Keywords

Rosa mini L; in vitro Culture; MS Medium; Organogenesis of Shoots and Roots; IAA; Pyridine; Pyrimidine; Ivin; Methyur and Kamethur.

Introduction

The rose is rightfully considered the most elegant and incredibly beautiful flower belonging to the genus Rosa L. and family Rosaceae [1-3]. The genus Rosa L., with over 200 species and thousands of cultivars grown worldwide, includes both wild rose species and oil rose species, which are widely used as garden ornamental plants and as a raw material for the production of rose essential oils, water and alcohol extracts of rose used in the perfumery, pharmaceutical and food industries [2-5]. Rose water and alcohol extracts enriched in secondary metabolites such as flavonoids (e.g., flavones, flavonols, anthocyanins) and rose essential oils containing monoterpenes and sesquiterpenes have therapeutic properties and can be used such as a respiratory antiseptic, anti-inflammatory, mucolytic, expectorant, decongestant, antioxidant, antiviral and anticancer agents for the prevention and treatment of serious diseases [3-5]. The fruits of wild roses, called rose hips, are exceptionally rich in vitamin C, 60 times the amount of vitamin C in citrus fruit, a significant amount of iron, calcium, and phosphorus [6].

Shrub or spray miniature roses with small flowers are in great demand among florists, designers and amateur gardeners for a beautiful bush and abundant flowering [4, 7, 8]. The Lydia variety, bred by the specialists of the Dutch company in 1995, is a small plant with abundant flowering and the formation of large flower rosettes.It is one of the most popular varieties of miniature shrub roses belonging to the floribunda category [8]. The Lydia variety is divided into 3 sub-varieties: Lovely Lydia, Spray White Lydia, and Floribunda Classic Lydia. The main advantages of the Lydia variety are frost resistance, immunity to fungal pathologies and pests, lack of capriciousness in growing, flowering during the growing season [8]. Growing Lydia roses is not difficult; the main thing is to observe the regime of watering, feeding and lighting [8]. This crop is planted in the second half of spring indoors, outdoors in containers, or outdoors in the soil well lit by the spring sun, but without direct sunlight and protected from drafts and strong winds [8].

Rose propagation methods mainly include vegetative methods such as cutting, grafting, layering, budding, and rarely seed propagation [7, 9]. As a rule, vegetative propagation and seed propagation methods are very time consuming and do not allow obtaining a healthy plant. Micropropagation through in vitro culture is widely used as an alternative biotechnological method to produce healthy plant clones that are genetically identical to the parent plant [9]. Rose propagation in vitro involves several stages such as initiation of aseptic cultures, proliferation and multiplication of shoots using apical buds, nodal stem segments, shoot tip segments, or leaf petioles through organogenesis or somatic embryogenesis, rooting of microshoots, acclimatization and establishment in the field [9-11].

The efficiency of rose organogenesis in vitro is influenced by various factors, including the type of plant explant, plant species, plant genotypes or cultivars, the composition of nutrient media used for plant cultivation and physical factors [10]. Plant hormones cytokinins such as BAP (6-benzyl aminopurine), 2-ip (2-isopentyladenine) used in high concentrations, auxins such as IBA (indole-3-butyric-acid), NAA (α-naphthalene acetic acid), IAA (indole-3-acetic acid), and gibberellic acid (GA3) used at low concentrations are the main factors affecting rose organogenesis in vitro [9-15]. At the same time, the optimization of nutrient media is carried out using plant hormones ortheir synthetic analogues, for example, a synthetic analogue of cytokininTDS (Thidiazuron), which make it possible to have a high stimulating effect on the regeneration of roses in vitro [10, 12, 14, 15]. A topical issue is the search for new synthetic analogues ofplant hormones, auxins and cytokinins, to increase the efficiency of rose organogenesis in vitro.

The main goal of our work is to study the effect of synthetic plant growth regulators Ivin, Methyur and Kamethuron the organogenesis of shoots and roots of miniature rose (Rosa mini L.) in vitro.

Materials and Methods

Plant growth regulators Ivin (N-oxide-2,6-dimethylpyridine), Methyur (sodium salt of 6-methyl-2-mercapto-4-hydroxypyrimidine) and Kamethur (potassium salt of 6-methyl-2-mercapto- 4-hydroxypyrimidine) were synthesized in the Department for Chemistry of Bioactive Nitrogen-Containing Heterocyclic Compounds, V.P. Kukhar Institute of Bioorganic Chemistry and Petrochemistry of the National Academy of Sciences of Ukraine (Table 1). The growth regulatory activity of synthetic plant growth regulators Ivin, Methyur and Kamethur was compared with the activity of plant hormone auxin IAA (2-(1H-Indol-3-yl) acetic acid) (Table 1).

