Somatic embryogenesis and evolution of phenolic compounds production in Teucrium polium L. subsp.
geyrii Maire cell suspensions
Meguellati H.1, Ouafi S.1, Saad S.1,2*, Harchaoui L.1 and Djemouai N.1,3,4
1. Laboratoire de Recherche sur les Zones Arides (LRZA), Faculté des Sciences Biologiques, Université des Sciences et de la Technologie Houari Boumediene (USTHB), BP32 El-Alia, 16111 Bab Ezzouar, Algiers, ALGERIA
2.Division Bioressources, Centre de Recherche Scientifique et Technique sur les Régions Arides (CRSTRA), Biskra, ALGERIA 3. Laboratoire de Biologie des Systèmes Microbiens (LBSM), Ecole Normale Supérieure de Kouba, B.P. 92, 16050 Kouba, Algiers, ALGERIA
4. Département de Biologie, Faculté des Sciences de la Nature et de la Vie et Sciences de la Terre, Université de Ghardaia, BP 455, 47000 Ghardạa, ALGERIA
*somiasaad89@gmail.com
Abstract
The valorization of Teucrium polium L. subsp. geyrii Maire, an endemic medicinal plant of Algeria was attempted. A somatic embryogenesis protocol was developed and two types of cell suspensions (brown and gray suspensions) were obtained. Weight evolution of the two suspensions revealed a latency phase, an exponential growth phase and a final stationary phase which was directly followed by cell degeneration at the end of the third month of culture. Total polyphenols estimation showed that brown suspensions contained the highest absolute content.
The production of flavones-flavonols, anthocyanidins and C-glycosides in the two types of cell suspensions remained low during the cell growth phase while it became active later during the stationary phase and was found to be more intense at the end of cell growth.
HPLC profiles indicated that the synthesis of the various phenolic fractions (flavones-flavonols, anthocyanidins and C-glycosides) took place unevenly in the two cell suspensions.
Keywords: Teucrium polium, Somatic embryogenesis, Cell suspension, Histological analysis, Polyphenols, HPLC.
Introduction
Biotechnology has experienced tremendous growth over the past years, as its development seems promising for the profitable production of complex molecules or the discovery of new active ingredients. Several studies have been carried out on the development and in vitro propagation of medicinal plants and on improving the production yield of active ingredients intended for the production of active compounds9.
Algeria is rich in plant genetic resources of medicinal and aromatic interest in north Africa37. Stimulation of secondary metabolic pathways is an important part of the plant’s control strategies in arid and semi-arid environments in particular. T. polium L. subsp. geyrii Maire is one of the endemic plants of the Hoggar region in the Tamanrasset
*Author for Correspondence
province (Algeria).
Teucrium polium has been used as a medicinal plant for more than 2000 years27. In traditional African medicine, this subspecies is used in times of stress. Its anti-stress and antioxidant properties can help reduce skin aging29. Furthermore, it is used to treat abdominal pain, indigestion, diabetes and urogenital diseases in the Middle East and the Mediterranean areas22,23,27.
Still, more studies are required for the biochemical study in order to better reveal these pharmaceutical potentials.
Moreover, the adoption of a biotechnological strategy will make it possible to meet the growing needs of consumers and the populations of this plant have dramatically decreased in the last few years in Tamanrasset (personnel observations) as in other regions such as Iran31.
Thus, wild populations are currently at risk of rapid eradication and extinction because of immoderate picking by the Targui population and irregular grazing by nomads. It is therefore essential to initiate in vitro cultures of T. polium L.
subsp. geyrii Maire to develop technologies that ensure a better yield of vegetative multiplication by somatic embryogenesis which is of great interest and allows the unlimited multiplication of the best individuals for the production of highly improved plants and at the same time a high rate of biologically active compounds. For this, we often use elicitors in the synthesis of target compounds that are added to the culture medium7.
