Plant Tissue Culture Techniques Research Articles

Green synthesis of copper nanoparticles using Sesbania aculeata to enhance the plant growth and antimicrobial activities

Green synthesis of copper oxide nanoparticles (CuONPs) was performed using Sesbania aculeata leaf extract. The characteristics of CuONPs were studied using the selected area electron diffraction analysis. The effect of nanoparticles on the plant growth including germination efficiency was studied in Brassica nigra at different concentrations (25, 30, 100 mg/100 ml) using plant tissue culture technique. The uptake of copper and potential metal-binding compounds (citrate, phytochelatins, nicotinamide) in xylem and phloem saps of B. nigra was examined. Copper uptake in xylem and phloem sap was found to be higher at 100 mg/100 ml concentration (2.2 ± 0.23 and 5.70 ± 0.9 µM, respectively) when compared to the control (1.0 ± 0.15 and 4.90 ± 0.7 µM) which could be toxic and inhibit the plant’s growth. 25 and 30 mg/100 ml were found to be safer. The root uptake of CuONPs in xylem was observed through the transmission electron microscopy analysis. Since the nanoparticles at higher dose inhibit plant’s growth, potassium leakage rate was analyzed and found to be higher with copper sulfate treatment (0.8 µg/g FW min−1) than with the CuONPs treatment (0.5 µg/g FW min−1) at 100 mg/100 ml concentration. Additionally, we evaluated the antimicrobial activity of CuONPs and the maximum zone of inhibition was found with Phoma destructiva (23 mm), Curvularia lunata (22 mm) at 40 µl concentration. This study shows that CuONPs synthesized using greener technology enhanced the plant growth of B. nigra at a lower dose (25 and 30 mg/100 ml) and act as a strong antimicrobial agent which can act as biofertilizer for both the crop protection and production.

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

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.

In Vitro Micropropagation of Rose (Hybrid Rosa spp.) through Plant Tissue Culture Technique

Roses are the most important cut flowers in the world. Tissue culture of rose has been improved since last twenty years, and exploited for various purposes from basic anatomical and physiological research to micropropagation from auxiliary buds, shoot tips, leaf explants, etc. Single bud nodal stem segments were surface sterilized and then cultured on MS medium supplemented with 30 g/l sugar, 6 g/l agar and different concentrations of BAP(1, 2, 3, 4 and 5mg/l). Among them, the most suitable concentration for shoot initiation and multiplication was 3mg/l BAP. And it also found that the best for shoot elongation occurred in MS medium supplemented with BAP 3 mg/l and GA3 1mg/l. The proliferated microshoots were transferred to root inducing media of half strength MS medium supplemented with 15 g/l sugar, 5 g/l agar and NAA (0.5, 1, 1.5, 2 mg/l). The best rooting was observed at NAA 1 mg/l concentration. The least response in root formation was found at NAA 0.5 mg/l supplementation on half strength MS media.

Effect of Coconut Water on Root Induction of Musa (AA Group) ‘KLUAI NAM THAI’ In Vitro

Kluai Nam Thai’ (Musa AA group) are rare in Thailand and it is at risk of extinction because of environmental changes and human behavior. Hence, the plant tissue culture technique is used for propagation. This research aimed to study the effect of coconut water on root induction of Kluai Nam Thai in vitro. The shootlets from laboratory were cultured on Murashige and Skoog (MS) medium supplemented with coconut water at concentrations 10, 15, 20, and 25% for 1 month. The experimental design was Completely Randomized Design (CRD). The results indicated that MS medium supplemented with 20% coconut water gave the highest average number of roots at 1.85 while MS medium supplemented with 10% coconut water gave the highest length of roots at 2.34 cm which were significantly different (p<0.01) from the other concentrations

In vitro regeneration and acclimatization of pineapple (Ananas comosus L. Merr. var. MD2)

