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.     

Optimization of Adventitious Root Culture for Production of Biomass and Secondary Metabolites in Prunella vulgaris L.

Adventitious root cultures of Prunella vulgaris L. were established in shaking flask system for the production of biomass and secondary metabolites. Adventitious root cultures were induced from callus cultures obtained from leaf explants on solid Murashige and Skoog (MS) medium containing combination of 6-benzyladenine (BA; 1.0 mg l^?1) and naphthalene acetic acid (NAA; 1.5 mg l^?1). Thereafter, 0.49 g inoculum was transferred to liquid MS medium supplemented with different concentrations of NAA (0.5–2.0 mg l^?1). Growth kinetics of adventitious roots was recorded with an interval of 7 days for 49 days period. Highest biomass accumulation (2.13 g/l) was observed in liquid medium containing 1.0 mg l^?1 NAA after 21 days of inoculation. However, other concentrations of NAA also showed similar accumulation pattern but the biomass gradually decreases after 49 days of inoculation. Adventitious roots were collected and dried for investigation of total phenolics (TP), total flavonoids (TF), and antioxidant activities. Higher TPC (0.995 GAE mg/g-DRB) and TFC (6.615 RE mg/g-DRB) were observed in 0.5 mg l^?1 NAA treated cultures. In contrast, higher antioxidant activity (83.53 %) was observed 1.5 mg l^?1 NAA treated cultures. These results are helpful in up scaling of root cultures into bioreactor for secondary metabolites production.     

A Comparative Study of Anti-Candida Activity and Phenolic Contents of the Calluses from Lythrum salicaria L. in Different Treatments

In the study, anti-Candida activity and phenol contents of Lythrum salicaria L. calli and wild species have been evaluated. The seeds of L. salicaria (Lythraceae), collected from Lahidjan City in the north of Iran, were cultured in Murashige and Skoog medium (MSM) with a supplement, gibberellin, to germinate. Callus inductions were performed from segments of seedling on MSM containing different concentrations of plant growth regulators, 2,4-dichlorophenoxyacetic acid (2,4-D) and 6-benzylaminopurine (BAP). The activity of calluses extracts, wild plant, gallic acid, and 3,3′,4′-tri-O-methylellagic acid-4-O-β-d-glucopyranoside (TMEG) as the main phenolic compounds against Candida albicans was assessed using cup plate diffusion method. The total phenols contents of calli and wild plant extracts were analyzed using Folin–Ciocalteu reagent. The callus formation in MSM supplemented with various concentrations of 2,4-D and BAP were 0–100 %. Anti-Candida activity of callus extract which obtained from MSM supplemented with 2,4-D and BAP (1 mg?dm^?3) was similar to the wild plant extract. Minimum inhibitory concentration values of gallic acid and TMEG were obtained as 0.312 and 2.5 mg?cm^?3, respectively. Gallic acid equivalent values in all treatments were from 0 to 288 μg GAE mg^?1. Phenolic contents of plant aerial parts (331?±?3.7 μg GAE mg^?1) and the callus, which developed in MSM including 1 mg?dm^?3 of both 2,4-D and BAP, showed the same phenolic value and exhibited anti-Candida extract activity.     

An Improved Micropropagation of Arnebia hispidissima (Lehm.) DC. and Assessment of Genetic Fidelity of Micropropagated Plants Using DNA-Based Molecular Markers

An efficient and improved in vitro propagation method has been developed for Arnebia hispidissima, a medicinally and pharmaceutically important plant species of arid and semiarid regions. Nodal segments (3–4 cm) with two to three nodes obtained from field grown plants were used as explants for shoot proliferation. Murashige and Skoog’s (MS) medium supplemented with cytokinins with or without indole-3-acetic acid (IAA) or naphthalene acetic acid was used for shoot multiplication. Out of different PGRs combinations, MS medium containing 0.5 mg l^?1 6-benzylaminopurine and 0.1 mg l^?1 IAA was optimal for shoot multiplication. On this medium, explants produced the highest number of shoots (47.50?±?0.38). About 90 % of shoots rooted ex vitro on sterile soilrite under the greenhouse condition when the base (2–4 mm) of shoots was treated with 300 mg l^?1 of indole-3-butyric acid for 5 min. The plantlets were hardened successfully in the greenhouse with 85–90 % 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 plants of A. hispidissima. Out of 40 (25 RAPD and 15 ISSR) primers screened, 15 RAPD and 7 ISSR primers produced a total number of 111 (77 RAPD and 34 ISSR) reproducible amplicons. The amplified products were monomorphic across all the micropropagated plants and were similar to the mother plant. To the best of our knowledge, it is the first report on the assessment of the genetic fidelity in micropropagated plants of A. hispidissima.     

