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A new and efficient method of direct shoot proliferation was developed here in two muskmelon genotypes (Cucumis melo L.), including 'Khatooni' and 'Sooski Sabz' (100%) using 4-day-old cotyledonary explants in vitro. The regeneration of muskmelon was evaluated on Murashige and Skoog (MS) medium supplemented separately with 17 treatments as combinations of two auxins (1-Naphthaleneacetic acid [NAA] and Naphthoxyacetic acid [NOA]) and four cytokinins (Benzylaminopurine [BAP], Thidiazuron [TDZ], Kinetin [KIN], and N-(2-Chloro-4-pyridyl)-N′-phenylurea [CPPU]). The treatments were experimented on 4- and 8-day-old cotyledon explants. A significant degree of organogenesis occurred in 4-day-old cotyledons (P < 0.01) than in 8-day-old cotyledons and shoots grew significantly (P < 0.01) by increasing the concentration of cytokinins in the media. The highest regeneration frequency was obtained in media supplemented with 0.6 mg/l BAP and 0.025 mg/l NOA. Derivatives were transferred to the greenhouse after complete adaptation. For the first time in muskmelon, genetic fidelity was evaluated in mother plants and in regenerated plants by inter simple sequence repeat (ISSR) markers. Ten primers amplified 112 well-resolved bands with an average of 11 bands per primer and ranged from 7 to 13 bands. According to the results of molecular analysis, no genetic variation was observed among the ex vitro and mother plants. The stability of the ploidy level through plant regeneration was verified by a flow cytometry analysis which indicated monomorphic patterns in the regenerated and mother plants. More than other treatments, the combination of BAP and NOA appeared more suitable for the tissue culture of muskmelon. [ABSTRACT FROM AUTHOR]

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

An efficient method of rapid micropropagation of Solanum khasianum Clarke was successfully established from the leaf, petiole, and nodal explants. The morphogenetic response of different concentrations of TDZ and BAP individually or in combination with auxins (IAA/IBA/2,4-D) was tested. Friable callus was obtained on different concentrations of BAP alone or in combination with IAA/IBA/2,4-D. Rapid multiple shoot induction was achieved from friable callus on MS medium supplemented with varying concentrations of TDZ and IBA. The leaf explants exhibited a high frequency of multiple shoots than petiole and nodal explants with an optimal percentage of response (92.73%), mean shoot number (53.5 ± 0.47), and shoot length (11.2 ± 0.53 cm) on MS medium augmented with TDZ (1.5 mg l−1) and IBA (1.5 mg l−1). Maximum rooting efficiency was achieved on MS medium with 1.5 mg l−1 IBA with 12.8 ± 0.36 mean number of roots. The in vitro rooted plants were acclimatized with a survival rate of 80%. The genetic fidelity of the regenerants assayed by the ISSR and the SCoT markers showed no genetic variation. The study examined the micropropagation responses of S. khasianum in the presence of various growth regulators and provided a simple and more suitable protocol adapted for the mass propagation of clones in this species. Key message: We have established a highly efficient micropropagation system for large scale production in Solanum khasianum. Evaluation of clonal fidelity by using ISSR and SCoT markers detected no somaclonal variations. The present study helps to the enhancement of potential alkaloids (solasodine) with the help of biotechnological tools. [ABSTRACT FROM AUTHOR]

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

Prunus africana is an endangered medicinal plant and hence new propagation methods are urgently required to increase its populations. Unfortunately, propagation through seeds is challenging due to its long flowering cycle and recalcitrant seeds. We developed a protocol for micropropagation using nodal segment explants. A woody plant medium supplemented with vitamins, 15 g L−1 sucrose, and 1.0 mg L−1 6-benzylaminopurine (BAP) supported the optimum rate (100%) of axillary shoot initiation. Supplementation with 15 g L−1 sucrose and 1.5 mg L−1 indole-3-acetic acid (IAA) provided the optimum rate (75%) of root initiation. Rooted plantlets were successfully planted in sterilized horticultural soil containing perlite (2:1 v/v) and the survival rate was 98% following acclimatization. The photosynthetic rate assessed using FlourPen FP110 series showed that the ratio of variable fluorescence to maximum fluorescence mean value for in vitro regenerated P. africana (0.830 ± 0.0008) was similar to that of the maternal P. africana plant (0.825 ± 0.005), indicating similarity in their photosynthetic performance; a pivotal process for growth and development. The Fourier transform near-IR (FT-NIR) spectrometer analysis of the in vitro regenerated and the maternal P. africana plant samples exhibited homogeneity in the absorbance peaks at 8,273, 6,344, and 4,938–4,500 cm−1 associated with lipids, starch, and proteins. The genetic fidelity of regenerated plants was confirmed using the randomly amplified polymorphic DNA (RAPD) technique. Our protocol is suitable for use in large-scale P. africana to meet the increasing demands for it in the global market.

Tetraclinis articulata (Vahl) Masters is an endangered tree growing in coastal and arid environments that is widely exploited by the timber and resin industry, among other applications. In this context, the use of in vitro techniques is highly encouraged for its propagation. We present a protocol for micropropagation using twigs from adult trees as a source of explants. The Schenk and Hildebrandt basal medium (SH) supplemented with 30 g L−1 sucrose, 6.5 g L−1 plant agar, 4.0 mg L−1 6-benzyladenine (BA), and 0.05 mg L−1 1-naphthaleneacetic acid (NAA) provided the optimum multiplication rate (90.48 ± 9.52 explants with basal shoots and 2.58 ± 0.29 basal shoots per explant). Application of activated charcoal (AC) or ½ Knop solution in a liquid overlay produced significantly longer shoots. Supplementation of solid media with indole-3-butyric acid (IBA) or NAA gave low rooting percentages (−1 AC improved rooting (40%) but rooting performance was optimal (66.7%) after a pulse treatment consisting of 4 h immersion in liquid SH medium without growth regulators, followed by 8 weeks of cultivation. Rooted microplants were successfully acclimatized (93.33%) in a peat moss and vermiculite mixture (1:1 v/v ratio). The genetic stability of the in vitro regenerated plantlets was confirmed using the randomly amplified polymorphic DNA (RAPD) technique. Explant survival and growth remained higher than 90% after 28 weeks of cold storage at both 4 °C and 10 °C. The protocol presented here allows for largescale T. articulata production and could be applied for both ex situ conservation strategies and industrial purposes.

