Plant Tissue Culture Research Articles

Impact of elicitors and precursor on quassinoids and canthin-6-ones production in adventitious root culture of Eurycoma harmandiana Pierre and improvement of their anti-inflammatory activity

The processes of elicitation and precursor are used to increase the production of secondary metabolites in plant tissue cultures. The roots of Eurycoma harmandiana Pierre, a herbal plant used in traditional Thai medicine, are highly sought after to treat fever and erectile dysfunction. Hence, alternative sources from in vitro plant tissue of interest are needed. This study investigated the growth kinetics of adventitious root culture of E. harmandiana and the effect of various elicitors, including yeast extract (YE), chitosan (CH), 2-hydroxypropyl-β-cyclodextrin (HPCD), and precursor tryptophan (T), to enhance the contents of quassinoids and canthin-6-ones in root biomass and culture media. Elicitors and precursor were added at the 10th week after subculturing, and the root biomass and culture media were harvested on days 7 and 14 after treatment. Canthin-6-ones accumulation on day 14 was up to 2-fold higher than that in the control when the root culture was treated with 200 µM T, and quassinoid accumulation was 3.6-fold higher when elicited with 2 mg/mL YE. 9-Hydroxy-canthin-6-one was the most abundant in the root biomass after addition of CH (200 ppm), while only eurycomanone and 9-hydroxy-canthin-6-one were found in the culture media. Additionally, on day 14, 9-methoxy-canthin-6-one was found in the media culture of the elicited root with HPCD, whereas it was not found after treatment with the other elicitors. Meanwhile, the morphology of adventitious roots changed after elicitation. The enhancement of quassinoids and canthin-6-ones compounds in the root biomass after elicitation led to stronger anti-inflammatory activity than in the non-treated roots owing to significantly inflammatory gene suppression. This study demonstrates the effective elicitation and precursor feeding of adventitious root cultures of E. harmandiana, which enhanced bioactive components and improved anti-inflammatory effects.

Nano-Management Approaches for Salt Tolerance in Plants under Field and In Vitro Conditions

Soil salinity is a serious global problem that threatens a high percentage of the global soils. Salinity stress can create ionic, oxidative, and osmotic stress, along with hormonal imbalances, in stressful plants. This kind of stress was investigated on agricultural productivity at different levels, starting in vitro (plant tissue culture), through hydroponics, pots, and field conditions. Several approaches were studied for managing salinity stress, including using traditional materials (e.g., gypsum, sulfur), organic amendments (e.g., compost, biochar, chitosan), and applied manufactured or engineered nanomaterials (NMs). Application of nanomaterials for ameliorating salinity stress has gained great attention due to their high efficiency, eco-friendliness, and non-toxicity, especially biological nanomaterials. The application of NMs did not only support growing stressful plants under salinity stress but also increased the yield of crops, provided an economically feasible nutrient management approach, and was environmentally robust for sustainable crop productivity. Nano-management of salinity may involve applying traditional nano-amendments, biological nanomaterials, nano-enabled nutrients, nano-organic amendments, derived smart nanostructures, and nano-tolerant plant cultivars. Producing different plant cultivars that are tolerant to salinity can be achieved using conventional breeding and plantomics technologies. In addition to the large-scale use of nanomaterials, there is an urgent need to address and treat nanotoxicity. This study aims to contribute to this growing area of research by exploring different approaches for nano-management of current practices under salinity stress under field and in vitro conditions. This study also raises many questions regarding the expected interaction between the toxic effects of salinity and NMs under such conditions. This includes whether this interaction acts positively or negatively on the cultivated plants and soil biological activity, or what regulatory ecotoxicity tests and protocols should be used in research.

