Effect of Water Stress Induced by Polyethylene Glycol on Growth,Proline Accumulation in <i>Agave americana</i> L.

The effect of water deficit was determined on both in vitro and soil seedling as well as in cells in suspension of Agave americana L. In order to do the establishment of cells, the formation of callus was induced; for it two auxins were evaluated: 2,4-dichlorophenoxyacetic acid (2,4-D) and 4-mino-3,5,6-trichloropicolinic acid (picloram) at three concentrations (0.25, 0.5 and 0.75 mg L⁻¹) in three explants (leaf, root and meristems) cultured in MS semisolid medium. The callogenesis response was related to the type and section of the explant, as well as the regulator used, and a cell suspension was established using 0.5 mg L⁻¹ naphthaleneacetic acid (NAA) + 0.5 mg L⁻¹ Benzylaminopurine (BAP). Seedlings were exposed to polyethyleneglycol (15% and 30% w/v) with a water potential of −0.87 and −2.67 MPa, respectively, under soil conditions. Water stress was applied through restricted irrigation. Fresh weight, root system growth, and chlorophyll concentration were some of the parameters that were affected by the effect of water deficit on A. americana L. Chlorophyll concentration values were significantly decreased by 15 at 30% PEG (19.6 SPAD units) compared to the control treatment. In in vitro plants, the highest concentration of proline was found in the roots, being the treatment with 30% polyethylene glycol where the highest concentration of this osmoregulator was obtained (62.5 mg g⁻¹ DW). Under restricted irrigation conditions, an increase in proline concentration was observed both in the aerial part (2.2 µg 100 g⁻¹ DW) and in the root system (1.8 µg 100 g⁻¹ DW). However, the concentrations found were approximately ten times greater, less than those found under in vitro conditions. Therefore, the accumulation of proline can be considered an indicator of stress in Agave Americana L. growth in vitro.     

Optimization of <i>Agrobacterium tumefaciens</i>-Mediated Genetic Transformation of Maize

Immature embryos of inbred maize (Zea mays) lines (H8183, H8184, and H8185) were used for Agrobacterium infection. We used the β-glucuronidase gene (GUS) as the target gene and the glufosinate resistance gene (bar) as the selection marker. We conducted research on several aspects, such as different genotypes, coculture conditions, screening agent concentrations, and concentrations of indole-3-butytric acid (IBA), 6-benzylaminopurine (6-BA), and ascorbic acid (Vc) in the differentiation medium. We optimized the genetic transformation system, and the obtained results indicated that among the three lines studied, the induction rate of H8185 was the highest at 93.2%, followed by H8184, with H8183 having the lowest induction rate (80.1%). The best coculture method was that using the N6 coculture medium layered with a sterile filter paper. Using orthogonal analysis, we found that the optimal combination of the three factors in the differentiation medium was A₃ (1 mg mL⁻¹ IBA), B₃C₁ (1.6 mg mL⁻¹ 6-BA), and D₃ (1.5 mg mL⁻¹ Vc). Through GUS staining analysis, Bar test-strip analysis, and polymerase chain reaction, five transgenic plants were finally obtained. This study established the optimal conditions for genetic transformation in maize.     

<i>In vitro</i> Germination and Micropropagation of <i>Aconitum vilmorinianum</i>: An Important Medicinal Plant in China