Numerous results obtained on cell cultures have opened the way to various applications and have also enriched knowledge of the secondary metabolism of plants. Indeed, cell cultures are axenic under the control of several parameters (the composition of the medium, temperature, agitation and photoperiod), they ensure the production of increasingly intense biomass regardless of the seasons10.
This work aims to study the kinetics of the production of total polyphenols and flavonoid compounds in cell suspensions of T. polium L. subsp. geyrii Maire during their growth evolution and a comparison of the flavonoid composition between the material cultivated in vitro and the mother plant which grows spontaneously in the Tamanrasset region (Algeria).
Material and Methods
Plant material: In April 2013, aerial parts at the flowering stage of T. polium L. subsp. geyrii Maire were randomly collected from Ilamen station, precisely in Chaaba Eghesmaden located in Tamanrasset province, Central Sahara of Algeria (longitude: 005 ° 29 30.6 E, latitude: 23 ° 14 26.9 N, altitude: 2055 m). The harvested plant was authenticated by researchers from the National Institute of Forestry Research (INRF) of Tamanrasset (Algeria) according to the work of Quézel and Santa.30 A voucher specimen was deposited in LRZA herbarium.
Three and a half months old calli were obtained from the in vitro culture of the aerial parts of T. polium L. subsp. geyrii Maire as published by Meguellati et al.22 The embryogenic calli of highly friable texture, brown-yellowish, or bronze colors were selected to serve as the material of choice for the establishment of cell suspensions.
Embryogenic callus histology: The obtained friable embryogenic calli were used for the histology study by light microscopy as described by Langeron18 and Gabe13. First, the obtained embryogenic calli were fixed in FAA (Formalin/glacial acetic acid/ethanol, 1: 1: 70.8, v/v/v) for 48 h, dehydrated through a graded ethanol series ethanol 70% to 100% and embedded in paraffin wax. The specimens were then sectioned at 7µm thickness using a rotary microtome (Leica) and double-stained with Periodic Acid Schiff (PAS) and Naphthol Blue Black (NBB)20. The obtained sections were observed under a light microscope to study and determine their nature and structure.
Initiation of cell suspensions: The obtained embryogenic calli that were obtained from the enriched medium in Kin/ANA and BAP/2,4-D were used for the establishment of suspension culture. The selected calli were transplanted into the same initial culture medium of Murashige and Skoog26 without agar-agar to avoid their metabolic destabilization22. Twenty milliliters of the prepared media were distributed in 100 ml Erlenmeyer flasks and autoclaved at 120 °C for 20 min. Then, one gram of the selected calli was finely separated into small cellular aggregates using a scalpel. Furthermore, they were transplanted onto the prepared liquid medium and were placed on a cell culture shaker at 85 tours/min of speed with the orbital shaker S1 in a culture chamber at 27 °C and under a photoperiod.
Weight evolution of cell suspensions: The liquid culture medium was renewed each week and the subcultures of the cellular aggregates were checked regularly to avoid their necrosis. This step is compulsory because it allows their multiplication after several cycles as a function of time. In sterile conditions, the transplanting process is done by filtration of the cell suspensions on a filter membrane and then quickly weighed before being transplanted into the new culture medium of identical composition to the initial medium. The weight evolution of the fresh cell masses was recorded and microscopic observations were made.
Phytochemistry study: This study was initiated by the extraction of polyphenolic fractions from the obtained cellular suspensions of T. polium L. subsp. geyrii Maire initiated from the obtained friable calli as described by Meguellati et al22 followed by their qualitative characterization by High-Performance Liquid Chromatography (HPLC).
For the study of cell suspensions, we took into account their weight growth as a function of the time estimated by weighing the cell suspension before each subculture as well as their relationship with the production of phenolic compounds. One gram of the cell suspensions was filtered through a filter membrane after each stage of their development and was subjected to the different extraction methods. Series of spectrophotometric assays in the first, second and third months of cultivation were carried out which corresponded to the first, second and third cycle of growth of these suspensions. The phytochemical study of cell suspensions related to their production of phenolic substances according to their weight and the different cycles of their growth was conducted.