Effect of graphene oxide nanoparticles on in vitro growth of Fragaria x ananassa (Cameron Highlands white Strawberry) and evaluation of genetic stability using DAMD and ISSR markers

Graphene oxide (GO) is a novel nanomaterial with distinct physical properties and significant biological applications. The use of GO in plant tissue culture offers several new properties and potential applications. This research is vital due to the growing need for innovative techniques to promote plant growth, improve plant productivity and mitigate challenges posed by environmental stressors. This study focused on the rare Cameron Highlands white strawberry plants (Fragaria x ananassa) and addressed issues such as callus production during direct shoot induction and hyperhydricity. The research aimed to investigate the effects of GO on the regeneration process and genetic stability of white strawberry plants and to use molecular markers to ensure that plants propagated in vitro are true to type. For this purpose, shoot tip explants were used and different concentrations of GO (0, 2.5, 5.0, 7.5, 10 mg/L) were added to the Murashige and Skoog (MS) medium for six weeks. The results showed that the optimum concentration for promoting the development of white strawberry seedlings was 7.5 mg/L of GO. The study also revealed that the addition of 7.5 mg/L GO in combination with 8 μM TDZ to the MS medium facilitated the induction of multiple shoots. Moreover, the clonal fidelity of the in vitro plants treated with GO showed a genetic similarity of over 97%. These results confirm that lower GO concentrations improve plant development and stability. Consequently, this nanomaterial has a positive effect on the growth of strawberry plants and is therefore well suited for strawberry tissue culture.

Establishment of An in Vitro Mass Propagation System for Dendrocalamus asper.

Dendrocalamus asper is a species of bamboo that has high commercial value and is the bamboo of choice for large scale agro-forestry plantations in the tropical regions of the world. Micropropagation using tissue culture is essential to generate uniform clones that are amenable to establishment in industrial agro-forestry projects for bamboo biomass, habitat restoration or in carbon sequestration. This paper reports on the establishment of D. asper invitro using commercially available seeds. The seeds were surface sterilized using three different chemical agents which were Sodium Hypochlorite (20%), Mercuric Chloride (0.1%) and Ethanol (70%) followed by shoot initiation on Murashige and Skoog (MS) medium supplemented with 6-Benzylaminopurine (BAP) with a concentration ranging from 1.0 - 5.0 mg/L. Propagules were multiplied on MS media supplemented with different concentration of IBA Indole-3-Butyric Acid (IBA) and Naphthalene Acetic Acid (NAA), and finally rooted and hardened in peat moss. The findings of our study indicate that the sterilization protocol eliminated all the plant pathogens, resulting in an axenic culture. Full strength MS medium supplemented with 5 mg/L BAP yielded the highest number of shoots (11.46 per explant) after four weeks of inoculation. The highest multiplication rate (3.95 shoots per explant) was obtained on MS medium supplemented with 3 mg/L BAP. The time required from initiation to hardening was 70 to 90 days, following which the plantlets were ready for field trials. The findings of this study will facilitate the establishment of plant tissue culture programmes dedicated to the production of D. asper locally, thus eliminating the need for imports and the possible entry of plant pathogens that can be detrimental to the local agro-forestry industry.

Increase in ENHANCER OF SHOOT REGENERATION2 expression by treatment with strigolactone-related inhibitors and kinetin during adventitious shoot formation in ipecac.

Increase of ENHANCER OF SHOOT REGENERATION 2 expression was consistent to treatment with kinetin, TIS108, and KK094 in adventitious shoot formation of ipecac. Unlike many plant species, ipecac (Carapichea ipecacuanha (Brot.) L. Andersson) can form adventitious shoots in tissue culture without cytokinin (CK) treatment. Strigolactone (SL) biosynthesis and signaling inhibitors stimulate adventitious shoot formation in ipecac, suggesting their potential use as novel growth regulators in plant tissue culture, but the molecular mechanism of their action is unclear. In this study, we compared the effects of SL-related inhibitors (TIS108 and KK094) and CKs (2iP, tZ, and kinetin) on adventitious shoot formation in ipecac. Exogenously applied SL-related inhibitors and CKs stimulated adventitious shoot formation. Combinations of SL-related inhibitors and kinetin also promoted adventitious shoot formation, but without additive effects. We also analyzed the expression of CK biosynthesis genes in ipecac. TIS108 increased the expression of the ipecac homolog of ISOPENTENYL TRANSFERASE 3 (CiIPT3) but decreased that of LONELY GUY 7 homolog (CiLOG7), presumably resulting in no change in 2iP-type CK levels. KK094 and kinetin increased CiLOG7 expression, elevating 2iP-type CK levels. Among pluripotency- and meristem-related genes, TIS108, KK094, and kinetin consistently increased the expression of ENHANCER OF SHOOT REGENERATION 2 homolog (CiESR2), which has a key role in shoot regeneration, in the internodal segment region that formed adventitious shoots. We propose that CiESR2 might be a key stimulator of adventitious shoot formation in ipecac.

