The poor rooting of in vitro-raised shootlets is a major bottleneck and severely affects the survival rate of plantlets in field conditions. Hedyotis biflora is used as a model plant to study the impact of Silicon nanoparticles (SiNPs) on in vitro adventitious root formation and their structural developments in this study. The in vitro proliferated shootlets cultured on ½ strength Murashige and Skoog (MS) medium containing 2.0 mg L−1 indole-3 butyric acid (IBA) and 3.0 mg L−1 SiNPs promoted the morpho-anatomical improvements in roots. Morphologically, the roots of SiNPs treatment were significantly superior, thick, and sturdy with increased biomass (1.298 g fresh weight and 0.614 g dry weight) and more in numbers (33.0 roots with 5.0 cm average length) from that of the fragile control roots (21.0 roots with 3.8 cm average length with reduced biomass of 0.735 g FW and 0.319 g DW). Light microscopic monitoring of roots revealed that SiNPs-derived roots were fully differentiated into periderm, exodermis, and secondary cortex and vascular tissues. Such structures were absent and only primary structures were detected in the control roots (derived from the medium without SiNPs). It was observed that the proportion of secondary xylem was significantly higher in the SiNPs-treated roots than the control roots, which correlated positively with the adaptation of roots toward field conditions. Furthermore, the acclimatization efficiency and survival of SiNPs-derived plantlets were higher (100

A novel and effective direct shoot regeneration technique from stomatal complexes was developed for a highly sought after medicinal plant Coleus forskohlii. Meta-Topolin was more efficient than 6-benzylaminopurine (BAP) in promoting direct organogenesis from leaf surface. Highest direct organogenesis was achieved from in vitro leaf explants on Murashige and Skoog (MS) medium augmented with meta-Topolin at the concentration of 2.0 mg/L. The regenerated leaves were fixed at weekly intervals and sectioned longitudinally to evaluate the anatomical developments. The results of light microscopic analysis suggested that the in vitro developed stomatal complexes showed new altered cell divisions, after a week of incubation, and the rapid periclinal divisions resulted in the formation of a meristematic dome. Distinct regions of meristematic activity were visible in the second week characterized by promeristems with small and densely stained parenchymatous cells. Promeristems lead to the development of a distinct tunica layer, which grew into shoot-like protuberances converting into adventitious shoots after 4 weeks of incubation. A clear three phases of shoot organogenesis viz. induction (7 days), initiation and organization (14 days), and shoot formation (28 days onwards) were identified. The shoots were proliferated (21.8 shoots/ single leaf explant) and elongated (5.0 cm length) using half-MS medium fortified with 0.5 mg/L mT and indole-3 acetic acid (IAA) at 0.1 mg/L. Notable rhizogenesis of the elongated shoots was achieved when cultured on half-MS medium containing indole-3 butyric acid (IBA) at the concentration of 1.5 mg/L. About 92.0% of the plantlets survived upon field transfer. Start Codon Target (SCoT) markers were employed to examine the regenerated plants' genetic uniformity with the mother plant, similar banding patterns confirming the genetic uniformity. Hence, the developed protocol of direct in vitro regeneration of C. forskohlii using mT can be used for the commercial production.

Thunbergia erecta (Benth.) T. Anderson (Acanthaceae) is an ornamental shrub. Viable seeds are not produced due to genetic incompatibility constraining propagation. An in vitro culture method is developed for the production of plants for the species. Surface-sterilized explants exhibited bud breaking on Murashige and Skoog (MS) medium + 2.0 mg L−1 6-benzylaminopurine (BAP). Multiple shoots (6.0 shoots/node, each measuring 3.6 cm average length) differentiated in 4 weeks of culture. Maximum (7.2 shoots/explants; each with 4.0 cm length) amplification occurred on MS medium + 1.0 mg L−1 BAP and 0.25 mg L−1 indole-3 acetic acid (IAA). Microscopic studies of foliage/leaves revealed abnormalities in stomata, trichomes, and rudimentary tissue systems on this medium. The incorporation of 2.0 mg L−1 Silicon nanoparticles (SiNPs) in the culture medium doubled the rate of shoot amplification and allowed the development of promoted leaves with well-differentiated tissue systems and functional stomatal complexes. About 98.0

