Comparison Study of Allelochemicals and Bispyribac-Sodium on the Germination and Growth Response of Echinochloa crus-galli L.

The phytotoxic effects of two allelochemicals (trans-cinnamic acid and syringaldehyde) at different concentrations (1000, 100, 10, and 1 µM) on seed germination, seedling growth, and physiological and biochemical changes of Echinochloa crus-galli L. were tested by comparison to a commercial herbicide ‘Nominee’ (that is, 100 g/L bispyribac-sodium). trans-Cinnamic acid and the herbicide inhibited seed germination completely at 100 µM, whereas for syringaldehyde, complete inhibition required 1000 µM. However, with 100 µM syringaldehyde, the seed germination of the test species was 53% of the control. Allelochemicals and the herbicide delayed seed germination and significantly affected the speed of germination index (S), speed of cumulative germination index (AS), and coefficient of germination rate (CRG). The roots were more affected when nutrients were not added to the growth bioassay. In general, with the increasing concentration of allelochemicals from 100 to 1000 µM, the inhibitory effects increased. Via microscopy analysis, we found leaf blade wilting and necrosis at concentrations above 100 µM in allelochemical-treated plants. Roots of E. crus-galli treated with 1000 µM allelochemicals had black points on root nodes but had no root hairs. The anatomy of roots treated with allelochemicals (1000 µM) showed contraction or reduction of root pith cells as well as fewer and larger vacuoles compared to the control. The allelochemicals also showed remarkable effects on seedling growth, SPAD index, chlorophyll content, and free proline content in a pot culture bioassay, indicating that trans-cinnamic acid and syringaldehyde are potent inhibitors of E. crus-galli growth and can be developed as herbicides for future weed management strategies.

     

Profile of Plant Hormones and their Metabolites in Germinated and Ungerminated Canola (Brassica napus) Seeds Imbibed at 8°C in either GA4+7, ABA, or a Saline Solution

Abscisic acid (ABA) and gibberellins (GAs) are two major phytohormones that regulate seed germination in response to internal and external factors. In this study we used HPLC-ESI/MS/MS to investigate hormone profiles in canola (Brassica napus) seeds that were 25, 50, and 75% germinated and their ungerminated counterparts imbibed at 8°C in either water, 25 μM GA₄₊₇, a 80 mM saline solution, or 50 μM ABA, respectively. During germination, ABA levels declined while GA₄ levels increased. Higher ABA levels appeared in ungerminated seeds compared to germinated seeds. GA₄ levels were lower in seeds imbibed in the saline solution compared to seeds imbibed in water. Ungerminated seeds imbibed in ABA had lower GA₄ levels compared to ungerminated seeds imbibed in water; however, the levels of GA₄ were similar for germinated seeds imbibed in either water or ABA. The ABA metabolites PA and DPA increased in seeds imbibed in either water, the saline solution, or ABA, but decreased in GA₄₊₇-imbibed seeds. In addition, ABA inhibited GA₄ accumulation, whereas GA had no effect on ABA accumulation but altered the ABA catabolism pathway. Information from our studies strongly supports the concept that the balance of ABA and GA is a major factor controlling germination.     

Abscisic acid regulates seed germination of Vellozia species in response to temperature

• The relationship between the phytohormones, gibberellin (GA) and abscisic acid (ABA) and light and temperature on seed germination is still not well understood. We aimed to investigate the role of the ABA and GA on seed germination of Vellozia caruncularis, V. intermedia and V. alutacea in response to light/dark conditions on different temperature.
• Seeds were incubated in GA (GA₃ or GA₄) or ABA and their respective biosynthesis inhibitors (paclobutrazol – PAC, and fluridone – FLU) solutions at two contrasting temperatures (25 and 40 °C). Furthermore, endogenous concentrations of active GAs and those of ABA were measured in seeds of V. intermedia and V. alutacea during imbibition/germination.
• Exogenous ABA inhibited the germination of Vellozia species under all conditions tested. GA, FLU and FLU + GA₃ stimulated germination in the dark at 25 °C (GA₄ being more effective than GA₃). PAC reduced seed germination in V. caruncularis and V. alutacea, but did not affect germination of V. intermedia at 40 °C either under light or dark conditions. During imbibition in the dark, levels of active GAs decreased in the seeds of V. intermedia, but were not altered in those of V. alutacea. Incubation at 40 °C decreased ABA levels during imbibition in both V. caruncularis and V. alutacea.
• We conclude that the seeds of Vellozia species studied here require light or high temperature to germinate and ABA has a major role in the regulation of Vellozia seed germination in response to light and temperature.