Metabolite bioprospection and expression analysis of patchoulol synthase gene in different in vitro systems of Pogostemon cablin: An important medicinal aromatic plant

Pogostemon cablin (patchouli) is one of the most prized medicinal herbs cultivated worldwide. However, with changes in the bioclimatic patterns and nematode infections in its root, the availability of quality planting material for essential oil extraction has become a challenging task in recent times. In the present research, a synergistic effort based on the identification of the optimal gene expression level of the patchoulol synthase (PS) along with fresh weight (FW), an effective strategy was formulated for the bioprospection of specialized metabolites of patchouli via plant tissue culture based in vitro approaches. The comparative expression levels of the PS gene in various tissues grown in semisolid as well as suspension cultures provided vital insights into its role in the biosynthesis of patchoulol and its associated compounds. Comparatively, cell suspension derived biomass exhibited better efficacy in terms of antioxidant activity (IC50 = 27.88 μg/ml), sun protection factor (SPF = 4.49–20.20), accumulation of both volatile and non-volatile metabolites profiled through ultra performance liquid chromatography and gas chromatography-mass spectrometry (GC-MS) along with up regulated PS gene activity (5.01 FC). Interestingly, semisolid culture media derived callus extract also exhibited potent activities establishing the novelty of the approach. The findings illustrate the utilization of aromatic cytokinin – meta-Topolin as a potent elicitor in synergy with conventional elicitors like methyl jasmonate and synthetic growth regulators such as thidiazuron. The current approach also provides vital, reproducible, sustainable and cost-effective method for the bioprospection of prized metabolites in the callus and cell suspension cultures with the goal of up-scaling to pilot scale via coordinated use of next generation bioreactor approaches.

Uma nova abordagem para micropropagação de begônias por explantes de botões florais

Abstract Begonias grown in greenhouses are susceptible to devastating disease caused by pathogenic bacteria and fungi, decreasing the quality of propagated material. Plant tissue culture provides an alternative for rapid propagation of healthy Begonia material. The present study was undertaken to develop the protocol of micropropagation of three Begonia species and one hybrid from inflorescence explants. Male flower buds with part of pedicel restricted to 1 mm have been cultured in vitro on 6 variants of modified N6 media. Adventitious shoot organogenesis has been shown to occur from both pedicel and receptacle tissues under the action of any type of cytokinin applied, whereas BA and 2-iP triggered mainly the direct organogenesis, while TDZ proceeding morphogenic events through the stage of callus formation. For the culture establishment in vitro the most effective was the medium, supplemented with 1.5 µM 2-iP + 0.54 µM NAA with the addition of 40 mg L-1 adenine sulfate, contributed to the highest shoot regeneration from floral explants of all begonias studied. Histological analysis of adventitious buds pathways approved that their induction occurs under the treatment directly from the subepidermal cells. Morphological analysis performed after plantlets adaptation to the greenhouse conditions showed no morphological or bloom variations in the progeny, derived from the begonias inflorescence. The suggested technique considered as a practical step toward obtaining the uniform planting material for the propagation of economically valuable Begonia plants.