Muntingia calabura L. is a potent medicinal plant. Because of that several researchers reported phytochemical and pharmacological studies, but only minimal information is obtainable on tissue culture and genetic stability evaluation studies. Therefore, in the present investigation we attempted to establish a reliable direct and indirect regeneration study through leaf and node explants on MS medium containing cytokinins BAP and KN (0.5-3.0 mg l-1) in combination with auxins IAA and NAA (0.5 mg l-1). The breakage of bud and shoot initiation were noticed with the initiation of callus at lower concentrations of auxin alone at 0.5 mg l-1. 2,4-D was found to be good in callus induction from leaf and nodal explants. A problem of browning callus was prevented by regular subculturing of callus cultures. Leaf explants exhibited maximum number of shoots (32±0.88a) with shoot length (7.6±0.17a) and nodal explants obtained optimal number of shoots (26±0.88a) with shoot length (9.6±0.30a) on MS medium supplemented with BAP (2 mg l-1) and IAA (0.5 mg l-1). Half strength MS medium fortified with IBA (2.0 mg l-1) was effective and achieved 70% of rooting. These welldeveloped plantlets were shifted to pots containing soil and vermicompost in 1:1 ratio for acclimatization. The acclimatized plants were field transferred with survival rate of 85%. The ISSR and RAPD markers analysis revealed the genetic stability of in vitro regenerated plants with the mother plant. [ABSTRACT FROM AUTHOR]

Prunus africana is an endangered medicinal plant and hence new propagation methods are urgently required to increase its populations. Unfortunately, propagation through seeds is challenging due to its long flowering cycle and recalcitrant seeds. We developed a protocol for micropropagation using nodal segment explants. A woody plant medium supplemented with vitamins, 15 g L−1 sucrose, and 1.0 mg L−1 6-benzylaminopurine (BAP) supported the optimum rate (100%) of axillary shoot initiation. Supplementation with 15 g L−1 sucrose and 1.5 mg L−1 indole-3-acetic acid (IAA) provided the optimum rate (75%) of root initiation. Rooted plantlets were successfully planted in sterilized horticultural soil containing perlite (2:1 v/v) and the survival rate was 98% following acclimatization. The photosynthetic rate assessed using FlourPen FP110 series showed that the ratio of variable fluorescence to maximum fluorescence mean value for in vitro regenerated P. africana (0.830 ± 0.0008) was similar to that of the maternal P. africana plant (0.825 ± 0.005), indicating similarity in their photosynthetic performance; a pivotal process for growth and development. The Fourier transform near-IR (FT-NIR) spectrometer analysis of the in vitro regenerated and the maternal P. africana plant samples exhibited homogeneity in the absorbance peaks at 8,273, 6,344, and 4,938–4,500 cm−1 associated with lipids, starch, and proteins. The genetic fidelity of regenerated plants was confirmed using the randomly amplified polymorphic DNA (RAPD) technique. Our protocol is suitable for use in large-scale P. africana to meet the increasing demands for it in the global market. [ABSTRACT FROM AUTHOR]

Atropa acuminata Royle ex Lindl. (family Solanaceae) is a critically endangered therapeutic herb, known for its immense therapeutic potential against innumerable ailments. In view of protecting this species from brink of extinction, present work focuses upon development of an effectual micropropagation protocol via indirect organogenesis using root-derived callus. Best callusing was observed on Murashige and Skoog (MS) medium augmented with 10 µM Thidiazuron (TDZ), where 99% of root explants induced whitish-green and compact calluses within 45 days. Upon sub-culturing on same medium (10 µM TDZ), these callus pieces differentiated an average of 5.54 ± 0.65 shoots having 2.38 ± 0.37 cm of length in 71.5% of the explants, after 28 days. MS + 1 µM indole-3-butyric acid (IBA) resulted in optimum rooting response where an average of 8.08 ± 0.19 roots having an average length of 7.28 ± 0.11 cm were produced. Micropropagated plants with well-developed and thick roots were acclimatized under greenhouse conditions. Genetic fidelity of ex vitro acclimatized plants using start codon targeted (SCoT) and sequence-related amplified polymorphism (SRAP) markers, revealed 96% similarity between mother plant and micropropagated plants. These plants showed higher yield of phenols, flavonoids and exhibited better antioxidant (DPPH and phosphomolybdenum assay) activities in comparison with mother plant. Higher levels of photosynthetic pigments and antioxidant enzymes were also observed in these plants as compared to the mother plant. Besides, quantification of atropine and scopolamine using high-performance liquid chromatography (HPLC) in different plant parts of acclimatized plantlets revealed higher amount of both the alkaloids in leaves. The present protocol shall be gainfully employed for large-scale micropropagation, conservation and extraction of biotherapeutic molecules for commercial purposes. [ABSTRACT FROM AUTHOR]

An in vitro regeneration system for propagation of a valuable medicinal tree, Pterocarpus marsupium, using shoot tip (ST) explants derived from 7-d-old axenic seedlings has been successfully developed. Murashige and Skoog (MS) medium containing cytokinins (BA, mT, or Kn) and auxins (NAA, IAA) in different concentrations and combinations showed to have a marked stimulatory effect on the regeneration output. Of cytokinins, meta-topolin (mT 7.0 μM) proved optimum dose for multiple shoot induction yielding 6.50 ± 0.49 shoots per explant with mean shoot length (4.00 ± 0.16 cm) in 70% cultures after 6 wk. Whereas, supplementation of low concentration of auxin (1.0 μM NAA) with optimal cytokinin (7.0 μM mT) favored enhanced shoot induction with increased percent response. At this level, a maximum of 13.54 ± 0.34 shoots per explant with (5.30 ± 0.10 cm) mean shoot length were recorded in 80% cultures after 12 wk. Thereafter, microshoots were isolated and their basal end treated with high doses of indole-3-butyric acid (250 μM) for 5 d followed to transplant onto various potting substrates for ex vitro root formation. A maximum of 3.63 ± 0.0.08 roots per microshoot and mean root length of 3.59 ± 0.07 cm with highest rooting frequency (67.7%) was observed after 4 wk of transplantation. During ex vitro rooting-cum-acclimatization, various physiological parameters were found to be fluctuating initially for 35 d; thereafter, an increasing trend was observed in net photosynthetic rate, intercellular CO2 concentration, and chlorophyll a/b estimation. Overall, the successfully acclimatized regenerants when transferred in natural condition showed about 96.7% survival rate after 3 mo. Assessment of genetic integrity of tissue culture raised plantlets was accomplished by DNA–based ISSR primers. A total of 35 bands with an average of 4.38 bands per primer were scored in monomorphic banding pattern. Thus, it is apparent that the mode of in vitro regeneration protocol was suitable for obtaining true-to-type and disease-free plants. [ABSTRACT FROM AUTHOR]

A droplet-vitrification cryopreservation protocol has been successfully developed for Aranda Broga Blue orchid hybrid using protocorm-like bodies (PLBs). However, maximum growth regeneration percentage was recorded at 5% only based on previous report. Thus, to improve growth recovery of cryopreserved PLBs, cryopreservation stages were supplemented with ascorbic acid, tested at 50, 100 and 150 mg/L. However, results demonstrated that exogenous ascorbic acid was not favorable in regeneration of cryopreserved explants (maximum value of 1.67 % with 50 mg/L ascorbic acid supplementation). Total soluble protein and various antioxidant enzyme activities such as catalase (CAT), superoxide dismutase (SOD) and ascorbate peroxidase (APX) were evaluated after each cryopreservation stages in conjunction with the application of exogenous ascorbic acid. Addition of antioxidant must be carefully evaluated and its application may not guarantee successful growth recovery. RAPD and SCoT molecular analysis confirmed the genetic stability of regenerated cryopreserved PLBs as no polymorphism was detected compared to control PLBs culture.

