Establishment and analysis of tissue and fast-growing normal root cultures of four Gentiana L . species , rare highland medicinal plants

N. M. Drobyk, V. M. Mel’nyk, L. R. Hrytsak © 2018 N. M. Drobyk et al.; Published by the Institute of Molecular Biology and Genetics, NAS of Ukraine on behalf of Biopolymers and Cell. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited UDC 577.127: 582.923.1 + 58.085


Introduction
Gentiana L., the largest genus of Gentianaceae Juss.family, includes about 400 species [1,2].In Ukraine Gentiana genus is represented by 10 species.Most of them are spread throughout the highland regions in the Carpathians [3,4].Gentians are extensively used for traditional and folk medicine in the world and, therefore, are currently the subject of intensive phytochemical investigations.Their medicinal activity results from several classes of secondary metabolites produced and stored in various parts of the plants.These are such compounds as alkaloids, iridoids, xanthones, flavonoids, carbohydrates, aromatic and nitrogen-bearing compounds, pectic substances, ascorbic acid, catechines etc. [5,6].
Flavonoids and xanthones are of special interest as they are characterized by a wide spectrum of influence on humans.
Flavonoids have anti-inflammatory, antispasmodic, capillary strengthening, choleretic, diuretic, anti-sclerotic, antineoplastic, anti-oxidant, detoxifying and other effects.They affect the enzyme systems, immune and metabolic processes, thicken membranes, prevent storage of free radicals in tissues, improve the motor activity, secretory and absorbing functions of alimentary canal, etc. [5,6].
Due to the uncontrolled use of roots for glucoside extraction and for liquor manufacturing, some of the species, like G. lutea are close to extinction in their natural habitats.G. lutea and related gentians are under protection as endangered species in most European countries [12], which is one more reason for expediency of their introduction and growing in vitro culture.
In vitro culture of tissues and organs is one of the alternative sources of medicinal raw material with limited natural store.At the same time, some changes in secondary metabolism take place under the conditions of growing in vitro.These include a lack of cell differentiation in callus and suspension cultures, differences of the biosynthetic potential of tissue and organ cultures derived from the various types of explants and plant genotypes, and structural and functional rearrangements of the cell genome in culture [13].During the subcultivation process or in the initial callus cultures in many cases one can observe a considerable decrease of BAS and their synthesis may regenerate only after formation of some morphogenic structures, i. e. after the restoration of organism regulation level [13].Therefore, for biosynthesis and accumulation of valuable secondary metabolites, along with the non-morphogenic tissue culture a morphogenic culture, in particular isolated roots, is promising.
Scientific literature describes the isolated roots ability to synthesize the biologically active substances immanent to roots of whole plants [14].To increase the productivity of isolated root culture, the transformation due to Agrobacterium rhizogenes is used.Herewith the so called "hairy roots" are obtained that are characterized by a high growth rate in the phytohormones free medium, the genetic stability and capacity of intense synthesis of valuable secondary metabolites [14,15].Such method of obtaining isolated root culture was used for gentians [16][17][18][19].The biosynthetic activity was investigated in some gentians hairy root cultures [20][21][22][23][24].
The objective of this work was to obtain tissue and isolated root cultures from some Gentiana species and to study peculiarities of their growth and content of flavonoids and xanthones in them.