     

A rapid effect of applied brassinolide on abscisic acid concentrations in <Emphasis Type="Italic">Brassica napus</Emphasis> leaf tissue subjected to short-term heat stress

Three-week old canola (Brassica napus L.) seedlings grown at 20/16°C (day/night) were subjected to short-term (4 and 8 h) heat stress (45°C) or maintained at a normal temperature of 20°C. Half of the plants under each treatment received a 10⁻⁶ M solution of brassinolide (BL) 1 h prior to beginning the temperature treatments. The concentration (ng/g dry weight) of endogenous abscisic acid (ABA) was subsequently determined in young leaves via the stable isotope dilution method. Applied BL had no effect on endogenous ABA for plants maintained at normal temperatures. However, ABA concentration was significantly elevated by heat stress alone and doubled by heat stress + BL. These results suggest that the well-known enhancement of tolerance to high temperature stress that can be obtained by BL or 24-epi-BL applications may be caused by a brassinosteroid-induced elevation in endogenous ABA concentration.     

Germination response of black and yellow seed coated canola (<Emphasis Type="Italic">Brassica napus</Emphasis>) lines to chemical treatments under cold temperature conditions

Seed quality is a key critical component to produce well established and vigorous seedlings under cool soil (<10°C) conditions experienced in Western Canada. A simple, relatively quick germination assay is required to separate small differences in seed germination which can have a significant impact on seedling growth. It has long been established that phytohormones regulate seed germination: abscisic acid inhibits germination whereas gibberellins enhance germination. We investigated the effects of ABA, GA, ethylene and inhibitors of these phytohormones alone and in combination on the germination rate of a black and a yellow seed canola (Brassica napus) imbibed at 8°C. The effects of either saline solutions, osmotic solutions, fusicoccin or testa on the germination of canola seeds imbibed at 8°C were also investigated. This temperature is representative of the soil temperatures experienced in the early spring of Western Canada. The two canola seed lines, especially the yellow seed line, were very sensitive to increasing concentration of saline solutions at 8°C, but not at 23°C; however, iso-osmotic solutions that reduced water potential were more inhibitory. The seed coat (testa) including the endosperm was a major factor affecting the germination rate of the yellow seed line at 8°C, however, GA₄₊₇ overcame the inhibitory effect of the testa, whereas ABA exacerbated it. Fusicoccin was more stimulatory to germination than GA₄₊₇, however, unlike GA₄₊₇, it was unable to overcome the inhibitory effect of paclobutrazol, a GA biosynthesis inhibitor. Fluridone, an ABA biosynthesis inhibitor, was unable to overcome the inhibitory effects of a saline solution suggesting that the inhibitory effect was not due to elevated ABA levels. Ethylene, a stimulator of germination did not appear to be involved in the germination of these two lines. Controlled deterioration at 35°C, 85% RH could be either partially or completely overcome by exogenous GA₄₊₇. This study demonstrated the effect of hormones, salinity and testa on the germination of canola seeds under less than ideal environmental conditions.     

