Showing papers on "Murashige and Skoog medium published in 2021"

Meta-topolin and liquid medium mediated enhanced micropropagation via ex vitro rooting in Vanilla planifolia Jacks. ex Andrews

Abstract: An advanced micropropagation protocol has been developed for the global spice crop Vanilla planifolia using meta-topolin [mT, 6-(3-hydroxybenzylamino) purine] for the first time. Among the two types of cytokinins [mT and BAP (6-benzylaminopurine)] experimented, healthy shoot development and growth in terms of shoot numbers (5.0 ± 0.20/explant) with shoot length (4.3 ± 0.17 cm) were noted on agar-gelled Murashige and Skoog’s (MS) medium supplemented with 1.0 mg L−1 mT. The regenerated shoots were multiplied in liquid medium using the continuous shake-flask culture method in an incubator shaker. The liquid MS medium containing 0.5 mg L−1 mT + 0.25 mg L−1 α-Naphthalene acetic acid (NAA) agitated at 60 rpm resulted in the production of 62.0 ± 0.31 shoots (3.8 ± 0.11 cm length) per explants per culture vessel after 3rd subculture, and this is the best rate of shoot proliferation among the prevailing reports on V. planifolia. Further, the transverse sections of the leaves revealed that the mT medium derived leaves were physically sturdy and physiologically more active with developed anatomical parameters, such as higher proportions of palisade and spongy parenchyma tissue, xylem and phloem elements, and abundant chloroplasts than the shoots raised on BAP. The leaves collected from the shoots of mT containing medium possessed the higher amount of chlorophylls (380.0 μg g−1 fresh weight) and carotenoids (52.0 μg g−1 fresh weight) under in vitro conditions. The concurrent ex vitro rooting and acclimatization method has been adopted to reduce the cost and time in large scale cloning of V. planifolia. All the shoots (100%) were rooted in soilrite™, when pulse treated with 100 mg L−1 NAA for 4 min. The ex vitro rooted plantlets were successfully acclimatized in a greenhouse. The regenerated clones’ genetic uniformity and stability with the donor plant were tested using Start codon targeted polymorphism and detected no somaclonal variation. All the plantlets were survived in the soil transplantation, confirming the production of genetically, physically, and physiologically stable plantlets of V. planifolia. An improved in vitro regeneration protocol was developed for Vanilla planifolia. It is the first report revealing the positive effect of meta-topolin and liquid medium (shake-flask culture method) for enhanced proliferation of shoots in this orchid. The mT derived leaves showed better anatomical features and retained high level of photosynthetic pigments. The in vitro raised shoots were rooted ex vitro and successfully transplanted in the field.

Meta-topolin and liquid medium enhanced in vitro regeneration in Scaevola taccada (Gaertn.) Roxb

Abstract: Scaevola taccada (Gaertn.) Roxb. is a hemi-sclerophyllous littoral shrub of the family Goodeniaceae. It is a salt-tolerant plant and used in soil reclamation and coastal landscaping programs to control beach erosion. The present article reports a reproducible in vitro regeneration system using meta-topolin (mT) through liquid medium employing nodal segment cultures. The highest number of axillary shoots (5.8 ± 0.29) was differentiated on Murashige and Skoog’s (MS) medium containing meta-topolin (1.5 mg L−1). The amplification of shoots (49.8 ± 0.37) and fresh growth was achieved by recurrent subcultures on liquid MS medium supplemented with 0.25 mg L−1 mT and 0.15 mg L−1 indole-3-acetic acid (IAA) in the presence of additives (50 mg L−1 ascorbic acid + 25 mg L−1 citric acid, adenine sulphate and l-arginine). Rooting and acclimatization was simultaneously achieved using concurrent ex vitro rooting and acclimatization (CEVRA) technique by pulse treating the shoot base with 300 mg L−1 IBA for 5 min. The in vitro developed shoots represented a maximum percentage (100%) of rooting in autoclaved soilrite in a greenhouse. After 2 mo, the plantlets were established in the field with cent percentage survival success. The genetic fidelity analysis using inter simple sequence repeats (ISSR) DNA molecular markers developed maximum of 55 monomorphic bands and revealed the genetic homogeneity of in vitro regenerated plantlets with the mother plant.

Influence of meta-topolin on in vitro propagation and foliar micro-morpho-anatomical developments of Oxystelma esculentum (L.f.)Sm

Abstract: 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 using meta-Topolin. The influence of meta-Topolin on micro-morpho-anatomical developments has been proved through the foliar micro-morpho-anatomical analysis for the first time.

Micropropagation of Ginger ( Zingiber officinale Roscoe) 'Bentong' and Evaluation of Its Secondary Metabolites and Antioxidant Activities Compared with the Conventionally Propagated Plant.

