Somatic Embryogenesis in Catharanthus Roseus: Proteomics of Embryogenic and Non-embryogenic Tissues; and Genome Size Analysis of Regenerated Plant

Mujib, A.; Malik, Moien Qadir; Bansal, Yashika; Syeed, Rukaya; Ejaz, Bushra; Mamgain, Jyoti

doi:10.1007/978-3-030-89269-2_4

Cited by 3 publications

(3 citation statements)

References 97 publications

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“…The promotive effect of NAA + BAP in the formation of shoots was reported in several plant species, like in Artemisia annua [ 28 ] and Asparagus cochinchinensis [ 29 ]. The rooting of regenerated shoots was obtained on IBA- or NAA-augmented MS medium in Tylophora ; the same was described in other different plant species [ 30 , 31 ].…”

Section: Discussionmentioning

confidence: 97%

Elicitation Induced α-Amyrin Synthesis in Tylophora indica In Vitro Cultures and Comparative Phytochemical Analyses of In Vivo and Micropropagated Plants

Mamgain,

Mujib,

Bansal

et al. 2023

Plants

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Tylophora indica (Burm. f.) Merrill is an endangered medicinal plant that possesses various active agents, such as tylophorinine, kaempferol, quercetin, α-amyrin and beta-sitosterol, with multiple medicinal benefits. α-amyrin, a triterpenoid, is widely known for its antimicrobial, anti-inflammatory, gastroprotective and hepatoprotective properties. In this study, we investigated the metabolite profiling of tissues and the effects of cadmium chloride and chitosan on in vitro accumulation of alkaloids in T. indica. First, the callus was induced from the leaf in 2,4-D-, NAA- and/or BAP-fortified MS medium. Subsequent shoot formation through organogenesis and in vitro roots was later induced. Gas chromatography–mass spectrometry (GC–MS)-based phytochemical profiling of methanolic extracts of in vivo and in vitro regenerated plants was conducted, revealing the presence of the important phytocompounds α-amyrin, lupeol, beta-sitosterol, septicine, tocopherol and several others. Different in vitro grown tissues, like callus, leaf and root, were elicited with cadmium chloride (0.1–0.4 mg L−1) and chitosan (1–50 mg L−1) to evaluate the effect of elicitation on α-amyrin accumulation, measured with high-performance thin layer chromatography (HPTLC). CdCl2 and chitosan showed improved sugar (17.24 and 15.04 mg g−1 FW, respectively), protein (10.76 and 9.99 mg g−1 FW, respectively) and proline (7.46 and 7.12 mg g−1 FW), especially at T3 (0.3 and 25 mg L−1), in the leaf as compared to those of the control and other tissues. The antioxidant enzyme activities were also evaluated under an elicitated stress situation, wherein catalase (CAT), superoxide dismutase (SOD) and ascorbate peroxidase (APX) displayed the highest activities in the leaf at T4 of both of the two elicitors. The α-amyrin yield was quantified with HPTLC in all tested tissues (leaf, callus and root) and had an Rf = 0.62 at 510 nm wavelength. Among all the concentrations tested, the T3 treatment (0.3 mg L−1 of cadmium chloride and 25 mg L−1 of chitosan) had the best influence on accumulation, irrespective of the tissues, with the maximum being in the leaf (2.72 and 2.64 μg g−1 DW, respectively), followed by the callus and root. Therefore, these results suggest future opportunities of elicitors in scaling up the production of important secondary metabolites to meet the requirements of the pharmaceutical industry.

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Section: Discussionmentioning

confidence: 97%

Elicitation Induced α-Amyrin Synthesis in Tylophora indica In Vitro Cultures and Comparative Phytochemical Analyses of In Vivo and Micropropagated Plants

Mamgain,

Mujib,

Bansal

et al. 2023

Plants

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“…The ploidy status of anther-derived calli was examined using the flow cytometry method as described by Galbraith [43]. A total of 3 samples of anther-derived callus were randomly chosen, along with a reference standard of diploid leaves of C. roseus with a known 2C DNA content of 1.51 pg [44]. Approximately 50 mg of callus was added to a Petri plate having 1.0 mL ice-cold Galbraith's buffer (nuclei isolation buffer) and finely macerated with the help of a surgical blade.…”

Section: Flow Cytometric Analysismentioning

confidence: 99%

Phytochemical Composition and Detection of Novel Bioactives in Anther Callus of Catharanthus roseus L.

