Callus Induction Frequency Research Articles

Impact of Zinc oxide nanoparticles on biosynthesis of Thymoquinone in cell cultures of Nigella sativa

The rising market interest for Nigella sativa (Black seeds) necessitates the development of cultivation strategies to enhance metabolites production. Zinc oxide nanoparticles (ZnO-NPs) have drawn global attention as efficient and bio safe elicitors for in vitro cultures, to enhance secondary metabolites production in medicinal plants. In this study, ZnO-NPs were utilized for establishment of callus and cell cultures in black seeds for the first time. Hypocotyl explants were cultured on Murashige and Skoog (MS) media with varying levels of ZnO-NPs, resulted in callus induction and biomass formation. Optimal response in callus growth parameters were observed when explants were grown on MS media supplemented with 60mg/L ZnO-NPs, resulting in 71.2% callus induction frequency, 28.2g/L fresh biomass, 9.7g/L dry biomass, and 63% water content. A substantial increase in callus growth was observed when ZnO-NPs were combined with 6-Benzylaminopurine (BA) at ratio of 45:1.5mg/L, resulting in 91.2% callus induction frequency and 42.2g/L fresh biomass. In cell suspension cultures, ZnO-NPs alone at 45mg/L produced optimum callus biomass (60.9g/L). However, in combination with BA, callus biomass did not increase significantly in cell cultures. Maximum accumulation of total phenolic content (TPC: 26.8mg GAE/g DW; Gallic acid equivalent dry weight) and total flavonoid content (TFC: 19.5mg QE/g DW; Quercetin equivalent dry weight) was observed in cell cultures treated with higher concentration (70mg/L) of ZnO-NPs in the 5th week of the growth curve. Moreover, ZnO-NPs incremented substantially the Phenylalanine lyase (PAL), Superoxide dismutase (SOD) and Peroxidase (POD) enzyme activities in cell cultures. Nonetheless, Reverse Phase High Performance Liquid Chromatography (RP-HPLC) analysis indicated peak production of tymoquinone (168.5mg/g FW) in cell cultures treated with 45mg/L ZnO-NPs alone. This study offers a promising approach for commercial production of Nigella sativa biomass and bioactive metabolites.

Determination of phosphinothricin and paromomycin selective concentrations for obtaining transgenic spelt plants

Aim. To determine the selective concentrations of phosphinothricin and paromomycin for the selection of transgenic plants of spelt wheat. Methods. Shoot apical meristem culture, mature embryo culture, Agrobacterium-mediated genetic transformation. Results. Isolation and cultivation of shoot apical meristems of seedlings from three spelt genotypes and mature embryos from three other genotypes were carried out. A high frequency (from 80 to 100 %) of callus induction from explants was observed. It was shown that the addition of 5 mg/l of phosphinothricin or 100 mg/l of paromomycin to the culture medium almost completely inhibited plant regeneration compared to the control. After Agrobacterium-mediated genetic transformation of calli with a vector containing the phosphinothricin-N-acetyltransferase gene, regeneration of spelt shoots for one genotype was observed on a selective medium with 5 mg/l phosphinothricin. Conclusions. The selective concentrations of herbicide and antibiotic for obtaining transgenic spelt wheat plants with the corresponding marker genes are 5 mg/l for phosphinothricin and 100 mg/l for paromomycin.

Comparison Between Maize Inbred and Hybrid Lines for Their Amenability to Agrobacterium Mediated Transformation and In Vitro Plant Regeneration

