Soaking Seeds In Water Research Articles

The effect of a doubled glutathione level on parameters affecting the germinability of recalcitrant Acer saccharinum seeds during drying

Approximately 20% of plant species, including silver maple (Acer saccharinum L.), produce seeds that are sensitive to desiccation, which is reflected in their poor storage potential and viability. In the search for a compound that can improve seed recalcitrance, freshly harvested seeds were soaked in either 2.5 mM reduced glutathione (GSH) or water and desiccated to comparable water levels of 55–20%. We examined the impact of a doubled endogenous level of glutathione on the seed germination capacity, the activity of enzymes involved in glutathione metabolism, the cell membrane components and integrity, reactive oxygen species, and ascorbate levels. GSH treatment resulted in slower dehydration and a higher germination capacity. The increased glutathione was mainly consumed by glutathione S-transferase, leading to more efficient detoxification, and by dehydroascorbate reductase (DHAR), accelerating the ascorbate regeneration. As a result, the cellular environment became more reduced, and protection of the membrane structures was enhanced. The ameliorated membrane integrity was manifested via a lower electrolyte leakage and a lower lipid peroxide level despite the higher level of hydrogen peroxide (H2O2) detected in the GSH-treated seeds. The degradation of phospholipids (PLs) was less intense and related to the phosphatidylinositol (PI) level, which is the precursor of the phospholipase D cofactor, whereas in water-soaked seeds, PL degradation was promoted by H2O2. The germination capacity of the dehydrated seeds depended primarily on the level of H2O2, lipid hydroxyperoxides, electrolyte leakage, GSH, the half-cell reduction potential of glutathione, PI, and the activity of DHAR and γ-glutamylcysteine synthetase. Interestingly, H2O2 affected all of the parameters. The germination of GSH-boosted seeds was strongly impacted by the pool of ascorbate, the half-cell reduction potential of ascorbate, and the glutathione peroxidase activity. In general, germination was DHAR activity-dependent. A strong negative correlation was detected in the water-soaked seeds, whereas a strong positive correlation was detected in the GSH-treated seeds. The enhanced level of glutathione likely improved the efficiency of the ascorbate–glutathione cycle, confirming its effect on seed germinability after dehydration.

English

Hydropriming of pearl millet seeds was tested during two growing seasons in Burkina Faso. A total of 32 field experiments were distributed equally between two agro-ecological zones: The Northern zone receiving on average less than 600 mm annual precipitation and the Central zone receiving 600 to 900 mm. Hydropriming was performed by soaking of seeds in water for 6 h, followed by air-drying overnight. In the Northern zone, an increase of both emergence and yield was observed for hydroprimed seeds in both years of testing. This was reflected by a higher yield observed in 13 out of 16 field experiments, increased median yield (+159 kg/ha; p<0.0053) and an increase of the relative yield by +29% as a field average (p<0.000054). In contrast, in the Central zone a net negative effect on crop emergence was observed in both years, and only 5 out of 16 field experiments showed a yield increase for hydroprimed seeds. Meteorological data confirmed the difference in rainfall between the two zones. Hydropriming by 6 h of soaking and drying of seeds overnight appears as a simple method to increase yield of pearl millet significantly in the most arid out of two agro-ecological zones tested in Burkina Faso. Drying of seeds overnight is a novel agronomically feasible approach, allowing a full day for subsequent sowing. Key words: Seed-priming, hydro-priming, Sahel, millets, location-dependent effect.