Short-chain dehydrogenase/reductase,PsDeHase, from opium poppy: putative involvement in papaverine biosynthesis

Embryogenic synseeds were prepared in Albizia lebbeck by encapsulating cotyledon stage somatic embryos derived from in vitro maintained embryogenic cultures in different types of Ca-alginate beads. The germination rate of somatic embryos was affected significantly by the bead type, matrix composition and germination substrate. A matrix made of 3% Na₂-alginate complexed with 100 mM CaCl₂·2H₂O for a hardening period of 20 min provided uniform encapsulation of somatic embryo. Among different types of synseeds, type IIA, wherein somatic embryos encapsulated in a single layer of Ca-alginate matrix composed of MS medium supplemented with 2 g L^?1 activated charcoal and 1.0 µM gibberellic acid (GA₃) as reconstituted endosperm, was found to be the most efficient type having maximum germination rates (88.6?±?0.51%). Incorporation of GA₃ in the alginate beads stimulated greater germination of somatic embryos as against GA₃ supplementation in the germination substrate. Further, viability studies on short term cold (4 °C) storage of different types of embryogenic synseeds revealed that double layered synseeds (DLS) were found comparatively more robust to withstand longer storage durations than single layered synseeds as evident by greater germination rates of the former after 4–8 weeks of refrigerated storage. Also, the elevated levels of antioxidative enzymes (superoxide dismutase, catalase, ascorbate peroxidase and glutathione reductase) and leaf proline content in the plantlets derived from DLS reveals the possible role of alginate coatings in conferring alleviation to low temperature stress generated during different storage durations. Similar Inter simple sequence repeat profiles of embryogenic synseeds derived plantlets and mother tree nullifies the possible occurrence of somaclones, thereby establishing the efficacy of synseed technology for clonal propagation of A. lebbeck germplasm.

     

Influence of an abscisic acid (ABA) seed coating on seed germination rate and timing of Bluebunch Wheatgrass

Semi‐arid rangeland degradation is a reoccurring issue throughout the world. In the Great Basin of North America, seeds sown in the fall to restore degraded sagebrush (Artemisia spp.) steppe plant communities may experience high mortality in winter due to exposure of seedlings to freezing temperatures and other stressors. Delaying germination until early spring when conditions are more suitable for growth may increase survival. We evaluated the use of BioNik? (Valent BioSciences LLC) abscisic acid (ABA) to delay germination of bluebunch wheatgrass (Pseudoroegneria spicata). Seed was either left untreated or coated at five separate rates of ABA ranging from 0.25 to 6.0 g 100 g^?1 of seed. Seeds were incubated at five separate constant temperatures from 5 to 25°C. From the resultant germination data, we developed quadratic thermal accumulation models for each treatment and applied them to 4 years of historic soil moisture and temperature data across six sagebrush steppe sites to predict germination timing. Total germination percentage remained similar across all temperatures except at 25°C, where high ABA rates had slightly lower values. All ABA doses delayed germination, with the greatest delays at 5–10°C. For example, the time required for 50% of the seeds to germinate at 5°C was increased by 16–46 d, depending on the amount of ABA applied. Seed germination models predicted that the majority of untreated seed would germinate 5–11 weeks after a 15 October simulated planting date. In contrast, seeds treated with ABA were predicted to delay germination to late winter or early spring. These results indicate that ABA coatings may delay germination of fall planted seed until conditions are more suitable for plant survival and growth.     

Effect of a membrane interactive peptide on plant cells of canola (Brassica napus) and two fungal pathogens

Summary A membrane interactive peptide was toxic to microspores, pollen and protoplasts of canola in the 1–5 µM concentration range. Similarly, at 5.0 µM the peptide completely inhibited germination of conidia ofVerticillium albo-atrum; however, when tested with conidia of a virulent isolate of blackleg (Leptosphaeria maculens), a fungal pathogen of canola, much higher levels (>30 µM) of the peptide were required to reduce or arrest germination and growth of the conidia. When testing the relative toxicities of novel peptides on plant cells and their pathogens, pollen germination is a simple, rapid and reliable alternative to protoplasts.     

Smoke-saturated Water and Karrikinolide Modulate Germination,Growth, Photosynthesis and Nutritional Values of Carrot (Daucus carota L.)

