Frequency Of Callus Research Articles

Synthesis and characterization of Lanthanum Oxide nanoparticles using Citrus aurantium and their effects on Citrus limon Germination and Callogenesis

The plant extract-mediated method is eco-friendly, simple, safe, and low-cost, using biomolecules as a reducing agent to separate nanoparticles. Lanthanum (La) is a rare earth metal that positively affects plant growth and agriculture. Citrus limon is a leading citrus fruit with many varieties. Conventional vegetative propagation methods depend on season, availability of plant material and are time-consuming. It is the main reason for limiting the acceptance of new varieties. So, In-vitro propagation of the lemon method is practiced overcoming all these problems. Lanthanum oxide nanoparticles (La2O3-NPs) were synthesized using plant extract of C. aurantium. Ultraviolet (UV)-Visible Spectroscopy, Scanning Electron Microscopy (SEM), Transmission Electron Microscopy (TEM), Fourier Transform Infrared (FTIR) spectroscopy, and Thermal Gravimetric Analysis (TGA) were used to characterize the synthesized La2O3-NPs. Fabricated La2O3-NPs were oval and spherical, with an average size of 51.1 nm. UV-visible absorption spectra of La2O3-NPs were shown at a sharp single peak at 342 nm and FTIR showed stretching frequency at 455 cm−1-516 cm−1. In the TGA outcome, mass loss was 9.1%. In vitro experiments demonstrated that La2O3-NPs significantly enhanced the germination and growth of C. limon seeds, achieving an 83% germination rate at 5 mg/L concentration, with uncoated seeds showing root initiation at 10 days and shoot formation at 15 days. Furthermore, La2O3-NPs effectively stimulated callus induction and maturation, with optimal responses observed in media containing MS and 2 mg/L 2,4-D, resulting in a maximum callus frequency of 100% from leaves and 87.5% from shoots at 5 mg/L concentration. These findings underscore the potential of La2O3-NPs to improve seed germination rates, seedling vigor, and callogenesis efficiency, suggesting their promising integration into agricultural practices for sustainable crop production, especially in suboptimal growing conditions. Future research is recommended to explore the mechanisms and broader applications of La2O3-NPs across various plant species and environments.

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

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.

Development of an in vitro regeneration system from immature inflorescences and CRISPR/Cas9-mediated gene editing in sudangrass

BackgroundSudangrass (Sorghum sudanense) is a major biomass producer for livestock feed and biofuel in many countries. It has a wide range of adaptations for growing on marginal lands under biotic and abiotic stresses. The immature inflorescence is an explant with high embryogenic competence and is frequently used to regenerate different sorghum cultivars. Caffeic acid O-methyl transferase (COMT) is a key enzyme in the lignin biosynthesis pathway, which limits ruminant digestion of forage cell walls and is a crucial barrier in the conversion of plant biomass to bioethanol. Genome editing by CRISPR/Cas9-mediated mutagenesis without a transgenic footprint will accelerate the improvement and facilitate regulatory approval and commercialization of biotech crops. Methods and resultsWe report the overcome of the recalcitrance in sudangrass transformation and regeneration in order to use genome editing technique. Hence, an efficient regeneration system has been established to induce somatic embryogenesis from the immature inflorescence of two sudangrass cultivars on four MS-based media supplemented with different components. Our results indicate an interaction between genotype and medium composition. The combination of Giza-1 cultivar and M4 medium produces the maximum frequency of embryogenic calli of 80% and subsequent regeneration efficiency of 22.6%. Precise mutagenesis of the COMT gene is executed using the CRISPR/Cas9 system with the potential to reduce lignin content and enhance forage and biomass quality in sudangrass. ConclusionA reliable regeneration and transformation system has been established for sudangrass using immature inflorescence, and the CRISPR/Cas9 system has demonstrated a promising technology for genome editing. The outcomes of this research will pave the road for further improvement of various sorghum genotypes to meet the global demand for food, feed, and biofuels, achieving sustainable development goals (SDGs).

