mgL-1 BAP Research Articles

Axillary shoot proliferation and regeneration of red rose [pisiformis (christ.) D. Sosn. An under thread extinction species endemic to Turkey

In vitro propagation of red rose [Rosa pisiformis (Christ.) D. Sosn.] has great importance in rapid proliferation of species with medicinal features and in culture of healthy plants free from deseases. After a succesfully pre-sterilization procedure, experiments were maintained by two-phase: multiplication phase and rooting of microshoots phase. Micropropagation of Rose was improved, using its nodal segments under different combination of BAP (0, 1.0, 2.0, 0.6 mg/l), NAA (0, 0.1, 0.2, 0.01 mg/l) on Driver and Kuniyuki (DKW) and MS medium in multiplication phase and using distinct compositions of IBA (0, 1.0, 3.0, 5.0 mg/l) and DKW medium in rooting phase. The maximum number of shoots/per plant (22.6) with the highest number of leaves (222.6) were obtained in DKW medium supplemented with 0.6 mgl-1 BAP and 0.01 mg/l NAA. The leaf explants were also used to occur callus in MS with different combinations of BAP and NAA. Good callus formation was obtained with 1:2 ratio of BAP:NAA combination in MS medium. Furthermore, the highest root induction (100%) was achieved in DKW medium consisting of 1 mg/l IBA. The rooted plantlets were transferred to the field after acclimatized in greenfield conditions. The present investigation presents an in vitro protocol for R. pisiformis. Bangladesh J. Bot. 52(4): 949-958, 2023 (December)

Callus induction and proliferation of Centella asiatica L. generated from leaves and petioles in the presence of Dicamba and BAP

Centella's need for industrial raw materials is high as a medicinal plant. These needs can be met through rapid multiplication using tissue culture techniques. In this study, induction and proliferation for a callus of centella cv. Castina 3 was conducted in the MS basal medium plus 4 mgL-1 Dicamba withand enriched with 7 concentrations of BAP (0, 0.1, 0.3, 0.5, 0.7, 0.9, and 1.1 mgL-1). Two kinds of explant were used, i.e., leaf and petiole. The results revealed that the addition of BAP in MS plus Dicamba medim stimulated better and produced a higher callus growth rate, both from leaf and petiole explants, than that media with Dicamba alone. Furthermore, 4 mgL-1 Dicamba + 1.1 mgL-1 BAP had a friable callus in the induction phase and a friable-compact callus in the proliferation phase. From this finding, it can be considered to use a combination of 4 mgL-1 Dicamba with 1.1 mgL-1 BAP in callus induction and proliferation for Centella rapid multiplication.

The of Effects of Light and a Combination of Growth Regulators on the Induction of Somatic Embryogenesis in Orchid Rhynchostylis gigantea (LindI.) Ridl.

The squirrel-tail orchid (Rhynchostylis gigantea) belongs to the Orchidaceae family. This orchid is indigenous to Southeast Asia and is scented and arranged in a bouquet of dangling stems. Its uniqueness makes it commercially desirable. In vitro culture has been utilized for orchid proliferation for a very long time to aid in the development of orchid seedlings. However, not all orchid species react the same way. Through somatic embryogenesis without gamete fusion, in vitro culture techniques can produce new plants during the entire embryonic phase. Endogenous and exogenous hormones, as well as light, influence the success of embryogenesis. Determining the optimal environmental parameters (light, combination and concentration of growth regulators, and their interaction) for inducing somatic embryogenesis from leaf explants in R. gigantea is the purpose of the present study. The leaves of R. gigantea clone 19 were utilized as explants in this investigation, utilizing a completely random factorial design. The first factor is a combination of growth regulator types and concentrations, and the second factor is light. Explants that are alive and growing at a rapid rate indicate that the in vitro culture is successful. Light and the combination of growth regulators significantly affected the percentage of viable explants, the beginning date of callus formation, and the number of embryogenic calluses produced. The interaction of two components (light and a combination of growth regulators) did not affect the three characteristics, with the exception of the proportion of somatic embryogenesis. Incubation in the dark is the optimal environment for initiating somatic embryogenesis in explants. The optimal combination of plant growth regulators for inducing somatic embryogenesis was 0.5 mgL-1 TDZ and 0.1 mgL-1 BAP. Bright light and a concentration of 1.0 mgL-1 TDZ + 0.1 mgL-1 BAP were the optimal interaction conditions for the induction of somatic embryogenesis in R. gigantea.