The present study aimed to determine the foliar micro-morpho-anatomical features of in vitro cultured Gardenia jasminoides J. Ellis (Rubiaceae) to compare the effect of exogenous supplementation of growth regulators (cytokinins; 6-benzylaminopurine and Kinetin), in order to attenuate heterotrophic nutrition (in vitro) induced structural disorders in the proliferating shoots. Murashige and Skoog’s (MS) medium supplemented with 2.0 mg L−1 6-benzylaminopurine (BAP) in combination with 0.15 mg L−1 indole-3-acetic acid (IAA) was detected optimal for axillary bud induction. Nutrient medium containing 0.5 mg L−1 BAP and 0.25 mg L−1 IAA was found appropriate combination for proliferation of multiple shoots, and yielded 37.2 shoots (per explant) with 7.45 cm average length after 2nd subculture (8 weeks). Supplementation of NAA with cytokinins resulted in callus formation. The proliferation of shoots on kinetin (Kn) and IAA combination resulted in the formation of fragile shoots and the leaves with increased structural impairments like underdeveloped photosynthetic, vascular, and ground tissue systems, non-functional stomata, and reduced vein density. Comparatively, BAP and IAA treatment favoured healthy shoot proliferation and development of stable tissue systems with reduced structural abnormalities. Half strength MS medium augmented with 3.0 mg L−1 indole-3-butytric acid (IBA) was the optimal medium for root induction (32.0 roots per shoot with 3.5 cm in length). The regenerated plantlets showed 97

Cymbopogon citratus (DC.) Stapf (lemongrass or citronella) is a herb of therapeutic potential highly sought by various industries due to the presence of essential oil. In vitro propagation of commercial crops is highly desir-able; it is an alternative approach for the production of quality planting materials. But the plantlets encounter certain morpho-structural and physiological disorders under in vitro conditions resulting in suppressed growth and reduced survival. The present study aimed to analyze the effect of silicon nanoparticles (SiNPs) on the in vitro morphometric and structural developments, rooting, and acclimatization efficiency in C. citratus. Murashige and Skoog (MS) medium supplemented with 4.0 mg/L of 6-benzylaminopurine (BAP) was effective for bud breaking. The shoot proliferation was further accelerated with the addition of 0.25 mg/L NAA (alpha-naphthalene acetic acid) in subsequent subcultures (after 3rd subculture, 21.0 shoots with 4.9 cm length per explants per culture vessel). Such shoots were characterized by thin leaf blades, reduced leaf area, and shoot tip necrosis, which negatively affected the life of cultures, ex vitro rooting, and acclimatization efficiency. Microscopic evaluation of those leaves showed underdeveloped stomata, epidermis, and reduced photosynthetic and vascular tissues. The incorporation of optimal concentration of SiNPs (40 mg/L) with the optimized growth regulators (4.0 mg/L BAP + 0.25 mg/L NAA) significantly increased shoot proliferation (38.0), shoot length (6.4 cm), robust leaf, and biomass (5.2 g fresh weight; 1.9 g dry weight). Shoot tip necrosis was reversed by the addition of SiNPs, and the developed shoots were morphologically thick with well-developed tissue systems. These C. citratus shoots per-formed better during ex vitro rooting (100% rooting, 8.0 roots/shoot) when treated with indole-3-butyric acid (IBA) at the concentration of 200 mg/L and acclimatization.