Ananas comosus L. Merr. is asexually propagated through the conventional method from its crowns, slips, hapas, and suckers. Plant tissue culture technique is applied for mass production of clonal pineapple within a shorter period compared to conventional propagation. Hence, this research aims to study the effect of coconut water and optimum sucrose concentration for in vitro growth performance of pineapple. In addition, this research also aims at optimizing the fertilization mechanism for the acclimatization of clonal pineapple ex vitro. The in vitro regeneration was performed by adding a different volume of coconut water and concentration of sucrose into the prepared culture media supplemented with 2.00 mg/L 6-Benzylaminopurine (BAP) and 1.00 mg/L Naphthaleneacetic acid (NAA). For acclimatization method, two different types of fertilizer served as treatments which were compound fertilizers (NPK 15:15:15) and controlled-release fertilizer (AJIB CRF, NPKMg 10:5:18:3). The planting media consisted of topsoil, sand, and compost composition in a ratio of 2:1:2. Results showed that culture media contained 20.00 mL/L coconut water obtained the highest number of shoots (45.33±7.56). On the other hand, culture media supplemented with 30.00 g/L sucrose concentration showed the highest number of shoots (47.25±10.81). Meanwhile, the acclimatization method found that controlled-release fertilizer (AJIB CRF), NPKMg (10:5:18:3) showed about 90% of survival rate. The mean value for the number of leaves and leaves length (cm) in acclimatized clonal pineapple using Controlled-release fertilizer (AJIB CRF) were 5.48±0.96 and 6.00±1.04, respectively. Therefore, the addition of coconut water, optimum sucrose concentration, and the suitable type of fertilizer in both in vitro and ex vitro promote a better growth performance of clonal pineapple.

Tissue culture-induced DNA methylation in crop plants: a review.

Plant tissue culture techniques have been extensively employed in commercial micropropagation to provide year-round production. Tissue culture regenerants are not always genotypically and phenotypically similar. Due to the changes in the tissue culture microenvironment, plant cells are exposed to additional stress which induces genetic and epigenetic instabilities in the regenerants. These changes lead to tissue culture-induced variations (TCIV) which are also known as somaclonal variations to categorically specify the inducing environment. TCIV includes molecular and phenotypic changes persuaded in the in vitro culture due to continuous sub-culturing and tissue culture-derived stress. Epigenetic variations such as altered DNA methylation pattern are induced due to the above-mentioned factors. Reportedly, alteration in DNA methylation pattern is much more frequent in the plant genome during the tissue culture process. DNA methylation plays an important role in gene expression and regulation of plant development. Variants originated in tissue culture process due to heritable methylation changes, can contribute to intra-species phenotypic variation. Several molecular techniques are available to detect DNA methylation at different stages of in vitro culture. Here, we review the aspects of TCIV with respect to DNA methylation and its effect on crop improvement programs. It is anticipated that a precise and comprehensive knowledge of molecular basis of in vitro-derived DNA methylation will help to design strategies to overcome the bottlenecks of micropropagation system and maintain the clonal fidelity of the regenerants.

The effects of paclobutrazol on enhancing tolerance of Plantago major L. to cadmium stress in vitro

The plant tissue culture technique has enabled us to study the tolerance of cells, tissues, and plant organs and cultures to different stresses that plants suffer from. The in vitro culture facilitates the ability to stimulate the production of secondary metabolites. The present study investigated the influence of paclobutrazol (PBZ) pre-treatment on the growth, physiological, morphological, biochemical traits, and phenolic compounds production in callus cultures of Plantago major L. in a medium containing different concentrations of cadmium. The callus was cultured in a Murashige and Skoog (MS) medium with different concentrations of cadmium (0, 50, 100, 150, and 200 µM), with a PBZ concentration of 0 and 2 mg l-1. A factorial experiment based on a completely randomized design (CRD) was performed in triplicate. The results of the growth of the callus culture were obtained after 28 days. The increasing concentrations of cadmium were found to have a negative effect on some traits such as fresh weight (FW), dry weight (DW), browning intensity (BI), tolerance index (TI), electrolyte leakage (EL), and membrane stability index (MSI). The PBZ treatment reduced the effects of cadmium levels through the decrease in malondialdehyde (MDA), and hydrogen peroxide (H2O2). This was caused by a significant increase in superoxide dismutase (SOD) and ascorbate peroxidase (APX). Also, there was an increase in total phenol and flavonoids content in the callus grown on the tissue cultures supporting 200 µM cadmium compared to the other levels. The PBZ pre-treatment was a significant reducing factor to produce total phenolic and flavonoids content in P. major L. callus culture under the cadmium levels.

