Chlorella as the source of biologically active compounds for in vitro plant propagation

. The use of nutrient media with an organic composition is a potentially commercially effective way to avoid the use of expensive components of nutrient media while maintaining and increasing the biosynthetic potential of cell cultures of higher plants in vitro . A method has been developed for the in vitro propagating of various taxonomic groups plants. It has been shown that the medium after cultivating chlorella at ¼ MS is optimal for the in vitro plant propagation. The method is intended for in vitro cultivation of seeds, microcuttings and microshoots of plants of different taxonomic groups and can be used for accelerated propagation of valuable plant varieties and hybrids.


Introduction
As a rule, when cultivating cell cultures in vitro, various nutrient media differing in mineral composition are used.Moreover, the mineral composition of the nutrient medium may differ for plants of different taxonomic groups.For example, a nutrient medium containing mineral salts according to Murashiga and Skoog's recipes is used for in vitro cultivation of agricultural, medicinal, and some berry plants; WPM (Woody Plant Medium) nutrient medium is used for in vitro cultivation of tree species [1], and Hamburg or Nitsch nutrient medium is used to obtain haploid crop plants in vitro [2].However, the proposed nutrient media are not optimal for growing in vitro cell cultures of some plant species, and their modification is required for use in cell engineering.
Currently, there is searching for alternative nutrient media and organic biostimulants that reduce the cost of the biotechnological process and promote satisfactory growth of in vitro cell cultures of higher plants.The use of nutrient media with an organic composition is a potentially commercially effective method that allows one to abandon the use of expensive components of nutrient media while maintaining and increasing the biosynthetic potential of in vitro cell cultures of higher plants [2].
A unique organic product is chlorella (Chlorella vulgaris) -a green microalgae containing more than 650 substances.Chlorella vulgaris is a spherical microscopic cell with a diameter of 2-10 µm.The microalgae chlorella contains a pool of biologically active compounds: about 50% protein, consisting of 18 essential amino acids; vitamins B1, B2, B3, B5, B6, A, E; a full complex of essential unsaturated fatty acids; 13 macro-and micronutrients important for health in organic form.
The rich composition creates the prerequisites for the widespread use of chlorella in various areas of human activity, which allows us to call chlorella the multifunctional element of the economic environment [3].
An important industry for the use of chlorella is a food production.Traditionally, chlorella is consumed in the form of powders or tablets, and there is also evidence that a live suspension of chlorella is commercially available.
Scientists have found that the complex of biologically active compounds of chlorella biomass has anti-inflammatory, antioxidant, antimicrobial and wound-healing effects [4].
Chlorella is cultivated in ponds or bioreactors, which differ in the degree of control and the ability to create special conditions for the growth of chlorella biomass.This microalgae has a relatively fast growth rate and responds to a set of conditions, and therefore it is possible to control the release of specific components, for example, lipids [5].
The rich composition of microalgae and, in particular, chlorella creates the prerequisites for their effective use as a substrate for the in vitro propagation of cultures [6,7] One study assessed the in vitro propagation and establishment of S. crispa (Schomburgkia crispa Lindley (Orchidaceae), an epiphytic species native to the Brazilian Cerrado) in a modified growth medium containing an extract of the microalgae Chlorella sorokiniana.Supplementation of WPM (Woody Plant Medium) with microalgae suspended in NPK medium, or as the supernatant resulting from the centrifugation of a culture in NPK medium, was analyzed.The extracts were added to WPM instead of distilled water.The compounds 6-benzylaminopurine (BAP) and indolebutyric acid (IBA) were used as reference in the in vitro multiplication and rooting of S. crispa, respectively.Both growth regulators were tested at 0, 2.5, and 5.0 mg L⁻¹.During in vitro multiplication of S. crispa, WPM supplemented with 5.0 mg L⁻¹ BAP favored the formation of more sprouts, whereas WPM containing 2.5 mg L⁻¹ IBA supplemented with microalgae extract stimulated in vitro rooting.Schomburgkia crispa explants cultivated in medium supplemented with microalgae suspension or the supernatant of C. sorokiniana showed growth similar to explants cultivated in WPM alone.Therefore, it is possible to use the microalga C. sorokiniana as a supplement and/or alternative to WPM for the in vitro cultivation of S. crispa [6].
The effect of microalgae Messastrum gracile and Chlorella vulgaris on the in vitro reproduction of Cattleya labiata orchids was studied in another research [7].The aim of this study was to evaluate the effect of two green microalgae (Messastrum gracile and Chlorella vulgaris) in comparison with plant growth regulators (6-benzylaminopurine, BAP; thidiazuron, TDZ; zeatin, ZEA) on the in vitro propagation of Cattleya labiata, an endangered orchid, using the thin cell layer (TCL) technique from protocorms.TCL sections were cultivated in MS/2 medium containing M. gracile extract (EM) and biomass (BM); C. vulgaris extract (EC) and biomass (BC); and BAP, TDZ, and ZEA in different concentrations.Subsequently, the explants were grown in MS/2 medium, with 2 g L−1 of activated charcoal, to induce elongation and roots formation.For acclimatization, plants were transplanted in trays using Sphagnum sp. as substrate.TCL explants showed a higher formation of protocorm-like bodies (PLBs) than entire protocorms.Explants cultivated in media supplemented with BM showed a high rate of PLB regeneration (59%) and high mean number of PLBs (4 per explant) and 85% survival after acclimatization of the plants.Supplementation with BAP stimulated similar morphogenic responses to those observed with BM and superior results obtained with ZEA and TDZ.Mass propagation of C. labiata plants was successfully achieved using TCL, and it is recommended to supplement the MS/2 medium with 4 g L −1 of BM or 4 mg L −1 of BAP.Microalgae extracts and biomasses are effective alternatives for in vitro propagation of C. labiata that can replace plant growth regulators, as they favored the formation of PLBs and plants.