Determination of total polyphenols content: The total content of polyphenols was assessed using the Folin- Ciocalteu reagent33. Three repetitions of 200 µl of cell suspension infusions were mixed with 1 ml of Folin- Ciocalteu reagent diluted 10 times in distilled water. After 5 min, 800 μl of 7.5% sodium carbonate (Na2CO3) was then added. After 30 min of incubation at room temperature, the total polyphenols content was determined by measuring the absorbance at 765 nm (UV-visible spectrophotometer, Cecil, CE2021, 2000 series) and results were expressed in mg of gallic acid equivalent per g of dry matter (GAE/g DM) using a gallic acid standard curve.
Preparation of the plant extracts: To extract the maximum of polyphenols from the obtained cell suspensions, we carried out several extraction methods. The experimental protocol that was used, was developed by Lebreton et al19 from the initial scheme of Bate-Smith et al4. The extraction first consisted of a 2N HCl acid hydrolysis (1 g of cell suspension/80 ml) at a high temperature (40 °C) which allows separation of the flavones-flavonols and phenolic acids in the ethereal phase (ExDi-Eth) and the C-glycosides and anthocyanidins in the butanolic phase (ExButOH). The two obtained phases were then evaporated under a ventilated hood and the dry residues are recovered in 3 ml of methanol.
The extracts were kept at 4 °C for qualitative and quantitative analysis.
HPLC analysis: Chromatographic analysis of the obtained extracts was performed using a Waters Alliance 2695 chromatography system equipped with a C18 column (20 X 4.6mm, 5µm) and a UV detector with bars of diodes surveyor. The extracts were analyzed using solvent system B (methanol) in solvent system A (0.1% phosphoric acid in water) with the following gradient: 1-10 min (30% B), 1-20
min (30 to 40% B) and 20-60 min (40 to 100% B). The injection volume was 20 µl and the flow rate was 1 ml/min.
Detection was performed between 250 nm and 365 nm for phenolic aglycones as well as phenolic acids and at 340 nm for C-glycosides.
Statistical analysis: The data were presented as Mean ± SD for bodyweight evolution results of cell suspensions.
Results
Somatic embryogenesis: Callus was induced from the different aerial parts of T. polium L. subsp. geyrii by somatic embryogenesis, the obtained calli generated in vitro plants on 2,4-D/BAP enriched minimal medium and established cell suspensions on minimal medium enriched by 2,4-D/Kin.
Histological analysis of embryogenic calli revealed cell clusters that were characterized by the dominance of dedifferentiated (meristematic) cells with very dense cytoplasm, rich in secretion products with high mitotic activity (Figure 1 B) and cells with a heterogeneous appearance with large, peripheral and differentiated parenchyma cells around small embryogenic cells full of protein and starchy reserves colored in blue by NBB and are delimited by the parietal mucus. These external cells have an
increasingly pronounced embryogenic ability (Figure 1 B) which implies a decrease in the accumulation of phenolic compounds.
The potentially friable and hydrated secondary callogenic embryos were the most suitable for the initiation of cell suspensions. Microscopic observations revealed the presence of diverse but non-specific structures. Among them, many fairly diffuse meristematic clusters and no developing embryo were spotted intended for the establishment of cell suspensions for the phytochemical study (Figure 2 A and B).
Microscopic analysis of cell aggregates after shaking:
The good quality of the starting calli is the first guarantee for the growth and evolution of the cell suspension. Thus, those of yellowish-brown color were selected (Figure 3 A and B).
All calli must have a highly friable consistency to allow good cellular dissociation in the liquid medium.
Small condensed cells were found at the periphery of the obtained cellular clusters. These small cells were round or elongate in shape with a central bulky nucleus that shows great mitotic activity (Figure 4 A and B).
A B
Figure 1: Embryogenic calli are formed of differentiated parenchyma cells (dC) and proembryos (PE).
