The article was published in journal “Biotechnology” (Kiev, Ukraine). 2012, Vol. 5, № 4, P. 65 – 73, e-mail: biotech@biochem.kiev.ua

CHARKOR GROWTH REGULATOR AS INDUCTOR OF BIOMASS ACCUMULATION IN THE CHICORY

«HAIRY ROOTS» CULTURES — PRODUCERS OF POLYFRUCTANS

V.A. TsygankovaInstitute of Bioorganic Chemistry & Petrochemistry, Natl. Acad. of Sci. of Ukraine, Kyiv, Ukraine

A. I. Yemets, A.P.GalkinInstitute of Food Biotechnology and Genomics, Natl. Acad. of Sci. of Ukraine, Kyiv, Ukraine

S.P. РоnоmаrenkoScience & Technology Center Agrobiotech, Natl. Acad. of Sci. and Min. of Ed. and Sci. of Ukraine, Kyiv, Ukraine

Key words: plant growth regulator Charkor, chicory «hairy roots», polyfructans, Dot-blot hybridization.

Abstract At the root culture of four lines chicory (Cichorium intybus L.), transformed by Agrobacterium rhizogenes, the considerable increase of polyfructan (PF) synthesis level is obtained in nutrient media with growth regulator Charkor. It is shown that adding growth regulator Charkor into medium with agar and liquid medium 1/2MS increased root mass (by dry mass) in 39–54 times relative to control depending on concentration of regulator (from 2,5 to 10 µL/L of medium). Influence of growth regulator Charkor on growth of root biomass was similar to action of auxin, in particular, indolebutyric acid. Adding of growth regulator Charkor to their mediums diminished the relative concentration of PF (mg/g of dry mass) in 2–8 times, that it is related to hastier root growth on such conditions. At the same time at cultivation of root at presence of growth regulator Charkor the general maintenance of PF (48–130 mg/g of total dry root mass grown for a 30 twenty-four hours) was well above than in the control. The method of Dot-blot hybridization showed a considerable difference in populations of cytoplasmic mRNA and small regulatory si/miRNA between control and experimental plants. It indicates on some changes in gene expression under the influence of the regulator.

INTRODUCTION

During the last decades, a considerable progress was achieved in development of biotechnological methods of cultivation in vitro transgenic plants: super producers of amino acids, peptides, proteins-enzymes, vitamins, as well as secondary metabolites (alkaloids, glycosides, steroids, phenol compounds, terpenoids, tannins, polysaccharides, pigments, essential oils, etc.), which are widely used in medical, pharmaceutical, perfume and food industries. The application in biotechnological practice of genetic engineering methods also allows producing the bacteria, yeasts and animal cells, that are "biofactories" for the large-scale production of proteins-hormones (insulin, somatotropin, interferon, etc.) and their use as medicinal preparations [1-3].

A principal problem for successful development of plant biotechnology is optimization of cultivation in vitro of isolated cells and tissues of transgenic plants for the intensification of

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synthesis of biologically active compounds in them. It is known that the cells of the transformed plants are able to produce secondary metabolites in vitro conditions in absence of the special growth regulators. However, the suspension and callus cell cultures can produce secondary metabolites in lower amounts than obtained from the whole plants [1-3].

Actually, the scientists focus their efforts on modification of existing nutrient media, and development of new ones by application of physiologically active compounds of chemical origin, which are effective substitutes of natural phytohormones. This strategy is used worldwide for application of traditional (known for more than half a century) synthetic substitutes of phytohormones with auxin and cytokinin activity (e.g., 2,4-D, NAA, CPAA, Kinetin, BA, BAP) [4-7], as well as novel recently synthesized compounds that were successfully used in plant cell and tissue cultures in vitro. Numerous experimental data [8-12] proved their ability to induce both the processes of cell dedifferentiation from highly specialized cells (callusogenesis), and stimulation of morphogenesis.