Materials and Methods
Plants of four Gentiana species were obtained from seeds harvested in different localities (Table 1), and grown in aseptic conditions in vitro.
For callus induction we used root explants of axenic plants of 9 genotypes of 4 Gentiana species (Table 1).Every experiment variant included testing 100-150 explants.The basic media used for callus induction and proliferation were B 5 (only for G. аsсlepiadea L.) [25], and MS-based medium [26] with full (MS) and twice decreased macro-and microsalts concentrations (MS/2), supplemented with various combinations and concentrations of plant growth regulators (PGR) including Кin, BAP, (2,4-D and NAA.The cultures were incubated in darkness at 25-26.5°С, with subculture every 4 weeks.The percentage of callus formation was recorded after 3 weeks of cultivation.Callus induction (CI) frequency was determined as follows: where Nc -number of explants developing callus; N -number of explants cultured.
To determine the optimal conditions for callus proliferation, 0.25-0.35g portions were separated from explants and placed on nutrient media with various combinations of BAP and 2,4-D (Fig. 1A).
The growth index (GI) according to callus fresh weight was determined after 21 days according to the formula: where M -callus weight after 21 days; minitial callus weight.
Fast-growing isolated root cultures were obtained from plants of different genotypes of four Gentiana species (Table 1).
Root apices each 1.5 cm long and 20-30 mg were used as initial explants for obtaining isolated root cultures, the inocula being taken from two-month axenic plants.Root cultures were obtained in two stages.At the first stage, the inocula were cultured for 2-3 weeks in MS/2 or B 5 (only for G. аsсlepiadea) liquid nutrient medium, supplemented with various concentrations of NAA and BAP or Кin (Fig. 1B).At the second stage, cultured roots with side rootlets were cultured in MS/2 or B 5 liquid medium without growth regulators for 2-3 weeks (Fig. 1C).The pH of both media was 5.6 ± 0.2 before autoclaving.
With regard to data concerning the calciphilous nature of the species, an increased CaCl 2 concentration was used for G. acaulis [27].
The volume of medium at both stages of culture was 50 ml, in 250 ml wide-mouth Erlenmeyer flasks with constant stirring on a shaker with 60-80 vibrations/min without light at 24±2 ºС.
The inocula before placing in the medium and isolated root cultures after 4-6 weeks of culture (the first and second culture stages) were weighed in axenic conditions with further determination of their growth index by fresh weight according to Eq. 2, where M -isolated root weight after 4-6 weeks of culture, and m -initial inoculum weight.
Additionally to growth index, the mean number of side rootlets per cultured inocula were determined as well as the mean length after the first and second stages of culture.Establishment and analysis of tissue and fast-growing normal root cultures of four Gentiana L. species, rare highland medicinal plants These parameters are important to characterize the isolated root cultures, as they indicate the ability of roots to grow continuously [27].Isolated tips of the main roots are not capable of growing in culture for a prolonged period and their continued use as inocula is accompanied by "ageing" of meristems resulting in death.This excludes the possibility to obtain sufficient biomass.By using the tips of side rootlets as inocula due to their number and vitality, the growth culture cycle is considerably extended.
The material for the research involved tissue cultures of root origin derived from plants of different genotypes at the 11-15th passages of growth (Table 1) and fast-growing root cultures, as well as wild plants of the target species.
Xanthones and flavonoids were quantified in isolated root cultures after the second stage of growth, in callus tissues at the end of the 11-15th passage, and in wild plants within the fruiting phase.To evaluate the biosynthetic activity of callus tissues, they were compared by the BAS content with shoots and roots of plants from natural localities.
Total xanthone amount was determined by the modified chromatospectrophotometric method [28].Mangiferin was used as a standard.The air-dried raw material was hydrolyzed in a mixture of acetone and water 1:1 (v:v; mixture А), containing 5 % HCl, on a water bath for 1 hour.Using micropipette with the investigated extract and standard mangiferin solution (0.03 ml of each), on the plate with cellulose of 20 × 20 cm in size we formed two-centimetre stripes three times.One more stripe was left clean and used as a negative control.After chromatography in a saturated 15 % solution of acetic acid, the plates were analyzed under ultraviolet light (360 nm).At the level of standard mangiferin spots of the stripes with the investigated extract there were marked zones, containing xanthones.The stripe with negative control had an equal in area spot of cellulose used for the preparation of control solution.All the marked areas of cellulose were quantitatively transferred and then desorbed in 10 ml of mixture A and periodically shaken for 15 minutes.The optical density of filtrated solutions was determined spectrophotometrically at 369 nm against a reference solution.
The total xanthone content was calculated from a standard mangiferin curve and expressed as the mg mangiferin equivalent per 1 g of dry weight.
Total flavonoid content was determined by a spectrophotometric method [28,29] with rutin as a standard.Samples were air dried at room temperature to constant weight.We extracted 1] g of dry raw material using 70 % ethanol for 30 minutes in a flask equipped with a reverse cooler on a boiling water bath.The solution obtained was filtered.1 ml of filtrate was placed into a 25 ml volume measuring flask, 5 ml of 2 % AlCl 3 solution in 95 % ethanol were added, and total volume was adjusted to 25 ml with 95 % (w:v) ethanol.Within 30 minutes, the optic density of the solution was measured with a spectrophotometer SPh-46 (410 nm).
The total flavonoid content was calculated from a calibration curve, and the result was expressed as the mg rutin equivalent per 1 g of dry weight.
The growth and biosynthetic characteristics of calli and root cultures were assessed on the basis of dry weight outcome per 1 l of nutrient medium, as well as on the flavonoid and xanthone content in dry material obtained from 1 l of nutrient medium (further -flavonoids and xanthones outcome from 1 l of nutrient medium).BAS (flavonoids or xanthones) per 1 l of nutrient medium (Х 3 ) was calculated according to the formula: where m dry -outcome of tissue culture or isolated root culture dry weight from 1 l of nutrient medium, g; Х 1 -flavonoid total content in % of dry weight of raw material; Х 2xanthone total content in % of dry weight of raw material.ANOVA dispersion analysis with Tukey's HSD (honestly significant difference) test was performed with use of Prism 6.In checking statistical hypotheses of the research the critical level of significance was equal to 0.05.
In some species the efficiency of callus formation on the same culture medium depended on the genotype.For example, in the case of G. asclepiadea and G. acaulis the difference between the highest CI of two different genotypes was approximately 20 % (Table 2).G. asclepiadea callus induced on МS and МS/2 media underwent necrosis after 4-5 subcultures.We could obtain stable and highly proliferating callus using B 5 medium only (Table 2).Callus induction was governed by the composition of MS culture medium.CI was up to 5 times higher on MS/2 than on standard MS medium in most samples.
The ratio and composition of PGR significantly affected the callus induction and proliferating activity.Callus obtained on MS with BAP and NAA had low CI (6-31 %) and short viability period.Only after one subculture multiple the necrosis in tissue was observed.The use of BAP (0.1 mg/l) and 2,4-D (0.5, 1 mg/l) stimulated the formation of pale yellow, friable callus with high proliferation potency.The highest callus induction frequency in most samples was obtained on the MS/2 medium supplemented with 0.1 mg/l BAP and 0.5 mg/l 2,4-D.However, in G.ac.T it was higher on the medium with 1 mg/l 2,4-D (Table 2).In the case of G. asclepiadea (G.asc.M genotype), the medium B 5 supplemented with 0.2 mg/l Kin stimulated CI more than 90 % (Table 2).
The МS/2 medium was optimal for callus proliferation of most species except for G. asclepiadea where the B 5 medium showed better results.We did not discover essential interspecies differences in the callus growth intensity (Fig. 2).The BAP and 2,4-D combination favored growth of all the cultures.Different genotypes of the same species efficiently proliferated in the same proliferation conditions (0.1 mg/l BAP and 0.5 mg/l 2,4-D).The active G. asclepiadea callus proliferation required double concentration of both cytokinin and auxin (0.2 mg/l BAP and 1 mg/l 2,4-D).
According to other authors, there is a lot of complexity in the callus initiation and proliferation for Gentiana species.It is supposed to be connected with a great number of phenolic compounds in these plants [30].For a rapid callus growth 2,4-D and BAP or Кin are usually used [30,31].
In our study we obtained callus from 9 genotypes of 4 gentian species.BAP and 2,4-D growth regulators were necessary for both the callus induction and growth.The MS/2 medium supplemented with 0.1 mg/l BAP and 0.5-1 mg/l 2,4-D was found to be optimal for the callus induction and proliferation in three species.In the case of G. asclepiadea the callus formation required the B 5 medium supplemented with double concentration of both cytokinin and auxin (0.2 mg/l BAP and 1 mg/l 2,4-D).