Abstract: ‘Bentong’ ginger is the most popular variety of Zingiber officinale in Malaysia. It is vegetatively propagated and requires a high proportion of rhizomes as starting planting materials. Besides, ginger vegetative propagation using its rhizomes is accompanied by several types of soil-borne diseases. Plant tissue culture techniques have been applied in many plant species to produce their disease-free planting materials. As ‘Bentong’ ginger is less known for its micropropagation, this study was conducted to investigate the effects of Clorox (5.25% sodium hypochlorite (NaOCl)) on explant surface sterilization, effects of plant growth regulators, and basal media on shoots’ multiplication and rooting. The secondary metabolites and antioxidant activities of the micropropagated plants were evaluated in comparison with conventionally propagated plants. Rhizome sprouted buds were effectively sterilized in 70% Clorox for 30 min by obtaining 75% contamination-free explants. Murashige and Skoog (MS) supplemented with 10 µM of zeatin was the suitable medium for shoot multiplication, which resulted in the highest number of shoots per explant (4.28). MS medium supplemented with 7.5 µM 1-naphthaleneacetic acid (NAA) resulted in the highest number of roots per plantlet. The in vitro-rooted plantlets were successfully acclimatized with a 95% survival rate in the ex vitro conditions. The phytochemical analysis showed that total phenolic acid and total flavonoid content and antioxidant activities of the micropropagated plants were not significantly different from the conventionally propagated plants of ‘Bentong’ ginger. In conclusion, the present study’s outcome can be adopted for large-scale propagation of disease-free planting materials of ‘Bentong’ ginger.

Influence of Light Conditions and Medium Composition on Morphophysiological Characteristics of Stevia rebaudiana Bertoni In Vitro and In Vivo

Abstract: We investigated the influence of different conditions (light composition and plant growth regulators (PGRs) in culture media) on the morphophysiological parameters of Stevia rebaudiana Bertoni in vitro and in vivo. Both PGRs and the light spectra applied were found to significantly affect plant morphogenesis. During the micropropagation stage of S. rebaudiana, optimal growth, with a multiplication coefficient of 15, was obtained in an MS culture medium containing 2,4-epibrassinolide (Epin) and indole-3-acetic acid (IAA) at concentrations of 0.1 and 0.5 mg L−1, respectively. During the rooting stage, we found that the addition of 0.5 mg L−1 hydroxycinnamic acid (Zircon) to the MS medium led to an optimal root formation frequency of 85% and resulted in the formation of strong plants with well-developed leaf blades. Cultivation on media containing 0.1 mg L−1 Epin and 0.5 mg L−1 IAA and receiving coherent light irradiation on a weekly basis resulted in a 100% increase in the multiplication coefficient, better adventitious shoot growth, and a 33% increase in the number of leaves. S. rebaudiana microshoots, cultured on MS media containing 1.0 mg L−1 6-benzylaminopurine (BAP) and 0.5 mg L−1 IAA with red monochrome light treatments, increased the multiplication coefficient by 30% compared with controls (white light, media without PGRs).

Establishment of an efficient in vitro propagation protocol for Sumac (Rhus coriaria L.) and confirmation of the genetic homogeneity.

Abstract: The conventional reproduction methods are not efficient for regeneration of Sumac (Rhus coriaria L.). The purpose of this work was to study the micropropagation of R. coriaria using lateral buds as explant in Murashige and Skoog (MS) medium with different concentrations of plant growth regulator (PGRs). Four concentrations of Benzylaminopurine (BAP) in combination with three concentrations of indol-3-butyric acid (IBA) and 1.0 mg/L gibberellic acid (GA3) were tested for establishment and shoot multiplication. For root induction, IBA was used at four levels combined with 0, 0.5 and 1 mg/L of naphthalene acetic acid (NAA) in full and half strength of MS medium. BAP at 2 mg/L with 1 mg/L IBA was best, with 88.88% of establishment. The highest shoot proliferation (12.30 ± 0.30) was obtained in medium fortified with 2 mg/L BAP plus 0.5 mg/L IBA and the highest shoot length (8.50 cm) was obtained at 3 mg/L BAP plus 1 mg/L IBA. The highest rooting (100%) was observed in 1/2-strength MS medium containing 1 mg/L IBA with 0.5 mg/L NAA. In conclusion, an efficient protocol with high rate of proliferation and rooting is described for R. coriaria, which can be used in massive propagation.