The cytoplasm of embryogenic cells is loaded with proteins (blue coloration by NBB) (x 100)
Figure 2: Microscopic appearance of calli with high friability potential and undifferentiated cells (UC) (x 100)
PE
PE
dC
UC
The inner parts of the cell aggregates showed good cell dissociation which allowed us to see solitary cells (Figure 4
C). The cellular debris decreased and the most widespread elements were mitotic dividing cells (Figure 4 D).
Figure 3: Appearance of friable calli intended for the establishment of cell suspensions.
A. Yellowish-brown friable calli grown on media enriched with BAP/2,4-D, B. Bronze-colored calli cultivated on medium enriched with Kin/ANA
Figure 4: Details of the microscopic appearance of cell aggregates grown in an agitated liquid medium.
A: Beginning of cell suspension establishment, B and C: Cells in intense mitotic divisions, D: Solitary rounded cells, CS: Lonely cell, Cad: Divided rounded cells, N: nucleus
Figure 5: Cell suspensions loaded with polyphenols at the end of their culture. A. Rounded cells loaded with polyphenols, B. Erlenmeyer flasks containing brown and gray suspensions
After 1 month of agitation in the culture medium, it is possible to observe at the level of the cell suspensions, cellular aggregates on which cells of rounded shape, charged with polyphenols which appear in the dense cytoplasmic content were found (Figure 5 A). Macroscopic observations of the cell suspensions after shacking revealed a change in the color of the culture medium. Indeed, those cultivated in a medium enriched in Kin/ANA gave a reddish-brown color while the cell suspensions cultivated in a medium enriched in BAP/2,4-D gave a greyish-brown color (Figure 5 B).
Weight evolution of cell suspensions: The results of the weekly weighing of the two types of cell suspensions as a function of time are shown in figure 6. The comparison of the means of the weights obtained in the two cell suspensions after one week in the agitated liquid culture medium revealed a significant reduction (p <0.05) in the weight of the two cell suspensions. This decrease could correspond to a lag phase which resulted in the non-adaptation to the new culture conditions (liquid medium, light and agitation). We recorded a decrease in the brown suspension and the gray suspension of the order of 0.86 g and 0.62 g respectively.
However, the weight gain was significantly remarkable during the second week in the gray suspension compared to the brown one. The cells try to adapt with intense activation of the cell divisions until reaching an exponential growth phase and then stabilizing at the fourth week statistically. It should be noted that the first two weeks did not ensure the homogeneous development of the suspensions because the standard deviations were very high. On the other hand, after the second week, the obtained distributions are rather homogeneous; the values of the SDs were weak. The fresh weight of the cell mass remained stable for the following 4 weeks and no significant difference was revealed (p> 0.05).
The average weight recorded during this period was 1.6 ± 0.3 g for the brown suspension and between 1.44 ± 0.1 g and 1.49 ± 0.1 g for the gray suspension.
From the third month of culture, the mass of cell aggregates fell progressively throughout the last four weeks of culture
and the weight of the cell suspensions did not stop decreasing, which probably corresponded to the start of cell lysis. The decrease in average weights was noticeable, going from 1.6 g/100 ml to 1 g/100 ml for the brown suspension and from 1.49 g/100 ml to 1.02 g/100 ml for the gray suspension. These results indicated the end of the culture of these cell suspensions.
Determination of total polyphenols content: The contents of the total polyphenols produced by the cell suspensions at the end of their culture are recorded in table 1. Cell suspensions have very high total polyphenols contents. We noted first of all that they produced different quantities, the brown suspension produced more polyphenols than the gray suspension with 3.1 ± 0.3 mg GAE/g DM and 1.8 ± 0.5 mg GAE/g DM respectively. We noted that the SDs were low showing that the synthesis of polyphenols was homogeneous.
Determination of different fractions: The most commonly used strategy when studying secondary metabolite production is to compare the growth and biosynthetic kinetics of secondary metabolites in a culture. The results of the assays (average contents of three measurements) for three months for the brown cell suspension are shown in figure 7. They show that after one month of culture in a stirred liquid medium, the assay of the flavones-flavonols fraction in the brown suspension revealed an average absolute content of the order of 0.003 mg/g FM. This small amount was produced during the first cycle of the suspension growth.
