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As in vitro culture environments can be mutagenic, it's challenging to produce true-to-true type plants from meristems. Therefore, the present study aimed to devlop an efficient protocol for micropropagation from shoot tip explants of medicinally important Aloe vera using different concentrations of A. vera leaf gel (AvG) as organic supplement to the culture media. Assessment of the genetic stability of A. vera in vitro multiplied plants compared to the donor plant is one of the most important pre-requisites especially before its use for commercial and medicinal purposes. For shoot multiplication, the highest number of axillary shoots recorded was 13.27 ±5.11 with an average length of 2.21 ±0.84 cm on M3 medium (MS + 0.2 mg L−1 of β-indole butyric acid (IBA) + 3 mg L−1 benzylamino-purine (BA) + 5% AvG). All three strength of MS medium (1, 1/2, 1/4) with different concentrations of AvG resulted in 100% root induction after 4 weeks of culture. All the rooted microshoots (100%) were successfully acclimatized. To provide a reliable conclusion on its nutritive supply to MS medium, the total phenolic (TPC), condensed tannins (TTC), flavonoid (TFC), and flavonol (TFlC) content as well as the antioxydant activity of AvG were investigated. TPC and TTC values were found to be 31.02 ± 0.46 μg gallic acid equivalent mg−1 of extract and 4.00 ± 0.96 μg catechin equivalent mg−1 of extract, respectively while TFC and TFlC were 13.58 ± 0.84 and 9.75 ± 0.58 μg rutin equivalent mg−1 of extract, respectively. The results of antioxydant activity of AvG following DPPH, FRAP and ABTS assays were equal to 12.50 ± 0.89; 49.59 ± 2.07 and 64.93 ± 3.23 µmol Trolox equivalent mg−1 of extract, respectively which may explain the positive role of AvG as supplement. The genetic stability of in vitro propagated A. vera plants was evaluated using 10 inter simple sequence repeat (ISSR), 10 start codon targeted (SCoT) and 10 simple sequence repeat (SSR) markers. Out of these, 5 ISSR, 3 SCoT and 6 SSR primers produced resolvable, reproducible and scorable bands. ISSR primers generated 32 amplification products ranging from 200 to 1040 bp in size with a mean number of 6.4 bands while SCoT primers generated 33 amplicons with an average of 11 bands in the range of 160–2410 bp. In the SSR products, 6 bands were observed, each primer produced one band ranging from 410 to 720 bp. All banding profiles were monomorphic proving that the stability of the micropropagated A. vera plants was maintained. This study proved the suitability of supplementing in vitro culture media with AvG to enhance direct regeneration through shoot tip explants of A. vera for large scale micropropagation applications.

This study represents an efficient preliminary protocol for in vitro mass production of two Paulownia species (Paulownia hybrid and Paulownia tomentosa) seedlings by using seed explant. Different concentrations of benzyladenine (BA) or Kinetin (Kin) (0.0, 2.0, 4.0, 6.0, 8.0 and 10.0 mg/L) were tested during multiplication stage. The number of shoots/explants was significantly increased with increasing either BA or Kin concentration; however, the shoot length significantly decreased. Data show that media fortified by BA (10 mg/L) combined with indole butyric acid (IBA) at 1.0 or 1.5 mg/L recorded the highest number of shoots/explant (9.13 and 9.25, respectively). After six weeks during the multiplication stage, data cleared that media fortified by benzyladenine (10 mg/L) combined with IBA at 0.5 mg/L recorded the highest shoot length (3.23 cm). The inclusion of indole butyric acid (IBA) or naphthalene acetic acid (NAA) at 1.0–1.5 mg/L to the medium significantly increased the number of roots/plantlets and the highest root length. The results indicated that IBA supplementation was more effective than NAA for in vitro rooting of both Paulownia species. The best treatment for multiplication was 10 mg/L and 8.0–10 mg/L BA for P. hybrid and P. tomentosa, respectively. Peat moss and sand (1:1, v/v) or peat moss and sand (1:2, v/v) were investigated as soil mixture during the adaptation stage. The results referred that Paulownia species plantlets were successfully survived (100 %) in soil mixture contained peat moss: sand (1:2, v/v). This mixture recorded the highest values of plantlet height and number of leaves/plantlets.

Micropropagation technique has been recommended for plant tissue rejuvenation/reinvigoration and the improvement of clonal plants production commercially. Concerning the limiting factors on in vitro cultivation of Eucalyptus grandis × E. urophylla hybrid clone, the effects of light intensity on in vitro introduction and multiplication phases were assessed. The experimental tissue was collected from ministumps cultivated in a semi-hydroponic system. The results of light intensity on in vitro introduction and multiplication were evaluated with four treatments (fluorescent lamp/40 µmol m−2 s−1, red/blue LEDs 20 µmol m−2 s−1, red/blue LEDs 40 µmol m−2 s−1, and red/blue LEDs 80 µmol m−2 s−1). The explants’ morphological, anatomical, and genetic characteristics were evaluated at 30 d in the in vitro introduction, and 12 subcultures for in vitro multiplication. Based on the results, the treatments F/L 40 µmol m−2 s−1 and R/B 80 µmol m−2 s−1 are the most suitable during in vitro introduction and multiplication of Eucalyptus grandis × E. urophylla. It provides a higher percentage of responsive explants, shoots per explant, vigor, photosynthetic pigment content, adaxial epidermis thickness, abaxial epidermis, mesophyll, palisade parenchyma, spongy parenchyma, and vascular tissues. Furthermore, it was found that the micropropagated plants are identical clones of the Eucalyptus grandis × E. urophylla selected tree; that is, there was no somaclonal variation during the twelve subcultures on in vitro multiplication.

Background: Plant tissue culture involves the use of explants obtained from plants to induce organogenesis with the help of plant growth regulators (PGRs). Micropropagation techniques provide a faster and economical solution to the limitations associated with traditional methods of plant cultivation. The present study focuses on the multiple shoot induction and proliferation of Ficus carica var. Black Jack. Factors that influence the growth of in vitro multiple shoots on the apical buds, which include growth media and PGRs, were investigated in this study. Different concentrations of cytokinins like 6-benzylaminopurine (BAP), Thidiazuron (TDZ), and Kinetin (Kin) were used on woody plant medium (WPM) for the optimization of media for multiple shoot induction and proliferation. Results: Apical buds of Ficus carica var. Black Jack growing in WPM supplemented with BAP produced the healthiest plantlets, with the highest number of multiple shoots. The most efficient medium composition which produced the highest number of multiple shoots (37.8) per growing explant was WPM supplemented with 20 µM BAP. Proliferated multiple shoots were efficiently rooted using WPM + 20 µM BAP + 8 µM indole-3-acetic acid (IAA). This optimized medium composition significantly enhanced the production of multiple, disease-free plantlets using single apical bud explants of Ficus carica var. Black Jack. Conclusions: In the present study the observations indicate that WPM supplemented with 20 µM BAP is the best-suited medium for organogenesis and multiple shoot culture of Ficus carica var. Black Jack, and this technique can be potentially applied for commercialization of the plant. Normal 0 false false false EN-US X-NONE X-NONE /* Style Definitions */ table.MsoNormalTable {mso-style-name:"Tabla normal"; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-parent:""; mso-padding-alt:0cm 5.4pt 0cm 5.4pt; mso-para-margin-top:0cm; mso-para-margin-right:0cm; mso-para-margin-bottom:8.0pt; mso-para-margin-left:0cm; line-height:107%; mso-pagination:widow-orphan; font-size:11.0pt; font-family:"Calibri",sans-serif; mso-ascii-font-family:Calibri; mso-ascii-theme-font:minor-latin; mso-hansi-font-family:Calibri; mso-hansi-theme-font:minor-latin; mso-bidi-font-family:"Times New Roman"; mso-bidi-theme-font:minor-bidi;}

Plantlets of Hemidesmus indicus (L.) R. Br. (Indian sarasaparilla) were developed in vitro in two different types of culture vessels and closure types—(1) culture bottles with polypropylene screw-caps and (2) conical flasks with cotton plugs both containing Murashige and Skoog medium with 1.0 mg L−1 6- benzylaminopurine, 0.5 mg L−1 kinetin, and 0.1 mg L−1 indole-3-acetic acid. Shoots proliferated in the bottles exhibited production of (i) a few leaves with reduced leaf area, (ii) wiry and brittle stem, and (iii) less number of shoots, whereas, the shoots developed in culture flasks were of superior characteristics with healthy leaves. The leaves developed in bottles revealed the presence of under-developed cuticle, lower palisade/spongy ratios, undifferentiated epidermal layers, abnormal and non-functional stomata, absence of periderm, and unorganized ground and vascular tissue systems preceded the development of hyperhydrated shoots. The use of a conical flasks culture system promoted the development of natural anatomical features in plantlets. Overall, the type of culture vessel remarkably influenced the growth and development of in vitro-derived plantlets of H. indicus which could be explored for commercial propagation of this multipotent medicinal plant. Compared with glass bottles, cotton plugged flasks improved shoots formation, morpho anatomical parameters, rooting, and survival of in vitro plantlets of Hemidesmus indicus.

