Leaf Callus Research Articles

Striking effects of melatonin on secondary metabolites produced by callus culture of rosemary (Rosmarinus officinalis L.)

In recent years, phytomelatonin has become even more important because of its numerous useful properties in plant metabolism. In this study, different concentrations of melatonin (MEL) (0, 100 and 200 µM) were used to investigate the changes in the accumulation of phenolic compounds, and the aromatic content of rosemary (Rosmarinus officinalis L.) leaf explants by callus culture. MS medium supplemented with 1.0 mg/l BAP and 2.0 mg/l NAA was used to induce callus production for determining the changes in secondary metabolites. Phenolic compounds such as rosmarinic acid, caffeic acid, p-coumaric acid and hesperidin and forty-five aromatic compounds were identified from rosemary leaf calli and measured by HPLC and GC/MS, respectively. 200 μΜ MEL increased the amount of rosmarinic acid (680 µg/g) compared to the control group (275 µg/g). However, 100 μΜ MEL increased the caffeic acid content (34 µg/g) two times higher than in the control group (17 µg/g). These findings were very remarkable for rosemary phenolic compound accumulation and it can be said that MEL could be a useful elicitor for rosemary. Totally, 200 μΜ MEL was more effective than 100 μΜ MEL in changing the aromatic content of rosemary calli. Also, some important aromatic compounds such as linalool, styrene and methional were only detected in MEL treated calli and not seen in the control group. The use and function of melatonin in callus culture systems as an elicitor to detect the change in secondary metabolites of rosemary leaf explants.

Diode Laser Radiation Effects in Initiation and Growth of Sun Flower Plant (Helianthus annuus L.) Callus

The research involved the studying of the effect of the red laser radiation at wave length 650 nm and power 50 mw/cm2 using a semi-connector laser (diode laser) for different periods of times 5,10,15,20 min. in the initiation and growth of callus sunflower (Helianthus annuus L.). Exposing of sunflower root, stem and leaf explants to laser radiation leads to clear increase in rate of growth indicators of calli (fresh weight of calli, dihydrofolate reductase activity and the amount of protein, DNA, RNA. folate) especially after 6o days of exposure. It was found that 20 min. duration was ideal to produce maximal activity of dihydrofolate enzyme giving value of 53.411µgm/min./mg protein and an increase in the amount of protein. DNA, RNA and folate to achieve 4.3 mg/gm, 23.311, 221,888, 1.2µgm fresh weight respectively after 60 days of leaf calli growth. These changes in growth indicators reflect on differentiation of stem calli to a group of white roots with dense root hairs after 35 days of stem explants exposure to 10 min. While leaf calli stimulated to form a number of vegetative branches after 50 days of exposure to 20 min. All shoots were rooted easily in hormone free medium. Plants continued to grow to form sunflower disks after 50 days of rooting process and growth. .

Effect of GO on the formation and differentiation of tobacco leaf callus and its antioxidant capacity

The present study was carried out to explore the effects of graphene oxide (GO) on callus induction, differentiation, metabolite biosynthesis and antioxidant capacity for tobacco leaves. Results showed that lower concentrations of GO (lower than 600 μg/mL) stimulate the differentiation of tobacco callus into buds and leaves. The content of chlorophyll, soluble sugar, soluble protein, and the activities of superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) all increased at lower concentrations of GO. In addition, the length of roots and stems of the regenerated tobacco callus also increased. However, GO induced an increase in malondealdehyde (MDA) content. The experimental results could provide valuable reference for other researches about interactions between carbon-based nanomaterials and plant in callus formation differentiation, metabolite biosynthesis and nanometer pollution.

