Cold Pretreatment Research Articles

Cold hardening modulates K+ homeostasis in the brain of Drosophila melanogaster during chill coma

Environmental temperature is one of the most important abiotic factors affecting insect behaviour; virtually all physiological processes, including those which regulate nervous system function, are affected. At both low and high temperature extremes insects enter a coma during which individuals do not display behaviour and are unresponsive to stimulation. We investigated neurophysiological correlates of chill and hyperthermic coma in Drosophila melanogaster. Coma resulting from anoxia causes a profound loss of K+ homeostasis characterized by a surge in extracellular K+ concentration ([K+]o) in the brain. We recorded [K+]o in the brain during exposure to both low and high temperatures and observed a similar surge in [K+]o which recovered to baseline concentrations following return to room temperature. We also found that rapid cold hardening (RCH) using a cold pretreatment (4°C for 2h; 2h recovery at room temperature) increased the peak brain [K+]o reached during a subsequent chill coma and increased the rates of accumulation and clearance of [K+]o. We conclude that RCH preserves K+ homeostasis in the fly brain during exposure to cold by reducing the temperature sensitivity of the rates of homeostatic processes.

Development of cultured microspores of maize in the presence of n-butanol and 2-aminoethanol

The aim of the present study was to examine whether the induction of maize microspore embryogenesis could be triggered by the application of biogenic alcohols, as was reported earlier in wheat. A single cross hybrid (A 18) raised in the phytotron was used as anther donor for shed microspore cultures after cold pretreatment. At the onset of culturing, anthers in liquid YP medium were treated with 0.2 or 0.4% n-butanol or with 2 mM aminoethanol (2-AE) for 6 or 18 hours.The treatments caused a drastic (approx. 50%) decrease in the viability of the microspores. After a few days of culture in medium containing neither n-butanol nor 2-AE, 9-13% of the microspores remained alive and capable of switching to the sporophytic pathway of development.Treatment with 0.2% n-butanol for 6 h considerably increased the frequency of symmetric nuclear divisions (more than 3×) and of induced microspores (2×). The embryo yield was also elevated by 10%. The results showed that n-butanol could be used to improve the androgenic response and microspore embryogenesis in maize, but not as efficiently as in wheat. Further examination will be required to find the reasons for the different behaviour of microspores of the two species.

The NF-κB-like factor DIF could explain some positive effects of a mild stress on longevity, behavioral aging, and resistance to strong stresses in Drosophila melanogaster

A mild cold stress can have positive effects on longevity, aging and resistance to severe stresses in flies (heat, cold, fungal infection), but the causes of these effects remain elusive. In order to know whether these effects could be explained by the DIF transcription factor (a NF-κB-like factor in the Toll innate immunity pathway), the Dif ( 1 ) mutant and its control cn bw strain were subjected to a pretreatment by cold. The DIF factor seems to be involved in the response to fungal infection after a mild cold stress and in the resistance to heat. However, DIF seems to have no role in the increased longevity of non-infected flies and resistance to a severe cold shock, because the cold pretreatment slightly increased longevity in females, mainly in Dif ( 1 ) ones, and resistance to a long cold shock in both sexes of these strains.

Effect of different pretreatment on callus formation from anther in balsam pear (Momordica charantia L.)

The effect of different pretreatment on callus induction from anther in three cultivars of balsam pear (Momordica charantia L.) was investigated. The results showed that cold pretreatment was the best for anther callus induction, and followed by heat shock, while centrifugation had no significant effect on callus induction from anther. Cold pretreatment for 24 h proved to be optimal as it significantly increased the callus induction rate, which varied with cultivars, reaching the maximum of 44.00, 51.10 and 72.67% for cv. Lvcui, Cuifei and Bixiu, respectively. Key words: Balsam pear (Momordica charantia L.), anther culture, callus induction, pretreatment.