Salicylic acid: An efficient elicitor of secondary metabolite production in plants

Thousands of structurally and chemically diverse phytochemicals called secondary metabolites are ubiquitously found in the plant kingdom. They are not essential for plant growth and development, yet they act as herbivore repellents and pollinator attractants. This fundamental role is facilitated via modulating the colour and fragrance of the plant/parts. However, they are economically important for humans in pharmaceuticals, nutraceuticals, food additives, and agrochemicals. Therefore, different ways and means have been explored to enhance their production and accumulation in the plants. Salicylic acid (SA) is one of the most important plant phenolics that affects seed germination, stomatal movements, pigment accumulation, photosynthesis, ethylene biosynthesis, heat production, enzyme activities, abscission reversal, nutrient uptake, flower induction, membrane functions, legume nodulation, metabolic activities and overall growth and development of the plants. Due to its hormone-like activity, SA has also been employed to different plant species, both in vivo and in vitro, to explore its role in the secondary metabolite synthesis and accumulation. These studies clearly demonstrate that SA can efficiently recuperate the biosynthesis of secondary metabolites in plants. Moreover, plant tissue culture technique per se elicits the secondary metabolites production. Supplementation of SA to the culture medium or short term exposure of the cultures to SA additively boosts the biosynthesis and accumulation of secondary metabolites. All this information is consolidated and presented here in detail to add a new dimension to the role of SA in plants as well as to explore a potent strategy to enhance the production and accumulation of secondary metabolites in plants.

REGENERACIÓN in vitro DE PLANTAS DE Tillandsia viridiflora (Beer) Baker POR ORGANOGÉNESIS DIRECTA

Tillandsia viridiflora (Beer) Baker es una bromelia epífita endémica de México y la destrucción de su hábitat y comercio ilegal han propiciado su inserción como especie amenazada en la NOM-059-SEMARNAT-2010. Para su aprovechamiento comercial como planta ornamental se requiere un sistema de propagación masiva. Las técnicas de cultivo de tejidos vegetales in vitro son una opción para producir gran cantidad de plantas. Los objetivos de este estudio fueron determinar las condiciones óptimas para regenerar plantas in vitro por organogénesis directa y aclimatarlas. La organogénesis se evaluó en medio Murashige-Skoog (MS) con 6-bencilaminopurina (BAP, 0.0 y 8.8 µM) y ácido naftalenacético (ANA, 0.0 y 2.1 µM) a partir de plántulas in vitro germinadas en medio MS con 50% de la concentración de sales. Grupos de brotes se colocaron en medio MS con ácido giberélico (AG3, 0 y 5.77 µM) para promover el alargamiento. El enraizamiento de brotes se evaluó en medio MS con 50% de la concentración de sales adicionado con ANA (0.00 y 2.15 µM) y ácido indol-3-butírico (AIB) (0.00 y 2.15 µM). En la aclimatación se evaluaron dos alturas de planta y dos tipos de sustrato: turba+perlita (1:1) y corteza de pino (Pinus). El estudio se estableció en un diseño completamente al azar y los datos obtenidos se analizaron con ANDEVA; para la comparación de medias se usó la prueba de Tukey. La germinación fue 82% a las cinco semanas. En la organogénesis se indujeron 10.4 brotes por explante con 6.65 µM de BAP y 0.5 µM de ANA y en la multiplicación se obtuvieron 13 brotes con 4.4 µM de BAP y 0.5 µM de ANA, ambos casos en 12 semanas. El alargamiento mayor (1.5 cm) de los brotes se indujo con 5.7 µM de ácido giberélico. El mayor número de raíces (2.4 raíces por planta) se indujo con 1.0 µM de ANA. En la aclimatación la supervivencia fue 100% con plantas de 4.5 y 6.5 cm plantadas en turba+perlita y corteza de pino.

