State-of-knowledge on Amburana cearensis (Fr. Allem.) A.C. Smith (Leguminosae: Papilionoideae) for genetic conservation in Brazil

This study explores the basis for conservation action on Amburana cearensis (Fr. Allem.) A.C. Smith (Leguminosae: Papilionoideae), a wide-ranging forest species occurring in Brazil, notably in the Caatinga and Cerrado regions, and other South American countries. It offers insight into the taxon through structured reviews of knowledge of its biology, ecology, silviculture and management. Widely scattered published reports are critically considered and efforts made to highlight and resolve contradictions and inconsistencies therein. Information about this species is scanty and scattered, particularly in respect to its biology and ecology. Because little effort has been applied to its domestication and improvement, knowledge of its silviculture and management is meagre. The taxon is typical of open and deciduous forest in the Caatinga and Cerrado. Gaps in current knowledge relevant for conservation are identified and steps to fill them proposed. Where in situ conservation proved to be more appropriate, recommendations are made for the location of additional protected areas. Ex situ and enrichment conservation action is highly recommended for specific parts of the range where resource losses are already so extensive that in situ measures alone are inadvisable. Provision for refining the limited management and conservation knowledge is made through highlighting priorities for study of the taxon. Finally, future action is discussed in the context of the infrastructure of the national conservation sector.     

Ultrastructural and biochemical investigations of protein mobilization of Mucuna pruriens (L.) DC. cotyledons and embryo axis

The mobilization of storage reserves, with particular emphasis on storage proteins of Mucuna pruriens (L.) DC., cotyledons, and embryo was investigated from the ultrastructural and biochemical points of view. Proteins and starch were the two main storage substances in cotyledons, and proteins and lipids were the main ones in the embryo. Embryo protein bodies were smaller and fewer in number than those of cotyledons. Structural and ultrastructural data determined between 24 and 48 h after imbibition and between 48 and 72 h after imbibition, the end of significant embryo and cotyledon protein mobilization, respectively, indicating more precocious storage protein mobilization in the axis than cotyledons. Moreover, storage protein mobilization in embryo and cotyledons occurred before the end of germination. Water soluble proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, producing 29 bands with molecular weights from 14 to 90 KDa. Embryo extract contained more proteins than cotyledon extract, contained seven characteristic bands, and showed a higher variability of the optical density trend than cotyledon.     

A seed respiration-based index of cold-sensitivity during imbibition in four macrothermal species

A research was carried out to evaluate the influence of temperature on seed respiration response of maize, cotton, grain sorghum and sunflower during imbibition, and to define reliable indices for a fast evaluation of cold-sensitivity at germination level in plants. The seed respiration activity was measured during seed imbibition at 25 °C (optimal) and 15 °C (suboptimal) constant temperatures, using a homemade respiration chamber adapted to an infrared gas analyzer. At 15 °C, sunflower and sorghum maintained high levels of seed germination (≥90 %), whilst this last dropped in cotton (36.7 %) and maize (27.8 %). With respect to this, cotton and maize seem to be cold sensitive during germination. Instantaneous seed respiration during imbibition versus temperature or thermal time could not be used as a good indicator for cold tolerance, since the levels of CO₂ recorded at 15 °C in cotton (higher than the other species) and maize (similar to that of sorghum and sunflower) did not correspond to adequate seed germination. Differently, the rates (b coefficient of linear regressions) of accumulation of CO₂ respired at optimal and suboptimal temperatures during the first hours of imbibition (up to approximately 24 h from the start of experiment), were significantly different in maize and cotton, whilst they did not differ in sorghum and sunflower. Therefore, the shift between slopes may represent a reliable index for seed cold-sensitivity assessment during early germination.     