Germination and seedling emergence responses of common bean and cowpea to plant extract seed treatments

SUMMARYThe present study was initiated to investigate the effect of crude plant extracts as seed treatments onPhaseolus vulgaris(common bean) andVigna unguiculata(cowpea) seed germination and emergence in the presence ofColletotrichum lindemuthianumandColletotrichum dematium,respectively. Common bean and cowpea seeds were treated with crude water and acetone extracts ofAgapanthus caulescensSpreng.,Allium sativumL.,Carica papayaL. andSyzygium cordatumHochst.ex Krauss at 5 and 15 mg/ml concentrations. Seeds treated with the synthetic fungicide fludioxonil+mefenoxam (the commercial product Celest®XL) represented the positive control, whereas dimethyl sulphoxide and water-soaked seeds represented negative controls. The rolled paper towel method of the International Seed Testing Association was used to investigate the effect of the treatments on seed germination. Mean emergence time (MET) was determined using seed inoculated with the respective pathogens. The changes in the ultrastructure of embryonic roots and the connecting tissues of embryo-cotyledon of common bean and cowpea treated withSyzygiumacetone extracts andAgapanthuswater extracts were investigated using transmission electron microscopy (TEM). High germination percentages of &gt;90% were observed in bean seeds from two production seasons treated with low concentrations of water extracts ofAllium, SyzygiumandAgapanthusand acetone extracts ofAllium, AgapanthusandCarica. These treatments also recorded high emergence percentages with low MET values, which were similar to the water control. Cowpea seeds treated withCaricawater extract had the highest germination and emergence.Syzygiumacetone was the only extract that gave higher germination and emergence in both IT93K5132 and PAN 311 varieties. Therefore,Caricawater andSyzygiumacetone extracts can be considered as potential bean and cowpea seed treatments. Generally, there were inconsistencies in terms of correlations of germination with emergence percentages in both cowpea and bean seed treated with plant extracts used in the study, which could be due to differences in vigour. The TEM study of embryo-cotyledon tissue of both species revealed thatSyzygiumandAgapanthusextract seed treatment may accelerate metabolic processes as evidenced by the presence of vacuoles, many cristae and few lipid bodies.

Seed Germination Response to Temperature, Cold Stratification Period, and Gibberellin Treatment in Spiraea fritschiana

To improve the germination of Spiraea fritschiana seeds for mass propagation, we evaluated the effect of a range of temperatures, cold stratification periods, and gibberellic acid (GA₃) treatments on three germination characteristics. Final germination percentage (FGP) increased as the temperature for seed germination increased, up to 30°C, while the mean germination time (MGT) and the mean number of days to 30% germination (T 30 ) decreased when seeds were germinated at 25-30°C. The optimum germination temperature of S. fritschiana seeds is approximately 30°C considering FGP, MGT, and T 30 together. FGP increased with the duration of cold stratification up to a period of 6 weeks, but declined after 8 weeks of cold stratification, as prolonged cold stratification can induce dormancy with a resultant decline in germination. Pretreatment with 6-8 weeks of cold stratification or soaking seeds in distilled water or 500 mg · L -1 GA₃ for 24 h accelerated and increased the germination of S. fritschiana seeds, regardless of temperature. However, further study might be required to evaluate the effect of GA₃ concentrations lower than 500 mg · L -1 on the promotion of germination in S. fritschiana seeds.

Variables influencing germination and initial survival of two critically endangered plants: Warea amplexifolia and Lupinus aridorum

One challenge of ex situ conservation is developing propagation methods that promote a high rate of survival and genetic diversity. Developing successful propagation methods is especially important for rare species to prevent their extinction. Clasping warea, Warea amplexifolia (Nutt.) Nutt. (Brassicaceae), and scrub lupine, Lupinus aridorum McFarlin ex Beckner (Fabaceae), are two rare species endemic to imperiled Florida sandhill and scrub habitats, respectively. We tested whether the collection site of seeds, seed stratification temperature, and several propagation methods influenced germination and initial survival of W. amplexifolia and L. aridorum. The collection site of seeds and type of pot influenced percent germination of W. amplexifolia, whereas soaking seeds in water and stratification temperature did not. The site where seeds were collected did not influence germination of L. aridorum and treating seedlings with salicylic acid, nitrogen, or salicylic acid and nitrogen sometimes reduced, but did not increase, initial survival of seedlings. Overall, our results will inform additional experiments on ex situ conservation and may be applicable to other herbs endemic to Florida.