After a slight decline in its growth which lasted about two weeks, there was a recovery in its development during the other two weeks of the first cycle. Following this growth where the gain of the fresh material was optimal, the cell suspension crossed a stationary phase, we noticed stability in its development. This phase corresponded to the second month of culture. In parallel, the absolute average content of flavones-flavonols was 0.006474 mg/g FM which was higher than that produced in the first cycle.
Table 1
Content of the total synthesized polyphenols by the studied cell suspensions Type of suspension Total polyphenols content
(mg EAG / g FM) Brown suspension
Gray suspension
3.1 ± 0.3 1.8 ± 0.5 Values are expressed as mean ± SD, n= 3.
Figure 6: Weight evolution of brown and gray suspensions. Values are expressed as mean ± SD, each measurement was repeated 3 times, the means between the columns followed by a different letter
are significantly different (p <0.05)
Figure 7: Evolution of the synthesis of the different phenolic fractions in brown suspension.
Values are expressed as mean ± SD, n= 3
a a
c
b b b b b
b
b b
b
a b
b
b b b b b
b b
b b
0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2
W 1 W 2 W 3 W 4 W 5 W 6 W 7 W 8 W 9 W 10 W 11 W 12
Weight (g)
Weeks (W)
Brown suspension
Gray suspension
0 0.01 0.02 0.03 0.04 0.05 0.06 0.07
1 month 2 month 3 month
Absolute content of different phenolic fractions (mg/g FM)
Time (month)
Flavones-flavonols Anthocyanidins C-glycosides
Figure 8: Evolution of the synthesis of the various phenolic fractions in the gray suspension.
Values are expressed as mean ± SD, n= 3
There was an increase in the production of this fraction during this period. The third growth cycle of this suspension was also in favor of the synthesis of these compounds that we recorded as content of 0.00897 mg/g FM. This was the maximum obtained amount during the three months of culture while the growth of mass cells tended to decrease during this cycle. The same observations for the other fractions were noted. Low production of anthocyanidins was observed during the first month of (0.0117 mg/g FM). This quantity increased during the second month or second cycle which corresponded to the stationary phase estimated as 0.01872 mg/g FM to reach the optimal value at the end of the third month (third cycle) with the highest value of 0.045084 mg/g FM.
The synthesis of C-glycosides followed the same trajectory as the other fractions. Their production was very low during the first month, it increased during the second month to reach its maximum value at the end of the third month, the noted levels were 0.0092 mg/g FM, 0.012975 mg/g FM and 0.0231 mg/g FM respectively. The contents of the phenolic compounds in the second suspension of a grayish color have kinetics of appearance identical to that observed in the brown suspension (Figure 8). In fact, during the first month which is marked by the beginning of the growth of cell mass (exponential phase), there is a small peak in the production of all the analyzed compounds (flavones-flavonols, anthocyanidins and C-glycosides) with their corresponding contents 0.01 mg/g FM, 0.004 mg/g FM, 0.015 mg/g FM respectively.
From the second month corresponding to the stationary growth phase, the start of a production peak was announced at all fractions where the dosage revealed the contents of
0.02184 mg/g FM of flavones-flavonols, 0.00624 mg/g FM of anthocyanidins and 0.01875 mg/g FM of C-glycosides while the maximum reached at the end of the third month.
This phase was marked by stopped growth cells with the exception of C-glycosides, their content tended to decrease during this last growth cycle.
During the first cycle, an increase in cell mass and a moderate biosynthesis of phenolic compounds are therefore observed simultaneously inside the cells for all the fractions in the two cell suspensions. It is possible that the biosynthesis threshold of these compounds inhibited cell growth and led to the stationary growth phase. Or this may be due to better adaptation to growing conditions.