Ruta chalepensis L., an evergreen shrub in the citrus family, is well-known around the world for its essential oils and variety of bioactivities, indicating its potential medicinal applications. In this study, we investigated the effect of different culture conditions, including plant growth regulators, media types, pH of the medium, and carbon sources, on in vitro regeneration from nodal explants of R. chalepensis. Following 8 weeks of culture, the highest percentage of regeneration (96.3%) and maximum number of shoots (40.3 shoot/explant) with a length of 4.8 cm were obtained with Murashige and Skoog (MS) medium at pH 5.8, supplemented with 3.0% sucrose and 5.0 µM 6-Benzyladenine (BA) in combination with 1.0 µM 1-naphthaleneacetic acid (NAA). For rooting, individually harvested shootlets were transferred on ½ MS (half-strength) supplemented with IAA (indole-3-acetic acid), IBA (indole 3-butyric acid), or NAA, and the best response in terms of root induction (91.6%), number of roots (5.3), and root mean length (4.9 cm) was achieved with 0.5 µM IBA after 6 weeks. An average of 95.2 percent of healthy, in vitro regenerated plantlets survived after being transplanted into potting soil, indicating that they were effectively hardened. DNA assays (PCR-based markers) such as random amplification of polymorphic DNA (RAPD) and directed amplification of minisatellite-region (DAMD) were employed to assess in vitro cultivated R. chalepensis plantlets that produced a monomorphic banding pattern confirming the genetic stability. Additionally, no changes in the flow cytometric profile of ploidy between regenerated plantlets and donor plants were detected. Regeneration of this valuable medicinal plant in vitro will open up new avenues in pharmaceutical biotechnology by providing an unconventional steadfast system for mass multiplication and might be effectively used in genetic manipulation for enhanced bioactive constituents.

Diplocyclos palmatus (L.) C. Jeffrey, commonly referred to as “Shivalingi” or “Lollipop climber” is a valuable medicinal plant with a climbing growth habit used in traditional medicine. It is reputed to have antiarthritic, anti-diabetic properties and to be useful in various skin and reproductive problems. Overexploitation of wild plants and low seed germination have resulted in the decline of the species in the wild. Thus, the present investigation was aimed to establish an effective in vitro propagation procedure for its large-scale production and conservation. Nodal explants, obtained from an established mother plant were grown on MS basal medium augmented with various cytokinins, alone or in combination with auxins, to study the morphogenic response. A maximum of 8.3 shoots/explants with an average shoot length of 7.2 cm were produced after six weeks on MS containing benzylaminopurine 5.0 µM + 1-naphthaleneacetic acid 2.0 µM. After 4 weeks of transfer, microshoots rooted well on a low nutrient medium of ½ MS + 1.0 µM indole-3-butyric acid, with a maximum of 11.0 roots/microshoot and an average root length of 7.4 cm. With an 80% survival rate, the regenerated plantlets were effectively acclimatized to natural conditions. DNA-based molecular markers were used to investigate the genetic uniformity. Scanning Electron Microscopic examination of leaves indicated the adaptation of the plantlets to natural, as evidenced by the formation of normal stomata. Gas chromatography-mass spectrometry analyses of mother and micropropagated plants were performed to identify essential secondary metabolites. The results obtained show that the in vitro propagation system can be adopted for preservation, large-scale production and secondary metabolites’ production in D. palmatus.

The present study describes the first successful report on an efficient in vitro protocol for direct and indirect organogenesis of an important medicinal and anticancerous plant, Solanum erianthum D. Don. An optimal response using leaf explants was achieved in half-strength Murashige and Skoog (MS)-medium supplemented with 5 mg L−1 naphthalene acetic acid for friable callus formation and 2 mg L−1 naphthaleneacetic acid and 0.5 mg L−1 6-benzylaminopurine amino purine for induction of compact callus. The maximum number of shoot buds (20.33 ± 11.55) was obtained from compact callus using indirect shoot organogenesis in MS medium supplemented with 0.5 mg L−1 thidiazuron among the four cytokinins tested, which included 6-benzyl amino purine, kinetin, 6-γ,γ-dimethylallylamino purine, and thidiazuron. Supplements of 3 mg L−1 6-benzyl amino purine played a crucial role in direct shoot bud formation from a leaf. The buds appeared as small protuberances, and finally developed into leafy shoots as the culture period progressed. Regenerated shoot buds were transferred to MS media that contained four cytokinins for shoot tip culture, in which 8 mg L−1 6-γ,γ-dimethylallylamino purine exhibited the best response (15 ± 2.52). Although efficient plant regeneration was achieved in the presence of all four tested cytokinins in the three organogenetic pathways, distinct differences exist in terms of shoot regeneration potential. Half-strength MS basal medium is recommended for root induction. An approximate 70% survival rate of regenerated plantlets was recorded following sequential transfer from culture conditions to the field. The genetic fidelity of the regenerated plants was evaluated using random amplified polymorphic DNA molecular markers, which showed a high number of uniform monomorphic bands. [ABSTRACT FROM AUTHOR]

High efficient and repeatable in vitro regeneration protocol was established from embryo axis, half-seed, axillary meristem, and cotyledonary node explants of chickpea. Various concentrations and combinations of various plant growth regulators (PGRs) were employed to induce multiple shoots, shoot elongation and rooting of shoots to obtain complete plantlets of chickpea. The pretreatment of seeds with 6-benzyl aminopurine (BAP) at 1.0 mg l−1 was found to significantly increase the multiple shoot regeneration from the all explants tested. Among three PGRs such as BAP, kinetin (KIN) and thidiazuron (TDZ) tested for multiple shoot induction; BAP at 2.0 mg l−1 produced the maximum number of shoots in all tested explants. The maximum number of shoots (48.80 shoots/explant) was attained from the embryo axis explant followed by half-seed (32.76 shoots/explant), axillary meristem (28.34 shoots/explant) and cotyledonary node explant (18.47 shoots/explant) on medium augmented with 2.0 mg l−1 BAP along with 0.05 mg l−1 Indole-3-butyric acid (IBA). The optimum percentage of shoot elongation response was recorded (96.68%) on medium fortified with IAA (0.05 mg l−1), GA3 (1.0 mg l−1) and BAP (1.0 mg l−1) with an average shoot length of 8.82 cm. The elongated shoots were successfully rooted in medium augmented with 2.0 mg l−1 IBA. The complete plants were acclimatized in the greenhouse with a survival rate of 72%. The plantlets regenerated from four explants appeared to be morphologically similar to mother plants. The genetic fidelity of in vitro regenerated plants was evaluated using Start Codon Targeted and Inter simple sequence repeats molecular markers. The in vitro regenerated plants from all four explants were found to be the true to type with their mother plant. The in vitro protocol presented in the study should offer as a feasible system for chickpea genetic transformation. Key message: An efficient and reproducible in vitro regeneration protocol was established for chickpea. Application of different concentrations and combinations of PGRs was found to enhance multiple shoot induction, shoot elongation, rooting and acclimatization of in vitro regenerated plants in field conditions, and further evaluated genetic fidelity using molecular markers. [ABSTRACT FROM AUTHOR]

