doi: 10.15389/agrobiology.2012.4.94eng

УДК 635.928:581.143.6:[573.6.086.83+577.21]


I.I. Litvinova1, E.A. Gladkov1, 2

With the aim of developing a culture method for efficient plant regeneration in vitro, the aseptically treated seeds of Lolium perenne L., Linum grandiflorum L., Brachycome iberidifolia L., Chrysanthemum carinatum L. and Trifolium repens L. were cultured on B5 and Murashige and Skoog (MS) media, containing different combinations of plant growth regulators to induce a callus. The highest percentage of calluses of Linum grandiflorum was observed on В5 with 2,4-D (6 mg/l) and МS with 2,4-D (6 mg/l) and kinetin (2 mg/l). In Lolium perenne L., MS with 2,4-D (4 mg/l) was used for the best callus formation. In Brachycome iberidifolia, В5 with 2,4-D (4 mg/l) and kinetin (2 mg/l) or B5 with 2,4-D (6 mg/l) and kinetin (2 mg/l) were the most effective conbinations. The best callus formstion for Chrysanthemum carinatum and Trifolium repens were achieved on MS supplemented with 6-BAP (1 mg/l) and IAA (0,1 1 mg/l) and В5 enriched with 2,4-Д (8 mg/l) and kinetin (2 mg/l), respectively. MS free of phytphormones, B5 with 6-BAP (1 mg/l) and NAA (0,1 mg/l), and 1/2MS with 6-BAP (2 mg/l) and NAA (0,1 mg/l) were identified as the optimal combination for plant regeneration for Lolium perenne L., Linum grandiflorum and Brachycome iberidifolia, respectively.

Keywords: Lolium perenne, Linum grandiflorum, Brachycome iberidifolia, Chrysanthemum carinatum, Trifolium repens, seeds, regeneration.


Mauritanian lawn looks a beautiful meadow composed of ornamental plants – meadow cereals and flowering grasses: perennial ryegrass, clover, cornflower, poppy, flax, bellflower, brachycome, chrysanthemums, daisies, asters, etc. (1), which are good honey plants, some are important forage and medicinal plants. Ryegrass is one of the most valuable forage cereals (1, 2). This grazing-tolerant plant produces high yield of green mass (mowed three times per growing season) while a relatively short vegetation period (65-70 days), it is commonly included in grass mix for pasture and hay fields in the north-western, western and central regions of the forest zone. Perennial ryegrass is grown for forage and hay (protein content in green mass – 3,2%, cellulose - 8%) (3); after mowing, its shoots and leaves rapidly regrow. The flax family Linnaceae are honey plants, a valuable source of vegetable oil, fiber, and forage. Some varieties of Chrysanthemum carinatum contain b-carotene in leaves and flowers providing its medicinal use as a source of this vitamin that enhances immunity. White creeping clover is a high-value forage and nectariferous plant that improves nutrition value of mixed forage of ryegrass, timothy and other grasses grown together (enriches forage with crude protein, fat, phosphorus, potassium, calcium, and reduced fiber content); it also stimulates productivity of a whole meadow by providing nitrogen for cereals (4). White clover is also used as a medicinal plant (5).
However, these plants are often sensitive to adverse environmental impacts. Stress-resistant forms can be obtained through biotechnological approaches; for example, it has been already developed forage bentgrass resistant to heavy metals (6, 7), wheat resistant to root flooding (8), and drought-resistant maize (9).
Cell selection operates on the cell culture. Callus formation and regeneration depend on many factors, such as a type of plant, composition of a culture medium, and conditions of culturing. Even closely related taxa, such as clover and alfalfa, in cell culture manifest different reactions to light, temperature, and phytohormones (10). Several Compositae have been already introduced in cell culture: for sunflower, it has been obtained an optimized method for genotype-independent regeneration from seeds in vitro (11), for stevia - conditions for callus culturing (12).
The purpose of this study was introduction in cell culture of perennial ryegrass, large-flowered flax, brachycome, chrysanthemum, and white clover.
Technique. The work was performed using the seeds of perennial ryegrass (Lolium perenne L.), large-flowered flax cv Goluboy (Linum grandiflorum L.), brachycome cv Golubaya Nezhenka (Brachycome iberidifolia L.), white creeping clover cv Belyi (Trifolium repens L.), chrysanthemum cv Radost’ (Chrysanthemum carinatum L.). The media for callus formation and plant regeneration were nutrient agar media of Murashige-Skoog (MS) (13) and Gamborg (B5) (14) with different content of sucrose and growth regulators: auxins (a-naphthaleneacetic acid (NAA), 2,4-dichlorophenoxyacetic acid (2,4-D), indole-3-acetic acid (IAA)), and cytokinins (6-benzylaminopurine (6-BAP), kinetin). For chrysanthemum and brachycome: regeneration media – half rate MS with different ratios of phytohormones. The seeds used as explants were subject to aseptic treatment: firstly alcohol 1-fold, then – sodium hypochlorite solution for 15-30 minutes (depending on the plant species), and finally washed three times with sterile distilled water. Optimum time of seed treatment was determined considering estimated viability and the proportion of infected primary explants.
The rate of callus formation was estimated as a percentage of resulting calli to the total number of explants placed on a nutrient medium, the rate of shoot formation - as the ratio of the number of calli with shoots to the total number of calli on morphogenic medium.
The confidence interval was calculated using Student’s t-test (reliability - 95%).
Results. To prepare cell culture of ryegrass the seeds were cultured on MS medium with different concentrations of 2,4-D (2 to 10 mg/l; seed treatment in sodium hypochlorite for 20 min since a significant amount of infection). The most intense development of callus occurred in media containing 2,4-D at the content 4 mg/l (58,1%) and 6 mg/l (54,3%) (Table 1). In these conditions, white and light-yellow calli were obtained in 18-21 days. Morphogenic calli developed in hormone-free MS medium; using nutrient media with hormones didn’t increase the rate of formation of such calli. Therefore, the best medium for callus formation was MS medium containing 4 mg/l 2,4-D , for regeneration – hormone-free MS medium, for rooting – half-rate hormone-free MS medium.