In experiments, explants of a miniature rose (Rosa mini L.), grown on a hormone-free MS (Murashige and Skoog) medium [16], divided up to 0.5 cm in size with 1-2 microbuds were used.These explants were divided into segments with an axillary bud, one or two leaf blades, and a stem no larger than 1,0 cm in size. Sterilization was carried out according to the scheme: treatment of explants in sodium hypochlorite solution - 20 min. followed by washing in sterile distilled water; sterilization with 70% ethanol (С2Н5ОН) - 4 sec. with washing in sterile distilled water; sterilization in 0.1% solution of Mercury (II) chloride (HgCl2) for 7 min. with 3 times washing with sterile distilled water.

Further, these segments were cultivated at an illumination of3000- 4000 lux, a temperature of 24 ± 2ºС, an air humidity of ~ 70%, and a 16-hour photoperiod for 28 days on control hormone-free MS medium, or modified MS medium containing either synthetic plant growth regulators Ivin, Methyur and Kamethuror plant hormone auxin IAA used at concentrations of 10-5M, 10-6M, 10-7M per 1 liter of MS medium. MS medium for explant cultivation was supplemented also with macro- and micro salts contained in MS basal medium [16], sucrose (0.3%), mesoinositol (100 mg/l) and agar (0.7%), medium pH – 5.7-5.8.

Parameters of organogenesis of miniature rose (Rosa mini L.) (average length of shoots (cm) per explant, length of roots (cm) per explant, average number of roots (pcs) per explant, and frequency (%) of shoot rooting, i.e. the average number of rooted shoots per number of explants) were measured on the 21st and 28th days of in vitro cultivation, respectively.

Statistical Analysis

All experiments were performed in three replicates. Statistical analysis of the data was performed using dispersive Student’s-t test with the level of significance at p≤0.05, the values are mean ± Standard Deviation (± SD) [17].

Results

The conducted studies showed that the use of synthetic plant growth regulators Ivin, Methyur and Kamethuras components of the MS medium had a positive effect on the formation and growth of shoots and rootsof miniature rose (Rosa mini L.) in vitro (Fig. 1 – Fig. 3).

Parameters of organogenesis of miniature rose (Rosa mini L.) in vitro, measured on the 21st and 28th days of cultivation, obtained on MS medium modified with synthetic plant growth regulators Ivin, Methyur and Kamethur at concentration of 10-5M, 10-6M and 10-7M, were generally similar to or higher than those obtained on the control hormone-free medium MS, or on the medium MS containing auxin IAA used at the same concentrations. The effect of synthetic plant growth regulators Ivin, Methyur and Kamethur on the organogenesis of shoots and roots of miniature rose (Rosa mini L.) in vitro varied depending on the concentrations of plant growth regulators (Table 2 – Table 4).

According to the parameters of length of shoots (cm) per explant, measured on the 21st and 28th days of cultivation, synthetic plant growth regulators showed the highest activity: Ivin at concentration of 10-6M (1.75±0,12 cm and 2.82±0,19 cm), Kamethurat concentrations of 10-5M (2.1±0.37 cm and 2.75±0.28 cm) and 10-6M (1.8±0.24 cm and 2.87±0.17 cm), and Methyur at concentrations of 10-5M (2.8±0.24 cm and 2.75±0.63 cm) and 10-7M (3.42±0.95 cm and 3.92±0.64 cm), respectively (Table 2 – Table 4).

Synthetic plant growth regulators showed the lowest activity in relation to the parameters of length of shoots (cm) per explant, measured on the 21st and 28th days of cultivation: Ivin at concentrations of 10-5M (1.88±0.47 cm and 2.83±0.33 cm) and 10-7M (1.73±0.66cm and 2.67±0.41 cm), Kamethurat a concentration of 10-7M (1.2±0.31cm and 2.25±0.33 cm) and Methyur at concentrations of 10-6M (1.42±0.16 cm and 1.87±0.16 cm), respectively (Table 2 – Table 4). Parameters of length of shoots (cm)per explant indicated above did not differ statistically than those obtained on the control hormone-free medium MS, measured on the 21st and 28th days of cultivation (1.0±0.57 cm and 2.17±0.33cm), respectively (Table 2 – Table 4).