Rheum emodi Wall. ex Meisn is an important medicinal herb used in treating type II diabetes mellitus, arthritis, inflammation, and fever. Roots and rhizomes of this plant possess anthraquinones showing anticancer, antiviral, anti-inflammatory, anti-ulcerogenic, immunosuppressive, and pro-apoptotic activities. In the present study, in vitro propagation and adventitious root establishment were achieved for the detection and quantification of important secondary metabolites. In micropropagation experiments, high rate of callus induction was obtained from mid rib (100%) as well as leaf explants (97.67 ± 1.20%). The highest percent shoot induction was observed from leaf explants (76.25 ± 2.39%) as compared to callus (66.67 ± 2.20%). Regenerated shoots showed 70% rooting response with healthy long roots (5.73 ± 0.15 cm). Adventitious root culture response from nodal and leaf explants was better in liquid medium (93.33 ± 1.67% and 73.13 ± 2.87%) compared to semi-solid medium (85.0 ± 2.89% and 76.67 ± 3.33%). The highest DPPH (2–2 diphenyl, 1-picrylhydrazyl) free radical scavenging activity (92.73 ± 0.07%) and total antioxidant activity (6.24 ± 0.07 µg ascorbic acid equivalent (AAE) mg−1) was recorded in in vitro roots. Maximum phenol content (14.70 ± 0.29 µg gallic acid equivalent (GAE) mg−1) was present in adventitious root culture whereas flavonoid content (82.37 ± 0.12 µg quercetin equivalent (QE) mg−1) in in vitro shoots. The identification and quantification of secondary metabolites in in vitro shoots, in vitro roots, and adventitious root cultures were evaluated via high-performance liquid chromatography-mass spectroscopy (HPLC–MS) analysis which revealed the presence of three anthraquinones, emodin, physcion, and chrysophanol, in adventitious root cultures. HPLC–UV detector analysis showed the highest emodin concentration (2.24%) in in vitro shoots. The present study gives a highly reproducible micropropagation protocol for R. emodi as well as provides an alternative source of anticancer compound, emodin, from in vitro shoots and root cultures.

Eugenia involucrata is an important Brazilian forest species, which it can be used for timber, such as firewood and charcoal, and non-timber purposes, for culinary and medicinal use. Considering the limitations of its seminal reproduction, vegetative propagation, particularly by micropropagation, becomes an alternative for plantlets production. In this study we evaluated the effect of the phytoregulators 6-benzylaminopurine (BAP) and 3-indolebutyric acid (IBA) on the in vitro multiplication stage of nodal segments. The experiment was carried out in a completely randomized design, in a 3 x 4 factorial array, the main factors were BAP (0; 0.1 or 0.2 µM) and IBA (0; 10; 20 or 30 µM). After 60 days of in vitro inoculation, evaluations on the the number of buds, number of shoots, number of leaves, callus formation (%) and number of roots per explant were carried out. BAP had not significant effect, however, the use of IBA alone favours the callogenesis and negatively affected leaves, shoots and roots formation. A moderate multiplication rate was obtained when considering in vitro bud formation. BAP and IBA are dispensable in in vitro multiplication of E. involucrata.

Micropropagation protocol of Oriental Hybrid Lilium cv. Ravenna was developed using bulb scale segments (Basal and Tip) as explants. Surface sterilization of healthy bulb scales with carbendazim 200 ppm for 30 min, then 0.1 percent mercuric chloride for 10 min, then 70% ethyl alcohol for 30 s was superior to all other treatments in recording highest culture asepsis (77.08%) and higher explant survival (86.12%). Explant survival was higher in basal segments (88.54%) compared to tip segments (85.52%). Highest culture establishment was recorded in basal scale segments (68.26%) followed by tip scale segments (55.21%). MS medium augmented with 0.50 mgl−1 Naphthalene acetic acid and 2.0 mgl−1. 6-Benzylamino Purine recorded maximum culture establishment (76.17%), highest bulblet number/explant (5.52) with maximum length of shoots (2.20 cm) and number of leaves (3.39). This treatment combination of growth regulators resulted in highest shoot proliferation (83.33%) along with maximum shoot number (2.41explant−1), shoot length (2.35 cm) and leaf number (5.44) of micro shoots during proliferation stage. Rooting of explants was superior with Indole-3-butyric acid compared to Naphthalene acetic acid. Highest rooting of 92.71% along with maximum number of primary roots shoot−1 (12.06), maximum primary root length (3.17 cm) was documented in Murashige and Skoog medium added with Indole-3-butyric acid 1.50 mgl−1 with best ex vitro survival rate (98.96%) of rooted plantlets during primary hardening in perlite + vermiculite (1:1) mixture.

Jatropha is a drought-tolerant plant producing seed oil for the biodiesel. Limitation to the development of jatropha is unavailability of high-yielding varieties and efficient in vitro regeneration system which is required for micropropagation. In this study, in vitro regeneration system from jatropha juvenile cotyledon was established. Firstly, concentrations of hormones in the MS medium were optimized and it was found that 1.5 mgL-1 benzyl adenine (BA) + 0.05 mgL-1 indole-3-butyric acid (IBA) + 0.5 mgL-1 thidiazuron (TDZ) turned out to be the best for shoot induction (3.82 ± 0.18 shoots/explant). Secondly, shoot induction medium was fortified with different concentrations of glutamine and adenine sulfate. It was found that 25 mgL-1 each of glutamine and adenine sulfate was the most effective, resulting to 9.09 ± 0.37 shoots/explant and 93.0% regeneration frequency. Regenerated shoots were cultured on medium containing 0.5 mgL-1 BA and different concentrations of gibberellic acid (GA3), 0.5 mgL-1 GA3 with 0.5 mgL-1 BA were found to be the best for shoot elongation (2.13 ± 0.18 cm). The highest frequency of root (40%) was observed on the medium with 0.5 mgL-1 IBA. The established procedures will be useful for the mass propagation and genetic transformation of elite jatropha genotypes. Key words: Adenine sulfate (Ads), benzyl adenine (BA), indole-3-butyric acid (IBA), in vitro regeneration, glutamine (Gln).

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.

Plant micropropagation comprises biotechnological tools used for the conservation and mass propagation of orchid species. However, there are few reports of its use for orchids of the subtribe Pleurothallidinae. The present study evaluated the effects of 6-benzylaminopurine (BAP) on shoots formation and development of Dryadella zebrina (Porsch) Luer. For this, seeds were in vitro germinated, and the plantlets were submitted to different concentrations of BAP (0, 3, 6, 9, 12, and 15 µM). The plantlets derived from the treatments 0, 6, and 15 µM of BAP were collected after 60 days in culture and subjected to light microscopy analysis. Our results indicated that BAP increased the formation of the new shoots of D. zebrina, especially in treatment with 6 µM, and its use is indicated for the in vitro multiplication phase. The anatomical analyses of the roots showed a deleterious effect of 15 µM BAP on the meristematic region, with the presence of more vacuolated cells in this zone. Our results represent the first successful report of in vitro propagation for the genus Dryadella and may serve as a basis for further studies of in vitro propagation of phylogenetically related species.

Leaf chlorosis is often a problem in micropropagated Rubus idaeus, which makes successful acclimatization difficult. We found that leaf bleaching becomes extreme when in vitro plants are exposed to continuous fluorescent light in the presence of 6-benzyl adenine (BA) or isopentenyl adenine (2iP). These cytokinins had a profound negative effect on the photosynthetic apparatus measured by the chlorophyll index (ChlIdx), photosystem II efficiency (Fv/Fm) and anthocyanin reflection index (AriIdx). Since calmodulin plays a role in the regulation of photosynthesis, we investigated the impact of calmodulin antagonist Chlorpromazine hydrochloride (CPZ) on this harmful bleaching effect. In cytokinin plus CPZ treated plants, ChlIdx was three times greater and Fv/Fm was increased 50% than in only cytokinins treated plants. while plants in hormone-free control medium had the highest ChlIdx and Fv/Fm. This study illustrates how drugs for humans, like the antipsychotic medication CPZ, can also be useful in other biological systems, such as in this case, where CPZ contributes to the quality of in vitro plants. Key Message: Calmodulin antagonist chlorpromazine hydrochloride can prevent leaf chlorosis in micropropagated raspberry culture, and can be used to optimize commercial raspberry micropropagation and contribute to delivery healthy in vitro plants.