Gas exchange rates and sucrose concentrations affect plant growth and production of flavonoids in Vernonia condensata grown in vitro

Abstract: Tissue culture enables the efficient clonal propagation of plants. This prevents the loss of genetic information in the natural environment and, in the case of medicinal plants, helps produce bioactive metabolites. Here, we used Vernonia condensata to understand how in vitro culture conditions impacted development, anatomy, physiological performance, and biosynthesis of flavonoids and phenolic compounds. The plants were grown in MS medium supplemented with 0, 15, or 30 g L−1 sucrose in flasks with lids without membrane (14 μL L−1 s−1 CO2 exchange rate) or with two membranes (25 μL L−1 s−1 CO2 exchange rate). Cultures were maintained for 35 days in a growth room under an irradiance of 50 μmol m−2 s−1, 16-h photoperiod, and 25 ± 2 °C. Plants exhibited greater growth, more photosynthetic pigments, and better leaf histological differentiation at a higher gas exchange rate and with sucrose supplementation. Despite the reduced stomatal density, a higher gas exchange augmented the photosynthetic rate and, consequently, increased the concentration of hexoses and starch while lowering the content of amino acids. Under conditions of lower gas exchange rates, plants grown in a medium with a higher concentration of sucrose showed an increase in phenolic compounds. However, the highest levels of these metabolites were found in plants grown under higher gas exchange rates. Thus, our findings show that the increase in gas exchange rates and sucrose supplementation are efficient strategies for obtaining plants with greater biomass and production of flavonoids. Vernonia condensata cultured in vitro under higher gas exchange and sucrose supplementation leads to more vigorous plants with increased production of bioactive compounds.

Silver nanoparticles as the sterilant in large-scale micropropagation of chrysanthemum

Abstract: Micropropagation has proven to be an effective method for large-scale plant production in a short time and a useful tool for plant breeding. Microbial contamination is one of the most difficult micropropagation challenges, resulting in reduced plant quality and loss of valuable stocks. Therefore, sterilization of culture media is a critical step in plant micropropagation. However, sterilized media might reduce the activity of plant growth regulators and nutritional components of culture media. The sterilization effects of silver nanoparticles (AgNP) on the growth of explants and culture media were examined. The treatment with 250 ppm AgNP for 15 to 20 min of 4-wk-old ex vitro leaves proved optimal for controlling the contamination. Furthermore, the Murashige and Skoog medium containing 4 ppm AgNP resulted in 100% medium disinfection (no contamination) after 4 wk of culture. The plantlets obtained from non-sterilized MS medium (NoM) containing 4 ppm AgNP and 4 g L−1 agar gave similar results as the control medium with 8 g L−1 agar and the absence of AgNP. Large scale culture systems using NoM in large plastic containers of two different sizes (NoM1 and NoM2) could produce quality plantlets. Chrysanthemum plantlets in the NoM1 system showed higher antioxidant enzyme activities of ascorbate peroxidase and superoxide dismutase than plantlets in the autoclaved medium. Furthermore, the plantlets from NoM were better acclimatized under greenhouse conditions than those from the autoclaved medium (AuM) system. The developmental stages (flower buds and blooming time) of NoM1 and NoM2 plantlets, were 1 wk earlier than those from the AuM system. The successful use of AgNP as a sterilizer and as a component of culture media would reduce the cost of micropropagation and improve plants' quality.

Short-term cold storage of encapsulated somatic embryos and retrieval of plantlets in grey orchid (Vanda tessellata (Roxb.) Hook. ex G.Don)

Abstract: Vanda tessellata (Roxb.) Hook. ex G.Don (grey orchid) is a horticultural and medicinally potent, and conservation prioritized orchid species. The present study investigates asymbiotic seed germination, callus induction, somatic embryogenesis, encapsulation of somatic embryos (SEs), and retrieval of plantlets of V. tessellata from the artificial seeds after short-term cold storage at − 4 °C for 12 months. Seeds cultured on full strength Murashige and Skoog’s (MS) medium containing 2.0 mg L−1 6-benzylaminopurine (BAP) had the highest frequency of asymbiotic seed germination (94%) as compared with half- and one-fourth strength MS media supplemented with various concentrations and combinations of 6-furfurylaminopurine (kinetin) and BAP. The protocorm like bodies (PLBs) gained dedifferentiation and proliferated into embryogenic calli on MS medium containing 2.0 mg L−1 BAP and 0.5 mg L−1 indole-3 acetic acid (IAA). Somatic embryos (SEs) were differentiated from the callus when cultured on MS medium in combination with 1.0 mg L−1 BAP and 0.5 mg L−1 IAA. The morpho-anatomical elucidations authenticated somatic embryogenesis and various developmental stages of SEs from embryogenic calli. Short-term cold storage of SEs was carried out by encapsulating with 2% sodium alginate and 100 mM calcium chloride and stored under dark at − 4 °C for 12 months to conserve this valuable germplasm. The stored artificial seeds were germinated (91%) on MS medium incorporated with additives and 0.5 mg L−1 each of BAP, kinetin, and IAA. Complete retrieval of plantlets was achieved in 8 week with formation of shoots (4.2 cm average length) and roots (2.5 cm). The well developed plantlets were acclimatized in the greenhouse with 90% survival rate. Vanda tessellata (grey orchid) is a horticultural and medicinally potent, and conservation prioritized orchid species. A reproducible protocol has been successfully established for asymbiotic seed germination, callus induction, somatic embryogenesis, encapsulation of somatic embryos, and retrieval of plantlets of V. tessellata from the artificial seeds after short-term cold storage at − 4 °C for 12 months.