The field of genetic engineering and molecular breeding utilizes maize immature embryos for transformation studies. However, there are hinderances such as the unavailability of immature embryos throughout the year and their low transformation frequencies which make them unsuitable for use at a commercial level. Among cereals, maize has prime importance due to its productivity, industrial products, animal feed and fodder. Regeneration capability in maize transformation studies varies due to differences in genetic makeup and explant source. In this study, we evaluate mature embryos as explants for in vitro plant regeneration. Six varieties including 3 hybrid lines (Pioneer 3025, SG 2002 and Neelam) and 3 inbred lines (NCML 107, CML 161 and FBF 3368) were screened for Agrobacterium mediated transformation amenability using mature maize embryos. The two inbred lines NCML 107 and CML 161 performed best showing 100% callus induction frequency, 93.33% and 86.66% transient GUS expression, and 43.75% and 13.3% regeneration frequency, respectively. Using split seed as an explant, better regeneration was observed in NCML 107 (14.86%) and CML 161 (7.5%) as compared to mature embryos, NCML 107 (10%) and CML 161 (2%). Here we report successful plant regeneration (regeneration medium supplemented with Kinetin + BAP) using split seeds as an explant. Our results demonstrate the capability of two elite maize inbred lines (NCML 107 & CML 161) for their amenability to Agrobacterium mediated transformation and better tissue culture response; hence can be selected in maize transformation studies for improving crop varieties for enhanced nutritional content and better adaptation.

An Efficient In Vitro Shoot Organogenesis and Comparative GC-MS Metabolite Profiling of Gaillardia pulchella Foug

Gaillardia pulchella Foug. is a widely studied plant because of its high pharmacological and ornamental value. The leaves of G. pulchella were used for inducing callus and subsequent plant regeneration as it is the primary source of phytocompounds. The purpose of the present investigation was to formulate an in vitro propagation method for Gaillardia by using leaf explants in MS (Murashige and Skoog) medium. The best callus induction was observed on high (2.0 mg/L) α-naphthalene acetic acid (NAA) and a low (0.5 mg/L) 6-benzylaminopurine (BAP) with callus induction frequency of 91.66%. The leaf callus also demonstrated high caulogenesis ability (95.83%), with an average 5.2 shoots/callus mass at 0.5 mg/L BAP and 2.0 mg/L NAA. Indole Acetic acid (IAA) at 1.0 mg/L had the maximum rooting percentage (79.17%) with 12.4 roots per shoot. Rooted plantlets were later transferred to greenhouse conditions, showing a survivability rate of 75–80%. The physiological parameters, i.e., phenolic compounds and the flavonoids’ level, in the DPPH assay were higher in leaves obtained in vitro compared to callus formed from leaves and field-obtained (mother) leaves. Gas chromatography–mass spectrometry (GC–MS) analysis of methanol extracts of leaves (in vivo and in vitro) and leaf callus presented a wide array of compounds. In callus extract, some 34 phytocompounds were identified. Some of them were 3-hydroxy-2,3-dihydromaltol (25.39%), isoamyl acetate (11.63%), palmitic acid (11.55%), 4-methyloxazole (7.54%), and 5-methoxypyrrolidin-2-one (7.49%). Leaves derived in vivo and in vitro had 45 and 28 phytocompounds, respectively, belonging to different classes like lignans, phenols, terpenoids, alkaloids and fatty acids, etc. Those findings demonstrated that the leaf derived callus and the leaves are the potential stable source of several compounds with medicinal importance. The developed protocol may provide an alternative source of compounds without affecting wild flora.

In vitro Propagation of Alocasia baginda 'Silver Dragon’ through Direct and Indirect Organogenesis

Direct and indirect in vitro organogenesis of Alocasia baginda 'Silver Dragon' was investigated using explants of shoot tips, leaf tissue, and petiole segments. For direct shoot induction from shoot tips, MS medium containing 1.5 mg/l BAP was shown to be the most efficient (80%), producing a maximum of 6.60 ± 0.93 shoots/explant with 3.66 ± 0.38 cm in length. Directly induced shoots were multiplied using a 2.5 mg/l BAP enriched medium, where the number and length of shoots/culture were 11.60 ± 1.12 and 9.20 ± 1.01 cm, respectively. Shoots gradually increased in number and length during the 1st three sub-culture cycles in the similar medium combination but declined after 3rd cycle. Following the 3rd sub-culture cycle, there were 14.80 ± 0.58 shoots per culture, with a shoot length of 9.90 ± 0.63 cm. The greatest frequency of callus induction was 97.67% for leaf tissue and 86.67% for petiole segments when they were cultured on the media containing 3.0 mg/l 2,4-D. Impressive results regarding indirect shoot organogenesis (90%) with maximum shoot number/unit callus (16.20 ± 1.62) and shoot length (11.06 ± 0.97 cm) were noted upon transferring the callus onto MS medium containing 3.5 mg/l BAP and 3% sucrose. Within 10-12 days, 100% rooting was observed in half-strength of gelled MS medium containing 2.0 mg/l IBA, with an average of 15.40 ± 1.17 roots/culture. After being acclimatized in a potting mixture containing garden soil, compost, coco peat, and moss in a 2:1:1:1 ratio, A. baginda 'Silver Dragon' exhibited a 100% survival rate. Established plantlets seem morphologically similar to mother plants and possess fully developed root and shoot systems, along with an adequate leaf number per plant. Plant Tissue Cult. & Biotech. 34(1): 35-47, 2024 (June)