Effective Contamination Agent For Tissue Culture Applications of Bacopa monnieri

Biocides and plant protection products have been used in plant tissue culture sterilization procedures since they have broad spectrum, are inexpensive and resistant to autoclave process. This study was conducted to determine the effects of contamination agents on tissue culture applications. MS medium was supplied with 0.5mg/l 6-benzylaminopurine and 0.1 mg/L indole-3-butyric acid. PPM and Contaminacide and their three dosages (0, 3 and 6 ml/L) were used for maintaining tissue culture aseptic conditions. Contamination rate and plant growth parameters were subjected. after lids remained open for three days in Bacopa monnieri micropropagation conditions. Although preservation-free and 3mlL-1 PPM-added media were contaminated, contamination did not occur in all dosages of Contaminacide and 6 ml/L PPM. In conclusion 3ml/L Contaminacide added media were superior in terms of contamination rate and plant height, fresh and dry weight (LSD;p

Improving Steviol Glycosides Production in Stevia Rebaudian Bertoni by Plant Tissue Culture Technology

Plant tissue culture techniques have made tremendous progress in the production of various active compounds of interest, including Steviol Glycosides in Stevia. Stevia rebaudiana is a perennial herbaceous plant known for its sweet-tasting leaves that contain active substances used as natural low-calorie sweeteners called Steviol glycosides. Because of its high sweetness and low-calorie nature, it is an alternative to sugar. However, conventional cultivation of Stevia plants for the production of Steviol Glycosides faces challenges such as long growth cycles, changes in chemical composition, and environmental determinants. Therefore, plant tissue culture is an alternative for the production of large quantities of Steviol Glycosides by altering various factors such as nutrient media, growth regulators, and response. Photovoltaic and tissue culture methods have been successfully used for plant propagation as well as the production of high-quality glycosides. Through techniques such as callus culture, organogenesis, and somatic embryo culture, large quantities of cotyledons can be rapidly established from small plant parts in a relatively short time. With further advances in tissue culture techniques and an understanding of the physiology and genetics of stevia, the potential for large-scale production of glycosides using Tissue culture methods is a promising technique for the commercial use of this natural sweetener.

Respon Pertumbuhan Tanaman Porang (Amorphophallus muelleri Blume.) Dengan Penambahan NAA (Naphthalene Acetic Acid) dan BAP (6-Benzyl Amino Purine) Pada Perbanyakan Secara In Vitro

Porang is one of the export crops that are much hunted in several countries. The increasing number of needs has resulted in limited porang seeds so that there is a need for propagation of porang plants with good quality, one of which is by propagation through tissue culture or in vitro. In order to propagate porang plants in vitro, this research tries to discover the ideal concentration of NAA and BAP. From August to December 2022, this study was conducted in the Plant Tissue Culture Laboratory of Jember State Polytechnic. The experiment employed a parametric statistical design with 2 treatments: 0.5 mg/L NAA + 1.5 mg/L BAP (P1) and 0.5 mg/L + 2.5 mg/L BAP (P2), with each treatment being repeated 10 times. According to the findings, the parameters of callus emergence time (46 HST) and callus diameter (1.51 cm) were best achieved by administering NAA and BAP at a dosage of 0.5 mg/L NAA + 2.5 mg/L BAP. According to the study aims, the optimal concentration for explant growth and development is believed to be 0.5 mg/l NAA + 2.5 mg/l BAP.

Light-emitting diode-induced ellagic acid production in Drosera tokaiensis inhibits advanced glycation end-product formation

Drosera tokaiensis (DT) is an insectivorous plant that effectively inhibits advanced glycation end products (AGEs) generated from non-enzymatic reactions between reducing sugars and protein amino groups using phenolic compounds, including ellagic acid, myricitrin, and quercitrin. The accumulation of AGEs increases during the pathogenesis of non-communicable diseases, such as diabetes and nephropathy. Therefore, the extracts of Drosera species could have preventive effects against such diseases. The development of light-emitting diodes (LEDs) and their use in plant cultivation and tissue culture can enhance secondary metabolite production by controlling the wavelength of light in which plants are cultivated. We evaluated how LEDs affected the production of AGE-inhibitory compounds in extracts isolated from DT tissue cultures. DT was cultured for two months in different light wavelengths under unified photosynthetic photon flex density and the resulting extracts were used to examine changes in phenolic compound content and the inhibition of AGE formation. Cultivation in monochromatic light from LEDs had no significant effect on plant weight compared to conventional fluorescent cultivation, except for red LED light. Ellagic acid content increased regardless of LED coloration and the production and accumulation of myricitrin and quercitrin were inhibited. The extracts isolated from plantlets cultured under LEDs showed the same level of AGE inhibition as those cultured under fluorescent light. Therefore, for anti-glycation activity, DT can be grown stably and at a low cost using LED lights when compared to conventional culture methods.

