Effects of seed storage on germination of two succulent desert halophytes with little dormancy and transient seed bank

Seeds of both Salsola imbricata and Haloxylon salicornicum have high germination level and germination speed, and form a transient seed bank in nature. The impacts of storage period and condition on germination level and speed were assessed in the two species. Storage for three months significantly increased both germination level and speed of seeds stored under the different conditions, compared to that of fresh seeds. In both species, nine months storage did not affect germination percentage in cold storage seeds, but completely inhibited it in field seeds. Storage for longer time in room and warm temperatures resulted in significant reduction or complete inhibition in the germination of the two species, so this was more pronounced in H. salicornicum. Storage significantly increased germinate rate index of seeds stored in all conditions till 17 months in S. imbricata and till 12 months in H. salicornicum. In both species, fridge storage had little effects on final germination and germination speed of seeds incubated at the different temperatures, compared to fresh seeds. However, room temperature and warm storages significantly reduced final germination and germination speed at the different temperatures, so the reduction was more pronounced at 35 °C, especially in H. salicornicum.     

Germination response and viability of an endangered tropical conifer Widdringtonia whytei seeds to temperature and light

The tropical conifer Widdringtonia whytei Rendle is an endangered species endemic to Mulanje Mountain in Malawi. A study was conducted for the first time under controlled conditions in order to assess the effects of temperature and light on germination and viability of W. whytei seeds. Seeds incubated at a constant temperature of 20 °C attained the highest cumulative germination percentage (100%) followed by 87% germination under fluctuating temperatures of 15 °C night/25 °C day. No seed germination occurred at temperatures below 15 °C. Seeds that failed to germinate at temperatures below 15 °C showed the highest (> 90%) viability compared to the seeds incubated at 25 °C (60%). Across temperature regimes, germination was significantly higher under light (44.7%) than dark (35.6%) conditions. It is concluded that temperature is one of the critical factors for germination of W. whytei seed. The ability of W. whytei seeds to germinate both in light and darkness implies that the species would unlikely form a persistent soil seed bank, an attribute which is common in species that survive in habitats frequently disturbed by fires.     

Germination responses of Croton macrostachyus (Euphorbiaceae) to various physico-chemical pretreatment conditions

Croton macrostachyus Hochst. ex Del. (Euphorbiaceae) is a multipurpose, deciduous, and medium sized tree of pantropic occurrence. Because the species has numerous useful qualities (e.g., establishment and growth in disturbed sites, drought tolerance, fast growth rate, copious litter/necromass production, suitability for agroforestry, and ability to attract avian frugivores), its speedy restoration has become increasingly critical. Germination studies were therefore conducted on seeds pooled from five widely located provenances with a view to supporting efforts geared toward the speedy propagation and restoration of this valuable tree species. Seed pretreatments were achieved using various dilution levels of plant-derived smoke–water (1:1, 1:10, 1:100 and 1:1000), as well as gibberellic acid (GA₃) or potassium nitrate (KNO₃) ranging in concentration from 0.1 to 100 μmol. The control was to use distilled water for seed pretreatment. Seeds were germinated under either illuminated (ca 60 μmol m^− 2 s^− 1; cool-white fluorescent lamp) or non-illuminated conditions. Experiments on the impact of seed storage durations, as well as storage temperatures were also conducted. The study found that germination percentage (GP: ca 90%), and mean germination time (MGT: 14 days) were significantly (P < 0.001) better when seeds were pretreated with smoke–water and germinated under non-illuminated conditions, than when these were pretreated with various concentrations of GA₃ or KNO₃ (GP and MGT of ca 65% and 20 days, respectively). Germination percentage (GP) and germination vigor (GV) declined with increasing storage-time for all storage temperatures, but GV's decline was faster for seeds stored at 22 °C than for those stored at 5 and 15 °C. On the other hand, mean germination time (MGT) increased significantly (P < 0.01) with seed storage-time of up to 8 months at 5, 15, and 22 °C, but the increase was more marked for seeds stored at 22 °C than for those stored at 5 and 15 °C. From these investigations, it is concluded that germination of C. macrostachyus seeds through use of smoke–water is faster, cheaper, and technically less demanding, compared to that of either GA₃ or KNO₃. The study also concludes that C. macrostachyus is intermediate between orthodox and recalcitrant seeds, and that it is non-photoblastic.     

