doi: 10.15389/agrobiology.2012.4.88eng

УДК 636.086.2/.3:581.143.6:[573.6.086.83+577.21]


E.V. Orlova1, A.Yu. Stepanova2

Basing on a somaclonal variability, the regenerants of Medicago glutinosa L. resistant to petroleum pollution were selected in vitro by an optimized scheme without an addition of selective agent to the media for induction of callus and regenerants. Such parameters as a survival rate for 56 days and plant height of the regenerants and the intact plants were compared. In 20 % of the regenerated plants the resistance was lower and in 65 % it was the same as compared with the control, and 15 % of the regenerated plants demonstrated a greater increase comparing to the other plants. It was shown regenerants contributed to a better purification of soil, polluted with petroleum as compared to intact plant grown from seeds.

Keywords: Medicago glutinosa L., somaclones (regenerants), petroleum.


Yellow lucerne is a promising forage plant commonly sown as improver of hayfields and pastures in southern territories with insufficient moisture. This crop produces high seed yields in unfavorable years, it shows longevity, good wintering, heat and drought resistance (1). Soils under lucerne plantations have accelerated accumulation of organic matter, improved physical and chemical properties, and enhanced development of oil-oxidizing bacteria (2).
Today, large areas of topsoil are contaminated with oil spills owing to increasing amount of car refills and traffic in motor roads. This problem is most urgent in locations of transit pipelines, chemical and oil-processing plants. In oil spills, soil particles are aggregated, soil porosity and density are disturbed, as well as soil aeration; it is poor in nitrogen and phosphorus, and can’t provide normal temperature and water regimes for plants; the structure of soil microbiota and composition of its species are violated as well (3, 4). Plants growing on oil spills show growth retardation, reduced contents of chlorophyll and carotenoids, low indices of seed germination, daily root growth, as well as delay or loss of particular phenophases, and later start of vegetation (5, 6). Most of plants are sensitive to oil contamination: 0,3% crude oil inhibit plant growth and development, while 5% oil is lethal for most of plants (7). So, vegetation in oil-contaminated lands is partly or completely destroyed, which makes important obtaining plants resistant to oil pollution.
Modern selection completes this task through biotechnology approaches based on in vitro cultured plant cells. This practice provides somaclonal variants including the ones resistant to various stresses. Such issues were shown by numerous publications (8-10), but none of them described resistance of somatoclones to oil pollution. Besides, oil can’t be used as a selective agent – it is hydrophobic and unevenly distributed in the agar medium to form micelles.
This study was performed in order to solve the following tasks: optimization of aseptic pre-sowing treatment of lucerne seeds, development of proper regeneration media, obtaining somatoclones-regenerants and evaluating their growth, resistance and phytoremediation potential under oil pollution of soil.
Technique. The object of investigation was yellow lucerne (Medicago glutinosa L.). The seeds were treated with commercial chlorine bleachener “Belizna” for 20, 30 and 40 minutes, then washed three times with sterile distilled water and placed on a solid nutrient medium Gamborg (B5) (11) with a half rate of macro- and micronutrients. Calli were obtained from cotyledons of sterile 1-week-old seedlings. Callus induction and growth were performed using complete Gamborg medium (B5) with the addition of nicotinic acid (1 mg/l), thiamine (10 mg/l), pyridoxine (1 mg/l), meso-inositol (100 mg/l), 2,4-dichlorophenoxyacetic acid (2,4-D, 2 mg/l), kinetin (0,2 mg/l), and sucrose (30 g/l). The calli were incubated in Petri dishes at 26 °C and natural illumination (daylight 16 hours), during subculturing the transfer to fresh medium was performed after 28-30 days. The obtained calli were cultured for 10 months. To stimulate regeneration, the calli were placed on B5 medium with 6-benzylaminopurine (6-BAP, 0,5 mg/l) instead of kinetin and 2,4-D. The medium was modified to improve the efficiency of embryogenesis: proline (500 mg/l) or arginine (600 mg/l) was added. Rhizogenesis was stimulated by transplanting somaclones into sterile containers of 100 ml on B5 medium (keeping half or full rate of macro- and micronutrients), and then introduced a-naphthyl acetic acid (NAA, 5 mg/l) or indole-3-butyric acid (IBA, 0,5 mg/l). The plants with developed roots before the transfer into vessels with soil were pre-incubated for 10-14 days in closed glass containers on a damp cotton wool.
Crude oil used in the experiment was obtained from the Moscow Oil Refinery Plant (MNPZ); characteristics of the oil: specific gravity – 0,83; total hydrocarbon content – 81,2% including n-alkanes – 9,3; isoalkanes – 7,9 ; naphthenes – 11,7; aromatic hydrocarbons – 64,0%; resin content – 3,1%; asphaltenes – 4,0%. To examine the response of regenerants to oil pollution in soil, the somaclones were planted in special containers filled with soil (100 g) thoroughly mixed with crude oil (at estimated final content 5%) and grown for 60 days. The control – lucerne plants grown from seeds. The experiments were conducted using 20 control plants and 20 regenerants tested in following variants: soil without oil (to assess hydrocarbon contamination of soil), soil + oil (to estimate the capability of indigenous microbiota for oil utilization), soil + oil + plants of seed origin (control), soil + oil + somaclones.
Oil content in soil was determined gravimetrically with chloroform extraction. Samples of soil were collected from different parts of the used containers and left to stay open until getting air-dry state. A sample of soil (2 g) was put in a glass flask, added 20 ml chloroform and mixed thoroughly. The extract was transferred through a paper filter into a round bottom flask of 100 ml volume with ground glass joint (pre-weighed with an accuracy of ± 1 mg). Chloroform extraction was repeated 3 times, at the end the extracts were combined in a total sample. Then chloroform was evaporated on a rotary evaporator with a water bath temperature of 58 °C. Oil content was found gravimetrically on analytical scale VPR-200 (Russia), up to the fourth significant figures.
Oil content in soil was calculated as ХF = (A/B) x 1000, where A - the amount of oil obtained when weighing, mg; B - weight of soil sample, g. The percentage of oil utilization during vegetation was determined by the formula:
where  Хк, Хн - respectively, final and initial content of oil. 
Statistical calculations were performed in Microsoft Excel. The text and tables present arithmetic means of the parameters  and their confidence intervals at 95% level of probability in Student’s T-test. Bars in figures correspond to the maximum values of confidence intervals.
Results. This work was the first evaluation of the resistance of somaclones to oil pollution in soil. Figure 1 shows the scheme of experiment when the somaclones of yellow lucerne were obtained from calli cultured on the media with different elemental composition.