Identification of Best Exposure Timings of Sodium Hypochlorite in Surface Sterilization of Different Explants in invitro Propagation of Annona muricata

The most crucial and delicate stage in plant tissue culture is the surface sterilization of the explant. An inappropriate concentration of sterilant may have deadly effects on explants by altering cell division and other metabolic processes that limit the growth and development of explants. Considering the explants observations and sterilization treatments of Sodium hypochlorite (4%), T6 (7 min), T7 (8 min) and T8 (9 min) are selected for green nodal explants as highest response, least days for initiation of response, good health with less contamination and moderate survival of explants. Whereas, T5 (6 min) T6 (7 min) and T7 (8 min) are selected for leaf with lower contamination with good health and survivability. Internode was not selected due to no response with higher contamination and less survivability. Brown nodal explants responded very less and less survivability with higher contamination as compared to other explants

Increase of the morpho-physiological responses of sugarcane plants in vitro (Saccharum spp. hybrids) using sodium silicate

Sugarcane (Saccharum spp. hybrids) is used as raw material in the agri-food and bioenergy industries. Plant tissue culture (PTC) allows production of many propagules in a relatively short time and limited space. Silicon (Si) is considered a non-essential element, and studies have demonstrated a positive impact on the development of plants obtained through PTC. This study aimed to evaluate the bio-stimulant effect of sodium silicate (Na2SiO3) on the in vitro propagation of sugarcane (CP 72-2086 and Mex 69-290). Plant tips were established for each variety. The shoots obtained were then cultivated in an MS medium supplemented with different concentrations of Na2SiO3 (0, 2.5, 5.0, and 10.0 mg L−1). After 30 d, morphometric analysis, quantification of photosynthetic pigments, and stomatal index measurements were performed. The results showed that 10.0 mg L−1 of Na2SiO3 had a positive effect on the morphology of both varieties. The CP 72-2086 variety had a 0.5 number of shoots, 6.36 leaves and 5.8 roots more compared to the control. In contrast, the Mex 69-290 variety had more sizable proportion of shoot length (1.42 times), root length (2.20 times), and dry weight (1.33 times) compared to the control. However, 2.5 and 5.0 mg L−1 of Na2SiO3 decreased the responses of variables evaluated in both varieties compared to the highest concentration. While the addition of Na2SiO3 in both varieties had a bio-stimulant effect on the content of photosynthetic pigments. Results obtained suggest that Na2SiO3 can be used as a bio-stimulant agent during the commercial micropropagation of sugarcane hybrids.

The effect of MgNPs synthesized by using green method on the produc-tion and proliferation of Capisum annuum callus culture In Vitro

Nanotechnology, consider as one of the newest important methods towards improve the production of callus using plant tissue culture. The objective of the current work was to create a unique callus induction methodology. and proliferation from Capisum annum in vitro. Therefor different concentrations of Mg nanoparticles were used which synthesized using green method. For callus inducement, young foliage, and stem from the sterile germinated seedlings using Sodium hypochlorite (NaOCL) 6% for 10 min were used as explants. They were cultivated in MS media supported by several combinations of kinetin and 2, 4- Dichlorophenoxyacetic. The maximum percentage responsive reached 100% from leaf explants at 2, 4-D 1.5 mg/L and Kin 1.0 mg/L. While 2, 4-D 1.5 mg/L and Kin 0.5 mg/L achieved the highest fresh weight for callus proliferation. The best MgNPs concentration for callus inducement and proliferation of capsicum leaves and stem explants was MS media supported with 1.0 and 0.3 ml/L respectively. While the highest proliferation mean value for callus was recorded at the concentration of 6.0 ml/L MgNPs in MS medium reached 6.1 gm. AFM photographs prove that MgNPs were in nanometer size and had the spheres and the rods shape. The average grain size of the MgNPs was 23.8 to 38.24 nm.

