In Vitro Regeneration of Stevia (Stevia rebaudiana Bertoni) and Evaluation of the Impacts of Growth Media Nutrients on the Biosynthesis of Steviol Glycosides (SGs)
Abstract
:1. Introduction
2. Materials and Methods
2.1. Plant Materials and Sterilization
2.2. Media Preparation
2.3. Surface Sterilization of Explants, Culture Establishment and Maintenance
2.4. Callus Induction and Characterization
2.5. Shoot Initiation
2.6. Root Induction
2.7. Steviol Glycosides (SGs) Extraction
2.8. HPLC Analysis
2.9. Extraction of Total RNA
2.10. Quantitative Reverse Transcription PCR (RT q-PCR) Analysis for UGT76G1, UGT74G1, and UGT85C2
2.11. Statistical Analysis
3. Results
3.1. Surface Sterilization of Explants
3.2. Callus Induction
3.3. Shoot Regeneration
3.4. Root Induction
3.5. HPLC Analysis for Steviol Glycosides (SGs)
3.6. Quality and Concentrations of RNAs Isolated from Stevia Leaves
3.7. RT q-PCR Analysis for UGT76G1, UGT74G1 and UGT85C2
4. Discussion
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Treatment | 2,4-D (mg/L) | Zeatin (mg/L) |
---|---|---|
T1 | 0.00 | 0.00 |
T2 | 0.10 | 0.10 |
T3 | 0.25 | 0.10 |
T4 | 0.50 | 0.10 |
T5 | 0.75 | 0.10 |
T6 | 1.00 | 0.10 |
T7 | 1.50 | 0.10 |
T8 | 2.00 | 0.10 |
Treatment | BAP (mg/L) | NAA (mg/L) |
---|---|---|
S1 | 0.00 | 0.00 |
S2 | 1.00 | 0.00 |
S3 | 1.00 | 1.00 |
S4 | 2.50 | 0.00 |
S5 | 2.50 | 1.00 |
S6 | 5.00 | 0.00 |
S7 | 5.00 | 1.00 |
S8 | 10.00 | 0.00 |
S9 | 10.00 | 1.00 |
Name (Accession No.) | Forward (F)/ Reverse (R) | Sequence (5′–3′) | Amplicon Size (bp) | Annealing Temperature (°C) |
---|---|---|---|---|
Actin (AF548026.1) | F | CTGAGAACTGAGGGCTAGGG | 187 | 70.5 |
R | AACCCAGCCTTGACCATTCC | |||
Aquaporin (DQ269455.1) | F | GGAGCCGCCGTAATCTACAA | 86 | 80 |
R | GCAATCGCCGCACCAATAAA | |||
Calmodulin (AF474074.1) | F | ATCCGCTCACCGACGATCA | 142 | 65 |
R | TGCAGTTCAGCTTCTGTTGG | |||
UGT76G1 (KM206772.1) | F | CTGCCAATGCCACCGTTATT | 94 | 52 |
R | TCATAAACCGTCTGACGCAGG | |||
UGT74G1 (AY345982.1) | F | TTCCAGTGCTTCAACGGTGG | 124 | 61 |
R | GTGAAGACCCAACGTGCTTG | |||
UGT85C2 (AY345978.1) | F | CGTTCGATGAGTTGGAGCCT | 181 | 61 |
R | AGCCACTGGAAACACTCTGG |
Step | Temperature | Duration | Cycle (s) |
---|---|---|---|
Initial activation | 95 °C | 2 min. | 1 |
Denaturation | 95 °C | 05 s | 40 |
Annealing | * 52–80 °C | 10 s | |
Extention | 72 °C | 15 s | |
Melt curve | 65–95 °C | 5 s for every increment of 0.5 °C |
15 Days after Culture Initiation (DACI) | 30 Days after Culture Initiation (DACI) | ||||||||
---|---|---|---|---|---|---|---|---|---|
Period of Time (Minute) | Period of Time (Minute) | ||||||||
5 | 10 | 15 | 20 | 5 | 10 | 15 | 20 | ||
Concentration of Clorox (%) | 0 | 11.11 ± 5.56 abc | 16.67 ± 0.00 ab | 5.56 ± 5.56 bc | 5.56 ± 5.56 bc | 11.11 ± 5.56 bc | 16.67 ± 0.00 b | 5.56 ± 5.56 bc | 11.11 ± 5.56 bc |