In vitro adventitious shoot regeneration from leaf explants of some apricot cultivars

ABSTRACT Apricot is one of the most valuable commercial fruits. In vitro propagation of apricot is very important for rapid multiplication of cultivars with desirable traits and production of cleaning up and virus-free plants. Low frequency of multiplication is the main limiting factor for traditional propagation methods. In this regard, the objective of our investigation was to study the morphogenetic capacity of apricot leaf explants of the promising cultivars ‘Iskorka Tavridy’, ‘Magister’ and ‘Bergeron’ for regeneration system development and solving some breeding questions. The source of explants was in vitro plants regenerated and cultured on QL medium. Leaves were maintained in the dark at 24±1 °C in thermostat for three-four weeks. Morphogenic callus and structures were mainly formed at the central and proximal parts of leaves on MS, QL and WPM media with 1.5 or 2.0 mg L-1 BAP and 1.5 or 2.0 mg L-1 IAA in different combinations, or TDZ (0.6 and 1.3 mg L-1). Callus with adventive buds was transferred to regeneration medium and placed into a growth chamber at 24±1 °C and 16-hour photoperiod with a light intensity of 37.5 μmol m-2 s-1. The best results were obtained when adaxial leaf surface was in contact with the culture medium. Frequency of leaf callus formation on MS medium with 1.5 mg L-1 BAP and 1.5 mg L-1 IAA was higher in the explants of ‘Iskorka Tavridy’ (80.0%) than in - ‘Bergeron’ (50.0%) and ‘Magister’ (36.7%). The best results of callogenesis for ‘Magister’ was obtained on MS medium with 1.3 mg L-1 TDZ (53.3%). Active microshoot regeneration in ‘Iskorka Tavridy’ cultivar was shown on MS medium with BAP and IAA and in ‘Magister’ cultivar - on MS medium with TDZ. Rhizogenesis was obtained on half strength MS medium with 2.0 mg L-1 IBA.

Indirect in vitro Regeneration of Viola canescens Wall. ex, Roxb. by using Leaf Calli

An efficient indirect plant regeneration protocol was developed for Viola canescens, an important medicinal herb used in broad spectra of diseases in number of folk medicines since aeon. Excessive use of this plant without any rehabilitating measure has led to decline its natural population. Present investigation reports the use of zeatin to regenerate the plant from the callus on MS following its acclimatization on the soil condition. Calli of the plant responded positively to zeatin and maximum number of shoots 13.07 ± 2.01 were obtained when 9.12 μM concentration of zeatin was used. Regenerated shoots were subsequently rooted with IBA on MS and half strength MS and showed maximum number of roots 14.13 ± 1.64 after 60 days when medium was fortified with 4.92 μM IBA, followed by transferring them to soil condition, acclimatization of the plantlet was carried in growth chamber and then finally to the field for their survival where it showed 80% survival.
 Plant Tissue Cult. & Biotech. 28(2): 215-222, 2018 (December)

Genome size analysis of field grown and tissue culture regenerated Rauvolfia serpentina (L) by flow cytometry: Histology and scanning electron microscopic study for in vitro morphogenesis

An efficient genetically stable regeneration protocol has been established in Rauvolfia serpentina (L) Benth ex. Kurz, an important medicinal plant, used for the treatment of various cardiovascular diseases. Here, in this present study, plants were regenerated through organogenesis (direct and indirect) and embryogenesis pathways. This is the first ever somatic embryo formation report of R. serpentina in liquid Murashige and Skoog (MS) medium from isolated root segments. The genetic fidelity of the regenerated plants was also analysed by flow cytometry. The 2C DNA content of the regenerated plants raised through organogenesis and somatic embryogenesis was measured along with field grown natural plants, which served as donor plant. The estimated 2C DNA levels are 2.08, 1.76 and 1.88 pg in direct, indirect and somatic embryo regenerated plants respectively while in field grown plant it was noted to be 1.94 pg in R. serpentina. This is perhaps the first ever 2C DNA estimation study in tissue culture raised plants along with natural population of Rauvolfia, an immensely important medicinal plant, present in India. We developed improved callus mediated shoot regeneration (indirect) protocol from leaf callus. Highest shoot regeneration percentage (66.10%) with 4.0 shoots / callus mass was noted on 2.0 mg l−1 BAP + 0.5 mg l−1 NAA added MS medium. The shoot tips and nodal explants on 2.5 mg l−1 BAP amended MS medium induced direct shoot buds, with highest shoot regeneration frequency (81.45%) from shoot tips. On 1.0 mg l−1NAA added MS medium maximum numbers of roots (8.87) were noted, indicating the effectiveness of NAA over IBA in rooting. Excised roots were cultured in agitated liquid MS medium and in 1.0 mg l−1 NAA added conditions somatic embryos were formed. The mature somatic embryos germinated on 1.5 mg l−1 BAP + 0.25 mg l−1 GA3 supplemented MS medium. The histology and scanning electron microscopic (SEM) evidences have been presented to describe the in vitro morphogenesis process. The well-rooted plantlets were acclimatized and transferred to soil successfully (79%). The flow cytometric analysis showed no significant variation in nuclear DNA contents, hence was observed to be genetically stable with their genome size similar to field grown Rauvolfia plants.