Ammonium nitrate (AN) is one of the major nitrogen sources of Murashige and Skoog (MS) medium. It is prohibited in various countries, including India because it is used in explosive manufacturing. Since MS is the most successful medium used for in vitro culture of many plant species, an attempt was made to achieve the composition of MS medium using ammonium hydroxide and nitric acid as an alternative to AN. This acid–base neutralization product AN, was further characterized by ATR-FTIR spectroscopy. Micropropagation of Musa acuminata cv ‘Grand Naine’ was tested using the alternate MS medium (AMS) and good mean shoot number was achieved. Shoot proliferation of M. acuminata cv ‘Grand Naine’ on AMS was significantly better than on normal MS medium. A 1-year production cycle of M. acuminata cv ‘Grand Naine’ was successfully accomplished with seven successive subcultures and rooting on AMS medium followed by satisfactory acclimation. To check broad cross-species utility of AMS for shoot proliferation, a range of species including Chlorophytum borivilianum, Dalbergia sissoo, Dregea volubilis and Plumbago zeylanica were tested. The in vitro shoot multiplication rate of these species on AMS was statistically not different from MS medium. These results indicate that AN can be replaced with ammonia hydroxide and nitric acid in preparing MS-based medium, without negatively affecting shoot proliferation/ rooting and it would be cost-effective too for micropropagation operations in comparison to commercially available MS medium. Commercial NH4NO3 could be replaced with HNO3 and NH4OH in tissue culture medium to follow the explosive regulations and was efficient for in vitro culture of various plant species.

Korean pine (Pinus koraiensis Sieb. et Zucc.) is a critical coniferous species ecologically. In addition, its fruits have numerous nutraceutical properties and its wood is high quality timber. However, this plant has a low propagation rate and is under risk of extinction. To accelerate breeding of the excellent seedlings and selection from Korean pine, determining methods to increase the number of Korean pine trees successfully is imperative. In this present study, using sprouted seeds as the initial explants, the de novo organogenesis system for this coniferous species has been successfully developed. After 30 days of incubation on Gupta and Durzan (DCR) medium containing 2 mg L−1 kinetin (KT) and 0.5 mg L−1 thidiazuron (TDZ), 92.67% of explants produced direct buds with an average of about 15 buds per explant. We confirmed the organogenic regeneration pattern by a scanning electron microscopy (SEM). For bud elongation after 60 days of culture, we obtained the highest mean length of 34.99 mm on DCR basal media supplemented with 6-benzyladenine (6-BA, 0.2 mg L−1), 1-naphthaleneacetic acid (NAA, 0.1 mg L−1), and activated charcoal (AC, 1 g L−1). The highest rooting rates of 20.74 and 21.48% were obtained within two months of culturing on 1/2 DCR medium (halved major elements) supplemented with 0.05 mg·L−1 NAA and 0.5 or 1 mg ·L−1 indole-3-butyric acid (IBA), respectively. Rooted shoots showed a survival rate of 90.28% after acclimatization in the substrate consisting of perlite, peat, and vermiculite (1:1:1). This protocol is a successful and efficient biotechnological approach to the micropropagation of Korean pine, and the results will be helpful to the clonal propagation and conservation of Korean pine. This study developed a useful protocol for multiplying plantlets from sprouted seed explants of Korean pine, an economically and ecologically important coniferous species.

Bulbophyllum odoratissimum (Sm.) Lindl. ex Wall. is widely used as traditional medicine for the treatment of tuberculosis, chronic inflammation and is listed as an endangered orchid due to over-exploitation and habitat destruction. The present study was carried out to establish a genetically stable in vitro regeneration protocol for mass multiplication using nodal segments and to determine phytochemical constituents, the antioxidant capacity of different parts (leaf, pseudobulb and root) of mother plants and acclimatized in vitro raised plants of B. odoratissimum. The synergistic effect of BA (4.0 mgl−1) and IBA (0.5 mgl−1) was found to be optimum for producing maximum shoot proliferation. IBA was found to be more effective for root proliferation than IAA, NAA and an average (3.08 roots/shoot) was obtained in 0.5 mgl−1 IBA. The well rooted plantlets were successfully acclimatized (more than 90% survival rate) in greenhouse, exhibiting normal development. The ISSR banding profiles confirmed high degree of genetic homogeneity among the clones. Extraction of all plant material from both mother plant and in vitro raised plant possess relevant TPC, TFC, radical scavenging activity (DPPH and ABTS) as well as total reducing power ability. HPLC chromatogram exhibited that phytochemical compounds detected in regenerated plants were similar to those present in mother plant and there was no remarkable difference. High regeneration frequency along with its genetic stability and secondary metabolite production validate the efficiency of this micropropagation protocol of B. odoratissimum for species recovery program as well as a potential source of commercial application.

The purpose of the study was to evaluate Chamerion angustifolium (L.) Holub genotypes for preliminary selection and further breeding programs aimed at obtaining a suitable industrial form for the pharmaceutical applications. Clonally propagated plants representing 10 genotypes of Ch. angustifolium were regenerated under in vitro conditions, hardened and planted in the field. Studies included an evaluation of shoot proliferation, phytochemical assessment of in vitro and ex vitro plants as well as investigations of intraspecies variability regarding four phenological stages: vegetative, beginning of blooming, full blooming, and green fruit phases. Quantitative and qualitative analyses of bioactive compounds were performed using high-performance liquid chromatography coupled with diode array detector and tandem mass spectrometer (HPLC–DAD–MS/MS) and high-performance liquid chromatography (HPLC) methods. The efficiency of shoot multiplication varied between genotypes from 8.12 to 21.48 shoots per explant. A high reproduction rate (> 20 shoots per explant) was recorded for four lines (PL_45, PL_44, PL_58, DE_2). Plants grown in vitro synthesized oenothein B (11.2–22.3 mg g−1 DW) and caffeic acid derivatives. Plants harvested from field contained the full spectrum of polyphenols characteristic for this species, and oenothein B and quercetin 3-O-glucuronide were the most abundant. The maximal content of oenothein B was determined in the vegetative phase of fireweed, while some flavonoids were found in the highest amount in full blooming phase. The results of analysis of variance indicated significant differences among genotypes in oenothein B, 3-O-caffeoylquinic acid and flavonoids accumulation in four phenological phases. PL_44 plants were characterized by high content of oenothein B and quercetin 3-O-glucuronide as well as a relatively high level of other flavonoids. Based on our phytochemical and micropropagation studies, PL_44 genotype was the best candidate for early selection and further breeding programs. Epilobium angustifolium genotypes differ in the shoot multiplication efficiency. Significant genotypic variation among tested genotypes was found in flavonoids, oenothein B and chlorogenic acid level in four developmental phases.

Bananas and plantains are among the most important food crops in Central and West Africa. Their plantation is lead to many problems. In the recent decades, biotechnology tools using in vitro culture technics are used for the mass and free disease plantlets production in order to increase the bananas production and the yield. The main way of in vitro tissue culture at this end is the direct organogenesis i.e., the ability of plant tissues to form various organs de novo by shoots or roots induction to differentiate from a cell or cell clusters. This review aims to summarize the main results obtained in the organogenesis of bananas and plantains (Musa spp.) under in vitro conditions and to identify the challenges during the process. The research articles used in this review show that micropropagation is a reliable alternative to conventional production system of bananas and plantains planting material. However, the use of the in vitro micropropagation for bananas and plantains entails choosing the optimal explant type and size according to objectives. Benzylaminopurine remains the preferred cytokinin for in vitro banana and plantain shoot proliferation, while the use of thidiazuron appears to be more and more common. Whichever cytokinin used, the optimal cytokinin concentration for shoot proliferation is genotype dependent. This review also focuses on the causes and control measures of the two major banana and plantain micropropagation constraints: lethal tissues browning/darkening and microbial contaminations. It showed that applying the suitable and available control measure, according to the evolution of culture, is necessary. All this available information on the in vitro conditions makes banana and plantain cultivars in vitro organogenesis possible.

Rheum spiciforme Royle is a high value medicinal herb restricted to NW Himalayas. The medicinal properties of Rheum include anti-oxidant, anti-microbial, antitumor, anti-inflammatory, anti-fungal, anti-atherosclerotic, anti-proliferative, hepatoprotective, and immuno-enhancing. The species is threatened and endemic which demands its conservation. In this context, we have developed a premiere efficient in vitro regeneration system for this herb. The seed germination displayed phenomenal increase when transferred from soil (13.6 ± 3.1%) to half-strength Murashige and Skoog (MS) medium (92.6 ± 1.3%) fortified with 0.005 mM gibberellic acid (GA3) and 1 mM potassium nitrate (KNO3) and calcium chloride (CaCl2) each with mean germination time (MGT) of 8.5 ± 1.8 d. Among four types of explants used for callusing, leaf explants responded highest with 87.3 ± 1.4% at 2 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 6-benzylaminopurine (BA) each. Nodal-segment-derived brown calluses exhibited significantly high regeneration (96.3 ± 1.6 %) at 8.0 μM BA and kinetin (KIN) each with 4.0 μM GA3. Leaf explants observed direct somatic embryogenesis which displayed maximum (91.0 ± 3.4%) germination at 25 μM BA, 1.0 μM NAA (naphthaleneacetic acid), 2.0 μM GA3, 50.0 μM glutamine (GTM), and adenine sulfate (ADS) each. Multiple shoot induction with mean number of 10.1 ± 2.6 shoots and elongation 4.2 ± 0.4 cm was observed at 12.5 μM BA and 0.5 μM NAA along with 25 μM GTM and ADS each. The rooted seedlings developed in half-strength liquid MS with 2.5 μM NAA were hardened and subsequently transferred to the field. The developed protocol could be utilized for various attributes which include development of large-scale micropropagation system as a conservation measure, Agrobacterium-mediated genetic transformation studies, and industrial production of important bioactive chemical constituents.