Bansal

Mujib

Mamgain

et al. 2023

Plants

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Catharanthus roseus L. (G.) Don is the most widely studied plant because of its high pharmacological value. In vitro culture uses various plant parts such as leaves, nodes, internodes and roots for inducing callus and subsequent plant regeneration in C. roseus. However, till now, little work has been conducted on anther tissue using plant tissue culture techniques. Therefore, the aim of this work is to establish a protocol for in vitro induction of callus by utilizing anthers as explants in MS (Murashige and Skoog) medium fortified with different concentrations and combinations of PGRs. The best callusing medium contains high α-naphthalene acetic acid (NAA) and low kinetin (Kn) concentrations showing a callusing frequency of 86.6%. SEM–EDX analysis was carried out to compare the elemental distribution on the surfaces of anther and anther-derived calli, and the two were noted to be nearly identical in their elemental composition. Gas chromatography–mass spectrometry (GC–MS) analysis of methanol extracts of anther and anther-derived calli was conducted, which revealed the presence of a wide range of phytocompounds. Some of them are ajmalicine, vindolinine, coronaridine, squalene, pleiocarpamine, stigmasterol, etc. More importantly, about 17 compounds are exclusively present in anther-derived callus (not in anther) of Catharanthus. The ploidy status of anther-derived callus was examined via flow cytometry (FCM), and it was estimated to be 0.76 pg, showing the haploid nature of callus. The present work therefore represents an efficient way to produce high-value medicinal compounds from anther callus in a lesser period of time on a larger scale.

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“…In this regard, flow cytometric method proves to be a handy technique for measuring nuclear 2C DNA content of plant cells [ 19 ], making it simple, fast and easy to determine the ploidy of micropropagated plants [ 20 ]. Recent investigations have been done to examine the ploidy levels in various plants, such as Allium sativum [ 21 ], different species of Zephyranthes [ 22 ] and Catharanthus roseus [ 23 ]. Additionally, the genetic homogeneity of tissue culture plants can also be carried out by using PCR-based DNA markers such as Random Amplified Polymorphic DNA (RAPD), Start Codon Targeted (SCoT), Inter Simple Sequence Repeat (ISSR) and Amplified Fragment Length Polymorphism (AFLP) [ 24 ].…”

Section: Introductionmentioning

confidence: 99%

Ploidy Status, Nuclear DNA Content and Start Codon Targeted (SCoT) Genetic Homogeneity Assessment in Digitalis purpurea L., Regenerated In Vitro

Bansal

Mujib

Siddiqui

et al. 2022

Genes

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Digitalis purpurea L. is a therapeutically important plant that synthesizes important cardiotonics such as digitoxin and digoxin. The present work reports a detailed and efficient propagation protocol for D. purpurea by optimizing various PGR concentrations in Murashige and Skoog (MS) medium. The genetic homogeneity of in vitro regenerants was assessed by the flow cytometric method (FCM) and Start Codon Targeted (SCoT) marker technique. Firstly, the seeds inoculated in full MS medium added with 0.5 mg/L GA3 produced seedlings. Different parts such as hypocotyl, nodes, leaves and apical shoots were used as explants. The compact calli were obtained on BAP alone or in combinations with 2, 4-D/NAA. The hypocotyl-derived callus induced somatic embryos which proliferated and germinated best in 0.75 mg/L BAP-fortified MS medium. Scanning electron microscopic (SEM) images confirmed the presence of various developmental stages of somatic embryos. Shoot regeneration was obtained in which BAP at 1.0 mg/L and 2.0 mg/L BAP + 0.5 mg/L 2,4-D proved to be the best treatments of PGRs in inducing direct and indirect shoot buds. The regenerated shoots showed the highest rooting percentage (87.5%) with 24.7 ± 1.9 numbers of roots/shoot in 1.0 mg/L IBA augmented medium. The rooted plantlets were acclimatized in a greenhouse at a survival rate of 85–90%. The genome size and the 2C nuclear DNA content of field-grown, somatic embryo-regenerated and organogenic-derived plants were estimated and noted to be 3.1, 3.2 and 3.0 picogram (pg), respectively; there is no alteration in ploidy status and the DNA content, validating genetic uniformity. Six SCoT primers unveiled 94.3%–95.13% monomorphic bands across all the plant samples analyzed, further indicating genetic stability among in vitro clones and mother plants. This study describes for the first time successful induction of somatic embryos from hypocotyl callus; and flow cytometry and SCoT marker confirmed the genetic homogeneity of regenerated plants.

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Somatic Embryogenesis in Catharanthus Roseus: Proteomics of Embryogenic and Non-embryogenic Tissues; and Genome Size Analysis of Regenerated Plant

Cited by 3 publications

References 97 publications

Elicitation Induced α-Amyrin Synthesis in Tylophora indica In Vitro Cultures and Comparative Phytochemical Analyses of In Vivo and Micropropagated Plants

Elicitation Induced α-Amyrin Synthesis in Tylophora indica In Vitro Cultures and Comparative Phytochemical Analyses of In Vivo and Micropropagated Plants

Phytochemical Composition and Detection of Novel Bioactives in Anther Callus of Catharanthus roseus L.

Ploidy Status, Nuclear DNA Content and Start Codon Targeted (SCoT) Genetic Homogeneity Assessment in Digitalis purpurea L., Regenerated In Vitro

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