Establishment of an Efficient in vitro Regeneration Protocol for Brassica campestris L. Var. Tori-7

The aim of the present investigation was to develop an efficient and reproducible in vitro regeneration protocol for Brassica campestris L. var. Tori-7. Brassica is one of the most popular and major oil yielding crop plants around the world including Bangladesh. For in vitro regeneration of this crop plant, MS medium supplemented with various concentrations of 6-benzylaminopurine (BAP) and naphthalene acetic acid (NAA) were used. The highest frequency of callus and shoot regeneration was obtained on MS medium supplemented with 3.0 mg/l BAP and 0.2 mg/l NAA from both cotyledonary leaf with petiole and hypocotyl explants. Elongation of shoots was also optimized on the same media composition. For inducing roots from the excised in vitro derived shoots, half strength of MS medium supplemented with 0.3 mg/l indole-3-butyric acid (IBA) was found to be more effective than other hormonal supplements applied. Following the development of effective roots, in vitro raised plantlets were successfully transplanted into soil where they produced flowers and fertile seeds. The plants of R1 generation was successfully established in natural environment. In vitro regeneration protocol of Brassica campestris var. Tori-7 established in this study could be used for future plant genetic transformation experiments. Plant Tissue Cult. & Biotech. 32(2): 237-246, 2022 (December)

In vitro regeneration and its histological characteristics of Dioscorea nipponica Makino

Dioscorea nipponica Makino is an optimal candidate to develop the diosgenin industry in North China. Due to its increasing demand in the medicine industry, it is urgent to apply new biotechnological tools to foster breeds with desirable traits and enhanced secondary metabolite production. The production of useful metabolites by the in vitro cultured rhizomes can be explored successfully for utilization by various food and drug industries. In this study, we reported callus formation and plantlet regeneration of the medicinal plant D. nipponica. Explants of leaves, stem segments and rhizomes of aseptic seedlings were cultured on Murashige and Skoog (MS) medium containing various combinations of auxin and cytokinin to find the optimal PGRs of each type of explant for callus induction and shoot regeneration of D. nipponica. The paraffin section technique was also used to observe of the morphogenesis of callus and adventitious bud. Explants of seeds and rhizomes formed calli at high frequency in all lines we examined. However, the explant of leaves rarely formed callus. Three kinds of callus were detected during the induction phase. Here, we describe three types of callus (Callus I–III) with different structure characteristics. Greenish in color and a nodule-like protrusion surface (Callus type III) were arranged more closely of cells with less interstitial substance, cell differentiation ability stronger than other callus types. The optimum combination was the maximum shoot differentiation frequency of 90% in callus derived from seeds cultured on MS medium with 2.0 mg L−16-BA + 0.2 mg L−1NAA. The shoot differentiation frequency (88.57%) of rhizome-induced callus was obtained by the combination of MS medium supplemented with 3.0 mg L−16-BA + 2.0 mg L−1NAA. 1/2 MS medium plus 0.5 mg L−1NAA resulted in a higher root regeneration frequency of 86.70%. In vitro propagated plantlets with healthy roots were domesticated and transplanted into small plastic pots containing sterile soil rite under greenhouse conditions with 80% survivability. Bud differentiation is mostly of exogenous origin, mostly occurring on the near callus surface. Therefore, it may be surmised that in vitro morphogenesis of D. nipponica is mainly caused by indirect organogenesis (adventitious bud).

Transcriptomic and physiological analyses reveal the acquisition of somatic embryogenesis potential in Agapanthus praecox

Somatic embryogenesis (SE) is an ideal cell model for plant cell totipotency study. The poor frequency of embryogenic callus (EC) induction limits the application of SE in many plants, such as Agapanthus praecox. Herein, we performed transcriptomic and physiological analyses with different callus (Ca) differentiation directions (SE and organogenesis) and stages (initial SE and repetitive SE) to better understand the physiological and molecular characteristics of the acquisition of embryogenic potential in A. praecox. A number of differentially expressed genes (DEGs) were significantly related to plant hormonal signaling transduction, starch and sucrose metabolism, and reactive oxygen species (ROS) responses. Compared to Ca, DEGs including heat shock protein, isoamylase, histone H4, alpha-1,2-mannosyltransferase, and sucrose synthase substantially changed expression levels in EC formation. Corresponding to DEGs, physiological indicators including indole-3-acetic acid, cytokinins, brassinosteroids, ethylene, abscisic acid, ROS, hydrogen peroxide, superoxide dismutase, catalase, starch and sucrose may be responsible for the acquisition of embryogenic potential in A. praecox. Moreover, plant growth regulators, carbon source combination, osmotic regulating substance, and DNA methylation inhibitors in the culture medium significantly affected the acquisition of SE potential. Altogether, our results suggested that plant hormone signal regulation, starch and sucrose metabolism, ROS scavenging, chromatin accessibility and DNA methylation jointly contributed to the activation of embryogenic ability in A. praecox.