EVALUATION OF HEPATOPROTECTIVE POTENTIAL OF LEAF AND LEAF CALLUS EXTRACTS OF ANISOCHILUS CARNOSUS (L) WALL.

<p>The study was carried out to evaluate the hepatoprotective activity of leaf and leaf callus extracts of <em>Anisochilus carnosus</em> (L) Wall. against alcohol induced toxicity using HepG2 cell line. Leaf explants were cultured on Murashige and Skoog solid medium supplemented with different growth regulators. Prior to the determination of hepatoprotective property leaf and leaf callus extracts were subjected to the toxic dose study. The degree of hepatoprotection of extracts was determined by measuring cell viability percentage by MTT assay. The preliminary phytochemical analysis of leaf and leaf callus was carried out by qualitative analysis. Maximum percentage of callus formation (98%) was obtained in MS medium fortified with 3 mg/l 2,4-D. HepG2 cells were pretreated with the different concentrations (below toxic dose) of leaf and leaf callus extracts for 72 hours followed by alcohol intoxication. Results revealed that ethanolic leaf extract pretreated HepG2 cells show 94% cell viability compared to the standard silymarin pretreated HepG2 cells which showed 81% cell viability. Leaf callus extracts also exhibited significant hepatoprotective activity where ethanolic callus extract pretreated HepG2 cells showed 86% viability after intoxication with alcohol. Results revealed that HepG2 cell viability percentage is dose dependent. Phytochemical studies revealed the presence of different secondary metabolites in leaf and leaf callus extracts. The bio-efficacy study confirms the presence of secondary metabolites of hepatoprotective nature in leaf and leaf callus of <em>A. carnosus.</em></p>

Production of squalene with promising antioxidant properties in callus cultures of Nilgirianthus ciliatus

There is a lot of market for squalene (C30H50) for industrial and therapeutic applications such as oil, biofuel, antioxidant, antimicrobial and anticancer agents. Squalene was used to be derived from liver oil of sharks and whales, but a sustainable alternative source such as plants is needed. An ayurvedic plant, Nilgirianthus ciliatus, was found to contain a limited amount of squalene. This study hypothesizes that squalene content in callus culture of N. ciliatus could produce an increased level of squalene. For efficient callus induction, various parameters such as explants, plant growth regulators, light and dark conditions were optimized. Leaf produced the highest frequency of callus induction (89.5%) with 2.3 fold enhanced squalene production on MS medium containing 4.0 mg l−1 2,4-dichlorophenoxyacetic acid and 0.4 mg l−1 benzyladenine. The wild and in vitro tissues were evaluated for metabolic profiles using GC–MS and antioxidant properties. The leaf callus exhibited increased level of total phenolic, flavonoid and squalene contents which confers the highest level of antioxidant (IC50 = 88.8 μg ml−1) activity compared to wild explants. The GC–MS profiles of wild and callus tissues showed twelve compounds, in which squalene was 9.3% in wild and 21.4% in callus. Yield of squalene was found to be 70.9 μg g−1 (dry weight) DW and 146.3 μg g-1 DW for wild and callus tissues, respectively. Furthermore, FT-IR analysis also confirmed the presence and increased level of squalene in callus compared to that of wild. This callus could be an ideal source for mass squalene production without depleting natural sources and simultaneously fulfilling the pharmacological demands.