Furthermore, the secondary metabolism remains dominant at the start of the stationary phase and is expressed by a significant stimulation of the synthesis of all the studied fractions while the primary metabolism responsible for growth is practically deactivated til the end of phase 3 which implies a fall in cell growth.
The collected data during three months of culture made it possible to identify the evolution of cell suspensions with an optimal period of production being between the beginning of the second month til the end of the third month of culture for practically all the studied compounds. All maximum values were measured during this period with an exceptional case of C-glycosides present in the brown cell suspension while its production was no longer favorable during the third cycle.
This could be explained either by release into the culture medium or by intracellular biodegradation.
We can confirm, on the other hand, that in our case there was an antagonism between the primary and secondary
0 0.005 0.01 0.015 0.02 0.025 0.03 0.035 0.04 0.045 0.05
1 month 2 month 3 month
Absolute content of different phenolic fractions (mg/g FM)
Time (month)
Flavones-flavonols Anthocyanidins C-glycosides
metabolism, so a negative correlation between the two metabolisms was noted. When cells divide, they do not immediately produce secondary metabolites, they start with the production of biomass and then they will synthesize the secondary metabolites. It should be noted that the quantity of anthocyanidins produced by the brown suspension during the three cycles (3 months) of its development remained the highest compared to the other fractions. We could say that these compounds were responsible for their coloring.
In contrast, in the greyish cell suspension, the absolute contents of flavones-flavonols were higher than those of anthocyanidins and C-glycosides. The nature of the culture medium can have an influence on the content of phenolic compounds, particularly in growth regulators (the other elements of the medium remained unchanged) which are in favor of the synthesis of such compounds. Indeed, the brown suspension was obtained in the medium enriched with Kin/ANA. The gray-colored suspension was cultured in the medium added to BAP/2,4-D.
Table 2
Phenolic compounds identified in the two types of cell suspensions of T. polium L. subsp. geyrii Maire
Phenolic compound Brown
suspension
Gray
suspension Retention time (min)
Hydroxybenzoic acid
Gallic acid X X 6.4
Syringic acid X X 7.5
p-hydroxy-benzaldehyd acid X X 7.8
Vanillin X X 8.6
Protocatechuic acid X X 10.4
m-anisic acid X - 11.6
Vanillic acid X - 17.5
Hydroxy- cinnamic acid
Caffeic acid X X 7.0
Chlorogenic acid X X 8.3
Hydroxy-cinnamic acid X X 8.7
Ferulic acid X X 9.2
Flavonol
Myricetin X X 11.7
Quercetin X - 12.5
Kempferol X X 13.5
Isorhamnetin X - 15.5
Flavone
Luteolin X X 12.0
Apigenin X - 14.7
Tricin X X 15.5
Flavanone Naringenin X X 10.5
C-glycosides
Naringenin-6-C-glucoside X X 10.5
Naringenin-di-C-glucoside X X 11.1
Myricetin monoglycoside X X 12.1
Orientin X X 13.3
Isoorientin X X 13.4
Kaempferol monoglucoside X X 13.8
Vitexin X X 14.4
Iso-vitexin X X 14.7
Methylxanthin
Cafein X X 6.2
Qualitative analysis by high-performance liquid chromatography: Table 2 summarizes the comparative results of the qualitative analysis of the fractions of flavonic aglycones and C-glycosides produced by the two cell suspensions of T. polium L. subsp. geyrii Maire at the end of the 12 weeks of culture of the cell suspensions. The obtained chromatograms have shown that the synthesis of these fractions is different. The brown suspension produced hydroxy-benzoic type phenolic acids as we have revealed the presence of gallic, syringic, p-hydroxy-benzaldehyde, chlorogenic, protocatechic, m-anisic and vanillic acids. All the mentioned hydroxy-benzoic type phenolic acids were revealed in the grey suspension with the exception of m- anisic and vanillic acids. Furthermore, the phenolic acids of the cinnamic series were found in the two types of cell suspensions.