Proteomic and biochemical analysis of maize anthers after cold pretreatment and induction of androgenesis reveals an important role of anti-oxidative enzymes

In stress conditions, microspores and young pollen grains can be switched from their normal pollen development toward an embryogenic pathway via a process called androgenesis. Androgenic embryos can produce completely homozygous, haploid or double-haploid plants. This study aimed to investigate changes in the abundance of protein species during cold pretreatment and subsequent cultivation of maize anthers on induction media using gel-based proteomics. Proteins upregulated on the third day of anther induction were identified and discussed here. Simultaneous microscopic observations revealed that the first division occurred in microspores within this period. Using 2-D electrophoresis combined with MALDI TOF/TOF MS/MS analysis 19 unique proteins were identified and classified into 8 functional groups. Proteins closely associated with metabolism, protein synthesis and cell structure were the most abundant ones. Importantly, ascorbate peroxidase, an enzyme decomposing hydrogen peroxide, was also upregulated. Isozyme analysis of peroxidases validated the proteomic data and showed increased peroxidase activities during androgenic induction. Further, the isozyme pattern of SOD revealed increased activity of the MnSOD, which could provide hydrogen peroxide as a substrate for in vivo peroxidase reactions (including ascorbate peroxidase). Together, these data reveal the role of enzymes controlling oxidative stress during induction of maize androgenesis.

Embryogenic callus induction and regeneration in anther culture of noug (Guizotia abyssinica (L.F) Cass.)

Two released noug varieties, Shambu and Fogera, were used for this experiment. Initially, two experiments were conducted to identify the appropriate morphological stage of capitula (buds) for harvesting maximum number of responsive anthers and to study the optimum cold pretreatment duration for callus induction from the anthers. Three culture media, MS, B 5 and NN, were employed for callus induction. MS medium, with nine different combinations of KN and IAA, was used for shoot regeneration study. Three different concentrations of IBA and growth regulator-free MS medium were used for the rooting experiment. The results showed that capitula, fully or slightly covered by sepal having whitish-green or greenish-yellow anthers, were the optimum stages of harvesting capitula; callus was efficiently induced when the capitula were pre-treated at 4 ° C for 24 hours. The types of calli induced significantly (P≤0.05) varied among the three media. Embryogenic calli were mainly produced in NN and B 5 media, while varying in texture and colour. High percentage of embryogenic calli (80%) was induced on NN medium from the variety Shambu, followed by b 5 medium showing 40% inductions for both varieties. Among nine different KN and IAA combinations, 2 mg/l KN combined with 1 mg/l IAA, was the most efficient for shoot regeneration. It was also found that 0.5 mg/l IBA was optimum for root induction. In the growth room as well as in the glasshouse, the survival rate of regenerants was generally better for the variety Shambu than for Fogera.

Cold pre-treatment is effective for 1-MCP efficacy in ‘Tsugaru’ apple fruit

1-Methylcyclopropene (1-MCP) treatment maintains apple fruit quality during storage, but its efficacy is dependent on a number of conditions. ‘Tsugaru’ apples are a major early season cultivar in Japan, but because ‘Tsugaru’ fruit produce abundant ethylene, they have a short shelf-life, and efficacy of 1-MCP is not as high with ‘Tsugaru’ as with other cultivars. To improve 1-MCP efficacy, ‘Tsugaru’ fruit were pre-cooled at −1 °C or −3 °C for 24 h before 1-MCP treatment. Ethylene production decreased with the cold treatment, resulting in better storage after 1-MCP treatment. Although ethylene production was low at the end of 24 h of the cold pre-treatment, expression of ACS1, the ethylene receptor genes ERS1, ETR1(a), ETR1b, ETR2 and ETR5, and the cell wall degradation-related gene PG1 all increased with a 24 h cold treatment. It is assumed that these elevated gene expression levels were not caused by ethylene, but more directly by cold stimulus. Thus, a short period of cold stimulus suppresses ethylene production, but induces expression of some genes. 1-MCP treatment was more effective with some initial fruit chilling.