Effects of drought stress on seed germination of ornamental sunflowers

The response to drought stress on germination was investigated on three hybrids of ornamental sunflower, ‘Hadar’, ‘Pazit’, and ‘Zohar’. Different levels of water potential [Ψ: 0.0 (control), ?0.15, ?0.30, ?0.45, ?0.60, ?0.75, and ?0.90 MPa] were adopted using polyethylene glycol-6000 (PEG6000) at four germination temperatures (15, 20, 25, and 30 °C). Final germination percentage, mean germination time, germination index, germination rate index, and germination stress tolerance index were used to evaluate the genotype response to PEG-induced water stress. Shoot and root length and fresh and dry weight were measured on seeds germinated at 20 °C under the different levels of water potentials. During germination, the three ornamental sunflowers showed to be more sensitive to suboptimal temperature than to supraoptimal. Decreasing water potential of imbibition solution progressively inhibited and delayed seed germination. Among cultivars, ‘Hadar’ and ‘Pazit’ performed better at temperature lower than 30 °C. ‘Zohar’ showed a lower sensitivity to PEG-induced water stress at all temperature conditions. Water stress during seed germination depressed the following seedling growth under favourable conditions. As a result, shoot and root length and fresh and dry weight was significantly lower in seedlings from seed germinated at ψ ≤ 0.45 MPa.     

Permeability of seed coats to water as related to drying conditions and metabolism of phenolics

The seed coat of Pisum elatius is normally impermeable to water. When seeds are dried in the absence of oxygen their coats are totally permeable to water. Structural differences are observed between permeable and impermeable seed coats. In the genus Pisum, species with normally impermeable seed coats have a high content of phenolics and of catechol oxidase, while seed coats of P. sativum contain very little catechol oxidase and have a very low content of phenolics. Such differences are not noted in the cotyledons. We hypothesized that during dehydration of seeds, oxidation of phenolic compounds in seed coats through catalysis of catechol oxidase in presence of O₂ might render the seed coats impermeable to water.     

Germination biology and seedling growth of Clusia hilariana Schltdl., a dominant CAM-tree of drought-prone sandy coastal plains

The spatial and temporal fluctuations of water availability can be an obstacle for recruitment of many species in the restinga and might restrict seed germination and seedling growth in specific regeneration safe-sites. Clusia hilariana is one of the most dominant species of Restinga de Jurubatiba. This species has a high proportion of seedling establishment occurring inside the tanks of soil bromeliads underneath vegetation patches. Given the thin seed coats, the fast germination time and seed dispersal of C. hilariana during the dry season, we hypothesized that their major regeneration niche (the tanks of soil bromeliads) is related to susceptibility of seed germination and also seedling growth to low water availability. To test this hypothesis, seeds were germinated under decreasing water potentials using PEG 6000 solutions and seedlings were grown under varying water regimes. The percentage of seed germination progressively decreased at lower water potentials. After 38 days in ?1.0 MPa no seeds germinated. However, approximately 90% of seeds germinated when transferred to Ψ = 0 MPa. The relative growth rates of seedlings of C. hilariana did not differ between water treatments. Thus, the major regeneration niche of C. hilariana is not a consequence of a high sensitivity of seeds and seedlings to water shortage. Nonetheless, C. hilariana showed an array of seed and seedling traits that may help to overcome establishment constraints of the harsh environment of restingas.     

Osmoconditioning prevents the onset of microtubular cytoskeleton and activation of cell cycle and is detrimental for germination of Jatropha curcas L. seeds

• Jatropha curcas is an oilseed crop renowned for its tolerance to a diverse range of environmental stresses. In Brazil, this species is grown in semiarid regions where crop establishment requires a better understanding of the mechanisms underlying appropriate seed, seedling and plant behaviour under water restriction conditions. In this context, the objective of this study was to investigate the physiological and cytological profiles of J. curcas seeds in response to imbibition in water (control) and in polyethylene glycol solution (osmoticum).
• Seed germinability and reactivation of cell cycle events were assessed by means of different germination parameters and immunohistochemical detection of tubulin and microtubules, i.e. tubulin accumulation and microtubular cytoskeleton configurations in water imbibed seeds (control) and in seeds imbibed in the osmoticum.
• Immunohistochemical analysis revealed increasing accumulation of tubulin and appearance of microtubular cytoskeleton in seed embryo radicles imbibed in water from 48 h onwards. Mitotic microtubules were only visible in seeds imbibed in water, after radicle protrusion, as an indication of cell cycle reactivation and cell proliferation, with subsequent root development. Imbibition in osmoticum prevented accumulation of microtubules, i.e. activation of cell cycle, therefore germination could not be resumed.
• Osmoconditioned seeds were able to survive re‐drying and could resume germination after re‐imbibition in water, however, with lower germination performance, possibly due to acquisition of secondary dormancy. This study provides important insights into understanding of the physiological aspects of J. curcas seed germination in response to water restriction conditions.