According to the parameters of length of roots (cm) per explant, measured on the 21st and 28th days of cultivation, synthetic plant growth regulator showed the highest activity: Kamethurat concentrations of 10-5М (2.75±0.28 cm and 2.83±0.33 cm), 10-6 М (2.08±0.39 cm and 3.15±0.33 cm) and 10-7M (1.55±0.44cm and 2.83±0.21cm), respectively (Table 2 – Table 4).

Synthetic plant growth regulators showed the lowest activity inrelation to the parameters of length of roots (cm) per explant, measured on the 21st and 28th days of cultivation: Ivin at concentrations of 10-5M (2.0±0.57 cm and 2.38±0.25 cm), 10-6M (1.5±0.36 cm and 2.28±0.23 cm) and 10-7M (1.03±0.17cm and 2.92±0.47 cm), and Methyur at concentrations of 10-5M (1.4±0.48 cm and 1..88±0.24 cm), 10-6M (1.67±0.33 cm and 1.9±0.1 cm) and 10-7M (0.82±0.16 cm and 2.08±0.3 cm), respectively (Table 2 – Table 4). Parameters of length of roots (cm) per explant indicated above did not differ statistically than those obtained on the control hormone-free medium MS, measured on the 21st and 28th days of cultivation (1.17±0.7cm and 2.0±0.57 cm), respectively (Table 2 – Table 4).

According to the parameters of number of roots (pcs) per explant,measured on the 21st and 28th days of cultivation, synthetic plant growth regulators showed the highest activity: Ivin at concentrations of 10-5M (6.0±1.39 pcs and 6.33±1.30 pcs) and 10-6М (6.16±1.37 pcs and 6.33±1.3 pcs), Kamethurat concentrations of 10-5M (5.8±1.13 pcs and 6.6±1.47 pcs), 10-6М (6.33±1.19 pcs and 7.83±2.11pcs) and 10-7М (5.88±1.28pcs and 6.19±1.32 pcs), Methyur at concentrations of 10-5M (7.5±1.49 pcs and 7.92±2.73 pcs) and 10-6М (5.95±1.25 pcs and 6.65±1.5 pcs), respectively (Table 2 – Table 4).

Synthetic plant growth regulators showed the lowest activity in relation to the parameters of number of roots (pcs) per explant, measured on the 21st and 28th days of cultivation: Ivin at a concentration of 10-7M (1.75±0.33pcs and 5.33±1.31 pcs) and Methyur at a concentration of 10-7M (3.83±0.94pcs and 3.5±0.67 pcs) (Table 2 – Table 4). Parameters of number of roots (pcs) per explant indicated above did not differ statistically than those obtained on the control hormone-free medium MS, measured on the 21st and 28th days of cultivation (3.0±1.39 pcs and 3.5±1.27 pcs), respectively (Table 2 – Table 4).

According to the parameters of the frequency (%) of shoot rooting,measured on the 21st and 28th days of cultivation, synthetic plant growth regulators showed the highest activity: Ivinat concentrations of 10-5M (67.75±0.93 % and 67.8±4.18 %) and 10-6M (80.17±2.74% and 80.83±3.05 %), Kamethurat concentrations of 10-5M (67.2±4.12 % and 73.75±3.69 %), 10-6M (73.67±2.51 % and 99.83±0.33 %) and 10-7M (60.67±2.61% and 67.17±1.85%), Methyurat concentrations of 10-5M (87.75±3.24 % and 87.33±5.10 %) and 10-7M (80.33±2.51% and 87.5±1.41%), respectively (Table 2 – Table 4).

Synthetic plant growth regulators showed the lowest activity in relation to the parameters of frequency (%) of shoot rooting, measured on the 21st and 28th days of cultivation: Ivinat concentrations of 10-7M (20.67±1.49% and 60.17±2.65%) and Methyurat concentrations of 10-6M (50.23±2.98 % and 60.5±1.92 %), respectively (Table 2 – Table 4). Parameters of frequency (%) of shoot rooting indicated above did not differ statistically or were lessthan those obtained on the control hormone-free medium MS, measured on the 21st and 28th days of cultivation (39.33±7.99 % and 53.17±4.84%), respectively (Table 2 – Table 4).

It has been shown that the effect of synthetic growth regulators Ivin, Methyur and Kamethur on the organogenesis of shoots and roots of miniature rose (Rosa mini L.) in vitro is similar or higher than the effect of the plant hormone auxin IAA used in the same concentrations 10-5M, 10-6M, 10-7M per 1 liter of MS medium (Table 2 – Table 4).

