Browsing by Author "Shailesh Kumar Tiwari"

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Development of plant tissue culture system for Nyctanthes arbor-tristis L. in a controlled environmental system from shoot-tip culture and genetic homogeneity evaluation utilizing inter simple sequence repeat (ISSR) and start codon targeted (SCoT) markers
(Springer Nature, 2025) Awadhesh Kumar Mishra; Kavindra Nath Tiwari; Pallavi Mishra; Rajesh Saini; Shailesh Kumar Tiwari; Sunil Kumar Mishra
An efficient in vitro shoot development profile is developed from the shoot-tip culture of N. arbor-tristis L. To evaluate cytokinin efficacy for manifold shoot regeneration from the shoot-tip, it was cultured on tissue culture Murashige and Skoog medium augmented with various strengths of cytokinin. The results emphasized that medium augmented with 5 mg l−1 BA was found most appropriate for the in vitro shoot development from the cultured explant. This media supported induction of the maximum shoot regeneration efficiency (75.33 ± 1.76), maximum number of shoots/explant (28.23 ± 0.52) and shoot length (4.53 ± 0.12 cm) without callus formation. The effect of variation in nitrogen content (KNO3 and NH4NO3) in medium on shoot regeneration potential was studied. The optimum regeneration from selected explant was achieved on medium compositions M3 (1900 mg l−1 KNO3 + 1650.00 mg l−1 NH4NO3) and M9 (1650.00 mg l−1 NH4NO3 + 1900 mg l−1 KNO3) respectively. The best in vitro root induction (69.50 ± 0.76%), the maximum roots/shoot (10.31 ± 0.82), and its optimum length (2.96 ± 0.14 cm) were achieved once microshoots were sub-inoculated on full-strength Murashige and Skoog medium after being pulse treated with 300 mg l−1 IBA for up to 30 min. The molecular authentication of regenerated shoots to the mother plant was confirmed using the inter simple sequence repeat (ISSR) and start codon targeted (SCoT) primers. Thus, this protocol may aid the consistent production of quality planting stocks, which can be used for transformation study and in other ethnomedicinal purposes. © The Author(s), under exclusive licence to Springer Nature B.V. 2025.
Direct shoot regeneration from cotyledonary node of Uraria picta (Jacq.) Desv. ex DC., an important plant of dashmula drugs, and assessment of genetic fidelity, metabolic profiling, and anti-diabetic activity
(Elsevier B.V., 2024) Jyoti Dixit; Pooja Verma; Pallavi Mishra; Kavindra Nath Tiwari; Shailesh Kumar Tiwari; Sunil Kumar Mishra; Jasmeet Singh
Uraria picta root is used in the polyherbal product ‘Dashmula’. Its exploitation for formulation preparation has depleted its availability, leading to medicine adulteration. Direct shoot regeneration from the cotyledonary node holds promise as a source of raw material. This study aimed to develop a regeneration protocol for U. picta and validate its genetic and metabolic fidelity. The seeds of U. picta showed low germination rates, prolonged dormancy, and poor viability. Root exploitation in the wild poses a threat to its availability in nature. Seedling's derived cotyledonary nodes cultured on B5 medium supplemented with BAP (0.5–3 mg L−1), Kinetin (0.5–3 mg L−1), Thidiazuron (0.01–1 mg L−1), and meta-Topolin (0.1–4 mg L1). To address hyperhydricity in regenerated shoots, cotyledonary nodes were cultured on high-agar concentration media. Microshoots were exposed to IBA solution (50–800 mg L−1) pulse treatment for rooting. Tissue-cultured plants genetic fidelity was assessed using ISSR and SCoT markers, while metabolic fidelity was studied with HRMS. The chlorophyll content, antioxidant, and antidiabetic activity of micropropagated plants were evaluated. The highest shoot regeneration frequency, with a maximum of 6.57±0.278 shoots per explant, was achieved using 2 mg L−1 meta-Topolin. The shoots were elongated, had expanded leaves, and were hyperhydrated. BAP (2 mg L−1) induced a maximum of 9.83±0.333 shoot buds per explant. BAP caused explant browning, profuse callus formation, dwarfing, and hyperhydric shoots. Hyperhydricity was alleviated with a higher agar concentration (1 %). IBA (400 mg L−1) induced a maximum of 2.18±0.090 roots per shoot and a root length of 9.23±0.033 cm. Tissue-cultured and mother plants exhibited clonal fidelity, similar metabolite and chlorophyll content, strong antioxidant activity, and equal efficacy for inhibiting α-amylase and α-glucosidase. This method can propagate elite clones of U. picta and offer its improvement via genetic transformation. © 2024 Elsevier B.V.