     

Antioxidant activities of different parts of Gnetum gnemon L.

Analyses on biological activities of Gnetum gnemon were done to determine the total phenolic and antioxidants of the plant. Four parts of G. gnemon were used in this study, which were leaf, bark, twig, and seeds of the plant. All parts were extracted in methanol, ethanol, hexane, chloroform and hot water using reflux. The total phenolic content of the plant extracts were determined by using Folin-Ciocalteu method. The results demonstrated that the bark from hot water extract showed the highest total phenolic at 10.71?±?0.01 mg GAE/ FDW, while the lowest was chloroform extract of seed at 2.15?±?0.01 mg GAE/ FDW. The antioxidant activity of the plant extracts were determined by using DPPH and FRAP assays, respectively. The DPPH results showed that all plant extracts demonstrated weak free radical scavenging activity tested at the final concentration of 300 μg/ml. In contrast, the methanolic twig extract showed strong reducing power activity (FRAP) at 83.55?±?1.05%, while the hot water seed extract showed the least activity at 41.86?±?4.22% tested at the final concentration of 300 μg/ml. However, there were no correlation between total phenolics and both antioxidant assays tested.     

Proteolytic and Trypsin Inhibitor Activity in Germinating Jojoba Seeds (Simmondsia chinensis)

Changes in proteolytic activity (aminopeptidase, carboxypeptidase, endopeptidase) were followed during germination (imbibition through seedling development) in extracts from cotyledons of jojoba seeds (Simmondsia chinensis). After imbibition, the cotyledons contained high levels of sulfhydryl aminopeptidase activity (APA) but low levels of serine carboxypeptidase activity (CPA). CPA increased with germination through the apparent loss of a CPA inhibitor substance in the seed. Curves showing changes in endopeptidase activity (EPA) assayed at pH 4, 5, 6, 7, and 8 during germination were distinctly different. EPA at pH 4, 5, 6, and 7 showed characteristics of sulfhydryl enzymes while activity at pH 8 was probably due to a serine type enzyme. EPA at pH 6 was inhibited early in germination by one or more substances in the seed. Activities at pH 5 and later at pH 6 were the highest of all EPA throughout germination and increases in these activities were associated with a rapid loss of protein from the cotyledons of the developing seedling.     

Development of seed coat-imposed dormancy during seed maturation in Cynoglossum officinale

The relationship between seed phenolics and appearance of seed coat–imposed dormancy during seed development in Cynoglossum officinale L. was studied. Up to 24 days after anthesis, seeds failed to germinate upon imbibition in Petri dishes at 25°C. At 44 days after anthesis, seeds were fully germinable; removal of seed coats did not improve their germination or O₂ uptake. At 72 days after anthesis, mature seeds at the base of the cyme did not germinate unless their coats were removed. Removal of seed coat also stimulated O₂ uptake at this harvest date. The methanol-soluble phenolic content of the seeds increased during the early stages of seed development, in both the seed coat and the embryo. As seed development continued, the methanol-soluble phenolic content of the embryo stabilized, but that of the seed coat declined. This decline was associated with an increase in the thioglycolic acid–soluble phenolics, presumably lignins, in the seed coat. These results suggest that polymerization of methanol–soluble phenolics into lignins in the seed coat during later stages of seed development renders the seed coat of C. officinale impermeable to 03, and thus keeps the seed dormant.     

