Beneficial Effect of Acetic Acid on the Xylose Utilization and Bacterial Cellulose Production by Gluconacetobacter xylinus

In this work, acetic acid was found as one promising substrate to improve xylose utilization by Gluconacetobacter xylinus CH001. Also, with the help of adding acetic acid into medium, the bacterial cellulose (BC) production by G. xylinus was increased significantly. In the medium containing 3 g l⁻¹ acetic acid, the optimal xylose concentration for BC production was 20 g l⁻¹. In the medium containing 20 g l⁻¹ xylose, the xylose utilization and BC production by G. xylinus were stimulated by acetic acid within certain concentration. The highest BC yield (1.35 ± 0.06 g l⁻¹) was obtained in the medium containing 20 g l⁻¹ xylose and 3 g l⁻¹ acetic acid after 14 days. This value was 6.17-fold higher than the yield (0.21 ± 0.01 g l⁻¹) in the medium only containing 20 g l⁻¹ xylose. The results analyzed by FE-SEM, FTIR, and XRD showed that acetic acid affected little on the microscopic morphology and physicochemical characteristics of BC. Base on the phenomenon observed, lignocellulosic acid hydrolysates (xylose and acetic acid are main carbon sources present in it) could be considered as one potential substrate for BC production.     

Enhanced Glucosamine Production with Actinomucor elegans Based on Stimulating Factor of Methanol

Glucosamine (GlcN) is a major and valuable component in the cell wall of fungi. In this study, the cell wall was treated via a two-stage alkali and acid process, and chitin and chitosan were fully deacetylated, partially depolymerized, and converted to GlcN oligosaccharides. Then, the oligosaccharides were analyzed by high performance liquid chromatography. The influences of Actinomucor elegans on GlcN production in a flask culture were investigated to achieve an optimum yield of GlcN. The experimental result showed that cultivation in condition of pH 6.0, 100 mL working volume (500 mL flask), 10 % (v/v) inoculum concentration, at 28 °C and 200 rpm for 6 days yielded highest dry cell weight (DCW) which was 23.43 g L⁻¹, with a GlcN concentration of 5.12 g L⁻¹. Methanol as stimulating factor was found to exert the best effect in concentration of 1.5 % (v/v). With addition of methanol into medium, the DCW increased from 23.69 to 32.42 g L⁻¹, leading to maximum GlcN concentration of 6.85 g L⁻¹ obtained. Here, the methanol addition may be useful for industrial production of GlcN, and may also be meaningful for the production of other fine chemicals by filamentous fungi.     

Plant Growth Promoting Bacteria from Cow Dung Based Biodynamic Preparations

Indigenous formulations based on cow dung fermentation are commonly used in organic farming. Three biodynamic preparations viz., Panchagavya (PG), BD500 and ‘Cow pat pit’ (CPP) showed high counts of lactobacilli (10⁹ ml⁻¹) and yeasts (10⁴ ml⁻¹). Actinomycetes were present only in CPP (10⁴ ml⁻¹) and absent in the other two. Seven bacterial isolates from these ferments were identified by a polyphasic approach: Bacillus safensis (PG1), Bacillus cereus (PG2, PG4 PG5), Bacillus subtilis (BD2) Lysinibacillus xylanilyticus (BD3) and Bacillus licheniformis (CPP1). This is the first report of L. xylanilyticus and B. licheniformis in biodynamic preparations. Only three carbon sources—dextrose, sucrose and trehalose out of 21 tested were utilized by all the bacteria. None could utilize arabinose, dulcitol, galactose, inositol, inulin, melibiose, raffinose, rhamnose and sorbitol. All the strains produced indole acetic acid (1.8–3.7 μg ml⁻¹ culture filtrate) and ammonia. None could fix nitrogen; but all except B. safensis and B. licheniformis could solubilize phosphorous from insoluble tri-calcium phosphate. All the strains except L. xylaniliticus exhibited antagonism to the plant pathogen Rhizoctonia bataticola whereas none could inhibit Sclerotium rolfsi. In green house experiment in soil microcosms, bacterial inoculation significantly promoted growth of maize; plant dry weight increased by ~21 % due to inoculation with B. cereus (PG2). Results provide a basis for understanding the beneficial effects of biodynamic preparations and industrial deployment of the strains.