Materials and methods
Chlorella was grown on a nutrient medium containing different concentrations of compounds according to the Murashige-Skoog recipe (¼ MS, ½ MS, 1 MS, 1.5 MS).After sowing chlorella into the nutrient medium, the optical density of the solution was determined (optical density was measured on a Cary-50 spectrophotometer, Varian, USA).
Chlorella was grown at a temperature of 24±1℃, a 16-hour photoperiod, and illuminated with white fluorescent lamps with a lighting intensity of 3 thousand lux for 5 days.After 5 days, the optical density of the suspension was measured again to determine the increase in biomass, and the growth index (I) and specific growth rate (µ) were calculated using the formulas: where Xmax and X0 are the maximum and initial values of optical density, units, X2 and X1 are the value of optical density (mm) at time t2 and t1, days, respectively.
The lowest growth index of the chlorella suspension was noted on the ¼ MS medium, and the highest -on the 1.5 MS medium, as well as in the control variant with distilled water.These variants of nutrient media on which chlorella was grown were used in further studies as a basis.6-benzylaminopurine (BAP) 1.0 mg/L and Indole-3-acetic acid (IAA) 0.5 mg/L were added to all media.As a control option, we used the MS nutrient medium, on which the microalgae suspension had not previously been grown, as well as pure distilled water, to which similar hormones were added (BAP 1.0 mg/l and IAA 0.5 mg/l).

Results and discussion
The results of measurements of optical density, growth index (I) and specific growth rate (µ) of a chlorella suspension using various concentrations of nutrients in the MS medium are presented in Table 1.The initial appearance of chlorella suspensions before cultivation and after cultivation are presented in Figures 1 and 2. As a result of the studies, it was established that when the studied objects were cultivated on nutrient media in which a suspension of chlorella was previously grown, active growth of the main and lateral shoots was observed.The data obtained were similar to the results noted when growing microcuttings on MS medium (Figures 3-8).Moreover, the best results for the growth of microshoots were obtained when using ¼ MS, on which chlorella was previously grown.When using a nutrient medium based only on distilled water, yellowing of the microcuttings and further death of the explants were observed.

Conclusion
The method of in vitro cultivating of different taxonomic groups plants by the growing plant explants (seeds, microcuttings, microshoots) on a nutrient medium containing a suspension of the microalgae Chlorella vulgaris can be used for accelerated in vitro propagation of valuable plant varieties and hybrids.This can be achieved by using a chlorella suspension as the basis of a nutrient medium, which is initially obtained on a hormone-free nutrient medium containing ¼ of the norm of macrosalts according to Murashiga and Skoog's prescription; after 5 days of cultivation, 20 g/l sucrose, 8 g/l agar, 6-benzylaminopurine at a concentration of 1 mg/l and indolyl-3-acetic acid at a concentration of 0.5 mg/l should be added to the suspension, and this plant medium can be used for cloning plants at the micropropagation stage.

Table 1 .
Results of measurements of optical density, growth index (I) and specific growth rate (µ) of a chlorella suspension using different concentrations of nutrients in the MS medium