Indirect Organogenesis of Calendula officinalis L. and Comparative Phytochemical Studies of Field-Grown and In Vitro-Regenerated Tissues
Abstract
:1. Introduction
2. Materials and Methods
2.1. Explant Collection and Sterilization
2.2. Callus Induction and Growth Conditions
2.3. Shoot Organogenesis via Indirect Method
2.4. Root Initiation and Acclimatization
2.5. Preparation of Extracts
2.6. Biochemical Attributes
2.6.1. Estimation of Total Phenolic Content
2.6.2. Estimation of Total Flavonoid Content (TFC)
2.6.3. Determination of Free Radical Scavenging Activity by DPPH Assay
2.7. GC–MS Analyses
2.8. Statistical Analysis
3. Results
3.1. Callus Induction and Proliferation
3.2. Shoot Organogenesis via Indirect Method
3.3. Rooting and Acclimatization
3.4. Total Phenolic Content (TPC), Total Flavonoid Content (TFC), and DPPH-Scavenging Activity
3.5. GC–MS Analysis
4. Discussion
5. Conclusions
Author Contributions
Funding
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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PGRs | Concentrations (mg/L) | Callusing Frequency (%) | Fresh Biomass (g) |
---|---|---|---|
Control | 0 | 0 f | 0 e |
NAA + BAP | 0.5 + 0.5 | 88.89 ± 5.56 ab | 3.67 ± 0.8 ab |
0.5 + 0.1 | 61.11 ± 5.55 bcd | 2.70 ± 0.6 bcd | |
0.5 + 2.0 | 55.55 ± 14.69 | 2.50 ± 0.3 bcde | |
1.0 + 0.5 | 72.22 ± 14.69 abc | 3.30 ± 0.6 abc | |
1.0 + 1.0 | 94.44 ± 5.56 a | 4.40 ± 0.4 a | |
2,4-D + BAP | 0.5 + 0.5 | 38.89 ± 11.11 def | 1.60 ± 0.7 cde |
0.5 + 1.0 | 27.78 ± 5.56 ef | 1.30 ± 0.6 de | |
0.5 + 2.0 | 11.12 ± 5.55 f | 0.90 ± 0.2 e | |
1.0 + 0.5 | 27.78 ± 14.69 ef | 1.10 ± 0.5 de | |
1.0 + 1.0 | 44.44 ± 5.56 cde | 1.93 ± 0.1 cde |
PGRs | Concentration (mg/L) | Frequency of Organogenesis (%) | Mean No of Shoot /Callus Mass |
---|---|---|---|
Control | 0 | 0 | 0 |
NAA + BAP | 0.5 + 0.5 | 0 d | 0 d |
0.5 + 1.0 | 55.55 ± 05.5 bc | 1.67 ± 0.3 c | |
0.5 + 2.0 | 27.77 ± 11.1 cd | 0.67 ± 0.3 d | |
1.0 + 0.5 | 72.22 ± 20.1 ab | 2.67 ± 0.3 b | |
1.0 + 1.0 | 88.89 ± 05.6 a | 3.33 ± 0.3 a |
PGRs | Concentration | Rooting (%) | Mean Root Numbers/Shoot |
---|---|---|---|
control | 0 | 0 e | 0 e |
IBA | 0.5 | 77.77 ± 5.56 ab | 9.4 ± 1.4 ab |
1 | 94.44 ± 5.56 a | 12.3 ± 0.5 a | |
2 | 55.55 ± 14.70 bc | 8.2 ± 1.1 b | |
IAA | 0.5 | 49.01 ± 9.62 c | 6.7 ± 1.8 bc |
1 | 38.89 ± 5.56 cd | 4.4 ± 0.9 cd | |
2 | 22.21 ± 5.55 de | 2.1 ± 0.1 d |
Sample Type | Total Phenolic Content (mg GAE/g DW) | Total Flavonoid Content (mg QE/g DW) | DPPH Scavenging Activity (%) |