Resistance Saffower (Carthamus tinctorius L.) Callus and its branches to salinity Stress

The study was implemented in the year 2013-2014 in the tissue culture laboratory of the field crops department/ faculty of agriculture and forestry for the purpose of producing callus and saline resistant plants , the results showed capability to initiation leaves callus with high levels which was 100% at present 2,4-D alone or its interaction with BA in medium of MS, which supported by sodium chloride salt levels (0.0, 0.05, 0.1 and 0.2 Molar) except a concentration (0.3 molar) which significantly reduced the initiation ratio. The estemation results of proline acomulation in leaves callus as an indicator of salinity resistance showed that the proline content was significantly higher at all levels of salinity used (0.05-0.3 molar) for more than 30 μg/g frish whight compared to 2.3 and 2.8 μg / g After 4 and 8 weeks of treatment any more than 10 times its presence in control tretment. Salinity resistance results showed that branches forming from callus of shoot tip and leaf showed resistance to salinity after four weeks of transfer of branches to growth medium and multiplication which suplimented with sodium chloride salt. Although the number of branches decreased slightly in the fourth week, they were able to grow and showed tolerance to the medium content of 0.2 molar NaCl, while no significant differences were observed in the number of branches during the four weeks on the saline medium (0.1 molar). The number of branches arising from leaf callus did not differ significantly in all medium (0.0-0.2) and showed continuous resistance for four weeks. Results of interaction salt with the plant part showed that increased salinity led to the reduction of branches with a significantly increased concentration of the tip stem and were not significant for the branches of the callus leaves. The effect of the time showed that it was not significant in reducing the number of branches in the two parts of the plant. It was also noticed that there was an increase in the length of the branches of each of the stem tip and leaves callus in the medium containing (0.1 molar NaCl). In high concentrations of salt (0.2 molar) It continued to grow in all saline medium, suggesting that salts may be resistant to saline stress, possibly for genetic mutations that have adapted to growth in these medium,

Comparative study of anti-angiogenic and cytotoxic activity of leaf and leaf callus silver nanoparticles of Tephrosia villosa Pers.

Comparative study of anti-angiogenic and cytotoxic activity of leaf and leaf callus silver nanoparticles of Tephrosia villosa Pers.

English

The study was aimed to standardise a protocol for the in vitro mass propagation of Rhinacanthus nasutus (L) Kurz, an anticancer shrub. Leaf, node and inflorescence explants were inoculated onto the Murashige and Skoog’s (MS) medium enriched with different combinations and concentrations of growth regulators. Maximum callusing percentage was achieved in leaf explants in MS medium supplemented with 5 mg/l 2,4-D. Multiple shoots were achieved from leaf, node and inflorescence explants with maximum of 25±0.42 (5 mg/l BAP + 2.5 mg/l IAA), 11±0.87 (5 BAP + 2.5 mg/l IAA) and 8±0.56 (3 mg/l BAP + 1.5 mg/l IAA) shoots, respectively. For in vitro rhizogenesis, elongated micro shoots were aseptically transferred to the half strength MS liquid medium with maximum number of 8±0.89 roots per shoot achieved in 1 mg/ml IAA fortified MS medium. The in vitro rooted micro shoots were acclimatised under laboratory conditions for two weeks by transferring to polycups containing sterile soil, sand and vermiculite (1:1:1). After two weeks, hardened plantlets were transferred to the green house for two weeks and then finally to the garden with 95% survivability. Key words: Rhinacanthus nasutus, multiple shoots, leaf callus, node, inflorescence, rhizogenesis.

English

English

Antibacterial Property and Molecular Docking Studies of Leaf Calli Phytochemicals of Bridelia scandens Wild.

Antibacterial Property and Molecular Docking Studies of Leaf Calli Phytochemicals of Bridelia scandens Wild.

Comparison of the Metabolic Behaviors of Six Systemic Insecticides in a Newly Established Cell Suspension Culture Derived from Tea ( Camellia sinensis L.) Leaves.

The use of an in vitro cell suspension to study insecticide metabolism is a simpler strategy compared to using intact plants, especially for a difficult matrix such as tea. In this study, a sterile tea leaf callus was inoculated into B5 liquid media with 2,4-dichlorophenoxyacetic acid (2,4-D, 1.0 mg L-1) and Kinetin (KT, 0.1 mg L-1). After 3-4 subcultures (28 days each), a good cell suspension was established. Utilizing these cultures, the metabolic behaviors of six insecticides, including two organophosphates (dimethoate, omethoate) and four neonicotinoids (thiamethoxam, imidacloprid, acetamiprid, and imidaclothiz) were compared. The results showed that thiamethoxam, dimethoate, and omethoate were easily metabolized by tea cells, with degradation ratios after 75 days of 55.3%, 90.4%, and 100%, respectively. Seven metabolites of thiamethoxan and two metabolites of dimethoate were found in treated cell cultures using mass-spectrometry, compared to only two metabolites for thiamethoxam and one for dimethoate in treated intact plants.