Aconitum vilmorinianum, a well-known traditional Chinese herb, is recently being threatened by overexploitation and environment disturbance. This study was conducted to provide propagation methods through in vitro germination and explant cultivation. Germination was stimulated up to 66.00% on Murashige and Skoog (MS) medium containing 2.0 mg L⁻¹ 6-benzylaminopurine (BAP), 0.1 mg L⁻¹ 1-napthaleneacetic acid (NAA), and 30 g L⁻¹ sucrose. Three bacteria (Pantoea agglomerans, Erwinia persicina, and Pseudomonas tolaasii) would be responsible for consistent contamination during germination. The latter two were effectively eradicated after disinfected. The influence of explant types and hormone combinations on direct and indirect organogenesis was evaluated in the present work. The frequency of shoot induction from axillary bud explants was 100% on the MS fortified with 2.0 mg L⁻¹ BAP and 0.3 mg L⁻¹ NAA. Shoots multiplication was optimized on MS medium supplemented with 0.1 mg L⁻¹ thidiazuron (TDZ) and 0.1 mg L⁻¹ NAA. High callus induction percentage (96.67%) was obtained from stem segments on MS medium with 2.0 mg L⁻¹ 2,4-D, then successfully regenerated into shoots on MS medium in the presence of 0.1 mg L⁻¹ TDZ and 0.2 mg L⁻¹ NAA. The present work could be useful for the utilization and conservation of this valuable species.     

Rapid Propagation of <i>Rhynchostylis retusa in Vitro</i>

An efficient regeneration system of Rhynchostylis retusa was established to provide technical reference for the application of tissue culture tube seedlings in production. The mixtures of callus and protocorm from aseptic germination were used as explants. The optimal media of each stage was selected for callus proliferation, protocorm occurrence and growth, rejuvenation and rooting via a single, complete combination and orthogonal experiment. The results showed that the optimal medium for callus proliferation, protocorms occurrence and growth was 1/2 Murashige and Skoog (MS) medium adding 50 g·L⁻¹ banana puree, 0.1 mg·L⁻¹ α-naphthaleneacetic acid (NAA), 1.5 mg·L⁻¹ 6-benzylaminopurine (6-BA) and 1.0 mg·L⁻¹ kinetin (KT) with 17.33 proliferation coefficient of callus and 19.63 occurrence coefficient of buds after 90 days. Then the buds occurred from protocorm were cultured on 1/2 MS medium including 100 g·L⁻¹ banana puree, 1.0 mg·L⁻¹ NAA, 2.0 mg·L⁻¹ 6-BA and 0.05 mg·L⁻¹ KT, in which the proliferation coefficient of callus was 10.32 and occurrence coefficient of buds reached 17.87. In the further subculture, the same medium was simultaneously used for callus proliferation, protocorm occurrence and bud growth. The plantlets developed roots in 1/2 MS medium containing 70 mL·L⁻¹ coconut water and 1.5 mg·L⁻¹ NAA with 100% rooting rates after 90 days. The survival rate was more than 90% after domestication and transplantation. This regeneration protocol will provide technique foundation for protecting wild resource and developing artificial cultivation.     

Effects of Nano-Cerium Oxide on Seed Germination and Seedling Growth of <i>Vitex negundo</i>

Cerium oxide nanoparticles (CeO₂NPs) are likely to have dramatic impacts on plant performances, yet the effects of CeO₂NPs on seed germination and seedling growth have not been fully explored. In this study, the seed germination and seedling growth of subshrub species Vitex negundo under different concentrations of CeO₂NPs (low-1 mg/L, medium-100 mg/L, high-500 mg/L) have been discussed. Results showed that: (1) The seed germination rate reduces by 11.25% and 2.5% under the low and medium concentrations of CeO₂NPs, respectively, but increased by 7.08% under the high concentration; (2) CeO₂NPs had significant effects on the growth traits (root length, shoot height and biomass) of seedlings, being the highest under the medium concentration and the lowest under the highest concentration; (3) The superoxide dismutase activity was the maximum (355.91 U/g), but the protein concentration was the minimum (3.85 ug/mL) under the high concentration of CeO₂NPs. Our results indicated that the effects of CeO₂NPs on seed germination and seedling growth are concentrationdependency, i.e., low and medium concentrations inhibited while high concentration promoted seed germination, however, seedling growth showed opposite responses. Therefore, appropriate CeO₂NPs concentrations are beneficial to the seed germination and seedling growth of Vitex negundo and improve the physiological performance of seedlings and enhance their adaptability to environmental adversity.     