Reproductive ecology of Aloe plicatilis,a fynbos tree aloe endemic to the Cape Winelands,South Africa

While the pollination ecology of many Aloe species is well-documented, knowledge on aloe seed ecology, and hence aloe reproductive ecology in its entirety is limited. The aim of this study was to investigate the reproductive ecology of Aloe plicatilis, a Cape fynbos tree aloe endemic to the Cape Winelands, South Africa. Results from a pollinator exclusion experiment conducted at an A. plicatilis population on Paarl mountain suggests pollination primarily by insects, although bird visitation significantly increased seed set/fruit indicating possible co-pollination with insects. The species' long-tubed flowers and production of concentrated nectar, with observations of malachite sunbirds as the most common avian visitors to A. plicatilis flowers indicate the importance of long-billed specialist avian nectarivores as floral visitors. Analysis of the relationship between plant size and inflorescence production for five populations combined revealed a significant, positive linear relationship between plant size and the logarithm of the number of inflorescences/plant. Natural fruit and seed set determined for three populations (1325, 27,930 and 251,616 seeds/population) suggests low reproductive output compared to several other Aloe species. The smallest (31 individuals) and least dense (75 plants/ha) A. plicatilis population produced the lowest seed set/plant (128 seeds) and per population (1325 seeds), suggesting an Allee effect. Evaluation of seed dispersal potential showed that potential dispersal distances were approximately three times the canopy height; however, the occurrence of A. plicatilis on mountains isolated from more continuous mountain ranges on which the species also occurs suggests the possibility of long-distance dispersal by strong, gusty, summer winds. Soil seed bank samples collected from 13 populations yielded close to zero seedling emergence, indicating the absence of persistent seed banks. A. plicatilis seeds stored under ambient laboratory conditions for 3, 18 and 24 months were germinated in an environmental control chamber and a laboratory. High percentage germination was recorded for 18- and 24-month-seed (86 and 80%, respectively), while germination of 3-month-old seeds was three times lower, suggesting the need for after-ripening. Germination of fresh and one-year-old seed under ambient nursery conditions at the Karoo Desert National Botanical Garden in Worcester yielded emergence percentages of 67 and 44%, respectively, and were therefore less successful than germination under more controlled conditions. This is the first known study to investigate the reproductive ecology of a tree aloe species and that of a Cape fynbos aloe. The study highlights the importance of further studies on aloe seed ecology, particularly for rare and threatened species.     

Effects of seed maturation time and dry storage on light and temperature requirements during germination in invasive Prosopis juliflora

The effects of time of seed maturation and dry seed storage and of light and temperature requirements during seed incubation on final germination percentage and germination rate were assessed for the invasive shrub Prosopis juliflora (Sw.) D.C., grown under desert environmental conditions of the United Arab Emirates (UAE). Seeds were collected from Fujira on the northern coast of the UAE at different times during the growing seasons (autumn, winter and spring) and were germinated immediately and after 8 months of dry storage under room temperature (20±3 °C). Seeds were germinated at three temperatures (15, 25 and 40 °C) in both continuous light and darkness. The results showed significant effects for time of seed collection, seed storage, light and temperature of seed incubation and many of their interactions on both germination percentage and rate. Fresh seeds matured during autumn and winter germinated significantly greater at 40 °C and in light than at lower temperatures and in dark. Storage significantly increased germination percentage and rate; the increase was greater for seeds matured during winter than for seeds matured during spring. This indicates that dormancy breakage was greater in seeds of winter than seeds of spring. The need for high temperature to achieve greater germination was significantly reduced after seed storage, especially for seeds matured in autumn and winter.     