Effect of Different Concentration of Thiamine Hydrochloride (Vitamin-B1) on Growth and Yield of Mustard (Brassica junceaL.) var. Varuna

One pot experiment was conducted according to simple randomised block design on Mustard (Brassica juncea L.) Var. Varuna to establish the most suitable concentration for soaking of mustard seeds in thiamine hydrochloride solutions of concentrations, that is, 0.01, 0.02, 0.03, 0.06, 0.09, 0.12% on the performance of mustard. The seeds were soaked in thiamine hydrochloride solutions for 4 h. Water-soaked seeds were treated as control. Uniform basal dose of N90, P30 and K30 fertilizers were given to the soil before filling in the pots at the rate of NPKkg/ha. The soaked seeds were sown in clay pots containing autoclaved sandy loam soil and compost in 3:1 mixture. The parameters such as shoot dry weight, root dry weight, leaf area, crop growth rate (CGR), relative growth rate (RGR) and net assimilation rate (NAR) were calculated from 40 to 120 days after sowing. Number of pods per plant, 1,000 seed weight, yield, biological yield and harvest index were measured at harvest. Soaking concentration of 0.03% of thiamine hydrochloride was found to be the best in comparison to any other concentration of the treatment for most of the parameter studied. In growth parameters, leaf area, plant dry weight, root dry weight showed significant results of the early stages of sampling [that is, 40–80 Days after sowing (DAS)]. CGR also followed the same trend, however, RGR and NAR did not follow the same at any stage of the sampling. Yield parameters like pod number per plant, biological yield and seed yield registered significant result. Thus, it was concluded that soaking of mustard seeds in 0.03% thiamine hydrochloride ensured better growth and yield.

Breaking seed dormancy in Cupressus atlantica Gaussen, an endemic and threatened coniferous tree in Morocco

Cupressus atlantica Gaussen (Cupressaceae) is an endemic and endangered coniferous tree geographically restricted to the N’Fis valley in South-Western Morocco. Like many forest species, C. atlantica exhibits dormancy which delays and reduces germination. To improve seed germination, different pre-treatments were conducted on C. atlantica seeds after storage for different periods (one, two and five years) including: scarification with sandy paper; soaking seeds in hot distilled water at 60°C and 80°C for 15 min and soaking seeds for 48 h in a gibberellic acid (GA3) at 1,000 and 2,000 mg·L−1. Results showed that scarification with sandy paper increased the germination rate of Atlas cypress by up to 67%, indicating that the species possess essentially an exogenous dormancy (physical dormancy) due to the hard seed coat (hardseededness). Exogenous application of gibberellic acid (GA3) at 1,000 mg·L−1 was also effective in breaking seed dormancy and germination induction. These two treatments induced faster speed germination expressed by low number of days to first germination (8–10 days) and low values of mean germination times (MGT). However, germination rate, under any treatment, is greatly dependent on the year of seed collection. Seeds collected in year 2004 gave the highest value, suggesting that even after five years of storage, the germination capacity of C. atlantica seeds could remain high. This observation is very interesting in the ex-situ conservation of such endemic and endangered species where the production of seeds is irregular over the years.

Utilization of seaweed Sargassum myriocystum extracts as a stimulant of seedlings of Vigna mungo (L.) Hepper

The effect of seaweed liquid fertilizer (SLF) of Sargassum myriocystum on germination, growth and biochemical constituents of Vigna mungo was studied. Different concentrations (5%, 10%, 25%, 50% and 75%) of SLF were prepared using distilled water. A total of 100 seeds were soaked for each SLF concentration for a 24-h period and were later placed in various Petri plates and watered regularly. Water-soaked seeds were used as controls. Seed germination was 98% for the 10% SLF soaked seeds. Similarly, shoot length (21.13 cm seedling-1), root length (12.19 cm seedling-1), fresh weight and dry weight (3.910 and 0.320 g seedling-1) recorded at 10% concentration of S. myriocystum treated seedlings were the highest. Besides, the chlorophyll (0.547 mg g-1 fr. wt.) and carotenoid (0.359 mg g-1 fr. wt.) contents in leaves recorded at 10% SLF soaked seedlings were the highest. The 10% SLF treated seedlings showed the highest content of protein (1.898 and 1.508 mg g–1 fr. wt.), amino acid (0.370 and 0.306 mg g–1 fr. wt.), reducing sugar (6.031 and 3.448 mg g-1 fr. wt.), total sugar (8.414 and 7.055 mg mL-1 fr. wt.), alpha-amylase activity (1.403 and 1.380 μg min-1 mg-1 protein) and beta-amylase activity (1.099 and 1.029 μg min-1 mg-1 protein) of shoots and roots respectively. The seaweed extract showed better response at lower concentration while higher concentrations of seaweed extract showed a decreasing trend.