Fast-growing root culture
MS/2 medium supplemented with 0.1 mg/l of BAP appeared to be the most effective for the isolated root formation and growth in G. lutea, G. punctata cultures at the first culture stage (Table 3).
Use of 0.1 mg/l of Kin was the best for the G. acaulis and G. asclepiadea cultures.The samples from different geographic locations for two gentian species required the same concentrations of NAA: 0.3 mg/l for G. asclepiadea, 0.5 mg/l for G. punctata.However, for G. lutea efficient concentrations of NAA to form isolated roots constituted 1 or 2 mg/l, depending on genotype (Table 3).The intensive growth of G. acaulis isolated roots on MS/2 was significantly stimulated by doubled concentration of СаСl 2 (440 mg/l).In contrast to other species, G. asclepiadea produced isolated roots only on the B 5 medium supplemented with 0.1 mg/l Kin and 0.3 mg/l NAA (Table 3).
After the first culture stage the mean number of side rootlets per inoculum significantly varied: 84-101 for G. lutea, 95 for G. acaulis, 35-42 for G. punctata and 35-44 for G. asclepiadea.After the second culture stage the mean root size was the longest for G. acaulis and G. lutea (23 mm and 22.4-30.8mm, respectively) and the shortest in G. punctata and G. asclepiadea (17.4-18.8mm and 18.2-18.6mm, respectively; Table 3).
Use of two-stage cultivation provided formation of a considerable number of side rootlets (in some cases over 100 per inoculum), an essential increase of side rootlets length (up to 30 mm) and their rapid growth (GI reached 926.5) (Table 3).
The highest yield of isolated roots (225 g of biomass per 1 l of medium) was obtained for the G. lutea cultures from Troyaska mountain samples.It is equal to the root mass of 10-12-year-old plant in nature [3].GI of this culture was 926.5 and corresponded to GI obtained by other authors after the root transformation by Agrobacterium rhizogenes.For example, GI of nine clones of   The growth index of G. punctata roots of non-transgenic culture constituted 140.7,GI of transgenic -45.3-287.7 for one strain and 33.8-1907.2 for the other one [21].For the obtained by us non-transgenic culture of this species (G.p.Tr i G.p.Br) these values were 203.4 and 192.8 respectively.In the research of transgenic culture of the Gentiana dinarica Beck.isolated roots, the maximal GI reached 46.7 [32].For the obtained by us G. acaulis culture -close to the G. dinarica speciesthis value equalled 324.
The yield of transgenic culture of the Gentiana macrophylla L. isolated roots was 130-260 g/l on the 30 th day of cultivation [23].The yield range of the investigated non-transgenic cultures of gentian isolated roots, obtained by us, was 42.4-225.5g/l.
During the investigation of transgenic G. punctata roots, it was found that their length was 38.8 and 45.2 mm, whereas our results for non-transgenic culture of the same species constituted 17.4 and 18.8 mm.Thus, the root length of transgenic culture is bigger, whereas the number of side roots is considerably bigger in the obtained by us non-transgenic cultures (35 and 42 against 0 and 1.54).After 35 days the biomass of explants, cultivated on media with different concentration of sucrose, showed a 1.5-3.4times increase [18].
Fast-growing root cultures of four Gentiana species plants were obtained and their growth was studied, in particular quantity and length of side rootlets, growth index and biomass production.The comparison of growth parameters of the obtained non-transgenic root cultures to those of the investigated transgenic ones and other Gentiana species showed similar and in some cases even higher productivity indices of our cultures.We found that GI of the isolated root cultures was significantly higher (60-300 times) than GI of proliferating calli of the same species.