Vitex negundo L. (Lamiaceae) is an aromatic species used to cure several illnesses worldwide. The plant is a panacea of herbal formulations with several of its metabolites exhibiting significant pharmacological properties. The present study is an attempt to find the impact of silicon nanoparticles (SiNPs) on inducing in vitro flowering in V. negundo . The floral morphology of in vitro and in vivo plants was evaluated using standard light microscopy techniques. SiNPs were tested at concentrations ranging from 50 mg L −1 to 300 mg L −1 by supplementation in the optimized shoot proliferation medium (Murashige and Skoog’s medium + 0.5 mg L −1 6- benzylaminopurine + 0.25 mg L −1 indole-3 acetic acid). The morphometric evaluation indicated 100 mg L −1 SiNP promoted flower bud induction within 2 weeks of the culture of shoots. The floral buds thus formed indicated the presence of distinct calyx, corolla, androecium, and gynoecium. A maximum of 28 flowers were found born on inflorescences by the 8th week of culture. The flowers remained pedicellate, pale purple, bisexual, and zygomorphic. The calyces were greenish-purple, pale-purple corolla, and didynamous-dithecous anthers. The ovary was globose, superior, and the style was linear with a short unequally bifid stigma. The stereomicroscopic analysis of floral morphology revealed that the flowers developed in vitro were almost similar to the mother plant except that the size was smaller. The pollens were viable, and the seed set occurred on pollination under in vitro conditions. To date, there is no published scientific report on the impact of SiNPs on in vitro flower induction in V. negundo and the comparative floral and pollen morphology. The present study confirms that the application of SiNPs could induce year-round in vitro flowering, and significantly improve in vitro rooting and acclimatization response of V. negundo plantlets.

Vitexnegundo L. (Lamiaceae) is an aromatic species used to cure several illnesses worldwide. The plant is a panacea of herbal formulations with several of its metabolites exhibiting significant pharmacological properties. The present study is an attempt to find the impact of silicon nanoparticles (SiNPs) on inducing invitro flowering in V.negundo. The floral morphology of invitro and invivo plants was evaluated using standard light microscopy techniques. SiNPs were tested at concentrations ranging from 50 mg L-1 to 300 mg L-1 by supplementation in the optimized shoot proliferation medium (Murashige and Skoog's medium + 0.5 mg L-1 6- benzylaminopurine + 0.25 mg L-1 indole-3 acetic acid). The morphometric evaluation indicated 100 mg L-1 SiNP promoted flower bud induction within 2 weeks of the culture of shoots. The floral buds thus formed indicated the presence of distinct calyx, corolla, androecium, and gynoecium. A maximum of 28 flowers were found born on inflorescences by the 8th week of culture. The flowers remained pedicellate, pale purple, bisexual, and zygomorphic. The calyces were greenish-purple, pale-purple corolla, and didynamous-dithecous anthers. The ovary was globose, superior, and the style was linear with a short unequally bifid stigma. The stereomicroscopic analysis of floral morphology revealed that the flowers developed invitro were almost similar to the mother plant except that the size was smaller. The pollens were viable, and the seed set occurred on pollination under invitro conditions. To date, there is no published scientific report on the impact of SiNPs on invitro flower induction in V.negundo and the comparative floral and pollen morphology. The present study confirms that the application of SiNPs could induce year-round invitro flowering, and significantly improve invitro rooting and acclimatization response of V.negundo plantlets.

Ceropegia juncea Roxb. is a medicinally important, rare, and endemic climber plant species. The present study optimized in vitro regeneration under the influence of different cytokinins and auxins on the morpho-struc-tural changes/disorders during adventitious shoot induction. The highest shoot number (4.2 shoots/node explant with 3.0 cm length) was noted on Murashige and Skoog (MS) basal medium augmented with 1.5 mg L-1 Meta-Topolin (mT) treatment. Nutrient medium fortified with 1.0 mg L-1 mT and 0.5 mg L-1 NAA (a-Naphthalene acetic acid) resulted in the proliferation of a maximum number of shoots (33.0 shoots with an average shoot length of 6.2 cm after 3rd sub-culture). Comparatively, 1.0 mg L-1 BAP (6-benzylaminopur-ine) and 0.5 mg L-1 NAA treatment resulted in the proliferation of 25.0 shoots with 4.0 cm length after 3rd subculture. Morphometric and microscopic analyses were performed after 3rd sub-culture under in vitro conditions to study the effects of growth regulators. The shoots developed on 1.0 mg L-1 BAP and 0.5 mg L-1 NAA possessed poorly developed anatomical features such as a thin cuticle, unorganized epidermal layer, reduced chlorophylls and tissue density, and low cell wall thickness of xylem vessels and phloem fibers. The leaves and stems produced on 1.0 mg L-1 mT and 0.5 mg L-1 NAA treatment had a larger area of tissue sys-tems, thick cuticle, organized epidermal layer with thick anticlinal walls, and increased cell wall thickenings of xylem and phloem tissues. Further, the shoots generated in treatments including mT and NAA rooted with the highest rooting percentage (98.3%) on half-strength MS medium supplemented with 2.0 mg L-1 NAA (6.2 roots with 4.0 cm length). Hence, the structural developments in the shoots derived from mT and NAA showed greater commitments to cellular differentiation along with the proliferation of shoots and survival (94.0%). Meta-Topolin in the medium moderately alleviated the in vitro-induced structural disorders and sup-ported rooting and hardening procedures for the continuous supply of quality plantlets.(c) 2023 SAAB. Published by Elsevier B.V. All rights reserved.