Advancing Hemidesmus indicus propagation and conservation: Somatic embryogenesis, histology, metabolite assessment and genetic stability

Hemidesmus indicus L. R. Br. is a commonly traded ethnomedicinal plant, and a method has been developed to produce somatic embryos (SEs) from calli grown from the leaves of the plant. With a maximum callus induction frequency of 91.66 %, pink-red friable proembryogenic calli were generated on Murashige and Skoog (MS) and 5.0 µM 2, 4-dichlorophenoxyacetic acid (2, 4-D) from the leaf explant (LE). With the aim of achieving a high success rate of 95.00 % for the induction of SEs, with a yield of 91.33 SEs per 100 mg of callus, the proembryogenic calli were transferred to fresh culture medium containing MS and 5.0 µM 6-benzylaminopurine (BA). This induction medium allowed the embryos to develop to the torpedo stage, but their further growth into emblings required a different culture medium. ½ MS with 5.0 µM BA and 1.0 µM gibberellic acid (GA3) resulted in 99.16 % maturation and 39.66 emblings regeneration out of 40 immature embryoids, respectively. With a maximum survival rate of 91.67 %, healthy emblings were successfully acclimatized using Soilrite. DNA-based molecular markers (RAPD and ISSR) were used to confirm the genetic similarities of the in vitro grown plants. Histological examinations were performed to determine the ontogeny, morphology, and development of somatic embryoids. Gas chromatography and mass spectrometry (GCMS–) were performed for both the mother and the in vitro generated plant to analyse the root for secondary metabolites. In the physiological and biochemical studies, enzymes such as superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT), and glutathione reductase (GR) were found to increase in vitro derived H. indicus plants during different acclimatization periods (0, 7, 14, 21 and 28 days). With increasing acclimatization time, net photosynthetic rate (PN) and total chlorophyll show a decrease at first, then an increase. The protocol provides high callus induction rates, efficient somatic embryogenesis, successful maturation and regeneration, robust acclimatization, genetic fidelity, detailed insight into development, and screening of secondary metabolites.

Genetic diversity assessment and in vitro propagation of some date palm (Phoenix dactylifera L.) varieties

The evaluation of genetic diversity is crucial for breeders to develop strategies and improve the resilience, quality, and adaptability of the date palm. In this study, the genetic diversity of three date palm varieties was performed using ISSR-PCR molecular markers to determine its relationship with in vitro propagation response of these varieties. The molecular profiling was performed using ISSR-PCR. A total of 49 loci were produced by the PCR reactions, 38 of which were polymorphic while 11 were monomorphic. The level of polymorphism revealed by ISSR-PCR varied from 33.33% to 100%. The three date palm varieties were grouped into two clusters based on the results of cluster analyses that used morphological data and molecular profiles. Cluster I comprised the ‘Barhy’ variety and Cluster II included ‘Magdoul’ and ‘Amri’ varieties. The clustering analyses revealed the independence of the ‘Barhy’ variety in its characteristics from the other varieties based on either morphological or molecular data. The results of in vitro propagation showed that the ‘Amri’ variety exhibited the highest callus induction frequency (86.28%), callus weight (2.33 g), number of somatic embryos (9.32), number of shoots (14.62), number of roots (4.11), root length (4.63 cm), shoot length (13.61 cm) followed by ‘Magdoul’ and ‘Barhy’ varieties. The ‘Amri’ variety had the shortest callus induction period, at 23.26 days while the ‘Barhy’ variety exhibited the longest period of callus induction (28.55). It was deduced from the study that the ISSR marker reproduced trustworthy patterns of bands to determine the genetic diversity among different date palm varieties that are considered the cornerstone for the genetic improvement of date palms. The understanding of the relationship between genetic diversity and in vitro propagation response of date palm is essential for ensuring the long-term sustainability of its crop. This will facilitate better conservation and development of new date palm varieties that fulfil the needs of farmers and consumers.