Alteration in the callogenesis, tropane alkaloid formation, and gene expression in Hyoscyamus niger under clinorotation.

This study aimed to investigate the effects of clinorotation induced by 2-D clinostat on the growth, tropane alkaloid production, gene expression, antioxidant capacity, and cellular defense responses in the callus tissue of Hyoscyamus niger. Callus induction was conducted by putting hypocotyl explants in the MS culture medium supplemented with 1 mgL-1 2,4-D and 1 mgL-1 BAP growth regulators. The sub-cultured calli were placed on a clinostat for 0, 3, 7, and 10 days (2.24 × 10-5 g on the edge of the callus ring). Clinorotation significantly increased callus fresh weight, dry weight, protein, carbohydrate, and proline contents compared to the control, and their maximum contents were obtained after 7 and 10 days. H2O2 level enhanced under clinorotation with a 76.3% rise after 10 days compared to control and positively affected the atropine (77.1%) and scopolamine (69.2%) productions. Hyoscyamine 6-beta hydroxylase and putrescine N-methyltransferase gene expression involved in the tropane alkaloid biosynthesis were upregulated markedly with 14.2 and 17.1-folds increase after 10 days of clinorotation, respectively. The expressions of jasmonic acid, mitogen-activated protein kinase, and ethylene-responsive element-binding transcription factor were upregulated, and the activity of peroxidase and catalase showed a 72.7 and 80% rise after 10 days. These findings suggest that microgravity can enhance callogenesis by stimulating the ROS level, which can impact the antioxidant enzymes, tropane alkaloid formation, and gene expression.

In vitro Propagation and Genetic Assessment by using Markers in Potato (Solanum tuberosum L.) cv Kufri Ganga

The purpose of this study was to determine the best types and concentrations of plant growth regulators to use during different stages of micropropagation, as well as to assess the genetic fidelity of tissue culture raised plants of a cv Kufri Ganga using reliable markers. According to the data on shoot bud initiation, a combination of 0.25 mgl-1 BAP and 0.5 mgl-1 Kinetin results in the greatest number of shoots per explant (3.4±0.11) in the shortest amount of time (3.2±0.09) days. The maximum number of in vitro shoots per shootlet (4.7±0.15) was recorded when auxins were combined with cytokinin (MS medium + 0.25 mgl-1 BAP + 0.01 mgl-1 IAA) for shoot proliferation. Root initiation was observed in (2.2±0.10) days on MS medium fortified with 1.5 mgl-1 NAA. The maximum number of in vitro roots per shoot (14.2±0.31) was observed when MS media was fortified with 2.0 mgl-1 IBA. Molecular markers were used to assess genetic fidelity in plants grown in vitro. Monomorphic DNA banding patterns were found in all tissue culture raised plants and mother plants, indicating true to type planting material. This protocol for tissue culture propagation, as well as testing its genetic fidelity, may be beneficial for the successful application of better conservation of germplasm.