An in vitro micropropagation protocol for Nyctanthes arbor-tristis was developed through the culture of seedling-derived nodal explants. The highest direct shoot induction rate, the mean number of shoots per explant and average shoot length was obtained on MS medium supplemented with 5 mg/l 6-benzylaminopurine (BAP). Full-strength MS medium was found better than other concentrations for organogenesis. Their lower or higher concentration from optimum level was inhibitory for shoot regeneration. Among different carbon sources tested for organogenesis, MS medium supplemented with 87.64 mM sucrose was superior to other carbon sources for in vitro morphogenesis. Best rooting of microshoots was achieved on one-quarter strength MS medium supplemented with 2 mg/l indole-3-butyric acid (IBA). Under this treatment, maximum mean number of roots/shoot and mean root length were 9.40 ± 0.70 and 2.47 ± 0.25 cm, respectively. The genetic fidelity among regenerates and donor plant has been confirmed by ISSR markers. [ABSTRACT FROM AUTHOR]

A simple and efficient protocol developed for plantlet regeneration through in vitro somatic embryogenesis in three ornamental bananas. In vitro propagation, especially somatic embryogenesis has been a method of choice to obtain homogenous planting material in ornamental bananas. In the present investigation, true-to-type plants via somatic embryogenesis were achieved in three ornamental bananas viz. Musa laterita, Musa beccarii and Musa velutina using immature male flower bud as explants. The evaluation was carried out at two stages of somatic embryogenesis, induction, and maturation of somatic embryos. Embryogenic calli desiccated up to 2 h at 25 ± 1 °C resulted in higher frequencies of embryo induction and maturation compared to non-desiccated embryos. The plantlets generated were hardened, and the genetic fidelity of the plantlets was confirmed using SRAP markers. It was observed that banana plants derived via somatic embryogenesis displayed normal phenotype as that of plants grown from a single elite mother plant. In the present study, the successful establishment of plants from male flower bud was achieved in 6 to 7 months which is a relatively short duration compared to other reports involving different species of banana. The protocol developed could be useful for large-scale micropropagation studies in these commercially important ornamental bananas. [ABSTRACT FROM AUTHOR]

The objective of this study was to develop DNA fingerprints of potato progenies by SSR markers for genetic fidelity purpose. Interspecific potato somatic hybrid progenies, developed earlier by crossing somatic hybrids with Indian potato varieties, were used in this study. A total of 81 genotypes (progenies and parents) were analysed by two wellcharacterized potato SSR markers (STU6SNRN and STIIKA), and developed a data matrix for genetic fidelity purpose and other applications. High polymorphism was observed in STIIKA (PIC: 0.98) than STU6SNRN (PIC: 0.95), and also higher number of alleles was observed in STIIKA (22) than STU6SNRN (7). In STU6SNRN, alleles size 179, 182, 190 and 200 bp were predominant; whereas in STIKA, alleles size 195, 201, 221, 235, 242 and 245 were observed frequently in more than 50 per cent of the genotypes. Diversity analysis showed a clear distinction among the genotypes based on the Jaccard dissimilarity coefficient by the Neighbour-joining tree method using the DARwin software. Our study suggests that SSR fingerprints would be of practical utility to strengthen genetic fidelity of these somatic hybrids. Further, SSR fingerprints would also be valuable resources in resolving issues related to identification of true-to-type clone during field evaluation in multiple environments. [ABSTRACT FROM AUTHOR]

The present investigation was carried out to establish an efficient and reproducible micropropagation protocol for the production of morphologically, genetically and chemically uniform plants of Curcuma zedoaria. Axillary bud explants of C. zedoaria were inoculated into MS basal medium supplemented with various combinations and concentrations of 6-benzyladenine (2.2–22.2 µM, BA), kinetin (2.3–23.2 µM, Kin), indole-3-acetic acid (2.9–11.4 µM, IAA), α-naphthalene acetic acid (2.7–10.2 µM, NAA) and adenine sulphate (33.9–203.6 µM, Ads). Almost 95% of rhizome buds sprouted on MS medium supplemented with 13.3 μM BA, 5.7 μM IAA and 63.9 μM Ads giving rise to an average of 12.89 ± 0.02 shoots within 6 weeks. However, the maximum number of roots (25.8 ± 0.07 roots per explant) was obtained on half strength MS medium supplemented with 7.4 µM of IBA after 4 weeks of inoculation. Morphological characteristics were similar in both conventionally propagated and micropropagated plants. Additionally, genetic homogeneity of in vitro plants was further confirmed through ISSR and flow cytometry analysis. A total of 27 ISSR primers were screened, out of which 13 ISSR primers generated 58 monomorphic and reproducible bands thereby confirming the genetic uniformity of obtained plants. The mean 2C DNA content of the mother plant (2.96 pg) was similar to that of in vitro derived plants (3.07 pg). Gas chromatography-mass spectrometry (GC–MS) analysis showed similarity in the qualitative profile of chemical constituents of essential oil and high-performance liquid chromatography analysis revealed no significant differences in curcumin content in the tissue culture regenerants and mother plants of C. zedoaria. Therefore, the present micropropagation protocol could be effectively employed to generate true to type plantlets of C. zedoaria. [ABSTRACT FROM AUTHOR]

To enhance the multiplication rate in Musa acuminata Colla (banana; 'Grand Nain') organogenesis, higher amounts of thiamine along with different sugar types and concentrations were evaluated at the proliferation phase. Thiamine at 1, 10, 50, 100, and 200 mg L−1 was compared with 0.1 mg L−1 thiamine found in conventional Murashige and Skoog (MS) medium. Maximum proliferation of banana was induced with 100 mg L−1 thiamine. Additionally, 15, 30, and 45 g L−1 sucrose, glucose, fructose, and sorbitol combined with regular and optimal levels of thiamine were tested. Glucose at 30 g L−1 most improved shoot proliferation alone and enhanced shoot proliferation further, when combined with 100 mg L−1 thiamine, followed by sucrose and fructose, whereas sorbitol completely inhibited growth and caused tissue browning. All evaluated vegetative traits were significantly affected by sugar type and concentration, and thiamine levels, unlike the photosynthetic pigments. Moreover, genetic stability of the plants recovered from the enhanced protocol was confirmed by inter-simple sequence repeats (ISSR) and randomly amplified polymorphic DNA (RAPD) analysis. A total of 230 bands generated by both marker types were monomorphic for the randomly selected regenerated plants, compared with their mother plant. Thus, the proliferation medium supplemented with 30 g L−1 glucose and 100 mg L−1 thiamine could be recommended for banana organogenesis. Results herein are of great importance and helpful in enhancing the commercial in vitro propagation protocols of banana, without the need of increasing the number of subcultures, which can cause somaclonal variation. [ABSTRACT FROM AUTHOR]

Neolamarckia cadamba is one of the fast-growing tree species selected for industrial tree plantations (ITPs). As demand and supply of true-to-type planting material are increasing, micropropagation of elite N. cadamba planting material is required for the sustainable development of ITPs. However, somaclonal variation among tissue culture-derived plantlets is a bottleneck in micropropagation. The present study described the genetic fidelity of in vitro regenerated N. cadamba plantlets from first, second, third and sixth subcultures. These plantlets were obtained from axillary shoot multiplication and repeated subcultures of microshoots with three weeks interval period, which initially developed from nodal explants. By using five random amplified length polymorphism (RAPD) and two inter-simple sequence repeats (ISSR) primers, a total of 9,334 bands and 2,760 bands were amplified respectively, from a total number of 164 tested plantlets. The banding profiles for each primer was highly uniform, and the DNA bands were monomorphic across all tissue culture-derived plantlets from every four subcultures compared to the stock plants. A target gene-specific marker was also employed to detect single nucleotide polymorphism (SNP) within the targeted genomic sequence of Susy gene. There was no SNP detected from all the analysed plantlets. The current findings ascertained the efficiency and reliability of the N. cadamba micropropagation protocol at least up to six subculture cycles for mass production of true-to-type plantlets. [ABSTRACT FROM AUTHOR]