5 | 11.11 ± 5.56 abc | 0.00 ± 0.00 c | 0.00 ± 0.00 c | 0.00 ± 0.00 c | 11.11 ± 5.56 bc | 0.00 ± 0.00 c | 0.00 ± 0.00 c | 0.00 ± 0.00 c | |
10 | 16.67 ± 9.62 ab | 0.00 ± 0.00 c | 0.00 ± 0.00 c | 0.00 ± 0.00 c | 16.67 ± 9.62 b | 0.00 ± 0.00 c | 0.00 ± 0.00 c | 0.00 ± 0.00 c | |
15 | 16.67 ± 9.62 ab | 5.56 ± 5.56 bc | 0.00 ± 0.00 c | 0.00 ± 0.00 c | 16.67 ± 9.62 b | 5.56 ± 5.56 bc | 0.00 ± 0.00 c | 0.00 ± 0.00 c | |
20 | 0.00 ± 0.00 c | 22.23 ± 5.56 a | 0.00 ± 0.00 c | 0.00 ± 0.00 c | 0.00 ± 0.00 c | 38.89 ± 5.56 a | 0.00 ± 0.00 c | 0.00 ± 0.00 c | |
30 | 5.56 ± 5.56 bc | 0.00 ± 0.00 c | 0.00 ± 0.00 c | 0.00 ± 0.00 c | 5.56 ± 5.56 bc | 0.00 ± 0.00 c | 0.00 ± 0.00 c | 0.00 ± 0.00 c |
15 Days after Culture Initiation (DACI) | 30 Days after Culture Initiation (DACI) | ||||||||
---|---|---|---|---|---|---|---|---|---|
Period of Time (Minute) | Period of Time (Minute) | ||||||||
5 | 10 | 15 | 20 | 5 | 10 | 15 | 20 | ||
Concentration of Clorox (%) | 0 | 55.56 ± 5.56 ab | 38.89 ± 5.55 bc | 55.57 ± 20.03 ab | 66.68 ± 9.63 a | 66.67 ± 9.62 a | 66.67 ± 0.00 a | 66.67 ± 9.62 a | 66.67 ± 9.62 a |
5 | 66.68± 0.00 a | 16.67 ± 9.62 de | 5.56 ± 5.56 e | 27.78 ± 5.56 dc | 72.22 ± 5.55 a | 38.89 ± 14.70 b | 55.56 ± 5.56 de | 27.78 ± 5.55 bc | |
10 | 38.89 ± 5.55 bc | 11.11 ± 5.56 de | 16.67 ± 9.62 de | 11.11 ± 5.56 de | 38.89 ± 5.56 b | 11.11 ± 5.56 cde | 22.22 ± 5.55 bcd | 11.11 ± 5.56 cde | |
15 | 16.67 ± 9.62 de | 5.56 ± 5.56 e | 11.11 ± 5.56 de | 38.89 ± 11.11 bc | 22.22 ± 11.11 bcd | 5.56 ± 5.56 de | 22.22 ± 5.55 bcd | 38.89 ± 11.11 b | |
20 | 5.56 ± 5.56 e | 27.78 ± 5.56 dc | 0.00 ± 0.00 e | 5.56 ± 5.56 e | 11.11 ± 5.56 cde | 27.78 ± 5.55 bc | 11.11 ± 5.56 cde | 11.11 ± 5.56 cde | |
30 | 0.00 ± 0.00 e | 5.56 ± 5.56 e | 0.00 ± 0.00 e | 0.00 ± 0.00 e | 5.56 ± 5.56 de | 16.67 ± 9.62 cde | 0.00 ± 0.00 e | 0.00 ± 0.00 e |
15 Days after Inoculation (DACI) | 30 Days after Inoculation (DACI) | ||||||||
---|---|---|---|---|---|---|---|---|---|
Period of Time (Minute) | Period of Time (Minute) | ||||||||
5 | 10 | 15 | 20 | 5 | 10 | 15 | 20 | ||
Concentration of Clorox (%) | 0 | 16.67 ± 0.00 cd | 27.78 ± 5.56 bc | 22.23 ± 5.56 bcd | 11.11 ± 5.56 cd | 5.56 ± 5.56 de | 0.00 ± 0.00 e | 11.11 ± 5.56 cde | 5.56 ± 5.56 de |
5 | 5.56 ± 5.56 d | 50.01 ± 16.67 a | 11.11 ± 11.11 cd | 22.22 ± 14.70 bcd | 0.00 ± 0.00 e | 27.78 ± 5.55 bc | 22.22 ± 11.11 bcd | 16.67 ± 9.62 bcde | |
10 | 16.67 ± 9.62 cd | 38.89 ± 5.55 ab | 5.56 ± 5.56 d | 5.56 ± 5.56 d | 22.22 ± 5.55 bcd | 0.00 ± 0.00 e | 16.67 ± 0.00 bcde | 5.56 ± 5.56 de | |
15 | 16.67 ± 0.00 cd | 22.23 ± 5.56 bcd | 11.11 ± 5.56 cd | 16.67 ± 0.00 cd | 33.33 ± 9.62 b | 27.78 ± 5.55 bc | 11.11 ± 5.56 cde | 27.78 ± 5.55 bc | |
20 | 27.78 ± 5.56 bc | 16.67 ± 0.00 cd | 50.01 ± 9.62 a | 11.11 ± 5.56 cd | 61.11 ± 5.56 a | 11.11 ± 5.56 cde | 72.23 ± 5.56 a | 27.78 ± 5.55 bc | |
30 | 5.56 ± 5.56 d | 11.11 ± 5.56 cd | 16.67 ± 9.62 cd | 11.11 ± 5.56 cd | 66.67 ± 9.62 a | 0.00 ± 0.00 e | 16.67 ± 9.62 bcde | 55.56 ± 5.56 a |