IN VITRO PLANT REGENERATION IN LOCAL AUS RICE THROUGH MATURED SEED CULTURE

An investigation was carried out to decipher the effect of genotypes and concentrations of growth regulators for callus induction and subsequent plant regeneration from mature seeds of rice. Two local rice cultivars viz. Nayanmony and Kalomona and two growth regulators viz. 2, 4-D and NAA were used to evaluate their performance. The seeds were cultured under aseptic condition onto MS basal medium at varying concentrations and combinations of 2, 4-D (1.0, 1.5 and 2.0 mgL- 1) and NAA (0.0, 0.5 and 1.0 mgL-1) for callus induction. For plant regeneration MS medium fortified with 0.5 mgL- 1 NAA and 3.0 mgL-1 BAP was applied. Between the two cultivars Nayanmony showed better responses in callus induction frequency (91.38%), weight of callus (0.90 g)as well as diameter of callus (7.48 mm). Among the combinations 2, 4-D (1.5 mgL-1) alone produced the best callus (95.16%) and the highest weight of callus (1.88 g) but diameter of callus (8.27 mm) was the highest from 2, 4-D 1.0 mgL-1 +0.5 mgL-1 NAA. It was observed that both genotype and growth regulator significantly affected callus induction and plant regeneration. The medium supplemented with 2, 4-D 1.5 mgL-1 + NAA0.0 mgL-1 exhibited better performance in callus induction (99.33%) and weight of callus (1.92 g) in cultivar Nayanmony whereas Kalomona was better for diameter of callus (8.38 mm) at 2, 4-D 1.0 mgL-1 + NAA0.5 mgL-1. The cultivar Nayanmony performed better regarding plant regeneration (88.15%) compared to Kalomona (86.67%). Plantlets derived from the 2, 4-D 1.5 mgL-1+ NAA 0.0 mgL-1 showed the best results for survival. Therefore, it is suggested that application of MS medium supplemented with 2,4-D 1.5 mgL-1 and NAA 0.0 mgL-1 is advantageous to accomplish overall efficiency of callus induction, subsequent plant regeneration and survival from seeds of Nayanmony cultivars. Thus, selection of better responsive cultivar like Nayanmony and MS medium fortified with 1.5 to 2.0 mgL-1 2, 4-D would be beneficial in transformation attempts to improve the crop.

Genetic similarity, morphological, and biochemical changes in micrografted grapes explants of Flame Seedless x Freedom

The explants were used as a rootstock of the Freedom grape cultivar and Flame Seedless cultivar as a scion in vitro micrografting. The in vitro obtained explants were considered as explants and cultured on MS media containing 0.4 mgL-1 BAP. Six methods of micro grafting; sterile wire tie, sterile metal needle, wax, aluminum foil, gel and callus were used. Survival percentage, number of shoots and explants, shoot length, number of leaves per shoot, rooting percentage, root length, and callus percentage were all recorded as morphological parameters. Biochemical analysis was performed to determine phenols; flavonoids content and oxidising enzymes activity in Flame Seedless and Freedom. Results recorded that all methods of in vitro micrografting succeeded in yielding grafts between rootstock with scion with success in wax method followed by aluminium foil, sterile wire tie, sterile metal needle, gel, and callus methods in descending order. The best success rate of micrografting was obtained by the micro grafting method of wax as recorded with the best results of morphological criteria as the highest survival (83.88%), number shoots/explants (1.77), shoot length (1.66 cm), leaves/shoot (4.55), rooting (58.20%), root length (1.27 cm) and callus (55.55). The study suggests that this treatment might be a promising alternative to traditional grapevine propagation methods.

ISOLATION, CLONING AND AGROBACTERIUM MEDIATED TRANSFORMATION OF MYMV REPLICASE (REP) GENE IN VIGNA RADIATA (L.) WILCZEK FOR MYMV RESISTANCE

The binary plant cloning vector pCAMBIA1305.2 carrying the MYMV Replicase (Rep) gene was constructed and transformed into a competent Agrobacterium tumefaciens, strain LBA4404 and was used for co-cultivation of mungbean varieties, KKM-3, IC-39340-1, China mung and LM-1668 for imparting mungbean yellow mosaic virus (MYMV) resistance. The putative transformants (To generation) were selected on shooting media (SM) supplemented with 5 mgl-1 BAP, 50 mgl-1 hygromycin and 250 mgl-1 cefotaxime. The multiple shoots were inoculated on shoot elongation media (SEM) with 0.3 mgl-1 zeatin and 2.5 mgl-1 BAP for elongation and rooted on rooting media (RM), comprising half MS supplemented with 0.2 mgl-1 NAA. The GUS assay and genomic PCR analysis of the To transformants revealed, four positive putative transgenic lines for Rep gene specific primers and had a positive correlation with vector specific hptII primers. Likewise, VirG amplification revealed no Agrobacterium contamination in the apoplast of allthe putative To transformants.