Antifungal Potential of <i>Beauveria bassiana</i> on <i>Solanum lycopersicum</i> L. Infected with <i>Fusarium oxysporum</i> f. sp. <i>lycopersici</i>

The objective of this work was to evaluate the effect of Beauveria bassiana (Bb 1205) on controlling Fusarium oxysporum f. sp. lycopersici (Fol 17108) in tomato plants in greenhouse conditions. Inoculation of Bb 1205 was the most promising among the agronomic variables and expression of the activity of the enzymes β-1,3-glucanases and chitinases. Inoculation of Bb 1205 occurred at a concentration of 1 × 10⁸ conidia·mL⁻¹, which was administered onto the leaves, directly into the soil and via injection. Infection with Fol 17108 occurred with 1 × 10⁶ spores·mL⁻¹, which were added directly to the soil. Spectrophotometry was used for measuring agronomic parameters, namely activity of chitinases and β-1,3-glucanases in foliage and roots. When Bb 1205 was added to the soil, the chlorophyll index and aerial part length showed significant differences. In addition, it was determined that root length, fresh weight of foliage, flower, and fruit count increased 82 days after inoculation (dai). Chitinase activity induced by Bb 1205 in leaves and roots of tomato plants infected with Fol 17108 was observed when injected into the stem at 32 dai (41.8 and 11.6-fold, respectively). Inoculation on the foliage showed a 10-fold increase of β-1,3-glucanases in the roots after 82 dpi. As for leaves, a 3.8-fold increase was found when the stem was inoculated. In the different in vivo applications, Bb 1205 activated its defenses by expressing the chitinase enzymes and β-1,3-glucanase, thus reducing the damage caused by Fol 17108, demonstrating increase plant growth thereafter.     

Evaluation of Pre-Emergence and Post-Emergence Herbicides for Weed Management in <i>Miscanthus sacchariflorus</i> and <i>Miscanthus sinensis</i>

Miscanthus, is a promising bioenergy crop, considered superior to other bioenergy crops because of its higher water and nutrient use efficiency, cold tolerance, and higher production of biomass. Broadleaf weeds and grass weeds, cause major problems in the Miscanthus field. A field experiment was conducted in 2018 and 2019, to assess the effects of pre-emergence (alachlor and napropamide) and post-emergence herbicides (nicosulfuron, dicamba, bentazon, and glufosinate ammonium) on broadleaf and grass weeds in M. sinensis and M. sacchariflorus fields. The weed control efficiency and phytotoxicity of pre- and post-emergence herbicides were evaluated at 30 days after treatment (DAT) and compared to those of the control plots. The results showed wide variations in the susceptibility of the weed species to the treated herbicides. Treatment with nicosulfuron 40 g.a.i.ha⁻¹ provided the most effective overall weed control (with 10% visual injury), without affecting the height and biomass of neither Miscanthus species in the field. Post-emergence herbicides such as glufosinate ammonium 400 g.a.i.ha⁻¹ and dicamba 482 g.a.i.ha⁻¹ were effective and inhibited the growth and density of the majority of weeds to a 100%; however, they showed significant phytotoxicity (toxicity scale of 1–10) to both species of Miscanthus. The application of glufosinate ammonium caused severe injuries to the foliar region (90% visual injury) of both Miscanthus sps. Comparatively, M. sinensis showed a slightly higher tolerance to the herbicides nicosulfuron, bentazon and napropamide with 10% visual injury at the recommended dose than M. sacchariflorus. The present study clearly showed that infestation of broadleaf and grass weeds in Miscanthus fields can cause significant damage to the growth and biomass of Miscanthus and applying pre-emergence and post-emergence herbicides effectively controls the high infestation of these weeds.     