Integrated Proteomic and Metabolomic Analyses Provide Insights Into Acquisition of Embryogenic Ability in Agapanthus praecox.

Somatic embryogenesis (SE) is an ideal model for plant cell totipotency. Transition from somatic cells to embryogenic cells is the key to SE. The poor frequency of embryogenic callus (EC) induction has limited the application of SE in many plants, such as Agapanthus praecox. We performed large-scale, quantitative proteomic and metabolomic analyses with different callus differentiation directions (SE and organogenesis) and stages (initial SE and repetitive SE) to better understand the morphological, physiological, and molecular characteristics of the acquisition of embryogenic ability in A. praecox. Integrated proteomic and metabolomic analyses suggested that callus differentiation direction was potentially regulated by pathways related to carbohydrate and energy metabolism (fatty acid metabolism, pyruvate metabolism, glycolysis/gluconeogenesis, pentose and glucuronate interconversions, starch and sucrose metabolism, galactose metabolism, carbon fixation pathways in prokaryotes, carbohydrate digestion and absorption, and fructose and mannose metabolism), chromatin accessibility and DNA methylation, reactive oxygen species responses and resistance (ascorbate and aldarate metabolism), and plant hormonal signaling. As a validation, we found that carbon source combination and plant hormone regulation in the culture medium significantly affected the acquisition of embryogenic ability, thereby inducing EC. Interestingly, plant hormonal signaling-related genes showed different expression patterns significantly when callus cultured with different carbon sources. Thus, our results suggested that energy supply and hormone signal transduction seemed to cooperatively contribute to the activation of embryogenic ability. Altogether, this study revealed valuable information regarding the molecular and biochemical changes that occurred during EC induction and provided valuable foundation for comprehensive understanding of the mechanisms associated with SE and organogenesis in A. praecox.

Callus Induction of Fenugreek Trigonella Foenum-Graecum via Auxin Combined with Cytokinins Hormones, and Assessment of Toxicity via Brine Shrimp Assay

Trigonella foenum-graecum (TFG) is a significant leguminous plant with diverse pharmacological effects. However, the resistant character of this plant accounts for significant difficulties in vitro multiplication, justifying the necessity to try new techniques for in vitro propagation of this plant. Hence, this study reports the effects of BAP, NAA, and 2,4-D on in vitro callus formation of seeds. The toxicity properties of seeds, plant, and callus aqueous extracts of TFG were measured by brine shrimp assay (BSA). Callus index, frequency of callus, callus weight, and morphology of callus were recorded after 30 days of culture. No callus formation was observed in the absence of plant growth regulators. The maximum callus formation observed in the MS media containing 1.0 mg/l 2,4-D, the highest mean of the callus index (52±9.5) with 100% frequency and callus yield (0.52±0.08 g) in 30 days of culture. The highest mean of callus index (37±0 4.05) for combination hormones with 100% callusing and yield (0.37±0.02 g) in 30 days of culture by 1.0 mg/l BAA with 0.5 mg/l NAA. Seeds extract of TFG showed the highest toxicity (954.99 µg/ml), aqueous plant extract (1237.98 µg/ml), and aqueous callus extract (1801 µg/ml) from BSA. Comparing individual hormones, the highest amount of callus in TFG can be yielded 2,4-D hormone alone, and a combination of BAP and NAA can yield 100% callus.

Agrobacterium-Mediated High-Efficiency Genetic Transformation and Genome Editing of Chaling Common Wild Rice (Oryza rufipogon Griff.) Using Scutellum Tissue of Embryos in Mature Seeds.