15 Days after Inoculation (DACI) | 30 Days after Inoculation (DACI) | ||||||||
---|---|---|---|---|---|---|---|---|---|
Period of Time (Minute) | Period of Time (Minute) | ||||||||
5 | 10 | 15 | 20 | 5 | 10 | 15 | 20 | ||
Concentration of Clorox (%) | 0 | 16.67 ± 0.00 d | 16.67 ± 0.00 d | 16.67 ± 9.62 d | 16.67 ± 9.62 d | 16.67 ± 0.00 f | 16.67 ± 0.00 f | 16.67 ± 9.62 f | 16.67 ± 9.62 f |
5 | 16.67 ± 9.62 d | 33.34 ± 9.62 cd | 83.34 ± 9.62 a | 50.01 ± 9.62 bc | 16.67 ± 9.62 f | 33.33 ± 9.62 ef | 72.22 ± 5.55 abc | 55.56 ± 5.56 cd | |
10 | 27.78 ± 5.56 cd | 50.01 ± 9.62 bc | 77.79 ± 11.11 a | 83.35 ± 0.00 a | 22.23 ± 5.56 f | 88.89 ± 5.56 a | 61.11 ± 5.56 cd | 83.33 ± 0.00 ab | |
15 | 50.00 ± 0.00 bc | 66.68 ± 9.63 ab | 77.79 ± 5.56 a | 44.45 ± 11.11 bc | 27.78 ± 5.55 ef | 61.11 ± 5.56 cd | 66.67 ± 9.62 bc | 27.78 ± 5.55 ef | |
20 | 66.68 ± 9.63 ab | 33.34 ± 9.62 cd | 50.01 ± 9.62 bc | 83.34 ± 9.62 a | 27.78 ± 5.55 ef | 22.22 ± 5.55 f | 16.67 ± 0.00 f | 61.11 ± 5.56 cd | |
30 | 88.90 ± 5.55 a | 83.35 ± 5.56 a | 83.34 ± 9.62 a | 88.90 ± 5.55 a | 22.22 ± 5.55 f | 83.33 ± 9.62 ab | 83.33 ± 9.62 ab | 44.44 ± 5.56 de |
RNA | Before DNAse I Treatment | After DNAse I Treatment | ||
---|---|---|---|---|
Yield (ng/µL) | Purity(A260/A280) | Yield (ng/µL) | Purity(A260/A280) | |
0.00 MS | 390.8 | 1.90 | 139.5 | 2.03 |
0.25 MS | 395.3 | 1.91 | 138.9 | 2.02 |
0.50 MS | 418.4 | 1.94 | 159.0 | 2.03 |
0.75 MS | 311.5 | 1.89 | 151.3 | 2.03 |
1.00 MS | 357.5 | 1.96 | 178.3 | 2.04 |
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Ghose, A.K.; Abdullah, S.N.A.; Md Hatta, M.A.; Megat Wahab, P.E. In Vitro Regeneration of Stevia (Stevia rebaudiana Bertoni) and Evaluation of the Impacts of Growth Media Nutrients on the Biosynthesis of Steviol Glycosides (SGs). Agronomy 2022, 12, 1957. https://doi.org/10.3390/agronomy12081957
Ghose AK, Abdullah SNA, Md Hatta MA, Megat Wahab PE. In Vitro Regeneration of Stevia (Stevia rebaudiana Bertoni) and Evaluation of the Impacts of Growth Media Nutrients on the Biosynthesis of Steviol Glycosides (SGs). Agronomy. 2022; 12(8):1957. https://doi.org/10.3390/agronomy12081957
Chicago/Turabian StyleGhose, Asish Kumar, Siti Nor Akmar Abdullah, Muhammad Asyraf Md Hatta, and Puteri Edaroyati Megat Wahab. 2022. "In Vitro Regeneration of Stevia (Stevia rebaudiana Bertoni) and Evaluation of the Impacts of Growth Media Nutrients on the Biosynthesis of Steviol Glycosides (SGs)" Agronomy 12, no. 8: 1957. https://doi.org/10.3390/agronomy12081957
APA StyleGhose, A. K., Abdullah, S. N. A., Md Hatta, M. A., & Megat Wahab, P. E. (2022). In Vitro Regeneration of Stevia (Stevia rebaudiana Bertoni) and Evaluation of the Impacts of Growth Media Nutrients on the Biosynthesis of Steviol Glycosides (SGs). Agronomy, 12(8), 1957. https://doi.org/10.3390/agronomy12081957