<em>In vitro</em> seed propagation and mass multiplication of some magnificent Orchids of Northeast India

Eastern Himalaya owing to its specific gradient and varied climatic conditions has cradled the wonderful natural heritage of temperate and sub-tropical orchids. Large scale forest conversion and over exploitation has brought the orchid diversity to the brink of extinction. Increasing vulnerability of these species it is an urgent need for conservation. In vitro seed propagation is the popular multiplication technique and indispensable components for conservation and restoration of rare and endangered orchids. Study highlighted In vitro seed propagation of ten magnificent orchid species of Northeast India viz. Aerides multiflora, Cleisocentron pallens, Cymbidium aloifolium, Dendrobium aduncum, Dendrobium fimbriatum, Dendrobium lituiflorum, Dendrobium moschatum, Phalaenopsis mannii, Phaius tankerville and Rhynchostylis retusa. MS medium exihibited better proliferation of seeds followed by Mitra et al medium. Lower amount of BAP was most effective for transforming the spherules into fully developed protocorm like bodies. Study revealed that, culture media supplemented with 1.5, mgL-1 BAP recorded maximum number of shoot (3.9±0.88) with length of 5.56±0.58 cm. for A. multiflora. Highest number (11.2±1.75, 4.1±0.74) and elongation of shoot (4.75±0.58 cm, 5.13±0.85 cm) were recorded in 1.5 mgL-1 BAP and 0.5 mgL-1 IBA after 8 week of the culture for C. pallens and R. retusa respectively. However, media supplemented with 0.2% activated charcoal Powder, 0.2% potato extract, 0.2% yeast extract and 0.2% banana extract as natural additives showed excellent performance. MS medium supplemented with TDZ (1.5 mgL-1) was best phytohormons for shoot elongation of D. moschatum. Root establishment of majority of species found high when medium supplemented by IBA (1.5 mgL-1). Highest survivality of seedlings established in potting medium containing Brick chips: Charcoal: Sphagnum moss (1:1:2).

Comparison of Essential Oils Composition Between in-vitro Plantlets and Greenhouse Plants from Various Populations of Dracocephalum kotschyi Boiss.

Dracocephalum kotschyi species belongs to the family Lamiaceae, a perennial herbaceous medicinal plant that is native to Iran and is considered an endangered species. In-vitro plantlets (seven populations) were raised in MS medium supplemented with 0.1 mgl-1 BAP and 0.01 mgl-1NAA and the rooted plantlets were acclimatized successfully under greenhouse conditions.In- vivo plants (eight populations) were propagated under greenhouse condition. The essential oils were isolated by hydro distillation and identification of chemical compounds was done by a combination of capillary GC and GC-MS instruments. Twenty-five and forty compounds were identified in the different populations of in-vitro plantlet and in-vivo plant constituting 85.8%-99.68% and 85.1%-95.06% of essential oils, respectively. The major components of in-vitro plantlet on different populations were Verbenone (2.5%-82.47%), Geranyl acetate (28.35%-62.07%), Methyl geranate (0.98%-79.06%), Neral (0.64%-3.13%), Geranial (4.93%-14.39%), Limonene (0.37%-10.36%) and E-Anethole (7.62%-20.71%). The composition of essential oil from greenhouse plant populations were dominated by Neral (11.24%-74.80%), Limonene (0.17%-25.26%), Geraniol (0.54%-40.81%), Geranial (0.04%-9.15%), Methyl geranate (0.16%-28.48%), E-Anethole (0 %-0.1%) and Verbenone (0.39%-23.96%). The highest values of Neral, Limonene and Geraniol percentage were obtained from greenhouse conditions. In contrast, the maximum values of Verbenone, Geranyl acetate, Methyl geranate, Geranial, and E-Anethole were observed in the essential oils of in-vitro plantlets. This study demonstrated difference of chemical composition between in-vitro plantlets and greenhouse plants of different populations on D. kotschyi species. Also, new chemotypes of D. kotschyi has been introduced for further research.

