Analysis of secondary metabolites through callus cell suspension culture of black cumin (Nigella sativa L.) with PGR combination on MS media

This study aims to resolve the effect of using ZPT picloram and BAP and a combination of MS media on cell suspension culture and secondary metabolite compounds contained in black cumin (Nigella sativa L.) callus. The enquiry was performed at the Tissue Culture Laboratory, Faculty of Agriculture and Phytochemical Laboratory, Faculty of Pharmacy, Universitas Sumatera Utara, from February 2023 to July 2023. This study used a Totally Randomized Design (RAL) method amid 2 treatment elements and each treatment had 3 replications. The first treatment factor was ZPT concentration in MS medium (Z): Z1 (Picloram 0 ppm + BAP 1 ppm), Z2 (Picloram 1 ppm + BAP 1 ppm), Z3 (Picloram 1 ppm + BAP 2 ppm), Z4 (Picloram 2 ppm + BAP 2 ppm). The second factor is the composition of the MS (M) media: M1 (½ MS) and M2 (MS). Parameters observed were callus morphology, growth index, SCV (Settled Cell Volume), PCV (Peacked Cell Volume) and analysis of secondary metabolite content. The outcomes exposed that the arrangement of MS media and the PGR concentration of picloram and BAP had no expressive impact on all parameters. Z4 and M2 concentrations provided the most optimal growth.


Introduction
Black cumin (Nigella sativa L.) is an herbal plant as of the Ranunculaceae line originating from the Mediterranean and West Asia regions.Black cumin has a great nutritive substance, specificially essential oils (psymena, timoquinone) and greasy acids (palmitic, linoleic, and oleic acids), tocopherols, sterols [1].
The content and nutrients contained in black cumin have resulted in increasing demand for both dried and fresh seeds.The content and nutrients contained in black cumin have resulted in increasing demand for both dried and fresh seeds.
Based on these problems, the use of the tissue culture method is one solution that can be done to meet the needs of black cumin in Indonesia.Tissue culture is a procedure for isolating plant parts such as cells, tissues and organs grown underneath germ-free settings, so that these parts can multiply and regenerate into whole plants again.
Black cumin (Nigella sativa L.) customs MS media further often than WPM, even though this herbal is a yearly plant so it is also appropriate for consuming WPM media [3].Consequently, adjustment of the form of tissue culture forms with the addition of development officials such as auxin and cytokinins requirements to be completed to rise the proportion of successful black cumin proliferation in vitro.Secondary metabolites are compounds that are not used as a growth process, but as a form of selfdefense from the environment.Alkaloids, flavonoids, terpenoid saponins are included in the secondary metabolite group which is often found in plant extracts and found in bacterial extracts [4].
The use of tissue culture methods with callus culture and cell suspension culture is a fast method for obtaining results of derived metabolite materials [6,7].Callus culture is the first step to obtain cells that are young and actively dividing before being used for culturing the cells into the cell suspension culture stage to produce secondary plant metabolite materials.Callus culture can be used to select potential cells in plants that produce secondary metabolites, such as medicinal plants and aromatic plants, so it needs to be developed first using a callus culture method so that the desired secondary metabolites can be produced better [5,8].

Materials
The substantial used as explants was black cumin sterile callus.The compounds that will be cast-off in this study comprise Murashige and Skoog (MS) media, picloram, BAP, distilled water, methylated spirits, aluminum foil, filter paper, plastic wrap, scarphs, 70% alcohol.
The apparatuses used in this analysis were erlenmeyer, autoclave, Laminar Air Flow Cabinet (LAFC), sterilized box, analytic balance, hot plate with magnetic agitator, measure glasses, pipette, cutters, scalpel, pincers, Bunsen lamp, pH meter, shaker, centrifuge, and other apparatuses that sustenance this research

Methods
This study was carried out at the Tissue Culture Laboratory, Faculty of Agriculture, and Phytochemical Laboratory, Faculty of Pharmacy, Universitas Sumatera Utara, from February to July 2023.
This examines scheme used a factorial complete randomized strategy with treatment aspects containing of:

Measurement of Growth Parameters
Callus cell growth was measured based on callus morphology, IP, SCV and PCV values.Callus morphology was observed at the end of the study by visually observing the appearance of the suspended callus.Growth index (IP) measurements were carried out by comparing the wet weight after and before cultivation.SCV (Settled Cell Volume) is calculated by measuring the cell sediment fraction.The cell suspension was transferred into a measuring cup and allowed to settle for 30 minutes.PCV (Peacked Cell Volume) was carried out by transferring the callus cell suspension to a centrifuge tube.The cell suspension was centrifuged for 5 minutes, at 25 o C (room temperature) with a speed of 2.000 rpm.