Influence of Culture Medium Composition and Light Conditions on the Accumulation of Bioactive Compounds in Shoot Cultures of <Emphasis Type="Italic">Scutellaria lateriflora</Emphasis> L. (American Skullcap) Grown In Vitro

Methanolic extracts from in vitro grown Scutellaria lateriflora shoots cultured on five Murashige and Skoog (MS) medium variants supplemented with different combinations of 6-benzylaminopurine (BAP) and α-naphthaleneacetic acid (NAA) under different light conditions (monochromatic light, white light and no light) were analysed by HPLC for three groups of metabolites: flavonoids (26 compounds), phenolic acids and their precursors (19+2) and phenylethanoid glycosides (2). The analyses revealed the presence of baicalein, baicalin, wogonin, wogonoside, 3,4-dihydroxyphenylacetic acid and verbascoside. There was clear evidence of the influence of plant growth regulators and light conditions on the accumulation of the analysed groups of secondary metabolites. The amounts of the compounds changed within a wide range—for the total flavonoid content, 30.2-fold (max. 1204.3 mg·100 g^?1 dry weight (DW)); for 3,4-dihydroxyphenylacetic acid, 5.5-fold (max. 33.56 mg·100 g^?1 DW); and for verbascoside, 1.5-fold (169.15 max. mg·100 g^?1 DW). The best medium for the production of most of the compounds was the Murashige and Skoog variant with 1 mg l^?1 BAP and 1 mg l^?1 NAA. For verbascoside, the best ‘productive’ medium was the MS variant supplemented with 0.5 mg l^?1 BAP and 2 mg l^?1 NAA. The accumulation of the metabolites was stimulated to the greatest extent by blue light, under which the extracts were found to contain the highest total amount of flavonoids and the highest amounts of flavonoid glucuronides, baicalin and wogonoside, as well as of verbascoside. Their amounts were, respectively, 1.54-, 1.49-, 2.05- and 1.86-fold higher than under the control white light.     

In Vitro Propagation,Encapsulation, and Genetic Fidelity Analysis of Terminalia arjuna: a Cardioprotective Medicinal Tree

The present study described an improved and reproducible in vitro regeneration system for Terminalia arjuna using nodal segment explants obtained from a mature plant. Shoot tips excised from in vitro proliferated shoots were encapsulated in 3 % sodium alginate and 100 mM CaCl₂?2H₂O for the development of synthetic seeds which may be applicable in short-term storage and germplasm exchange of elite genotype. Shoot multiplication was significantly influenced by a number of factors, namely types and concentrations of plant growth regulators, medium composition, repeated transfer of mother explants, subculturing of in vitro regenerated shoot clumps, agar concentrations, and temperature. Maximum numbers of shoots (16.50?±?3.67) were observed on modified Murashige and Skoog (MMS) medium containing 0.5 mg l^?1 of benzylaminopurine (BAP) and 0.1 mg l^?1 of naphthalene acetic acid (NAA). To shortening the regeneration pathway, rooting of micropropagated shoots under in vitro condition was excluded and an experiment on ex vitro rooting was conducted and it was observed that the highest percentage of shoots rooted ex vitro when treated with indole-3-butyric acid (IBA, 250 mg l^?1)?+?2-naphthoxy acetic acid (NOA, 250 mg l^?1) for 5 min. The well-developed ex vitro rooted shoots were acclimatized successfully in soilrite under greenhouse conditions with 80 % survival of plants. Randomly amplified polymorphic DNA (RAPD) analysis confirmed that all the regenerated plants were genetically identical to the mother plant, suggesting the absence of detectable genetic variation in the regenerated plantlets. To the best of our knowledge, this is the first report on synthetic seed production as well as ex vitro rooting and genetic fidelity assessment of micropropagated shoots of T. arjuna.     

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.     

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.     

A Stable and Efficient Agrobacterium tumefaciens-Mediated Genetic Transformation of the Medicinal Plant Digitalis purpurea L.