Increased chilling tolerance and altered protein patterns in tomato seedlings following hardening of seeds or system in application

Plants have evolved complex mechanisms to perceive environmental cues and develop appropriate and coordinated responses to abiotic and biotic stresses. Considerable progress has been made towards a better understanding of the molecular mechanisms of plant response to a single stress. However, the existence of cross-tolerance to different stressors has proved to have great relevance in the control and regulation of organismal adaptation. In this study, we showed that cold pre-treatment (at 15, 10, 5 and 0 °C) enhanced chilling stress in two cultivars of tomato plants (Lycopersicon esculentum cv. ‘Tres Cantos’ and ‘Roma’), as indicated by de novo synthesis of stress proteins in the cold-treated plants than the control ones. The cold-treated ‘Roma’ seedlings were more resistant to chilling stress than ‘Cantos’. Active synthesis of 43 kDa heat shock proteins and some low molecular weight proteins took place after chilling stress in both cultivars. Seeds pre-treatment with systemin at three concentrations (10−6, 10−2 and 100 μM) or spraying the seedlings of two plants with the same concentrations leads to stressful polypeptides were detected as 16 kDa which is one from low molecular weight proteins were referred as vegetative storage proteins (VSPs). In ‘Tres Cantos’, the spraying of plants was more effective than the pre-soaking, while in ‘Roma’, the pre-soaking with systemin is more effective at low and moderate concentrations (10−6 and 10−2 μM), meanwhile, the spraying with systemin is more effective at high concentration (100 μM).

Non-deep complex morphophysiological dormancy in seeds of the Iberian Peninsula endemic geophyteMerendera montana(Colchicaceae)

AbstractHeretofore, no detailed account was available on seed dormancy and germination of a member of the Colchicaceae (Liliales). Thus, the primary aim of this study was to do an in-depth investigation of the temperature requirements for dormancy break and germination in seeds ofMerendera montana(Colchicaceae) at the embryo and whole-seed levels under near-natural temperatures in a non-heated frame shade-house and under controlled conditions in the laboratory. Mean embryo length in fresh seeds wasc. 0.57 mm and embryos had to grow to at least 1.30 mm before radicle emergence. Embryos grew to full size and seeds completed germination (radicles emerged) when they were stratified at 28/14°C for 60 d followed by a cool temperature for 60 d and then incubated at a cool temperature for 30 d. The optimum cool stratification temperature for dormancy-break was 10°C. Thus, after the moist pretreatment at 28/14°C+10°C, radicle emergence was>93% at all incubation temperatures (5, 15/4 and 20/7°C). In its natural habitat,M. montanaseeds are dispersed in June, the embryo elongates to full size in autumn and radicles emerge from early November to early February. Although the shoot does not emerge until March and April, it is not physiologically dormant. The shoot emerged from 80% of the radicle-emerged seeds in 13 d at 20/7°C without a previous cold pretreatment. Seeds ofM. montanahave non-deep complex morphophysiological dormancy, C1b1aB-C1a. This is the first study on seeds with complex MPD to show a delay in shoot emergence following root emergence despite the shoot being physiologically non-dormant.

Physiology, morphology and phenology of seed dormancy break and germination in the endemic Iberian species Narcissus hispanicus (Amaryllidaceae)

Only very few studies have been carried out on seed dormancy/germination in the large monocot genus Narcissus. A primary aim of this study was to determine the kind of seed dormancy in Narcissus hispanicus and relate the dormancy breaking and germination requirements to the field situation. Embryo growth, radicle emergence and shoot growth were studied by subjecting seeds with and without an emerged radicle to different periods of warm, cold or warm plus cold in natural temperatures outdoors and under controlled laboratory conditions. Mean embryo length in fresh seeds was approx. 1·31 mm, and embryos had to grow to 2·21 mm before radicle emergence. Embryos grew to full size and seeds germinated (radicles emerged) when they were warm stratified for 90 d and then incubated at cool temperatures for 30 d. However, the embryos grew only a little and no seeds germinated when they were incubated at 9/5, 10 or 15/4 °C for 30 d following a moist cold pre-treatment at 5, 9/5 or 10 °C. In the natural habitat of N. hispanicus, seeds are dispersed in late May, the embryo elongates in autumn and radicles emerge (seeds germinate) in early November; however, if the seeds are exposed to low temperatures before embryo growth is completed, they re-enter dormancy (secondary dormancy). The shoot does not emerge until March, after germinated seeds are cold stratified in winter. Seeds of N. hispanicus have deep simple epicotyl morphophysiological dormancy (MPD), with the dormancy formula C(1b)B(root) - C(3)(epicotyl). This is the first study on seeds with simple MPD to show that embryos in advanced stages of growth can re-enter dormancy (secondary dormancy).