The plant hormone auxin IAA showed the highest activity in relation tothe parameters of shoot and root organogenesis, measured on the 21st and 28th days of cultivation, according to the length of shoots (cm) per explantat concentrations of 10-7M (2.85±0.77cm and 2.93±0.32 cm)andfor the frequency (%) of shoot rootingat concentrations of 10-6M (60.33±1.4 % and 80.16±2.74 %) and 10-7M (93.17±1.55% and 93.17±1.55%), respectively (Table 2 – Table 4).

The plant hormone auxin IAA showedthe lowest activity in relation to the parameters of shoot and root organogenesis, measured on the 21st and 28th days of cultivation, in relation to the length of shoots (cm) per explantat concentrations of 10-5M (1.25±0.22 cm and 1.55±0.25 cm) and 10-6M (1.34±0.18 cm and 1.83±0.21cm), length of roots (cm) per explantat concentrations of 10-5M (0.58±0.17 cm and 0.8±0.24 cm), 10-6M (1.82±0.16 cm and 1.71±0.19 cm) and 10-7M (1.58±0.59cm and 2.0±0.36cm), number of roots (pcs) per explantat concentrations of 10-5M (3.2±1.14 pcs and 4.67±1.57 pcs),10-6M (2.83±0.94 pcs and 3.5±0.84 pcs) and 10-7M (5.67±1.73pcs and 6.16±1.85 pcs), and frequency(%) of shoot rooting at concentrations of 10-5М (47.0±2.88 % and 47.25±5.02 %), respectively (Table 2 – Table 4). Parameters of organogenesis indicated above did not differ statistically or were lessthan those obtained on the control hormone-free medium MS, measured on the 21st and 28th days of cultivation in relation to the length of shoots (cm) (1.0±0.57 cm and 2.17±0.33cm), length of roots (cm)(1.17±0.7cm and 2.0±0.57 cm), number of roots (pcs)(3.0±1.39pcs and 3.5±1.27 pcs), and frequency (%) of shoot rooting (39,33±7,99 % and 53.17±4.84%), respectively (Table 2 – Table 4).

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Thus, the obtained results showed both cytokinin- and auxin-like effectsof synthetic plant growth regulators Ivin, Methyur and Kamethuron theprocesses of elongation, division and differentiation of the isolated plant cells, which are the main processes of organogenesis of shoot and root meristems of miniature rose (Rosa mini L.) in vitro.

Analysis of growth regulating activity showed that synthetic plant growth regulators have a more powerful effect on the organogenesis of shoots and roots of miniature rose (Rosa mini L.) in vitro when used as components of the MS medium in concentrations: Ivin at concentrations of 10-5M and 10-6M, Kamethurat concentrations of 10-5M and 10-6M, Methyur at concentrations of 10-5M and 10-7M.

The plant hormone auxin IAA showed the highest effect on the organogenesis of shoots and roots of miniature rose (Rosa mini L.) in vitro when used as component of the MS medium at concentrations of 10-6M and 10-7M.

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Discussion

Rose micropropagation through in vitro culture refers to a method of asexual propagation that produces healthy plant clones that are genetically equal plants to the parent plant and to each other [9]. Since the 1970s, rose micropropagation protocols have been developed [18].

The apical buds, nodal stem segments, shoot tip segments, or leaf petioles are used as explants for proliferation and multiplication of shoots and roots [9-11]. To enhance the organogenesis of stems, leaves and roots,MS basal medium [16] supplemented with plant hormones cytokinins and auxins is used [9-15, 18]. Cytokinins BAP, 2-ip, and TDS, and auxins IAA, NAA, and IBA are most widely used for shoot and root organogenesis [9-15, 18]. The ratio of plant hormones auxins and cytokinins affects organogenesis towards the proliferation of shoots or roots. Usually, high concentrations of cytokinins and low concentrations of auxins or gibberellic acid GA3 promote shoot proliferation through direct organogenesis or somatic embryogenesis [9-15, 18]. To promote root organogenesis, modified MS medium with a high content of mineral salts, with or without the addition of auxins at low concentrations, is used [9-15, 18].