Development of Sinningia magnifica (Otto & A. Dietr.) Wiehler (Gesneriaceae) tissue culture for in vitro production of quinones and bioactive molecules

Quinones and phenolic glycosides display several biological activities and has been reported in Sinningia species (Gesneriaceae family). The plant tissue culture technique is an excellent tool for metabolism process studies and metabolites extraction. The aim of this work was the callogenesis induction and the secondary callus culture optimization in Sinningia magnifica (Otto & A. Dietr.) Wiehler, varying different types and concentrations of growth regulators, light, antioxidant, and culture medium. These conditions may influence the potential and the morphology of the induced callus, and thus the production and variation of secondary metabolites. Our studies found that for S. magnifica, the best method was fixed using Murashige and Skoog medium culture, in the absence of light, at a concentration of 8.0 mgL−1 of 6-Benzylaminopurine and of 2,4-dichlorophenoxyacetic acid, with a callus development reaching a plateau in about 45 days. The potent antioxidant activity of calli was compared with the in natura plant and demonstrated the greater variety of secondary metabolites. 7-hydroxy-6-methoxy-tectoquinone and 7-hydroxy-6-methoxy-α-dunnione were found in higher quantities in in vitro plants culture compared with the in natura plant, demonstrating the importance of using other methods in order to improve the secondary metabolites production and the advantages of this source for bioactive compounds development and exploitation.

Creation of new forms of feeding cereal grasses from A to Z

Aim. To create a new cultivars of perennial cereal grasses with the high feeding and seed productivity, longevity, drought resistance and freezing tolerance in order to increase the quality of livestock super-elite seeds production. DNA certification of economically valuable properties estimation. Methods. Hybridization, a partially known combination. Embryo culture method from immature caryopsis with completion of growing in a nutrient medium. Techniques of plant tissue culture (initiation, multiplication, rooting and adaptation in vitro plants to ex vitro conditions. DNA certification was performed using SSR (Simple Sequence Repeat) marker system. Results. The new competitively cultivar of Agropyron cristatum "Malarytsky" have been created. The system of the cultivar and parental forms registration in the form of molecular genetic passports is presented. Conclusions. The cultivar of Agropyron cristatum "Malarytsky" was turned over to the State Institution “The State Inspection for Testing and Protection of Plant Varieties” for state testing and for inclusion in the State Register of Varieties. The economic efficiency of this new feeding culture cultivating for Belarus and countries with sharply continental climate is presented.
 Keywords: Agropyron cristatum, hybridization, cul populations, in vitro, water-soluble carbohydrates, DNA certification, molecular genetic passports, cultivar.

In Vitro Responses of Plant Growth Factors on Growth, Yield, Phenolics Content and Antioxidant Activities of Clinacanthus nutans (Sabah Snake Grass).

Clinacanthus nutans, commonly known as Sabah snake grass, is one of the more important medicinal plants in Malaysia’s herbal industry. C. nutans has gained the attention of medical practitioners due to its wide range of bioactive compounds responsible for various biological activities, such as anti-cancer, anti-venom and anti-viral activities. Due to its high pharmacological properties, the species has been overexploited to meet the demands of the pharmaceutical industry. The present study was conducted to establish a suitable in vitro culture procedure for the mass propagation of C. nutans. Murashige and Skoog (MS) basal medium, supplemented with different types of cytokinins, auxins, basal medium strength and sucrose concentrations, were tested. Based on the results, a full-strength MS basal medium supplemented with 12 µM 6-benzylaminopurine (BAP) and 30 g/L sucrose was recorded as the best outcome for all the parameters measured including the regeneration percentage, number of shoots, length of shoots, number of leaves and fresh weight of leaves. In the analysis of the phenolics content and antioxidant activities, tissue-cultured leaf extracts assayed at 100 °C exhibited the highest phenolic content and antioxidant activities. The propagation of C. nutans via a plant tissue culture technique was recorded to be able to produce high phenolic contents as well as exhibit high antioxidant activities.