Genetic transformation is an important strategy for revealing gene function, and it is used extensively in both functional genomics study and molecular breeding of rice. Demand for its application in wild Oryza species is rising for their extensive genetic diversity. However, genetic transformation of wild Oryza accessions with AA genome using calli induced from scutellum tissue of embryos in mature seeds has not been successfully established. In the present study, we used Chaling common wild rice (CLCWR) (Oryza rufipogon Griff.) with AA genome to successfully establish an Agrobacterium-mediated genetic transformation system based on scutellum tissue of embryos in mature seeds. The calli from embryos in mature seeds of CLCWR were easy to be induced and regenerated. The callus induction rate and texture were optimum under 2.5 mg/L 2,4-D. The optimal hormone combination used for regeneration was 2 mg/L ZT + 0.1 mg/L NAA. Studies on genetic transformation and genome editing showed that the transformation efficiency was 87–94%, the efficiency of single genome editing and multiplex genome editing were about 60–70% and 20–40%, respectively. Compared with Nipponbare (Nip), CLCWR had higher Hygromycin-resistant callus frequency and transformation efficiency. Taken together, our study establishes a highly efficient transformation system for common wild rice with AA genome and provides a good rice material for de novo domestication by genome editing in the future.

In vitro regeneration of shoot and roots of the wild folkloric medicinal plant Ammannia baccifera L. via indirect organogenesis from leaf explant cultures

Ammannia baccifera L. is a wetland medicinal herb that belongs to the family Lythraceae. This plant is very notable to have a wide scope in pharmacological, therapeutic and medicinal properties. The current examination imparts an effective, proficient and fast strategy of commercial proliferation to satisfy the folklore medicinal and pharmacological demands of the market. A large-scale micropropagation of Ammannia baccifera L was done through a meticulous and a well-organized procedure. The leaf segments were cultured on MS medium augmented with different concentrations (0.5–2.5 mg/l) of 6- benzylaminopurine (BAP), (0.5- 2.0 mg/l) 1- naphthaleneacetic acid (NAA) and (1.0- 2.0 mg/l) 2,4- Dichlorophenoxyacetic acid (2,4-D) and were used for callus initiation. BAP at 1.0 mg/l combined with 0.5 mg/l NAA was found to be the best concentration and combination for initiation of high frequency of reddish green nodular callus (95.00±1.87) and good response for leaf explants. For shoot regeneration, MS medium blended with BAP at 1.5 mg/l and IAA at 0.6 mg/l produces maximum percentage of shoots from the callus derived by leaf explant. The leaf segments produce maximum percentage shoots (90.00±1.52), total number of shoots per explant (26.33±1.45) and length of shoot (9.63±0.73 cm). However, healthy established shoots were cultured on MS medium fortified with 1.5 mg/l indole-3-butyric acid (IBA) along with 2.0 mg/l. Indole-3-acetic acid (IAA) was found to be best for the root initiation and production in the leaf segments (95±1.57), number of roots (19.33±0.88) and root length (8.63±0.61cm). The regenerated plantlets were relocated to paper cups filled with vermiculite:sand:soil in the proportion of 1:2:1 (w/w) and were then transferred into outdoor conditions.

An Efficient Agrobacterium-Mediated Transformation Method for Hybrid Poplar 84K (Populus alba × P. glandulosa) Using Calli as Explants.

A highly efficient Agrobacterium-mediated transformation method is needed for the molecular study of model tree species such as hybrid poplar 84K (Populus alba × P. glandulosa cv. ‘84K’). In this study, we report a callus-based transformation method that exhibits high efficiency and reproducibility. The optimized callus induction medium (CIM1) induced the development of calli from leaves with high efficiency, and multiple shoots were induced from calli growing on the optimized shoot induction medium (SIM1). Factors affecting the transformation frequency of calli were optimized as follows: Agrobacterium concentration sets at an OD600 of 0.6, Agrobacterium infective suspension with an acetosyringone (AS) concentration of 100 µM, infection time of 15 min, cocultivation duration of 2 days and precultivation duration of 6 days. Using this method, transgenic plants are obtained within approximately 2 months with a transformation frequency greater than 50%. Polymerase chain reaction (PCR), reverse transcription-PCR (RT-PCR) and β-galactosidase (GUS) histochemical staining analyses confirmed the successful generation of stable transformants. Additionally, the calli from leaves were subcultured and used to obtain new explants; the high transformation efficiency was still maintained in subcultured calli after 6 cycles. This method provides a reference for developing effective transformation protocols for other poplar species.