Synthetic plant growth regulators Ivin, Methyur and Kamethurwere previously studied by us in tissue culture of potato, tobacco and cherry to intensify plant organogenesis in vitro [19, 20]. In our earlier published work [19], it was found that Ivin (N-oxide- 2,6-dimethylpyridine) and its derivatives, which were used as components of culturemedia in concentration range from 0,05 mg to 10 mg per 1 liter, exhibit predominant cytokinin-like activity, affecting the processes of increasing the growth of callus biomass, delaying cell ageing, activating photosynthesis in plant cells and promoting organogenesis of potato and tobacco plants in vitro. The effect of Ivin and its derivatives on plant organogenesis was similar or higher than that of the plant hormone cytokinin Kinetin (6-furfurylaminopurine) used at the same concentrations. In our work [20] it was shown that the use of Methyurand Kamethurat concentrations of 10-5М - 10-8М as components of the MS medium improved the development of the root system on cherry microshoots. The plant growth regulating activity of Kamethur and Methyur was similar or higher than that of the auxin IBA, cytokinin BAP, and gibberellic acid.

This work is aimed at searching for new effective plant growth regulators capable of initiating organogenesis of miniature rose (Rosa mini L.) in vitro on MS medium without the addition of plant hormones auxins and cytokinins. The obtained results showed that synthetic plant growth regulators Ivin (N-oxide-2,6- dimethylpyridine), Methyur (sodium salt of 6-methyl-2-mercapto- 4-hydroxypyrimidine) and Kamethur (potassium salt of 6-methyl- 2-mercapto-4-hydroxypyrimidine) revealed both cytokinin - and auxin-like activity on the organogenesis of shoots and roots of miniature rose (Rosa mini L.) in vitro. Their growth regulating effect is similar to or exceeds that of the plant hormone auxin IAA. The physiological and molecular mechanisms of action of the synthetic plant growth regulators Ivin (N-oxide-2,6-dimethylpyridine), Methyur (sodium salt of 6-methyl-2-mercapto-4-hydroxypyrimidine) and Kamethur(potassium salt of 6-methyl- 2-mercapto-4-hydroxypyrimidine)on plant development are of the considerable interest. The results of our previous studies with synthetic low molecular weight heterocycles, derivatives of pyridine, pyrimidine, pyrazole, isoflavones, oxazolopyrimidine and oxazole showed their auxin- and cytokinin-like effects on the processes of elongation, division and differentiation of plant cells [21-30], which are the main processes of organogenesis of plant shoot and root meristems in vivo and in vitro, which are controlled by plant hormones [31, 32].

Our earlier studies of the molecular mechanisms of action of plant growth regulators Ivin and Methyur demonstrated their stimulating effect on gene expression at the level of transcription and translation of genetic information, due to which the time of plant ontogenesis is almost halved [33]. In addition, we came to the conclusion that synthetic plant growth regulators Ivin and- Methyur affect plant growth indirectly, through the endogenous pool of plant hormones [34].

The proposed mechanism of action of synthetic plant growth regulators directly, through a network ofplant hormone signaling pathway [35, 36], or indirectly, i.e. affecting the biosynthesis of plant hormones, their stability and metabolism [36], was discussed in the works of other authors [37-47].

Therefore, our further work will focus on study of molecular mechanism of action of synthetic plant growth regulators Ivin, Methyur and Kamethur.

Conclusion

Our studies showed that the use of synthetic plant growth regulators Ivin (N-oxide-2,6-dimethylpyridine), Methyur (sodium salt of 6-methyl-2-mercapto-4-hydroxypyrimidine) and Kamethur (potassium salt of 6-methyl-2-mercapto-4-hydroxypyrimidine)as components of the MS medium improves the organogenesis of shoots and roots of miniature rose (Rosa mini L.) in vitro. The growth regulatory activity of Ivin, Methyur and Kamethurwas compared with the activity of plant hormone auxin IAA (2-(1HIndol- 3-yl)acetic acid). The use of synthetic growth regulators Ivin, Methyurand Kamethurhad a positive effect on the formation and growth of shoots and roots, measured on the 21st and 28th days of cultivation. The parameters of average length of shoots (cm) per explant, length of roots (cm) per explant, number of roots (pcs) per explant, and frequency (%) of shoot rooting generally increased depending on the concentration of regulators. The effect of Ivin, Methyur and Kamethur,used at concentrationsof 10-5M, 10-6M, 10-7M per 1 liter of MS medium,on the organogenesis of shoots and roots of a miniature rose is similar or higher than that of the plant hormone auxin IAA used at the same concentrations. The application of synthetic plant growth regulatorsIvin, Methyur and Kamethur for micropropagation of the miniature rose (Rosa mini L.) through in vitro culture is proposed.