The flavones, flavonols were also produced unevenly in the two cell suspensions. For the flavonol class, the brown suspension contained myricetin, quercetin, kaempferol and isorhamnetin while myricetin and kaempferol were found in the gray suspension. Likewise, for flavones, the results of the qualitative analysis showed that the brown suspension accumulated more flavones. In addition, we have noticed that the extract of the gray suspension was devoided of apigenin. We found that the brown suspension is richer in flavones and flavonols than its counterpart. Naringenin is a compound in the family of flavanones, it is identified in the two cell suspensions. The comparative qualitative analysis of C-glycosides that was carried out from the obtained profiles of the two cell suspensions showed that these compounds are similarly synthesized in the two types of suspensions.
Discussion
In this study, we describe for the first time a reliable and very efficient methodology for the rapid production of embryogenic cell suspensions in T. polium L. subsp. geyrii Maire. The regeneration process that we have developed makes it possible to repeatedly produce vitro-plants through an indirect process of embryogenesis which involves the passage of primary explants through a cal stage. The initiation of proembryogenesis takes place during the induction of callogenesis.
Proembryogenic cells were visible on calli from inflorescences of T. polium L. subsp. geyrii Maire on media enriched in 2,4-D/BAP between the 90th and 100th day22. 2,4-D combined with BAP improved the percentage of calli by its effect on DNA synthesis36. This hormonal association can also trigger the differentiation of somatic cells into competent embryogenic cells11. The contribution of 2,4-D to the culture medium stimulates the induction of calli36. This growth regulator is generally favorable to the induction of friable calli used for the establishment of cell suspensions16.
Our results have shown that the success of the initiation of a good cell suspension is conditioned by obtaining friable
calli. Teixeira et al35 had also stressed the importance of using friable tissues, to install cell suspensions and that the growth rates could be multiplied by 9 when the suspensions are initiated with friable tissues. We could not compare our results with other works due to the lack of data available in this area and for the studied plant. However, it is certain that the proliferation of our cell suspensions goes through the same phases (latency, exponential and stationary growth) as many species. We can cite the examples of the date palm variety Degla Beida, Sweet potato (Ipomoea batatas L.
(Lam) and Aerva javanica5,38.
The quantitative analysis of the total polyphenols and the fractions by UV-visible spectrophotometric assays had the objective of determining the content of these phenolic compounds by comparing the productivity in vitro and in situ under normal conditions (without elicitation or addition of precursors). The average contents of the total polyphenols present in the aqueous extract of the two cell suspensions are significantly lower than those detected in the infused adult plant23. This may be due to the instability of cell cultures.
This same result was observed in the calli of Fagopyrum esculentum. Their productivity in polyphenolic compounds could be considered stable around the 63rd week of cultivation3. Furthermore, if we compare the contents of the different phenolic fractions of the extracts obtained from cell suspensions, we can say that the material cultivated in vitro is not very rich in phenolic compounds.
The study of the kinetics of the synthesis of the different fractions is the first contribution to the knowledge of the studied plant composition. The main limit, however, is the small amount of these metabolites produced during the described tests. It should be noted that the process is far from being optimized and we believe that the juvenile state of these tissues also has a lot to do with it. It would probably be advisable to play on certain conditions of culture (intensity and/or duration of lighting, variation of pH, elicitation to maximize the producing biomass etc.).
Tumova and Ostrozlik39 were able to demonstrate a marked increase in the production of flavonoids in cell suspensions of Ononis arvensis L elicited by adding jasmonic acid and iodo-acetic acid to the culture medium compared to the control (cell suspension without elicitors). Zhang et al40 studied the effect of temperature on the production of anthocyanidins in cell suspension cultures of Fragaria ananassa at a temperature between 15 and 35 °C. The maximum production was obtained at 20 °C, it is estimated at 270 mg/l. Zhao et al41 also reported the accumulation of anthocyanidins in the cell suspension of Vitis vinifera after the addition of jasmonic acid or light irradiation.