Plant-derived smoke is a positive regulator of seed germination and growth with regard to many plant species. Of the several compounds present in plant-derived smoke, karrikinolide or KAR₁ (3-methyl-2H-furo[2,3-c]pyran-2-one) is considered to be the major active growth-promoting compound. To test the efficacy of smoke-saturated water (SSW) and KAR₁ on carrot (Daucus carota L.), two separate pot experiments were simultaneously conducted in the same environmental conditions. SSW and KAR₁ treatments were applied to the plants in the form of aqueous solutions of variable concentrations. Prior to sowing, seeds were soaked in the solutions of SSW (25.8 µg L⁻¹_, 51.6 µg L⁻¹,103.2 µg L⁻¹ and 258.0 µg L⁻¹) and KAR₁ (0.015 µg L⁻¹, 0.150 µg L⁻¹, 1.501 µg L⁻¹ and 15.013 µg L⁻¹). Percent seed germination, vegetative growth, photosynthesis and nutritional values were the major parameters through which the plant response to the applied treatments was investigated. The results obtained indicated a significant improvement in all the plant attributes studied. In general, SSW (51.6 µg L⁻¹) and KAR₁ (1.501 µg L⁻¹) proved optimum treatments for most the parameters. As compared to the control, 51.6 µg L⁻¹ of SSW and 1.501 µg L⁻¹ of KAR₁ increased the percent seed germination by 58.0% and 54.4%, respectively. Over the control, the values of plant height and net photosynthetic rate were enhanced by 33.9% and 40.9%, respectively, due to 51.6 µg L⁻¹ of SSW, while the values of these parameters were increased by 25.2% and 34.0%, respectively, due to 1.501 µg L⁻¹ of KAR₁. In comparison with the control, treatment 51.6 µg L⁻¹ of SSW increased the contents of β-carotene and ascorbic acid by 32.7% and 37.9%, respectively, while the treatment 1.501 µg L⁻¹ M of KAR₁ enhanced these contents by 42.0% and 48.4%, respectively. This study provides an insight into an affordable and feasible method of crop improvement.

     

Comprehensive in vitro regeneration study with SCoT marker assisted clonal stability assessment and flow cytometric genome size analysis of Carthamus tinctorius L.: an important medicinal plant

An efficacious and reproducible in vitro regeneration technique for safflower was established using varying concentrations and composition of plant growth regulators (PGRs) supplemented Murashige and Skoog (MS) medium. Successful in vitro seed germination in half strength MS (H-MS) with 1.4 µM GA₃ resulted in procurement of sterile explants (cotyledons, apical meristems) for in vitro study. Callogenesis (2.2 µM BAP?+?2.7 µM NAA), indirect organogenesis of shoot buds (0.54 µM NAA?+?9.08 µM TDZ), somatic embryogenesis (2.2 µM BAP?+?5.4 µM NAA) and somatic embryo germinated plantlets (H-MS?+?1.4 µM GA₃?+?2.2 µM BAP?+?5.4 µM NAA) were successfully obtained. Histological study and scanning electron micrographs of embryogenic callus revealed pre-globular, heart-shaped and torpedo stages of dicot embryogeny. H-MS?+?8 µM NAA showed maximum rhizogenic response with a mean root and shoot length of 17.5 mm and 48.50 mm respectively in 2.2 µM BAP?+?0.54 µM NAA bearing an average of 9 capitula per plantlet with 70% post transplantation survival rate. True to type nature of the regenerates was confirmed using Start Codon Targeted (SCoT) marker, exhibiting 100% and 97.3% monomorphic bands for direct and somatic embryo regenerated plants respectively. Flow cytometry method (FCM) was employed for 2C DNA content analysis. The histogram peaks of 2C nuclear DNA content of in vitro regenerated safflower (direct and embryo derived) were similar to the peak of field grown donor plant. 2C nuclear DNA content of field grown, direct and somatic embryo regenerated C. tinctorius was 2.65?±?0.04 pg, 2.62?±?0.06 pg and 2.68?±?0.04 pg respectively, further verifying genetic homogeneity. All things considered, the above protocol is insusceptible to genetic alteration and can be used for large scale production and sustainable utilization of desired genotype.