Spatial Heterogeneity of Selected Soil Nutrients Related to <i>Torreya grandis</i> cv. <i>Merrillii</i> Plantation in Southeastern China

Chinese Torreya grandis (Torreya grandis cv. Merrillii) is a unique economic tree species in China. Intensive management related to application of chemical fertilizer and herbicides caused serious soil quality degradation of Chinese Torreya grandis plantations. Totally, 120 soil samples were collected from the main disbtributed areas of Chinese Torreya grandis in Southeastern China. In this area, soil pH values varied from 3.68 to 6.78, with a median value of 4.91, implying a trend of acidification. The average concentrations of organic matter, available nitrogen, available phosphorus and available potassium were 27.52 g kg⁻¹, 135.77 mg kg⁻¹, 15.12 mg kg⁻¹, and 153.43 mg kg⁻¹, respectively. The results of spatial analysis revealed that target variables had clear spatial patterns. The soil fertility was relevantly high in most of the study area, with soil nutrient imbalances existing. The stand age had a large effect on soil properties, which were also mainly influenced by human activities. Therefore, it is necessary to change the fertilization method for a sustainable management of Torreya grandis plantation.     

An Efficient Plant Regeneration System of <i>Hydrangea bretschneideri</i> Dipp via Stem Segments as Explants

Hydrangea bretschneideri Dipp is a highly popular ornamental plant for garden decoration. Genetic engineering technology has been successfully used in many plant species, but it is limited in Hydrangea. Here we established an efficient regeneration system by using stem segments as explants for the first time. In our study, the plant growth regulators (PGRs) were evaluated at the different regeneration processes, including axillary shoots regeneration and root induction. We found that the optimal concentration for axillary buds’ induction was 2.0 mgL⁻¹ 6-BA and 0.5 mgL⁻¹ 1 IAA, its highest induction rate was 70%. Moreover, the highest axillary shoots proliferation coefficient was 10.7 on the Murashige and Skoog (MS) medium with 2.0 mgL⁻¹ 6-benzyladenine (BA), 0.2 mgL⁻¹ indole-3-butyric acid (IBA), and 1.0 mgL⁻¹ gibberellin A3 (GA3). The highest frequency of root induction was 80.0 ± 0.06% by culturing the elongated shoots in 1/2 MS medium containing 0.1 mgL⁻¹ IBA. In summary, our study will provide an effective technology for large-scale propagation and important pathway for promoting the popularization and application of Hydrangea bretschneideri Dipp.     

Organic Amendments Improve Plant Morpho-Physiology and Antioxidant Metabolism in Mitigating Drought Stress in Bread Wheat (<i>Triticum aestivum</i> L.)

Due to the unpredictable climate change, drought stress is being considered as one of the major threats to crop production. Wheat (Triticum aestivum L. cv. BARI Gom-26) being a dry season crop frequently faces scarcity of water and results in a lower yield. Therefore, this experiment aims to explore the role of different organic amendments (OAs) in mitigating drought stress-induced damage. The pot experiment consisted of different organic amendments viz. compost, vermicompost and poultry manure @0.09 kg m⁻² soil, biochar @2.5% w/w soil and chitosan @1% w/w soil which was imposed on the plants grown under both well-watered and drought conditions. Results showed that drought stress reduced plant height (15%), SPAD value (16%), relative water content (13%), number of spikelet spike⁻¹ (17%), number of grains spike⁻¹ (12%), and 100-grain weight (18%). Organic amendments act as a protectant and reduce drought stress-induced damages by enhancing the morpho-physiological and yield attributes. Vermicompost enhanced SPAD value by 18%, number of spikelets spike⁻¹ by 20%, number of grains spike⁻¹ by 17%, whereas poultry manure increased plant height by 16% under drought condition compared to control plant. Unlike other OAs applied, vermicompost was proved to be capable of reducing the higher lipid peroxidation and proline content raised by drought condition. Drought stress-induced increment of catalase, ascorbate peroxidase and glutathione reductase activities were also efficiently modulated by the organic amendment application. The present study concluded that OAs play significant roles in alleviating drought stressinduced damages by improving the morpho-physiological attributes and among the different types of OAs used vermicompost performed better which in addition ceased the production of reactive oxygen species.     