---|---|---|---|
Callus | 1.43 ± 0.04 c | 8.25 ± 0.2 b | 22.72 ± 4.11 b |
Field-grown leaf | 8.51 ± 0.2 b | 15.55 ± 0.3 a | 28.07 ± 3.11 ab |
In vitro leaf | 10.28 ± 0.1 a | 16.04 ± 0.2 a | 39.93 ± 4.81 a |
S. No. | R. Time | Area% | Name | Molecular Formula | Molecular Weight |
---|---|---|---|---|---|
1 | 4.763 | 0.52 | 1,2-butanolide | C4H6O2 | 86 |
2 | 5.02 | 0.19 | 2-propylheptanol | C10H22O | 158 |
3 | 5.551 | 0.92 | 5-methyifurfural | C6H6O2 | 110 |
4 | 5.83 | 0.56 | pyranone | C6H8O4 | 144 |
5 | 6.186 | 0.43 | 1,4-diazabicyclo[2.2.2]octane | C6H12N2 | 112 |
6 | 6.933 | 0.68 | ethyl methylacetoacetate | C7H12O3 | 144 |
7 | 7.604 | 3.66 | melamine | C3H6N6 | 126 |
8 | 8.006 | 0.25 | levoglucosenone | C6H6O3 | 126 |
9 | 8.568 | 6.35 | pyranone | C6H8O4 | 144 |
10 | 9.153 | 0.26 | 5-methoxypyrrolidin-2-one | C5H9NO2 | 115 |
11 | 9.397 | 0.22 | isoamyl trimethylacetate | C10H20O2 | 172 |
12 | 9.927 | 30.23 | 5-hydroxymethylfurfural | C6H6O3 | 126 |
13 | 10.193 | 0.4 | 3-hexene-2,5-dione | C6H8O2 | 112 |
14 | 11.132 | 1.21 | ethyl 3-hydroxy-4-pentenoate | C7H12O3 | 144 |
15 | 13.119 | 1.69 | xanthosine | C10H12N4O6 | 284 |
16 | 13.717 | 3.3 | levoglucosan | C6H10O5 | 162 |
17 | 15.043 | 1.48 | 1,6-anhydro-beta-d-glucofuranose | C6H10O5 | 162 |
18 | 16.506 | 0.29 | tetradecanoic acid | C14H28O2 | 228 |
19 | 17.26 | 0.23 | neophytadiene | C20H38 | 278 |
20 | 18.17 | 0.22 | methylpalmitate | C17H34O2 | 270 |
21 | 18.396 | 0.82 | palmitoleic acid | C16H30O2 | 254 |
22 | 18.609 | 7.66 | n-hexadecanoic acid | C16H32O2 | 256 |
23 | 19.108 | 0.47 | 1,4-naphthalenedione, 2-hydroxy-3-(1-propenyl) | C13H10O3 | 214 |
24 | 19.813 | 0.41 | linoleic acid, methyl ester | C19H34O2 | 294 |
25 | 19.871 | 0.38 | methyl petroselinate | C19H36O2 | 296 |
26 | 19.98 | 0.21 | phytol | C20H40O | 296 |
27 | 20.245 | 0.61 | 9,12-octadecadienoic acid (z,z)- | C18H32O2 | 280 |
28 | 20.296 | 0.81 | 13-tetradecenal | C14H26O | 210 |
29 | 20.48 | 1.17 | podocarpan-12-ol | C17H30O | 250 |
30 | 20.667 | 0.13 | 2-piperidinemethanol | C6H13NO | 115 |
31 | 21.292 | 1.48 | sclareolide | C16H26O2 | 250 |
32 | 21.796 | 0.28 | palmidrol | C18H37NO2 | 299 |
33 | 21.858 | 0.26 | 11-hexadecenal, (z)- | C16H30O | 238 |
34 | 22.642 | 0.48 | sclareolide lactol | C16H28O2 | 252 |
35 | 22.943 | 0.29 | 3-aminoheptane | C7H17N | 115 |
36 | 23.084 | 0.62 | 4-cyanobenzoic acid, undec-10-enyl ester | C19H25NO2 | 299 |