Impact of sterilants on culture establishment of indigenous Musa L. varieties: a step forward for conservation.

In this paper, we are focusing on study of the impact of sterilization on culture establishment of indigenous Musa variety to restore plant diversity and its importance in human life. Biodiversity supports biological system profitability where every species has a significant task to carry out. Biodiversity of plants guarantees an asset for new nourishment harvests and prescriptions. The biodiversity conservation can start from in situ and ex situ techniques. In situ strategies secure the plants in their common natural surroundings, where as ex situ techniques include safeguarding and upkeep of plant species through plant tissue culture technique. Re-establishing Indigenous plant territory is fundamental for conservation of biodiversity. Indigenous plants are those that present normally in a place where they developed. They are the biological premise whereupon the life of animals and human depends. Traditional medicines from plants are significant part of Indian culture. Banana cultivars nowadays are triploid (AAA, AAB, ABB) cross breeds of two species, Musa acuminate Colla (AA) and Musa balbisiana Colla (BB), which separately contribute the A and B genomes. The M. balbisiana which is normally present in North-Eastern belt of India has high nutrients and resistance to biotic and abiotic stresses. Nanjangud rasabale has been found in parts of Mysuru district, known for its stand-out smell, flavor, taste, and time span of sensible convenience anyway incredibly viewed as a jeopardized grouping; red bananas are phenomenal anyway concerning supplement C and potassium, they are better than yellow ones; Meitei Hei is acclaimed for its enjoyableness and incredible alluring quality. It is a cold permissive grouping thriving incredible even at 12-15°C. The accomplishment of plant tissue culture convention relies upon explant sterilization procedure. Determination of sterilant and time span of introduction are additionally basic on the grounds that the living material should not lose their natural action and just contaminants should be wiped out during cleansing. Surface sanitization of explant is a procedure which includes the drenching of explants into suitable convergence of compound sterilants for a predetermined time bringing about the foundation of a contamination-free culture.

Conservation protocols for Ensete glaucum, a crop wild relative of banana, using plant tissue culture and cryopreservation techniques on seeds and zygotic embryos

Ensets (Ensete spp., family Musaceae), are multipurpose plants with great versatility and potential as a food security crop, although they are cultivated only in Ethiopia and few other regions of Africa. Currently due to loss of habitat, intensification of agriculture, genetic diversity of both wild and domestic ensets is at high risk of erosion. Ensete glaucum (Roxb.) Cheesman, a less-known and under-utilized crop wild relative of banana, is found as natural strands in North-eastern regions of India. No report exists on seed conservation or cryopreservation of E. glaucum and the present work was undertaken to fill this knowledge gap. Mature fruits of two seeded accessions of E. glaucum (MZU-DBT-T15 and HSB-8) were collected from plants in natural populations from two geographically distinct North-eastern region of India. Seed morphology, moisture content (MC) of seed and embryo, viability and germination of excised embryos, desiccation and cryosensitivity were studied. Fresh seed (31% MC) viability measured through in vitro germination of excised embryos was not significantly different in both accessions (87–89%). Seeds could tolerate desiccation up to 5–10% MC without significant loss in germination potential (83–84%) and safely stored up to 1 year at room temperature (RT), without loss of viability. Air dehydrated seeds (10 ± 2% MC) and embryos (12 ± 2% MC) were cryopreserved (in liquid and vapour phase of liquid nitrogen). High post-thaw recovery was achieved in both tissues i.e. seeds (80.2 ± 2.2%) and embryos (83.1 ± 2.6%) by in vitro culture of excised embryos on MS medium + 2 µM gibberellic acid + 1 µM 6-benzyl amino purine (BAP) + 1 µM ascorbic acid. The seedlings converted into multiple shoots (15–20 shoots/explant) within 2 months of culture on medium with MS salts + 10 µM BAP + 1 µM indole-3-acetic acid (IAA) + 1 µM ascorbic acid. This study is the first successful report of cryopreservation of E. glaucum using simple air dehydration. Using the method devised, seeds and zygotic embryos of the two accessions of E. glaucum are safely conserved in the Cryogenebank, while in vitro plantlets are maintained in the In Vitro Genebank of ICAR-NBPGR, New Delhi, India. This study is the first successful report of cryopreservation of Ensete glaucum using seeds and zygotic embryos