Effect of cytokinin and MS medium composition on efficient shoot proliferation of Nyctanthes arbor-tristis L. through cotyledonary node explant and evaluation of genetic fidelity and antioxidant capacity of regenerants
(Elsevier B.V., 2019) Awadhesh Kumar Mishra; Kavindra Nath Tiwari; Pallavi Mishra; Shailesh Kumar Tiwari; Sunil Kumar Mishra; Rajesh Saini
Nyctanthes arbor-tristis L. is an important member of family Oleaceae. An efficient shoot proliferation response (21.53±0.58 shoots/explant) from cotyledonary node was achieved on Murashige and Skoog (MS) medium supplemented with 22.19 µM 6-benzyl amino purine (BAP). To optimize the role of ammonium and nitrate on shoot proliferation, the explants were cultured on modified MS media (MS-1 to MS-12). Among these media, MS-3 and MS-9 were found most suitable for shoot-proliferation than others. The microshoots after indole 3-butyric acid (IBA) pulse treatment (1968.11 µM) for 30 min, when sub-cultured on ½ strength MS basal medium were successfully rooted with a maximum frequency of root induction (77.66±1.45), the maximum number of roots/ shoot (10.66±0.88) and maximum root length (1.60±0.05 cm). In-vitro rooted plants were acclimatized and transplanted outside into field with 80 percent survival-rate after 90 days. The genetic-fidelity was confirmed by monomorphic nature of inter-simple sequence repeat (ISSR) marker pattern. The ethanol extract of leaves of regenerated plants contained good amount of phenol (118.00±0.82 µg/mg gallic acid equivalent) and flavonoid (17.34±0.64 µg/mg rutin equivalent) and possess strong antioxidant activity. The EC50 value for 2, 2-diphenyl-1-picrylhydrazyl (DPPH) free radical, superoxide radical (SOD) scavenging activity and lipid peroxidation (LPO) inhibition activity was 339.98±3.27, 755.80±75.50 and 689.67±2.46 µg/ml respectively. Hence, these findings suggest that regenerated plants can be used as a therapeutic and inhibitory agent against free radical-induced disease progression. © 2019 SAAB
Factors affecting the efficiency of in vitro regeneration from seedling-derived nodal explants of Nyctanthes arbor-tristis L. and evaluation of genetic fidelity
(Taylor and Francis Ltd., 2020) Awadhesh Kumar Mishra; Kavindra Nath Tiwari; Pallavi Mishra; Shailesh Kumar Tiwari; Sunil Kumar Mishra; Jasmeet Singh
An in vitro micropropagation protocol for Nyctanthes arbor-tristis was developed through the culture of seedling-derived nodal explants. The highest direct shoot induction rate, the mean number of shoots per explant and average shoot length was obtained on MS medium supplemented with 5 mg/l 6-benzylaminopurine (BAP). Full-strength MS medium was found better than other concentrations for organogenesis. Their lower or higher concentration from optimum level was inhibitory for shoot regeneration. Among different carbon sources tested for organogenesis, MS medium supplemented with 87.64 mM sucrose was superior to other carbon sources for in vitro morphogenesis. Best rooting of microshoots was achieved on one-quarter strength MS medium supplemented with 2 mg/l indole-3-butyric acid (IBA). Under this treatment, maximum mean number of roots/shoot and mean root length were 9.40 ± 0.70 and 2.47 ± 0.25 cm, respectively. The genetic fidelity among regenerates and donor plant has been confirmed by ISSR markers. © 2019, © 2019 Societá Botanica Italiana.
Germplasm conservation of economically important medicinal plant Nyctanthes arbor-tristis L. through encapsulation technique and maintenance under slow growth condition
(Springer Science and Business Media B.V., 2022) Awadhesh Kumar Mishra; Kavindra Nath Tiwari; Pallavi Mishra; Sunil Kumar Mishra; Shailesh Kumar Tiwari
An efficient encapsulation and slow growth conservation protocol was developed for Nyctanthes arbor-tristis L. an antiviral medicinal plant of the family Oleaceae. A gel matrix with 3% sodium alginate and 100 mM calcium chloride (CaCl2⋅2H2O) was found best for the encapsulation of nodal segments. The viability and shoot development potential of encapsulated nodal segments was optimized. Encapsulated nodal segments stored at 4 °C and 24 °C remained viable for up to 90 days and showed shoot development potential 42.89 ± 6.04% and 33.53 ± 7.15% respectively. Nodal segments maintained under slow growth conditions up to 180 days on one-eighth strength Murashige and Skoog (MS) medium supplemented with 0.5% sucrose was suitable for satisfactory viability (40.28 ± 2.04%), while further addition of 0.5 mg/l abscisic acid supported 40.36 ± 1.01% viability of the nodal segments. The best rooting response was achieved on half-strength MS medium supplemented with 4 mg/l indole-3-acetic acid. The field survival of rooted plants was 65%. The clonal fidelity of in-vitro derived plantlets was studied with start codon targeted primer profile, which showed the same banding mobility patterns as the source parent plant. The maximum banding profile was monomorphic and consistent, confirming the clonal stability of regenerated plants. The method developed will permit the in-vitro conservation of this species and facilitate an easy exchange of plant germplasm. © 2022, The Author(s), under exclusive licence to Springer Nature B.V.