1. The rate of callus formation (%) in perennial ryegrass (Lolium perenne L.) and large-flowered flax (Linum grandiflorum L.) cv Goluboy depending on the content of 2,4-D in nutrient medium (X±х)

2,4-D, mg/l

MS medium

B5 medium

Perennial  ryegrass



















Large-flowered  flax



















Note. MS and В5 — respectively, Murashige-Scoog and Gamborg medium (empty cells – the medium wasn’t used)

Seeds of large-flowered flax were placed on MS and B5 media with various concentrations of 2,4-D, in some cases kinetin  was added (2 mg/l). The optimum media for callus formation (Table 1) were B5 with 2,4-D (6 mg/l) (60,0%) and MS with 2,4-D (8 mg/l) (75,4%). In these conditions, light-yellow or light-green calli of medium density was formed in 14-20 days. High frequency of callus formation was also observed on B5 medium with 2,4-D (4 mg/l) (56,4%) and MS with 2,4-D (6 mg/l) (57,1%) (Table 1). This was the first work on obtaining the cell culture of large-flowered flax, so the authors based upon available literature data about other flax species. For large-flowered flax, callus formation was hormone-dependent: it enhanced with increasing the content of auxin, which was consistent with reported facts of common fiber flax (15).
For regeneration to plants, the calli were transplanted on media with NAA (0,1-1 mg/l) and 6-BAP (1,2-2 mg/l). The maximum rate of formation of morphogenic calli was observed on B5 medium with 1 mg/l BAP and 0,1 mg/l NAA (Table 2). During the culturing, a part of callus cells became green and regenerating calli were obtained at the 2nd-3rd passage. These phytohormones were reported to cause plant regeneration from calli of fiber flax and oil flax (16, 17); in the case of fiber flax, the older were cotyledon explants, the higher concentration of NAA was needed for shoot formation (15). In the case of oil flax (most of genotypes), the optimum concentrations of 6-BAP and NAA for regeneration of shoots on hypocotyl segments ranged within, respectively, 1-2 mg/l and 0,02-0,1 mg/l (18, 19).