Anthocyanidin biosynthesis was improved by 8.5-fold compared to the control culture in the dark while cell growth was inhibited.
The effect of the nature of growth regulators on the stimulation of a particular compound appears to be robust.
Constabel et al8 studied the influence of ANA and cytokinins on the production of anthocyanidins by a cell suspension of Haplopappus gracilis. These authors showed that there is a concentration threshold for growth regulators (2,4-D or ANA) which allowed the accumulation of anthocyanins and that benzyladenine and kinetin in high doses improved this accumulation and reduced growth. This justifies our results on the levels of anthocyanidins produced in our brown cell suspensions cultivated in the culture medium rich in Kin/ANA which were superior to those of the other fractions.
In contrast, Matsumoto et al21 found that there were no large variations in the growth rate of a Populus cell suspension when they added 2,4-D, ANA or AIA to culture media but noted that 2,4-D inhibited the synthesis of anthocyanins unlike ANA and AIA. This agrees with our obtained results on the gray suspensions cultivated on medium-enriched by BAP/2,4-D where the anthocyanidin contents were not high enough compared to the other obtained fractions. Cell cultures can have a concentration a thousand times higher than that of the whole plant, thanks to the overproducing embryos and the use of a culture medium favoring production12.
When the accumulation of metabolites takes place during the exponential phase, the development of processes ensuring optimal growth will make it possible to obtain higher quantities of metabolites. This production model is achieved in particular by cell suspensions from Berberis synthesizing berberine and cell suspensions from Phytolacca americana synthesizing bethacyanins15,32. The rotenoid production curve followed cell growth in Tephrosia vogelii17. Similarly, for the maximum amount of furocoumarins in the cell suspensions of Ruta graveolens L, it was reached at the end of the exponential phase24.
On the other hand, when production and growth are decoupled, it is interesting to keep the cells in a state of non- proliferation after growth28. This confirms that it is the inhibition or stopping of growth that puts the development of defense mechanisms in competition with the primary functions of cells. This antagonism has already been noted by many authors such as Haluk and Bousta14 who reported that the production of thujaplicin-type tropolones in cell cultures of Thuja plicata Donn was not correlated with cell growth. The concentration of resveratrol in the vine culture medium varied roughly in the opposite direction of the biomass7.
We have identified chlorogenic acid and p-hydroxy- benzaldehyde acid in the obtained cell suspensions of our plant while they were absent in the adult plant22. This shows that the destabilization of cell metabolism during in vitro cultures can open up new biosynthetic pathways.34 We were able to detect a new peak corresponding to syringic acid.
This acid did not exist in other in vitro cultures such as calli and vitro-plants from the different explants of the aerial parts
of Teucrium polium L. subsp. geyrii Maire, nor in the adult plant22. Thus, we can say that the in vitro production of secondary metabolites is largely dependent on biological factors and the environment. These are determined by the composition of the culture medium and the in vitro culture conditions (agitation, liquid medium and growth regulators etc.).
Indeed, the production of secondary metabolites of interest in a confined environment (culture chamber, or greenhouse) is currently part of metabolic engineering. According to Bourgaud6, this emerging biotechnology offers great production potential when it comes to producing secondary metabolites with remarkable properties. These metabolites would be harvestable regardless of the season, climatic conditions and biological variability2.
Conclusion
This work presents an approach to the development of bioactive products of T. polium L. subsp. geyrii Maire involving the use of two in vitro culture techniques namely somatic embryogenesis and the establishment of cell suspensions. The development of regeneration by the technique of somatic embryogenesis could allow the rapid production of quality plants for the propagation of this endemic and medicinal subspecies. In cell suspensions, it is often effective to first allow cell growth because the optimal conditions are not always optimal for the production of secondary metabolites.
Acknowledgement
The authors acknowledge both, the Ministry of Higher Education and Scientific Research of Algeria and the University of Sciences and Technology Houari Boumediene, Algiers-Algeria for supporting the project N°F00220130059.
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(Received 06th January 2022, accepted 08th March 2022)