Morpho-anatomical and physiological changes of Indian sandalwood (Santalum album L.) plantlets in ex vitro conditions to support successful acclimatization for plant mass production

Abstract: Santalum album L. (Indian sandalwood) is an economically important but vulnerable tropical tree species. Cultures were established via direct shoot regeneration from axillary buds on Murashige and Skoog (MS) medium supplemented with 2.5 mg L−1 6-benzylaminopurine (BAP). The shoots were multiplied using MS medium containing 1.0 mg L−1 BAP and 0.5 mg L−1 indole-3 acetic acid and rooted on half strength MS medium containing 1.0 mg L−1 indole-3 butyric acid. The rooted plantlets were hardened and acclimatized in greenhouse using soilrite® and cocopeat (1:1) mixture. The concentrations of photosynthetic pigments were analyzed and detected less under in vitro conditions (6.05 μg g−1 FW) as compared to the 4 weeks old hardened (6.91 μg g−1 FW) and 12 weeks old acclimatized plantlets (7.8 μg g−1 FW) under greenhouse (ex vitro) environment. The anatomical evaluation of plantlets at subsequent stages of propagation suggested that the in vitro raised plantlets possessed structural abnormalities such as underdeveloped cuticle, unorganized tissue systems, reduced mesophyll tissues, fewer vascular elements and mechanical tissues, and loosely arranged thin walled paranchymatous ground tissues, which were slowly repaired during ex vitro hardening and acclimatization process to validate the developmental adaptation of micropropagated plantlets for maximum survival in the field (98.0% survival rate). The findings could help in the optimization of high-frequency commercial micropropagation of S. album for year-round production, and supply of this economically prominent vulnerable plant species to the farmers and the industries that rely on it. The anatomical and physiological features at subsequent stages in micropropagation of Santalum album elucidated the in vitro anomalies and ex vitro repair mechanism for improved survival in field.

The development of callus and cell suspension cultures of Sabah Snake Grass (Clinacanthus nutans) for the production of flavonoids and phenolics

Abstract: Clinacanthus nutans (Burm.f.) Lindau is a valuable medicinal plant that has gained interest as a side treatment for cancer in Southeast Asia. Phenolic and flavonoid compounds identified in this plant have been linked to anti-cancer properties. However, quantification of such metabolites in plants varies depending on cultivation methods and conditions resulting in inconsistent yield. This study aims to establish cell suspension culture of C. nutans, evaluate the accumulation of phenolics, flavonoids and to assess the antioxidant activity of extracts from in vitro cultures. Callus was induced from leaf explants of C. nutans and proliferated on Murashige & Skoog (MS) medium supplemented with different combinations of plant growth regulators (2,4-dichlorophenoxyacetic acid, 6-benzylaminopurine and kinetin). Assessment of growth kinetics for the suspension culture was performed. The total phenolic, flavonoid contents and antioxidant activity of the extracts were evaluated followed by high-performance liquid chromatography (HPLC) to detect selected flavonoids. MS medium supplemented with 0.25 mg/L 2, 4-D and 0.25 mg/L BAP was optimal for callus induction, proliferation and suspension cultures. The highest total phenolic content was obtained from suspension cells (55.35 mg GAE/g DW) whereas leaf showed the highest flavonoid content (25.13 mg QE/g DW). Leaf extract demonstrated the strongest antioxidant activity with the lowest IC50 value (117.42 μg/mL). HPLC analysis revealed the presence of catechin, luteolin, quercetin and kaempferol in the suspension cells and the leaf. The present study indicated that cell suspension cultures are able to accumulate higher phenolic compounds and possess all four selected flavonoids similar to the outdoor grown plant.

