In vitro multiplication in Kaempferia galanga linn

Kaempferia galanga is an important medicinal plant that is facing threat of extinction owing to indiscriminate and unsustainable harvesting in the wild. Conventional breeding is difficult in this plant, and in vitro multiplication is important to conservation and propagation. Leaf and rhizome explants of Kaempferia were aseptically cultured on MS medium with various combinations of indole-3-acetic acid (IAA), benzyl amino purine (BAP), napthalene acetic acid (NAA), 2-4-dichlorophenoxy acetic acid (2,4-D) and kinetin at concentrations ranging from 0.5 to 2.5 mg/L. High-frequency organogenesis and multiple shoot regeneration was induced from rhizome explants on MS medium supplemented with 0.5 mg/L of IAA and 2.5 mg/L of BAP. Rooting was induced in MS medium with 0.5 mg/L of IAA and 2 mg/L of BAP.     

Shoot Multiplication and Callus Induction of Labisia pumila var. alata as Influenced by Different Plant Growth Regulators Treatments and Its Polyphenolic Activities Compared with the Wild Plant

This study aims to investigate whether the in vitro-cultured L. pumila var. alata has higher antioxidant activity than its wild plant. An 8-week-old L. pumila var. alata nodal segment and leaf explants were cultured onto Murashige and Skoog (MS) medium supplemented with various cytokinins (zeatin, kinetin, and 6-benzylaminopurine (BAP)) for shoot multiplication and auxins (2,4-dichlorophenoxyacetic acid (2,4-D) and picloram) for callus induction, respectively. The results showed that 2 mg/L zeatin produced the optimal results for shoot and leaf development, and 0.5 mg/L 2,4-D produced the highest callus induction results (60%). After this, 0.5 mg/L 2,4-D was combined with 0.25 mg/L cytokinins and supplemented to the MS medium. The optimal results for callus induction (100%) with yellowish to greenish and compact texture were obtained using 0.5 mg/L 2,4-D combined with 0.25 mg/L zeatin. Leaves obtained from in vitro plantlets and wild plants as well as callus were extracted and analyzed for their antioxidant activities (DPPH and FRAP methods) and polyphenolic properties (total flavonoid and total phenolic content). When compared with leaf extracts of in vitro plantlets and wild plants of L. pumila var. alata, the callus extract displayed significantly higher antioxidant activities and total phenolic and flavonoid content. Hence, callus culture potentially can be adapted for antioxidant and polyphenolic production to satisfy pharmaceutical and nutraceutical needs while conserving wild L. pumila var. alata.     

Growth kinetics and diosgenin estimation from callus cultures of Costus speciosus (Koen. ex. Retz.)

The present investigation reports the growth kinetics and diosgenin accumulation in callus cultures of Costus speciosus. Effect of explants, media and plant growth regulators was evaluated with respect to callus induction and growth. Out of the two explants viz pseudostem and seed, pseudostem showed maximum callus induction frequency of 90% on MS medium. The fresh weight of callus was maximum (9-folds) on 28th day on 1.0 mg/L picloram containing medium. The callus obtained was white compact hard (WCH). For growth kinetics study pseudostem derived callus was transferred on different media supplemented with 1.0 mg/L picloram. All phases of growth were seen in callus inoculated on all the three media except the absence of stationary phase on MS and SH media. MS medium proved to be the best for maximum biomass accumulation (9-fold) on 28th day of culture and callus in post-exponential phase showed maximum diosgenin accumulation (33 ppm).     