The study reports a low-cost micropropagation protocol for Nardostachys jatamansi, a high value, critically endangered Himalayan herb. Shoot-bud bearing rhizome segments of the plant were made to sprout and elongate on 0.8% agar-gelled Murashige and Skoog's medium containing 3% sucrose. The sprouted shoots were then multiplied on agar-gelled as well as liquid MS medium containing various concentrations and combinations of kinetin (Kn) and 1-phenyl-3- (1,2,3-thiadiazol-5-yl) urea (thidiazuron or TDZ). Maximum number of shoots developed per explant after 30 days on 100 ml agar gelled (6.7 shoots) and 30 ml liquid (6 shoots) MS medium containing 0.5 mg l−1 Kn and 0.75 mg l−1 TDZ. Although rooting of in vitro shoots was attempted on full as well as half-strength MS medium containing different auxins, rooting was achieved in 100% shoots (3.07 roots/ shoot) after 30 days on half-strength MS medium supplemented with 1.0 mg l−1 indole-3- acetic acid (IAA). Different potting mixtures were used to acclimatize the micropropagated plantlets under polyhouse condition, but a maximum of 83% survival was recorded in sand and sand plus soil. The genetic fidelity of the in vitro plantlets was also assessed using Start Codon Targeted Polymorphism (SCoT) and Directed Amplification of Minisatellite DNA (DAMD) markers, and a total of 8.03% polymorphism was recorded. Eventually, the cost of micropropagation was reduced by 6.4 times during shoot multiplication and further by half during rooting. The present micropropagation method developed for N. jatamansi can have potential applications in medicinal plant-based industry.

Micropropagation of orchids are often through the production of Protocorm Like Bodies (PLBs) and this research conducted a study to discover the optimal condition for the growth of Protocorm Like Bodies (PLBs) and also plantlets regeneration of Grammatophyllum speciosum Several aspects such as different carbon sources (sucrose, maltose) and the presence of charcoal alongside with the variety of concentration of agar were taken into account for the optimization. This study discovered that the 30 g/L of sucrose in 1 mg/L of BAP was superior in producing heavy PLBs. However, the trend differs in the number of plantlets where maltose (5 g/L) is seen to produce a higher number of plantlets compared to sucrose. Charcoal plays an important role in the growth of PLBs where the heaviest PLBs were recorded in the medium supplemented with 0.20 % of charcoal along with 4.5 g of agar. Vice versa relationship was recorded for the number of plantlets where without charcoal in 4.5 g of agar produce the highest number of plantlets.

Micropropagation was successfully established in the pineapple orchid Dendrobium densiflorum for its conservation and future utilization in the anticancer drug discovery. Micropropagation was carried out via seeds culture on the MS medium. Furthermore, antioxidant and anticancer activities were explored through DPPH and MTT assays. For culture initiation, maximum seed germination (84%) was achieved on half-strength MS medium supplemented with 10% coconut water. Full-strength MS medium supplemented with 15% coconut water was found suitable for highest number of shoot formation from protocorms, while a maximum number of roots were developed on micro-shoots grown on the full-strength MS medium supplemented with 1.5 mg/L IBA. The combination of cocopeat, pine bark and sphagnum moss in the ratio of 2:1:1 was the best substrate used in acclimatization stage resulting in 92% survivability of acclimatized plantlets. Extract of wild plant’s stems (DDW) at 475.28 μg/mL concentration inhibited the 50% DPPH free radicals. Therefore, DDW at 369.16 and 470.97 µg/mL concentrations inhibited the growth of 50% cervical cancer cells (HeLa) and glioblastoma cells (U251) respectively. This research highlighted the micropropagation of D. densiflorum to be utilized in conservation and its potential anticancer drugs to be discovered in future.

lbgBackground and Objective:l/bg The cat whiskers plant (ligOrthosiphon aristatusl/ig Blume Miq) is a plant that has been widely used as raw material for traditional medicine. The population of white-purple varieties of ligO. aristatusl/ig is decreasing efforts to maintain the white-purple ligO. aristatusl/ig need to be done keeping in mind its potential as raw material for traditional medicine. This study aims to determine the composition of a suitable medium in growing plantlet ligO. aristatusl/ig white-purple varieties and the content of its secondary metabolites. lbgMaterials and Methods:l/bg The internode explants were induced on MS medium added by various combinations of zeatin and 2,4-Dichlorophenoxyacetic acid (2,4-D). Root induction was carried out on shoots formed on MS medium with Indole-3-Butyric Acid (IBA). The acclimatization process was carried out using soil media. Determination of secondary metabolite levels was carried out on ligO. aristatusl/ig (ligin vitrol/ig culture) and wild-type plants aged ten months using high-performance liquid chromatography (HPLC). lbgResults:l/bg MS+BAP 2ppm+NAA3 ppm media was the optimal medium for growing shoots in leaf explants. Media MS+zeatin 3 ppm+2,4-D 2 ppm produced good shoot growth on internode explants. The best root induction occurred in MS+IBA media of 0.75 ppm. The acclimatization process was successful on shoots originating from the internode, while those from leaf explants had not succeeded in growing and developing. lbgConclusion:l/bg The levels of rosmarinic acid and sinensetin in the white-purple variety ligO. aristatusl/ig (ligin vitrol/ig culture) were 1.08 and 1.62% w/w and higher than those of wild varieties.

Carnation is an important cut flower in the floriculture industry. A micropropagation method for carnation cv. ‘Irene’ has been standardized. Nodal plant explants were grown in Murashige and Skoog (MS) medium containing plant growth regulators (PGRs). Determination of clonal fidelity of regenerated plantlets was performed with the help of SSR markers. Early formation of micro-shoots with maximum regeneration was noted in the half MS medium in combination of benzyl amino purine (BAP) thidiazuron (TDZ) and silver nitrate (AgNO3) in the concentrations 1.5, 0.25 and 0.5 mg L−1, respectively; while BAP (2.5 mgL−1), TDZ (1.0 mg L−1) and AgNO3 (1.5 mg L−1) produced maximum length of the micro-shoots. Quick induction of roots and maximum number of roots per culture were achieved in half MS basal rooting medium supplemented with 1.5 mg L−1 of indole-3-butyric acid (IBA) and 0.5 mg L−1 of naphthalene acetic acid (NAA). Simple sequence repeat (SSR) markers for clonal fidelity analysis showed uniform genetic fidelity among the proliferated plantlets and the mother plant lines. The protocol can be suitable for commercial cultivation of carnation plants.

Endophytic microorganisms is a major constrain to the establishment and growth of tissue culture plants. We report the use of copper sulfate (CuSO4.5H2O) to eliminate the endophytic bacteria in Philodendron selloum in vitro cultures. Contaminated shoots were cultured onto Murashige and Skoog (MS) medium containing 5 mg/L BA and supplemented with different concentrations of copper sulfate at 0, 35, 70 and 140 mg/L for 6 weeks. Copper sulfate at 70 mg/L completely eliminated the endogenous bacteria without decline in plant growth. However, 35 mg/L copper sulfate was optimal for maximum shoot multiplication (25), survival percentage (100%) and growth of plants. Antioxidant enzymes activity of catalase, peroxidases, and polyphenol oxidase were increased because of copper sulfate treatments. Conversely, electrolyte leakage was decreased at low copper sulfate (≤70 mg/L). Randomly amplified polymorphic DNA (RAPD) analysis revealed that plantlets exposed to different levels of copper sulfate were not genetically different from control plants.