Novel growth regulators of chemical origin [13] (synthesized at the IBCP and the IOC) are widely used in agricultural practice in Ukraine. In our earlier experiments with the cultures of the isolated plant cells of tobacco (Nicotiana tabacum L.), potato (Solanum tuberosum L.) and tomato of two cultivars (Lycopersicon esculentun L. and L. peruvianum), it was shown that Ivin (2,6-dimethylpyridine-N-oxide), Triamelon (tris-(2,2-trimethylammonium-methylphosphate) iodide), D-107 regulator (1-acetylamino-1-acetylthio-2-oxo-2-phenylethane), and regulator N 2622 (derivative of tetrahydrothiophene dioxide) can effectively substitute natural phytohormones auxins and cytokinins [14]. In subsequent research, a phenomenon of the synthetic growth regulators effect mediated through increase of endogenous phytohormones (auxins and cytokinins) pool was discovered [15]. Later the scientists worldwide have carried out similar experiments with the objective to study a possibility of PGRs application in isolated cell and tissue cultures of different plants (i.e., Arachis hypogeae L., Pelargonium hortorum Bailey, Humulus lupulus L., Phaseolus radiatus L., etc.) of new inducers of morphogenesis: synthetic substitutes of cytokinin, TDS (thidiazuron, derivative of thiocarbamide) [8-12]; and auxin, BSAA (3-(benzo[b]selenienil)acetic acid) [16]. The obtained data proved a considerable increase of endogenous phytohormones (auxins and cytokinins) levels in plant cells cultivated on these media.

New PGRs of natural origin are Poteitin, Zeastimulin, Charkor, Biolan, Biogen and Radostim, developed in the IBCP in cooperation with “Agrobiotech”. They contain products of metabolism of symbiotic fungus-endophyte of ginseng roots Panax ginseng M., cultivated in vitro conditions (e.g., mixture of amino acids, carbohydrates, fatty acids, polysaccharides, phytohormones and microelements) that regulate plant growth processes, in particular, stimulate seed germination and promote the productivity of a number of agricultural crops, as well as their resistance to a wide range of pathogenic and parasitic organisms [17, 18]. Charkor [19] is the most effective stimulant among these regulators of root formation in young plants of fruit and baccate cultures, decorative trees, flowers, and medicinal plants. For this reason, a possibility to use this regulator not only in agriculture, but also in biotechnology in vitro, especially for induction of synthesis of secondary metabolites in the hairy root cultures of medicinal plants, is of theoretical and practical interest.

Chicory (Cichorium intybus L.) is an important agricultural and medicinal culture. Its medical properties are caused due to presence of bioactive polyfructans (PF). Inulin polysaccharide is the most widespread among them. It has relatively low molecular mass: ~5000-6000 Da [20-23]. Earlier, the scientists of the Institute of Cell Biology and Genetic Engineering (ICBGE) have obtained the cultures of chicory “hairy roots” using Agrobacterium rhizogenes-mediated transformation with genes of tubercular antigen ESAT6 (esxA), Ag85 (fbpBΔTMD) and human leucocyte interferon ifn-a2b, and have determined the inulin content in the transformed roots [24]. The obtained transgenic roots differed in increasing of PF content that was probably a consequence of genetic transformation due to stress.

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The objective of work was verification of possibility to intensify PF synthesis in “hairy root” culture of chicory (Cichorium intybus L., Palla Rossa cultivar), transformed by Agrobacterium rhizogenes, with the use of Charkor.

MATERIALS AND METHODS

In the experiments, we used the “hairy root” chicory (Cichorium intybus L., Palla Rossa cultivar) culture of four lines (N 2, 6, 14, and 21), obtained by Agrobacterium rhizogenes-mediated transformation of cotyledons’ explants with vector pCB161 (selective gene npt-II, objective gene ifn-alfa2b). This experimental material was kindly provided by Dr. N.A. Matveeva from the ICBGE [24].

The terminal root explants of 10 mm size were cultivated on agar and ½ MS liquid media (MS medium [25] with the concentration of macroelements diminished twice) at +24 °C for 30 days. We added Charkor in concentrations: 0; 2.5; 5 and 10 µL per 1 L of the medium. For comparison, we also conducted experiments with the traditional PGRs: we added separately 0.5 mg/L of Kinetin, IBA, BAP, and NAA to ½ MS medium. The root cultivation was conducted in similar conditions.

We determined initial root mass (m0), root mass after 30 days of cultivation (mi); increase of root mass (Δm), dry mass (mi-dry), relative PF content (mg/g of dry root mass), total PF content (mg/g of total dry mass of roots grown for 30 days).