Callogenesis and somatic embryogenesis of <i>Oryza sativa</i> L. (cv. MARDI Siraj 297) under the influence of 2, 4-dichlorophenoxyacetic acid and kinetin

<abstract> <p>Callogenesis and embryogenesis are integral parts of many tissue culture procedures for genetic manipulation in rice. However, the efficiency of both processes is largely dependent on the media constituent especially the plant growth regulators (PGRs) due to the genotype-dependent nature of <italic>in vitro</italic> culture protocols. Therefore, this study investigates the effect of two PGRs; 2, 4-dichlorophenoxyacetic acid (2, 4-D) and kinetin (Kin) on callus growth and somatic embryogenesis of an important Malaysian rice cultivar (<italic>Oryza sativa</italic> L. cv. MARDI Siraj 297). Mature rice seeds explants were inoculated in Murashige & Skoog (MS) medium supplemented with different combinations of 2, 4-D (0 to 3.5 mg/L) and Kin (0 to 0.5 mg/L) to induce callogenesis. Parameters for callus growth such as fresh weight (FW), callus induction frequency (CIF), embryogenic callus frequency (ECF), regeneration frequency (RF), number of plantlets per callus (PPC), callus texture and callus color were observed after 35 days of inoculation. The results show that the maximum callus growth was achieved in MS medium supplemented with combination of 2.0 mg/L 2, 4-D and 0.2 mg/L Kin, represented by the highest FW (211 mg), CIF (95%), ECF (90%), RF (100%) and PPC (22 plantlets); along with friable callus texture. Low concentration of 2, 4-D (0 to 0.5 mg/L) in the presence or absence of Kin promotes root growth instead of callus, while high concentrations (above 3.0 mg/L) retard the callus formation. The embryogenic calli from this optimized PGRs combination were successfully formed shoots in MS medium supplemented with 2 mg/L BAP and 1 mg/L NAA, followed by rooting in PGRs-free MS medium. This finding provides an efficient protocol for callogenesis and somatic embryogenesis of MARDI Siraj 297, since this is the first published report regarding somatic embryogenesis induction of this cultivar.</p> </abstract>

Fiber-Optic Bragg Grating Sensors for Biomechanical Analysis of Fracture Healing

Despite the complexity of bone callus formation, the evaluation of fracture healing is usually performed based on x-ray visual inspection, a subjective and imprecise method. The main objective of this study is to access the evolution of bone callus. Tests were performed using a synthetic femur and an in vivo subject (preliminary results). Fiber Bragg Grating (FBG) sensors were glued to the Schanz pins of a circular external fixator for the analysis of static and dynamic strain. Three materials of different densities were inserted in the fracture site (synthetic femur) to simulate the formation of bone callus. The results from the static tests with the synthetic femur show that the strain measured on the Schanz pins presented an expected deformation pattern, according to a previous simulation. It was possible to differentiate bone callus stiffness through strain detected in the Schanz pins. The results from the dynamic tests allowed to find the first resonant frequency of each bone callus, with a difference of 4 Hz between them, measured by the FBG sensor. In the preliminary in vivo tests, the FBG sensor results demonstrated its technical viability to detect the bone callus healing stage. Nonetheless, a comprehensive study using a higher number of subjects is necessary. Being a noninvasive technology, it has the potential to become an important tool to assist medical staff in bone healing analysis.

Genetic variation of doubled haploids derived from anther culture of M1 red rice plants

Abstract. Nurmansyah, Setyadi AH, Fatumi NC, Fatmawati Y, Wulandari RA, Purwantoro A. 2021. Genetic variation of doubled haploids derived from anther culture of M1 red rice plants. Biodiversitas 22: 4923-4929. The doubled haploid (DH) technology when integrated with induced mutation could accelerate development of local rice cultivars with several desirable traits. Anthers from the first generation of mutant (M1) plants were utilized for DH production. However, the effectiveness of inducing and fixing mutation requires detailed evaluation with the help of molecular techniques for its accuracy and reliability than that of morphological or biochemical assessments. The objective of this research was to develop and detect genetic variation of DH plants derived from anther culture of M1 plants. Seeds of local red rice cultivar, Cempo Abang, were treated with four gamma-ray doses (0, 100, 200, 300 Gy) and planted to produce the M1 plants. Panicles at the booting stage of the M1 plants were collected and used as donor anthers. The present study suggested that gamma irradiation treatments indirectly could increase callus formation. However, it also reduced the number of callus producing plantlets due to higher frequency of brown calli. Eleven spontaneous DH plants obtained in the study along with parental plants were assessed to determine the occurrence of genetic variation using six RAPD primers. The RAPD primers generated 51 bands, of which 34 alleles or 66.7% were polymorphic with an average of 5.6 polymorphic alleles per primer. The genetic similarity among parent and 11 DH lines based on Jaccard’s similarity index ranged from 0.622 to 0.902. The DNA polymorphism among the DH plants demonstrated the effect of gamma irradiation to create genetic variation. Therefore, this method could be used as an alternative for rice breeding programs especially to develop preferred traits in the local rice cultivars.

