 and 80.0%) and new
shoot numbers (1.5, 1.3 and 1.1) were obtained, respectively. Fresh
weight, height, numbers of leaf and root of new shoots and initial
explants were discussed.
Abstract: Bone remodeling occurs by the balanced action of
bone resorbing osteoclasts (OC) and bone-building osteoblasts.
Increased bone resorption by excessive OC activity contributes
to malignant and non-malignant diseases including osteoporosis.
To study OC differentiation and function, OC formed in
in vitro cultures are currently counted manually, a tedious
procedure which is prone to inter-observer differences. Aiming
for an automated OC-quantification system, classification of
OC and precursor cells was done on fluorescence microscope
images based on the distinct appearance of fluorescent nuclei.
Following ellipse fitting to nuclei, a combination of eight
features enabled clustering of OC and precursor cell nuclei.
After evaluating different machine-learning techniques, LOGREG
achieved 74% correctly classified OC and precursor cell
nuclei, outperforming human experts (best expert: 55%). In
combination with the automated detection of total cell areas,
this system allows to measure various cell parameters and most
importantly to quantify proteins involved in osteoclastogenesis.