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In vitro propagation of female Ephedra foliata Boiss. & Kotschy ex Boiss.: an endemic and threatened Gymnosperm of the Thar Desert

Ephedra foliata Boiss. & Kotschy ex Boiss., (family – Ephedraceae), is an ecologically and economically important threatened Gymnosperm of the Indian Thar Desert. A method for micropropagation of E. foliata using nodal explant of mature female plant has been developed. Maximum bud-break (90 %) of the explant was obtained on MS medium supplemented with 1.5 mg l⁻¹ of benzyl adenine (BA) + additives. Explant produces 5.3 ± 0.40 shoots from single node with 3.25 ± 0.29 cm length. The multiplication of shoots in culture was affected by salt composition of media, types and concentrations of plant growth regulators (PGR’s) and their interactions, time of transfer of the cultures. Maximum number of shoots (26.3 ± 0.82 per culture vessel) were regenerated on MS medium modified by reducing the concentration of nitrates to half supplemented with 200 mg l⁻¹ ammonium sulphate {(NH₄) ₂SO₄} (MMS3) + BA (0.25 mg l⁻¹), Kinetin (Kin; 0.25 mg l⁻¹), Indole-3-acetic acid (IAA; 0.1 mg l⁻¹) and additives. The in vitro produced shoots rooted under ex vitro on soilrite moistened with one-fourth strength of MS macro salts in screw cap bottles by treating the shoot base (s) with 500 mg l⁻¹ of Indole-3-butyric acid (IBA) for 5 min. The micropropagated plants were hardened in the green house. The described protocol can be applicable for (i) large scale plant production (ii) establishment of plants in natural habitat and (iii) germplasm conservation of this endemic Gymnosperm of arid regions.     

Formulation of a protein-free medium based on IPL-41 for the sustained growth of Drosophila melanogaster S2 cells

An animal protein-free medium was developed for Drosophila melanogaster S2 (S2AcGPV2) cells genetically modified to produce the rabies virus G glycoprotein (GPV). IPL-41, used as a basal medium, was supplemented with yeastolate, carbohydrates, amino acids and lipids aiming initially to reduce and further to eliminate the need of fetal bovine serum. The S2AcGPV2 cells were fully capable of growing in serum-free supplemented IPL-41 medium containing 6 g L⁻¹ yeastolate ultrafiltrate, 10 g L⁻¹ glucose, 3.5 g L⁻¹ glutamine, 0.5 g L⁻¹ fructose, 2 g L⁻¹ lactose, 0.6 g L⁻¹ tyrosine, 1.48 g L⁻¹ methionine and 1% (v/v) lipid emulsion, reaching 19 × 10⁶ cells mL⁻¹. Maximum specific growth rate and cell productivity were 0.025 h⁻¹ and 0.57 × 10⁵ cells mL⁻¹ h⁻¹, respectively. Glucose and lactose were consumed during cell culture, but not fructose. Lactate concentration generally decreased during cell culture, while ammonium concentration reached 167 mg L⁻¹, however, without noticeable deleterious effects on cell growth. GPV concentration values achieved were, however, modest in the proposed medium formulation.     

The influence of Agrobacterium rhizogenes on induction of hairy roots and ß-carboline alkaloids production in Tribulus terrestris L.