Ecophysiological requirements on seed germination of a Mediterranean perennial grass (Stipa tenacissima L.) under controlled temperatures and water stress

Stipa tenacissima L. (alpha grass) steppes are one of the most representative ecosystems in arid Mediterranean ecosystems. On the one hand these steppes, which are perpetually exposed to climate and strong anthropogenic pressure, have undergone severe degradation. On the other hand, the ability of S. tenacissima to regenerate naturally is significantly reduced. In this study the germination response and seedling emergence of S. tenacissima are examined in relation to the main environmental factors (water stress and temperature) under laboratory-controlled conditions. The main aim of this paper was to investigate the influence of temperature over a temperature range (10 °C–30 °C) and water stress induced by the solutions of polyethylene glycol (PEG)-6000 (0 to − 1.6 MPa) for a period of 30 days, on the germination behavior of S. tenacissima seeds. The results showed that temperatures between 10° and 20 °C seem to be favorable for the germination of this species, with optimum temperatures among accessions found in 20 °C. When seeds were water-stressed, germination severely decreased at − 0.8 MPa, indicating that the accession resistance limits to the water stress, and was completely inhibited at − 1.6 MPa. Consequently, the final germination percentage (FGP) decreased and the mean time germination (MTG) increased. Based on the empirical data of the germination rate, we estimated that the parameters of the thermal time and hydrotime models showed different values in all accessions which proves the difference between accession adaptive capacities.     

Seed viability of five wild Saudi Arabian species by germination and X-ray tests

Our objective was to evaluate the usefulness of the germination vs. the X-ray test in determining the initial viability of seeds of five wild species (Moringa peregrina, Abrus precatorius, Arthrocnemum macrostachyum, Acacia ehrenbergiana and Acacia tortilis) from Saudi Arabia. Usually several days were required to determine the viability of all five species via germination tests. However, X-ray test will give immediate results on filled/viable seeds. Seeds of all species, except Acacia ehrenbergiana and Acacia tortilis showed high viability in both germination (96–72% at 25/15 °C, 94–70% at 35/25 °C) and X-ray (100–80%) test. Furthermore, there was a general agreement between the germination (19%, 14% at 25/15 °C and 17% and 12% at 35/25 °C) and X-ray (8%, 4%) tests in which seed viability of Acacia ehrenbergiana and Acacia tortilis was very low due to insect damaged embryo as shown in X-ray analysis. Seeds of Abruspreca torius have physical dormancy, which was broken by scarification in concentrated sulfuric acid (10 min), and they exhibited high viability in both the germination (83% at 25/15 °C and 81% at 35/25 °C) and X-ray (96%) tests. Most of the nongerminated seeds of the five species except those of Acacia ehrenbergiana and Acacia tortilis, were alive as judged by the tetrazolium test (TZ). Thus, for the five species examined, the X-ray test was proved to be a good and rapid predictor of seed viability.     

Determination of seed viability of eight wild Saudi Arabian species by germination and X-ray tests

Our purpose was to evaluate the usefulness of the germination vs. the X-ray test in determining the initial viability of seeds of eight wild species (Salvia spinosa, Salvia aegyptiaca, Ochradenus baccatus, Ochradenus arabicus, Suaeda aegyptiaca, Suaeda vermiculata, Prosopisfarcta and Panicumturgidum) from Saudi Arabia. Several days were required to determine viability of all eight species via germination tests, while immediate results on filled/viable seeds were obtained with the X-ray test. Seeds of all the species, except Sa.aegyptiaca, showed high viability in both the germination (98–70% at 25/15 °C, 93–66% at 35/25 °C) and X-ray (100–75%) test. Furthermore, there was general agreement between the germination (10% at 25/15 °C and 8% at 35/25 °C) and X-ray (5%) tests that seed viability of Sa.aegyptiaca was very low, and X-ray analysis revealed that this was due to poor embryo development. Seeds of P.farcta have physical dormancy, which was broken by scarification in concentrated sulfuric acid (10 min), and they exhibited high viability in both the germination (98% at 25/15 °C and 93% at 35/25 °C) and X-ray (98%) test. Most of the nongerminated seeds of the eight species except those of Sa.aegyptiaca were alive as judged by the tetrazolium test (TZ). Thus, for the eight species examined, the X-ray test was a good and rapid predictor of seed viability.     