Fig. 1. Scheme of consistent changes in composition of a culture medium (based on Gamborg В5) used to obtain somaclones-regenerants of yellow lucerne (Medicago glutinosa L.): A — callus formation medium, B — regeneration medium, C — rooting medium; 2,4-D — 2,4-dichlorophenoxyacetic acid, 6-BAP — 6-benzylaminopurin, NAA — a-naphthylacetic acid.

Experimentation with tissue cultures in vitro necessitates aseptic material with high viability; usually it is fixed by treatment with chlorine-containing agents - hypochlorite of calcium or sodium (in this work –  a commercial bleachener “Belizna” keeping 4-7% sodium hypochlorite). For yellow lucerne, an optimal time of seed treatment was established - 20 min (Table 1). A longer treatment contributed to reduced viability of the seeds without any notable impact on the efficiency of sterilization.

1. The yield of viable aseptic material after seed treatment with chlorine-containing commercial preparation “Belizna” of seeds of yellow lucerne (Medicago glutinosa L.)

Time of seed treatment, min

Total number of seeds, pcs.

Number of seeds, pcs. (proportion, %)

aseptically viable

aseptically non-viable




35 (87,5)

3 (7,5)

2 (5,0)



30 (71,4)

10 (23,8)

2 (4,8)



31 (77,5)

8 (20,0)

1 (2,5)



35 (77,8)

9 (20,0)

1 (2,2)

Obtaining callus tissue from cotyledons of 10-day-old seedlings of lucerne is commonly performed on Gamborg medium containing macro-, micronutrients, and vitamins (B5 medium), as well as on Schenk-Hildebrandt and Murashige-Skoog media (12, 13). Callus induction is caused by addition of 2,4-D and kinetin (14) (the ratio selected individually in respect to the genotype), in some cases NAA or IAA must be added. In the authors’ preliminary experiments the base medium for obtaining the calli was established: B5 with 2,4-D (2 mg/l) and kinetin (0,2 mg/l).
To induce regeneration, the calli were transplanted  on B5 medium containing 6-BAP (0,5 mg/l) with or without the addition of amino acids. It is known that amino acids (L-proline, L-alanine, L-glutamine, L-asparagine, L-serine, L-lysine, and L-arginine) increase the efficiency of embryogenesis and the yield of somaclones (15, 16). In the authors’ experiments, transplantation of the calli on the regeneration medium provided the increase in number of morphogenic calli on all types of media (Table 2). However, the shoots (with following transformation into viable plants-regenerants) on morphogenic calli were most often developed only on the medium with amino acids (Fig. 2).