A proficient and reliable in vitro plant regeneration protocol was established for pea by utilizing cotyledonary node as an explants, which excised from 3-days old imbibed seeds. The present research work explains the positive role of polyamines (PAs) accompanied by plant growth regulators (PGRs) on the multiple shoot induction and rooting in pea plant regeneration. The highest multiple shoots (65.1 shoots/explant) was attained from the cotyledonary node explant on Murashige and Skoog (MS) medium accompanied with 20 mg l−1 spermidine (SPD) along with 1.5 mg l−1 6-benzyladenine (BA). Moreover, the highest elongation of multiplied shoots (10 cm length/shoot) was noted on MS medium enriched with 1 mg l−1 of gibberellic acid (GA3). The elongated shoots produced the highest number of roots (33.66 roots/shoot) on MS medium augmented with 30 mg l−1 putrescine (PUT) along with 0.6 mg l−1 1-naphthaleneacetic acid (NAA). Rooted plants were hardened and acclimatized in the greenhouse with an existence rate of 98%. The standardized procedure with the use of PAs has enhanced the multiple shoot induction to threefold higher than the plants raised from PGRs treatments. The regenerated pea plants revealed the fivefold enhanced photosynthetic pigment, twofold enhanced antioxidant profile and the substantial growth in chloroplast count have been achieved in optimized SPD and BA supplemented MS medium compared to control plant. Start Codon Targeted polymorphism and Random Amplified Polymorphic DNA molecular markers recognized the genetic purity of the in vitro regenerated pea plants for their true type of nature. Key message: An enhanced and consistence in vitro regeneration protocol was established for pea, by the application of polyamines along with PGRs, which significantly improved the multiple shooting, shoot elongation, rooting, total chlorophyll and antioxidant profile of regenerated plants, further not hampering their genetic fidelity. [ABSTRACT FROM AUTHOR]

Korean arbor vitae (KAV; Thuja koraiensis Nakai) is a critically endangered coniferous tree in Korea. Here, we report the somatic embryogenesis (SE) and cryopreservation system that can be used for micropropagation of KAV and long-term storage of KAV cultures. To induce SE in KAV, the influence of the developmental stage of zygotic embryos and the effect of basal medium on embryogenesis induction were examined. The developmental stage of zygotic embryos had a significant effect on the embryogenesis induction (P < 0.0001). The highest frequency of embryogenesis induction occurred in megagametophytes with zygotic embryos at precotyledonary (P) and late embryogeny (L1) stage (36%). The highest frequency of embryogenesis induction was obtained on initiation medium containing IM basal salts with 2.2 μM 6-benzylaminopurine and 4.5 μM 2,4-dichlorophenoxyacetic acid (35%). The effect of abscisic acid (ABA) on production of somatic embryos was tested. The highest number of somatic embryos per 50 mg of embryogenic tissue was achieved on maturation medium with levels of 100 μM ABA (24.0 ± 2.4). The effect of cryopreservation treatment to embryogenic tissues on the maturation capacity of somatic embryos was also tested. No significant differences between noncryopreservation and cryopreservation treatment were observed (P = 0.1896), and the highest mean number of somatic embryo per 50 mg of embryogenic tissues was obtained in noncryopreserved cell line (28.17 ± 5.66). Finally, the genetic identities of the plantlets regenerated from non- and cryopreserved embryogenic cell lines were verified and there was no genetic variation in the regenerated plantlets from cryostored embryogenic cell lines. This study is the first report on SE and the successful cryopreservation of embryogenic culture of the genus Thuja. [ABSTRACT FROM AUTHOR]

Crinum malabaricum , Lekhak & S.R. Yadav (Amaryllidaceae) is a critically endangered bulbous plant endemic to India. Establishing an efficient plant regeneration protocol is necessary for its conservation and large-scale propagation. In the present study, regeneration was achieved through somatic embryogenesis from callus produced on MS media supplemented with various different concentrations of 2,4-D alone and in combination with N6-benzyl-adenine (BA). Different advanced stages of embryo development (globular, torpedo and cotyledonary) and maturation were obtained on MS medium, with combinations of picloram and thidiazuron (TDZ). A high rate of somatic embryos (55.89 ± 0.60) was obtained after eight weeks. SEM examinations showed the occurrence of cell clusters (embryogenic) which converted to somatic embryos. Well-developed cotyledon embryos were successfully germinated on PGRs free full and half strength MS medium, however, 94.03% of somatic embryos germinated on half-strength MS medium supplemented with 1.0 mg L−1 gibberellic acid. The true-to-type genetic conformity of regenerated plants was examined by SCoT, ISSR and RAPD primers based molecular analysis. The amplification bands produced by SCoT, ISSR and RAPD primers applied were monomorphic across all the regenerated plants. This confirmed their genetic homogeneity compared to the mother plant and also demonstrated the reliability of our somatic embryogenesis system for C. malabaricum. A rapid method for phytochemical analysis based on LC-ESI/MS exhibited better separation and analysis of galanthamine and lycorine from methanolic extracts of in vitro raised plants derived from somatic embryogenesis. The protocol developed should be helpful in reintroduction, restoration and ex situ conservation of this valuable plant in the natural condition as well as in the pharmaceutical sectors. [Display omitted] • An efficient somatic embryogenesis system was developed in Crinum malabaricum. • Different concentrations and combinations of plant growth regulators influenced the development of somatic embryos. • The clonal fidelity of somatic embryo-derived plants was confirmed by molecular markers. • Galanthamine and lycorine were efficiently identified by using LC-ESI-MS/MS analysis. • This study will be helpful for large scale clonal propagation and production of secondary metabolites. [ABSTRACT FROM AUTHOR]

This study was conducted to compare the growth and yield of one of the commercial hybrid coffee cultivars (Coffea congensis x Coffea canephora) of robusta coffee established from somatic embryogenesis as well as conventional seedlings. Results indicated no statistically significant differences in the growth pattern or the cumulative yield between the somatic embryogenesis derived plants and the seedlings. The genetic fidelity of somatic embryogenesis derived plants and the mother plant was tested using sequence related amplified polymorphism (SRAP) markers. A total of 24 SRAP primers were employed for DNA analysis which produced a total of 153 clear, distinct and reproducible amplicons of variable size. Out of 24 SRAP primers, 9 primers produced amplification patterns which are identical between the mother plants and plants derived from somatic embryogenesis. Cluster analysis revealed more than 95% genetic similarity between the somatic embryogenesis derived plants and the mother plants indicating a high degree of genetic fidelity. The present study clearly demonstrates the usefulness of SRAP markers in genetic fidelity analysis of coffee.