Effect of combined cold pretreatment and heat shock on microspore cultures in broccoli

With 1 figure and 3 tables AbstractApplied stress is often used to induce embryogenesis in microspore culture. Cold pretreatment has been used in cereal crops, Brassica napus and Brassica rapa microspore cultures, but seldom attempted in Brassica oleracea microspore culture. In this study, the effect of cold pretreatment (4°C) on embryogenesis in microspore culture and ploidy of regenerants resulting from those cultures was investigated in three broccoli cultivars ‘Green Elite’, ‘Green Star’ and ‘TI‐111’. Cold pretreatment for 1, 2 or 3 days was effective when applied alone using these three cultivars, but the frequency of embryos derived from microspores was low. The combination of cold pretreatment (4°C) for 1 or 2 days and heat shock (32.5°C) for 1 day significantly enhanced microspore embryogenesis efficiency, especially with ‘TI‐111’ for which it was increased by 63–72‐fold. In addition, the combined treatment resulted in higher diploid frequency of the regenerated population compared to traditional microspore culture protocol which typical only uses heat shock at 32.5°C for 1 day in broccoli.

Androgenic potential of different tomato genotypes induced by colchicine and cold pretreatments

The androgenic potential of four tomato lines (Micro-Tina, Micro-Gemma, Moneymaker and MsK8 as parents) and their hybrids were studied through colchicine (250 mg/l) and cold (4°C for 72 h) pretreatments of isolated anthers. The pretreated anthers were cultured on induction medium. Statistical analysis showed that there was significant differences between genotypes for the studied traits. The lines MsK8 and Micro-Gemma had a considerably higher frequency of callus formation (CF) and shoot regeneration (SR) as well as greater callus diameters (CD) than that of other parents. The highest frequency of CF and SR belonged to hybrid MsK8 × Micro-Tina, and the longest CD possessed by Micro-Tina × Micro-Gemma. The mean values for hybrid’s CF, CD, and SR were significantly higher than those of the parents. In total, heterosis (hybrid vigor) was observed for the studied traits and heterozygous genotypes had a higher frequency of androgenic potential compared with homozygous parents.

Effects of colchicine and cold duration pretreatments on androgenesis responses of tomato (Lycopersicon esculentum Mill) via anther culture

The aim of the present study was to optimize the in vitro chromosome-doubling procedure by colchicine pretreatment in tomato anther culture. Anthers of 1.7–2.0 mm long were isolated at prophase-metaphase I and pretreated by different concentrations of colchicine (0, 250, 500 and 750 mg/l) at 4°C for 36 and 72 hrs under dark condition. After colchicine pretreatment, anthers were transferred to a colchicine free medium to callus and shoot induction. Analysis of variance showed that percentage of callus and shoot induction (PCI and PSI) were not influenced by cold pretreatment durations. However, significant differences were observed between cold durations for the number of regenerated (NRP) and the percentage of doubled haploid plants (PDH) (p < 0.05). Androgenesis responses were affected by different colchicine concentrations and interactive colchicine x cold durations (p < 0.05). Maximum PCI, PSI, NRP and PDH were belonged to 250 mg/l colchicine and 72 h cold duration. However, colchicine concentrations above than 250 mg/l had negative effect on androgenesis responses of anther cultures in both cold durations.

Androgenic response of tomato (Lycopersicon esculentum Mill.) lines and their hybrids to anther culture