Callus induction and regeneration in sugarcane under drought stress.

Tissue culture methods are useful in assessing the tolerance of various stresses due to the ease of controlling stress under in vitro conditions. This study aimed to investigate the response of sugarcane genotyps to drought stress using calli as a model system. For inducing sugarcane callus, the medium of Murashige and Skoog (MS) was used with different mannitol concentrations (100, 200, and 300 mM) to measure their effects on callus frequency, the day of callus initiation, embryogenic potential, relative growth rate (RGR), water and proline contents, K+ and Na+ contents, as well as the formation of shoot and roots for three sugarcane genotypes (e.g., GT 54-9, G 84-47, and pH 8013). The RAPD-PCR analysis was carried out using five oligonucleotide primers to identify the genetic variation among sugarcane genotypes. The results indicated that the degree of callus proliferation varied from 70 − 86%. The highest value of callus proliferation, PGR, shoot formation was recorded for the genotype GT 54-9 compared to the other two genotypes (G 84-47 and pH 8013). Calli treated with 100 mM mannitol showed the highest RGR, proline and waer contents for the genotype GT 54-9, while, those treated with 300 mM recorded the lowest values of these parameters for the genotype pH 8013. The genotype G 84-47 collected highest Na+ content, while the genotype pH 8013 collected highest K+ content. The results of this study recommend preference for GT 54-9 genotype, which is considered the most promising genotype, showing more tolerance to drought stress based on all studied traits.

Indirect somatic embryogenesis and genetic homogeneity assessment in Plectranthus bourneae Gamble an endemic plant to Western Ghats of Tamil Nadu, India

An effective somatic embryogenesis protocol was established for <i>Plectranthus bourneae</i> an Endemic plant to Western Ghats of Tamil Nadu, India. High frequency (77.28 %) of embryogenic callus was achieved from leaf explants on Murashige and Skoog (MS) medium supplemented with 0.5 mg/L 2,4- dichlorophenoxyacetic acid (2,4-D). The embryogenic callus was subcultured in a combination of 0.5 mg/L 2,4-D and 50 mg/L glutamine (GLU) which produced somatic embryos(84.22 %). Different stages of embryos (globular, heart, torpedo and cotyledonary stage) were observed. Maturation and the frequency of somatic embryo germination increased when transferred onto half-strength MS medium containing 0.5 mg/L gibberellic acid (GA3) and 0.5 mg/L 6-benzyladenine (BA). Plantlets were acclimatized successfully with 90 % survival. Histological studies revealed the development of primary and secondary embryos. The genetic fidelity of mother plants and in vitro raised plants was established by inter-simple sequence repeat (ISSR) markers. The established protocol assists the rapid production of true-to-type plants by somatic embryogenesis and hence could provide a valuable target material for genetic transformation experiments.

Indirect somatic embryogenesis and genetic homogeneity assessment in Plectranthus bourneae Gamble an endemic plant to Western Ghats of Tamil Nadu, India

An effective somatic embryogenesis protocol was established for <i>Plectranthus bourneae</i> an Endemic plant to Western Ghats of Tamil Nadu, India. High frequency (77.28 %) of embryogenic callus was achieved from leaf explants on Murashige and Skoog (MS) medium supplemented with 0.5 mg/L 2,4- dichlorophenoxyacetic acid (2,4-D). The embryogenic callus was subcultured in a combination of 0.5 mg/L 2,4-D and 50 mg/L glutamine (GLU) which produced somatic embryos(84.22 %). Different stages of embryos (globular, heart, torpedo and cotyledonary stage) were observed. Maturation and the frequency of somatic embryo germination increased when transferred onto half-strength MS medium containing 0.5 mg/L gibberellic acid (GA3) and 0.5 mg/L 6-benzyladenine (BA). Plantlets were acclimatized successfully with 90 % survival. Histological studies revealed the development of primary and secondary embryos. The genetic fidelity of mother plants and in vitro raised plants was established by inter-simple sequence repeat (ISSR) markers. The established protocol assists the rapid production of true-to-type plants by somatic embryogenesis and hence could provide a valuable target material for genetic transformation experiments.