Increasing Amounts of Secondary Metabolites and Medicinal Compounds in Callus Culture of Moringa Oleifera (Lam.) Using Abiotic Elicitors

Moringa oleifera Lam. tree has often been considered a medicinal and economical plant for its nutritional and medicinal properties. This study is intended to use plant tissue culture and elicitation technologies to increase secondary metabolites in the callus cultures of the M. oleifera. Young nodal segments were cultured on Murashige and Skoog (MS) medium supplemented with different concentrations of plant growth regulators (PGRs). A combination of 1.5 mg/l NAA plus 0.5 mg/l TDZ resulted in a high callusing rate (70%) with the largest size. Various concentrations of Salicylic acid (SA) and Methyl jasmonate (MeJa) were individually added to the growth medium to evaluate their impact on the biomass of callus cultures, DPPH antioxidant activity, and accumulation of some flavonoids. Results indicated that all elicitors doses reduced weights and growth index of callus. While DPPH assay recorded high significant values at high concentrations of SA and MeJa compared to other treatments. HPLC analysis revealed that every concentration of SA and MeJa used led to rising flavonoids content, including Baicalin, Rutin, Hesperidin, Rosmarinic acid, Quercitrin, Quercetin, and Kaempferol. It should be noted that 20 mg/l MeJa gave the highest flavonoid content compared to the control and other treatments. In conclusion, the in vitro elicitation by SA or MeJa can improve beneficial biological substances like flavonoids and other antioxidant compounds in Moringa callus cultures.

Multifaceted Role of Nanomaterials in Modulating In Vitro Seed Germination, Plant Morphogenesis, Metabolism and Genetic Engineering.

The agricultural practices of breeding, farm management and cultivation have improved production, to a great extent, in order to meet the food demands of a growing population. However, the newer challenges of climate change, global warming, and nutritional quality improvement will have to be addressed under a new scenario. Plant biotechnology has emerged as a reliable tool for enhancing crop yields by protecting plants against insect pests and metabolic engineering through the addition of new genes and, to some extent, nutritional quality improvement. Plant tissue culture techniques have provided ways for the accelerated clonal multiplication of selected varieties with the enhanced production of value-added plant products to increase modern agriculture. The in vitro propagation method has appeared as a pre-eminent approach for the escalated production of healthy plants in relatively shorter durations, also circumventing seasonal effects. However, there are various kinds of factors that directly or indirectly affect the efficiency of in vitro regeneration like the concentration and combination of growth regulators, variety/genotype of the mother plant, explant type, age of seedlings and other nutritional factors, and elicitors. Nanotechnology as one of the latest and most advanced approaches in the material sciences, and can be considered to be very promising for the improvement of crop production. Nanomaterials have various kinds of properties because of their small size, such as an enhanced contact surface area, increased reactivity, stability, chemical composition, etc., which can be employed in plant sciences to alter the potential and performance of plants to improve tissue culture practices. Implementing nanomaterials with in vitro production procedures has been demonstrated to increase the shoot multiplication potential, stress adaptation and yield of plant-based products. However, nanotoxicity and biosafety issues are limitations, but there is evidence that implies the promotion and further exploration of nanoparticles in agriculture production. The incorporation of properly designed nanoparticles with tissue culture programs in a controlled manner can be assumed as a new pathway for sustainable agriculture development. The present review enlists different studies in which treatment with various nanoparticles influenced the growth and biochemical responses of seed germination, as well as the in vitro morphogenesis of many crop species. In addition, many studies suggest that nanoparticles can be useful as elicitors for elevating levels of important secondary metabolites in in vitro cultures. Recent advancements in this field also depict the suitability of nanoparticles as a promising carrier for gene transfer, which show better efficiency than traditional Agrobacterium-mediated delivery. This review comprehensively highlights different in vitro studies that will aid in identifying research gaps and provide future directions for unexplored areas of research in important crop species.