     

The Occurrence of Vascular Cavities and Specialized Parenchyma Cells in the Roots of Cool-season Legumes

The seeds of 20 legume species were grown in the greenhouse or in growth chambers at different temperatures. Under warm temperature conditions (above 15 °C), six species, pea (Pisum sativum), broadbean (Vicia faba), chickpea (Cicer arietinum), lentil (Lens culinaris), wild lupine (Lupinus latifolius), and soybean (Glycine max), formed cavities in the vascular cylinder of their primary roots, which in turn became filled by the ingrowth of specialized parenchyma cells (SP cells). When these species were grown at low temperature (below 15 °C), however, a “normal” vascular cylinder formed in the primary roots with late-maturing metaxylem vessel members differentiating in the center. These species were all cool-season legumes except soybean, a warm-season legume, which sometimes also formed cavities and SP cell ingrowths. The occurrence of cavities and SP cells therefore was restricted to the cool-season legumes (except soybean) when they were grown under warm temperature conditions. The position and size of cavities varied among these species. Pea and broadbean usually formed large, axially elongated cavities in the central vascular cylinder, or in the xylem poles. Others formed smaller cavities of various lengths.     

Novel Peptides Inhibit Zika NS2B-NS3 Serine Protease and Virus Replication in Human Hepatic Cell Line

Zika virus (ZIKV) is a Flavivirus associated with several neurological complications. Currently, there are no vaccines or cures available and an efficient antiviral treatment is urgently needed to combat ZIKV infection. Herein, we targeted ZIKV NS2B-NS3 serine protease with short peptides to inhibit ZIKV replication in human hepatic cell line (WRL-68). The short peptide inhibitors were designed using Hyperchem 8.0.10 software. Docking energy and binding configuration were calculated using HADDOCK webserver. ZIKV NS2B-NS3 protease was produced as a recombinant single peptide in Escherichia coli and the protease activity was examined by measuring the cleavage of a fluorescent substrate in the presence of the peptides or aprotinin as a standard protease inhibitor. Computational analysis revealed that the short peptides, AYA2 and AYA9, exhibited lower docking energy to ZIKV protease than aprotinin. Both peptides also possessed lower half maximal inhibitory concentration (IC₅₀), 30.9 and 22.1 µM respectively, against ZIKV protease activity when compared to aprotinin (35.4 µM). Interestingly, AYA2 and AYA9 exhibited minimal cytotoxic effects in WRL-68 cells and showed considerable inhibition against ZIKV replication in vitro at half maximal effective concentration (EC₅₀) of 40.73?±?2.3 µM and 34.65?±?1.8 µM respectively. Fusion of these two peptides to MAP30 peptide substantially reduced the IC₅₀ of ZIKV protease inhibition to 1.1 µM and inhibited ZIKV replication at EC₅₀ of 0.5157?±?0.03 µM. In sum, we reported novel peptides that effectively inhibited ZIKV replication in vitro. This study represents a cost-effective strategy of developing peptide inhibitors by shortening the peptides and producing them in recombinant form.

     

Nitrate,abscisic acid and gibberellin interactions on the thermoinhibition of lettuce seed germination