Impact of silver nanoparticles as antibacterial agent derived from leaf and callus of Celastrus paniculatus Willd

BackgroundCelastrus paniculatus Willd. is a rich source of numerous active constituents such as celapanigin, celapagin, malkangunin, celapanin, zeylasteral, pristimerin, and zeylasterone which render medicinal properties to its various parts. Therefore, the present work provides a protocol for the synthesis of AgNPs from in vivo leaf and in vitro developed callus extract of C. paniculatus and both the extracts have great antibacterial potential, which may give immense support for the drug preparation using AgNPs prepared from this valuable medicinal plant.ResultsHigh frequencies of calli induced from leaves and its counterpart, i.e., the natural part leaf were selected as the experimental materials for the green synthesis. The collected data exhibited gradual color variations started with whitish color, creamish, and after 8 weeks it ultimately turns into a solid mass of brownish callus. The silver nanoparticles (AgNPs) were synthesized using in vivo fresh leaves and in vitro developed callus extracts with an indication of brown colored complex. Further confirmation of AgNPs synthesis in both the samples was done using UV-visible spectral analysis followed by X-ray diffraction (XRD) analysis, Fourier transformation infrared spectroscopy (FTIR), and scanning electron microscopic (SEM) analysis.ConclusionThe antibacterial activity of both extracts reflected the presence of the zone of inhibition in both gram-positive and gram-negative bacteria. AgNPs derived from callus extract showed better results with 24.00 ± 1.00 mm zones of inhibition. This protocol deserves the recognition of the antibacterial potential of AgNPs synthesized from CP plant extracts.

Exploring potential of copper and silver nano particles to establish efficient callogenesis and regeneration system for wheat (Triticum aestivum L.)

ABSTRACT In vitro recalcitrance of wheat to regeneration is the major bottleneck for its improvement through callus-based genetic transformation. Nanotechnology is one of the most dynamic areas of research, which can transform agriculture and biotechnology to ensure food security on sustainable basis. Present study was designed to investigate effects of CuSO4, AgNO3 and their nanoparticles on tissue culture responses of mature embryo culture of wheat genotypes (AS-2002 and Wafaq-2001). Initially, MS-based callus induction and regeneration medium were optimized for both genotypes using various concentrations of auxin (2,4-D, IAA) and cytokinins (BAP, kinetin). The genotypes differed for embryogenic callus induction and regeneration potential. Genotype AS-2002 yielded maximum embryogenic calli in response to 3.0 mg/l 2,4-D, whereas Wafaq-2001 offered the highest embryogenic calli against 3.5 mg/l 2,4-D supplemented in the induction medium. Genotype AS-2002 showed maximum regeneration (59.33%) in response to regeneration protocol comprising 0.5 mg/l IAA, 0.3 mg/l BAP and 1.0 mg/l Kin, while Wafaq-2001 performed best in response to 0.5 mg/l IAA, 0.3 mg/l BAP and 1.5 mg/l Kin with 55.33% regeneration efficiency. The same optimized basal induction and regeneration medium for both genotypes were further used to study effects of CuSO4, AgNO3 and their nano-particles employing independent experiments. The optimized induction medium fortified with various concentrations of CuSO4 or CuNPs confirmed significant effects on frequency of embryogenic callus. Addition of either 0.020 mg/l or 0.025 mg/l CuSO4, or 0.015 mg/l CNPs showed comparable results for embryogenic callus induction and were statistically at par with embryogenic callus induction of 74.00%, 75.67% and 76.83%, respectively. Significantly higher regeneration was achieved from MS-based regeneration medium supplemented with 0.015 mg/l or 0.020 mg/l CuNPs than standard 0.025 mg/l CuSO4. In another study, the basal induction and regeneration medium were fortified with AgNO3 or AgNPs ranging from 1 to 7 mg/l along with basal regeneration media devoid of AgNO3 or AgNPs (control). The maximum embryogenic calli were witnessed from medium fortified with 3.0 mg/l or 4.0 mg/l AgNPs compared with control and rest of the treatments. The standardized regeneration medium fortified with 5.0 mg/l AgNO3 or 3.0 mg/l AgNPs showed pronounced effect on regeneration of wheat genotypes and offered maximum regeneration compared with control. The individual and combined effect of Cu and Ag nanoparticles along with control (basal regeneration media of each genotype) was also tested. Surprisingly, co-application of metallic NPs showed a significant increase in embryogenic callus formation of genotypes. Induction medium supplemented with 0.015 mg/l CuNPs + 4.0 mg/l AgNPs or 0.020 mg/l CuNPs + 2.0 mg/l AgNPs showed splendid results compared to control and other combination of Cu and Ag nanoparticles. The maximum regeneration was achieved by co-application of 0.015 mg/l CuNP and 4.0 mg/l AgNPs with 21% increment of regeneration over control. It is revealed that CuNPs and AgNPs are potential candidate to augment somatic embryogenesis and regeneration of mature embryo explants of wheat.