Gibberellic acid and thidiazuron promote micropropagation of an endangered woody tree (Pterocarpus marsupium Roxb.) using in vitro seedlings

Abstract: Pterocarpus marsupium Roxb., which belongs to the Fabaceae family, is a promising herbal drug producing forest tree widely known as the ‘Indian Kino’. An effective regeneration protocol using in vitro seedlings of P. marsupium under the gibberellic acid and thidiazuron regimen is described in the present study. Gibberellic acid was also tested to have an effect on in vitro rhizogenesis from microshoots. Combination of gibberellic acid and thidiazuron in culture media improved the germination percentage, multiplication and subsequent elongation of shoots. Murashige and Skoog medium containing 0.50 µM gibberellic acid with 0.50 µM thidiazuron was found to be the most effective treatment for producing the highest number of shoots per seedling (23.0) with an average shoot length 5.14 cm in 85% cultures, after 8 weeks. For in vitro rhizogenesis, 100 μM of indole 3-butyric acid was treated for 5 days in basal ends of isolated microshoots. Thereafter, pretreated microshoot was transferred to gibberellic acid (0.50 µM) supplemented MS medium. This treatment was found to be the most effective for in vitro root formation, where 10.54 roots with an average root length of 4.60 cm per microshoot in 80% cultures were recorded, after 4 weeks. During acclimatization photosynthetic traits and their attributes such as net photosynthetic rate, stomatal conductance, intercellular CO2 concentration, and Chlorophyll content were analyzed and these attributes were found to be fluctuating initially for 35 days, thereafter, an increasing trend was recorded up to 84 days of acclimatization. The well acclimatized plantlets were shifted to fields where they grew normally without any morphological changes with an 86.7% of survival. Genetic integrity in micropropagated plants was validated by DNA-based ISSR primers. These primers were amplified in monomorphic banding pattern suggesting a high-level of genetic uniformity in micropropagated plants. Overall, an effective and high throughput plant regeneration method has been developed for obtaining true-to-type regenerants of P. marsupium. The present study revealed the high throughput regeneration of P. marsupium through in vitro seedlings method which may be an alternative tool for mass propagation and germplasm conservation of woody trees directly from seed, thus reducing an explant preparation and recovery of seedlings capable of multiple shoot formation under the regimes of gibberellic acid and thidiazuron. Both the growth regulators are involved in the improvement of plantlet regeneration processes as well as maintenance of physiological stresses during in vitro cultures.

Efficient in vitro plant regeneration from leaf-derived callus and genetic fidelity assessment of an endemic medicinal plant Ranunculus wallichianus Wight & Arnn by using RAPD and ISSR markers

Abstract: Ranunculus wallichianus is a medicinally important plant and an endemic species to the Western Ghats of South India. An efficient and reliable indirect regeneration protocol system for R. wallichianus was developed from leaf explants in the present investigation. Leaf explants were cultured on both full-strength and half-strength MS (Murashige & Skoog) medium supplemented with different concentrations (0.5 mg L− 1 to 3.0 mg L− 1) of 2,4-D and NAA. Among the different concentrations tested, the highest percentage of yellowish-green compact nodular callus formation was observed on a half-strength MS medium with 2.0 mg L− 1 of 2, 4-D. Then, the in vitro raised organogenic callus was cultured on a half-strength MS medium containing various concentrations (0.5 mg L− 1 to 3.0 mg L− 1) of BA, KIN, and TDZ with 0.5 mg L− 1 NAA and 10 % CW for in vitro shoot regeneration. The highest percentage of regeneration response (97 %) and a maximum number of shoots formation (11.1 ± 0.13 shoots/culture with 9.2 ± 0.21 cm mean shoot length) were obtained from MS medium containing 2.5 mg L− 1 BA with 0.5 mg L− 1 NAA and 10 % CW. The well elongated in vitro raised shoots were effectively rooted in a half-strength MS medium with 2.5 mg L− 1 IBA + 250 mg L− 1 activated charcoal. The well-rooted plantlets were successfully hardened and acclimatized with a survival rate of 94 %. Clonal fidelity of in vitro raised plantlets was assessed by using DNA-based RAPD and ISSR molecular markers. A total of 56 and 47 monomorphic bands were obtained from RAPD and ISSR markers respectively. This present in vitro propagation protocol system could be effective for the conservation of R. wallichianus with their genetic purity and its further investigations. The present study develops a protocol for mass multiplication of Ranunuculus wallichianus through indirect organogenesis and evaluates the clonal fidelity of regenerants by using RAPD and ISSR markers.

Indirect somatic embryogenesis and regeneration of Fraxinus mandshurica plants via callus tissue