Effects of Barium Stress in <i>Brassica juncea</i> and <i>Cakile maritima</i>: The Indicator Role of Some Antioxidant Enzymes and Secondary Metabolites

Soil contamination by toxic trace metal elements, like barium (Ba), may stimulate various undesirable changes in the metabolic activity of plants. The plant responses are fast and with, direct or indirect, generation of reactive oxygen species (ROS). To cope with the stress imposed by the ROS production, plants developed a dual cellular system composed of enzymatic and non-enzymatic players that convert ROS, and their by-products, into stable nontoxic molecules. To assess the Ba stress response of two Brassicaceae species (Brassica juncea, a glycophyte, and Cakile maritime, a halophyte), plants were exposure to different Ba concentrations (0, 100, 200, 300 and 500 μM). The plants response was evaluated through their morphology and development, the determination of plant leaves antioxidant enzymatic activities and by the production of plants secondary metabolites. Results indicated that the two Brassicaceae species have the ability to survive in an environment containing Ba (even at 500 μM). The biomass production of C. maritima was slightly affected whereas an increase in biomass B. juncea was noticed. The stress imposed by Ba activated the antioxidant defense system in the two species, noticed by the changes in the leaves activity of catalase (CAT), ascorbate peroxidase (APX) and guaicol peroxidase (GPX), and of the secondary metabolites, through the production of total phenols and flavonoids. The enzymatic response was not similar within the two plant species: CAT and APX seem to have a more important role against the oxidative stress in C. maritima while in B. juncea is GPX. Overall, total phenols and flavonoids production was more significant in the plants aerial part than in the roots, of the both species. Although the two Brassicaceae species response was different, in both plants catalytic and non-catalytic transformation of ROS occurs, and both were able to overcome the Ba toxicity and prevent the cell damage.     

Doubled Haploid Production Using an Improved Anther Culture Protocol for Sorghum [<i>Sorghum bicolor</i> (L.) Moench]

Sorghum [Sorghum bicolor (L.) Moench] can benefit from accelerated breeding and release of improved varieties through doubled haploid technology. The technology has been used in speeding up the breeding of other major cereals such as wheat, maize and rice, for which generally widely applied optimised protocols exist. A reproducible protocol for the crop, that can overcome genotype dependency and other species-specific challenges such as phenolic exudation is however lacking. This study aimed at sorghum doubled haploids production thereby contributing to the development of an improved protocol. From the 28 hybrid genotypes, both F₁ registered- and experimental hybrids involved, this study successfully produced haploids from five genotypes and subsequently, four confirmed doubled-haploid lines on W14mf medium or its modification with 1.0 gl⁻¹ L-proline, 1.0 gl⁻¹ L-asparagine and 1.0 gl⁻¹ KH₂PO₄. Medium 190-2Cu was used for regeneration and rooting, which occurred successfully, if the calli were transferred on to it less than 7 days after induction, and temperature was maintained at 25˚C under light condition. Genotype dependency was not wholly overcome; however, sorghum’s high tillering ability and abiotic stress tolerance were observed to contribute to attainment of haploid plantlets. Spontaneous diploids producing seeds at rates of upto 80.5% were obtained, therefore eliminating the need for colchicine duplication.     