He‐Ne Laser‐Induced Improvement in Biochemical, Physiological, Growth and Yield Characteristics in Sunflower (Helianthus annuus L.)

The water-soaked seeds of sunflower were exposed to low power continuous wave He-Ne laser irradiation of energies 0, 100, 300 and 500 mJ to evaluate the effect on various biochemical, physiological, growth and yield parameters of sunflower. The experiments which consisted of four replicates arranged in a completely randomized design (CRD) were carried out under the greenhouse conditions. The physiological attributes like, photosynthetic rate (A), transpiration rate (E), intrinsic CO(2) concentration (C(i) ), stomatal conductance (g(s)), chlorophyll a and b contents, relative membrane permeability and leaf water (ψ(w)), osmotic (ψ(s)) and turgor (ψ(p)) potentials, relative water contents and leaf area increased significantly as compared to control due to He-Ne treatment of seeds. The activities of superoxide dismutase, peroxidase and catalases and contents of total soluble proteins, malondialdehyde, proline and leaf total phenolic also increased due to laser treatment. Significant increase in growth parameters of sunflower like shoot fresh and dry masses, root fresh and dry masses, root and shoot lengths, number of leaves per plant and stem diameter has also been observed. The contents of K, Ca and Mg in shoot and root were also increased and an overall increase of up to 28.12% was observed due to laser treatment.

The protective role of selenium in recalcitrant Acer saccharium L. seeds subjected to desiccation

Freshly harvested silver maple ( Acer saccharinum L.) seeds were soaked in either sodium selenite (10 mg/L) or water for 6 h. After washing and air drying, seeds were desiccated at 22 °C at a RH of 45–50% to comparable water levels from 50 to 12%. Germination capacity was significantly higher in seeds treated with selenium and desiccated [from 50 to 40, 35 and 30% of water content (WC)] than in water-soaked seeds. At 20% WC, the seeds from both treatments had low viability (approximately 20%). The electrolyte leakage and the MDA content were significantly lower in the embryonic axes of seeds soaked in selenite than in seeds soaked in water. We also found that the activity of glutathione peroxidase (GPX) of embryonic axes from selenium-treated seeds that were not desiccated, or from seeds that were desiccated to 40 and 35% WC, was significantly higher than that of non-treated axes. No difference in GPX activity was detected in cotyledons. This was confirmed by activity staining of GPX after native PAGE of proteins extracted from embryonic axes and cotyledons. An increase in glutathione reductase (GR) activity was also observed in embryonic axes of seeds treated with selenium and dried to 35 and 30% WC compared to non-treated samples. Selenium appeared to have no such effect on cotyledons.

Effects of Different Doses of Low Power Continuous Wave He–Ne Laser Radiation on Some Seed Thermodynamic and Germination Parameters, and Potential Enzymes Involved in Seed Germination of Sunflower (Helianthus annuus L.)