We have developed an efficient transformation system for Tribulus terrestris L., an important medicinal plant, using Agrobacterium rhizogenes strains AR15834 and GMI9534 to generate hairy roots. Hairy roots were formed directly from the cut edges of leaf explants 10–14 days after inoculation with the Agrobacterium with highest frequency transformation being 49 %, which was achieved using Agrobacterium rhizogenes AR15834 on hormone-free MS medium after 28 days inoculation. PCR analysis showed that rolB genes of Ri plasmid of A. rhizogenes were integrated and expressed into the genome of transformed hairy roots. Isolated transgenic hairy roots grew rapidly on MS medium supplemented with indole-3-butyric acid. They showed characteristics of transformed roots such as fast growth and high lateral branching in comparison with untransformed roots. Isolated control and transgenic hairy roots grown in liquid medium containing IBA were analyzed to detect ß-carboline alkaloids by High Performance Thin Layer Chromatograghy (HPTLC). Harmine content was estimated to be 1.7 μg g⁻¹ of the dried weight of transgenic hairy root cultures at the end of 50 days of culturing. The transformed roots induced by AR15834 strain, spontaneously, dedifferentiated as callus on MS medium without hormone. Optimum callus induction and shoot regeneration of transformed roots in vitro was achieved on MS medium containing 0.4 mg L⁻¹ naphthaleneacetic acid and 2 mg L⁻¹ 6-benzylaminopurine (BAP) after 50 days. The main objective of this investigation was to establish hairy roots in this plant by using A. rhizogenes to synthesize secondary products at levels comparable to the wild-type roots.     

Ethanol production from sweet sorghum juice using very high gravity technology: Effects of carbon and nitrogen supplementations

Ethanol production from sweet sorghum juice by Saccharomyces cerevisiae NP01 was investigated under very high gravity (VHG) fermentation and various carbon adjuncts and nitrogen sources. When sucrose was used as an adjunct, the sweet sorghum juice containing total sugar of 280 g l⁻¹, 3 g yeast extract l⁻¹ and 5 g peptone l⁻¹ gave the maximum ethanol production efficiency with concentration, productivity and yield of 120.68 ± 0.54 g l⁻¹, 2.01 ± 0.01 g l⁻¹ h⁻¹ and 0.51 ± 0.00 g g⁻¹, respectively. When sugarcane molasses was used as an adjunct, the juice under the same conditions gave the maximum ethanol concentration, productivity and yield with the values of 109.34 ± 0.78 g l⁻¹, 1.52 ± 0.01 g l⁻¹ h⁻¹ and 0.45 ± 0.01 g g⁻¹, respectively. In addition, ammonium sulphate was not suitable for use as a nitrogen supplement in the sweet sorghum juice for ethanol production since it caused the reduction in ethanol concentration and yield for approximately 14% when compared to those of the unsupplemented juices.     

Kinetic and Thermodynamic Characterization of Glucoamylase from Colletotrichum sp. KCP1

Extracellular glucoamylase of Colletotrichum sp. KCP1 produced through solid state fermentation was purified by two steps purification process comprising ammonium sulphate precipitation followed by gel permeation chromatography (GPC). The Recovery of glucoamylase after GPC was 50.40 % with 19.3-fold increase in specific activity. The molecular weight of enzyme was found to be 162.18 kDa by native-PAGE and was dimeric protein of two sub-units with molecular weight of 94.62 and 67.60 kDa as determined by SDS-PAGE. Activation energy for starch hydrolysis was 26.45 kJ mol⁻¹ while temperature quotient (Q₁₀) was found to be 1.9. The enzyme was found to be stable over wide pH range and thermally stable at 40–50 °C up to 120 min while exhibited maximum activity at 50 °C with pH 5.0. The pKa₁ and pKa₂ of ionisable groups of active site controlling V_max were 3.5 and 6.8, respectively. V_max, K_m and K_cat for starch hydrolysis were found to be 58.82 U ml⁻¹, 1.17 mg (starch) ml⁻¹ and 449 s⁻¹, respectively. Activation energy for irreversible inactivation (E_a(d)) of glucoamylase was 74.85 kJ mol⁻¹. Thermodynamic parameters of irreversible inactivation of glucoamylase and starch hydrolysis were also determined.     