In this study, we developed a rapid and efficient method for in vitro propagation and Agrobacterium tumefaciens-mediated transformation of Digitalis purpurea L. (syn. foxglove), an important medicinal plant. Mature leaf explants of D. purpurea were used for 100 % adventitious shoot regeneration on Murashige and Skoog (MS) medium supplemented with 1 mg L^?1 thidiazuron (TDZ) (a cytokine) and 0.1 mg L^?1 1-naphthaleneacetic acid (NAA) (an auxin). Transformation was achieved by inoculating leaf explants with the A. tumefaciens strains GV2260/pBI121 or GV3101/pBI121. The binary vector pBI121 contained the reporter β-glucuronidase gene (GUS) and kanamycin selection marker nptII. Kanamycin-resistant shoots were regenerated directly on the selection medium 4–6 weeks after co-cultivation. Approximately, 52.2 and 60 % of kanamycin-resistant shoots transformed with Agrobacterium strains GV2260 and GV3101, respectively, showed strong GUS staining by histochemical assay. Furthermore, PCR and Southern blot analysis confirmed the presence of nptII and GUS on the chromosome of the transformed D. purpurea plants, and stable GUS expression was detected in the transformants by RT-PCR analysis. This efficient method of shoot regeneration and genetic transformation of D. purpurea will provide a powerful tool to increase and produce valuable components such as digitoxin, digoxin, and digoxigenin in D. purpurea through improved secondary metabolic pathways via a biotechnological approach.     

Effect of Elicitors and Precursors on Azadirachtin Production in Hairy Root Culture of Azadirachta indica

The present study involved strategies for enhancement in in vitro azadirachtin (commercially used biopesticide) production by hairy root cultivation of Azadirachta indica. Improvement in the azadirachtin production via triggering its biosynthetic pathway in plant cells was carried out by the exogenous addition of precursors and elicitors in the growth medium. Among the different abiotic stress inducers (Ag⁺, Hg⁺², Co⁺², Cu⁺²) and signal molecules (methyl jasmonate and salicylic acid) tested, salicylic acid at 15 mg l^?1 of concentration was found to enhance the azadirachtin yield in the hairy roots to the maximum (up to 4.95 mg g^?1). Similarly, among the different biotic elicitors tested (filter-sterilized fungal culture filtrates of Phoma herbarium, Alternaria alternata, Myrothecium sp., Fusarium solani, Curvularia lunata, and Sclerotium rolfsii; yeast extract; and yeast extract carbohydrate fraction), addition of filter-sterilized fungal culture filtrate of C. lunata (1 %?v/v) resulted in maximum azadirachtin yield enhancement in hairy root biomass (up to 7.1 mg g^?1) with respect to the control (3.3 mg g^?1). Among all the biosynthetic precursors studied (sodium acetate, cholesterol, squalene, isopentynyl pyrophosphate, mavalonic acid lactone, and geranyl pyrophosphate), the overall azadirachtin production (70.42 mg l^?1 in 25 days) was found to be the highest with cholesterol (50 mg l^?1) addition as an indirect precursor in the medium.     

Pilot-Scale Culture of Hypericum Perforatum L. Adventitious Roots in Airlift Bioreactors for the Production of Bioactive Compounds

Hypericum perforatum L. (St. John’s Wort) is an important medicinal plant which is widely used in the treatment for depression and irritable bowel syndrome. It is also used as a dietary supplement. Major bioactive phytochemicals of H. perforatum are phenolics and flavonoids. Quality of these phytochemicals is dramatically influenced by environmental and biological factors in the field grown plants. As an alternative, we have developed adventitious root cultures in large-scale bioreactors for the production of useful phytochemicals. Adventitious roots of H. perforatum were cultured in 500 l pilot-scale airlift bioreactors using half-strength Murashige and Skoog medium with an ammonium and nitrate ratio of 5:25 mM and supplemented with 1.0 mg l^?1 indole butyric acid, 0.1 mg l^?1 kinetin, and 3 % sucrose for the production of bioactive phenolics and flavonoids. Then 4.6 and 6.3 kg dry biomass were realized in the 500 l each of drum-type and balloon-type bioreactors, respectively. Accumulation of 66.9 mg g^?1 DW of total phenolics, 48.6 mg g^?1 DW of total flavonoids, 1.3 mg g^?1 DW of chlorogenic acid, 0.01 mg g^?1 DW of hyperin, 0.04 mg g^?1 DW of hypericin, and 0.01 mg g^?1 DW of quercetin could be achieved with adventitious roots cultured in 500 l balloon-type airlift bioreactors. Our findings demonstrate the possibilities of using H. perforatum adventitious root cultures for the production of useful phytochemicals to meet the demand of pharmaceutical and food industry.     

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.     