In-vitro Callogenesis and Screening of Antimicrobial Activity of Callus and Seed of Caesalpinia bonducella F.: A Threatened Medicinal Plant of Western Ghats

Aim: Caesalpinia bonducella Flem is a dioecious scrambling woody liana of Caesalpinoideae, a subfamily of Leguminosae. The plant is threatened and distributed in the deciduous forests of the Western Ghats of India. Being an important medicinal plant Caesalpinia bonducella F. attracted many scientists to exploit various activities associated with a number of phytoconstituents. The present study was undertaken to evaluate the most suitable media and suitable concentrations of plant growth regulators for in vitro Callogenesis and screening of antimicrobial activity of callus and seed of Caesalpinia bonducella.
 Materials and Methods: Callus was initiated from stem explants, on 1x and 0.5x MS medium plus supplements. The effects of plant growth regulators on callus cultures were studied and observations were made. The in vitro antibacterial activity was performed by using extracts of callus and seed of Caesalpinia bonducella in petroleum ether and methanol against multidrug resistance organisms. The organic extracts of seed and callus of the plant at concentrations of 0.02 mg/ml - 0.1 mg/ml were taken and their activities were measured. 
 Results: The combination of 2.5 mgL-1 2,4-D with 2 mgL-1 BAP, resulted in the highest frequency and the highest mean percentage of callus formation (2.35 ± 0.294) with yellow friable callus. The results revealed that all the extracts had a variable degree of antibacterial activity.
 Conclusions: It was observed that 2,4-D at 2.5 mgL-1 in combination with BAP,2.0 mgL-1 BAP resulted in early initiation, highest induction percentage, with frequency highest mean percentage of callus formation, Antimicrobial tests with methanol and diethyl ether extract of Caesalpinia bonducella seed powder against the clinical isolates showed the zone of inhibition for all the pathogens tested with concentration of methanolic extract of C. bonducella seed powder.

Plant tissue culture of Nicotiana tabacum cv. TAPM 26 and its minimum inhibition against herbicide-Dalapon

Current study is to establish a basic plant tissue culture of Nicotiana tabacum TAPM 26 and test the plant tissue on resistancies against 2,2 DCP an active ingredient in herbicide-Dalapon. During micropropagation, the surface sterilization method was ascertained on seeds of tobacco. HgCl2 was used to disinfect tobacco seeds at different concentrations (0.05 gL-1, 0.2 gL-1, 0.5 gL-1 and 1.0 gL-1) within three minutes. About 70% seeds were survived when exposed to 0.05 gL-1 of HgCl2, whereas, no seeds were germinated when sterilized at concentrations above 0.05 gL-1 of HgCl2. To optimize an efficient protocol of shoots and callus formation during in vitro regeneration, explant types and plant growth were studied. Growth regulators NAA (0.1 mgL-1, 0.2 mgL-1, 0.5 mgL-1, 1.0 mgL-1 and 2.0 mgL-1) and BAP (1.0 mgL-1, 2.0 mgL-1, 3.0 mgL-1 and 4.0 mgL-1) were used. The explants types were one month old leaves and two weeks old cotyledons. The maximum numbers of shoots per explants were obtained from cotyledon with combination 0.1 mgL-1 NAA and 1.0 mgL-1 BAP. The highest callus fresh weight was achieved when NAA 0.5 mgL-1 with BAP 1.0 mgL-1 after four weeks. Thus, the highest number of shoots produced per explants from leaves culture on the MS media containing 0.2 mgL-1 NAA and 4.0 mgL-1 BAP. The best callus fresh weight was obtained with combination of 1.0 mgL-1 NAA and 1.0 mgL-1 BAP by using leaves explant. Finally, Dalapon (5 gL-1, 10 gL-1, 15 gL-1 and 20 gL-1) were applied onto leaves and cotyledon cultures of N. tabacum to check on the minimum concentration of inhibition. The minimum concentration of inhibition of leaves and cotyledon cultures of N. tabacum was at 5 gL-1 of 2,2DCP but not at 10 gL-1, 15 gL-1 and 20 gL-1. This investigation will shed alight for future studies on transgenic tobacco resistant against Dalapon