The effect of plant growth regulators, carbon sources and gelling agent on the callus regeneration of Malaysian rice MR219 (Oryza sativa L.)

The development of a highly efficient tissue culture system for indica rice is crucial to speed up the implementation of gene editing in breeding programmes as well as to explore the roles of indica genes. However, because indica rice types are recalcitrant to in vitro response, there are only a few reports on the successful transformation process on indica rice due to recalcitrant response to in vitro response. The present study attempted to identify the effects of plant growth regulator (PGR), carbon sources and different concentrations of gelling agent on embryogenic callus induction and rice regeneration of Oryza sativa var MR219. The PGR used were 1 or 2 mg/L 6-benzylaminopurine (BAP) and 1-Naphthaleneacetic acid (NAA) and the carbon sources were 30 g/L sucrose and maltose. Three different gelling agent concentrations (3 mg/L, 6 mg/L or 9 mg/L) were evaluated. The highest frequency of friable callus induction (87%) was observed in callus induction media (CI) containing Murashige and Skoong media (MS) supplemented with 3 mg/L 2,4-dichlorophenoxy acetic acid (2,4-D) and 0.3 mg/L kinetin. Histological studies of the calli showed the presence of embryogenic calli with small intercellular spaces compared to non-embryogenic areas which developed on the hormone MS media. The highest regeneration frequency (50%) was achieved when friable calli were cultured on MS medium containing 3% sucrose, 1 mg/L BAP, and 1 mg/L NAA with 9% phytagel. The callus grown on sucrose media showed the highest number (66.13%) of albino plantlets, while the number of albino plantlets on maltose media was smaller (20%). In conclusion, this optimised mature embryo-derived callus of MR219 rice is responsive to regeneration and could be amenable to genetic transformation studies.

Callus induction studies in various explants in different genotypes of sesame (Sesamum indicum L.)

The goal of this study was to develop a suitable protocol for callus induction in sesame, Sesamum indicumwhich can be further used for regeneration studies. The experiments were carried out using three explants viz.,cotyledonary leaf, hypocotyls and primary leaf at different hormonal combinations and concentrations to studycallus induction. There was significant difference in the response of explants to different hormone treatmentsalthough there is no genotypic difference with regard to explants. Results showed that callus induction wassignificantly influenced by explants type. Hypocotyls responded well for callus induction in most of the genotypes.The callus induction frequency was significantly reduced (36.5%) when 0.2 mg BA was used along with 0.2 mg/lNAA. Replacing this hormonal combination with 2.0mg/l BA and 0.2 mg/l NAA greatly improved the callusinduction frequency (51.4%).

Effect of Plant Growth Regulators on Callus Induction of Black Rice (Oryza sativa L.)

Aims: Black rice of Manipur is known for its aroma and nutritional quality. As per the reports on tissue culture of rice, callus induction response of traditional cultivars was much lower than the modern high yielding varieties. Considering the importance of the black rice, and effort was taken to standardized callus induction protocol of black rice of Manipur.
 Study Design: Complete Randomized Design.
 Place and Duration of Study: Department of Seed Science and Technology, Faculty of Agriculture, Uttar Banga Krishi Viswavidyalaya, Pundibari, Cooch Behar 736165, West Bengal, India, between June 2020 and July 2023.
 Methodology: Mature seeds of two black rice varieties used in this work. The seeds were dehusked manually, washed with running tap water. Then sterilized with 1% bavistin and again washed with running tap water. Seeds were surface sterilized with 0.2% HgCl2 for 15 minutes. Sterilised seeds were rinsed with sterile distilled water inside laminar air flow hood. The seeds were blotting dry using autoclaved tissue paper and sterile seeds were cultured with the scutellum pointing up wards on callus induction medium fortified with different concentrations and combinations of plant growth regulators.
 Results: MS medium fortified with 2,4-D was found to be better than picloram and TDZ to induce callus and the 2,4-D @ 1.00 mg/L and 2,4-D 2.00 mg/L was found to be the best concentrations. Callus health was also good when MS medium invigorated with 2,4-D.
 Conclusion: An efficient embryogenic callus induction and plantlet regeneration protocol for black rice was established by using mature seeds. In callus induction studies, with different concentrations of PGR, 2,4-D, higher callus induction frequency was observed with 1.00 mg/L 2,4-D and 2,4-D @ 2 mg/L for both rice varieties.