In this study, water-soaked seeds of sunflower were exposed to He-Ne laser irradiation of different energies to determine whether or not He-Ne laser irradiation caused changes to seed thermodynamic and germination parameters as well as effects on the activities of germination enzymes. The experiment comprised four energy levels: 0 (control), 100, 300 and 500mJ of laser energy and each treatment replicated four times arranged in a completely randomized design. The experimentation was performed under the greenhouse conditions in the net-house of the Department of Botany, University of Agriculture, Faisalabad. The seed thermodynamic parameters were calculated according to seed germination thermograms determined with a calorimeter at 25.8°C for 72h. Various thermodynamic parameters of seed (ΔH, (ΔS)(e), (ΔS)(c), (ΔS)(e) /Δt and (ΔS)(c) /Δt) were affected significantly due to presowing laser treatment. Significant changes in seed germination parameters and enzyme activities were observed in seeds treated with He-Ne laser. The He-Ne laser seed treatment resulted in increased activities of amylase and protease. These results indicate that the low power continuous wave He-Ne laser light seed treatment has considerable biological effects on seed metabolism. This seed treatment technique can be potentially employed to enhance agricultural productivity.

Factors affecting the efficacy of pressure inactivation of Escherichia coli O157:H7 on alfalfa seeds and seed viability

The application of high hydrostatic pressure technology as a seed decontamination technology was evaluated. Alfalfa seeds inoculated with approximately 10 5 CFU/g of Escherichia coli O157:H7 were subjected to oscillatory pressure treatments at 600 MPa and 20 °C for up to five cycles with a holding time of 2 min/cycle. However, oscillatory pressurization was not able to eliminate E. coli O157:H7. The application of pressure treatment at 600 MPa for 2 min at 20 °C in the presence of chemicals such as calcium hypochlorite, calcium hydroxide, lactic acid or sodium acid sulfate was subsequently investigated and it was demonstrated that this “multiple hurdle” approach was unable to decontaminate alfalfa seeds. Soaking seeds prior to pressure treatment was found to play a critical role on enhancing the pressure inactivation of E. coli O157:H7; seeds soaked in water for 60 min followed by treatment at 600 MPa for 2 min at 20 °C were decontaminated and had a germination rate of 91% which was 4% lower than that of the untreated seeds (not statistically significant, P > 0.05). It was further demonstrated that a process involving soaking seeds in water for ≥ 10 min followed by treatment at 600 MPa for 15 min at 20 °C was equally effective with respect to E. coli O157:H7 elimination and viability retention of the seeds.

Dependence of rate of germination of teak (&lt;i&gt;Tectona grandis&lt;/i&gt;) seeds on sources, periods of soaking in water and pericarp treatments

A study was conducted to determine suitable sources of teak (Tectona grandis) seeds and methods of treating the seeds to promote higher rate of germination, with the objective to supply large quantities of seedlings for developing commercial teak plantations in Ghana. The field work involved seed collection, seed pericarp treat-ments and soaking of seeds in water before sowing. Teak seeds collected from Abofour yielded higher number of germinated seeds than seeds from Jema, possibly because of higher annual rainfall at Abofour. The germinat-ion was enhanced when the seeds coat pericarps were removed, and the number of germinated teak seeds was highest when the seeds were not soaked in water. The highest percentage germination (77.7 %) among all treat-ment combinations was obtained for seeds from Abofour, without pericarp and soaking in water. Logistic model was used to determine the number of germinated teak seeds for a period of time, and the coefficient of determi-nation (R2) exceeded 96 %. Journal of Applied Science and Technology Vol. 13 (1 & 2) 2008: pp. 108-113

Film coating of seeds with Bacillus cereus RS87 spores for early plant growth enhancement

The plant growth-promoting rhizobacterium Bacillus cereus RS87 was previously reported to promote plant growth in various crops in both greenhouse and field trials. To apply as a plant growth promoting agent with practical use, it is essential to ease the burden of routine preparation of a fresh suspension of strain RS87 in laboratory. The objectives of this study were to investigate the feasibility of film-coating seeds with B. cereus RS87 spores for early plant growth enhancement and to reveal the indoleacetic acid (IAA) production released from strain RS87. The experiment consisted of the following 5 treatments: nontreated seeds, water-soaked seeds, film-coated seeds, seeds soaked with vegetative cells of strain RS87, and film-coated seeds with strain RS87 spores. Three experiments were conducted separately to assess seed emergence, root length, and plant height. Results showed that both vegetative cells and spores of strain RS87 significantly promoted (P < or = 0.05) seed emergence, root length and plant height over the control treatments. The strain RS87 also produced IAA. In conclusion, the film coating of seeds with spores of B. cereus RS87 demonstrated early plant growth enhancement as well as seeds using their vegetative cells. IAA released from strain RS87 would be one of the mechanisms for plant growth enhancement.