Synergistic effects of Vermi-Co extracts mixture with cytokinins on shoot regeneration of Lindernia minima (Benth.) Mukerjee—An endangered medicinal plant

The present investigation illustrates the synergistic growth effect of Vermi-Co (Vermicast and Coconut-water) extracts mixture with cytokinins on in vitro shoot regeneration of Lindernia minima. Due to less distribution threat status, this plant species has been considered as an endangered plant by IUCN (International Union for Conservation of Nature). Hence, it is necessary to conserve and to develop an efficient plant regeneration protocol for the rapid multiplication of L. minima. The nodal explants were inoculated on (¼) quarter-strength MS (Murashige and Skoog) medium supplemented with different concentrations (0.5 – 2.5 mg L-1) of BA (6-Benzyladenine), KIN (Kinetin) and TDZ (Thidiazuron) combined with various percentages of (5%, 15%, and 25%) Vermi-Co extracts mixture. Among the concentrations tested, 1.5 mg L-1 TDZ with 15% of Vermi-Co extracts mixture produced the maximum percentage of response with the highest (19.5 ± 0.20 shoots/explant) number of shoots and (10.2 ± 0.14 cm) shoot length. Then, the well-elongated shoots were transferred to rooting medium (quarter-strength MS) supplemented with 0.5 – 3.0 mg L-1 of IBA (Indole -3- butyric acid) and IAA (Indole -3- acetic acid). The highest percentage of response (98%) and maximum number of roots were observed in 1.5 mg L-1 IBA with 11.5 ± 0.29 roots per shoot and 6.6 ± 0.28 cm root length. Finally, well-developed plants were successfully transferred to the natural field condition with a survival rate of 97%. The present research revealed that, the combined growth stimulating effect of Vermi-Co extracts mixture combined with cytokinins increased the number of shoots and shoot length on shoot regeneration of L. minima from nodal explants. Our findings will be very useful for regeneration of Lindernia minima through plant tissue culture.

High-throughput MALDI-MSI metabolite analysis of plant tissue microarrays.

A novel metabolomics analysis technique, termed matrix-assisted laser desorption/ionization mass spectrometry imaging-based plant tissue microarray (MALDI-MSI-PTMA), was successfully developed for high-throughput metabolite detection and imaging from plant tissues. This technique completely overcomes the disadvantage that metabolites cannot be accessible on anintact plant tissue due to the limitations of the special structures of plant cells (e.g. epicuticular wax, cuticle and cell wall) through homogenization of plant tissues, preparation of PTMA moulds and matrix spraying of PTMA sections. Our study shows several properties of MALDI-MSI-PTMA, including no need of sample separation and enrichment, high-throughput metabolite detection and imaging (>1000 samples per day), high-stability mass spectrometry data acquisition and imaging reconstruction and high reproducibility of data. This novel technique was successfully used to quickly evaluate the effects of two plant growth regulator treatments (i.e. 6-benzylaminopurine and N-phenyl-N'-1,2,3-thiadiazol-5-ylurea) on endogenous metabolite expression in plant tissue culture specimens of Dracocephalum rupestre Hance (D.rupestre). Intra-day and inter-day evaluations indicated that the metabolite data detected on PTMA sections had good reproducibility and stability. A total of 312 metabolite ion signals inleaves tissues of D. rupestre were detected, of which 228 metabolite ion signals were identified, they were composed of 122 primary metabolites, 90 secondary metabolites and 16 identified metabolites of unknown classification. The results demonstrated the advantages of MALDI-MSI-PTMA technique for enhancing the overall detection ability of metabolites in plant tissues, indicating that MALDI-MSI-PTMA has the potential to become a powerful routine practice for high-throughput metabolite study in plant science.