Effects of sample handling,processing, storage,and hemolysis on measurements of key energy metabolites in ovine blood

Four experiments were conducted using mature Suffolk ewes to evaluate the effects of blood sample handling, processing and storage on measurements of the energy metabolites, β-hydroxybutyrate, total cholesterol, glucose, l-lactate, nonesterified fatty acid (NEFA), and triacylglycerol. In experiment 1 the effects of anticoagulants on metabolite measurements and packed-cell volume (PCV) were evaluated. Blood samples (n=12) were collected into one of four evacuated blood-collecting tubes: no anticoagulant (SER; yields serum), and plasma-yielding sodium heparin (HEP), sodium fluoride and potassium oxalate (NAF), and tripotassium ethylenediaminetetraacetic acid (K₃EDTA). Anticoagulant affected (P<0.05) metabolite values, with total cholesterol, triacylglycerol, and l-lactate highest in SER, and glucose highest in HEP; NEFA and β-hydroxybutyrate measurements were not affected (P>0.10) by anticoagulant. In addition, among the plasma-yielding tubes, PCV was highest in HEP and lowest in NAF (P<0.01). Experiment 2 investigated the effects of repetitive freezing-thawing cycles of plasma on metabolite levels. Blood samples (n=20) were collected using NAF tubes, and plasma was stored at −20 °C and thawed in a water bath (21 °C) 0, 1, 2, and 3 times within 18 h after collection. Compared with fresh samples (Thaw 0), by thaw 2, l-lactate increased (P<0.01) 5%, and glucose and total cholesterol decreased (P<0.001) 13 and 4%, respectively. Plasma NEFA increased 10% (P<0.01) between thaws 0 and 1, but returned to fresh levels (Thaw 0) with subsequent freeze-thaw cycles (P<0.05). Plasma β-hydroxybutyrate decreased (P<0.01) between thaws 0 and 1, but there was no further decline with subsequent freeze-thaw cycles (P<0.05). Experiment 3 evaluated the effects of plasma storage temperature (−20 °C versus −80 °C) and length (0–180 days) on metabolite levels in blood samples (n=12) collected in NAF tubes. All plasma metabolites were affected by storage length (day effect, P<0.01) but only total cholesterol values were affected by storage temperature, with values being higher in plasma stored at −20 than −80 °C (P<0.01). Glucose measurements were higher in samples stored at −20 °C for up to 30 days, but were higher thereafter in samples stored at −80 °C (storage length × temperature effect, P<0.01). Experiment 4 evaluated the effects of anticoagulant (SER versus NAF) and whole blood storage (4 °C) for 0, 1, 3, and 7 days on metabolite concentrations. Glucose was higher (P<0.0001) in NAF, possibly the result of the presence of the glycolytic inhibitor, sodium fluoride, whereas l-lactate, NEFA, total cholesterol and β-hydroxybutyrate were higher in SER (P<0.01). Total cholesterol, l-lactate, and NEFA increased, whereas β-hydroxybutyrate decreased with days in storage (P<0.01). Development of hemolysis in the samples artifactually elevated glucose and NEFA values by as much as 41 and 230%, respectively. Attention to proper blood handling, processing, and storage procedures, and avoidance of hemolysis are important in blood clinical analyses and in the proper interpretation of experimental results.     