37 | 23.255 | 1.05 | 1-heptacosanol | C27H56O | 396 |
38 | 23.45 | 9.95 | 2-palmitoylglycerol | C19H38O4 | 330 |
39 | 23.826 | 3.28 | copalic acid | C20H32O2 | 304 |
40 | 24.683 | 1.24 | oleoyl chloride | C18H33ClO | 330 |
41 | 24.885 | 2.29 | 17-pentatriacontene | C35H70 | 490 |
42 | 25.131 | 3.19 | glycerin 1-monostearate | C21H42O4 | 358 |
43 | 25.697 | 0.32 | 9-octadecenamide | C18H35NO | 281 |
44 | 25.975 | 0.42 | squalene | C30H50 | 410 |
45 | 26.413 | 0.29 | 1-cyclohexene-1-butyraldehyde, 2,6,6-trimethyl- | C13H22O | 194 |
46 | 27.313 | 0.19 | hexacosanoic acid, methyl ester | C27H54O2 | 410 |
47 | 28.631 | 0.15 | stigmasta-4,7,22-trien-3.alpha.-ol | C29H46O | 410 |
48 | 29.568 | 0.15 | stigmasterol acetate | C31H50O2 | 454 |
49 | 30.478 | 0.41 | vitamin e | C29H50O2 | 430 |
50 | 32.617 | 0.25 | ergost-5-en-3-ol | C28H48O | 400 |
51 | 33.221 | 4.47 | stigmasterol | C29H48O | 412 |
52 | 34.745 | 2.82 | gamma-sitosterol | C29H50O | 414 |
53 | 35.153 | 0.38 | fucosterol | C29H48O | 412 |
S. No. | R. Time | Area% | Name | Molecular Formula | Molecular Weight |
---|---|---|---|---|---|
1 | 8.570 | 1.72 | pyranone | C6H8O4 | 144 |
2 | 13.083 | 6.40 | guanosine | C10H13N5O5 | 283 |
3 | 13.360 | 0.98 | 2-tridecynyl 2,6-difluorobenzoate | C20H26F2O2 | 336 |
4 | 15.060 | 0.25 | megastigmatrienone 4 | C13H18O | 190 |
5 | 15.554 | 0.16 | 4,6,6-trimethyl-bicyclo[3.1.1]heptan-2-ol | C10H18O | 154 |
6 | 15.982 | 0.17 | tetradecanal | C14H28O | 212 |
7 | 16.508 | 0.14 | undecanoic acid | C11H22O2 | 186 |
8 | 17.193 | 0.37 | tetrahydrogeranyl acetate | C12H24O2 | 200 |
9 | 17.265 | 13.94 | neophytadiene | C20H38 | 278 |
10 | 17.321 | 0.41 | hexa-hydro-farneso | C15H32O | 228 |
11 | 17.715 | 3.24 | neophytadiene | C20H38 | 278 |
12 | 18.173 | 0.19 | pentadecanoic acid, 14-methyl-, methyl ester | C17H34O2 | 270 |
13 | 18.370 | 0.30 | 11,14,17-eicosatrienoic acid, methyl ester | C21H36O2 | 320 |
14 | 18.595 | 3.44 | pentadecanoic acid, 14-methyl-, methyl ester | C16H32O2 | 256 |
15 | 19.749 | 0.36 | phytol isomer | C20H40O | 296 |
16 | 19.817 | 0.32 | linolic acid | C18H32O2 | 280 |
17 | 19.876 | 0.35 | 6-octadecenoic acid, methyl ester, (z)- | C19H36O2 | 296 |
18 | 19.983 | 12.41 | phytol isomer | C20H40O | 296 |
19 | 20.230 | 0.33 | 9,12-linoleic acid | C18H32O2 | 280 |
20 | 20.302 | 1.15 | 9,12-octadecadienoic acid | C18H32O2 | 280 |
21 | 21.291 | 1.24 | 2-formylhexadecane | C17H34O | 254 |