Effect of seed soaking with thiols on the antioxidant enzymes and photosystem activities in wheat subjected to water stress

Photosystem 1 and 2 and antioxidant enzyme activities were determined in wheat (Triticum aestivum L. cv. Sonalika) leaves. Seedlings from both control seeds and seeds soaked in solutions like dithiothreitol, thioglycollic acid and thiourea were subjected to water stress induced by polyethylene glycol. Photosystem 1 and 2 activities were less inhibited by water stress due to seed soaking with sulphydryl compounds. The changes in activities of antioxidant enzymes induced by water stress were higher in seedlings from thiol-pretreated seeds than from water-soaked seeds.

Mature embryo axis-based high frequency somatic embryogenesis and plant regeneration from multiple cultivars of barley (Hordeum vulgare L.)

A highly reproducible regeneration system through somatic embryogenesis from the excised mature embryos (MEs) of dry seeds of a range of European barley cultivars was developed. By minimizing the germination of plated MEs, primary callus could be obtained with high frequency which permitted efficient embryogenesis and regeneration of a large number of green plants. Different approaches were tested to reduce or prevent normal germination: (i) the use of a well defined balance of maltose and 2,4-D in the induction medium, (ii) soaking of seeds in water containing 2,4-D solution, (iii) direct culture of excised embryonic axes, (iv) longitudinally bisected MEs giving two halves, and (v) complete removal of the elongated main shoot including any roots within a week of culture initiation. Culturing of bisected MEs and whole embryonic axes gave the best responses with respect to large amounts of callus combined with minimal germination. The incorporation of BAP at low levels in the medium was found to be most effective for embryogenesis and the maintenance of long-term morphogenic capacity (more than 11 months up to now). This procedure allows the complete regeneration of plants in 16-20 weeks, from the initial isolation of MEs through all the steps to the development of plants ready to be transferred to the soil. The protocol was first developed for cv. Golden Promise and successfully applied to commercial cultivars. All cultivars tested formed embryogenic callus, with overall rates ranging from 22-55% and an average number of green plants per embryogenic callus from 1.5 to 7.5 across the genotypes.

Soyasapogenol A and B Distribution in Soybean (Glycine max L. Merr.) in Relation to Seed Physiology, Genetic Variability, and Growing Location

An efficient analytical method utilizing high-performance liquid chromatography (HPLC)/evaporative light scattering detector (ELSD) was developed to isolate and quantify the two major soyasaponin aglycones or precursors in soybeans, triterpene soyasapogenol A and B. Soaking of seeds in water up to 15 h did not change the content of soyasapogenols. Seed germination had no influence on soyasapogenol A content but increased the accumulation of soyasapogenol B. Soyasapogenols were mainly concentrated in the axis of the seeds as compared with the cotyledons and seed coat. In the seedling, the root (radicle) contained the highest concentration of soyasapogenol A, while the plumule had the greatest amounts of soyasapogenol B. In 10 advanced food-grade soybean cultivars grown in four locations in Ontario, total soyasapogenol content in soybeans was 2 +/- 0.3 mg/g. Soyasapogenol B content (1.5 +/- 0.27 mg/g) was 2.5-4.5-fold higher than soyasapogenol A content (0.49 +/- 0.1 mg/g). A significant variation in soyasapogenol content was observed among cultivars and growing locations. There was no significant correlation between the content of soyasapogenols and the total isoflavone aglycones.