Abstract: Somatic embryogenesis of Fraxinus mandshurica has the problems of low somatic embryo (SE) yield, unsynchronized SE development, and a high percentage of deformed SEs. We aimed to improve F. mandshurica SE production by synchronizing SE development, improving SE quality, and inducing root formation to obtain complete regenerated plants. Cotyledons of immature zygotic embryos of F. mandshurica were induced to form callus and then SEs. The SE induction percentage from explants differed among 32 mother trees, and the one with the highest SE induction percentage (29.8%) was used for further experiments. The highest callus induction percentage was 94.2% on ½-strength Murashige and Skoog medium (MS½) supplemented with 0.15 mg·L−1 naphthalene acetic acid. The highest callus proliferation coefficient (240.5) was obtained on McCown’s Woody Plant Medium containing 0.1 mg·L−1 6-benzyl adenine and 0.15 mg·L−1 2, 4-dichlorophenoxyacetic acid. The highest number of SEs (1020.5 g−1 fresh weight) was obtained on MS½ medium supplemented with 1 mg·L−1 6-benzyladenine. The highest number of cotyledon embryos (397/g fresh weight) was obtained by incubating materials on medium containing 1 mg·L−1 abscisic acid and then applying a drying treatment. The cotyledon embryos were milky white, uniformly sized (average length 4.7 mm), and 80% of them were normal. The SE rooting percentage on ½MS medium containing 0.01 mg·L−1 NAA was 37.5%. Overall, the germination percentage of SEs was 26.4%, and complete regenerated plants were obtained after transplanting and acclimation. These results provide more possibilities for the preservation and breeding of F. mandshurica.

Plant regeneration through direct and indirect organogenesis, phyto-molecular profiles, antioxidant properties and swertiamarin production in elicitated cell suspension cultures of Swertia minor (Griseb.) Knobl

Abstract: The present study reports an optimized protocol for high frequency in vitro plant propagation through direct and indirect organogenesis, phytochemical accumulation, molecular profiling and antioxidant evaluation for micropropagated Swertia minor, a promising alternative to industrially important Swertia chirayita. Moreover, the study also aimed at enhancing the production of antidiabetic and anti-obesity drug swertiamarin using an alternative technology of elicitated cell suspension cultures. Different types, concentrations and combination of cytokinins and auxins showed their effects during various in vitro growth stages. A combination of BAP (3.0 mg/l) and TDZ (1.0 mg/l) had a dominant role in promoting multiple shoot proliferation with production of an average of 19.1 ± 0.95 shoots/node in 85% response. MS medium added with IBA (2.0 mg/l) showed optimal response for in vitro rooting (9.2 ± 0.56 roots/shoot). In order to establish genetic stability, molecular marker-based profiling of micropropagated plants were done and 'monomorphic banding pattern were identical to the mother plants. 2,4-D (2.0 mg/l) supported the maximum callus induction and proliferation rate (95%). The wild-grown plants showed higher polyphenols content and antioxidant activities as compared to callus and in vitro derived plantlets. However, chitosan-treated (25 ppm) methanolic extract of cell biomass accumulated in cell suspension cultures produced higher contents of swertiamarin (1.45 mg/g DW) than salicylic acid and methyl jasmonate. The described protocol can be effectively used for the large-scale propagation, exploitation of active compounds and will serve as potential alternative to S. chirayita for fulfillment of over-growing industrial requirements. The present investigation addressed in vitro regeneration, callus culture, somatic embryogenesis, molecular profiling, secondary metabolite production, cell suspension culture studies for first time in Swertia minor.

High-frequency in vitro propagation and assessment of genetic uniformity and micro-morphological characterization of Origanum majorana L. –A highly traded aromatic herb

Abstract: Origanum majorana (L.) (family Lamiaceae) is a multipurpose medicinal plant with high demand across the pharmaceutical, cosmetic, food, beverage, and perfume industries that has raised concern for its mass propagation through in vitro culture techniques. The present report describes an effective in vitro propagation system for an aromatic medicinal plant Origanum majorana L. The Murashige and Skoog's (MS) medium supplemented with 1.0 mg L-1 6-benzylaminopurine (BAP) initiated a maximum of 4.4 ± 0.22 micro-shoots from the nodal segment explants. The regenerated shoots were further proliferated (98.0 ± 0.22 shoots per explant/culture vessel) on MS medium augmented with 0.5 mg L-1 BAP and 0.25 mg L-1 α-naphthalene acetic acid (NAA). Individual shootlets were rooted on a half-strength MS medium with 1.0 mg L-1 indole-3 butyric acid (IBA) (4.8 ± 0.20 roots/shoots). The rooted plantlets were transplanted into eco-friendly paper cups containing soilrite®: manure; sand (2:1:1) and maintained in the greenhouse for hardening. Inter simple sequence repeats (ISSR) molecular markers were utilized to investigate the somaclonal variations between in vitro developed plants and the mother plant of O. majorana. The band patterns of regenerated plantlets have shown genetic uniformity with the donor plant. The foliar micro-morphological features under in vitro and ex vitro conditions were compared with the mother plant and elucidated morphologically stable plantlets development through gradual acclimatization. This is the foremost report on ISSR molecular marker-based genetic fidelity studies and foliar micro-morphological evaluation of micropropagated plants of O. majorana.