Gardenia jasminoides J. Ellis (Cape jasmine) is a horticultural plant. It is difficult to cultivate species due to pathogen sensitivity; hence, the demand for this species is gradually increasing. Thus, micropropagation would be a convenient tool to produce quality planting material, but the higher rate of mortality constitutes a constraint to large-scale propagation. Seismic stress is one of the abiotic stresses that promote growth, structural stability, and positively affect plantlet development. The objective of the present study was to evaluate the effect of moderate seismic stress under in vitro conditions on the proliferation of shoots, biochemical attributes (photosynthetic pigments, carbohydrates constituents, free amino acids, etc.), and foliar anatomical stability of plantlets G. jasminoides. Results showed the superiority of moderate seismic stress (75 rpm for 10 min) in the improved proliferation of shoots, development of functional stomata, elimination of anatomical abnormalities, improved photosynthetic pigments and rooting efficiency, and finally increased survival percentage of plantlets after transplantation into field conditions. These findings provided evidence for the positive role of the optimised seismic stress in regulating the morpho-anatomical and biochemical traits in G. jasminoides which is an indication of the development of diverse adaptive strategies by the micropropagated plantlets to abiotic stress.

Authors report the application of silver nanoparticles (AgNPs) in the plant tissue culture system for effective and reproducible in vitro propagation of Gaillardia pulchella cv. ‘Torch Yellow’ for the year-round production of quality planting materials for the first time. Murashige and Skoog (MS) medium augmented with 1.0 mg L−1 of 6-benzylaminopurine (BAP) resulted in the highest percentage of bud break response (98.0

The present study aimed to analyze the effect of silicon nanoparticles (SiNPs) on the in vitro proliferation of shoots, biomass production, foliar micro-structural features, and root-inducing ability in Vitex trifolia L. Bud breaking was achieved from axillary meristems on 2.5 mg L-1 of 6-benzylaminopurine (BAP) in Murashige and Skoog's (MS) medium. The shoots were proliferated (18.0 & PLUSMN; 0.39 shoots) on 0.75 mg L-1 BAP with 0.2 mg L-1 1-Naphthaleneacetic acid (NAA). Among the varied concentrations of Aerosil300 (hydrophilic silica) used, 6.0 mg L-1 SiNPs with optimal growth regulators into the medium had a positive impact on shoot proliferation (24.0 & PLUSMN; 0.30 shoots) and fresh biomass production. The light microscopic evaluation showed that the leaves developed without SiNPs possessed underdeveloped cuticle, trichomes, stomata, epidermal cells, and photosynthetic and vascular tissues. The SiNPs treatment resulted in well-differentiated tissue systems and favored an increase in width of the lamina, differentiation of palisade and spongy tissues, thickening of cell walls, increased density of trichomes, organized guard cells in stomata, and improved xylem and phloem tissues. The shoots were effectively rooted on half-strength MS medium containing 3.0 mg L-1 NAA, and the shoots derived from SiNPs treatment had the highest rooting efficiency (8.0 roots per shoot). The findings highlight that the use of SiNPs significantly promoted tissue systems and morphology of V. trifolia, which favored the survival of plantlets under in vivo conditions.& COPY; 2023 SAAB. Published by Elsevier B.V. All rights reserved.