2. Efficiency of morphogenesis in callus culture of yellow lucerne (Medicago glutinosa L.) depending on composition of inducing medium

Medium variant

Total number of calli, pcs.

Morphogenic calli

number, pcs.

% (Х±х)

B5 + 6-BAP (0,5 mg/l)




B5 + 6-BAP (0,5 mg/l) + arginine (600 mg/l)




B5 + 6-BAP (0,5 mg/l) + proline (500 mg/l)




Note. 6-BAP — 6-benzylaminopurin. Composition of media – see Technique.

Fig. 2. Efficiency of regeneration of plants from cultured calli of yellow lucerne (Medicago glutinosa L.) depending on composition of inducing medium: А  B5 + 6-BAP (0,5 mg/l), Б  B5 + 6-BAP (0,5 mg/l) + arginine (600 mg/l), В  B5 + 6-BAP (0,5 mg/l) + proline (500 mg/l); а a calli with shoots, б — plants-regenerants.

abscissa –  Medium variant
ordinate – Proportion, %

So, B5 with proline addition was established as the best nutrient medium providing the highest yield of morphogenic calli  (54%) and plants-regenerants (23%).
According to the literature, oil content in soil used in this experiment for growing lucerne somaclones and control samples (5%) is considered as very high and lethal for most of plant species (16). Testing the survival rate of experimental plants for 56 days (Fig. 3), it was observed the survival of 20% regenerants worse than in control (after planting in the “oil spill” plants stopped to grow, quickly turned yellow and died within the first 7 days), while 65% ones maintained viability similar with control. At the end of the experiment, the control group exceeded somaclones in survival rate (80%).
Among the regenerants-survivors there was a notable group of 15% plants that looked more viable than control (most of the leaves remained green); they showed a greater increase in plant height (220 ± 7,2% above the initial in each group) compared with control plants (110 ± 5,1%) and other somaclones (160 ± 6,7%). So, all plants-somaclones were divided into three groups: the ones with resistance to oil contamination in soil below the control, at the level, and above the control.
The maximum reduce of oil content in soil was observed in the variant with somaclones. Thus, the degree of oil degradation in the soil without plants was 15,07 ± 3,5%, while in the variants with control plants and somaclones – respectively, 48,98 ± 1,8% and 56,90 ± 1,4%. Probably, the latter was associated with activity of the most viable regenerants (the abovementioned15%).

Fig.3. Survival of control plants (a) of yellow lucerne (Medicago glutinosa L.) and plants-regenerants (b) obtained from callus tissue without the use of selective agent, after 56 days growing in soil keeping 5% crude oil.  

Rhyzodegradation of oil is the main known mechanism of its phytoremediation by yellow lucerne grown in oil-contaminated soil. In this case, oil is destructed by soil microorganisms, and plant exudates stimulate proliferation of soil microbiota (17, 18). It was obvious that the most viable somaclones of yellow lucerne provided better rhyzodegradation of oil.
Thus, the authors have established the optimized conditions for callus induction and plant regeneration of yellow lucerne Medicago glutinosa L. This work was the first evaluation of yellow lucerne somatoclones in respect to resistance to oil contamination in soil and their capacity for oil destruction, which has confirmed that resistant somaclonal forms were derived without the use of a selective agent (oil) but owing to expressed individual somaclonal variation. The best somaclones-survivors in “oil spill” (15%  of total tested) manifested better growth than both the rest of regenerants and control. In following experiments this group of somaclones provided more efficient phytoremediation of oil-contaminated soil than control plants of seed origin.