Germination of lettuce seeds has obvious thermoinhibition, but the mechanism for thermoinhibition of seed germination is poorly understood. Here, we investigated the interactions of nitrate, abscisic acid (ABA) and gibberellin on seed germination at high temperatures to understand further the mechanism for thermoinhibition of seed germination. Our results showed that lettuce (Lactuca sativa L. ‘Jianye Xianfeng No. 1’) seeds exhibited notable thermoinhibiton of germination at ≥17°C in darkness, and at ≥23°C in light, but the thermoinhibited seeds did not exhibit secondary dormancy. Thermoinhibition of seed germination at 23 or 25°C in light was notably decreased by 5 and 10 mM nitrate, and the stimulatory effects were markedly prevented by nitric oxide (NO) scavenger 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide. The sensitivity of seed germination to exogenous ABA increased with increasing temperature. Thermoinhibition of seed germination was markedly decreased by fluridone (an inhibitor of ABA biosynthesis) and GA₃, and was increased by diniconazole (an inhibitor of the ABA-catabolizing enzyme ABA 8′-hydroxylase) and paclobutrazol (an inhibitor of GA biosynthetic pathway). The effect of fluridone in decreasing thermoinhibition of seed germination was obviously antagonized by paclobutrazol, and that of GA₃ was notably added to by fluridone, and that of nitrate was antagonized by paclobutrazol, diniconazole and ABA and was added to by GA₃ and fluridone. Our data show that thermoinhibition of lettuce seed germination is decreased by nitrate in a NO-dependent manner, which is antagonized by ABA, diniconazole and paclobutrazol and added by fluridone.     

Symbiont transmission and reproductive mode influence responses of three Hawaiian coral larvae to elevated temperature and nutrients

Elevated temperatures and nutrients are degrading coral reef ecosystems, but the understanding of how early life stages of reef corals respond to these stressors remains limited. Here, we test the impact of temperature (mean ~ 27 °C vs. ~ 29 °C) and nitrate and phosphate enrichment (ambient, + 5 µM nitrate, + 1 µM phosphate and combined + 5 µM nitrate with 1 µM phosphate) on coral larvae using three Hawaiian coral species with different modes of symbiont transmission and reproduction: Lobactis scutaria (horizontal, gonochoric broadcast spawner), Pocillopora acuta (vertical, hermaphroditic brooder) and Montipora capitata (vertical, hermaphroditic broadcast spawner). Temperature and nutrient effects were species specific and appear antagonistic for L. scutaria and M. capitata, but not for P. acuta. Larvae survivorship in all species was lowest under nitrate enrichment at 27 °C. M. capitata and L. scutaria survivorship increased at 29 °C. However, positive effects of warming on survivorship were lost under high nitrate, but phosphate attenuated nitrate effects when N/P ratios were balanced. P. acuta larvae exhibited high survivorship (> 91%) in all treatments and showed little change in larval size, but lower respiration rates at 29 °C. Elevated nutrients (+N+P) led to the greatest loss in larvae size for aposymbiotic L. scutaria, while positive growth in symbiotic M. capitata larvae was reduced under warming and highest in +N+P treatments. Overall, we report a greater sensitivity of broadcast spawners to warming and nutrient changes compared to a brooding coral species. These results suggest variability in biological responses to warming and nutrient enrichment is influenced by life-history traits, including the presence of symbionts (vertical transmission), in addition to nutrient type and nutrient stoichiometry.

     

Anti-lipopolysaccharide Factor from Crucifix Crab Charybdis feriatus,Cf-ALF2: Molecular Cloning and Functional Characterization of the Recombinant Peptide

Antilipopolysaccharide factors (ALFs) are important effectors of innate immunity in crustaceans with broad spectrum antimicrobial activity. Present study deals with the molecular and functional characterization of a 98-amino acid ALF isoform from, crucifix crab, Charybdis feriatus termed as Cf-ALF2. The ALF isoform Cf-ALF2 exhibits characteristic features of an AMP including a cationic net charge of + 9 and a total hydrophobic ratio of 34%. Recombinant peptide rCf-ALF2 showed remarkable antimicrobial activity against Gram-negative and Gram-positive bacteria especially against Staphylococcus aureus (minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of 5 µM) and Escherichia coli (MIC 10 µM and MBC 20 µM). Using scanning electron microscopy, bacterial membrane blebbing, disruption, and cell content leakage were observed in peptide treated E. coli. The recombinant peptide was found to be non-hemolytic and non-cytotoxic in NCI-H460 cell line at the highest tested concentration (20 µM). Thus, this study identified a novel isoform of ALF from C. feriatus and revealed the potent antimicrobial property of the recombinant peptide Cf-ALF2 and the future prospects of using the peptide for therapeutic applications in the future.