Temperature requirements for dormancy break and seed germination vary greatly among 14 wetland Carex species

We evaluated dormancy loss in seeds of 14 Carex species (C. atherodes, C. brevior, C. comosa, C. cristatella, C. cryptolepis, C. granularis, C. hystericina, C. lacustris, C. pellita, C. scoparia, C. stipata, C. stricta, C. utriculata, C. vulpinoidea) under growing season and stratification conditions and determined the temperature requirements for germination. Seeds were germinated for 1 year at a diel temperature regime (5/1 °C, 14/1 °C, 22/8 °C, or 27/15 °C) or a seasonal regime (seeds moved among the four diel regimes to mimic seasonal temperatures). All species had conditionally dormant seeds at maturity. The optimal temperature for germination of most species was 27/15 °C. The 14 species were grouped by their seed viability, dormancy, and germination with a Seed Regeneration Index (SRI; range 0–1) using the results of this study and a previously published paper on stratification effects on Carex seed dormancy and germination. The eight species that had an SRI value >0.5 (C. brevior, C. comosa, C. cristatella, C. cryptolepis, C. hystericina, C. scoparia, C. stipata, C. vulpinoidea) had high seed viability (>60%) and required little to no stratification to germinate readily over a broad range of temperatures. The six species with an SRI value <0.5 (C. atherodes, C. granularis, C. lacustris, C. pellita, C. stricta, C. utriculata) generally had low seed viability (<50% and often <1%) and required stratification or particular temperatures (35/30 °C or 5/1 °C for C. stricta; 35/30 °C for C. utriculata; 27/15 °C for C. atherodes, C. lacustris, C. pellita; 5/1 °C for C. granularis) for germination ≥50%. These six species will require more attention from restoration practitioners to ensure that there are sufficient viable seeds to meet revegetation goals, that dormancy break is achieved, and that seeds are sown when temperatures are optimal for germination. The different seed germination syndromes that we found for these Carex species likely contribute to variable seed bank formation and emergence patterns, and species coexistence.     

Stability of the intra- and extracellular toxins of Prymnesium parvum using a microalgal bioassay

Prymnesium parvum produces a variety of toxic compounds, which affect other algae, grazers and organisms at higher trophic levels. Here we provide the method for development of a sensitive algal bioassay using a microalgal target, Teleaulax acuta, to measure strain variability in P. parvum toxicity, as well as the temporal stability of both the intracellular and the extracellular lytic compounds of P. parvum. We show high strain variation in toxicities after 3 h incubation with LC₅₀s ranging from 24 to 223 × 10³ cells ml⁻¹. Most importantly we prove the necessity of testing physico-chemical properties of P. parvum toxins before attempting to isolate and characterize them. The extracellular toxin in the supernatant is highly unstable, and it loses significant lytic effects after 3 days despite storage at −20 °C and after only 24 h stored at 4 °C. However, when stored at −80 °C, lytic activity is more easily maintained. Reducing oxidation by storing the supernatant with no headspace in the vials significantly slowed loss of activity when stored at 4 °C. We show that the lytic activity of the intracellular toxins, when released by sonication, is not as high as the extracellular toxins, however the stability of the intracellular toxins when kept as a cell pellet at −20 °C is excellent, which proves this is a sufficient storage method for less than 3 months. Our results provide an ecologically appropriate algal bioassay to quantify lytic activity of P. parvum toxins and we have advanced our knowledge of how to handle and store the toxins from P. parvum so as to maintain biologically relevant toxicity.     

Germination characteristics of the grass weed Digitaria nuda (Schumach.)