The article presents the results of research work on optimizing the composition of the nutrientmedium of raspberry varieties “Bryanskaya Divo” and “Polka” for clonal micropropagation. Studies haveshown that the sterilizing agent and the time of explant processing are also important for isolating explantsof raspberry varieties. As a result of the study, the effectiveness of treatment with 0.1% mercury(II) chlorideagainst saprophytic microflora for 5 minutes is known. Murashige and Skoog (MS) medium, containing 0.5mg/l of benzylaminopurine (BAA), 0.1 mg/l of indolyl-3-fatty acid (IMS), 0.2 mg/l of gibberellic acid (HA),0.1 mg/l of thiamine (vitamin B1), 0.5 mg/l of pyridoxine (vitamin B6) and nicotinic acid (vitamin PP), 1 mg/lof ascorbic acid, 30 g/l sucrose, 100 mg/l mesoinosite, 2 mg/l glycine, and 10 mg/l iron chelate. are usedas the nutrient medium necessary for the introduction of plants in vitro. The average rate of regeneration ofexplants sterilized and introduced into the culture medium was 95%. During the research work, taking intoaccount the possibility of the presence of latent pathogenic microflora in explants, we examined them in aspecial nutrient medium Viss, poured into Petri dishes. Bacterial damage in the specified medium containing10 g / l sucrose, 15 g/l MdSO47H2O, 8 g/l casein hydrolysate, 2 g/l KN2RO4, 4 g/l yeast extract and 6 g / lgelwright was 4-5%. The MS medium containing 0.5 mg/l, 0.1 mg/l IMS, 100 mg/l mesoinosite, 2 mg/lglycine, and 5 mg/l iron chelate was optimal and effective for clonal micropropagation of raspberry varieties.

Banana is a horticultural plant with very high potentials, which contains carbohydrates and vitamins that are useful in fulfilling people's food and nutritional needs. Hence, this study aims to produce superior banana seedlings and develop a protocol for their mass production using a plant in vitro culture technique. This was a two stage-experiment i.e. microshoot production and plantlet formation. The result showed that Gebyar cultivar produced more shoots than the Kepok Kuning cultivar, with an average of 4.25 microshoots explant-1. However, Kepok Kuning produced more leaves than Gebyar, with an average of 4.64 leaves plantlet-1. Banana shoots cultured on the media containing Indole-3-acetic acid (IAA) at a concentration of 2.5 µM produced the highest leaves number. Meanwhile, those cultured on the media containing 1-Naphthalenesacetic acid (NAA) at a concentration of 7.5 µM produced the highest roots number. A Murashige and Skoog (MS) medium supplemented with 6-Benzylaminopurine (BAP) up to 30 µM and the one supplemented with 7.5 μM of NAA are suitable for Kepok Kuning and Gebyar cultivars micropropagation with regard to microshoot induction and plantlet formation, respectively.

A reliable micropropagation protocol was developed for Red Maple tree for the first time in Iraqi Kurdistan Region to introduce this newly entered species to the local community. At initiation stage, healthy and vigorous cultures were established following a critical disinfestation protocol by bleach (Sodium hypochlorite) at 2.5 % for 20 minutes. The obtained results show that shoots multiplication was successfully enhanced by recording the highest number of shoots and leaves per explant when 0.5 mg.L-l BA was added to WPM by recording 2.08 shoots/ explant and 13.7 leaves/ explant, respectively. Whereas, the longest shoots were obtained when 1.5 mg.L-l BA was added by reaching 1.86 cm. For root formation, IBA was more effective than NAA by giving the best rooting parameters. The highest number of roots per explant (3.00 roots/ explant), the longest roots (5.87 cm) and the highest rooting percentage (100%) were obtained when 0.1 mg.L-l IBA was added to WPM. The half-light intensity (50 feet/ candle) showed better rooting abilities of microshoots than full light intensity conditions (100 feet/ candle) by raising the number of roots from 3.00 to 4.00 roots/ explants, increasing the mean length of roots from 5.87 cm to 6.37 cm when the comparing between the highest rooting parameters at full light illumination. A 100% survival rate was achieved from the acclimatized plantlets in the greenhouse conditions without any abnormal morphological characteristics. It can be recommended that this important plant can be propagated by tissue culture technique toward mass production in the local area.

A biotechnological system for the production of plants biomass and phenylpropanoids of maqui was developed in photomixotrophic TIBs. The in vitro maqui multiplication was evaluated using combinations of TDZ and BAP in TIBs 1L capacity. Treatment of MS basal supplemented with TDZ 1 mg/L shows the best results for the variables fresh weight and multiplication rate. Photomixotrophic conditions were standardized in media with 3%, 2%, 1%, 0% sucrose at a light intensity of 100 µM m−2 s−1. The treatments reduced in sucrose (1 and 2%) and air supplemented with 0.4 MPa CO2 do not differ significantly in biomass production (fresh weight per cluster of plants) compared to the control treatment with sucrose 3% and standard air. Treatment with ABA (1 m/l) induced the production and accumulation of phenylpropanoids metabolites in maqui cultures bioreactors. Phenylpropanoids in in vitro biomass of maqui and culture medium from TIBs were determined in parallel with control samples from wild plants and mature fruits. After the third day of analysis, not significant differences in polyphenols and anthocyanin contents were verified between the treatments of maqui in TIBs + ABA and controls. The non-significant differences in the contents of polyphenols and anthocyanin were maintained until the 15 days of analysis. The antioxidant capacity comparing samples of maqui in bioreactors and wild plants showed no significant differences by the ORAC test from day 5 to day 15 of the study. Results originate an optimized methodology that was scale to SETIS™ bioreactors of 5L capacity. For the first time maqui micropropagation has been established in TIBs followed by elicitation of phenylpropanoids metabolites. Contents of metabolites from maqui in TIBs do not differ from those of wild fruits, while antioxidant capacity of phenylpropanoids was preserved during the study.

Jasmonates (JAs), such as jasmonic acid and its methyl ester, are lipid-derived compounds with signal functions in plant growth and development, as well as in responses to stress. JAs are widely distributed in plants as natural plant growth regulators. JAs do not work independently but work as a part of a complex signaling network with other phytohormones. They are deployed to induce response during wounding and are often used for elicitation and stimulation of secondary metabolites production in different in vitro culture systems. Application of JAs seems to be promising during different steps of the micropropagation system for different species. JAs stimulate proliferation rate of shoots, roots, callus and induce microtubers and bulblets formation. However, negative effects of JAs on the condition of plant tissues are also reported, e.g. leaf senescence, reduced growth and inhibited somatic embryogenesis. This review summarizes the current knowledge of the application and properties of jasmonates under in vitro conditions in terms of cell division, explant growth, proliferation ability, storage organ formation and stress response. The review summarized in detail the results achieved for plants cultivated in vitro in the presence of jasmonates and their possible mechanisms of action.

Graphical abstract, An efficient protocol for in-vitro propagation of an important ornamental crop, Petunia hybrida Vilm. Cv. “Bravo” was developed. The explants that were used to carry out the experiment were Leaf segments, nodal segments and shoot tips. Nodal segments recorded highest per cent asepsis followed by shoot tips and leaf segments. Asepsis was found to be highest when the explants were sterilized with Fungicide (Carbendazim) 0.02% for the duration of 30 min followed by 0.1% HgCl2 for duration of 10 min and then ethanol 70% for 10 s. Longer duration of the sterilant treatment showed more necrotic effects on the explants, thus mercuric chloride treatment when given for 5 min proved to be more effective in terms of survival of the explants. Maximum establishment per cent was recorded in Murashige and Skoog (MS) media fortified with BAP (1.5 mg L−1) and IBA (0.5 mg L−1) in shoot tips and nodal segments, i.e. 97.90 and 95.74% respectively. Callus was efficiently induced and developed when PGR amalgamation of BAP (0.1 mg L−1) and 2,4-D (1.5mg L−1) was used. Kinetin at the concentration of 2.0 mg L−1 along with IBA at 0.5mg L−1 recorded highest callus regeneration in both leaf and internodal segment derived callus. Maximum proliferation percent of shoots (97.90%), highest number of shoots (20.50 explant−1) and maximum length of shoot (2.70 cm) was recorded in PGR combination of IBA and BAP both at 0.5 mg L−1 concentration level. Rhizogenesis was recorded to be highest in the MS media containing IBA 1.00 mg L−1. Best hardening media which recorded maximum survival per cent 92.50% was noticed on the media formulation comprised of equal ratio of perlite and vermiculite mix, under poly house conditions.

The addition of cytokinin drastically increases shoot proliferation in Melaleuca. Individual in vitro shoots previously treated with BA showed better rooting development than BA-free culture medium. Reduction of the osmotic potential in the culture medium decreased dehydration of micropropagated plantlets. Melaleuca alternifolia, known as tea tree, is an Australian medicinal plant widely used in the cosmetic and pharmaceutical industries due to its antibacterial and antifungal properties. Propagation of Melaleuca is limited due to low rates of seed germination and multiplication and the poor rooting of stem cuttings. Thus, micropropagation can be an alternative to the propagation of this woody medicinal species. In this study, different concentrations of 6-benzyladenine (BA) (0, 0.55, 1.11 and 2.22 µM) were tested during the in vitro multiplication phase. It was observed that even the lowest concentration of BA (0.55 µM) could drastically increase the multiplication rate in Melaleuca, compared to the BA-free treatment, due to multiple shoot proliferation. At the rooting stage, in the culture medium without phyto-regulators, individual shoots previously treated with BA had a higher rooting percentage (91–97%) and considerable height growth compared to those of the control treatment (without BA) (66%). However, none of the in vitro plantlets survived to acclimatization stage due to excessive and rapid dehydration of the plantlets under ex vitro conditions, making it the most challenging phase for the micropropagation of Melaleuca. Therefore, a second experimental setup was designed, which included treatments with sucrose, sucrose + sorbitol and sucrose + mannitol in the culture medium at − 0.2170, − 0.3255 and − 0.4340 MPa, respectively, to determine the effects of these osmotic agents on the development of Melaleuca in rooting and acclimatization stages. Sorbitol with sucrose at Ψπ = − 0.4340 decreased stomatal density in leaves and reduced dehydration of plantlets under ex vitro conditions, but was not enough to provide successful plantlets acclimatization.