Effect of Explant Type and Phytohormone Concentration on Micropropagation of Citrus

Citrus fruits are among the most popular fruits in the world, thanks to their energizing flavor and health advantages.In this work, the ideal levels of activated charcoal and plant growth regulators were determined for micropropagation of lemon. Citrus limon (Osbeck Family: Rutaceae) has been successfully regenerated in vitro using both direct and indirect organogenesis in the current study. For indirect organogenesis, callus was stimulated and multiplied from leaf explants of in vitro grown seedlings on Murashige and Skoog (MS) medium with 6-Benzylaminopurine (BAP) alone or in conjunction with -naphthalene acetic acid (NAA). The placement of explants on MS medium containing BAP alone or in combination with NAA was done for direct organogenesis. For root multiplication, various concentrations of NAA were applied to well-developed microshoots. The frequency of callus induction with BAP(4µl) + NAA(0.2µl) was observed to be 94.5%. When the plants started to root, MS medium treated with 2.0 µl of IBA and 30 g/l of sucrose showed abundant root development. In order to quickly multiply disease-free potential material and take over wide regions in a short amount of time, it may be necessary to establish mature micropropagation methods for new elite lemon cultivars. This phenomena is therefore of fundamental economic relevance. In this study, it was shown that Microshoots were best rooted (90.8%) when IBA (2.0 µl) was treated for 48 hours with activated charcoal.

Optimizing Micropropagation Protocol of Rose (Rosa hybrida L.) cv. ‘Double Delight’

An experiment was conducted to develop a suitable in vitro plant regeneration technique for Rosa hybrida L. cv. ‘Double Delight’ using nodal segments and leaf tissues as explants through direct and indirect organogenesis. Aseptic explants were inoculated onto gelled MS medium contained various strengths of plant growth regulators alone and in combinations for callus and direct shoot induction. MS medium containing 2.0 mg/l BAP was found to be the most effective for direct shoot induction from nodal explants (87.5%) as it produced the maximum number of shoots per explant. The highest callus induction frequency was found to be 80% in leaf tissue, 72% in nodal segments and 96% in leaf and intermodal segments of in vitro raised plantlets in MS medium containing 2.5 mg/l 2, 4-D. The best percentage of indirect shoot organogenesis (93.33%) with maximum number and length of shoot were found when the different explant-derived callus was transferred in 3.0 mg/l BAP supplemented medium. The direct and indirectly induced shoots were multiplied on MS medium containing 3.0 mg/l BAP, where the average number and length of shoots per culture were 7.20 ± 0.80 and 5.22 ± 0.47 cm, respectively. Maximum rooting (80%) was observed in ½-strength of gelled MS medium containing 15.0 g/l sucrose with 1.0 mg/l IBA. Plantlets with the proper root system were then placed in a polybag with a 1:1:1 ratio of sand, garden soil, and compost, and they had a survival rate of about 76%. Plant Tissue Cult. & Biotech. 33(1): 25-36, 2023 (June)

Androgenesis in tomato (Solanum lycopersicum L.)-Effect of genotypes, microspore development stage, pre-treatments and media composition on induction of haploids