The effect of various pre-treatments and their interaction with temperature on cumulative percentage and the rate of germination were evaluated for Digitaria nuda. Stored and fresh seeds were pre-treated with either 0.02 M KNO₃, soaked in water for 24 h (priming), sterilized with 0.5% NaOCl or heat treated at 60 °C. Seeds were germinated at constant temperatures of 25 and 30 °C and fluctuating temperature regimes of 25/10 and 30/15 °C. The effect of pre-chilling on germination of stored and fresh seed was evaluated at 30/15 °C, and seed emergence in two soil types at different burial depths (0, 0.5, 1, 2, 3, 4, 5 and 6 cm) was also determined. The pre-treatment of stored seed with KNO₃ resulted in the highest germination percentage (100%), whereas the pre-treatment of fresh seed with water for 24 h gave the best germination (99%), at constant temperatures of 25 and 30 °C. Pre-chilling of seed increased germination by more than 30%. Emergence from clay loam soil was greater compared with the emergence from sandy loam soil. Total seedling emergence decreased exponentially with increasing burial depths with only 5% of seed germinating from a burial depth of 6 cm. Results from this study showed that germination requirements are species specific and knowledge of factors influencing germination and emergence of grass weed seed can assist in predicting flushes in emergence allowing producers to implement control practices more effectively.     

Cold storage of the egg parasitoids Trissolcus basalis (Wollaston) and Telenomus podisi Ashmead (Hymenoptera: Scelionidae)

Adults of Trissolcus basalis and Telenomus podisi were stored either at 15 or 18 °C after their immature development had been completed at 18 or 25 °C. Longevity of the parasitoids in the storage temperatures was evaluated, as well as fecundity and longevity following their return to 25 °C after different periods in reproductive diapause. Temperature during immature development influenced female longevity and highest mean longevity was obtained for females that developed to the adult stage at 25 °C and then were stored at 15 °C (ca. 13 months for T. basalis and 10 months for Te. podisi). For adults of T. basalis that developed at 25 °C, storage periods of 120 or 180 days at 15 or 18 °C did not affect fecundity. The fecundity of T. basalis females that developed at 18 °C and were stored for 120 days at 15 or 18 °C was not affected; however, after remaining for 180 days, fecundity was reduced in ca. 30 and 50%, respectively. Storage of Te. podisi adults at 15 or 18 °C significantly reduced fecundity. It is concluded that adults of T. basalis can be stored in the adult stage at 15 or 18 °C between two soybean crop seasons for mass production purposes, aiming the biological control of stink bugs.     

Effect of cryopreservation and microencapsulation of lactic acid bacterium Enterococcus faecium MC13 for long-term storage

The aim of this work was to investigate the effect of cryoprotectants on the survival of probiotic bacterium Enterococcus faecium MC13 during freeze drying and storage. The maximum relative cell viabilities were observed when cells were freeze dried and stored at −20 °C, which is optimum temperature for the preservation of E. faecium. At all storage temperatures, trehalose was found to be retaining the highest relative cell viability than other cryoprotectants. In addition, alginate–chitosan capsules were produced to encapsulate E. faecium with the aim of enhancing survival of probiotic cells and keeping the probiotic during exposure to the harsh gastro-intestinal conditions. Encapsulation of probiotic into alginate–chitosan capsules found to be retaining higher survival of probiotic cells (4.342 ± 0.26 Log CFU mL⁻¹) at −20 °C for six months. Microencapsulated cells were resistant to simulated gastric (pH 2.0) and intestinal fluids (pH 7.5), resulting in significantly enhanced survival when compared with free cells. During in vivo treatment, capsules were broken and probiotic cells were directly released into the intestinal tract of rat. This result showed that microencapsulation of E. faecium MC13 with alginate and a chitosan coating offers an effective means of delivery of viable cells to the colon and maintains their survival during the adverse gastro-intestinal conditions.     

Temperature requirements for seed germination of Pereskia aculeata and Pereskia grandifolia

Pereskia aculeata and Pereskia grandifolia have been studied widely due to their high nutritional and therapeutic values. However, little is known about the biological requirements of their seeds for the various germination factors. Thus, this experiment aimed to evaluate the thermal effects on the germination of these species at the temperatures of 24 °C, 27 °C, 30 °C, 33 °C and 36 °C. After verification of the existence of differences in the performance of germination, a non-linear regression was carried out, relating the germination to temperature and identifying its point of maximum efficiency. We found that the lowest synchronization indexes of germination were observed close to 30 °C. The best germination response of the P. aculeata and P. grandifolia was observed at 30 °C and 33 °C, respectively, with greater germination strength and fewer days to attain 63.21% of germinations. The results obtained from the germination of P. aculeata and P. grandifolia can be described by the Weindull distribution model with three parameters, as proposed by Carneiro and Guedes (1992).     