Vitex negundo L. var. cannabifolia (Sieb. et Zucc.) Hand.-Mazz. is an important medicinal plant rich in bioactive compounds. In vitro tissue culture can be used to produce high-quality starting materials for commercial cultivation. However, the micropropagation technique, especially the comprehensive evaluation of regenerants at the metabolite level, is not well known in this plant species. Employing dormant buds as explants, we developed direct and callus mediated indirect propagation protocols for this species and then evaluated the antioxidant and cytotoxic activities of regenerants. The highest multiple shoot induction frequency of 100%, with a proliferation coefficient of 20.3, was achieved using Murashige and Skoog basal medium with 4.4 µM 6-benzylaminopurine and 1.1 µM 1-naphthylacetic acid. The same medium supplemented with 31.5 µM copper sulfate was optimal for callus differentiation, with a shoot regeneration rate of 81.10%. Inter-simple sequence repeat analysis showed a high genomic fidelity of in vitro regenerants. Chemical, physiological, and cytotoxic analyses indicated that in vitro propagated plants at the adult stage are indistinguishable from wild-grown plants. In particular, the cytotoxicity of V. negundo var. cannabifolia to breast cancer cell line SUM159 reveals the potential of this species for use in treating human breast cancer, with cytotoxic compounds beyond vitexin also apparently observed. Our results indicate that V. negundo var. cannabifolia can be reliably micropropagated in vitro. Moreover, the regenerants have high cytotoxic activity against highly metastatic human breast cancer. We developed efficient micropropagation protocols for Vitex negundo var. cannabifolia, and found the regenerants were consistent with wild-grown plants according to the assessments at the genomic, metabolic, physiological, and cytotoxic levels. We also found the high anticancer activity of V. negundo var. cannabifolia, and confirmed that there are other cytotoxic compounds other than vitexin.

Somatic embryogenesis in Coffea spp. is important for mass production and genetic engineering. Coffee is an exceptional case, as somatic embryogenesis can be induced by applying a cytokinin such as 2iP as the only plant growth regulator. 1-(2-(2-Hydroxyethyl)phenyl)-3-(3-(trifluoromethoxy)phenyl)urea (3TFM-2HE) and 1-(3-bromo-5-(trifluoromethoxy)phenyl)-3-(2-(2-hydroxyethyl)phenyl)urea (3TFM,5Br-2HE) are two newly designed cytokinin oxidase/dehydrogenase inhibitors (CKX) from the diphenylurea group. We used a Coffea arabica leaf disks bioassay to demonstrate the indirect somatic embryo induction potential of these compounds. The leaf disks were incubated on modified Murashige and Skoog (MS) semi-solid medium in which 3TFM-2HE or 3TFM,5Br-2HE were combined with N6-(2-isopentenyl)adenine (2iP). Although these compounds do not possess intrinsic cytokinin activity, they enhanced the activity of 2iP, resulting in direct somatic embryogenesis after seven weeks. The best results were obtained with 1 µM 3TFM-2HE and 5 or 10 µM 2iP. Maturation of somatic embryos into fully developed plants took place on medium supplemented with 0.5 µM kinetin and the somatic embryos developed true leaves and a root system.

Date palm plantlets produced in vitro using tissue culture technique in a greenhouse face a great challenge that may lead to their death due to different ambient environmental conditions, moreover the slow growth rate of the survived tissue culture derived date palm plants during acclimatization are critical problems facing the tissue culture as a commercial technique of date palm propagation. ​Therefore, the success of date palm micropropagation is strongly linked to the success and quality of plantlets in the greenhouse. This study aimed to evaluate the effect of three different cultural systems on survival percentage, plant length, number of leaves and estimation photosynthetic pigments in greenhouse in the first experience as well as effect of foliar spray and watered three concentrations of Sorbitol (0, 15, 30 and 45 g l-1) and salicylic acid at concentrations (0, 20, 40 and 60 mg l-1) on some vegetative growth parameters such as survival percentage, leaf width and growth vigor in the secondary experience. Findings indicated that using both the hydroponic (first culture system) and the traditional systems, with the addition of Barbary-Plante (third culture system) during the acclimatization stage gave highest values of survival percentage, plant length, number of leaves and chlorophyll a+b content compared to the second culture system, where the lowest values were recorded. Also spraying and irrigation with sorbitol at 30 gl-1 plus salicylic acid at 40 mg l-1 giving the highest values of survival percentage, leaf width and growth vigor (91%, 2.73 cm, 4, respectively) compared to other treatments.

Stevia rebaudiana is a herbaceous perennial plant with great global demand due to its beneficial steviol glycosides(SGs) content. Current conventional breeding technique is unable to cater the need for more S. rebaudiana planting materials. Therefore, an improved in vitro shoot regeneration protocol was developed for S. rebaudiana by using chitosan. The highest fresh weight of plant (0.586 ± 0.176 g/explant), dry weight of plant (0.056 ± 0.02 g/explant) and plant height (4.94 ± 1.17 cm/explant) with maximum number of leaves (25.33 ± 6.95 /explant) were observed on explants grown in optimun treatment of MS basal medium supplemented with 1.0 mg/L of 6-benzyaminopurine (BAP) and 60 mg/L of low molecular weight (MW) chitosan after 4 weeks of culture. Scanning electron microscopy (SEM) analysis showed that new shoot primordia and shoot bud formation can be seen as early as day 3 and 5 of cultures on optimun treatment. Further biochemical assays showed that total phenolic acid content, total protein content and total hydrolyzed sugar content were recorded higher in explants cultured in optimun treatment as compared to control media. In contrast, total chlorophyll content and total flavonoids content were reduced in optimum treatment. Meanwhile, no significant difference in antioxidant activity was observed. All cultures from the optimal treatment were successfully regenerated, acclimatized and grew well with 100% survival rate. Taken together, an enhanced and efficient shoot regeneration protocol of S. rebaudiana was successfully developed which will be useful for rapid and large-scale micropropagation in future.

This study was carried out to reformulate half-strength Murashige and Skoog (MS) basal medium, one of the standard media for orchid tissue culture for protocorm-like bodies (PLB) culture of Dendrobium Sabin Blue. Initial PLB fresh mass of 0.5 g was inoculated in various culture treatments for six weeks of culture period. Alteration of half MS medium yielded a modified medium without Fe-EDTA and containing half-strength macronutrients, double-strength micronutrients, full-strength vitamins and 30 g/L sucrose. The modified medium enhanced the proliferation of PLBs by 23% (0.299 ± 0.017 g) as compared half MS medium (0.243 ± 0.012 g) on a dry mass basis. PLB suspension culture was established using liquid medium of the revised formula for a period of 7 weeks. Supplementing the modified medium with quarter-strength of Fe-EDTA is beneficial for long-term culture of PLBs. Improvement of PLB biomass through the use of modified culture medium devoid of plant growth regulators reduces production cost for micropropagation and secondary metabolite production. Besides, it can reduce the likelihood of somaclonal variations related to the use of plant growth regulators in the culture medium.

An in vitro propagation protocol has been developed for Berberis asiatica, an important Himalayan medicinal shrub. Significantly higher in vitro seed germination (50%) was obtained in Murashige and...

Background Switchgrass (Panicum virgatum L.) is an important bioenergy and forage crop. The outcrossing nature of switchgrass makes it infeasible to maintain a genotype through sexual propagation. Current asexual propagation protocols in switchgrass have various limitations. An easy and highly-efficient vegetative propagation method is needed to propagate large natural collections of switchgrass genotypes for genome-wide association studies (GWAS). Results Micropropagation by node culture was found to be a rapid method for vegetative propagation of switchgrass. Bacterial and fungal contamination during node culture is a major cause for cultural failure. Adding the biocide, Plant Preservative Mixture (PPM, 0.2%), and the fungicide, Benomyl (5 mg/l), in the incubation solution after surface sterilization and in the culture medium significantly decreased bacterial and fungal contamination. In addition, “shoot trimming” before subculture had a positive effect on shoot multiplication for most genotypes tested. Using the optimized node culture procedure, we successfully propagated 330 genotypes from a switchgrass GWAS panel in three separate experiments. Large variations in shoot induction efficiency and shoot growth were observed among genotypes. Separately, we developed an in planta node culture method by stimulating the growth of aerial axillary buds into shoots directly on the parent plants, through which rooted plants can be generated within 6 weeks. By circumventing the tissue culture step and avoiding application of exterior hormones, the in planta node culture method is labor- and cost-efficient, easy to master, and has a high success rate. Plants generated by the in planta node culture method are similar to seedlings and can be used directly for various experiments. Conclusions In this study, we optimized a switchgrass node culture protocol by minimizing bacterial and fungal contamination and increasing shoot multiplication. With this improved protocol, we successfully propagated three quarters of the genotypes in a diverse switchgrass GWAS panel. Furthermore, we established a novel and high-throughput in planta node culture method. Together, these methods provide better options for researchers to accelerate vegetative propagation of switchgrass.