2. Proportion of calli with shoots (%) in large-flowered flax (Linum grandiflo-rum L.) cv Goluboy depending on the content of 6-BAP and NAA in nutrient medium  (X±х)

Variant 6-BAP + NAA, mg/l

MS medium

В5 medium

1 + 0



1 + 0,1



1 + 0,5



2 + 0,1



2 + 0,5



1 + 1,0



Note. MS and В5 — respectively, Murashige-Scoog and Gamborg medium

Then plants-regenerants were transplanted on hormone-free MS medium for culturing and further transplantation on a rooting medium (Figure, A). It’s notable that although the maximum yield of calli was recorded in the variant of B5 medium with 2,4-D (6 mg/l) and MS with 2,4-D (8 mg/l), the calli formed on MS medium with 2,4-D (6 mg/l) and kinetin (2 mg/l) exhibited better maintenance of regenerative capacity.



Plants-regenerants of large-flowered flax (Linum grandiflorum L.) cv Goluboy (A) and brachycome   (Brachycome iberidifolia L.) cv Golubaya nezhenka (B) obtained in vitro.

Brachycome was another species introduced in cell culture for the first time, so the authors used nutrient media MS and B5 known for cell culture of some other Compositae. These two with a combination of auxin and cytokinin were found to be optimum nutrient media for callus formation in brachycome (Table 3): B5 with 2,4-D (6 mg/l) and kinetin (2 mg/l) (50,1% ) and MS with 2,4-D (6 mg/l) and kinetin (2 mg/l) (55,4%); resulting calli were light-yellow medium dense. It was observed intense seed germination in variants with 2,4-D at concentrations of 1, 2 and 4 mg/l.

3. The rate of callus formation in brachycome (Brachycome iberidifolia L.) cv Golubaya nezhenka and white creeping clover (Trifolium repens L.) cv Belyi depending on the content of 2,4-D in B5 medium (X±х)

Variant 2,4-D + kinetin, mg/l


White creeping clover



















1 + 2



2 + 2



4 + 2



6 + 2



8 + 2



10 + 2



Note В5 — Gamborg medium

Brachycome regenerants were obtained on x MS with NAA (0,1 mg/l) and 6-BAP (2 mg/l) (62,3%) (Fig., B); decreasing the concentration of 6-BAP up to 1 mg/l significantly reduced the rate of regeneration (20%). The notably high regenerative capacity was observed in the calli obtained on B5 medium with 2,4-D (4 mg/l) and kinetin (2 mg/l) (44,6%).
According to the literature, cell culture of white clover can be prepared using a modified Gamborg medium (10), which provided callus formation in about 2/3 genotypes of the initial population of this species. In the authors’ experiments, white clover calli were obtained on B5 medium with various concentrations of 2,4-D and its combination with kinetin (Table 3). Addition of kinetin to the medium increased the rate of formation of callus cells (Table 3). The optimum medium for callus formation (60,2%) was B5 with 2,4-D (8 mg/l) and kinetin (2 mg/l).
To obtain callus tissue of chrysanthemums, the seeds were planted on MS medium containing 6-BAP, kinetin, IAA and NAA in different combinations and concentrations. Intense callus formation occurred in presence of 6-BAP and IAA, the maximum (78,2%) - on MS medium with 6-BAP (1 mg/l) and IAA (0,1 mg/l). This fact is consistent with available reports about increased growth of calli and formation of dense callus tissue on nutrient media containing IAA (20, 21). At the same time, it was known that culturing chrysanthemum petals on media with IAA and 6-BAP resulted in a friable callus not showing further differentiation to meristem centers and development of shoots (16). However, in this work, MS medium with two phytohormones provided the development of dense calli capable of morphogenesis, which then were transplanted on ½ MS with 6-BAP (0,5 mg/l) and after 2-3 passages developed to morphogenic calli (24,8%).
Optimized nutrient media were used to obtain callus cultures and plants resistant to copper. Depending on plant species and variety, inhibiting action of copper was recorded at its concentration in the medium of 10-30 m/l, while selective concentrations of copper were established at 20, 30 and 40 mg/l.
Thus, in this work the authors have selected the optimum nutrient media for callus formation from seed tissues and regeneration of plants of perennial ryegrass, large-flowered flax, brachycome, chrysanthemum and white clover.


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1Moscow State University for Engineering Ecology, Moscow 105066, Russia4;
2K.A. Timiryazev Institute of Plant Physiology, Moscow 127276, Russia, e-mail:

Received April 5, 2011