Hormonal and temperature regulation of seed dormancy and germination in <Emphasis Type="Italic">Leymus chinensis</Emphasis>

Fluctuating temperature plays a critical role in determining the timing of seed germination in many plant species. However, the physiological and biochemical mechanisms underlying such a response have been paid little attention. The present study investigated the effect of plant growth regulators and cold stratification in regulating Leymus chinensis seed germination and dormancy response to temperature. Results showed that seed germination was less than 2 % at all constant temperatures while fluctuating temperature significantly increased germination percentage. The highest germination was 71 % at 20/30 °C. Removal of the embryo enclosing material of L. chinensis seed germinated to 74 %, and replaced the requirement for fluctuating temperature to germinate, by increasing embryo growth potential. Applications of GA₄₊₇ significantly increased seed germination at constant temperature. Also, inhibition of GA biosynthesis significantly decreased seed germination at fluctuating temperatures depending upon paclobutrazol concentration. This implied GA was necessary for non-dormant seed germination and played an important role in regulating seed germination response to temperature. Inhibition of ABA biosynthesis during imbibition completely released seed dormancy at 20/30 °C, but showed no effect on seed germination at constant temperature, suggesting ABA biosynthesis was important for seed dormancy maintenance but may not involve in seed germination response to temperature. Cold stratification with water or GA₃ induced seed into secondary dormancy, but this effect was reversed by exogenous FL, suggesting ABA biosynthesis during cold stratification was involved in secondary dormancy. Also, cold stratification with FL entirely replaced the requirement of fluctuating temperature for germination with seeds having 73 % germination at constant temperature. This appears to be attributed to inhibition of ABA biosynthesis and an increase of GA biosynthesis during cold stratification, leading to an increased embryo growth potential. We suggest that fluctuating temperature promotes seed germination by increasing embryo growth potential, mainly attributed to GA biosynthesis during imbibitions. ABA is important for seed dormancy maintenance and induction but showed less effect on non-dormant seed germination response to temperature.     

Environmental cues for germination of the invasive bunch grass <Emphasis Type="Italic">Pennisetum ciliare</Emphasis> (L.) Link

Responses of seed germination to air temperature, water potential, light, and smoke were studied in the laboratory for seeds of the invasive bunch grass Pennisetum ciliare (L.) Link (syn. Cenchrus ciliare L.; buffel grass). First introduced to North America during the mid-twentieth Century for establishing pastures, this African bunch grass has become an invasive species of concern. Across all the experiments conducted, a low germination was observed for P. ciliare fascicles that never exceeded 30 % at 21 days after sowing. Optimal day/night air temperatures for germination, controlled with an environmental chamber, were 25/15 and 30/20 °C, while extreme temperatures of 15/5 and 45/35 °C inhibited germination. By sowing seeds of P. ciliare under different water potentials, created with aqueous solutions of polyethylene glycol, an optimum of ?0.03 MPa led to the highest germination, while no germination was observed at ?1.0 MPa. Monochromatic optical filters were utilized to germinate seeds under various wavelengths, of which red (650 nm) and far red (730 nm) led to the highest germination. In addition, seeds that were incubated in the dark had higher germination than those incubated under white light. Incubation in smoke water, which can stimulate germination of pyrophytic species, resulted in a marginal inhibition of germination compared with imbibition with distilled water.     

The Damaging Effect of Water on Dry Pea Embryos During Imbibition

When pea seeds were imbibed in water without their seed coats,vital staining revealed that cells on the abaxial surface ofthe cotyledons were dead. No damage occurred on the surfaceof cotyledons when the seeds were imbibed intact, or beneaththe testa when only half of the testa was removed. Cell deathoccurred as a result of rapid water uptake within the first2 min of imbibition, since reducing the rate of imbibition insolutions of Carbowax 4000 lessened the damage. Cell death wasrestricted to the outer layers of the cotyledons; inner tissuesremained alive. These observations supported the hypothesisthat rapid early leakage during imbibition of dry embryos resultedfrom the death of cells caused by the physical disruption ofmembranes. Imbibition damage resulted in reduced respirationand germination, a decline in the rate of food reserve transferfrom the cotyledons to the growing axis, and a lower growthrate in the seedlings produced. Greater sensitivity of embryosto imbibition damage at low temperature, and similarities betweenfeatures of imbibition damage and chilling injury led to thesuggestion that so-called chilling injury is the result of imbibitiondamage rather than the effects of low temperature.     