Background In vitro culture of olive, as an economically valuable tree, has fundamentally a genotype-dependant low micropropagation rate which needs to be improved in already established and newly released cultivars. Various plant tissue culture media, planting systems and growth factors were evaluated in two promissing Iranian olive cultivars ˈAminˈ and ˈMeshkatˈ and the commercial Spanish cultivar ˈArbequinaˈ. Results The results showed that cultivars have their specific optimal media, i.e. ˈAminˈ in the MS with 4 mg/L zeatin, ˈArbequinaˈ in the OM with 1 mg/L zeatin, and ˈMeshkatˈ in the OM and MS with 2 mg/L zeatin, which produced significantly a higher number of axillary shoots than other media. The results also indicated a significant improvement in the growth indices of ˈAminˈ (number of axillary shoots) when cultured using periodical mini bioreactor (PMB) in the VS medium. In comparison with VS, OM did not reveal any significant differences when both culturing systems (PMB and semi-solid media (SSM)) were used. Regarding the effect of carbon source and light intensity, mannitol and 2000 cd sr m−2 greatly enhanced ˈArbequinaˈ growth indices (main shoot length and growth quality). The results of genetic stability of callus induced shoots (CIS) and meristem induced shoots (MIS) revealed that 2C DNA value assessed by partec flow cytometery (FCM) had 0.01, 0.03 and 0.08 pg discrepencies in ˈAminˈ, ˈArbequinaˈ and ˈMeshkatˈ, repectively. The Amplified Fragment Length Polymorphism (AFLP) results also indicated that the cultivars were classified regardless of the micropropagation origin (CIS or MIS), except for ˈArbequinaˈ. The AFLP findings showed that ˈArbequinaˈ had the highest dispersal (7–38%) in CIS and MIS, while the Iranian cultivar of ˈMeshkatˈ (5–9%) had the highest stability. Conclusions This study indicated the importance of in vitro growth parameters for improving the micropropagation indices of olive cultivars. It showed that optimized protocols (OM, PMB, zeatin, mannitol and 2000 cd sr m−2) co-produced larger calli resulting in indirect organogenesis. Based on FCM and AFLP analysis, it can be concluded that true-to-typeness of micropropagated olive was cultivar-dependent.

Hylocereus polyrhizus is a climbing vine cactus species that grows in tropical area. Recently, it has attracted the attention of public as it provides numerous benefits to humans in term of health especially for those who prioritize health over mostly anything. The objective of this research was to assess the effects of different type of cytokinins which were 6-Benzylaminopurine (BAP) and Kinetin in different concentration with combination of waste coconut water and sucrose on enhancing the in vitro multiplication of H. polyrhizus. It was found that BAP had higher effect in shoot multiplication than Kinetin as the highest number of shoots was produced at 3.67± 0.67 when supplemented with 3.0 mg/L of BAP whereas for Kinetin, it only produced 1.75±0.38 shoots and without cytokinin was 1.17±0.01. However, Kinetin had a slightly better effect in shoot elongation than BAP as Kinetin produced shoots with longer length than BAP. It was found that the longest length produced by Kinetin was 2.51±0.06 while BAP was 2.43±0.05 cm and without cytokinin was 2.18±.0.01.For the production of roots, low concentration of cytokinin in range 0.3 mg/L to 1.0 mg/L provides the best number of roots. In conclusion, addition of cytokinin has significant effect in enhancing the growth and multiplication of vegetative H. polyrhizus.

In clinical treatment, Pleione bulbocodioides (Franch.) Rolfe (PB) has been widely used as an anti-tumor drug. However, low productivity impeded its extensive application. The lack of endosperm in the seeds of PB led to a low natural germination rate, causing inefficient production and long reproductive cycle. Improving production and retaining clonal fidelity are the challenges in its efficient micropropagation. This study is to optimize medium and evaluate clonal fidelity on induction and differentiation of Protocorm-like Body (PLB) from PB. The effects of plant growth regulators (PGR) and natural additives on the formation and proliferation of protocorms from seeds and the induction and proliferation of PLBs were investigated. The ultra-structural were performed via a scanning electron microscope (SEM) and transmission electron microscope (TEM) and clonal fidelity assessment was conducted using histochemical assays, DNA electrophoresis analysis and Inter-simple sequence repeat (ISSR) molecular marker analysis. The optimum medium for inducing protocorm was that the Murashige and Skoog (MS) medium supplemented with 2.0 mg/L 2,4-D, 2.0 mg/L KT (Kinetin), 2.0 mg/L AgNO3, and 50 mL/L coconut water in the orthogonal experiments and validation experiments, and for inducing PLBs was that MS medium with 0.3 mg/L indole-3-butyric acid (IBA), 0.3 mg/L KT, 100 mg/L potato extract, and 1 g/L activated charcoal. The SEM and TEM showed the ultra-structural of PB had no change from protocorm to PLBs, and the DNA electrophoresis showed the total DNA did not change in different stages of PB by induction and differentiation and ISSR analysis revealed no DNA polymorphisms included bulb, PLBS, and the differentiated tissue of PLBs. In conclusion, the DNA hereditary character and ultra-structural of PB was retained using our optimum medium induction and differentiation of PLBs from PB, which will be useful for the efficient micropropagation popularization for PB. The present work reports the DNA hereditary stability character and optimum medium for induction and differentiation of PLBs from Pleione bulbocodioides (Franch.) Rolfe (PB), which will be useful for the efficient micropropagation popularization for PB.

Arbutus unedo L. is a Mediterranean species used for fruit production, it is tolerant to drought and shows regeneration ability following forest fires. Mycorrhizal plants with Tuber borchii add resilience and value. This study aims to test the effect of quercetin on mycorrhizal synthesis between T. borchii and A. unedo. Two genotypes selected for fruit production and hydric stress tolerance, were micropropagated for mycorrhizal synthesis, accomplished during ex vitro rooting in perlite, using lyophilized spores of T. borchii suspended in culture media with different quercetin levels (0–10 µM). Six months after inoculation, plants were transferred to pots and maintained in nursery. Ten and 12 months after inoculation, roots were morphological examined and molecularly characterized using ITS1-5.8SITS2 rDNA region and specific primers. Results showed that mycorrhizae establishment was dependent on studied factors (genotype, quercetin level, and culture medium) and their interaction (genotype X culture medium). Quercetin levels up to 2.0 µM favored mycorrhizae establishment and plant growth, although levels superior to 4 µM showed a toxic effect. Quercetin showed to be an efficient factor on inducing mycorrhiza thriving independent of the genotype. Morphological observations and molecular analysis confirmed the permanence of the fungus association 10 and 12 months after inoculation.

The paper presents the results of 2018-2019 research on improving the technology of growing planting material of bog cranberry (Vaccinium oxycoccos L.) of Krasa Severa, Severyanka, Virussaare varieties on the basis of in vitro. Studied was the effect of the concentrations of growth regulators in the composition of the nutrient medium according to Anderson's recipe on the reproduction and subsequent rooting of micro cuttings, as well as the duration of cultivation and adaptation of micro plants depending on partial pruning of shoots. It has been established that at the stage of introduction into in vitro culture, sterilization of explants with 33% hydrogen peroxide in an exposure of 5-8 minutes with washing in 5 portions of sterile distillate gives 60-80 % of viable shoots. The optimum phase of plant development for the successful introduction of in vitro culture is the swelling of buds. Cultivation of micro cuttings was carried out in a light room at a temperature of 25±2 °С, a photoperiod of 16 hours. The duration of each subculturing was 30-60 days. For the stage of actual micropropagation on Anderson's nutrient medium, an increase in the dose of cytokinin 6-benzylaminopurine (6-BAP) from 0.2 to 0.5 mg/l and an increase in the duration of cultivation from 30 to 60 days contributed to a significant increase in the multiplication factor on average for the tested cranberry varieties.According to the efficiency of micropropagation, the varieties Virussaare and Krasa Severa were distinguished – 9.3-12.0 pcs/stalk, respectively. At the rooting stage, the use of a root-forming reagent of indolyl-3-acetic acid (IUK) in doses of 0.2, 0.5 and 1.0 mg/l in the composition of Anderson's nutrient medium did not affect the quality of root formation and the length of shoots of Virussaare micro-plants. No significant varietal differences in the root-forming ability of microcuttings were found. The tendency of better rooting of micro cuttings was observed in the Virussaare variety (90.3 %) compared to the Severyanka (85.7 %) and Krasa Severa (79.3 %) varieties. Micro plants of the Krasa Severa cultivar were characterized by the longest shoots, the total number of roots was less, but their length was longer in comparison with other cultivars. For the adaptation stage, a substrate from a mixture of lowland peat and sphagnum moss was used (1:1). The efficiency of adaptation of micro plants of cranberry varieties when cutting the tip of the shoots was 100 %. Pruning of micro plants shoots contributed to the formation of more side shoots and better development of the aboveground part of the plants.