Micropropagtion of Terminalia bellerica from nodal explants of mature tree and assessment of genetic fidelity using ISSR and RAPD markers

The present study reports an efficient in vitro micropropagation protocol for a medicinally important tree, Terminalia bellerica Roxb. from nodal segments of a 30 years old tree. Nodal segments taken from the mature tree in March-April and cultured on half strength MS medium gave the best shoot bud proliferation response. Combinations of serial transfer technique (ST) and incorporation of antioxidants (AO) [polyvinylpyrrolidone, PVP (50 mg l⁻¹) + ascorbic acid (100 mg l⁻¹) + citric acid (10 mg l⁻¹)] in the culture medium aided to minimize browning and improve explant survival during shoot bud induction. Highest multiplication of shoots was achieved on medium supplemented with 6-benzyladenine (BA, 8.8 μM) and α-naphthalene acetic acid (NAA, 2.6 μM) in addition to antioxidants. Shoot elongation was obtained on MS medium containing BA (4.4 μM) + phloroglucinol (PG, 3.9 μM). Elongated shoots were transferred to half strength MS medium containing indole-3-butyric acid (IBA, 2.5 μM) for root development. The acclimatization of plantlets was carried out under greenhouse conditions. The genetic fidelity of the regenerated plants was checked using inter simple sequence repeats (ISSR) and randomly amplified polymorphic DNA (RAPD) analysis. Comparison of the bands among the regenerants and mother plant confirmed true-to-type clonal plants.     

Role of nitrogen and magnesium for growth,yield and nutritional quality of radish

Nitrogen (N) affects all levels of plant function from metabolism to resource allocation, growth, and development and Magnesium (Mg) is a macronutrient that is necessary to both plant growth and health. Radish (Raphanus sativus L.) occupies an important position in the production and consumption of vegetables globally, but there are still many problems and challenges in its nutrient management. A pot trial was conducted to investigate the effects of nitrogen and magnesium fertilizers on radish during the year 2018–2019. Nitrogen and magnesium was applied at three rates (0, 0.200, and 0.300 g N kg⁻¹ soil) and (0, 0.050, and 0.100 g Mg kg⁻¹ soil) respectively. The experiment was laid out in a completely randomized design (CRD) and each treatment was replicated three times. Growth, yield and quality indicators of radish (plant height, root length, shoot length, plant weight, total soluble sugar, ascorbic acid, total soluble protein, crude fiber, etc.) were studied. The results indicated that different rates of nitrogen and magnesium fertilizer not only influence the growth dynamics and yields but also enhances radish quality. The results revealed that the growth, yield and nutrient contents of radish were increased at a range of 0.00 g N. kg⁻¹ soil to 0.300 g N. kg⁻¹ soil and 0.00 g Mg. kg⁻¹ soil to 0.050 g Mg. kg⁻¹ soil and then decreased gradually at a level of 0.100 g Mg. kg⁻¹ soil. In contrast, the crude fiber contents in radish decreased significantly with increasing nitrogen and magnesium level but increased significantly at Mg₂ level (0.050 g Mg. kg⁻¹ soil). The current study produced helpful results for increasing radish quality, decreasing production costs, and diminishing underground water contamination.     

Biodegradation of Phenanthrene by Pseudomonas putida and a Bacterial Consortium in the Presence and in the Absence of a Surfactant

This study describes the biodegradation of phenanthrene in aqueous media in the presence and in the absence of a surfactant, Brij 30. Biodegradations were performed using either Pseudomonas putida DSMZ 8368 or a bacterial consortium Pyr01 isolated from one PAHs-polluted site. P. putida degraded phenanthrene to form 1-hydroxy-2-naphthoic acid (1H2Na) as the major metabolite. LC–MS analysis revealed the production of complementary intermediates in the presence of Brij 30, showing intense ions at mass-to-charge ratios (m/z) 97 and 195. Higher phenanthrene biodegradation rate was obtained in the presence of Brij 30. Conversely, in the case of Pyr01consortium, the addition of Brij 30 (0.5 g L⁻¹) had a negative effect on biodegradation: no phenanthrene biodegradation products were detected in the medium, whereas a production of several intermediates (m/z 97, 195 and 293) was obtained without surfactant. New results on phenanthrene metabolism by P. putida DSMZ 8368 and Pyr01 consortium in the presence and in the absence of Brij 30 we obtained. They confirm that the knowledge of the effect of a surfactant on bacterial cultures is crucial for the optimization of surfactant-enhanced PAHs biodegradation.