Phytochemical Analysis and Establishment of Embryogenic Cell Suspension and Agrobacterium-mediated Transformation for Farmer Preferred Cultivars of West African Plantain (Musa spp.).

Banana and plantain are among the foremost staple food crops providing food and livelihood to over 500 million people in tropical countries. Despite the importance, their production is hampered due to several biotic and abiotic stresses. Plant tissue culture techniques such as somatic embryogenesis and genetic transformation offer a valuable tool for genetic improvement. Identification and quantification of phytochemicals found in banana and plantain are essential in optimizing in vitro activities for crop improvement. Total antioxidants, phenolics, flavonoids, and tannins were quantified in various explants obtained from the field, as well as in vitro plants of banana and plantain cultivars. The result showed genotypic variation in the phytochemicals of selected cultivars. The embryogenic cell suspensions were developed for three farmer-preferred plantain cultivars, Agbagba, Obino l’Ewai, and Orishele, using different MS and B5-based culture media. Both culture media supported the development of friable embryogenic calli (FEC), while MS culture media supported the proliferation of fine cell suspension in liquid culture media. The percentage of FEC generated for Agbagba, Obino l’Ewai, and Orishele were 22 ± 24%, 13 ± 28%, and 9 ± 16%, respectively. Cell suspensions produced from FECs were successfully transformed by Agrobacterium-mediated transformation with reporter gene constructs and regenerated into whole plants.