22 | 21.513 | 1.46 | 3-cyclopentylpropionic acid, 2-dimethylaminoethyl ester | C12H23NO2 | 213 |
23 | 21.793 | 0.23 | hexadecanoyl-chloride- | C16H31ClO | 274 |
24 | 22.938 | 0.73 | 3-cyclopentylpropionic acid, 2-dimethylaminoethyl ester | C12H23NO2 | 213 |
25 | 23.001 | 2.92 | 3-cyclopentylpropionic acid, 2-dimethylaminoethyl ester | C12H23NO2 | 213 |
26 | 23.088 | 0.90 | 4-cyanobenzoic acid, undec-10-enyl ester | C19H25NO2 | 299 |
27 | 23.260 | 3.08 | n-tetracosanol-1 | C24H50O | 354 |
28 | 23.455 | 16.06 | 2-palmitoylglycerol | C19H38O4 | 330 |
29 | 23.859 | 0.60 | globulol | C15H26O | 222 |
30 | 24.689 | 1.69 | oxalic acid, monoamide, n-allyl-, hexadecyl ester | C21H39NO3 | 353 |
31 | 24.890 | 4.62 | 1-heptacosanol | C27H56O | 396 |
32 | 24.965 | 0.65 | ethyl linolate | C20H36O2 | 308 |
33 | 25.137 | 4.31 | octadecanoic acid, 2,3-dihydroxypropyl ester | C21H42O4 | 358 |
34 | 25.709 | 0.44 | 9-octadecenamide | C18H35NO | 281 |
35 | 25.978 | 2.38 | squalene | C30H50 | 410 |
36 | 26.971 | 2.32 | 1-heptacosanol | C27H56O | 396 |
37 | 28.267 | 1.31 | 8,14-cedrane oxide | C15H24O | 220 |
38 | 29.143 | 1.01 | gamma-tocopherol | C28H48O2 | 416 |
39 | 30.495 | 2.09 | vitamin e | C29H50O2 | 430 |
40 | 33.227 | 3.75 | stigmasterol | C29H48O | 412 |
41 | 34.758 | 1.58 | gamma-sitosterol | C29H50O | 414 |
S. No. | R. Time | Area% | Name | Molecular Formula | Molecular Weight |
---|---|---|---|---|---|
1 | 12.999 | 12.18 | guanosine | C10H13N5O5 | 283 |
2 | 13.365 | 0.82 | 2,6-difluorobenzoic acid | C20H26F2O2 | 336 |
3 | 13.717 | 0.10 | 1-[2-bromoethenyl] adamantane | C12H17Br | 240 |
4 | 13.749 | 0.10 | gamma-cadinene | C15H24 | 204 |
5 | 15.984 | 0.09 | tridecanal | C13H26O | 198 |
6 | 17.192 | 0.28 | 3,7-dimethyloctyl acetate | C12H24O2 | 200 |
7 | 17.265 | 10.47 | neophytadiene | C20H38 | 278 |
8 | 17.320 | 0.26 | hexa-hydro-farnesol | C15H32O | 228 |
9 | 17.520 | 1.81 | neophytadiene | C20H38 | 278 |
10 | 17.715 | 2.60 | 3,7,11,15-tetramethyl-2-hexadecen-1-ol | C20H40O | 296 |
11 | 18.175 | 0.33 | pentadecanoic acid, 14-methyl-, methyl ester | C17H34O2 | 270 |
12 | 18.590 | 0.83 | n-hexadecanoic acid | C16H32O2 | 256 |
13 | 19.816 | 0.60 | 9,12-octadecadienoic acid, methyl ester | C19H34O2 | 294 |
14 | 19.875 | 0.54 | 6-octadecenoic acid, methyl ester, (z)- | C19H36O2 | 296 |
15 | 19.985 | 7.18 | phytol | C20H40O | 296 |
16 | 20.294 | 0.60 | 9,12-octadecadienoic acid (z,z)- | C18H32O2 | 280 |
17 | 20.677 | 0.12 | 9,12-octadecadienoic acid, methyl ester | C19H34O2 | 294 |