Piper nigrum: Micropropagation, Antioxidative enzyme activities, and Chromatographic Fingerprint Analysis for Quality Control

A reliable in vitro regeneration system for the economical and medicinally important Piper nigrum L. has been established. Callus and shoot regeneration was encouraged from leaf portions on Murashige and Skoog (MS) medium augmented with varied concentrations of plant growth regulators. A higher callus production (90 %) was observed in explants incubated on MS medium incorporated with 1.0 mg?L^?1 6-benzyladenine (BA) along with 0.5 mg?L^?1 gibberellic acid after 4 weeks of culture. Moreover, a callogenic response of 85 % was also recorded for 1.0 mg?L^?1 BA in combination with 0.25 mg?L^?1 α-naphthalene acetic acid (NAA) and 0.25 mg?L^?1 2,4-dichlorophenoxyacetic acid or 0.5 mg?L^?1 indole butyric acid (IBA) along with 0.25 mg?L^?1 NAA and indole acetic acid. Subsequent sub-culturing of callus after 4 weeks of culture onto MS medium supplemented with 1.5 mg?L^?1 thiodiazoran or 1.5 mg?L^?1 IBA induced 100 % shoot response. Rooted plantlets were achieved on medium containing varied concentrations of auxins. The antioxidative enzyme activities [superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and ascorbate peroxidase (APX)] revealed that significantly higher SOD was observed in regenerated plantlets than in other tissues. However, POD, CAT, and APX were higher in callus than in other tissues. A high-performance liquid chromatography (HPLC) fingerprint analysis protocol was established for quality control in different in vitro-regenerated tissues of P. nigrum L. During analysis, most of the common peaks represent the active principle “piperine.” The chemical contents, especially piperine, showed variation from callus culture to whole plantlet regeneration. Based on the deviation in chromatographic peaks, the in vitro-regenerated plantlets exhibit a nearly similar piperine profile to acclimated plantlets. The in vitro regeneration system and HPLC fingerprint analysis established here brought a novel approach to the quality control of in vitro plantlets, producing metabolites of interest with substantial applications for the conservation of germplasm.     

Plantlet Regeneration from Callus Cultures of Selected Genotype of <Emphasis Type="Italic">Aloe vera</Emphasis> L.—An Ancient Plant for Modern Herbal Industries

Aloe vera L., a member of Liliaceae, is a medicinal plant and has a number of curative properties. We describe here the development of tissue culture method for high-frequency plantlet regeneration from inflorescence axis-derived callus cultures of sweet aloe genotype. Competent callus cultures were established on 0.8% agar-gelled Murashige and Skoog’s (MS) basal medium supplemented with 6.0 mg l⁻¹ of 2,4-dichlorophenoxyacetic acid (2,4-D) and 100.0 mg l⁻¹ of activated charcoal and additives (100 mg l⁻¹ of ascorbic acid, 50.0 mg l⁻¹ each of citric acid and polyvinylpyrrolidone, and 25.0 mg l⁻¹ each of l-arginine and adenine sulfate). The callus cultures were cultured on MS medium containing 1.5 mg l⁻¹ of 2,4-D, 0.25 mg l⁻¹ of Kinetin (Kin), and additives with 4% carbohydrate source for multiplication and long-term maintenance of regenerative callus cultures. Callus cultures organized, differentiated, and produced globular embryogenic structures on MS medium with 1.0 mg l⁻¹ of 2,4-D, 0.25 mg l⁻¹ of Kin, and additives (50.0 mg l⁻¹ of ascorbic acid and 25.0 mg l⁻¹ each of citric acid, l-arginine, and adenine sulfate). These globular structures subsequently produced shoot buds and then complete plantlets on MS medium containing 1.0 mg l⁻¹ of 6-benzylaminopurine and additives. A hundred percent regenerated plantlets were hardened in the greenhouse and stored under an agro-net house/nursery. The regeneration system defined could be a useful tool not only for mass-scale propagation of selected genotype of A. vera, but also for genetic improvement of plant species through genetic transformation.     

In Situ Biphasic Extractive Fermentation for Hexanoic Acid Production from Sucrose by Megasphaera elsdenii NCIMB 702410

Hexanoic acid production by a bacterium using sucrose as an economic carbon source was studied under conditions in which hexanoic acid was continuously extracted by liquid–liquid extraction. Megasphaera elsdenii NCIMB 702410, selected from five M. elsdenii strains, produced 4.69 g l^?1 hexanoic acid in a basal medium containing sucrose. Production increased to 8.19 g l^?1 when the medium was supplemented by 5 g l^?1 sodium butyrate. A biphasic liquid–liquid extraction system with 10 % (v/v) alamine 336 in oleyl alcohol as a solvent was evaluated in a continuous stirred-tank reactor held at pH 6. Over 90 % (w/w) of the hexanoic acid in a 0.5 M aqueous solution was transferred to the extraction solvent within 10 h. Cell growth was not significantly inhibited by direct contact of the fermentation broth with the extraction solvent. The system produced 28.42 g l^?1 of hexanoic acid from 54.85 g l^?1 of sucrose during 144 h of culture, and 26.52 and 1.90 g l^?1 of hexanoic acid was accumulated in the extraction solvent and the aqueous fermentation broth, respectively. The productivity and yield of hexanoic acid were 0.20 g l^?1 h^?1 and 0.50 g g^?1 sucrose, respectively.     