REGENERACIÓN DE PLANTAS DE Agave marmorata Roezl, VÍA EMBRIOGÉNESIS SOMÁTICA

<p><strong>Background. </strong><em>Agave marmorata</em> Roezl is a monocot plant of the Asparagaceae family, which is mainly used in the elaboration of the alcoholic drink called mezcal. This species is propagated by seeds or stolons (hijuelos), but its reproduction is slow because its maturation stage can last up to 35 years until flowreing. Therefore, efficient and alternative propagation methods should be used to ensure the permanence of the resource. Biotechnology, in particular plant tissue culture techniques, represents a viable alternative for the propagation of this species, however, until now, there are no previous reports on the spread of <em>A. marmorata</em> Roezl, via somatic embryogenesis. <strong>Objective. </strong>Evaluate various treatments to regenerate seedlings of this species through somatic embryogenesis. <strong>Methodology.</strong> Callus induction was obtained from mature seeds established in MS (Murashige and Skoog, 1962) at 25%, supplemented with vitamins L2 (Phillips and Collins, 1979), 5 mg L<sup>-1</sup> of 2,4-dichlorophenoxyacetic (2 , 4-D), 3 mg L<sup>-1</sup> of 6-benzyladenine (BA), 60 g L<sup>-1</sup> of sucrose, 8 g L<sup>-1</sup> of agar and adjusted to a pH of 5.7 ± 0.1. These calluses were sectioned into small portions weighing approximately 0.25 g and were incubated in an MS medium at 50% of their concentration with 30 g L<sup>-1</sup> of sucrose, 8 g L<sup>-1</sup> of agar and subjected to eight concentrations of two plant growth regulators (RCV): BA (0.0, 2.0, 6.0 and 10 mg L<sup>-1</sup>) and 2,4-D (0.0 and 5 mg L<sup>-1</sup>), alone or combined, giving a total of eight treatments. Those explants that responded to the embryogenic callus formation were subcultured to 50% MS medium, 30 g L<sup>-1</sup> of sucrose and gelled with 8 g L<sup>-1</sup> agar where two RCV were evaluated independently (0.1 mg L<sup>-1</sup> of 2,4-D or 3 mg L<sup>-1</sup> of AG<sub>3</sub>) and two culture conditions: 16 h light and 8 h dark and complete darkness, giving a total of 24 treatments. The variables evaluated in this work were: percentage of callus induction, percentage of embryogenic structures, number and growth of regenerated seedlings of <em>A. marmorata</em> Roezl at 255 ddic. <strong>Results.</strong> The best response was observed 120 days after starting the culture (ddic) in treatment five with 0.1 mg L<sup>-1</sup> of 2,4-D under light conditions where the pre-treatment was with 10 mg L<sup>-1</sup> of BA, obtaining 19.4 somatic embryos per explant. Maturation of somatic embryos in 50% MS medium was achieved with 30 g L<sup>-1</sup> of sucrose and 8 g L<sup>-1</sup> of agar, without RCV. The 100% of the regenerated seedlings survived and grew under greenhouse conditions. <strong>Implications.</strong> The results of this study contribute to a better understanding of the importance of a pretreatment with high concentrations of cytokinin (BA) and culture conditions, for the regeneration of <em>Agave marmorata</em> Roezl plants, via somatic embryogenesis. This can assist in the initiation of germplasm genetic improvement and <em>in vitro</em> conservation programs. <strong>Conclusions. </strong>The factors evaluated here were important in the induction and expression of embryogenic structures of this species, making possible the regeneration of seedlings of <em>A. marmorata </em>Roezl, from the formation of somatic embryos.</p>

Biotechnological advancements in Catharanthus roseus (L.) G. Don.

Catharanthus roseus (L.) G. Don, also known as Madagascar periwinkle or Sadabahar, is a herbaceous plant belonging to the family Apocynaceae. Being a reservoir for more than 200 alkaloids, it reserves a place for itself in the list of important medicinal plants. Secondary metabolites are present in its leaves (e.g., vindoline, vinblastine, catharanthine, and vincristine) as well as basal stem and roots (e.g., ajmalicine, reserpine, serpentine, horhammericine, tabersonine, leurosine, catharanthine, lochnerine, and vindoline). Two of its alkaloids, vincristine and vinblastine (possessing anticancerous properties), are being used copiously in pharmaceutical industries. Till date, arrays of reports are available on in vitro biotechnological improvements of C. roseus. The present review article concentrates chiefly on various biotechnological advancements based on plant tissue culture techniques of the last three decades, for instance, regeneration via direct and indirect organogenesis, somatic embryogenesis, secondary metabolite production, synthetic seed production, clonal fidelity assessment, polyploidization, genetic transformation, and nanotechnology. It also portrays the importance of various factors influencing the success of in vitro biotechnological interventions in Catharanthus and further addresses several shortcomings that can be further explored to create a platform for upcoming innovative approaches. KEY POINTS: • C. roseus yields anticancerous vincristine and vinblastine used in pharma industry. •In vitro biotechnological interventions prompted major genetic advancements. • This review provides an insight on in vitro-based research achievements till date. • Key bottlenecks and prospective research methodologies have been identified herein.