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Efficient Open Fermentative Production of Polymer-Grade L-Lactate from Sugarcane Bagasse Hydrolysate by Thermotolerant Bacillus sp. Strain P38

Lactic acid is one of the top 30 potential building-block chemicals from biomass, of which the most extensive use is in the polymerization of lactic acid to poly-lactic-acid (PLA). To reduce the cost of PLA, the search for cheap raw materials and low-cost process for lactic acid production is highly desired. In this study, the final titer of produced L-lactic acid reached a concentration of 185 g·L⁻¹ with a volumetric productivity of 1.93 g·L⁻¹·h⁻¹ by using sugarcane bagasse hydrolysate as the sole carbon source simultaneously with cottonseed meal as cheap nitrogen sources under the open fed-batch fermentation process. Furthermore, a lactic acid yield of 0.99 g per g of total reducing sugars was obtained, which is very close to the theoretical value (1.0 g g⁻¹). No D-isomer of lactic acid was detected in the broth, and thereafter resulted in an optical purity of 100%, which exceeds the requirement of lactate polymerization process. To our knowledge, this is the best performance of fermentation on polymer-grade L-lactic acid production totally using lignocellulosic sources. The high levels of optically pure l-lactic acid produced, combined with the ease of handling and low costs associated with the open fermentation strategy, indicated the thermotolerant Bacillus sp. P38 could be an excellent candidate strain with great industrial potential for polymer-grade L-lactic acid production from various cellulosic biomasses.     

Detection of Ammonia-Oxidizing Archaea in Fish Processing Effluent Treatment Plants

Ammonia oxidation is the rate limiting step in nitrification and thus have an important role in removal of ammonia in natural and engineered systems with participation of both ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB). However, their relative distribution and activity in fish processing effluent treatment plants (FPETPs) though significant, is hitherto unreported. Presence of AOA in sludge samples obtained from FPETPs was studied by amplification and sequencing of thaumarchaeal ammonia monooxygenase subunit A (AOA-amoA) gene. Different primer sets targeting 16S rRNA and AOA-amoA gene were used for the detection of AOA in FPETPs. Phylogenetic analysis of the gene revealed that the AOA was affiliated with thaumarchaeal group 1.1a lineage (marine cluster). Quantitative real time PCR of amoA gene was used to study the copy number of AOA and AOB in FPETPs. The AOA-amoA and AOB-amoA gene copy numbers of sludge samples ranged from 2.2 × 10⁶ to 4.2 × 10⁸ and 1.1 × 10⁷ to 8.5 × 10⁸ mg⁻¹ sludge respectively. Primer sets Arch-amoAF/Arch-amoAR and 340F/1000R were found to be useful for the sensitive detection of AOA-amoA and Archaeal 16S rRNA genes respectively in FPETPs. Their presence suggests the widespread occurrence and possible usefulness in removing ammonia from FPETPs which is in line with reports from other waste water treatment plants.

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Sweet Sorghum Juice and Bagasse as Feedstocks for the Production of Optically Pure Lactic Acid by Native and Engineered <Emphasis Type="Italic">Bacillus coagulans</Emphasis> Strains

Sweet sorghum is a bioenergy crop that produces large amounts of soluble sugars in its stems (3–7 Mg ha^?1) and generates significant amounts of bagasse (15–20 Mg ha^?1) as a lignocellulosic feedstock. These sugars can be fermented not only to biofuels but also to bio-based chemicals. The market potential of the latter may be higher given the current prices of petroleum and natural gas. The yield and rate of production of optically pure d-(?)- and l-(+)-lactic acid as precursors for the biodegradable plastic polylactide was optimized for two thermotolerant Bacillus coagulans strains. Strain 36D1 fermented the sugars in unsterilized sweet sorghum juice at 50 °C to l-(+)-lactic acid (～150 g L^?1; productivity, 7.2 g L^?1 h^?1). B. coagulans strain QZ19-2 was used to ferment sorghum juice to d-(?)-lactic acid (～125 g L^?1; productivity, 5 g L^?1 h^?1). Carbohydrates in the sorghum bagasse were also fermented after pretreatment with 0.5 % phosphoric acid at 190 °C for 5 min. Simultaneous saccharification and co-fermentation of all the sugars (SScF) by B. coagulans resulted in a conversion of 80 % of available carbohydrates to optically pure lactic acid depending on the B. coagulans strain used as the microbial biocatalyst. Liquefaction of pretreated bagasse with cellulases before SScF (L + SScF) increased the productivity of lactic acid. These results show that B. coagulans is an effective biocatalyst for fermentation of all the sugars present in sweet sorghum juice and bagasse to optically pure lactic acid at high titer and productivity as feedstock for bio-based plastics.     