     

Adventitious Shoot Induction from Internode and Root Explants in a Semiparasitic Herb Monochasma savatieri Franch ex Maxim

A highly efficient protocol for the induction of adventitious shoots from young internode and root explants of a semiparasitic medicinal herb Monochasma savatieri Franch ex Maxim was developed. MS basal medium supplemented with 5 µM thidiazuron (TDZ) induced 32 adventitious shoots/explant, which was double the number obtained using the same concentration of 6-benzyladenine (BA). Hyperhydric shoots were observed when 10 µM of any cytokinin was added to MS media. Use of any cytokinin at 2.5 µM produced an average of 14–21 adventitious shoots/root explant. Shoots formed roots in vitro more effectively than α-naphthaleneacetic acid when indole-3-butyric acid and indole-3-acetic acid were used at 1.0 µM. Two-month-old rooted plantlets were transplanted to vermiculite and 70% survived after 4 months.

     

Flow cytometry and start codon targeted (SCoT) genetic fidelity assessment of regenerated plantlets in Tylophora indica (Burm.f.) Merrill

Tylophora indica (Burm.f.) Merrill. is a pharmacologically important plant, popular for alkaloidal and non-alkaloidal richness. Large scale propagation of T. indica is difficult in the wild as the seeds are small and the frequency of germination is very poor. In the present study, the genome size estimation of in vitro regenerated (indirect, direct and somatic embryo mediated) T. indica was made by flow cytometric method. Clonal fidelity of the regenerants was assessed using a start codon targeted (SCoT) molecular marker. Initially, the explants were inoculated on Murashige and Skoog basal medium supplemented with various concentrations of plant growth regulators like 2,4-dichlorophenoxy acetic acid (2,4-D), Kinetin, 6-benzyl amino purine (BAP) and 1-naphthalene acetic acid either singly or in combinations. The highest callus induction frequency (87.75%) was obtained in 6.7 µM 2,4-D added MS medium which metamorphosed into progressive stages (globular, heart, torpedo, and cotyledonary) of embryos. Mature and healthy somatic embryos efficiently germinated into plantlets on 8.8 µM BAP?+?1.4 µM GA₃ enriched MS medium. Histological and scanning electron microscopic study confirmed the above developing stages. The regenerated shoots were rooted best in 2.45 µM Indole-3-butyric acid supplemented solid MS medium. The plants were hardened and acclimatized with 90% survivability. The flow cytometric 2C DNA content of indirect, direct and somatic embryo derived plants was 1.896 pg, 1.940 pg and 1.926 pg respectively, very similar to the mother plant (1.928 pg). SCoT marker generated a high percentage of monomorphic bands (94%) revealing similarity with the mother plant, thus ensuring genetic fidelity. To the best of our knowledge, this is perhaps the first ever report of 2C DNA content estimation and SCoT marker based genetic homogeneity study in T. indica.

     

The in vitro germination and storage characteristics of Keteleeria fortunei var. cyclolepis pollen provide a reference for cross breeding

Keteleeria fortunei var. cyclolepis is an ideal tree species for mountain afforestation, timber forests, and landscaping. Its pollination process can be affected by the rainy season, making it difficult to pollinate the massive female cones, which leads to a high abortion rate and low quality of seeds. Here, we observed the pollen morphology of K. f. cyclolepis using scanning electron and light microscopes, investigated the characteristics of its in vitro germination by the detached method, and explored the effect of different storage temperatures and times on the pollen germination rate and the activity of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT). Our results indicated that the pollen of K. f. cyclolepis is a five-cell pollen, comprising one noumenon and two air sacs, both of which were oval in polar view. The optimal condition for pollen germination of K. f. cyclolepis was 240 g/L sucrose + 70 mg/L CaCl₂ + 210 mg/L H₃BO₃ at 24 °C and pH 6.0, resulting in a germination rate of 45.0%. The effects of different storage temperature and time on pollen germination rate varied significantly. The best storage temperature was − 80 °C, at which the germination rate was 20.9% after 365 days of storage, and the activity of three protective enzymes remained relatively high, representing relatively strong antioxidation and antiaging activity. Stepwise regression analysis showed that SOD was the main factor affecting the pollen germination rate of K. f. cyclolepis. The function of the three protective enzymes differed under various temperatures, for example, SOD served as a sensitive protective enzyme at room temperature, − 20 °C and − 80 °C, whereas both SOD and CAT served as sensitive protective enzymes at 4 °C.