Data analysis
The examine records occurred analyzed using a completely randomized design (RAL) and research data from the results of expressively changed variants will be verified handling the Duncan Multiple Range Test (DMRT) at the 5% level.

Callus Morphology
Created on the remarks, the callus morphology results obtained are accessible in table 1 as monitors.Founded on the marks of observations, it is identified that there is an effect of a combination of MS media and PGR picloram and BAP on callus color and texture.Table 1 shows that the combination treatment of MS media and PGR picloram and BAP produced different callus colors and textures [7].

Growth Index
Based on the observations, the callus growth index results were obtained which are existing in table 2 as monitors.2, it is identified that the MS media combination treatment produces the same growth index, namely 0.04 grams.The combination treatment of PGR Picloram and BAP showed that the Z4 treatment produced the highest progress mark with an average of 0.05 g and Z1 and Z3 produced the lowest growth index, namely 0.03 g.

SCV (Settled Cell Volume)
Based on the observations, the SCV callus results were obtained which are accessible in table 3 as tracks.Based on Table 3 it is identified that in the MS media combination treatment it showed that the M2 treatment produced the highest SCV with an average of 0.32 ml.The combination treatment of PGR Picloram and BAP showed that treatment Z4 produced the highest SCV with an average of 0.38 ml and Z1 and Z2 produced the lowest SCV with an average of 0.23 ml.

PCV (Peacked Cell Volume)
Based on the observations, the PCV results of callus were obtained which are existing in table 4 as follows.4, it is proven that the MS media combination treatment shows that the M2 treatment produces the highest PCV with an average of 0.25 ml.The combination treatment of PGR Picloram and BAP showed that treatment Z4 produced the highest PCV with an average of 0.33 ml and Z1 produced the least PCV with an average of 0.16 ml [9].

Flavonoid Test
Founded on Fig. 1, it is known that the highest levels of flavonoids were found in the Z3M1 handling with an average level of 2.2009 mg/g and the lowest levels of flavonoids were found in the Z2M2 dealing with a regular level of 1.2665 mg/g.Based on Fig. 2, it is known that the highest tannin content was found in the Z3M1 handling with an average level of 0.9133 mg/g and the lowest content was found in the Z2M2 with an average level of 0.4869 mg/g.Accordance with [7,9,10] that the increase in secondary metabolites due to the presence of PGR to activates plant immune signaling pathways.

Conclusion
The concentration of the most optimal combination of Z4M2 (Picloram 2 ppm + BAP 2 ppm, MS) in terms of growth index with an average of 0.053 g, SCV (Settled Cell Volume) 0.5 ml, and PCV (Peacked cell volume) 0.43 ml.The combined concentration of Z3M1 has the highest flavonoid content with an average level of 2.2009 mg/g and tannin content with an average level of 0.9133 mg/g.
Factor I: Differences in PGR dilution in MS media, namely: Z1 = Picloram 0 ppm dan BAP 1 ppm Z2 = Picloram 1 ppm dan BAP 1 ppm Z3 = Picloram 1 ppm dan BAP 2 ppm Z4 = Picloram 2 ppm dan BAP 2 ppm Factor II: Differences in the composition of MS media are: M1 = ½ MS M2 = MS Research consists of: Number of repetitions : 3 Number of treatments : 8 Number of bottles : 24 Number of all samples : 24

Table 1 .
Callus morphology of black cumin with PGR combination on MS media Note: H = green, K = yellowish, C = chocolate

Table 2 .
Black cumin callus growth index with a combination of PGR on MS media

Table 3 .
SCV of black cumin callus with a combination of PGR on MS media

Table 4 .
PCV of black cumin callus with a combination of PGR on MS media