Alterations in Microrhizome Induction, Shoot Multiplication and Rooting of Ginger (Zingiber officinale Roscoe) var. Bentong with Regards to Sucrose and Plant Growth Regulators Application

Abstract: Ginger (Zingiber officinale Roscoe) var. Bentong is a monocotyledon plant that belongs to the Zingiberaceae family. Bentong ginger is the most popular cultivar of ginger in Malaysia, which is conventionally propagated by its rhizome. As its rhizomes are the economic part of the plant, the allocation of a large amount of rhizomes as planting materials increases agricultural input cost. Simultaneously, the rhizomes’ availability as planting materials is restricted due to the high demand for fresh rhizomes in the market. Moreover, ginger propagation using its rhizome is accompanied by several types of soil-borne diseases. Plant tissue culture techniques have been applied to produce disease-free planting materials of ginger to overcome these problems. Hence, the in vitro-induced microrhizomes are considered as alternative disease-free planting materials for ginger cultivation. On the other hand, Bentong ginger has not been studied for its microrhizome induction. Therefore, this study was conducted to optimize sucrose and plant growth regulators (PGRs) for its microrhizome induction. Microrhizomes were successfully induced in Murashige and Skoog (MS) medium supplemented with a high sucrose concentration (>45 g L−1). In addition, zeatin at 5–10 µM was found more effective for microrhizome induction than 6-benzylaminopurine (BAP) at a similar concentration. The addition of 7.5 µM 1-naphthaleneacetic acid (NAA) further enhanced microrhizome formation and reduced sucrose’s required dose that needs to be supplied for efficient microrhizome formation. MS medium supplemented with 60 g L−1 sucrose, 10 µM zeatin and 7.5 µM NAA was the optimum combination for the microrhizome induction of Bentong ginger. The in vitro-induced microrhizomes sprouted indoors in moist sand and all the sprouted microrhizomes were successfully established in field conditions. In conclusion, in vitro microrhizomes can be used as disease-free planting materials for the commercial cultivation of Bentong ginger.

meta-Topolin-induced enhanced biomass production via direct and indirect regeneration, synthetic seed production, and genetic fidelity assessment of Bacopa monnieri (L.) Pennell, a memory-booster plant

Abstract: The present study reports enhanced in vitro mass propagation, synthetic seed production and its regeneration, acclimatization, and genetic fidelity assessment of in vitro regenerants of Bacopa monnieri (L.) Pennell; a medicinal plant renowned for its memory-enhancing property. For the first time, meta-Topolin (mT) as well as N6-benzyladenine (BA) and kinetin at their variable concentrations (0.5, 1. 1.5, 2 and 2.5 mg/l) were supplemented individually in Murashige and Skoog (Physiol Plant 15:473–497, 1962) (MS) basal medium to induce multiple shoots from shoot tip explants, initially collected from two-month-old vegetatively propagated plants. The best result in terms of multiplication (~ 7 shoots/explant) and biomass accumulation (~ 451.3 mg fresh weight, ~ 104.3 mg dry weight) was obtained in MS medium supplemented with 1 mg/l mT. For rooting of in vitro shoots, supplementation of 1.5 mg/l IAA and 0.5 mg/l IBA in MS media resulted in induction of the maximum number (~ 20) and highest length (~ 5.5 cm) of roots, respectively. Most efficient indirect regeneration and biomass production from green and hard basal calli (induced spontaneously in BA-supplemented media) was recorded in a simple MS medium. Synthetic seeds were produced from in vitro shoot tips using 2.5% sodium alginate and 75 mM calcium chloride solutions and were successfully regenerated in half-strength MS medium. Six-week-old in vitro regenerants were transferred to different substrates for acclimatization, among which cocopeat was found to be the best exhibiting the highest survival and growth in external conditions. Finally, molecular marker-(ISSR) and phenology-based genetic fidelity assessment of the in vitro regenerants exhibited that the in vitro regenerants were true-to-type in nature within themselves as well as with the mother plant.

Efficient in vitro organogenesis, micropropagation, and plumbagin production in Plumbago europaea L.

Abstract: In this study, an efficient method for in vitro regeneration of Plumbago europaea was developed using direct and indirect organogenesis. Accordingly, micropropagation and regeneration were obtained on Murashige and Skoog (MS) medium supplemented with different concentrations and combinations of plant growth regulators. The effects of explant type and plant growth regulators on shoot organogenesis of P. europaea were evaluated. For the nodal explants, MS medium containing 0.5 mg/l TDZ (11.62 shoots per node) was the best medium for high frequency of micropropagation. In comparison, the highest percentage of direct organogenesis (70%) and number of shoots per explants (14.6) were acquired for the internode explants using 0.5 mg/l TDZ and 0.1 mg/l IAA. The obtained data revealed that TDZ is the most effective cytokinin for the direct shoot organogenesis. The highest indirect organogenesis rate was observed using 2 mg/l BA and 0.1 mg/l NAA for the internode explant. The maximum number of roots was distinguished on ½ MS medium containing 0.5 mg/l IBA (6.42). The rooted plantlets were gradually hardened and acclimatized under ex vitro conditions. As an important outcome, the active compound plumbagin was found mainly in the root tissues of the micro-propagated and regenerated plantlets. Taken all together, this study achieved a successful protocol for in vitro regeneration of P. europrea and could be considered for large-scale multiplication of this important medicinal plant.