For determination of total PF content, the roots were dried at 90 ºC for 10 min, and then at room temperature to constant mass. PF content was determined by a method based on the ability of keto-saccharose to be decolorized by 0.1 % ethanol solution of resorcinol in acidic medium [26]. 5 mL of distilled water, 5 mL of 0.1 % ethanol solution of resorcinol, and 5 mL of concentrated hydrochloric acid were added to 100 mg of dry root material, and then it was heated on a water bath at 80 ºC for 20 min. A solution was cooled and the intensity of color was measured on a KPK-2 Photoelectric Colorimeter with a green color-filter (λ=540 nm). PF concentration was determined by calibration with the use of fructose.

The molecular-genetic experiments with objective to determine the homology level in mRNA populations of control and transformed by A. rhizogenes chicory roots, which were grown on the nutrient media with Charkor, were conducted by the method of DOT-blot (point) hybridization of mRNA of control roots with [32P]-cDNA of experimental roots [27, 28]. For this purpose, we isolated total RNA preparation from the cells of control and experimental roots according to the methods described in [29, 30]. Polymerity of isolated total RNA preparations was analyzed by 1.5 % agarose gel electrophoresis in presence of 7 M urea according to Locker's method [31] (gels were saturated by solution of ethidium bromide before photographing RNA fractions with UV lamp). The separation of poly(A)+mRNA and poly(A)–mRNA was conducted on the oligo(dT)-cellulose column with the following precipitation of high-molecular poly(A) –

mRNA from eluate by 10 % solution of polyethylene glycol (M.W.=8000) with 0.5 M NaCl at -22 °C for 24 h. si/miRNA was precipitated by equal volume of 96 % ethanol. Elution of poly(A)+mRNA from the oligo(dT)-cellulose column was conducted by 2-3 volumes of a buffer containing 10 mM Tris-HC1 (pH=7.5), 1 mM EDTA, and 0.05 % SDS [27, 28]. Poly(A)+mRNA was precipitated from a column after elution by 96 % ethanol.

For the synthesis of single-stranded cDNA, we used a buffer solution containing 100 mM Tris-HCl (pH=8.3), 10 mM MgCl2, 140 mM KCl, 100 µg/mL oligo(dT)12-18 primer, 2 mM methylmercuric hydroxide, 20 mM β-mercaptoethanol, 1 mM vanadyl-ribonucleoside complexes, or 0.5 units/µL of RNase, 1 mM solution of all four dNTP, 100 µg/mL poly(A)+RNA, 400-800 units/mL of reverse transcriptase, and [α-32P]-dCTP (800 Ci/mM) at 42 °C [27, 28].

For [32P]-cDNA transferring to the filters, it was dissolved at a concentration of 20 µg/mL (2xSSC) in a buffer containing 0.3 M NaCl and 0.03 M sodium citrate (pH=7.0). For

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hybridization, we applied 1 mL of [32P]-cDNA solution to modified and activated cellulose filters (Whatman 50, 2-aminophenyl-thioether paper, which forms covalent bond with applied DNA or RNA, unlike ordinary cellulose or nitrocellulose filters, which form only hydrogen bonds). It allows avoiding the loss of nucleic acids at washing [27, 32, 33].

For hybridization of cDNA with mRNA on the filters, they were placed into small bottles for determination of radioactivity, which were filled with solution of 2xSSC, with tenfold excess of mRNA to cDNA [27, 28]. In each test, we placed the filters with DNA from a genetically remote source in separate small bottles for control of hybridization specificity. Filters without DNA were also used for estimation of size of nonspecific sorption on the filters. Small bottles with filters were tightly closed and placed in a thermostat for hybridization.

Hybridization was conducted at 66 °C, however, during the work with nucleic acids from different sources; we took into account that the RNA molecules with more stable secondary structure (due to number of GC pairs higher than in the total mRNA preparation) required higher temperatures. An optimal temperature and maximal level of hybridization were chosen by measuring the level of mRNA hybridization with homologous and heterologous cDNA. Hybridization levels were compared in different time intervals during the day.

After hybridization completion, the small bottles with filters were cooled; the filters were removed and washed in a funnel on every side by 50 ml of 2xSSC. Then the filters were placed in a solution of 2xSSC, which contained RNase in a concentration of 20µg/mL. After incubation with RNase at the room temperature for 1 h, the filters were washed by solution of 2xSSC, and then by 96% ethanol. Radioactivity of the samples was determined on Millipore AP-15 glass filters in toluene scintillator in a Beckman LS 100C scintillation counter.