2,4-D Hyper Accumulation Induced Cellular Responses of <i>Azolla pinnata</i> R. Br. to Sustain Herbicidal Stress

In the present experiment with ongoing concentration (0 µM, 100 µM, 250 µM, 500 µM and 1000 µM) of 2,4-D, the responses of Azolla pinnata R.Br. was evaluated based on cellular functions. Initially, plants were significantly tolerated up to 1000 µM of 2,4-D with its survival. This was accompanied by a steady decline of indole acetic acid (IAA) concentration in tissues with 78.8% over the control. Membrane bound H⁺ -ATPase activity was over expressed within a range of 1.14 to 1.25 folds with activator (KCl) and decreased within a range of 57.3 to 74.6% in response to inhibitor (Vanadate) application. With regards to IAA metabolism, plants recorded a linear increase with wall bound oxidase activity up to maximum concentration of 2,4-D. The variations were more moderated when wall bound IAA-oxidase recorded a linear increase proportionate to the 2,4-D concentrations. This was more extended with the presence of different isoforms of IAA-oxidase which was much more pronounced with distinct polymorphisms of expressed proteins, however, not independent to the 2,4-D concentrations. Polyamines like spermine, spermidine and putrescine (spm, spd and put) were not consistent in concentration with the dosages of 2,4-D. Besides these, plants were induced to apoplastic NAD(P)H oxidase activity maximally by 1.6 folds under 500 µM 2,4-D over control. Still, putrescine responded more or less consistently and recorded maximally 11.9 folds at 500 µM 2,4-D as compared to the control. NAD(P)H oxidase activity recorded maximally 1.6 folds against control and remain consistent throughout the concentrations of 2,4-D. GPX along with APX were more linear in responses through the concentration of 2,4-D except CAT as compared to control. On enzymatic antioxidative activity, peroxidases (GPX and APX) were overexpresed in a similar manner except for catalase with a non-significant rise. In stabilization of cellular redox, glutathione reductase attended maximum value by 2.45 folds at 1000 µM evidenced with significant variations in protein polymorphism. The sensitivity of 2,4- D also appeared in Azolla with a maximum loss of nucleic acids as documented by the comet assay. Moreover, the Azolla might have some DNA damage protective activity as evident using frond extract with plasmid nick assay. Therefore, Azolla plants with its cellular responses is evident to sustain against the 2,4-D herbicidal stress and may be granted in bio remediation process for the contaminated soil.     

Effects of Chemical Products on Fire Blight Suppression,and Fruit Production and Quality in Apple (<i>Malus domestica</i> Borkh.) cv. Golden Glory

Antibiotics are widely used in fire blight management programs, yet there are no studies that demonstrate the evaluation of their efficacy in Mexico. Therefore, the present study was conducted to investigate the effects of the active ingredients in five commercial products (Kasumin^® 2L, Agrygent Plus^®, Agricultural Terramycin^®, Agrimicin^® 100, and Actigard^®) on fire blight suppression, and fruit yield and quality of apple (Malus domestica Borkh.) cv. Golden Glory. The experiment was conducted in a commercial orchard using a completely randomized block design, with six treatments: (1) Oxytetracycline [Ox], 110 mg L⁻¹; (2) Kasugamycin [Kas], 4.7 mL L⁻¹; (3) Oxytetracycline + Gentamicin [Ox + Gen], 48 mg L⁻¹+12 mg L⁻¹; (4) Streptomycin + Oxytetracycline [Str + Ox], 90 mg L⁻¹+9 mg L⁻¹; (5) Acibenzolar-S-methyl [ASM], 70 mg L⁻¹; and (6) Control, only water, with four replications, and three 11-year-old trees as an experimental unit. Variables of infection including flowers, shoots and fruits, yield and fruit quality were evaluated. All treatments suppressed infection in flowers, shoots, and fruits. ASM provided the highest levels of reduction of flower and shoot infection, while Kas had the least effect on the reduction of infection in these variables. The Ox + Gen treatment had the greatest suppression of fruit infection, and the best results on fruit yield and quality, followed by Ox and ASM. This is the first study conducted to evaluate the efficacy of the active ingredients of five commercial products used for the management of fire blight in apple trees in Mexico.     