Micropropagation of Drypetes roxburghii (Wall.) Hurusawa—a valuable medicinal plant through root culture and evaluation of its genetic fidelity and metabolomics
(Springer, 2025) Rajesh Saini; Awadhesh Kumar Mishra; Pallavi Mishra; Praveen Kumar Shukla; Jyoti Vishwakarma; Kavindra Nath Tiwari; Shailesh Kumar Tiwari; Jasmeet Singh
Drypetes roxburghii (Wall.) Hurusawa (Euphorbiaceae) is a valuable endemic medicinal plant. The International Union for Conservation of Nature Red Data Book classified it under threatened species. The seeds exhibit low viability, prolonged dormancy, and a low germination rate. In the current study, direct shoot regeneration from the root explant was achieved, which helps in the conservation of germplasm. Root explants were cultured on woody plant as well as Murashige and Skoog medium containing 6-benzylaminopurine (0.5–5.0 mg L−1), kinetin (0.5–5.0 mg L−1), meta-Topolin (0.5–5.0 mg L−1), thidiazuron (0.1–1.5 mg L−1), and N-(2-chloro-4-pyridyl)-N′-phenyl urea (0.1–1.0 mg L−1). The 2.0 mg L−1meta-Topolin supplemented in WP medium was found most effective for induction of maximum shoot responding frequency (100 ± 0.0), number of shoots/explant (6.47 ± 0.21), and mean shoot length (4.54 ± 0.19 cm). The direct shoot organogenesis was observed under scanning electron microphotographs. Similarly, histological observations confirmed its direct origin from pericycle tissues. The microshoots showed the best rooting on Murashige and Skoog media containing indole-3-butyric acid (3.0 mg L−1) with a maximum percent responding frequency (95 ± 2.89), numbers of roots/shoot (4.41 ± 0.06), and mean root length (6.10 ± 0.10 cm). Plants were well acclimatized and grown in the field. The genetic homogeneity among mother and micropropagated plants was established using inter-simple sequence repeat, start codon targeted markers, and DNA content (2C) analysis with the help of flow cytometry. Regenerated plants exhibited good antioxidant activity. Both plants showed similar metabolites based on Fourier transform infrared and high-resolution mass spectrometry analyses. This protocol can be used for conservation, multiplication, and genetic improvement by transforming the elite clones. © The Society for In Vitro Biology 2025.
Organogenesis from Leaf Tissue of Spondias pinnata (L. f.) Kurz, SEM study and Genetic Fidelity Assessment by ISSR and ScoT
(Springer Science and Business Media B.V., 2021) Pooja Jaiswal; Nishi Kumari; Sarvesh Pratap Kashyap; Shailesh Kumar Tiwari
In vitro raised plantlets were obtained from nodal tissue through direct organogenesis and they served as donor plants for the collection of leaf explants. Leaf explants were inoculated on Murashige and Skoog’s medium with different concentrations of 2,4-Dichlorophenoxyacetic acid (2,4-D). Hundred percent callogenesis was observed on medium supplemented with 5 mg l−1 2,4-D. For the multiplication of cells (proliferation), calli were transferred to either basal medium or media containing different types and concentrations of cytokinins. Proliferation was observed maximum on media containing 0.5 mg l−1 BAP or 1.0 mg l−1 BAP. Shoot differentiation from calli took place on media supplemented with BAP in combinations with TDZ or ZN. Initiation of organogenesis was observed in calli within two weeks of their subculture on differentiation medium. Shoot differentiation was maximum, when calli proliferated on medium having 1 mg l−1 BAP were transferred to the medium containing 1 mg l−1 BAP and 0.5 mg l−1 TDZ. Organogenic responses after four weeks of subculture on above differentiation medium were as such: number of shoots per explants (25.33 ± 0.88), number of shoots per calli replicate (3.67 ± 0.33) and maximum shoot length (3.43 ± 0.20 cm). Medium supplemented with 2.5 mg l−1 NAA was most responsive for rooting of shoots (54.16 ± 1.39%). About 62.5% plantlets survived after hardening and 54.17% plantlets got acclimatized. All acclimatized plants were transferred to field condition successfully. Formation of unipolar shoots and their multicellular attachment with callus were observed by scanning electron microscopy. To confirm the genetic fidelity of micropropagated plants, five micropropagated plants derived from different leaf explants and two mother plants (randomly selected from micropropagated plants raised from nodal explants) were subjected to molecular analysis. The genetic fidelity of in vitro regenerated plants was assessed by using SCoT and ISSR molecular markers. 12.5% polymorphism was reported in both studies, which may be due to callus mediated regeneration of shoots. © 2021, The Author(s), under exclusive licence to Springer Nature B.V.