A micropropagation protocol via direct shoot organogenesis from Streptocarpus x hybridus Voss. leaf explants was established in this study. The shoot induction of three Streptocarpus cultivars (‘Snow White’, ‘Black Panther’ and ‘Slumber Song’) was successfully achieved on Murashige and Skoog (MS) medium supplemented with 0.2 mg L-1 -indole-3-acetic acid (IAA) and 0.2 mg L-1 thidiazuron (TDZ). In proliferation stage, the effects of two combinations of plant growth regulators -PGR- (V1-0.2 mg/L-1 IAA + 0.5 mg/L-1 BAP and V2-1.0 mg L-1 NAA + 0.2 mg L-1 TDZ) on shoot number and length were examined. The results suggest that PGRs combinations significantly influenced shoot proliferation and root induction in all Streptocarpus cultivars. Among the treatments, 0.2 mg L-1 (IAA) in combination with 0.5 mg L-1 6-benzylaminopurine (BAP) were the most effective for in vitro shoot multiplication and rooting. The in vitro rooting percentage was also determined before subjecting the plantlets to the acclimatization process. Due to acclimatization, Streptocarpus plantlets showed a very high rate of survival (90%). The generated PCR-RAPD profiles for the selected in vitro-raised plants and donor plants were similar which indicates the clonal or true-to-type nature of the progenies.

An efficient in vitro direct shoot regeneration system has been described for Lycium chinense Miller using leaf explants. Influence of various parameters such as growth regulator concentration, explant type, effect of basal salt type, Murashige and Skoog (1962) medium (MS), Schenk and Hildebrandt (1972) medium (SH), Gamborg et al. (1968) medium (B5), and carbon sources (sucrose, maltose, and fructose) on the regenerating shoots has been studied. Micromorphological studies and genetic fidelity of regenerated shoots were assessed and compared with those of the donor plants. Among the different concentrations of plant growth regulator (PGRs) tested, MS supplemented with lower concentration of 6-benzylaminopurine (BAP) (0.5 mgL−1) and thidiazuron (TDZ) (0.5 mgL−1) increased the frequency of shoot. Comparatively, indole-3-butyric acid (IBA) was more effective in the regeneration and growth of the root system. A higher number of root formation (6.67 ± 1.25) was observed when the rooting medium comprised half-strength MS salts supplemented with 3% sucrose. The surviving plantlets were gradually transferred to the greenhouse and natural soil. More than 90% of the plantlets survived and matured within 85 days. Similarity in the band patterns produced by inter simple sequence repeat (ISSR) primers confirmed the genetic stability and uniformity between the regenerated and donor plants. The present optimized direct shoot regeneration system may be useful for mass propagation and improving the genetic traits in L. chinense.

Begonias are ornamental indoor plants and also cultivated for landscaping. Begonia malabarica Lam., found naturally in the hilly regions of South India and Sri Lanka called by the tribals as Narayana Sanjeevi, i.e. possess miraculous therapeutic properties. The present study targets in vitro culture, field establishment and evaluation of its genetic uniformity using anatomical, molecular markers followed by callus induction and establishment of cell suspension. Explants such as shoot tip and leaves from in vitro germinated seeds were cultured on Murashige-Skoog medium. Maximum callus induction was obtained in Murashige-Skoog medium with 0.3 mg L-1 2, 4-D + 0.5 mg L-1 BAP, i.e. 90.8 ± 2.8% with 2.5 ± 0.1 g callus growth. In vitro suspension of cells and their growth rate were also analysed. RAPD and ISSR experiments were carried to confirm the genetic fidelity among in vitro and in vivo plants. Twelve RAPD and seven ISSR primers raised 119 amplicons. Genomic DNA amplification showed similarity between the in vivo and the in vitro generated plantlets. The overall results revealed the uniformity of the in vitro raised B. malabarica plantlets in terms of histoanatomical genetical features and substantiated the assumption that in vitro micropropagation is the secure mode for mass propagation of true to type plants without any somaclonal variations. [ABSTRACT FROM AUTHOR]

Morphological variation, genotypic stability and genetic distances among micropropagated tea [Camellia sinensis (L.) Kuntze] cv. 'Kolkhida' plantlets after 7 years of propagation in vitro and among plantlets regenerated from callus were compared. Fifteen individuals, randomly chosen amongst a set of 100 plantlets regenerated from callus, and ten individuals, randomly chosen from a set of 100 micropropagated plants were used in the study. Morphological descriptors showed higher variability among callus-derived regenerants than in micropropagated plants. Flow cytometry analysis showed no significant variability of nuclear DNA content among micropropagated plants, however changes in DNA indicated aneuploidy in three out of ten callus-derived regenerants. ISSR analysis showed lesser genetic distances among micropropagated plantlets and maternal cultivar 'Kolkhida' compared with callus derived plantlets. Yet, there is some degree of genetic instability after long-term micropropagation. Results confirmed the hypothesis of genetic instability of some species during long term in vitro conservation. Key message: Tea vegetative explants plants were propagated in vitro for 7 years. A comparison of tea plantlets derived from callus with plantlets from long-term in vitro propagation revealed genetic variability and confirmed the hypothesis of genotype instability of tea plant. [ABSTRACT FROM AUTHOR]

Meta-topolin (mT), a benzyladenine analog [N 6-(3-hydroxybenzylamino) purine] is a highly active cytokinin. The present study evaluates the efficiency of two aromatic cytokinins, mT and BA for inducing in vitro regeneration in a woody legume Pterocarpus marsupium (Roxb.) using cotyledonary node (CN) explants. Of the two cytokinins tested, mT-derived cultures resulted in better shoot multiplication and rhizogenesis than BA. Among the different doses of mT, maximum shoot (9.58 ± 0.30) induction per explant and average shoot length (4.12 ± 0.05 cm) were recorded on Murashige and Skoog (Physiol Plant 15:473–497, 1962) medium containing 7.5 µM mT, after 6 weeks of culture. The combined effect of cytokinin and auxin was tested, auxin was mixed with optimum doses of BA or mT separately and the effect of combination was studied. Among the cytokinin–auxin combinations, the highest number of shoots (17.44 ± 0.25) per explant and average shoot length (5.72 ± 0.18 cm) were achieved on MS medium containing 7.5 µM mT with 1.0 µM αnaphthalene acetic acid in 85% of the cultures after 12 weeks. Meta-topolin alone or in combination with auxin has shown an increase in the quality and number of shoots in comparison to BA. In vitro rhizogenesis in individually regenerated microshoots was carried out on half-strength MS medium augmented with 1.0 µM indole-3-butyric acid via a two-step procedure method. After 4 weeks, 7.35 ± 0.11 roots per shootlet with an average root length of 4.54 ± 0.10 cm were recorded in mT-derived microshoots. The well-developed plantlets were acclimatized in a separate batch of single CN explant-derived plantlets. About 80% survival rate was recorded for mT-derived plantlets. Biomass and photosynthetic pigments were also improved in mT-derived plantlets, when compared with the BA derived. Analysis of genetic homogeneity of ten micropropagated plantlets was done through RAPD. Out of 40 RAPD primers, 29 primers produced clearly scorable monomorphic bands, thus exhibiting complete genetic uniformity among in vitro regenerated plantlets. [ABSTRACT FROM AUTHOR]