18 | 21.291 | 0.66 | 11-dodecen-2-one | C12H22O | 182 |
19 | 21.512 | 0.99 | octanoic acid, 2-dimethylaminoethyl ester | C12H25NO2 | 215 |
20 | 21.630 | 0.44 | glycidyl palmitate | C19H36O3 | 312 |
21 | 22.938 | 0.55 | fumaric acid, 2-dimethylaminoethyl nonyl ester | C17H31NO4 | 313 |
22 | 22.999 | 1.58 | 3-cyclopentylpropionic acid, 2-dimethylaminoethyl ester | C12H23NO2 | 213 |
23 | 23.086 | 3.35 | 4-cyanobenzoic acid, undec-10-enyl ester | C19H25NO2 | 299 |
24 | 23.259 | 5.94 | 2-ethylbutyric acid, eicosyl ester | C23H46O2 | 354 |
25 | 23.455 | 23.59 | 2-monopalmitin | C19H38O4 | 330 |
26 | 23.857 | 0.21 | alpha-selinene | C15H24 | 204 |
27 | 24.180 | 0.56 | glycerol.beta.-palmitate | C19H38O4 | 330 |
28 | 24.685 | 4.12 | 4-cyanobenzoic acid, tridecyl ester | C21H31NO2 | 329 |
29 | 24.889 | 3.36 | eicosyl heptafluorobutyrate | C24H41F7O2 | 494 |
30 | 25.030 | 2.04 | 1-monolinolein | C21H38O4 | 354 |
31 | 25.139 | 5.13 | octadecanoic acid, 2,3-dihydroxypropyl ester | C21H42O4 | 358 |
32 | 25.705 | 0.77 | 9-octadecenamide, (z)- | C18H35NO | 281 |
33 | 25.976 | 2.52 | squalene | C30H50 | 410 |
34 | 26.969 | 1.02 | 1-heptacosanol | C27H56O | 396 |
35 | 27.326 | 0.36 | eicosanoic acid, methyl ester | C21H42O2 | 326 |
36 | 28.257 | 0.42 | cysteamine sulfonic acid | C2H7NO3S2 | 157 |
37 | 29.138 | 0.42 | gamma-tocopherol | C28H48O2 | 416 |
38 | 30.491 | 0.90 | vitamin e | C29H50O2 | 430 |
39 | 33.218 | 1.36 | stigmasterol | C29H48O | 412 |
40 | 34.759 | 0.78 | beta-sitosterol | C29H50O | 414 |
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Fatima, T.; Mujib, A.; Bansal, Y.; Dewir, Y.H.; Mendler-Drienyovszki, N. Indirect Organogenesis of Calendula officinalis L. and Comparative Phytochemical Studies of Field-Grown and In Vitro-Regenerated Tissues. Agronomy 2024, 14, 1743. https://doi.org/10.3390/agronomy14081743
Fatima T, Mujib A, Bansal Y, Dewir YH, Mendler-Drienyovszki N. Indirect Organogenesis of Calendula officinalis L. and Comparative Phytochemical Studies of Field-Grown and In Vitro-Regenerated Tissues. Agronomy. 2024; 14(8):1743. https://doi.org/10.3390/agronomy14081743
Chicago/Turabian StyleFatima, Tooba, A. Mujib, Yashika Bansal, Yaser Hassan Dewir, and Nóra Mendler-Drienyovszki. 2024. "Indirect Organogenesis of Calendula officinalis L. and Comparative Phytochemical Studies of Field-Grown and In Vitro-Regenerated Tissues" Agronomy 14, no. 8: 1743. https://doi.org/10.3390/agronomy14081743