Experimental studies were conducted on the effects of seismic stress on (a) in vitro shoot proliferation and (b) induction of stress tolerance in Dioscorea pentaphylla L. cultivated in vitro. The morphometric traits in plantlets developed under in vitro seismic stress set at 60 rpm for 8.0 min twice a day for 4 weeks promoted the number of shoots per explant (21.0 shoots), shoot length (6.8 cm average length), leaf area (2.3 cm × 2.0 cm), and biomass over control. Improvement was recorded in rooting response, acclimatization, and survival percent of the plants. Leaves of control plants exhibited stomatal abnormalities, the lowest defense trichomes, and raphides. These negative traits could be amended by seismic stress. We suggest counter-stress mitigating in vitro environment-caused stresses enabling mass and clonal propagation/production of climate change-resilient crop plants. This novel approach of experimental research offers new dimensions in the fields of plant production biology/biotechnology. In vitro moderate seismic stress improved shoot proliferation, biomass, rooting response, development of stress-tolerant microstructures, and acclimatization efficiencies in Dioscorea pentaphylla.

An improved in vitro propagation system has been developed for Santalum album L. using meta-Topolin for the first time. Direct organogenesis (100 %) was achieved by induction of axillary buds and a maximum of 8.2 shoots with 4.0 cm length was developed from nodal explants on Murashige and Skoog's (MS) medium containing 1.0 mg L-1 meta-Topolin (mT). The MS medium supplemented with 0.5 mg L-1 mT and 0.25 mg L-1 alpha-naphthalene acetic acid (NAA) exhibited maximum shoot proliferation potential (135 shoots with 7.9 cm length per explant) after the 3rd subculture. Further, the combination of mT and NAA resulted in the production of higher levels of free amino acids [33.6 mg g(-1) dry weight (DW)], soluble sugar (37.4 mg g(-1) DW), and reducing sugar (45.0 mg g(-1) DW) in the leaves than the BAP and NAA combinations. The efficiency of mT has also been highlighted in the foliar structural developments. Comparative anatomy of leaves showed the presence of structural variations influenced by the type of cytokinins used. BAP and NAA-derived leaves revealed the presence of under-developed cuticle, unorganized tissue systems, undifferentiated mesophyll tissues, and under-developed mechanical and vascular tissues. Whereas, mT and NAA-derived leaves exhibited comparatively thick cuticle, collenchymatous hypodermis, well-differentiated mesophylls, and increased vascular elements. Roots were induced ex vitro under the greenhouse conditions when the shoot-bases were treated with 500 mg L-1 indole-3-butyric acid (IBA) for 10 min. These plantlets were hardened in soilrite (R) and cocopeat (1:1) mixture and a 100 % survival rate was achieved in the field. Additionally, the Start codon targeted (SCoT) markers analysis demonstrated that the genetic homogeneity was maintained during in vitro development of plantlets. The application of mT can be an effective growth regulator for in vitro propagation of S. album, as it favoured enhanced and quick production of genetically uniform clones.