In vitro culture and adventitious rhizogenesis of Bambusa tulda Roxb. in agro-climatic conditions of Bihar, India

Bambusa tulda is highly economical bamboo, playing an important role as non-timber raw material for large number of products. Forest resource of this bamboo is depleting at a high rate and hence to meet its growing demand in commercial sector, technique for industrial production is required. The present experimental studies were carried out for rapid and mass clonal propagation of B. tulda covering two major aspects of propagation i.e. estab-lishment of aseptic culture and adventitious rhizogenesis. Organogenetic mode of regeneration of plant tissue culture technique has been employed for massive production of this bamboo species. The treatment of explant with Dettol (0.4%) for 10 mins and Bavistin (0.2%) for 30 mins followed by Ethyl alcohol (70%) for 35 seconds and HgCl2 (0.1%) for 10 mins resulted with maximum number of survival of aseptic cultures. The effect of MS medium+ additives+ NAA (0.25mg/l) supplemented with TDZ (0.5mg/l) showed maxi-mum (96%) shoot initiation. Maximum root growth (8.8cm) and number (approx 7 roots/ clump) was observed in ½ strength of MS with IBA (4mg/l) supplemented with coumarin (4mg/l). Seventh sub-culture cycle/ passage was the most suitable and the earliest possible passage of cultures (sub-culturing) for generation of profused in vitro rooting. The establishment of aseptic in vitro cultures and profused adventitious root induction derived from above studies will further help in industrial production of quality planting material of B. tulda within short span of time.

Saving threatened plant species: Reintroduction of Hill's thistle (Cirsium hillii. (Canby) Fernald) to its natural habitat.

Hill’s thistle (Cirsium hillii (Canby) Fernald) is a perennial plant endemic to the Great Lakes region of North America. Hill’s thistle is listed as threatened in Ontario and Canada where it is found in globally rare alvar habitats. The main objective of this study was ex-situ conservation of Hill’s thistle using in vitro culture techniques and reintroduction of micropropagated plants back to their natural habitat in Bruce Peninsula National Park, Ontario, Canada. Two out of twenty-nine available seeds were successfully germinated under in vitro condition. An efficient micropropagation protocol was optimized with 100% survival during acclimatization of plantlets in the greenhouse. Three hundred micropropagated plants were reintroduced to twelve different sites within Bruce Peninsula National Park in June and July 2017. Plants were monitored for survival, rosette growth, and flowering on all sites from 2017–2019. After four months of planting, 67 to 99% of the plants were alive in different sites and 90 to 99% of them survived over winter. In the following years, shoot regeneration and flowering were observed on most sites. This study further confirms the benefit of plant tissue culture techniques to ensure revival of Hill’s thistle ecological biodiversity through the reintroduction of micropropagated plants. This approach consisting of the components of conservation, propagation, and reintroduction (CPR) may potentially serve as a model for saving and enriching other species at risk.

Chitosan effects on the elevation of essential oils and antioxidant activity of Carum copticum L. seedlings and callus cultures under in vitro salt stress

Chitosan is a natural nontoxic biopolymer, which is indicated as an aid for reducing oxidative injury caused by salt stress. To explore the potential effects of chitosan on the production of secondary metabolites and antioxidant activity of Carum copticum L., commonly known as Ajwain, seedlings and callus under in vitro salt stress, total phenolics, protein content, antioxidant enzyme activities, and essential oil contents were measured. Two-week-old C. copticum was cultured on MS medium containing 0, 10 and 20 mg L−1 chitosan and 0 and 100 mM NaCl. Calli induced on MS medium supplemented with 4 μM benzyl amino purine and 1 μM 2, 4-dichlorophenoxyacetic acid were cultured on MS medium containing 4 μM BAP, 1 μM 2, 4-D, and different concentrations of chitosan with 100 mM NaCl. The total phenolics, protein content, and antioxidant enzyme activities significantly increased in callus cultures and seedlings under NaCl treatment, except for the root phenolic content. On the other hand, chitosan suppressed phenolic accumulation and antioxidant enzyme activities under NaCl treatment. Furthermore, both NaCl and chitosan enhanced the contents of thymol and p-cymene in seedlings and calli. No significant difference was observed between calli and seedlings. Regarding the effects of chitosan, 20 mg L−1 chitosan found to be most effective on phenol content with increased production of thymol and p-cymene, and it could serve as a favorable excipient for the pharmaceutical industry. These results suggest that plant tissue culture techniques are a promising approach for improving the production of target secondary metabolites through abiotic elicitors supplemented to the medium.