Quality assessment of Riptortus pedestris (Hemiptera: Alydidae) eggs cold-stored at different temperature and relative humidity regime

Supplementation of host resource can be more economical method for the biological control of insect pest compared to direct release of adult parasitoids. Periodical release of non-viable cold-stored eggs of Riptortus pedestris (Fabricius) (Hemiptera: Alydidae) has been found to enhance parasitism of this pest in soybean fields. To find the optimum environmental conditions for cold storage of these host eggs, we evaluated nine different combinations of temperature (2, 6, and 10 °C) and relative humidity (high 90–95%, medium 70–75%, and low 30–35%). After 30 d of cold-storage, eggs were weighed and held at 26.6 °C and 75% relative humidity for 8 d before testing. To test the eggs’ suitability as hosts following cold storage, females of Ooencyrtus nezarae Ishii (Hymenoptera: Encyrtidae) were released individually onto batches of eggs, and parasitization rates and the development, emergence, sex ratio, adult longevity, and size of parasitoid progeny were examined. Eggs stored at high relative humidity showed less weight loss than those stored at low relative humidity. The number of eggs parasitized was highest (5.9/15) on eggs stored at 6 °C and high relative humidity. Developmental times and adult emergence were optimal on host eggs stored at 2 °C and high relative humidity. A significantly lower proportion of eggs produced male parasitoids when eggs were stored at 2 or 6 °C. Adult longevity was not affected by egg storage conditions, but adult size of progeny decreased in eggs stored at 10 °C. In conclusion, eggs of R. pedestris stored below 6 °C and with a high relative humidity maintained the best quality for parasitization by O. nezarae.     

First results from conservation studies of chlorophyllous spores of the Royal fern (Osmunda regalis, Osmundaceae)

Pteridophytes spore banks are a promising ex situ conservation tool used to increase the chances of survival of ferns, in fact that large quantities of germplasm with high genetic variation can be conserved in a small space with low economic and technical costs. However, methods to maintain the viability of chlorophyllous spores during storage are less understood.The aim of this study was to investigate the influence of long term storage on the viability of Royal Fern spores, which were stored under different conditions derived from various combinations of temperature and degrees of hydration. Survival and germination tests were performed after 1 and 28 months of storage. Our results showed the highest survival percentages for spores stored under Normal humidity at subzero temperatures (T = ? ?20 °C). These spores received no pre-treatment, dehydration, or cryoprotectants, which resulted in fast germination and gametophyte development which seemed to be stimulated by low temperatures.     

Evaluation of Tris–citric acid,skim milk and sodium citrate extenders for liquid storage of Punjab Urial (Ovis vignei punjabiensis) spermatozoa

The Punjab Urial (Ovis vignei punjabiensis) is an endangered subspecie of ovidae, distributed as small scattered populations in the forest belt of the Himalayan foothills of Pakistan and in the areas enclosed by the Indus and the Jhelum rivers. The present study was conducted to evaluate the liquid storage of Punjab Urial spermatozoa in different extenders for use in future in situ conservation activities. Semen was collected by electro-ejaculation from three captive Punjab Urial rams. Suitable ejaculates of individual animals were pooled and divided into three aliquots for dilution with the experimental extenders (Tris–citric acid, skim milk and sodium citrate) at 37 °C. Extended semen was cooled from 37 °C to 5 °C in 2 h, and stored for three days at 5 °C. Sperm motility (%), viability (%; live/dead), acrosome integrity (%) and plasma membrane integrity (%) were assessed on days 1, 2 and 3 of storage. On day 1, sperm motility, viability as well as acrosome and plasma membrane integrity were similar (p > 0.05) in all three experimental extenders. On day 2, sperm motility, viability, acrosome and plasma membrane integrity were higher (p < 0.05) in Tris–citric acid extender compared to sodium citrate based extender. On day 3 of storage, the values of motility, viability and acrosome integrity were higher (p < 0.05) in Tris–citric acid extender than in skim milk and sodium citrate based extenders. In conclusion, Tris–citric acid extender appears to be a better option compared with skim milk and sodium citrate extenders for liquid storage of Punjab Urial semen.     