     

Novel avenues for passion fruit in vitro regeneration from endosperm culture,and morpho-agronomic and physiological traits of triploid Passiflora cincinnata Mast. emblings

The present study describes a new regeneration system based on somatic embryogenesis from mature endosperm Passiflora cincinnata Mast. cultures. Moreover, the morpho-agronomic and phenological traits, as well as enzymatic activity of regenerated triploid emblings are compared to those of diploids. Mature endosperms were cultured on Murashige and Skoog medium supplemented with various concentrations (4.5–45.2 µM) of 2,4-dichlorophenoxyacetic acid (2,4-D) and 4.5 μM 6-benzylaminopurine (BA). No plant growth regulators were included in the control group. Embryogenic calli were observed only in treatments supplemented with 13.6 and 18.1 µM 2,4-D?+?4.5 µM BA, with the highest number of somatic embryos per explant and regenerated plants (emblings) obtained with 18.1 µM 2,4-D. Most emblings (70%) were triploid (2n?=?3x?=?27), with a DNA amount (4.38 pg) similar to that of endosperm and 1.5 times greater than in diploid P. cincinnata seedlings (2n?=?2x?=?18), that contained 2.98 pg of DNA. While the number of organs and/or structures was akin to that in diploids, triploid emblings generally exhibited larger and longer vegetative and floral structures. The flower lifespan was also slightly altered by triploidy, nectar concentration was 27% higher, and the activity of plant defense enzymes β-1,3-glucanase and polyphenol oxidase was 29.8% and 22.1% higher. This study describes a new regeneration system for the production of phenotypic variants of this ornamental passion fruit species, opening new perspectives for future studies on genetic passion fruit breeding.

     

Somatic embryogenesis and shoot organogenesis from the hypocotyl slices and free radical scavenging activity of regenerants of collard greens (Brassica oleracea L. var. acephala)

Brassica oleracea var. acephala is an important leafy vegetable that has been widely consumed as a high-nutrient, low-calorie food. Because of the plant’s biennial and self-incompatibility nature, biotechnological approaches are alternative way for propagation and breeding improvements. Since tissue culture studies have been focused in other B. oleracea representatives, the aim of the present study was to achieve effective regeneration protocol distinctive for collard greens, and evaluate the total phenolic content and antioxidant activity of regenerants. The effect of 3 cytokinins [thidiazuron (TDZ), 6-benzyladenine (BA) and 6-furfuryladenine (kinetin, KIN)] at increasing concentrations (0, 5, 7.5, 10, 20 or 30 µM) in combination with tenfold lower concentration of 1-naphtaleneacetic acid (NAA) (0, 0.5, 0.75, 1, 2 or 3 µM, respectively) on the regeneration from hypocotyl slices was studied. Histological analysis revealed the two regeneration pathways, somatic embryogenesis and shoot organogenesis, simultaneously occurred in the same explant, regardless of the cytokinin/NAA combinations used. The regeneration frequency of 95.9%, with 7.5 morphogenic structures regenerated per explant, and the healthy appearance of regenerated plants indicated the optimal combination 20 µM TDZ?+?2 µM NAA. TDZ at 5 µM provided the high somatic embryo proliferation rate by generation of secondary embryos (7.79) along with the lowest rate of their abnormalities. Embryo-developed plants were successfully acclimatised (above 90%). The plants regenerated and proliferated on TDZ-containing media had higher total phenolic content that correlated with the highest free radical scavenging activity (IC₅₀?=?19.09 µg ml^??1).