In vitro Callus Induction and Regeneration of Popular Indica Rice Genotypes

Five rice cultivars viz., ASD 16, White Ponni, Pusa Basmati 1, Pusa Sugandh 4 and Pusa Sugandh 5 belonging to subspecies indica were compared for its ability in callus formation and regeneration. In this experiment, the different parameters viz., the effect of hormones (2,4-D and kinetin), organic supplement (coconut milk O1-CM 100 mll-1, O2-CM 75 mll-1, O3-CM 50 mll-1), explants (seed and immature embryo), media (MS and N6), carbon source (sucrose and maltose) using five genotypes on callus response was studied. The effect of hardening methods was also assessed. Results showed that for enhanced callus induction was with MS medium supplemented with 2.0 mgl-1 2, 4-D + 0.5 mgl-1 kinetin + 30 gl-1 maltose irrespective of explants used. Addition of 100 ml l-1 coconut milk was found have improvement in callus response. The performance of immature embryo was better than seed for callus induction, emrbyogenic callus formation, rhizogenic callus formation and regeneration. MS media provided superiority over N6. Among the genotypes Pusa Basmati 1 rendered outstanding performance in callus behavior. The treatment combination MS + 2.5 mgl-1 BAP + 0.5 mgl-1 NAA + 1.0 mgl-1 KN gave the highest organogenesis response and regeneration of plantlets. Hardening in mist chamber was recognized as the best method to give the highest per cent of regenerated plant lets.

Development and regeneration of somatic embryos from leaves-derived calli of Coffea liberica

Abstract. Ardiyani F, Utami ESW, Purnobasuki H, Paramita SA. 2020. Development and regeneration of somatic embryos from leaves-derived calli of Coffea liberica. Biodiversitas 21: 5829-5834. Coffea liberica is an important and potentially commercial plant with a high economic value from the Coffea genus. Therefore, the availability of planting material is needed to increase productivity and ensure the sustainability of its farming. Somatic embryogenesis is a powerful propagation method used to produce clonal plants from limited genetic material. In the present research, we have shown that C. liberica could be successfully regenerated in vitro via somatic embryogenesis from leaves derived embryogenic callus. These calli were cultured on Murashige Skoog (MS) medium added with 1 mgL-1 BAP or in combination with 2.4 D (0.5, 1.0, 1.5 and 2 mgL-1) for embryo development induction. Furthermore, the medium containing only BAP was best for embryo development induction after culturing for 12 weeks, with the highest number of cotyledonary stage embryos (17.8%) and producing a total of embryo (20.2). Following cotyledonary stage embryo were cultured on new MS medium containing 0.5 mgL-1 BAP, 0.5 mgL-1 IAA, 0.5 mgL-1 NAA only, and 0.5 mgL-1 BAP in combination with 0.5 mgL-1 IAA or 0.5 mgL-1 NAA. Interestingly, the results showed that cotyledonary stage embryos were converted into complete plants at all treatment, but the MS medium containing 0.5 mgL-1 BAP was found to be the most effective in promoting regeneration with 2.6 leaves per-plantlet and height of 5.2 mm. Based morphological analysis confirm that the development of somatic embryo from leaves-derived calli of Coffea liberica started with the formation of embryo globular, heart, torpedo, cotyledonary stages, and finally conversion of cotyledonary embryo into complete plant.

Callus Production Protocol for Aegle marmelos (L.) Corr.: As a Tool for Extraction of Secondary Metabolites

Background: Aegle marmelos (L.) Corr. is an important medicinal and a fruit tree belongs to the family Rutaceae possessing numerous valuable secondary metabolites. The growing commercial importance for secondary metabolites has led to a great demand in the pharmaceutical industry in recent years. Therefore, an efficient callus production protocol was developed as a tool for extracting valuable secondary metabolites from Aegle marmelos.Methods: For seeds, callus induction was observed under three conditions as with seed coat, after removing seed coat and split into two halves after removing seed coat. For callus multiplication, 1cm2 pieces of initiated calli were used. These explants were established in MS medium supplemented with combinations of 2, 4 D either with BAP or Kinetin. All experiments were arranged according to the completely randomized design (CRD) with 20 replicates at the Tissue Culture Laboratory, Department of Crop Science, Faculty of Agriculture, University of Ruhuna, Sri Lanka, for a period of 1 year. Percentage of fungal and bacterial contaminations and percentage of bleached explants were observed to select the best explant/s. Percentage of responded explants were observed to select the best condition for callus induction and quality of callus. Growth of callus was observed visually by giving a score. Best hormonal combination for callus multiplication was observed as fresh weight and dry weight of callus produced under each treatment.Result: High quality callus with higher growth was observed in all combinations of BAP and 2, 4 D tested: ranging from 0.5 mgL-1 to 1.5 mgL-1 BAP and 1.0 mgL-1 to 2.0 mgL-1 2,4 D in Murashige and Skoog (MS) medium. Initiated calli were further multiplied in MS medium supplemented with 2,4 D combined with either BAP or Kinetin. Highest amount of callus biomass was recorded in the MS medium with 0.5 mgL-1 2, 4 D and 1.0 mgL-1 Kinetin (132.58 gL-1 fresh weight). The optimized protocol could be used to produce higher amount of callus in order to extract secondary metabolites from Aegle marmelos (L).