Effect of Various Growth Hormone Concentration and Combination on Callus Induction,Nature of Callus and Callogenic Response of Nerium odorum

Nerium odorum, Linn. (Apocynaceae) is an important evergreen shrub. It is heat, salinity and drought tolerant. Plants with milky sap have medicinal value, mainly cardenolides, flavonoids and terpenes. It is used for wastewater purification and for restoration of riparian woodlands. In view of these facts, the study was conducted for micropropagation of N. odorum. Murashige and Skoog (MS) media supplemented with different concentrations (0.5–10.0 mg/l) of 1-naphthaleneacetic acid (NAA), 2,4-dichlorophenoxyacetic acid (2,4-D), 6-benzylaminopurine (BAP) and kinetin (Kin) were used singly and in combinations. Among all the growth hormones, 2,4-D was the best for callus induction (75 % in stem and 79 % in leaf) and in combination 2,4-D and BAP (78 % in stem and 81 % in leaf). The day of callus induction started from the 19th to the 37th day. This variation is due to the differences in culture conditions and the age of explants. The fresh and dry weight and moisture content showed good growth of callus, which is used in further studies of alkaloid production. Micropropagation of this plant allows the production of clones at a fast rate and in continuous manner. This work can lead to the development of an efficient protocol for callus induction and other issues.     

Antioxidant potential in callus culture of Artemisia amygdalina Decne

This study was conducted to analyse the free radical scavenging potential of callus obtained from nodal segments and leaf explants of Artemisia amygdalina Decne. The explants were inoculated on MS medium augmented with various concentrations of BAP, Kn, NAA and 2,4-D for callus induction. In this study, 12.42?g of callus developed from the leaf explant on MS (NAA 10?+?BAP 7.5?μM) and 8.81?g of callus developed from nodal explant on NAA 2?μM+BAP 2?μM. Callus raised from both explants on all treatments seemed non-regenerative but BAP 2?μM produced 7.33 shoots and BAP 15?μM produced callus and 5 shoots per nodal segment. Callus was analysed for antioxidant activity via DPPH, riboflavin photoxidation and DNA damage assays. Methanol and aqueous extracts show more scavenging in DPPH, deoxyribose assay and in contrast, petroleum ether and ethyl acetate extracts show higher activity in riboflavin photoxidation assay. Tocopherol, ascorbic acid and BHT were used as controls.     

Selection and regeneration of groundnut plants resistant to the pathotoxic culture filtrate ofCercosporidium personation through tissue culture technology

Callus cultures were established from immature leaf expiants ofArachis hypogaea on MS medium supplemented with 2.0 mg/L of NAA and 0.5 mg/L of BAP of the susceptible cultivars namely VRI-2 and TMV-7. Three-week-old calli were subjected to mutagenic treatments (gamma rays: 50–250 Gy and EMS: 5–25 mM). Mutagen-treated calli were subcultured to fresh medium containing various concentrations (25–100% v/v) of pathotoxic culture filtrates. Calli were challenged in vitro with pathotoxic culture filtrate of the fungal pathogen and were assessed by visible growth ratings expressed as the percent response to the doses/concentrations of mutagen. Selected mutagen-treated calli showed resistance in vitro on media containingCercosporidium personatum pathotoxic culture filtrate. Resistance calli were then transferred to MS regeneration medium supplemented with BAP (2.0 mg/L) and NAA (0.5 mg/L) for shoot bud regeneration. The progeny of the plants produced 13 disease-resistant plants (R₂) in both the cultivars. Among the eight R₂ populations studied, 70.2–82.5% of the plants exhibited enhanced resistance. This study suggested that groundnut plants with resistance to C.personatum can be selected     

In Vitro Plant Propagation of Catharanthus roseus and Assessment of Genetic Fidelity of Micropropagated Plants by RAPD Marker Assay

An investigation was carried out to develop an efficient micropropagation protocol for Catharanthus roseus. Experiments were conducted to optimize suitable media for in vitro shoot multiplication and root induction. Out of the different media compared for in vitro shoot multiplication, Murashige and Skoog (MS) medium supplemented with 1 mg/l of 6-benzylaminopurine and 0.2 mg/l α-naphthaleneacetic acid showed better response in terms of the emergence of shoots from axillary buds as well as proliferation and multiplication of shoots. The shoots when placed on half strength of MS medium having 1 mg/l indole 3-butyric acid and 0.25 % charcoal showed cent percent root induction with maximum number of roots per shoot (4.2) as well as maximum root length (1.72 cm). Further, clonal fidelity of the in vitro-raised plants was carried out using randomly amplified polymorphic DNA marker and results indicated that all the tissue culture-derived plants are true-to-type and there were no somaclonal variations among these plants.     