In vitro regeneration and assessment of genetic fidelity of acclimated plantlets by using ISSR markers in PPR-1 (Morus sp.): An economically important plant

Abstract Mulberry is a tree species; hence, it is difficult to carry out the indirect generation. In view of this limitation, in present study an indirect regeneration protocol was developed for in vitro clonal propagation of sericulturally important superior temperate mulberry variety cv. Pampore-1 (PPR-1) by using leaf, petiole and nodal based callus. Initially friable callus (mean fresh weight) was induced in maximum amounts (288.2 ± 21.09; 248.2 ± 16.52 & 138.4 ± 04.25) from leaf, petiole and nodal explants on Murashige and Skoog (MS) media supplemented with 5.0 μM/L, 7.5 μM/L of 2,4-Dichlorophenoxyacetic acid (2,4-D) and 12.5 μM/L of Naphthalene acetic acid (NAA), respectively, after 3 weeks of culture. Best response of shoot regeneration from the induced callus with maximum frequency (14.8 ± 0.82) was observed in leaf callus on MS media supplemented with 2.5 μM/L of Thidiazuron (TDZ) + 2.5 μM/L of 6-Benzyl amino purine (BAP). The regenerated shoots were rooted with maximum rooting frequency (96 ± 02.2) on MS media with 7.5 μM/L concentration of Indole-3-Butyric acid (IBA). The raised plantlets were then hardened using 1:1:2 ratio of farmyard manure, sand and garden soil, then gradually they were transferred and acclimatized to field conditions. The survival rate of in vitro raised PPR-1 plantlets in field conditions is about 82%. Genetic homogeneity between the micropropagated plants and mother plant was confirmed by carrying out the inter simple sequence repeats (ISSR) primer based polymerase chain reaction (PCR) analysis. Among the ten ISSR primers used, two primers, i.e., M5 and M8 has given good amplification with clear, distinct and scorable DNA bands which were monomorphic across the micropropagated plantlets and the mother plant. Hence, the in vitro regenerated PPR-1 mulberry plantlets were confirmed as clonally uniform and genetically stable.

Proteases from Calotropis gigantea stem, leaf and calli as milk coagulant source

Abstract Background Universal demand for cheese keeps the search for appropriate enzymes from plants mimicking animal rennet action in scientific focus. Objective To associate distribution of milk clotting potential and profile of whole/κ-casein hydrolysis by Calotropis gigantea stem, leaf and respective calli crude enzymes (CE). Materials and methods Milk clotting activity and index were assayed for CE. Caseinolytic activity (CA) was evaluated spectrophotometrically. 0.5 CA units of CE and Enzeco® were used for studying whole/κ-casein hydrolysis pattern by Tricine SDS-PAGE. Inhibition studies were performed for enzyme characterisation. Results Traditionally propagated (TP) stem and its callus CE exhibited high specific milk clotting activity (1297.30±0.2 U/mg of protein and 926.74±44.13 U/mg of protein, respectively) and milk clotting index (103.562±1.162 and 79.365±4.93, respectively). Comparison of whole casein hydrolytic pattern by 0.5 CA units of CE revealed closer resemblance between leaf callus and Enzeco®. However, κ-casein specificity analysis revealed TP leaf to be closely mimicking the performance by Enzeco®. Conclusion Study suggests CE from TP leaf to be a potential vegetable coagulant to work as an effective and low-cost milk clotting mediator in cheese industry.