In vitro regeneration technique in Rauwolfia serpentina and quantification of reserpine

The reliable technique for the indirect regeneration of R. serpentina was standardised in the present investigation. The secondary metabolites extracted from callus using methanol and analysed using Thin Layer Chromatography (TLC) and UV-Vis Spectrophotometer. The in vitro induction of callus influenced by several factors such as media composition and explants quality. From the various media combinations of 2, 4-D and BAP the frequency of highest callus induction (94.67%) was observed on MS + 2.0 mgL-1 2.4-D + 1.0 mgL-1 BAP when leaf disc used as explant. Throughout the organogenic callus induction, different calli were observed having variation in colour and texture. The shoot regeneration frequency was observed highest (98.33%) in 4.5 mgL-1 BAP + 0.5 mgL-1 NAA. Maximum shoots (15.33 per callus) were observed in leaf callus. The rooting was induced (95.33%) in in vitro regenerated shoots in Murashige and Skoog (MS) medium which contain 1.0 mgL-1 NAA + 0.1 mgL-1 BAP + 1 gL-1 activated charcoal (AC). The in vitro regenerated plantlets through callus culture were hardened in the greenhouse with 75 per cent establishment. The quantity and quality of reserpine was measured in callus and root of R. serpentina. The TLC reveals the accumulation of reserpine at callus as well as roots of cultivated plant. Spectrophotometric estimation showed that concentration of reserpine observed in callus extract was higher (6.8 µgml-1) than the root extract (6.4 µgml-1).

An easy and reliable method for establishment and maintenance of tissue cultures of Nicotiana tabacum cv TAPM 26

In vitro regeneration of Nicotiana tabacum was efficiently achieved using growth regulators combination supplemented into semi-solid MS medium using leaves from 1-month-old plant as explants. The growth regulators used were NAA for auxin (0.0, 1.0, 2.0, and 3.0 mgL-1) and BAP (0.0, 0.5, 1.0, 2.5, and 5.0 mgL-1). The effects of growth regulator combination were assessed based on a number of callus formation, shoots formation and fresh callus weight. The maximum number of callus formation was 100 % at five hormone combination, observed at the fourth week after culture. Maximum number of shoots produced per explant was 21.4 shoots at 1.0 mgL-1 BAP + 3.0 mgL-1 NAA, after 6 weeks of culture. The maximum callus fresh weights were obtained at 0.5 mgL-1 BAP + 1.0 mgL-1 NAA after 6 weeks (9.92 g). The best combination for shoots regeneration of Nicotiana tabacum was 1.0 mgL-1 BAP + 3.0 mgL-1 NAA. And the best combination for heaviest callus production was 0.5 mgL-1 BAP + 1.0 mgL-1 NAA.

Challenges in micropropagation of Bael [Aegle marmelos (L.) Corr.] from leaf Disk Explants and methods for its adoption amongst progressive farmers and farm women

The present study was performed to define tissue culture techniques for micropropagation of bael cvs. through leaf disk explants from 25 days old in vitro grown plantlets on Knop’s medium supplemented with 0.5 mgl-1 BAP. Leaf disk explants were cultured on various concentrations of growth regulators (BAP, KIN and NAA). Knop’s medium supplemented with 1.0 mgl-1 BAP, 0.5 mgl-1 KIN and 0.5 mgl-1 NAA produced maximum percentage of shoot regeneration (46.66%) and maximum per cent multiple shoot (33.33%) was found in cv. Mirzapuri on Knop’s medium supplemented with 1.0 mgl-1 BAP+0.5 mgl-1 KIN +0.5 mgl-1NAA. Maximum mean number of shoots per explants (1.20) was recorded in cv. Mirzapuri at 1.0 mgl-1 BAP+0.5 mgl-1 KIN+0.5 mgl-1NAA whereas maximum shoot length (0.95cm) was observed in cv. Mirzapuri on Knop’s medium supplemented with 1.0mgl-1 BAP+0.5mgl-1KIN+0.5mgl-1NAA.