Genetic Stability, Phenolic, Flavonoid, Ferulic Acid Contents, and Antioxidant Activity of Micropropagated Lycium schweinfurthii Plants.

Abstract: Lycium schweinfurthii is a Mediterranean wild shrub rich in plant secondary metabolites. In vitro propagation of this plant may support the production of valuable dietary supplements for humanity, introduction of it to the world market, and opportunities for further studies. The presented study aimed to introduce an efficient and reproducible protocol for in vitro micropropagation of L. schweinfurthii and assess the genetic stability of micropropagated plants (MiPs) as well as to estimate phenolic, flavonoid, ferulic acid contents, and the antioxidant activity in leaves of micropropagated plants. Two DNA-based techniques, random amplified polymorphic DNA (RAPD) and inter-simple sequence repeats (ISSR), and one biochemical technique, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), were used to assess the genetic stability in MiPs. Spectrophotometric analysis was performed to estimate total phenolic and flavonoid contents and antioxidant activity of MiPs leaves, while ferulic acid content was estimated using high-performance thin-layer chromatography (HPTLC). Sufficient shoot proliferation was achieved at MS (Murashige and Skoog) medium supplemented with 0.4 mg L−1 kinetin and rooted successfully on half-strength MS medium fortified with 0.4 mg L−1 Indole-3-butyric acid (IBA). The Jaccard’s similarity coefficients detected in MiPs reached 52%, 55%, and 82% in the RAPD, ISSR, and SDS-PAGE analyses, respectively. In the dried leaves of MiPs, the phenolic, flavonoid, and ferulic acid contents of 11.53 mg gallic acid equivalent, 12.99 mg catechin equivalent, and 45.52 mg were estimated per gram, respectively. However, an IC50 of 0.43, and 1.99 mg mL−1 of MiP dried leaves’ methanolic extract was required to scavenge half of the DPPH, and ABTS free radicals, respectively. The study presented a successful protocol for in vitro propagation of a valued promising plant source of phenolic compounds.

Optimization of efficient direct organogenesis protocol for Punica granatum L. cv. Kandhari Kabuli from mature leaf explants

Abstract: Punica granatum L. is an important horticultural fruit crop with high medicinal and economic value. Its rising commercial demand necessitates the production of high-quality planting material. Here, we describe an efficient protocol for direct organogenesis in Punica granatum L. cv. Kandhari Kabuli from mature leaf explant. The optimized sterilization procedure for explant includes sequential treatment with 70% ethanol (0.75 min), 0.2% Bavistin (15 min), and 0.5% sodium hypochlorite (2 min), which resulted in 83% axenic cultures. The accumulation of phenolics was effectively controlled by subculturing of leaf explants three to four times at a regular interval of 24 h. The organogenic capability of leaf segments was investigated on full-strength Murashige and Skoog (MS) medium supplemented with different plant growth regulators (PGRs), including the cytokinins 6-benzylaminopurine (BAP) and thidiazuron (TDZ) alone or in combination with α-naphthaleneacetic acid (NAA). BAP promoted the greatest morphogenic response as compared to that from TDZ. However, the greatest frequency of shoot induction (43%) was achieved on MS medium supplemented with 10 μM BAP and 2.5 μM NAA under dark incubation for 2 wk. Furthermore, micro-shoot proliferation and elongation were achieved on multiplication medium consisting of MS medium supplemented with 9.0 μM BAP, 2.5 μM Kinetin (KN), and 0.5 μM gibberellic acid (GA3) up to the third subculturing. However, further subculturing resulted in vitrification. A hormone-free medium containing 300 mg L−1 activated charcoal (AC) was found to be effective to reduce vitrification and promote shoot multiplication. In vitro rooting was carried out on the ½ MS basal medium containing 500 mg L−1 AC using shoots from different subculture passages. Successive subculturing tends to have a positive effect on in vitro rooting and increased rooting up to 70.62%. Well-rooted plantlets were acclimatized successfully in the small plastic pots containing sterilized sand and later shifted to the soil. This optimized protocol can be routinely used for rapid large-scale propagation of pomegranate and is a prerequisite for trait improvement via genetic engineering.