Xanthone and flavonoid contents in cultured tissues
The results of the analysis showed the diversity of total xanthone and flavonoid amounts in callus cultures of different species (Tables 4, 5).
A comparison of calli from G. acaulis and G. lutea, derived from plants from different localities, allowed establishing essential distinctions in the quantity of xanthone (Table 4).Thus, in the G.ac.Т callus tissue, the amount of this class of compound exceeded 4.4 times an analogous value in the G.ac.Reb culture.G.l.R and G.l.Tr calli synthesized 2.6 and 2.5 times more xanthones in comparison with the G.l.P culture.Xanthone content in calli was generally less than in shoots of intact plants (with the exception of G. asclepiadea), but greater than in roots.In the G.punctata cultures the amount of BAS was less than in roots (Table 4).Similar results were obtained for the Gentianella austriaca shoot cultures.It was shown by high-performance liquid chromatography, that the shoot cultures contained nearly two times less xanthones than the plants growing in nature [33].
For nine tissue cultures, the highest value of total flavonoids was characteristic of G.ac.Т (Table 5).This value was rather high in the G. lutea and G.ac.Reb calli.The G. punctata and G. acaulis calli derived from plants of various localities, differed considerably by the amount of flavonoids (Table 5).In the G.ac.Т callus there were 1.8 times more flavonoids compared to the G.ac.Reb culture.Flavonoid content in all tissue cultures was considerably less than in shoots of intact plants.In some calli, namely G.p.Tr, G.l.Tr and G.l.P, the amount of these BAS exceeded that in roots (Table 5).Dry weight outcome after four weeks of callus growth varied within 11.6 to 29.3 g/l (Fig. 3).This value was the highest for the G.ac.Reb tissue cultures, and the lowest for G. asclepiadea.Assessment of the biosynthetic characteristics of gentian calli showed their ability to accumulate from 50 to 249 mg of xanthones per 1 l of nutrient medium (see Eq. 3).This value was the highest for the G.l.R, G.l.Tr and G.ac.T tissue cultures, and the lowest -in the G.asc.P callus (Fig. 3).The gentian tissue cultures are able to accumulate 37-279 mg of flavonoids per 1 l of cultural medium (see Eq. 3).This value was the greatest in the G. acaulis calli (Fig. 3), but lowest in the tissue cultures of G. asclepiadea.The given data prove that in most callus cultures, the growth conditions favoured both biomass production and BAS (xanthones and flavonoids) synthesis.However, in some cases, in the G.asc.P callus for example, low values were characteristic of dry biomass and BAS.