For hybridization of 33P-labelled si/miRNA with mRNA (through cDNA) of control roots, the si/miRNA preparations of high purity were isolated from control and experimental chicory roots grown on nutrient media with adding Na2HP33O4 and Charkor as described in [34]. The statistical processing of experimental data was conducted by the Student's method.

RESULTS AND DISCUSSION

Studying four lines (N 2, 6, 14, and 21) of transgenic chicory roots (Cichorium intybus L., Palla Rossa cultivar), we found their different sensitivities to the effect of Charkor (Fig. 1). Although addition of the growth regulator into agar medium resulted in acceleration of root mass growth in all four lines, the best results were obtained in the line N 21. In this line, the root growth did not differ notably at different PGR concentrations in the range of 2.5-100 µL/L, but it was significantly more intensive than in the control plants. For the line N 6, an insignificant growth of root mass was observed at increase of the PGR concentration from 2.5 to 10.0 µL/L. The minimal increase of root mass (measured on dry mass) was observed in control experiments on ½ MS medium without addition of growth regulators. After Charkor addition, the root mass increased in 39-54 times (line N 21), depending on the PGR concentration in the medium (2.5-10 µL/L).

Meanwhile, a statistically reliable correlation between concentrations of PGR and its impact on root mass growth was not found (Fig. 2). The highest relative PF content was observed in roots cultivated in ½ MS medium (control, the slowest growth). Addition of Charkor reduced relative PF content (in mg/g of dry mass) in 2-8 times, in particular, the minimal concentration of the regulator (2.5 mg/L) reduced relative PF content in the highest degree (Fig. 2). Possibly it was caused by faster root growth in these conditions.

The highest total PF content (48-130 mg/g of total dry root mass grown for 30 days) was obtained at root cultivation in the presence of Charkor. Addition of this regulator increased the total PF content in 9-11 times compared to the control plants. As one can see on the diagrams (Fig. 3), it occurred due to increase of the root mass. Thus, for increasing of PF content in chicory “hairy roots”, the application of this growth regulator in concentrations of 2.5-10 µL/L is advisable.

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The biological activities of Charkor and other traditional growth regulators (i.e., BAP, IBA, NAA, Kinetin) were compared on two lines (N 6 and 21) of chicory “hairy root” cultures. Obtained results proved significant acceleration of transgenic root growth on media containing auxins (NAA and IBA) that resulted in increase of total PF content (Fig. 4). The maximum increase of root mass was observed after adding of IBA (0.5 mg/L) into ½ MS nutrient medium. A similar effect was observed at the root cultivation on ½ MS medium with Charkor (5.0 µL/L). Less notable increase of these indexes was observed after adding of Kinetin and BAP (in concentration of 0.5 mg/L) into the medium.

At determination of the homology level in populations (sets) of cytoplasmic mRNA-transcripts isolated from the cells of control and experimental chicory roots (lines N 6 and 21) obtained on nutrient media with Charkor using method of the DOT-blot hybridization of [32P]-cDNA with mRNA, the reliable differences in the mRNA populations (Table) were found. Significant changes in population indexes (in % of homology) of small regulatory si/miRNA were also found by the DOT-blot hybridization of 33P-labelled si/miRNA with mRNA between the control and experimental samples of chicory “hairy roots” (lines N 6 and 21) grown on nutrient media with Charkor.