Callus induction from leaf and plant regeneration of Sedum plumbizincicola

To establish the efficient regeneration system and lay foundation for transgenic system of Sedum plumbizincicola in this research, the young leaves of S. plumbizincicola as explants, several factors affecting callus induction, bud differentiation, and plant regeneration were studied, and then a regeneration system was established. An MS medium supplemented with 300 mg·L-1 hydrolyzed casein was used as the basic medium. Different concentrations of hormone combinations was designed. Results showed that the best medium for leaf callus induction was the basic medium with 1.00 mg·L-1 2, 4-Dichlorophenoxyacetic acid (2, 4-D) and 0.30 mg·L-1 6-Benzylaminopurine (6-BA) added having a callus induction rate of 92.01%. The best medium for bud differentiation was the basic medium supplemented with 0.10 mg·L-1 2, 4-D and 1.00 mg·L-1 6-BA having a callus differentiation rate of 27.44%. A suitable medium for bud proliferation was the basic medium with 0.10 mg·L-1 2, 4-D and (0.50-1.00) mg·L-1 6-BA added. Also, a suitable rooting medium was the basic medium with 1.00-2.00 mg·L-1 3-indolebutyric acid (IBA) added. These findings laid a tissue culture foundation for the establishment of a transgenic system with S. plumbizincicola.

Phytochemical Analysis and In vitro Antimicrobial activities of various extracts of Decalepis arayalpathra

The present study was aimed to investigate the phytochemical, antioxidant, antimicrobial activity of in vivo (root, stem and leaf), and in vitro derived leaf callus of Decalepis arayalpathra. The explants were cultured on MS medium supplemented with different concentration and combination of auxins and cytokinins for callus induction. The total phenolic, flavonoid and tannin contents in different solvent extracts were analysed using standard methods. Antibacterial and antifungal activity of different solvent extracts was carried out using the agar well diffusion method and food poison technique. Maximum callus induction was noticed on MS media supplemented with 2, 4-D (9.05µM/L) and BAP (4.44 µM/L) + NAA (2.69 µM/L) respectively. Total phenolic and flavonoid contents were higher in methanolic extracts of root and stem compared to other extracts. Antimicrobial activities was significantly higher in methanolic extract of root, leaf, stem and callus followed by aqueous and chloroform extract. The maximum antibacterial activity was observed in Klebsiella pneumoniae, Staphylococcus aureus and Escherichia coli in methanolic extracts of stem, leaf and callus. Our results demonstrated the phytochemical and antimicrobial activities of D. arayalpathra and further studies have to be carried out to identify the novel compounds.

PROPAGATION STRATEGIES OF TRIBULUS TERRESTRIS L. A PORTENTOUS MEDICINAL PLANT EMPLOYING TISSUE CULTURE TECHNOLOGY

A protocol for micropropagation of Tribulus terrestris, an important medicinal herb was established using juvenile explants viz., leaf, node and internode. All the explants were tested for callus induction on Murashige and Skoog’s (MS) medium, supplemented with BAP, NAA and 2,4-D. Among the three explants leaf explant responded well (98%) for the callus induction in the MS medium composted with BAP and NAA (4.0 and 0.5 mg/L) followed by the nodal segments (58.75%) in the same medium. Maximum number of shoot induction from the callus of leaf derived explants (91.1%) was perceived on MS medium fortified with BAP 4.0 mg/L and NAA (0.5 mg/L). Moreover, root elongation and profuse rooting percentage (77.19%) were achieved when the well-grown shoots were cultured on MS media supplemented with IAA (2.0 mg/L) for leaf callus derived shoots. The regenerated plantlets were hardened and established at 80% survival rate in hardening media encompassed with red soil, sand and vermicompost in the ratio of 1:1:1 by volume.

Analysis of genetic variations in Musa paradisiaca cv Karibale-Monthan using AFLP markers

In the present study, the genetic variations of leaf calli regenerants of Musa paradisiaca cv Karibale-Monthan has been analyzed using AFLP markers. The highest (86.66%) frequency of compact callus formation was obtained when leaf explants were cultured on MS media, containing 3mg/L 2,4-D and 0.5 mg/L BAP. The optimum shoot formation and multiplication (5.70±1.49) was achieved from the leaf derived callus on MS medium fortified with 3mg/L BAP+0.5mg/L TDZ. The 3-4 cm small shoots produced distinct roots on MS media containing 0.5mg/l NAA with 0.2% activated charcoal. The AFLP analyses produced 1012 monomorphic bands from total 1094 bands with 7.5% polymorphism between mother plant and callus derived regenerants. The percentage of monomorphism for individual primer combinations was very high and varied from 90.3% (EcoRI-AG×MseI-CCG) to 97.6% (EcoRI-AT×MseI-CGA). The result showed lack of polymorphism is due to genetic integrity of the plants that was not altered indifferent to the hormonal treatment.