Interaction of <i>Acaena elongata</i> L. with Arbuscular Mycorrhizal Fungi under Phosphorus Limitation Conditions in a Temperate Forest

The aim of this study was to analyze the performance of Acaena elongata colonized by arbuscular mycorrhizal fungi (AMF) to different phosphorus (P) concentrations, as a measure of AMF dependency. A. elongata, is a species from soils where P availability is limited, such as temperate forests. Our research questions were: 1) How do different P concentrations affect the AMF association in Acaena elongata, and 2) How does the AMF association influence A. elongata’s growth under different P concentrations? A. elongata’s growth, P content in plant tissue, AMF colonization and dependency were measured under four P concentrations: control (0 g P kg⁻¹ ), low (0.05 g P kg⁻¹ ), intermediate (0.2 g P kg⁻¹ ) and high (2 g P kg⁻¹ ) in different harvests. A complete randomized block design was applied. A. elongata’s growth was higher under -AMF in intermediate and high P concentrations, and the lowest growth corresponded to +AMF in the low and intermediate P concentration. We observed a negative effect on the root biomass under +AMF in intermediate P concentration, while the P concentration had a positive effect on the leaf area ratio. The AMF colonization in A. elongata decreased in the highest P concentration and it was favored under intermediate P concentration; while the low and the high concentrations generated a cost-benefit imbalance. Our results suggest that the performance of some plant species in soils with low P availability may not be favored by their association with AMF, but a synergy between AMF and intermediate P concentrations might drive A. elongata’s growth.     

Benzyl Amino Purine and Gibberellic Acid Coupled to Nitrogen-Limited Stress Induce Fatty Acids,Biomass Accumulation,and Gene Expression in <i>Scenedesmus Obliquus</i>

The need for renewable energy sources makes microalgae an essential feedstock for biofuels production. The molecular aspects and the response to nitrogen (N)-limited conditions with a phytohormone stimulus in microalgae have been slightly explored. In this work, Scenedesmus obliquus was used as a study model to analyze the effect of benzyl amino purine (BAP) and gibberellic acid (GA) coupled to nitrogen limitation on cell growth, biomass and fatty acids. The selected 10^-5 M BAP increased the biomass by 1.44-fold, and 10^-6 M GA by 1.35-fold. The total lipids also increased by 2.8 and 1.11-fold, respectively. The 10^-5 M BAP and
10^-6 M GA addition to S. obliquus cultures at different initial nitrogen percentages (N-0, N-25, and N-50) showed a significant increase in cell growth and biomass productivity compared to the unstimulated cultures. BAP N-0 and GA N-0 produced the highest lipid yields with 55% and 50%, respectively. The lipid profile analysis revealed an increase, particularly in C18:1 and C16:0 fatty acids. Gene expression analysis showed an over-expression of acyl carrying protein (ACP), stearoyl-ACP desaturase (SAD), fatty acid acyl-ACP thioesterase (FATA), and diacylglycerol acyltransferase (DGAT) genes, which were mainly induced by nitrogen limitation. Furthermore, BAP and GA produced a significant over-expression on these genes in the N-replete cultures. This study shows that BAP and GA, coupled to N limitation stress, can be used to increase the biomass and lipid production in S. obliquus for sustainable biofuels.     

<i>In Vitro</i>-Propagation of <i>Agave tequilana</i> Weber cv. azul in a Temporary Immersion System