Acceleration of Adventitious Shoots by Interaction Between Exogenous Hormone and Adenine Sulphate in Althaea Officinalis L.

In the current study attempts were made to investigate the effects of three different phases of callus induction followed by adventitious regeneration from leaf segments (central and lateral vein). Callus induction was observed in Murashige and Skoog’s (MS) medium supplemented with 15.0 μM 2,4-dichloro phenoxy acetic acid (2,4-D). Adventitious shoot buds formation was achieved on MS medium supplemented with 7.5 μM 2,4-D and 20.0 μM AdS in liquid medium as it induced 19.2?±?0.58 buds in central vein explants. Addition of different growth regulators (cytokinins—6-benzyladenine, kinetin and 2-isopentenyl adenine alone or in combination with auxins—indole-3-acetic acid, indole-3-butyric acid and α-naphthalene acetic acid, improved the shoot regeneration efficiency, in which 5.0 μM 6-benzyl adenine along with 0.25 μM α-naphthalene acetic acid was shown to be the most effective medium for maximum shoot regeneration (81.3 %) with 24.6 number of shoots and 4.4?±?0.08 cm shoot length per explant. Leaf culture of central veins led to better shoot formation capacity in comparison to lateral vein. Rooting was readily achieved on the differentiated shoots on 1/2 MS medium augmented with 20.0 μM indole-3-butyric acid. The plants were successfully hardened off in sterile soilrite followed by their establishment in garden soil with 80 % survival rate.     

Optimization of Factors Influencing Microinjection Method for Agrobacterium tumefaciens-Mediated Transformation of Tomato

A simple and efficient protocol for Agrobacterium-mediated genetic transformation of tomato was developed using combination of non-tissue culture and micropropagation systems. Initially, ESAM region of 1-day-old germinated tomato seeds were microinjected for one to five times with Agrobacterium inoculums (OD₆₀₀?=?0.2–1.0). The germinated seeds were cocultivated in the MS medium fortified with (0–200 mM) acetosyringone and minimal concentrations of (0–20 mg?L^?1) kanamycin, and the antibiotic concentration was doubled during the second round of selection. Bacterial concentration of OD₆₀₀?=?0.6 served as an optimal concentration for infection and the transformation efficiency was significantly higher of about 46.28 %. In another set of experiment, an improved and stable regeneration system was adapted for the explants from the selection medium. Four-day-old double cotyledonary nodal explants were excised from the microinjected seedlings and cultured onto the MS medium supplemented with 1.5 mg?L^?1 thidiazuron, 1.5 mg?L^?1 indole-3-butyric acid, 30 mg?L^?1 kanamycin, and 0–1.5 mg?L^?1 adenine sulphate. Maximum of 9 out of 13 micropropagated shoots were shown positive to GUS assay. By this technique, the transformation efficiency was increased from 46.28 to 65.90 %. Thus, this paper reports the successful protocol for the mass production of transformants using microinjection and micropropagation techniques.     

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.     

Growth Parameters,Photosynthetic Performance,and Biochemical Characterization of Newly Isolated Green Microalgae in Response to Culture Condition Variations

This work aimed to characterize two native microalgal strains newly isolated from South Mediterranean areas and identified as Chlorella sorokiniana ES3 and Neochloris sp. AM2. The growth properties and biochemical composition of these microalgae were evaluated in different culture media (Algal, BG-11, f/2, and Conway). Among the tested media, nitrate- and phosphate-rich Algal medium provided the maximum biomass productivities (85.5 and 111.5 mg l^?1 day^?1 for C. sorokiniana and Neochloris sp., respectively), while the nitrate- and phosphate-deficient f/2 medium resulted in the highest lipid productivities (24.1 and 35.8 mg l^?1 day^?1 for C. sorokiniana and Neochloris sp., respectively). The physiological state of both microalgae was investigated under different light and temperature levels using the pulse amplitude-modulated fluorometry. The better photosynthetic efficiency of C. sorokiniana was obtained at 23 °C with a light saturation of 156 μE m^?2 s^?1, while that of Neochloris sp. was achieved at 15 °C with a light saturation of 151 μE m^?2 s^?1. The analysis of fatty acid profile and biodiesel parameters revealed that C. sorokiniana, cultivated in Algal and f/2 media, can be considered as a suitable candidate for high-quality biodiesel production.