The androgenic ability of four tomato (Lycopersicon esculentum Mill.) lines (Micro-Tina, Micro-Gemma, Moneymaker and MsK8 as parents) and their hybrids (obtained as half diallel crosses) were studied through anther culture, The isolated anthers were cultured on three culture media including M1 (Park et al., 2001), M2 (Summers, 1997) and M3 (Evans et al. 1986) exposed to two pretreatments (26 ± 2°C as a control and at 4°C for 48 h as a cold pretreatment). Callus formation and callus growth (measured by callus diameter) occurred in the both pretreated anthers, but no callus produced shoot regeneration on the non-pretreated anthers. Statistical analysis showed significant differences between genotypes, culture media and their interactions for the studied traits. The highest callus formation was on M1 and M2 media, among which the longest callus diameter and the highest shoot regeneration were on M1 medium. The lines MsK8 and Micro-Gemma had a considerably higher frequency of callus formation and shoot regeneration and longer callus diameters than the other parents. On M1 medium, the highest frequency of callus formation and shoot regeneration belonged to hybrid MsK8 × Micro-Tina, and the longest callus diameter observed on hybrid Micro-Tina × Micro-Gemma. The mean values of callus formation, callus diameter, and shoot regeneration of hybrids in different culture media were significantly higher than those of the parents. In other words, heterosis (hybrid vigor) for the mentioned traits was observed on different culture media. Therefore, heterozygous genotypes (the hybrids) had a higher frequency of androgenic response than their parents.

ENHANCEMENT OF MATURATION AND GERMINATION OF SOMATIC EMBRYOS OF PERSIAN WALNUT (JUGLANS REGIA L.) BY GELLAN GUM, COLD AND PGR

One of the potentially desirable propagation strategies for mass production of walnut cultivars, selected hybrids, or genetically engineered genotypes is somatic embryogenesis. Maturation and germination of somatic embryos of walnut has not been optimized yet. In this study, maturation of somatic embryos of Persian walnut (Juglans regia L.) was evaluated on media containing various concentrations of Gelrite (0.2%, 0.3%, 0.4% and 0.5%) and PEG (0.0%, 3.0%, 5.0% and 7.5%) in the presence of 2 mg L -1 ABA and 3.0% sucrose. Gelrite was more effective in stimulating the production of mature somatic embryos than PEG The highest number of cotyledonary (70.0%) and normal (50.0%) SEs was obtained with 0.3% Gelrite without PEG Media containing 0.2% Gelrite (control) resulted in the highest secondary embryogenesis. With increasing concentrations of Gelrite, the number of matured somatic embryos was decreased. The combination of Gelrite and PEG was less effective for maturation of somatic embryos. The Gelrite 0.5% and PEG 7.5% caused the highest number of abnormal somatic embryos. For germination of matured somatic embryos, after growing the SEs on maturation medium for four weeks, four treatments including basal medium without PGR, basal medium with PGR, basal medium with cold pretreatment (four weeks at 4°C), basal medium with PGR and cold pretreatment were tested. The highest amount of normal germination and conversion to plantlets (55.0%) was achieved using 0.3% Gelrite and cold pre-treatment and PGR.

Germination characteristics and sporeling success of Azolla filiculoides Lamarck, an aquatic invasive fern, in a Mediterranean temporary wetland

Spore germination characteristics of Azolla filiculoides Lamarck, a recent invader of the Doñana Mediterranean wetlands, have been quantified experimentally. Batches of spores were subjected to different storage conditions, salinity concentrations, phosphorus availability and drought effects. Cold pre-treatment did not stimulate spore germination, whereas drought, a characteristic different from Azolla's native habitat, reduced it (40 ± 7 vs 13 ± 5). Germination declined with the increase of salinity (r = −0.50, notably above 5000 μS cm −1), whereas phosphorus availability had no significant effect.

Plant development of &lt;i&gt;Ranunculus asiaticus&lt;/i&gt; L. tuberous roots is affected by different temperature and oxygen conditions during storage period

The objective of this research was to carry out comparative agronomical trials of eleven genotypes of Ranunculus asiaticus L. and to evaluate the effect on their physiological traits and agronomic features of storage conditions before planting. The effect of temperature and oxygen concentration during storage of the tuberous roots was evaluated. The cold pre-treatment accelerated flower initiation and develoopment in all genotypes. Storage of tuberous roots for one year at 2 °C kept their sprouting ability at satisfactory level (around 90%). Qualitative and quantitative differences have been observed in different genotypes at the level of root carbohydrate pool and cell structure. Atmosphere composition had no significant effects on the stored propagation units.