Xanthone and flavonoid content in the isolated root cultures
The root cultures obtained from G. lutea, G. punctata, G. acaulis and G. asclepiadea synthesize large amount of flavonoids and xanthones as compared to calli (Tables 4, 5).The greatest flavonoid content (14.3 mg (RUT)/g DW) was found in the G. acaulis root cultures; the highest values of xanthones (12.1-14.8mg (MAN)/g DW) were characteristic for the isolated roots of G. lutea and G. acaulis.
Comparison of root cultures derived from plants from different localities showed that there were no significant distinctions of BAS content for G. lutea and G. punctata (Tables 4, 5).
It is shown that in isolated root cultures of G. lutea and G. acaulis the amount of xanthones was smaller compared to shoots, but larger than in roots.The amount of xanthones in the culture of G. punctata isolated roots was For notations, see Table 1 smaller than in both roots and shoots of the plants from natural populations (Table 4).
In all root cultures investigated the amount of flavonoids was considerably smaller than in shoots, but larger or practically the same as in roots (Table 5).
Similar results were obtained for extracts from the Withania somnifera Dunal isolated cultured roots.It was shown by high-performance liquid chromatography that the content of alkaloid withanolides in cultured roots constituted 4 mg/g of their dry weight and exceeded the analogous parameter value in roots in vivo (3 mg/g of dry weight) [34].
After 4-6 weeks of growth the dry weight of isolated cultured roots varied from 7.3 to 34.5 g/l (Fig. 4).This parameter value was higher for G.l.Tr roots in culture.The least isolated root growth was characteristic of G. punctata.
The isolated root cultures are characterized by their capacity to accumulate 50.3-318.2mg of flavonoids and 40.6-424.3mg of xanthones per 1 l of nutrient medium (see Eq. 3).The productivity values by both BAS amounts were the highest for the G.l.Tr cultured roots, when comparing them with rather high values for the G.ac.T, G.l.P and G.l.R isolated roots (Fig. 4).
Thus, the growth conditions for most of isolated root cultures favoured the BAS synthesis in biomass accumulation.However, for the G. punctata cultures both these parameters were low.
Generalization of the results concerning flavonoid and xanhone accumulation in gentian calli and isolated root cultures showed the following: 1) most of gentian cultures are able to accumulate flavonoids and xanthones in amounts, which in some cases reached 1.5 % of dry weight; 2) in calli tissues and isolated root cultures, the amount of BAS was greater or close to that in roots of plants, but considerably less than in shoots; 3) in most of the isolated root cultures analyzed there were more flavonoids and xanthones than in calli, although the difference of the BAS content be- For notations, see Table 1 tween the two types of cultures was insignificant; 4) BAS amount in both morphogenic and non-morphogenic cultures depended on the genotype of the plant donor; 5) a correlation was found between flavonoid and xanthone content in vitro.
Growth conditions enable the biomass accumulation as well as the flavonoid and xanthone synthesis in callus and isolated root cultures.The above growth and biosynthesis parameters of cultures prove that gentians are potential material as a source for obtaining valuable secondary metabolites.In general, the essential distinctions were not found for biomass and BAS per 1 l of nutrient medium between callus tissues and corresponding root cultures.However, for the latter we established high growth index values (192-926) that exceeded by 60-300 times those for callus cultures.Obtaining and growing isolated roots are less labour-consuming and methodically less complicated than initiating and maintaining callus.To gain biomass and BAS synthesis for isolated roots 4-6 weeks are required, whereas for stable tissue cultures 11-15 passages are necessary, each passage lasting 4 weeks.Isolated root can be cultured in liquid nutrient media without agar or growth regulators at the second stage of culture.All these factors reduce the material costs for the isolated root cultures and improve their availability in comparison to callus tissues.