Doubled haploid (DH) technology remarkably accelerates the crop breeding by obtaining homozygous lines in a single generation. The present study was targeted in generating haploid plants through androgenesis. Anthers from immature flower buds of six tomato genotypes viz., LE-1230, LE-1236, LE-1256 TLCV 2, PKM 1 and TNAU tomato hybrid CO 3 were used for induction of haploids. A preliminary study based on callus induction frequency (CIF), more than 5% was helpful in short listing flower bud size, pre-treatments and growth regulator combinations. Subsequently, anthers from two different sized flower buds (4 and 6 mm length), dissected either from fresh or pre-treated flower buds (2 and 5 days in dark at 4 °C or gamma irradiated) were inoculated in MS medium fortified with different growth regulators for callus induction. Among the genotypes, TLCV 2 had recorded the maximum CIF (38.80%) from anthers of 4 mm long flower buds followed by TNAU tomato hybrid CO 3 (34.70%). Throughout the study, anthers from 4 mm long flower buds responded the best for callus induction. Among the pre-treatments, anthers from gamma irradiated flower buds recorded the highest CIF (31.90%) when compared to others. Cold shock (4 °C) in dark to flower buds for 2 days had improved the CIF of anthers when compared to fresh in LE 1230, LE 1238, TLCV2 and TNAU tomato hybrid CO 3, but when the cold shock was increased to 5 days, invariably there was a reduction in CIF in all the six genotypes. TA 8 (MS + 2iP (0.5 mg L-1) + NAA (0.5 mg L-1)) medium was found to be the best for maximum CIF in LE 1230 and PKM1, TA1 (MS + 2iP (1.0 mg L-1) + IAA (2.0 mg L-1)) in LE 1238, LE 1256 and TNAU tomato hybrid CO 3 and TA7 (MS + 2iP (0.5 mg L-1) + Kinetin (1.5 mg L-1) + NAA (1.0 mg L-1)) for TLCV 2 genotypes. The callus induced was sub cultured at monthly intervals in the same medium for proliferation and later transferred to regeneration medium. A good number of shoots got regenerated only from anther calli of TNAU hybrid CO 3 that was sub cultured in MS medium fortified with Zeatin (0.5 mg L-1). The clumps of shoots induced were separated and inoculated in MS medium supplemented with GA3 (0.5 mg L-1) for shoot elongation. After 4-6 weeks, the elongated shoots were transferred to half strength MS medium enhanced with IBA (1 mg L-1). Profuse rooting from the base of the shoot was noticed in 4-5 weeks. The stomatal count with leaves from the diploid plants and in vitro plants observed were 3-4 and 1 respectively indicating the haploidy nature of in vitro plants.

Optimization of Pre-treatment Incubation Period on Callus Induction Response in Anthers of Selected Rice Genotypes

Background: The microspores of five tropical japonica and five indica rice genotypes were subjected to androgenic studies. The effect of growth regulators on callus induction were studied to improve the anther culture efficiency. Methods: The cold pre-treatment of panicles at 10°C were done at different days of intervals viz., 5, 8, 10 and 12 days. The microspores at uninucleate stage were selected and dusted after pre-treatment. The anthers were cultured in N6 basal media supplemented with casein hydrolysate (250 mg/L), proline (250 mg/L), silver nitrate (100 mg/L), maltose (50 g/L) and growth regulators. Result: The 8 days of cold pre-treatment initiated calli in most of the ten genotypes. The days taken for callus induction varied with genotype from 32-55 days. The callus induction frequency ranged from 1.41 to 5.12%. The responsive genotypes (Azucena, Palawan, Nira) on callus induction were studied for their regeneration potential. Background: The microspores of five tropical japonica and five indica rice genotypes were subjected to androgenic studies. The effect of growth regulators on callus induction were studied to improve the anther culture efficiency.

Establishment of a suitable regeneration protocol for rapid propagation of Piper nigrum L. through in vitro culture