Thidiazuron-Induced Changes in Biomass Parameters,Total Phenolic Content,and Antioxidant Activity in Callus Cultures of Artemisia absinthium L.

Callus culture of Artemisia absinthium L. was established for enhanced production of phenolics and higher antioxidant activity. Callus was induced from seed-derived leaf explants, incubated on to MS media supplemented with thidiazuron (TDZ; 0.5–5.0 mg/l) either alone or in combination with α-naphthalene acetic acid (NAA; 1.0 mg/l). These callus cultures were investigated for their growth kinetics, total phenolic content, and antioxidant activity on weekly basis for a period of 49 days. Maximum dry biomass accumulation of 8.73 g/l was observed on day 42 in response to 1.0 mg/l TDZ and 1.0 mg/l NAA. Furthermore, maximum level of total phenolic content of 8.53 mg GAE/g DW and highest 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity of 72.6 % were observed in calli formed in response to 1.0 mg/l TDZ on day 42. The results showed a positive correlation of total phenolic content and DPPH radical scavenging activity in most of the callus cultures of A. absinthium L.     

In vitro accelerated mass propagation and ex vitro evaluation of Aloe vera L. with aloin content and superoxide dismutase activity

An innovative protocol on accelerated in vitro propagation and acclimatisation was developed in Aloe vera L. Culture was initiated with rhizomatous stem where Murashige and Skoog (MS) medium fortified with 0.5?mg?L(-1) α-naphthalene acetic acid and 1.5?mg?L(-1) N(6)-benzylaminopurine (BAP) promoted earliest shoot induction. Maximum shoot multiplication was achieved in MS medium supplemented with 2.5?mg?L(-1)BAP. The best in vitro rooting was observed in the MS medium with 0.5?mg?L(-1) indole-3-acetic acid plus 2?g?L(-1) activated charcoal. The simple acclimatisation process, primarily with a combination of sand and soil (1?:?1 v/v) and finally with a blend of sand, soil and farm yard manure (2?:?1?:?1 v/v), ensured a 98% survival rate. Overall, 192 true-to-type plantlets were achieved from a single explant within 85 days. Morphologically, in vitro generated plants performed better than conventionally propagated plants; nevertheless the similarity in aloin content, gel content and superoxide dismutase activity was corroborated.     

Callus induction and plant regeneration from different explants of Actinidia deliciosa

In this study, an efficient procedure was developed for callus induction and regeneration of kiwifruit (Actinidia deliciosa) using different organs of shoots developed under in vitro conditions. Effects of explants source and media (M₁, 1.0 mg l⁻¹ BA + 2.0 mg l⁻¹ 2,4-D–M₂, 1.0 mg l⁻¹ NAA + 2.0 mg l⁻¹ 2,4-D) on initiation of callus were examined in order to obtain callus for organogenesis. The best callus for plant regeneration was obtained from leaf explants on Murashige and Skoog’s medium (MS) supplemented with M₂. Formation of callus from leaf of kiwifruit (A. deliciosa) was cultured in MS medium containing different concentration of N⁶-benzylaminopurin (BA; 0.0, 1.0, 2.0, 4.0, 6.0, 8.0 mg l⁻¹) for callus proliferation and plant regeneration. Although the first shoot formation was appeared in medium containing 6.0 and 8.0 mg l⁻¹ BA, the best shoots formation was obtained in medium with 4.0 mg l⁻¹ BA.     

Somatic Embryogenesis and Synthetic Seed Production—a Biotechnological Approach for True-to-Type Propagation and In Vitro Conservation of an Ornamental Bulbaceous Plant Drimiopsis kirkii Baker.