Accelerated aging-enhanced lipid peroxidation in bitter gourd seeds and effects of priming and hot water soaking treatments

Both priming (mixing seeds with moist vermiculite for 36 h at 20 °C) and hot water soaking (soaking seeds for 4 h in water at 40 °C) improved the percentage and mean emergence time (MET) of bitter gourd seeds at sub-optimal temperature of 20 °C. The amounts of MDA and total peroxide in imbibing primed and hot water-soaked seeds were reduced and the activities of several free radical and peroxide scavenging enzymes were increased in comparison with imbibing untreated seeds. Thus, the improved emergence performance might be partly contributable to decreased lipid peroxidation during imbibition and peroxide scavenging activities. The percentage of emergence was decreased and MET was increased by accelerated aging. Accelerated aging not only resulted in increased lipid peroxidation, but also in decreased activities of several free radical and peroxide scavenging enzymes. Both percentage and speed of emergence after accelerated aging were decreased to a greater extent in primed and particularly in hot water-soaked seeds than in control seeds. Moreover, lipid peroxidation was increased to a larger extent in pretreated than in untreated seeds after accelerated aging. Therefore, as in the case of pretreated seeds, the increased peroxide accumulation and the reduced enzyme activities appeared to be linked to the reduced emergence performance of aged seeds.

Effect of various indigenous processing methods on the ?-galactoside and mono- and disaccharide content of an Indian tribal pulse,Mucuna pruriens varutilis

The effect of various treatments, such as soaking in different solutions, cooking of raw and soaked seeds, autoclaving of raw and soaked seeds, dehusking of water-soaked seeds, fermentation of water-soaked/dehusked seeds, autoclaving of fermented seeds, dry heating of raw seeds and germination, on the levels of mono- and disaccharides and α-galactosides in two varieties of an Indian tribal legume, mucuna bean (Mucuna pruriens var utilis), was investigated. The levels of raffinose, stachyose and verbascose decreased under various treatments. Among the different soaking and cooking/autoclaving treatments, tamarind pulp extract soaking and sodium bicarbonate solution soaking followed by autoclaving procedures were the most effective for removing α-galactosides (68.4–70.9 and 68.5–68.9% respectively). The lowest reduction of α-galactosides (8.4–17.2%) was observed in dry-heated samples. Germination for more than 72 h resulted in the highest reduction of total α-galactosides (93.6 and 89.6% in white and black varieties respectively). During the germination process, glucose, fructose and sucrose concentrations increased significantly. © 2001 Society of Chemical Industry

Defense proteins from seed of Cassia fistula include a lipid transfer protein homologue and a protease inhibitory plant defensin

A novel trypsin inhibitor was extracted from the seeds of Cassia fistula by a process successively involving soaking seeds in water, extraction of the seeds in methanol, and extraction of the cell wall material at high ionic strength. The protease inhibitor (PI) was subsequently purified by chromatography on carboxymethylcellulose, gel filtration and reversed phase HPLC (RP-HPLC). Electrospray ionization mass spectrometry (ESMS) of the oxidized from of the PI yielded an average molecular mass of 5458.6±0.8 Da. Edman sequencing of the PI yielded a full-length 50 amino acid sequence inferred to contain eight cysteines and with a calculated average molecular mass (fully oxidized form) of 5459.3 Da, in agreement with the observed mass. The C. fistula seed PI is homologous to the family of plant defensins (γ-thionins), which have four disulfide linkages at highly conserved locations. The C. fistula PI inhibits trypsin (IC 50 2 μM), and is the first known example of a plant defensin with protease inhibitory activity, suggesting a possible additional function for some members of this class of plant defensive proteins. C. fistula seeds also contain a 9378 Da lipid transfer protein (LTP) homologue, other LTPs, a 7117 Da protein copurifying with PI activity and a 5144 Da defensin which does not inhibit trypsin. The complete sequence of the 5144 Da defensin was determined by Edman sequencing, yielding a calculated average molecular mass (oxidized form) of 5144.1 Da, in agreement with the mass observed by ESMS. The likely trypsin inhibitory residue on the 5459 Da defensin is Lysine-25, the corresponding amino acid being Tyrosine-25 in the homologous 5144 Da defensin that is not a trypsin inhibitor.