Improving the Production of Secondary Metabolites via the Application of Biogenic Zinc Oxide Nanoparticles in the Calli of Delonix elata: A Potential Medicinal Plant.

The implementation of nanotechnology in the field of plant tissue culture has demonstrated an interesting impact on in vitro plant growth and development. Furthermore, the plant tissue culture accompanying nanoparticles has been showed to be a reliable alternative for the biosynthesis of secondary metabolites. Herein, the effectiveness of zinc oxide nanoparticles (ZnONPs) on the growth of Delonix elata calli, as well as their phytochemical profiles, were investigated. Delonix elata seeds were collected and germinated, and then the plant species was determined based on the PCR product sequence of ITS1 and ITS4 primers. Afterward, the calli derived from Delonix elata seedlings were subjected to 0, 10, 20, 30, 40, and 50 mg/L of ZnONPs. The ZnONPs were biologically synthesized using the Ricinus communis aqueous leaf extract, which acts as a capping and reducing agent, and zinc nitrate solution. The nanostructures of the biogenic ZnONPs were confirmed using different techniques like UV-visible spectroscopy (UV), zeta potential measurement, Fourier transform infrared spectra (FTIR), X-ray diffraction (XRD), and scanning electron microscopy (SEM). Adding 30 mg/L of ZnONPs to the MS media (containing 2.5 µM 2,4-D and 1 µM BAP) resulted in the highest callus fresh weight (5.65 g) compared to the control and other ZnONP treatments. Similarly, more phenolic accumulation (358.85 µg/g DW) and flavonoid (112.88 µg/g DW) contents were achieved at 30 mg/L. Furthermore, the high-performance liquid chromatography (HPLC) analysis showed significant increments in gallic acid, quercetin, hesperidin, and rutin in all treated ZnONP calli compared to the control. On the other hand, the gas chromatography and mass spectroscopy (GC-MS) analysis of the calli extracts revealed that nine phytochemical compounds were common among all extracts. Moreover, the most predominant compound found in calli treated with 20, 30, 40, and 50 mg/L of ZnONPs was bis(2-ethylhexyl) phthalate, with percentage areas of 27.33, 38.68, 22.66, and 17.98%, respectively. The predominant compounds in the control and in calli treated with 10 mg/L of ZnONPs were octadecanoic acid, 2-propenyl ester and heptanoic acid. In conclusion, in this study, green ZnONPs exerted beneficial effects on Delonix elata calli and improved their production of bioactive compounds, especially at a dose of 30 mg/L.

In Vitro Culture Technology and Advanced Biotechnology Tools Toward Improvement in Gladiolus (Gladiolus species): Present Scenario and Future Prospects.

In the world's flower trade, gladiolus (Gladiolus spp.) is ranked first among bulbous flowers and eighth among cut flowers, with more than 30,000 different cultivars being grown. Mass multiplication and commercialization are restricted by the traditional propagation methods. However, the large-scale proliferation and improvement of the gladiolus have been accomplished with the aid of plant tissue culture and other biotechnological techniques. The current review includes a thorough examination of the growth and development parameters required for successful in vitro gladiolus development as well as cormel formation. Moreover, focus is being given to various techniques and methods such as in vitro cytogenetic stability and modification of chromosome number, in vitro mutagenesis and selection of pest resistance, in vitro identification and selection to develop virus-free germplasm, cryopreservation, synthetic seed technology, identifying virus diseases by RT-PCR, somaclonal variation, and protoplast and somatic hybridization. Molecular markers and their applications for genetic diversity analysis, relationships between different genotypes, and clonal stability analysis in Gladiolus species have been conducted by several research groups worldwide and are also being discussed.The article also covers efforts to enhance the functionality of plant phenotypes through genetic transformation. Future prospects for further improvement of ornamental gladiolus are also explored.Overall, the current review provides insight into the applications of basic and advanced biotechnological tools for gladiolus improvement.