An efficient plant regeneration protocol through indirect somatic embryogenesis pathway via callus had been developed from the leaf explant of an ornamental bulbaceous plant Drimiopsis kirkii. Optimum friable calli were induced on Murashige and Skoog (MS) basal medium supplemented with 3.0 mg/l of 2,4-dichlorophenoxyacetic acid and 1.0 mg/l of α-naphthalene acetic acid (NAA). On subculturing the callus on MS medium supplemented with 2.5 mg/l of thidiazuron (TDZ), 73.3 % of the cultures responded with 20.4?±?0.3 somatic embryos (SEs) per 500 mg callus at different stages of development after 6 weeks of culture. The highest response of 86.7 % with 28.3?±?0.5 embryos per 500 mg callus was observed on MS medium supplemented with 2.5 mg/l TDZ and 1.0 mg/l NAA. SEs were encapsulated in calcium alginate beads for the production of synthetic seeds (SSs) and their storability was investigated. The highest SS germination (93.3 %) was observed in 1.0 % sodium alginate followed by 86.7 % germination with 2.5 % sodium alginate. The SSs were stored at three different temperatures (4, 15, and 24?ºC) up to 6 months. The SSs kept at 15 °C showed 64.4 % germinability even after 4 months of storage. Both nonencapsulated and encapsulated SE-derived plants were successfully transferred to soil with 93.3 and 88.3 % survival rate accordingly. Randomly amplified polymorphic DNA (RAPD) analysis revealed that there were no somaclonal variations among the plants produced via somatic embryogenesis and they are true-to-type to their parental plant. These results confirmed the most reliable methods, which can be further used for genetic transformation studies as well as for mass propagation of ornamental D. kirkii at a commercial level.     

Effect of auxins and cytokinins on efficient plant regeneration and multiple-shoot formation from cotyledons and cotyledonary-node expiants of groundnut (Arachis hypogaea L.) by in vitro culture technology

Tissue cultures were established from cotyledon and cotyledonarynode segments ofArachis hypogaea L. on Murashige and Skoog (MS) medium supplemented with different concentrations of auxins (IAA, NAA, IBA, and 2, 4-D) and cytokinins (KIN and BAP). For callus initiation, high concentration of auxins and low concentration of cytokinins were used, whereas high concentration of cytokinins and low concentration of auxins were used for shoot-bud differentiation. Callus induction and shoot-bud regeneration frequency, however, varied with genotype, expiant, and the different plant-growth regulators combination in the medium. The shoot-bud multiplication was also influenced by genotype, explant type, and growth regulators. The combination of BAP and NAA produced more shoots than other combinations. The maximum number of shoots was obtained from cotyledonary-node segments on a medium containing BAP (5.0 mg/L) and IBA (1.0 mg/L). Rooting of regenerated shoots was achieved on a medium augmented with NAA or IBA (2.0 mg/L) in combination with KIN (0.5 mg/L). Rooted plantlets were successfully established in the soil, where 95% of them survived. Tissue-culture studies of these expiants suggests the shoots to be ofde nova origin, which would make the system suitable for gene-transfer technology.     

Shoot organogenesis from root-derived callus of Rhinacanthus nasutus (L.) Kurz. and assessment of clonal fidelity of micropropagted plants using RAPD analysis

An efficient regeneration system was established for an ethnomedicinal shrub Rhinacanthus nasutus from root-derived callus organogenesis. The root segments were cultured on MS medium supplemented with various concentrations of Kn (1.0–4.0 μM) alone or in combination with IBA (0.2–0.6 μM) or 2, 4-D (0.5–1.5 μM). The optimum frequency (94 %) of callus induction was recorded on MS medium supplemented with 3.0 μM Kn and 0.4 μM IBA. For shoot regeneration from callus, MS medium supplemented with different concentrations (1.0–7.0 μM) of BA or TDZ alone or in combination with NAA (0.2–1.0 μm) was employed. The highest frequency of shoot regeneration (91 %) and mean number of shoots (28.3) were observed on MS medium supplemented with 5.0 μM BA and 0.7 μM NAA. The shoots were excised and cultured on MS medium with 4.0 μM IBA produced 3.4 roots per shoot in 88 % cultures. Of the 65 plants transferred to soil 54 survived (83 %). The plants were transferred to field after successful hardening. RAPD analysis of the regenerated plants showed high similarity with the mother plant.     