1. Kirk J.L., Klironomos J.N., Lee H., and Trevors J.T., The Effects of Perennial Ryegrass and Alfalfa on Microbial Abundance and Diversity in Petroleum Contaminated Soil, Environ. Pollut., 2005, vol. 13, pp. 455-465.
2. Buyankin N.I., About the Reasons of Some Biological Features in Annual Plants in Case of Summer Sowing and Its Practical Use, S.-kh. biol., 2008, vol. 5, pp. 1-10.
3. Guzev V.S., Levin S.V., Seletsky G.I. et al., Role of Soil Microbiota in Recultivation of Oil-Contaminated Soil, in Mikroorganizmy i okhrana pochv (Microorganisms and Soil Protection), Moscow, 1989, pp. 121-150.
4. Gasheva M.N., Gashev S.N., and Soromotin A.V., State of Flora as a Criterion for Evaluating the Disturbance of Forest Biocenoses Caused by Oil Pollution, Ekologiya, 1990, vol. 2, pp. 77-78.
5. Adam G. and Duncan H.J., Effect of Diesel Fuel on Growth of Selected Plant Species, Environ. Geochem. Health, 2000, vol. 21, pp. 353-357.
6. Davydova I.Yu. and Pakhnenko-Durynina E.P., Response of Agricultural Plants to Oil Pollution in Soil, in Voprosy regional’noy geografii i geojekologii (Topics of Regional Geography and Geoecology), Krivtsov V.A., Ed., Ryazan, 2004, issue 4, pp. 119-129.
7. McGrath D., Oil Spillage on Grassland Effects on Grass and Soil, Farm Food Res., 1988, vol. 19, no. 5, pp. 28-29.
8. Iskakov A.R., Selection Value of Barley Plants Regenerated from the Culture of Somatic Tissues, in Mat. Resp. konf. “Problemy teoreticheskoy i prikladnoy genetiki v Kazakhstane” (Papers of Regional Sci. Conf. “Fundamental and Applied Aspects of Genetics in Kazakhstan”), Almaty, 1990, pp. 111-115.
9. Bobkov S.V., Sidorenko V.S., and Gurinovich S.O., Use of Biotechnology in Selection of Proso Millet (Panicum miliaceum L.), in Mat. II Mezhd. konf. “Biotekhnologiya v rastenievodstve, zhivotnovodstve, veterinarii” (Papers II Int. Sci. Conf. “Biotechnology in Plant Growing, Animal Husbandry, and Veterinary”), Moscow, 2000, p. 85.
10. Heinz D.J., Krishnamurthi M., Nickell L.G., and Maretzki A., Cell Tissue and Organ Culture in Sugarcane Improvement, in Applied and Fundamental Aspects of Plant Cell, Tissue and Organ Culture, Berlin: Springer-Verlag, 1977, pp. 3-17.
11. Gamborg O.L., Miller R.A., and Ojima K., Nutrient Requirements of Suspension Cultures of Soybean Root Cells, Exp. Cell Res., 1968, vol. 50, pp. 151-158.
12. Murashige T., and Skoog F., A Revised Medium for Rapid Growth and Bioassays with Tobacco Tissue Cultures, Physiologia Plantarum, 1962, vol. 15, pp. 473-497.
13. Shenk R.V. and Hildebrandt A.C., Medium and Techniques for Induction and Growth of Monocotyledonous and Dicotyledonous Plant Cell Culture, Can. J. Bot., 1972, vol. 50, pp. 199-204.
14. Atanassov A. and Brown D., Plant Regeneration from Suspension Culture and Mesophyll Protoplasts of Medicago sativa L., Plant Сell Tissue Organ Cult., 1984, vol. 3, no. 2, pp. 149-162.
15. Stuart D.A. and Strickland S.G., Somatic Embryogenesis from Cell Cultures of Medicago sativa L. I. The Role of Aminoacid Addition to the Regeneration Medium, Plant Sci. Let., 1984, vol. 34, pp. 165-174.
16. Duncan D.R., Williams M.T., Zehr B.T. and Widholm J.M., The Production of Callus Capable of Plant Regeneration from Immature Embryos of Numerous Zea mays Genotypes, Planta, 1985, vol. 165, pp. 322-332.
17. Susarla S., Medina V.F., and McCutcheon S.C., Phytoremediation: an Ecological Solution to Organic Chemical Contamination, Ecol. Eng., 2002, vol. 18, pp. 647-658.
18. Frick C.M., Farrell R.E., and Germida J.J., Assessment of  Phytoremediation as an In-Situ Technique for Cleaning Oil-Contaminated Sites, Canada: Petroleum Technology Alliance of Canada (PTAC), Calgary, 1999, pp. 10-50.


1Moscow State University for Engineering Ecology, Moscow 105066, Russia, e-mail:;

2K.A. Timiryazev Institute of Plant Physiology, RAS, Moscow 127276, Russia,

Поступила в редакцию
11 января 2012 года