Shoot culture of Bacopa monnieri: standardization of explant,vessels and bioreactor for growth and antioxidant capacity

Standardization of biomass production in different vessels and bioreactor using explants and media for growth, total phenolic content and antioxidant capacity of shoot culture of Bacopa monnieri is described. Maximum number of shoots per explant, higher explants response irrespective of the type of explants, and higher shoot length was obtained on MS medium containing BAP (2.5 mg l⁻¹) and IAA (0.01 mg l⁻¹) with 3 % sucrose. This medium was selected by varying BAP concentration and recorded optimal for shoot culture on gelled medium. The condition of 0.5 cm explant size and 20 explant/40 ml (1 explant/2 ml) was optimal for high explant response, number of shoots per explant regenerated and shoots length. Among the different vessels used, maximum growth index was achieved in Growtek bioreactor (10.0) followed by magenta box (9.16), industrial glass jar (7.7) and conical flask (7.2). The cultures grown in conical flask (100 ml) were used as control. The total phenolic content and antioxidant capacity of in vitro grown plants was higher to that recorded for in vivo material. Among in vitro regenerated plants, the activity was maximal in the tissues grown in 250 ml conical flask. The most critical function for vessels is to support the optimum profusion (growing area for maximum growth) of shoots and for B. monnieri, Growtek bioreactor supported 1980 shoots l⁻¹ medium as compared to control (938 shoots l⁻¹). Growtek bioreactor was considered effective system to produce B. monnieri biomass in culture without loss of antioxidant properties.     

Plant growth stimulation of wheat (Triticum aestivum L.) by inoculation of salinity tolerant Azotobacter strains

Five salinity tolerant Azotobacter strains i.e., ST3, ST6, ST9, ST17 and ST24 were obtained from saline soils. These Azotobacter strains were used as inoculant for wheat variety WH157 in earthen pots containing saline soil under pot house conditions, using three fertilizer treatment doses i.e., control (no fertilizer, no inoculation), 90 Kg N ha⁻¹ and 120 Kg N ha⁻¹. Inoculation with salinity tolerant Azotobacter strains caused significant increase in total nitrogen, biomass and grain yield of wheat. Maximum increase in plant growth parameters were obtained after inoculation with Azotobacter strain ST24 at fertilization dose of 120 kg N ha⁻¹ and its inoculation resulted in attaining 89.9 cms plant height, 6.1 g seed yield, 12.0 g shoot dry weight and 0.7 % total nitrogen. The survival of Azotobacter strain ST24 in the soil was also highest in all the treatments at 30, 60 and 90 days after sowing (DAS). However, the population of Azotobacter decreased on 90 DAS as compared to counts observed at 60 DAS at all the fertilization treatments.     

Temporal changes of soil physic‐chemical properties at different soil depths during larch afforestation by multivariate analysis of covariance

Soil physic-chemical properties differ at different depths; however, differences in afforestation-induced temporal changes at different soil depths are seldom reported. By examining 19 parameters, the temporal changes and their interactions with soil depth in a large chronosequence dataset (159 plots; 636 profiles; 2544 samples) of larch plantations were checked by multivariate analysis of covariance (MANCOVA). No linear temporal changes were found in 9 parameters (N, K, N:P, available forms of N, P, K and ratios of N: available N, P: available P and K: available K), while marked linear changes were found in the rest 10 parameters. Four of them showed divergent temporal changes between surface and deep soils. At surface soils, changing rates were 262.1 g·kg⁻¹·year⁻¹ for SOM, 438.9 mg·g⁻¹·year⁻¹ for C:P, 5.3 mg·g⁻¹·year⁻¹ for C:K, and −3.23 mg·cm⁻³·year⁻¹ for bulk density, while contrary tendencies were found in deeper soils. These divergences resulted in much moderated or no changes in the overall 80-cm soil profile. The other six parameters showed significant temporal changes for overall 0–80-cm soil profile (P: −4.10 mg·kg⁻¹·year⁻¹; pH: −0.0061 unit·year⁻¹; C:N: 167.1 mg·g⁻¹·year⁻¹; K:P: 371.5 mg·g⁻¹ year⁻¹; N:K: −0.242 mg·g⁻¹·year⁻¹; EC: 0.169 μS·cm⁻¹·year⁻¹), but without significant differences at different soil depths (P > 0.05). Our findings highlight the importance of deep soils in studying physic-chemical changes of soil properties, and the temporal changes occurred in both surface and deep soils should be fully considered for forest management and soil nutrient balance.     