Oxystelma esculentum (L. f.) Sm. (Apocynaceae) is a perennial medicinal climber, enormously explored in traditional and modern systems of medicines. Due to over-harvesting from the wild, this species is categorized as ‘rare’. Hence, this study was aimed to develop an in vitro regeneration system for O. esculentum as a conservation measure. Nodal explants were cultured on Murashige and Skoog (MS) medium supplemented with cytokinins [benzyladenine (BA) and meta-Topolin (mT)] for the establishment of cultures. The growth of shoots from the pre-existing meristems of explants was obtained on MS medium containing 4.14 µM mT with the highest mean number (7.25) of shoots. The maximum shoot proliferation frequency (124.8 shoots) was achieved on MS medium containing 2.07 µM mT and 1.42 µM indole-3-acetic acid (IAA). The foliar micro-morpho-anatomical stability of in vitro raised shoots (from BA and mT) was analyzed using light microscopy. Although the in vitro raised leaves possessed microscopic anomalies, the BA-derived leaves showed a higher degree of abnormalities, such as underdeveloped stomata with single guard cells, reduced vein and trichome density, poor deposition of cutin, and less differentiation of ground and vascular tissue systems. Comparatively, mT-derived in vitro leaves had functional stomata, a higher amount of cutin deposition, and remarkable developments in vascular and ground tissue systems. The regenerated shoots were rooted following pulse treating with 976.08 µM indole-3-butyric acid (IBA) under greenhouse (ex vitro) conditions. All the mT raised plantlets (100%) were survived in the field conditions. This study could suggest a highly efficient cytokinin (mT) for large-scale propagation and conservation of O. esculentum, which could ultimately help in the production of the quality shoots in terms of essential structural developments for successful rooting and hardening processes. A reproducible in vitro propagation protocol for Oxystelma esculentum was developed usingmeta-Topolin. The influence of meta-Topolin on micro-morpho-anatomical developments hasbeen proved through the foliar micro-morpho-anatomical analysis for the first time.

The anatomical and histochemical localization at subsequent stages of somatic embryogenesis in Santalum album revealed the direct development of somatic embryos from internode explants. Santalum album L. (Santalaceae) is a vulnerable tree species due to overexploitation for commercial, pharmaceutical and cosmetic industries, which necessitate improving the existing in vitro propagation systems for effective plant production and conservation. To date, there is no report on the histochemical basis of somatic embryogenesis in this species. Hence, this study aimed to induce direct somatic embryos (SEs) from the internode explant and to analyze the developmental anatomy and histochemical features during direct somatic embryogenesis. Somatic embryos were induced on Murashige and Skoog (MS) medium with 2.5 mg L−1 6-benzylaminopurine (BAP), 0.5 mg L−1 1-naphthaleneacetic acid (NAA), 50 ml L−1 deproteinized coconut water (CW) and 25 mg L−1 each of myo-inositol, adenine sulphate and l-arginine. Pre-treatment of explants with 50 mg L−1 citric acid and ascorbic acid for 0.5 h at 4 °C, and the addition of CW in the medium, favored high-frequency somatic embryogenesis from internode. The light microscopic analysis revealed the development of embryonic cells, vasculature and primary globular stage to matured dicotyledonous embryos. The contents of lignin, cellulose and starch were low in the embryogenic regions, whereas deposition of phenolic compounds, polyphenols, lipids and polysaccharides were found increased in somatic embryos. Embryos germinated well (98%) and the shoots were proliferated on MS medium supplemented with 1.0 mg L−1 BAP, 0.5 mg L−1 NAA and 50 ml L−1 CW. The well-developed shoots were rooted within four weeks on MS medium containing 1.5 mg L−1 indole-3-butyric acid (IBA) and successfully acclimatized in a greenhouse. The highest (97%) survival success was achieved in the field conditions. Thus, the present work has established a novel protocol for induction of somatic embryogenesis from internode explants complemented with studies of the histo-anatomical developments of the SEs. This could help to understand the embryogenic pathway and represent an alternative way of production plantlets for the conservation of S. album.

Justicia betonica L., family Acanthaceae is a widely used traditional folk medicinal herb. It is a unique source of jusbetonin and the leaves yield bluish purple dye. Traditionally, the plant is used to cure constipation, diarrhea, malaria, orchitis, pain, snake bite, stomach ache, vomiting etc. The whole plant possess significant biologically active principles like steroids, triterpenoids, alkaloids, saponins, glycosides, carbohydrates, gum and mucilage, proteins, fixed oils and fat, phenolics and tannins. It is reported to exhibit antiviral, antioxidant, anti-inflammatory, analgesic and antimalarial activities. The intention of the study was to endow an overview of the ethnomedicinal properties, phytochemistry and related pharmacological applications of J. betonica, and to make an authenticated evidence base for further research on this important medicinal plant.