In Vitro Propagation of Dioscorea alata var. purpurae

Dioscorea alata var. purpurae (Indian purple yam) is an important source of diosgenin, a triterpenoid that is used as a raw material in the synthesis of corticosteroid hormones. These drugs are used for the treatment of pharmacological conditions such as arthritis. This paper reports in vitro propagation of explants of various parts of Dioscorea—tuber, leaves, and nodes. Murashige and Skoog media supplemented with hormones and additives was used to get maximum callus initiation and shoot/root induction. All the cultures were maintained at 25?±?2 °C under cool-white fluorescent tubes with 16-h photoperiod. Callus initiation was observed from 8th to 11th day of inoculation, and subsequent root/shoot was initiated in nodal callus after 21 days. Hormones such as kinetin, indole-3-acetic acid, indole-3-butyric acid, α-naphthalene acetic acid, and thiadizuron did not show significant enhancement. Also, there was no need for supplementing additives (silver nitrate, glutamine, l-asparagine monohydrate, polyethylene glycol). Combination of 6-benzylaminopurine (0.2 ppm) and 2,4-dichlorophenoxyacetic acid (2 ppm) hormones gave the best results, and all parts of the plants gave similar callus induction.     

Plant Regeneration Through Callus Organogenesis and True-to-Type Conformity of Plants by RAPD Analysis in Desmodium gangeticum (Linn.) DC.

An efficient plant regeneration protocol was established for an endangered ethnomedicinal plant Desmodium gangeticum (Linn.) DC. Morphogenic calli were produced from 96 % of the cultures comprising the immature leaf explants on Murashige and Skoog (MS) medium supplemented with 2,4-dichlorophenoxyacetic acid (4.0 mg?l^?1) in combination with 6-benzylaminopurine (BA; 0.8 mg?l^?1). For callus regeneration, various concentrations of BA (1.0–5.0 mg?l^?1) or thidiazuron (TDZ; 1.0–5.0 mg?l^?1) alone or in combination with indole-3-acetic acid (IAA; 0.2–1.0 mg?l^?1) were used. Highest response of shoot regeneration was observed on MS medium fortified with TDZ (4.0 mg?l^?1) and IAA (0.5 mg?l^?1) combination. Here, 100 % cultures responded with an average number of 22.3 shoots per gram calli. Inclusion of indole-3-butyric acid in half MS medium favored rooting of recovered shoots. Out of 45 rooted plants transferred to soil, 40 survived. Total DNA was extracted from the leaves of the acclimatized plants of D. gangeticum. Analysis of random amplified polymorphic DNA using 13 arbitrary decanucleotide primers showed the genetic homogeneity in all the ten plants regenerated from callus with parental plant, suggesting that shoot regeneration from callus could be used for the true-to-type multiplication of this plant.     

Production of a new mucilage compound in Lepidium sativum callus by optimizing in vitro growth conditions

The mucilage in Lepidium sativum L. is considered a biologically active compound with diverse medicinal properties. Different explants (hypocotyls and leaf) were transferred to Murashige and Skoog (MS) medium supplemented with twelve different plant growth regulator combinations under two different incubations (light and dark). The best mucilage production from callus (36.76% g g^?1 dry weight) was obtained in the MS medium supplemented with 1 mg L^?1 of 2, 4-D and 2 mg L^?1 of BAP under the light condition. The mucilage produced by callus culture was nearly three times more than the mucilage yield of the seeds. The glucose, arabinose + mannose and galactose were 43.4 (mg g^?1 DW), 195.3 (mg g^?1 DW) and 86.2 (mg g^?1 DW) in the mucilage originated from seed, callus leaf and callus hypocotyl, respectively. The present study proposes an efficient method for producing large scales of mucilage with a favorable sugar aimed at food or pharmaceutical industries.     