Obtained differences in phenotype characteristics of control and experimental chicory roots, and also differences in molecular-genetic indexes (% of homology in populations of cytoplasmic mRNA), proved reprogramming of the plant genome under effect of Charkor: "switching on" a cascade of genes that were nonactive earlier, but have a similar function in multigene families or gene superfamilies of endogenous phytohormone biosynthesis, which enhance the biomass growth and promote the productivity, i.e., increase the total PF content in chicory roots. Each member (variant) of gene family is slightly different from the nucleotide sequence in regulatory, encoding, and noncoding areas of their structure, and is regulated by different external factors, i.e., growth regulators. These results presented in our earlier publications [15, 17, 30, 35, 36] are in good agreement with the data reported by other scientists [37 - 46]. Discovered phenotype differences (significantly higher “fluffiness” of chicory “hairy roots” in a medium with Charkor, that are similar to the adventitious plant roots for phenotypical signs) may be caused by PGRs’ induction of the synthesis of species-specific, root growth-regulating si/miRNA. Numerous literature data support this hypothesis [47- 51]. Authors of these works confirmed that the main function of small regulatory si/miRNA in the roots of both wild types and transgenic plants consists of selective arrest (silencing) of translation mRNA of a number of negative regulatory transcriptional ARF-factors (Auxin Response Factor) that inhibit signaling transduction of auxin phytohormones in the adventive plant roots. As a result, an improvement of signaling transduction of auxins occurs, and the adventitious root growth increases. Obviously, a notable increase of “hairy roots” (similar to the adventitious roots) growth in vitro conditions was caused by significant increasing of the rate of the endogenous phytohormones synthesis (in particular, auxins) by Charkor as well as the synthesis of species-specific si/miRNA, which regulates root growth and arrests translation of mRNA of some negative regulatory transcriptional ARF-factors. As a consequence, the signaling transduction of auxins was improved [47- 54].

CONCLUSIONS

Thus we consider advisable the application of Charkor at concentrations of 2.5-10 µL/L in ½ MS nutrient medium for obtaining higher PF contents in chicory “hairy roots”. In spite of relatively lower PF contents per unit of root mass, the total PF amount is higher due to growth activation and significant increase of total mass of chicory “hairy roots” (up to 9-11 times in respect to control samples after 30 days) at cultivation on medium with Charkor.

Comparative experiments with the traditional synthetic PGRs (BAP, IBA, NAA, and Kinetin) have showed that adding of Charkor to the nutrient medium had resulted in increase of

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total PF contents due to the significantly more intensive increase of root biomass in comparison with above mentioned regulators.

Using a molecular-genetic method of DOT-blot hybridization, we found considerable differences in the populations of cytoplasmic mRNA, and also in the populations of small regulatory si/miRNA isolated from cells of control and experimental transgenic chicory roots grown in nutrient media with Charkor. Obtained results proved that the genome reprogramming processes occurred in the cells of experimental plants under effect of this regulator through "switching on" cascade of genes with high activity under strong promoters in multigene families or gene superfamilies of phytohormone biosynthesis (in particular, auxins) that regulate gene expression in growing roots. It resulted in increase of biomass growth and productivity (total PF content) in the cultures of transgenic chicory roots.

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54. TsygankovaV.А., PonomarenkoS.P., GalkinA.P., Yemets A.I. Charkor growth regulator as inductor of biomass accumulation in the chicory “hairy roots” cultures – producers of polyfructans. Biotechnology (Kiev, Ukraine). 2012, 5 (4), p. 65–73, (biotech@biochem.kiev.ua)

FIGURES

Figure 1.Growth of transgenic chicory roots on ½ МС agar medium with Charkor:

line N 6 (1-4); line N 21 (5-8); line N 2 (9-12); line N 14 (13-16)

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Figure 2.Growth of transgenic chicory roots (line N 21) and PF synthesis in

½ МS liquid medium with Charkor

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Figure 3. Increase of root mass (А), relative (B) and total (C) PF content in

“hairy root” cultures (line N21) grown in ½ МС media with Charkor during 30 days

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Figure 4. Increase of root mass (A) and total PF content (B) in chicory “hairy roots” culture of two lines (N 21, left, and N 6 right) grown on media with regulators: Kinetin, BAP, NAA, IBA, and Charkor

TABLE

Table. Hybridization of mRNA (through cDNA) with mRNA and si/miRNA isolated from cells of chicory roots

RNA type PGR dilution, µL/LH2O“Hairy root” line

6 21

mRNA

Control 98 ± 1.6 98 ± 1.42.5 95 ± 1.8* 82 ± 1.5**5.0 93 ± 1.6** 78 ± 2.2**10.0 88 ± 1.7** 76 ± 1.4**

si/miRNA Control 97 ± 1.4 98 ± 1.62.5 94 ± 1.3* 86 ± 1.8**

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5.0 90 ± 1.8** 84 ± 1.4**10.0 86 ± 1.4** 82 ± 1.6**

The experiments were carried out in triplicate.*insignificant differences between experiment and control** considerable differences from control, р<0.05

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