An experiment was performed to establish a suitable regeneration protocol for the rapid propagation of Piper nigrum L. using nodal segments and leaf tissues as explants through direct and indirect organogenesis. After surface sterilization, several types of explant were inoculated onto gelled MS medium containing various concentrations and combinations of growth regulators for callus and direct shoot induction. The highest callus induction frequency was 92% and 84% in the case of leaf tissues and nodal segments, respectively, in gelled MS medium containing 2.0 mg/l 2,4-D. Multiple shoots (6.43±0.35 shoots per unit callus) were obtained when the calli from both explant types were cultured on MS medium containing 1.0 mg/l BAP. Nodal segments showed the best result (85%) in terms of direct shoot induction in MS medium supplemented with 1.5 mg/l BAP, where the highest number of shoots per explant was 5.03±0.69. The directly induced shoots were multiplied and elongated on MS medium containing 1.0 mg/l BAP and 0.5 mg/l IBA, the number and length of regenerated shoots per culture being 6.07±0.39 and 5.84±0.65 cm, respectively. The best response to root induction (86.67%) was observed when shoots were transferred to ½ strength of gelled MS medium supplemented with 1.5 mg/l IBA within 16-24 days, with 13.60±1.76 roots per shoot unit. The well-rooted shoots were successfully acclimated in a mixture of soil, sand, and compost (1:1:1) with a survival rate of 88%.
 Jahangirnagar University J. Biol. Sci. 11(1 & 2): 31-40, 2022 (June & December)

In vitro regeneration through organogenesis in Costus igneus–an important herbal insulin plant

Costus igneus is an important high-valued herbal insulin plant. An efficient protocol for in vitro plant regeneration through organogenesis from leaf and nodal explants was standardized. Explants were incubated in Murashige and Skoog (MS) medium amended with diverse levels of plant growth regulators (PGRs). Optimum frequency (68.25%) of adventitious shoot regeneration was obtained from nodal segments cultured onto MS medium containing 2.0 mg/l of 6-benzylaminopurine (BAP) with an average number of 5.0 shoots per explant. Further, MS medium containing 1.5 mg/l 2, 4- dichlorophenoxyacetic acid (2, 4-D) and 1.0 mg/l Thidiazuron (TDZ) produced a higher frequency of callus induction (55.22%) from leaf explants. Optimum shoot regeneration response (66.0%) through indirect organogenesis was observed from leaf-derived callus on MS medium fortified with BAP (2.0 mg/l) and kinetin (0.5 mg/l) with 6 shoots per callus with mean shoot length of 8.0 cm. The shootlets obtained from node and leaf cultures were rooted with a frequency of (63.52%) on MS medium with 1.0 mg/l indole-3-acetic acid (IAA). Plantlets obtained through organogenesis were successfully acclimatized in the greenhouse and field conditions. There were no significant differences among frequency of somatic plants regenerated through either direct or indirect organogenesis. The present study reports on successful plant regeneration through direct and indirect organogenesis in C. igneus. The regeneration protocols discussed here can be used effectively for large-scale multiplication, germplasm survival, pharmacological and genetic manipulation research.

Callus Induction and Plant Regeneration from Carum copticum and Assessment of Antioxidant Responses and Phytochemical Profiling by In Vitro Salinity Stress

Higher production of secondary metabolites is one of the adaptive responses to alleviate the impact of environmental injuries. In the present investigation, the production of these metabolites with medicinal importance induced by salinity in Carum copticum was investigated. To develop a better way for the production of medicinal substances, callogenesis and plant regeneration were analyzed, and seeds, calli, and/or regenerated seedlings were exposed to different concentrations of NaCl under in vitro culture conditions. The maximum frequency of callus induction was obtained on a medium supplemented with 0.25 mg L−1 2, 4-dichlorophenoxyacetic (2,4-D) and 1 mg L−1 benzyl amino purine (BAP) from stem explants. Plant regeneration with multiple shoots was obtained from pieces of callus transferred to MS medium fortified with 0.25 mg L−1 2,4-D and 1.5 mg L−1 BAP. Four weeks after treatment, salinity induced a substantial increase in the accumulation of reducing sugars and proline as compatible osmolytes and the activity of antioxidant enzymes. Total phenolics and anthocyanin significantly increased in all samples with increasing NaCl concentrations; however, the regenerated seedlings showed a reduction in these compounds at severe NaCl concentration compared to the control. Moreover, NaCl enhanced thymol, γ-terpinene, sabinene, and myrcene in the seedlings and calli, as well as carvacrol, limonene, and α-terpinene in the regenerated seedlings. These results suggest that salinity has a marked impact on improving the content of antioxidant metabolites and essential oils in C. copticum, whose callus might be the most salt tolerant in all tested samples.