In Mexico, there is a need to produce large quantities of plantlets for the establishment and replanting of blue (cv. azul) agave production areas. Most of these plots are within the origin denomination area (DOT, Spanish acronym) of the distilled product of this plant, known as tequila. The objective of this study was to develop an in vitro-propagation protocol for Agave tequilana Weber cv. azul using segmented stems in both: solid and liquid media. A disinfection and in vitro technique were developed to obtain shoots, through plantlets collected in commercial plots, which attained 100% surface-disinfection and budding rate. At the multiplication stage, the effects of 6-Benzylaminopurine (BA) (0.0, 4.4 and 13.2 μM) and kinetin (0.0, 9.4, 18.8 and 37.6 μM) were evaluated on lateral-shoot production of segmented sagittal stems. These were cultivated on Murashige & Skoog (MS) medium, with the addition of 3.0% sucrose and 8 g L⁻¹ agar. It was observed that BA and kinetin increased the number of shoots per explant, obtaining up to 18 and 26, respectively. Furthermore, it was found that just the sagittal segmentation of explants increased axillary budding. On the other hand, segmented-stem bases were grown in MS liquid medium with 3.0% sucrose, inside a RITA® system, programmed by a 5 min immersion step with a frequency of every 4 h. The effect of Indole−3-Acetic acid (IAA) (0.57, 2.9, 5.7 μM) was evaluated, while maintaining a concentration of BA (13.2 μM). It was observed that the greatest concentration of IAA led to the formation of more than 20 buds per explant. These results offer a new methodology to increase the efficiency of A. tequilana Weber cv. azul-in vitro multiplication by sagittal segmentation of stems and the addition of BA and/or IAA.     

Encapsulation of Immature Somatic Embryos of <i>Coffea arabica</i> L. for <i>in Vitro</i> Preservation

The present study aimed to develop a protocol for somatic embryogenesis and encapsulation of coffee embryos (Coffea arabica L.), for the conservation of genotypes with characteristics of commercial interest. Somatic embryos were induced from leaf explants in Murashige and Skoog medium (MS) supplemented with 1 mg · L⁻¹ of 2,4-dichlorophenoxiacetic acid (2,4-D) combined with 2 mg · L⁻¹ of benzyladenine (BA). Somatic embryos (SE) at the globular stage were encapsulated in a sodium alginate matrix; two treatments were tested: MS + 5 mg · L⁻¹ BA + 1 mg · L⁻¹ NAA + 3% (w/v) alginate, and MS + 7 mg · L⁻¹ BA + 5.7 mg · L⁻¹ indoleacetic acid (IAA) + 3% (w/v) alginate. Alginate was complexed with 100 mM calcium chloride (CaCl₂). Viability of the encapsulated SE was determined by staining with 0.01% fluorescein diacetate (FDA) after 0, 15, 30, and 45 days of storage at 4°C. Embryo viability was 100% in both treatments.     

High synergistic antibacterial,antibiofilm, antidiabetic and antimetabolic activity of Withania somnifera leaf extract-assisted zinc oxide nanoparticle

Nanotechnology is currently gaining immense attention to combat food borne bacteria, and biofilm. Diabetes is a common metabolic disease affecting majority of people. A better therapy relies on phytomediated nanoparticle synthesis. In this study, W. somnifera leaf extract-assisted ZnO NPs (Ws-ZnO NPs) was synthesized and characterized. From HR-TEM analysis, it has been found that the hexagonal wurtzite particle is 15.6 nm in size and − 12.14 mV of zeta potential. A greater antibacterial effect of Ws-ZnO NPs was noticed against E. faecalis and S. aureus at 100 µg mL⁻¹. Also, the biofilm of E. faecalis and S. aureus was greatly inhibited at 100 µg mL⁻¹ compared to E. coli and P. aeruginosa. The activity of α-amylase and α-glucosidase enzyme was inhibited at 100 µg mL⁻¹ demonstrating its antidiabetic potential. The larval and pupal development was delayed at 25 µg mL⁻¹ of Ws-ZnO NPs. A complete mortality (100%) was recorded at 25 µg mL⁻¹. Ws-ZnO NPs showed least LC₅₀ value (9.65 µg mL⁻¹) compared to the uncoated ZnO NPs (38.8 µg mL⁻¹) and leaf extract (13.06 µg mL⁻¹). Therefore, it is concluded that Ws-ZnO NPs are promising to be used as effective antimicrobials, antidiabetic and insecticides to combat storage pests.