Effect of initial freezing temperature on the semen characteristics of frozen-thawed ram spermatozoa in a semi-arid tropical environment

Ram spermatozoa are sensitive to extreme changes in temperature during the freeze-thaw process. The degree of damage depends on a combined effect of various factors including initial freezing temperature. The present study was conducted to observe the effect of initial freezing temperature on post-thawing motility of ram spermatozoa of native and crossbred rams maintained in a semi-arid tropical environment. Good quality semen obtained from native Malpura and crossbred Bharat Merino rams were pooled within breed and diluted at a rate of 1000 million spermatozoa per milliliter in TEST—yolk–glycerol extender. Diluted semen samples were loaded in 0.25 ml straws and cooled to −25, −75 or −125 °C freezing temperature at the rate of −25 °C/min under controlled conditions before plunging into liquid nitrogen for storage. The thawing of straws was performed at 50 °C in a water bath for 10 s and motility characteristics of the frozen-thawed spermatozoa were assessed by a computer-assisted spermatozoa analysis technique. Initial freezing temperature significantly affected the post-thawing motility of sperm in both the breeds. The post-thawing % motility and rapid motile spermatozoa were significantly higher at initial freezing temperature of −125 °C and lower at −25 or −75 °C. The percentage medium motile sperm were similar at all three initial freezing temperatures. The percentage of slow motile and linearity of sperm varied (P<0.01) between the different freezing temperatures. The curvilinear velocity, average path velocity and straight line velocity of spermatozoa were higher (P<0.01) at −125 °C than −25 or −75 °C. Although the lateral head displacement of spermatozoa did not vary significantly between the different initial freezing temperatures, the stroke frequency was significantly lower at −25 °C than −75 or −125 °C. Except for % linearity, the average path velocity and straight line velocity, other spermatozoa characteristics were not significantly different between breeds. The interaction between freezing temperature and breed was significant only for the % motility and linearity of the spermatozoa. The study indicates that initial freezing temperature has a significant effect on spermatozoa motility and velocity following post-thawing. The best motile spermatozoa following thawing were achieved at −125 °C freezing temperature.     

Performance of diapausing parasitoid wasps,Habrobracon hebetor,after cold storage

The ectoparasitoid Habrobracon hebetor (Say) (Hymenoptera: Braconidae) is an important potential biological control agent for lepidopterous pests of stored products. We investigated the effects of long-term cold storage of diapausing and nondiapausing H. hebetor on their performance after cold storage. Mortality during storage increased with increasing storage duration, and the mortality of diapausing females was lower than that of nondiapausing females after 8, 12, and 16 weeks of storage. Longevity, egg laying, number of progeny produced, and time to 50% egg laying were all reduced, as compared with the culture females when parasitoids were reared at conditions that do not induce diapause. But, for females reared at 20 °C at conditions that induce diapause, all of these quality parameters did not differ from those of culture insects when the storage duration was 8 weeks or less. The percentage of female F1 offspring was always lower for cold stored insects than for the culture insects. Presence of a male after cold storage did not impact any of the quality parameters measured. Thus, rearing parasitoids at 20 °C and 10L:14D and then storing them for up to 8 weeks at 5 °C would produce parasitoids that are similar to culture parasitoids, except that the percentage of females is lower than that in the cultures (36% vs. 52%).