Development of In Vitro Callus Induction and Regeneration Protocol for Newly Released Sugarcane (Saccharum officinarum L.) Genotypes from Inner Leaf Sheath

Improvement of sugarcane through conventional breeding takes longer time (8-10 years) to release new improved genotypes. Callus induction and regeneration protocol is a prerequisite for genetic improvement through genetic engineering. Hence, this study was initiated to develop a protocol for in vitro callus induction and regeneration of three sugarcane genotypes (C86-56, C132-81 and SP70-1284) using immature young leaf as a source of explants. Seed cane of 30 cm size having 2 nodes each were obtained from Metehara Research and Development, operating under Ethiopian Sugar Corporation. They were then brought to the National Agricultural Biotechnology Research center and planted in the greenhouse to serve as sources of explants. The treatments were arranged in a factorial experiment laid out in a completely randomized design. For callus induction MS medium supplemented with five levels of 2,4-D (2.0, 2.5, 3.0, 3.5 and 4.0 mg/l) was used. Immature leaves were collected from the green house grown plants 3 months after planting and were cultured on callus induction medium after sterilizing with 5% berekina for 15 minutes and then followed by 70% alcohol for 30 seconds under the biological safety cabinet. For plant regeneration four levels of BAP (0.5, 1.0, 1.5 and 2.0 mg/l) were used in combination with four levels of IBA (0.0, 0.25, 0.5 and 0.75 mg/l). Callus induction experiment was replicated four times and each replicate was represented by four explants per a culture jar and shoot regeneration experiment was replicated three times, 2 embryogenic calli per jar each for regeneration. Data were recorded for all parameters and analyzed using Statistical Analysis System (SAS) Software version 9.3. Means were separated using least significant differences (LSD) at 0.001 probability level. Analyses of variance indicated that days to callus initiation, frequency of callus induction and callus weight were affected significantly (p≤0.001) by genotype, 2, 4-D levels and their interaction. Genotype C132-81 was identified as the best for early callus initiation, high frequency of callus and callus weight. The highest percentage (92.31%) of callus initiation was recorded on medium supplemented with 3.0 mg/l of 2,4-D with higher callus weight (454.5 mg). Regeneration percentage, number of shoots, shoot length and number of leaves per shoot were also significantly (p≤0.001) affected by genotype, BAP, IBA and their interaction effect. Genotype C132-81 also recorded significantly higher number of shoots (43.44±0.5) on MS medium supplemented with 1 mg/l BAP in combination with 0.5 mg/l IBA. Genotype C86-56 gave higher shoot length (6.38±0.08) cm while C132-81 gave the higher number of leaves (8.24±0.06) at 2.0 mg/l BAP+0.25 mg/l IBA. In conclusion, 3.0 mg/l and 3.5 mg/l 2,4-D concentrations were found optimum for callus induction and development, while 1.5 mg/l BAP+0.5 mg/l IBA was the best for plant regeneration. Keywords : genotype, explant, callus induction, regeneration DOI: 10.7176/JBAH/11-3-05 Publication date: February 28 th 2021