Highlights • The results obtained in the present study are a step forward to develop improved genotype through hybridization in wild pomegranate. • Phenotypic study helps in the selection of best performing individuals of wild pomegranate for domestication. • Attack of fruit borer on fruits showed that controlled pollinated fruits are more prone as compare to fruits open pollinated sources. • The important hybrids obtained were tested for their paternity using 21 RAPD markers of which thirteen primers revealed the clue for hybridity. Abstract The study was conducted in locations Tatool (L 1) district Solan, Narag (L 2) and Daron (L 3) district Sirmour, H.P. The half sibs from Narag and Daron location performed best for seedling height, minimal fruit borer incidence and oval shape. The highest titratable acidity was recorded from genotype L 3 T 3 (Daron location) with elliptical shape which is beneficial for Anardana processing. The tree genotypes bearing ovate and elliptical shaped fruits can be preferred for better fruit size selection. High heritability followed by genetic advance was observed for percent germination and seedling at six month age. In cross L 2 T 2 X L 3 T 3 , reputed fresh weight of seed without aril and germination was better whereas L 1 T 1 X L 2 T 1 cross showed better fruit quality biochemical estimates. The fruit crosses viz. L 2 T 4 X L 2 T 1 and L 2 T 2 X L 2 T 1 were found to have lower incidences of the borer attack. Maximum value in fruit traits were obtained from open pollination as compared to controlled cross combination for all the characters except fruit weight and fresh seed weight with aril. The hybrids were tested for their paternity using 21 RAPD markers of which thirteen primers revealed that 7 hybrids out of 15 hybrids have similar pattern as their parent. [ABSTRACT FROM AUTHOR]

Abstract Multiplication of banana cvs. Grand Naine (Musa AAA, Cavendish-sub group) and Rasthali (Musa AAB, Silk-sub group) were attempted through somatic embryogenesis. The influence of position of male flower buds, amino acid supplements in the induction of somatic embryogenesis and field performance of embryogenic cell suspension (ECS) derived banana plants were studied. Differentiated immature male flower buds positioned at 6–8 th bract whorl as explants showed better callus induction and somatic embryogenesis. Supplementation with glutamine at 400 mg L−1 along with 20:20 g L−1sucrose: maltose in maturation media induced a 10-fold increase in somatic embryo formation compared to control. Cotyledonary stage somatic embryos desiccated for 2 h showed higher germination compared to non-desiccated embryos. The plantlets generated were hardened, and the genetic fidelity of the plantlets was confirmed using ISSR marker. To check the field performance of ECS derived plants, plantlets were hardened and planted in the field along with meristem and sucker. During the field growth, these ECS derived plants were morphologically similar to those of control plants. In this experiment, it was observed that ECS derived banana plants displayed normal phenotype as that of plants grown from meristem and sucker. The protocol developed could be useful highly for large-scale micropropagation or genetic manipulation studies in these commercially important banana cultivars. [ABSTRACT FROM AUTHOR]

Abstract This review article presents a consolidated explanation and provides a comprehensive description of various studies, carried out on in vitro culture and hairy root cultures of S. marianum which can be consider an alternative source of flavonolignans. To overcome the constrains of conventional propagation of silybum plant, tissue culture and advanced biotechnology proved to be an influential tool that can complement conventional breeding and accelerate silybum development. The present review is focused on biotechnological tools like in vitro culture, hairy root cultures and genetic fidelity of S. marianum which can be a potent tool for production of secondary metabolites from these cultures. [ABSTRACT FROM AUTHOR]

In the present study, a simple, eco-friendly, biogenic protocol for synthesis of silver nanoparticles (AgNPs) using leaf extract of in vitro banana plantlets (Musa acuminata Colla cv. Grand Nain) was established. The synthesized AgNPs were characterized by X-ray diffraction, transmission electron microscopy, scanning electron microscopy, and fourier transform-infrared spectroscopy. Shoot tips of stevia (Stevia rebaudiana Bert.) were cultured on three forms of regeneration medium (Murashige and Skoog basal medium + 0.5 mg. L−1 6-benzyladenine) i.e. 'semi-solid', 'liquid', and 'semi-solid followed by liquid' supplemented with 100–400 µM of AgNPs to study the effect of different concentrations of AgNPs along with different forms of medium on plant growth and rebaudioside-A content in stevia under in vitro conditions. Among different concentrations, 400 µM AgNP showed the best growth response irrespective of different forms of medium. Nonetheless, the overall best growth response was recorded in 'semi-solid followed by liquid' medium. High performance liquid chromatography analysis of regenerants showed gradual incline in rebaudioside-A content with the increase in AgNP concentrations. Regenerants from medium containing 400 µM AgNPs produced the highest rebaudioside-A of 334.8 mg. g−1 dry weight (>2-fold) while control ones produced 154.3 mg. g−1 dry weight. Genetic fidelity of AgNP-treated and control regenerants was confirmed using 20 inter simple sequence repeats and start codon targeted primers. The present study can be useful for commercial-scale propagation of stevia ensuring higher fresh and dry biomass as well as ameliorated rebaudioside-A production. • First report on green synthesis and characterization of silver nanoparticles (AgNPs) from in vitro grown banana leaf. • Among three forms, 'semi-solid followed by liquid' medium showed superior growth of stevia. • Ameliorated biomass and rebaudioside-A production with rising AgNP concentrations in stevia. • AgNP-treated regenerants via ISSR and SCoT markers confirmed 100% genetic fidelity. [ABSTRACT FROM AUTHOR]

An efficient micropropagation protocol for Rheum rhabarbarum L. was developed in this study. The in vitro rhubarb plants obtained in the multiplication stage (proliferation rate: 5.0 ± 0.5) were rooted in vitro (96% rooting percentage) and acclimatized ex vitro in floating perlite, with 90% acclimatization percentage. To assess the genetic fidelity between the mother plant and in vitro propagated plants, sequence-related amplified polymorphism (SRAP) markers were used. All banding profiles from the micropropagated plants were monomorphic and similar to those of the mother plant indicating 100% similarity. Regarding the polyphenolic profile, gallic, protocatechuic, p-hydroxybenzoic, vanillic, chlorogenic, caffeic, syringic, p-coumaric and ferulic acid were present in different amounts (2.3–2690.3 μg g−1 dry plant), according to the extracted matrix. Aglicons and glycosides of different classes of flavonoids were also identified. The rhizome extracts (both from in vitro and field grown plants) contained resveratrol, a stilbene compound with high antioxidant properties, ranging between 229.4 to 371.7 μg g−1 plant. Our results suggest that in vitro propagation of Rheum rhabarbarum L. represents a reliable alternative to obtain a large number of true-to-type planting material with high bioactive compound content of this valuable nutritional and medicinal species.