Genetic and nongenetic factors influencing callus induction inMiscanthus sinensis (Anderss.) anther cultures

Miscanthus sinensis is a promising species for biomass production. Influences of genetic and nongenetic factors on androgenesis induction efficiency were investigated. This is the first report on successful induction of pollen-derived callus in M. sinensis. The callus yield was strongly affected by genotype. A beneficial influence of cold pretreatment of spikes on androgenesis induction was observed. The highest yield of calli was obtained in cultures on a modified C17 medium. The results suggest that the high callus yield might be caused by the late culture initiation. The beginning of anther culture at the end of the flowering season caused a 17-fold increase in callus yield, in comparison to culture initiated at the height of the flowering season (August). It is likely, however, that the efficiency of androgenesis induction in the case of M. sinensis anther culture beginning in October could be related to a positive influence of growing donor plants in conditions of cooler and shorter day, i.e. 11-h day with temperature around 11xC and 13-h night with temperature around 5xC. Results of this study can significantly support the development of effective methods of M. sinensis haploidization, which could be used in crop improvement by breeding.

English

&nbsp; Isolated microspores of many plants can be induced&nbsp;in vitro&nbsp;to switch their developmental process from the gametophytic to a sporophytic pathway under appropriate conditions and produce haploid plants. This research reports the effects of cold pretreatment with or without either mannitol or chemical + heat and 5 embryo induction media (NPB-99, C17, W14, CHB-2 and P2) on embryo production, percentage of total and green plant regeneration in isolated microspore culture of an Iranian spring bread wheat cultivar (Falat). The results showed that combination of 21 days cold (4&deg;C) with mannitol (0.3 M) produced the highest number of embryos/spike while the combination of cold with chemical + heat produced the lowest number. In the case of total and green plant regeneration, &quot;7 days cold + mannitol&quot; was more superior than other pretreatments. NPB-99, W14 and C17 media produced the highest number of embryos/spike, while CHB-2 medium appeared to be a better medium for green plant regeneration. &nbsp; Key words:&nbsp;Hexaploid wheat, haploid, isolated microspore culture, pretreatment, embryo induction medium, cultivar &ldquo;Falat&rdquo;.

Effect of Low Temperature Pre-Treatment on Anther Culture in Selected Indica, Japonica Rice Varieties and their Inter Sub-Specific Hybrids

Effect of cold pre-treatment of panicles with reduced temperatures (of 5°C, 7°C, 8°C and 10°C) and exposure for different time durations (at weekly intervals up to 4 weeks) on anther culture of Japonica rice variety Hu lo tao, Indica rice varieties Bg 90-2, Bg 379-2 and their F1 hybrids for high frequency callus induction and plant regeneration was investigated. Anthers were cultured in N6 medium with 5% sucrose, 2 mg l-1 2,4 D and 0.5 mg l-1 Kinetin. The cultures were kept in the dark at 28 ± 2°C for callus induction. Induced calli of 1-2 mm diameter were transferred to half strength Murashige and Skoog (MS) medium supplemented with 2.0 mg l-1 Kinetin and 0.5 mg l-1 NAA. The effect of variety and cold pre-treatment was significant at 5% probability level. The Indica varieties had the lowest callus induction frequency (0.3% - 7.0%). Japonica and Japonica × Indica hybrids had high callus induction (12.3% - 70.0%). The F1 hybrid Hu lo tao × Bg 90-2 had comparatively high callus induction (31.0% - 70.0%) than the other genotypes. Japonica variety Hu lo tao and F1 hybrids had highest callus development at cold pre-treatment at 8°C for 14 and 21 days. The highest green plant regeneration was observed in F1 hybrid, Hu lo tao × Bg 90-2 followed by Japonica variety subjected to 8°C for 14 days. Very poor green plant regeneration was observed in Indica variety, Bg 90-2 when it was exposed to 7°C for 21 and 28 days. Variety Bg 379-2 did not produce any green plant at any treatment. The F1 hybrid Hu lo tao × Bg 379-2 produced lower green plants than F1 hybrid Hu lo tao × Bg 90-2. Cold pre treatment at 8°C temperature for 14 days was found to be most effective for anther culture in experimental genotypes used in the study. Key words: variety, cold pre-treatment, callus induction, plant regeneration, rice DOI: 10.4038/cjsbs.v38i1.1322 Cey. J. Sci. (Bio. Sci.) 38 (1): 11-16, 2009