Synthetic seed has the potential to be used as a cost-effective tool for propagation and storage of germplasm. In the present study, synthetic seeds were produced by encapsulating nodal segments of Allamanda cathartica in calcium alginate gel. The best gel complex was achieved using 3% sodium alginate and 100 mM calcium chloride (CaCl2·2H2O). Maximum conversion response of encapsulated nodal segments into plantlets was obtained on Murashige and Skoog (MS) medium with 3.0 μM meta-Topolin (mT) and 0.5 μM alpha-naphthalene acetic acid (NAA). Encapsulated nodal segments could be stored at low temperature (4 °C) up to 6 weeks with a survival frequency of 85%. The regenerated shoots rooted on half-strength MS medium containing 0.5 µM indole-3-butyric acid (IBA). Plantlets with adequate root and shoot systems were hardened off successfully with 90% success rate. Inter Simple Sequence Repeat (ISSR) markers were used to evaluate genetic stability among synthetic seed derived plantlets. The repeated high regeneration of plants from alginate-coated axillary buds and apparently good viability of the synseeds after four weeks of storage indicate that this is a viable strategy for the exchange of A. cathartica genetic resources. Synseed technology enhances the efforts of micropropagation and helps in the maintenance of rare, endangered and elite genotypes. Allamanda cathartica possesses high global market value due to the presence of phytoconstituent in leaves and roots of the plant. Plant has ornamental properties for its attractive funnel shaped yellow coloured flowers. Genetic stability of the regenerated plantlets were analyzed using PCR-based molecular markers.

Grafted fruit trees are made up of two parts, the scion and the rootstock. The rootstocks affect the growth and physiological and biochemical treats of scion. The use of grafting technique in vitro produces healthy plants and allows careful study of rootstock and scion interactions. The objective of this research was to achieve a successful protocol for micrografting of the late-flowering Sahand cultivar on two wild almond rootstocks (Badam Kohi and Arjan) and also to determine the best rootstock for mentioned cultivar. In this regard, a factorial experiment was conducted to investigate the effect of rootstock type, culture medium (MD1 = MS, MD2 = MS +0.49 µM IBA + 2.2 µM BA, MD3 = MS + 2.46 µM IBA + 0.44 µM BA) and supporting system (paper bridge and perlite) on micrografting success of ‘Sahand’ scion. The results showed that in micrograft combination of ‘Sahand’/Badam kohi, the highest grafting success (100%) and scion length (27.8 mm) were achieved on the MD2 medium with perlite. The Arjan rootstock increased electrolyte leakage and decreased total chlorophyll of the scion in compare to Badam Kohi rootstock. In the grafted combinations, the highest amount of superoxide dismutase, peroxidase and catalase activities were recorded in ‘Sahand’ on the Badam Kohi rootstock which were significantly higher than in the same scion on Arjan rootstock. It can be concluded that Badam Kohi is more suitable than Arjan as a rootstock for Sahand cultivar. In vitro wedge micrografting was successfully established for Sahand cultivar on wild almond rootstocks using IBA and BA combinations in culture media. Badam Kohi rootstock supported ‘Sahand’ scion by induced more antioxidant enzymes than Arjan rootstock.

Ninebark is a very popular ornamental shrub. Micropropagation is an efficient method for mass production of uniform plant material. This study was designed to develop and optimize conditions at all phases of ninebark micropropagation. For the multiplication stage, the Murashige and Skoog (MS) medium at full concentration and pH 5.8 was chosen as the basal medium. Sorbitol proved a more effective carbohydrate source than fructose, with no adverse effects on shoot vitrification or the medium itself. The best shoot production, both in number and length, was on the medium enriched with 2 and 3 mg·L−1 zeatin. High numbers of shoots were also obtained in treatments with 1 mg·L−1 6-benzyladenine (BA) or 2 mg·L−1 meta-Topolin (mT) in the basal medium. BA was the most cost-effective cytokinin. There was a positive effect of the gibberellic acid on proliferation: the highest shoot number per explant was produced in the presence of 1 mg·L−1 GA3. No effect of the culture age (up to 20 subcultures) on the percentage of regenerating explants was evident, and the highest numbers of shoots were obtained between passages 10 and 17. For rooting, the MS medium at half strength was used. The best rooting was at 1 mg·L−1 IBA. Spraying the in vitro rooted cuttings with abscisic acid (ABA) favored plant acclimation to the ex vitro conditions. Exvitro rooting, including the treatments with IBA and ABA, shortened the production time by approximately one third.

Dendrobium fimbriatum is an ornamental and medicinal orchid listed in the Red data book of IUCN. Phytohormones’ effect on the in vitro regeneration of the orchid was studied using Mitra medium supplemented with different growth regulators. KN produced effective shoot formation when present alone or in combination with IBA or NAA. The shooting was gradually increased when KN concentration was increased from 0.8 to 4.8 mg L−1, but the opposite response was observed with BAP at higher concentration (4.8 mg L−1). IBA either in combination with BAP or KN promoted effective root development and multiplication. Micropropagated orchids grown in the basal medium devoid of any phytohormone showed 100% monomorphism, while low genetic polymorphism of 1.52% (RAPD—Random Amplification of Polymorphic DNA), 1.19% (ISSR-Inter Simple Sequence Repeat) and 3.97% (SCoT—Start Codon Targeted) was exhibited among the regenerants propagated in the hormone enriched medium. UPGMA (Unweighted pair group method using arithmetic averages) dendrograms showed the grouping of mother plant (MP) with the in vitro regenerants. The principal coordinate analysis (PCoA) further confirmed the clustering patterns as determined by the cluster analysis. The study reported for the first time the successful in vitro propagation of Dendrobium fimbriatum and their genetic stability assessment using molecular markers.

Centella asiatica, a medicinal herb consumed as a food supplement and used in traditional and folklore medicine is being overexploited for its therapeutic properties. Lack of adequate cultivation and replacement practices could engender the depletion of the wild variety. Application of plant tissue culture has helped in the regeneration and preservation of several plants in-vitro. The present study reports media standardization for micropropagation and callus induction for the conservation of Centella asiatica using field-grown plants. In media standardization for callus induction, appreciable results were obtained where full strength MS (Murashige and Skoog) media was supplemented with 2 mg.L-1 IAA (Indole acetic acid) and a combination of 0.5 mg.L-1 Kn (Kinetin) with 1 mg.L-1 IAA while MS media supplemented with a combination of 0.5 mg.L-1 Kn and 1.5 mg.L-1 IAA was used for callus proliferation. Combination of BAP (6- Benzyl amino purine) and IAA from preliminary experimentation on micropropagation showed high shooting frequency and were further optimized with parameters such as sucrose and incubation period using Response surface methodology (RSM) to study their individual and interactive effects. A maximum shoot height of 3.8 cm was obtained from the optimized values after 13 days with both BAP and IAA at 0.5 mg.L-1 and sucrose at 6% with the R2 value at 0.73.

Solanum capsicoides All., is an important medicinal plant which is used as a source of ‘Kantakari’ (medicine for phlegm disorders) in India. The restricted distribution of the plant in Kerala along with its overexploitation necessitates the development of a protocol for in vitro propagation of S. capsicoides. The present study reports the effects of four different cytokinins (2 isopentenyl adenine (2iP), benzyl adenine (BAP), kinetin (KIN), and zeatin (ZEA)) in combination with indole 3-butyric acid (IBA) on indirect organogenesis of S. capsicoides from in vitro grown seedling explants such as cotyledon, shoot tip, and hypocotyl. Maximum shoot development was observed from cotyledon explants inoculated on MS medium supplemented with 2.46 μM IBA and 4.56 μM ZEA. An average of 17 ± 0.5 shoots per cotyledon with an average length of 0.53 ± 0.03 cm was observed. After subculture, an average of 20 ± 1 shoots was observed in cotyledon explants inoculated on Murashige and Skoog medium supplemented with 2.46 μM IBA and 6.84 μM ZEA. Clonal fidelity of regenerated plants was confirmed using DNA based molecular marker, intersimple sequence repeat (ISSR). Clonal fidelity test revealed 0% polymorphism and the PIC value 0 which indicate no diversity from seed-borne plants. The protocol thus developed can be used for rapid multiplication and conservation of S. capsicoides.