Callogenesis and plant regeneration of sweet orange cv. Washington Navel from floral organ cultures

Washington Navel orange (Citrus sinensis L.) can be infected with virus and virus like diseases that affect not only the production but also fruit quality and the plant’s longevity. For viral sanitation, Washington Navel regeneration was investigated in vitro via floral organ culture. Flowers were collected before opening from healthy Washington Navel trees kept under greenhouse. Floral organs (style/stigma and ovary) were cultured on Murashige and Skoog (MS) medium containing various plant growth regulators combinations of naphthalene acetic acid (NAA), 2,4-dichlorophenoxyacetic acid (2,4-D) and 6-benzylaminopurine (BAP). The highest rate of callogenesis (95%) was obtained from style/stigma explant cultures on MS medium enriched with 3 mgL-1 BAP, which also resulted in 100% rooted plantlets. Ovary cultures did not show any success on the culture medium with various plant growth regulators combinations. The acclimatization success of rooted plantlets by grafting on Citrus volkameriana rootstocks was about 83%. Thus, these results can be used for mass production of disease-free citrus plants and improve sanitation program of the local citrus genotypes in Tunisia.

Micro Propagation of Pterocarpus santalinoides Using Three Different Growth Media

Aims: Micro propagation of P. santalinoides was carried out in order to ascertain the most appropriate culture media for its micro propagation.
 Study Design: The experiment was laid out in different growth media in the laboratory.
 Place and Duration of Study: The micro propagation of Pterocarpus santalinoides was carried out at the Tissue culture laboratory of the University of Nigeria, Nsukka and lasted between July and October 2018.
 Methodology: Seeds from fresh and healthy ripe fruit which was cut open mechanically with the help of secateurs were gotten from Ai-kwu, Otukpa Local Government Area of Benue State, Nigeria. The seeds were air dried and used as explant. The explants were surface sterilized using NaOCl solution for 10 mins, rinsed with distilled water and then the soft seed coat were removed and the seeds were cultured under aseptic conditions on MS medium and other growth medium. Seeds of Pterocarpus santalinoides were inoculated on six different growth media with varying compositions. The media are MS, B5 and white’s without growth hormones (MSoo, B5oo, and WHoo), and each of them was supplemented with 3.0 mgl-1 BAP and 0.5 mgl-1 NAA (MSBN, B5BN, WHBN).
 Results: Seed germination improved in all the media studied. However, MS combinations gave the best result (90-93%). The maximum number of leaves and roots recorded was in MSBN (3.8 for leaves and 2 for roots) followed by MSoo (2.6) and WHBN (2.6). The leaf area was best for the MS combination (0.232 cm2) followed by the White’s combinations (0.154 cm2) and least for the B5 combinations (0.026 cm2) while shoot and root length was maximum in MSBN (4.28 cm for the shoot and 1.18 cm for the root) followed by WHBN (1.90 cm). The result for t-test revealed that there was a significant difference between the parameters studied for growth media with growth hormones and those without growth hormones. The recorded percentage germination rate for MS medium without growth hormone was 90.75±0.97 while MS medium supplemented with growth hormone was 93.25±0.25. B5 medium without growth medium was 60.25±0.50 and when supplemented with growth hormone, the value was 66.50±0.57. White medium without growth hormone had a value of 75.25±1.70 and when supplemented with growth hormone the value was 78.0±0.81.
 Conclusion: The growth rates of Pterocarpus santalinoides, in MS medium among other basal media (B5 and White) offers a compromise between all the growth parameters which indicates that variation of the basal medium composition could lead to enhanced Pterocarpus santalinoides regeneration efficiency.