Reactive oxygen species release, vitamin E, fatty acid and phytosterol contents of artificially aged radish (Raphanus sativus L.) seeds during germination

Seeds of Raphanus sativus L. subjected to accelerated ageing were investigated for reactive oxygen species (ROS) release and for content of vitamin E (tocopherol, TOC, and tocotrienol, TOC-3), fatty acids and phytosterols in seed coats, cotyledons and embryonic axes during germination. In unaged seeds, ROS release occurred mainly in seed coats of non-imbibed seeds and in seedlings (48?h of imbibition). TOC and TOC-3 were mainly represented by the ??-isoform, abundant in embryonic axes. Fatty acids were mainly found in cotyledons. In seed coat and embryonic axis, phytosterols consisted mainly of sitosterols. The effects of ageing were mainly visible in embryonic axes at 48?h of imbibition. Deterioration was associated with a decrease in fresh weight increase percentage, germination percentage, ??-TOC and total fatty acid content. An increase in ROS release from seed coats and in ??-TOC, ??-TOC, ??-TOC-3 content in embryonic axis was also observed. The use of ??-TOC and total fatty acids in embryonic axis as parameters of seed quality evaluation during storage was suggested.     

Seed germinability and longevity influences regeneration of <Emphasis Type="Italic">Acacia gerrardii</Emphasis>

Acacia gerrardii is the only native tree species of the Kuwaiti desert ecosystem. However, anthropogenic disturbances and harsh arid climate have contributed towards the disappearance of this keystone species from its habitat. In this study, effects of different seed pretreatments to break dormancy, water entry pathway, and ecology (seasonal timing) of dormancy loss and germination of A. gerrardii were investigated. Effects of mechanical scarification, hot water treatment (30 s, 1, 2, and 5 min), and concentrated acid scarification (10, 20, and 30 min) on germination percentage and rate (time to 50% germination and final germination) were also examined. Pretreatment with mechanical scarification produced the highest germination in the least time and 20 °C, 40% RH with 12 h of light (2370 Lux) were found to provide the best germination environment. Seeds were rapidly aged at 60% RH and 45 or 50 °C to determine longevity, and the results were analyzed using probit analysis. Times taken for viability of A. gerrardii seeds aged at 45 and 50 °C to fall to 50% (p₅₀) were 38.6 and 9.3 days, respectively, and therefore the seeds can be considered to have medium longevity. Experiments to find the water entry pathway in A. gerrardii indicated that the micropyle region was the primary point of water entry into the seed. Seed burial experiments indicated that though seed retention decreased over time, there was no significant decrease in number of viable seeds after 31 weeks. The findings of this study are important to nursery managers, seed banks, and those involved in conservation and restoration activities.     

In situ seed baiting to isolate germination-enhancing fungi for an epiphytic orchid,Dendrobium aphyllum (Orchidaceae)

Orchid conservation efforts, using seeds and species-specific fungi that support seed germination, require the isolation, identification, and germination enhancement testing of symbiotic fungi. However, few studies have focused on developing such techniques for the epiphytes that constitute the majority of orchids. In this study, conducted in Xishuangbanna Tropical Botanical Garden, Yunnan, China, we used seeds of Dendrobium aphyllum, a locally endangered and medicinally valuable epiphytic orchid, to attract germination promoting fungi. Of the two fungi isolated from seed baiting, Tulasnella spp. and Trichoderma spp., Tulasnella, enhanced seed germination by 13.6 %, protocorm formation by 85.7 %, and seedling development by 45.2 % (all P?Epulorhiza, another seed germination promoting fungi isolated from Cymbidium mannii, also enhanced seed germination (6.5 %; P?P?Trichoderma suppressed seed germination by 26.4 % (P?Tulasnella was the only treatment that produced seedlings. Light increased seed imbibition, protocorm formation, and two-leaved seed development of Tulasnella inoculated seeds (P?Tulasnella be introduced for facilitating D. aphyllum seed germination at the protocorm formation stage and that light be provided for increasing germination as well as further seedling development. Our findings suggest that in situ seed baiting can be used to isolate seed germination-enhancing fungi for the development of seedling production for conservation and reintroduction efforts of epiphytic orchids such as D. aphyllum.     