Regeneration-Based Quantification of Coumarins (Scopoletin and Scoparone) in <Emphasis Type="Italic">Abutilon indicum</Emphasis> In Vitro Cultures

Abutilon indicum exploited for its immense value has been propagated successfully through multiple shoot induction and somatic embryogenesis. Direct regeneration (8.20?±?0.83 shoots) was achieved from nodal explants using 0.5 mg/l kinetin (Kn) in MS media. The basal callus from nodal explants turned embryogenic on subsequent introduction of 0.2 mg/l TDZ into the Kn-supplemented media, giving rise to somatic embryos. The embryogenic potential of calli expressed in terms of embryo-forming capacity (EFC) increased from 8.15 EFC to 20.95 EFC after plasmolysis. The phytochemical analysis (HPLC) for the presence of scopoletin and scoparone has revealed a unique accumulation pattern, with higher levels of scopoletin during the earlier stages and scoparone in the later stages of development. The embryogenic calli contained the highest amount of coumarins (99.20?±?0.97 and 61.03?±?0.47 μg/gFW, respectively) followed by regenerated plant (9.43?±?0.20 and 36.36?±?1.19 μg/gFW, respectively), obtained via somatic embryogenesis. Rapid multiplication of A. indicum equipped with two potent coumarins is important in order to meet the commercial demand for combat against dreadful diseases, thereby providing a new platform for plant-based drugs and their manufacture on a commercial scale.     

In Vitro Propagation and Assessment of the Genetic Fidelity of Musa acuminata (AAA) cv. Vaibalhla Derived from Immature Male Flowers

An efficient in vitro propagation method has been developed for the first time for Musa acuminata (AAA) cv. Vaibalhla, an economically important banana cultivar of Mizoram, India. Immature male flowers were used as explants. Murashige and Skoog’s (MS) medium supplemented with plant growth regulators (PGRs) were used for the regeneration process. Out of different PGR combinations, MS medium supplemented with 2 mg L^?1 6-benzylaminopurine (BAP) + 0.5 mg L^?1 α-naphthalene acetic acid (NAA) was optimal for production of white bud-like structures (WBLS). On this medium, explants produced the highest number of buds per explant (4.30). The highest percentage (77.77) and number (3.51) of shoot formation from each explants was observed in MS medium supplemented with 2 mg L^?1 kinetin + 0.5 mg L^?1 NAA. While MS medium supplemented with a combination of 2 mg L^?1 BAP + 0.5 mg L^?1 NAA showed the maximum shoot length (14.44 cm). Rooting efficiency of the shoots was highest in the MS basal medium without any PGRs. The plantlets were hardened successfully in the greenhouse with 96 % survival rate. Random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) markers were employed to assess the genetic stability of in vitro regenerated plantlets of M. acuminata (AAA) cv. Vaibalhla. Eight RAPD and 8 ISSR primers were successfully used for the analysis from the 40 RAPD and 30 ISSR primers screened initially. The amplified products were monomorphic across all the regenerated plants and were similar to the mother plant. The present standardised protocol will find application in mass production, conservation and genetic transformation studies of this commercially important banana.     

Effects of different concentrations of benzylaminopurine on shoot regeneration and hypericin content in Hypericum triquetrifolium Turra

The current study was undertaken to determine the effects of different benzylaminopurine (BAP) concentrations on the accumulation of bioactive hypericin in Hypericum triquetrifolium Turra. via micropropagation. To achieve this objective, seeds of H. triquetrifolium Turra. were cultured on Murashige and Skoog (MS) medium supplemented with a BAP (0.5, 1.0 and 2.0), 3% sucrose and 5.5% agar. Apical tips of axenic germinated seeds were proliferated on a MS medium supplemented with BAP (0.0, 0.5, 1.0 and 2.0 mg L(-1)). The highest shoot number was obtained from a MS medium supplemented with a 2.0 mg L(-1) BAP. Hypericin percentages were found to be highest in a 1.0 mg L(-1) BAP supplemented medium. These results provide the indication that cytokinin BAP can change the chemical composition of H. triquetrifolium Turra.; thereby, seriously impacting the quality and the efficacy of natural plant products produced by an in vitro culture system for aseptic production of hypericin.