In vitro callogenesis essays to induce somatic embryogenesis of the Tunisian chickpea

The present study was the first report on the somatic embryogenesis of the Tunisian chickpea (Cicer arietinum L.) particularly supposed to be recalcitrant and difficult to manipulate in vitro. An efficient protocol has been developed for inducing indirect somatic embryogenesis derived from immature zygotic embryo axis, young leaflet and hypocotylar explants. Callogenesis was achieved on full-strength Murashige and Skoog's (1962) (MS) basal medium supplemented with two auxin/cytokinin combinations; 2,4-dichlorophenoxyacetic acid (2,4-D) and 6-furfurylamonopurine (KIN) or 1-naphthaleneacetic acid (NAA) and 6-benzylaminopurine (BAP) to establish the phytohormones requirement to promote the best induction of friable calli. For somatic embryos induction, embryogenic calli developed from zygotic embryo axes, leaflet and hypocotylar explants were cultured on MS full strength and MS/2, half strength, free of phytohormones media. This study found that all explants exhibited high frequency of callus induction. For immature zygotic embryo axes and young leaflet explants, media containing the combination of 2,4-D and KIN were best effective for inducing callogenesis and friable calli. The most important embryogenic callus percentages were obtained on the culture medium containing the highest concentrations of 2,4-D (2 mg L-1) and KIN (0.5 mg L-1). However, media containing the combination NAA and BAP were best effective for inducing embryogenic calli on hypocotylar explants. A maximal rate of embryogenic calli was reached on medium containing 3 mg L-1 NAA and 1 mg L-1 BAP. The highest number of somatic embryos was obtained with embryogenic calli derived from embryo axis explants and cultured on MS half strength free phytohormones medium. About 56% of somatic embryos converted successfully into fertile plantlets.

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.

IN VITRO PLANT REGENERATION IN LOCAL AUS RICE THROUGH MATURED SEED CULTURE

An investigation was carried out to decipher the effect of genotypes and concentrations of growth regulators for callus induction and subsequent plant regeneration from mature seeds of rice. Two local rice cultivars viz. Nayanmony and Kalomona and two growth regulators viz. 2, 4-D and NAA were used to evaluate their performance. The seeds were cultured under aseptic condition onto MS basal medium at varying concentrations and combinations of 2, 4-D (1.0, 1.5 and 2.0 mgL- 1) and NAA (0.0, 0.5 and 1.0 mgL-1) for callus induction. For plant regeneration MS medium fortified with 0.5 mgL- 1 NAA and 3.0 mgL-1 BAP was applied. Between the two cultivars Nayanmony showed better responses in callus induction frequency (91.38%), weight of callus (0.90 g)as well as diameter of callus (7.48 mm). Among the combinations 2, 4-D (1.5 mgL-1) alone produced the best callus (95.16%) and the highest weight of callus (1.88 g) but diameter of callus (8.27 mm) was the highest from 2, 4-D 1.0 mgL-1 +0.5 mgL-1 NAA. It was observed that both genotype and growth regulator significantly affected callus induction and plant regeneration. The medium supplemented with 2, 4-D 1.5 mgL-1 + NAA0.0 mgL-1 exhibited better performance in callus induction (99.33%) and weight of callus (1.92 g) in cultivar Nayanmony whereas Kalomona was better for diameter of callus (8.38 mm) at 2, 4-D 1.0 mgL-1 + NAA0.5 mgL-1. The cultivar Nayanmony performed better regarding plant regeneration (88.15%) compared to Kalomona (86.67%). Plantlets derived from the 2, 4-D 1.5 mgL-1+ NAA 0.0 mgL-1 showed the best results for survival. Therefore, it is suggested that application of MS medium supplemented with 2,4-D 1.5 mgL-1 and NAA 0.0 mgL-1 is advantageous to accomplish overall efficiency of callus induction, subsequent plant regeneration and survival from seeds of Nayanmony cultivars. Thus, selection of better responsive cultivar like Nayanmony and MS medium fortified with 1.5 to 2.0 mgL-1 2, 4-D would be beneficial in transformation attempts to improve the crop.