Orthosiphon stamineus has been widely used as traditional remedy for various illnesses and diseases, such as cardiovascular diseases and epileptic seizures. In this study, direct regeneration through nodal segment of this species was attempted using Kinetin (6-Furfurylaminopurine) and IAA (indole-3-acetic acid). Optimum regeneration media was identified as MS media supplemented with 2.0 mg L−1 Kin plus 0.5 mg L−1 IAA. This yielded the highest number of shoots (5.57 ± 0.42) and leaves (20.53 ± 1.91) per explant. Acclimatization of the resulting in vitro regenerants was successful in all potting mixtures tested. However, potting mixture PF (1:1:1 ratio of black soil/red soil/compost) was identified as the best medium for acclimatization of this species, as it yielded 100% survival percentage after 90 days of acclimatization. Ten in vitro regenerants of O. stamineus were randomly collected after the third subculture and subjected to genetic variation analysis using inter-simple sequence repeat (ISSR) markers. Out of 20 ISSR markers tested, 10 working primers were observed to produce satisfactory amplification of bands, with an average of 7.11 bands per primer. A total of 610 bands were produced by the 10 primers. The percentage of polymorphism was observed to be very low, yielding only 7.32% polymorphism among all samples. Jaccard dissimilarity analysis was also conducted and very low genetic distance (about 0.1) was found among the in vitro regenerants and between the regenerants with the mother plant, thus ascertaining the clonal nature of the plantlets produced in this study.

An effective and rapid method for propagating genetically true plantlets of a pharmaceutically important plant Bacopa monnieri L. was developed from thidiazuron (TDZ) pulse treated nodal segment explants. Explants treated for 24 h with different concentration of 0.0, 5.0, 10, 20 or 40 μM thidiazuron (TDZ) prepared in liquid Murashige and Skoog (MS) medium induces multiple shoot on MS basal medium without growth regulators. The highest frequency 95.3 ± 2.3% of regeneration and maximum number 43.7 ± 2.0 of shoots per explants with an average shoot height of 5.4 ± 0.29 cm were achieved after 08 weeks of culture from the explants treated with 20 μM TDZ. Simultaneously, the regenerated microshoots were rooted on the same growth regulator free media. Elongated shoots with roots were successfully acclimated to ex vitro condition with a 97% survival rate. Genomic stability of in vitro developed plants of B. monnieri was first time determined using flow cytometry and single primer amplification reaction (SPAR) methods viz., random amplified polymorphic DNA (RAPD), intersimple sequence repeat polymorphic DNA (ISSR) and directed amplification of minisatellite DNA (DAMD) and compared with donor plant. Nuclear DNA (nDNA) content of in vitro propagated B. monnieri plants has been estimated as 2.08 pg/2C based on the flow cytometric investigation of nuclei isolated from leaf tissues. No significant variations in nDNA content, plant genome size and ploidy level were observed between micropropagated plants and field grown donor plants. Likewise, the SPAR markers also revealed monomorphic banding profiles in micropropagated plantlets of B. monnieri and similar with to that of the donor plants verifying their genomic stability. [ABSTRACT FROM AUTHOR]

A micropropagation protocol via direct shoot organogenesis from Streptocarpus x hybridus Voss. leaf explants was established in this study. The shoot induction of three Streptocarpus cultivars ('Snow White', 'Black Panther' and 'Slumber Song') was successfully achieved on Murashige and Skoog (MS) medium supplemented with 0.2 mg L-1 -indole-3-acetic acid (IAA) and 0.2 mg L-1 thidiazuron (TDZ). In proliferation stage, the effects of two combinations of plant growth regulators -PGR- (V1-0.2 mg/L-1 IAA + 0.5 mg/L-1 BAP and V2-1.0 mg L-1 NAA + 0.2 mg L-1 TDZ) on shoot number and length were examined. The results suggest that PGRs combinations significantly influenced shoot proliferation and root induction in all Streptocarpus cultivars. Among the treatments, 0.2 mg L-1 (IAA) in combination with 0.5 mg L-1 6-benzylaminopurine (BAP) were the most effective for in vitro shoot multiplication and rooting. The in vitro rooting percentage was also determined before subjecting the plantlets to the acclimatization process. Due to acclimatization, Streptocarpus plantlets showed a very high rate of survival (90%). The generated PCR-RAPD profiles for the selected in vitro-raised plants and donor plants were similar which indicates the clonal or true-to-type nature of the progenies. [ABSTRACT FROM AUTHOR]

RAPD and ISSR molecular markers were employed to evaluate genetic fidelity of shoots of Achras sapota L. var. 'Cricket Ball' raised through nodal bud cultures. Out of 53 RAPD and 60 ISSR primers screened, 34 RAPD and 20 ISSR primers generated a total of 270 and 120 clear and scorable amplification products, respectively. All the shoot cultures analyzed at different culture passages showed similar RAPD and ISSR profiles as compared to that of mother plant. No genetic variation was observed in in vitro shoot cultures. The results indicated the genetic stability of the tissue culture raised shoot cultures of A. sapota and corroborate the assumption that axillary multiplication is the safest mode for multiplication of true to type plants without any somaclonal variation. [ABSTRACT FROM AUTHOR]

An efficient and reproducible protocol for in vitro plant regeneration was developed for Lawsonia inermis L. using cotyledonary node explant derived from axenic seedlings. Highest shoot proliferation frequency (ca 96.6%) was achieved on Murashige and Skoog's, 1962 (MS) basal medium supplemented with 8.88 μM 6-Benzyladenine (BA) + 2.68 μM Napthalene acetic acid (NAA). Up-scaling of shoots was carried out using in vitro nodes on MS medium supplemented with 4.44 μM BA. So overall, an average of 238 shoots was produced at 75 days. Of the four different forms of cotyledonary node explants evaluated, highest shoot multiplication was observed in cotyledonary node explant with two whole cotyledons. In vitro regenerated shoots were best rooted (ca 34.3 roots / shoot) on ½ MS medium devoid of any growth regulator. The plantlets were successfully acclimated in sand:soil:: 1:1and established in the garden soil. Random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) analysis revealed a homogeneous amplification profile for all micropropagated plants validating the genetic fidelity of the in vitro-regenerated plants and supporting the regeneration protocol for economic commercial exploitation. [ABSTRACT FROM AUTHOR]

An efficient mass propagation system for a rapid delivery of pro-vitamin A cassava varieties to farmers is essential for acquiring food sufficiency and reducing vitamin A deficiency in Africa. The study investigated multiple shoot inductions from axillary buds of three pro-vitamin A enriched cassava varieties using a combination of 10 mg/l 6-benzylaminopurine (BAP) and 0.1-0.5 mg/l naphthalene acetic acid (NAA). Growth, photosynthetic pigment content and molecular stability of the regenerated plants with random amplified polymorphic DNA (RAPD) markers are also assessed. Shootbud formation in TMS 30572 variety was found to be 8.9% higher than in the three pro-vitamin A varieties. But, the number of regenerated shoots was higher by 7.3% in pro-vitamin A varieties than in TMS 30572. The formation of roots was outstanding in UMUCASS 38. Root carotene contents of regenerated and mother plants were not different. The highest number of shootbuds per explant and frequency of regenerated shoots were obtained through a medium containing 10 mg/l BAP and 0.3 mg/l NAA. The RAPD analysis of regenerated plants showed a uniform profile of 1628 bands among the regenerants and mother plants. The study concluded that a combination of 10 mg/l BAP and 0.3 mg/l NAA enhances a multiple shoot induction with no genetic infidelity of regenerated plants. [ABSTRACT FROM AUTHOR]