Pressure-Driven Extrusion of Intracellular Substances from Bean and Pea Cotyledons during Imbibition

Intracellular substances leak from imbibing cotyledons of grain legumes during imbibition. This work reports the discovery of a biophysical process by which intracellular substances are driven from cotyledons during imbibition. Light and scanning electron microscopy were used to examine bean (Phaseolus vulgaris L.) and pea (Pisum sativum L.) cotyledons and the material released from them into imbibition water. A large fraction of the visible materials released from excised bean and pea cotyledons during the first 30 minutes of imbibition consisted of convoluted or helical streams of material which rapidly emerged from the cotyledons surfaces. Large streams of material from bean cotyledons contained starch grains and protein bodies, and smaller streams from bean and pea cotyledons probably contained protein bodies. The forms of streams were characteristic of a viscous fluid which had been forced by pressure through irregular orifices. The sites of extrusion from bean cotyledons were multicellular blisters which formed on the surfaces of imbibing cotyledons. In 6 hours, pea and bean cotyledons leaked from 1 to 11 micrograms protein per milligram of seed dry weight. The quantities of protein leaked primarily depended on cultivar.     

Positive responses of strawberry (<Emphasis Type="Italic">Fragaria</Emphasis> × <Emphasis Type="Italic">ananassa</Emphasis> Duch.) explants to salicylic and iron nanoparticle application under salinity conditions

Mucuna bracteata DC. ex Kurz is an important cover crop in plantations across the tropics. However, low germination rate and poor viability of Mucuna seeds pose significant challenges of using the seeds as starting material. To address these limitations, we have optimized seed germination conditions (such as scarification period, surface sterilization protocols and imbibition period) and in vitro propagation protocols for M. bracteata. We found that seeds treated with sulphuric acid for 30 min, imbibed for 6 h and incubated in dark conditions on a wet cotton roll (10 mL of sterile distilled water) supplemented with 0.1% activated charcoal produced the highest percentage of seed germination (44%) and seed vigor index. In vitro-derived cotyledonary nodes showed the highest number of shoots per explant (5.60) and rooting response (92.9%) when cultured on Murashige and Skoog medium containing 4.44 µM 6-benzylaminopurine and 10.7 µM 1-naphthaleneacetic acid, respectively. Of the 100 rooted plantlets acclimatized, 89.0% survived after 4 weeks of transplanting. Single sequence repeat and flow cytometry analysis were performed to confirm the genetic fidelity of the plants. Our protocol offers, for the first time, a simple and effective seed germination and scalable propagation procedures for M. bracteata. Furthermore, we have also estimated the genome size (1448?±?9 Mb) and DNA content (1.48?±?0.01 pg) for M. bracteata that can be used for future cytogenetic studies on genetic diversity and gene exchange.     

β-Estradiol Protects Embryo Growth from Heavy-Metal Toxicity in Germinating Lentil Seeds

Mammalian sex hormones spread in the environment from both natural and anthropogenic origin. In the present study, we found that treatment with β-estradiol (E) could improve embryo growth and alleviate unsuitable availability of nutrients imposed by cadmium and copper toxicity during germination of lentil (Lens culinaris Medik.) seeds. The length of embryonic axes decreased in the presence of 100 μM CdCl₂ or 200 μM CuCl₂. Addition of 10^?6 M E in the germination media could greatly reverse the inhibitory effect of heavy-metal (HM) stress on post-germination events. The cotyledons of E-treated seeds also tended to (1) retain higher protease and amylase activities, (2) breakdown more storage compounds (albumin, globulin, and starch), and (3) have higher contents of free amino acids and glucose than controls without added E (Cd or Cu applied individually). Further investigations showed that exposure to HM dramatically provoked the solute leakage in imbibition medium, whereas the combination of HM with E significantly reduced the loss of nutrients. Moreover, the seed Cd and Cu contents were not significantly different between the cotreatment of Cd or Cu with E and no treatment, meaning that E was not responsible for preventing HM accumulation in seed tissues.