Synergistic effect of BAP and GA3 on in vitro flowering of Guizotia abyssinica Cass.-A multipurpose oil crop

Apical and axillary buds of Guizotia abyssinica Cass., isolated from seedlings raised in vitro, were cultured. High frequency of shoot regeneration was achieved on MS medium with BAP (1 mgl⁻¹). Effect of BAP, Kn and GA₃ applied successively in culture on shoot regeneration and flower bud formation has been studied. The shoots differentiated in cultures elongated on this medium. These rooted subsequently on half strength MS medium. The shoots flowered in vitro on MS medium with a combination of BAP (0.1mgl⁻¹) + GA₃ (0.1 mgl⁻¹). The plantlets thus formed were successfully hardened with 90 % survival.     

Penconazole induced changes in photosynthesis, ion acquisition and protein profile of Mentha pulegium L. under drought stress

Effect of penconazole (PEN) treatment on drought-stressed Mentha pulegium L. plants was investigated. Six weeks after sowing, seedlings were grown under soil moisture corresponding to 100, 75, 50 and 25 % field capacity (FC) with or without PEN (15 mg l⁻¹) for 4 weeks. Results showed that the seedlings at 75 % FC showed maximum growth and water supply lower than 75 % FC was the threshold of drought-initiated negative effects on seedling growth. Drought stress significantly induced proline and carbohydrate contents and the decreased chlorophyll, photosynthesis parameters, soluble proteins and ion accumulations. Exogenous PEN increased the growth parameters, pigments, photosynthesis and ion accumulations in drought stressed and unstressed plants, but the effects of PEN were more significant under water deficit conditions. PEN also reduced the negative effects of drought by osmotic balance and protein accumulations. Electrophoretic patterns indicated that PEN treatment increased the intensity of some protein bands with the molecular weights of 30 kDa in shoot and 31 kDa in roots, and several new protein bands with the molecular masses between 116 and 14 kDa appeared in leaves, shoots and roots. These results suggest that the PEN application can be a useful tool in alleviation of effects of drought stress in M. pulegium plants.     

Micropropagation of Hedychium coronarium J. Koenig through rhizome bud

An optimized protocol was developed for in vitro plant regeneration of a medicinally important herb Hedychium coronarium J. Koenig using sprouted buds of rhizomes. The rhizomes with sprouted bud were inoculated on Murashige and Skoog (Physiol Plant 15:473–497, 1962) medium (MS) supplemented with either N⁶-benzyladenine (BA) alone (1.0–4.0 mg L⁻¹) or in combination with 0.5 mg L⁻¹ naphthalene acetic acid (NAA). Of these combinations, MS supplemented with a combination of 2.0 mg L⁻¹ BA and 0.5 mg L⁻¹ NAA was most effective. In this medium, best shoots (3.6) and roots (4.0) regeneration was observed simultaneously with an average shoot and root length of 4.7 cm and 4.2 cm respectively. Regeneration of shoots and roots in the same medium at the same time (One step shoot and root regeneration) reduced the time for production of in vitro plantlets and eliminates the media cost of rooting. Cent-percent (100 %) success in plant establishment was observed in both gradual acclimatization process as well as when plants were directly transferred to outdoor in clay pots containing a mixture of garden soil and sand (2:1) without any sequential acclimatization stage.