Conclusion
In vitro tissue and organs cultures of Gentiana species from the Ukrainian flora were obtained.The capacity to form morphogenic and non-morphogenic cultures and their growth depended on the original genotype, mineral composition and PGR content of nutrient medium.In some cases the efficiency of callus formation from root and stem explants rated up to 100 %.The highest GI for calli from root explants was within 2.1-3.1.The fast-growing root cultures were characterized by a high growth rate (growth index reached 927) and considerable yield of biomass.
The tissue and fast-growing root cultures are able to synthesize flavonoids and xanthones in amounts which in some cases reached 1.5 mg/g of dry weight.The amount of these BAS in calli and isolated roots was considerably less than in shoots of plants, but more or approximatly the same as in roots.The number of secondary metabolites investigated varied depending on the original plant genotype and culture type.The greatest productivity was in both types of G. lutea and G. acaulis cultures.Taking into consideration the outcome of gentian tissue cultures and roots as well as their capacity to synthesize flavonoids and xanthones, they have a great potential as an alternative source of these BAS.Compared to callus tissues, a preference should be given to the isolated root cultures that are characterized by a considerably higher growth index and less complicated growth pattern.

Fig. 1 .
Fig. 1.G. lutea callus (A) and isolated root culture at I (B) and II (C) stages of cultivation.

Fig. 2 .
Fig. 2. Growth index of root origin calli of gentians on most effective nutrient media.For notations, see Table1

Fig. 3 .
Fig. 3. Productivity of gentian tissue cultures by dry weight and biologically active substances (flavonoid and xanthone) per 1 l of nutrient medium.

Fig. 4 .
Fig. 4. Productivity of the gentian isolated root cultures by dry weight and biologically active substances per 1 l of nutrient medium.

Table 2 . Frequency of callus induction (%) from root explants of four species on different media
2 -The highest values of callus induction efficiency are in the bold type.3 -Callus induction was insignificant, planted explants underwent necrosis after 1-1.5 weeks of cultivation.а, b, c, d, e, f -the same Latin letters stand for unreliable difference of mean values for callus genesis from root explants of one genotype plants on different nutrient media according to Tukey's test.