Small heat shock protein responses in leaf tissues of wheat cultivars with different heat susceptibility

The effect of heat stress on soluble proteins extracted from leaf tissues of bread (Triticum aestivum cv. Gönen-98, tolerant; cv. Cumhuriyet-75, susceptible; genome ABD) and durum (Triticum durum cv. Ege-88, tolerant; cv. Ankara-98, susceptible; genome AB) wheat cultivars differing in sensitivity to high temperature was examined by two-dimensional gel electrophoresis. At acclimation (37°C) and acclimation→high temperature (37°C→50°C) treatments compared to control (25°C), evaluation of gels revealed 31 proteins to be differentially expressed in first leaves as a result of heat stress in heat-susceptible and heat-tolerant cultivars of bread and durum wheats. All of the increased or decreased proteins in amount, newly synthesized and/or disappeared were in low-molecular-weight (LMW, 16.1–24.0 kDa) and generally acidic character (pI 4.8–6.9). The responses of the four cultivars were compared: Twenty-two of 31 proteins were detected as newly synthesized LMW heat shock proteins (LMW HSPs = small HSPs). The number of these sHSPs was different in cultivars which have the same genome. In addition, the number of the sHSPs in heat-tolerant cultivars was higher than in heat-susceptible cultivars. Some of the sHSPs were specific to cultivar. Most of the sHSPs synthesized at 37°C were also detected at 37°C→50°C treatment. It is suggested that sHSPs have special importance in two points: Firstly, sHSPs in cultivars showed abundance and diversity. Secondly, these proteins may play an important role in the acquiring of thermal tolerance.     

Chilling stress and oxygen metabolizing enzymes in Zea mays and Zea diploperennis*

Abstract. The activities of five active-oxygen scavenging enzymes were compared for cold-lability and three were compared for chilling induction in two Zea genotypes of contrasting susceptibility to photoinhibition during chilling. Activities of superoxide dismutase (SOD, EC 1.15.1.1), ascorbate peroxidase (APX, EC 1.11.1.11), monodehydroascorbate reductase (MDHAR, EC 1.6.5.4), dehydroascorbate reductase (DHAR, EC 1.8.5.1), and glutathione reductase (GTR, EC 1.6.4.2) in leaf extracts from plants grown without chilling stress were assayed at 19°C and 5°C. Enzymes from the chilling-susceptible Z. Mays cv. LG11 had lower specific activities at 5°C than did enzymes from the chilling-tolerant Z. diploperennis, except for MDHAR where no significant differences were observed. The activities of SOD and APX from Z. diploperennis were double those of Z. mays at both assay temperatures. Monodehydroa-scrobate reductase and glutathione reductase activities in both species were reduced by 63–78% at a 5°C assay temperature. The dehydroascorbate reductase (DHAR) showed the greatest low-temperature lability losing 96% (Z. diploperennis) and 100% (Z. mays) of its activity at 5°C. To examine possible chilling-induced changes in levels of enzyme activity, plants of both Zea genotypes were transferred to growth chambers at 10°C at moderate light intensities. Glutathione reductase activity was found to increase within 24h in Z. diploperennis, but it decreased slightly in Z. mays. MDHAR activity decreased by 50% in Z. diploperennis but showed only a transient increase in activity in Z. mays.     

Effect of temperature onin vitro pollen germination in pigeonpea

Pollen of pigeonpea(Cajanus cajan (L.) Millsp.) cultivars H-77-216 and ICPL-151 were cultivatedin vitro at six different temperatures (12, 17, 22, 27, 32, 37 °C). Pollen of cv. H-77-216 started to germinate at 17 °C whereas the pollen of cv. ICPL-151 at 22 °C, the optimal temperatures were 22 and 27 °C, respectively. Pollen germination at different temperatures was found to be positively correlated with the tube length. Per cent pollen bursting increased with rising temperature. The indeterminate cv. H-77-216 showed a wide range of suitable temperatures (17 – 27 °C) for pollen germination while the determinate cv. ICPL-151 had optimum at 27 °C     

Efficient production of active form of recombinant cassava hydroxynitrile lyase using <Emphasis Type="Italic">Escherichia coli</Emphasis> in low-temperature culture

Overexpression and production of the high concentration of hydroxynitrile lyase from cassava (Manihot esculenta (MeHNL, EC 4.1.2.39)) were investigated. Hydroxynitrile lyase is a useful enzyme for the production of optically active cyanohydrin compounds. The production of MeHNL was increased by changing the rare codons of the original sequence of cassava MeHNL. However, most of the produced MeHNL was in the insoluble form. In order to increase the solubility of MeHNL, the effects of the cultivation temperature were investigated. When the cultivation temperature was reduced, the cell yield and the ratio of soluble MeHNL increased significantly. The enzyme activity and yield at low-temperature cultures (17 °C) were 850 times higher than those obtained at the optimum growth temperature of 37 °C. The rate of MeHNL production in the present study was calculated as 3,000 unit/h. Low-temperature cultivation was very effective in improving the productivity of the active form of MeHNL. Unlike the temperature-shift method, low-temperature cultivation has more potential for the large-scale production of MeHNL for the optically active cyanohydrin production. An erratum to this article can be found at     

Effect of low-temperature fermentation on yeast nitrogen metabolism

The aim of this study was to analyse the influence of low-temperature wine fermentation on nitrogen consumption and nitrogen regulation. Synthetic grape must was fermented at 25 and 13°C. Low-temperature decreased both the fermentation and the growth rates. Yeast cells growing at low-temperature consumed less nitrogen than at 25°C. Specifically, cells at 13°C consumed less ammonium and glutamine, and more tryptophan. Low-temperature seemed to relax the nitrogen catabolite repression (NCR) as deduced from the gene expression of ammonium and amino acid permeases (MEP2 and GAP1) and the uptake of some amino acids subjected to NCR (i.e. arginine and glutamine). Low-temperature influences the quantity and the quality of yeast nitrogen requirements. Nitrogen-deficient grape musts and low temperature are two of the main prevalent causes of sluggish fermentations and, therefore, the effects of both growth conditions on yeast metabolism are of considerable interest for wine making.     

Effects of temperature on germination and mitochondrial dehydrogenases in two soybean (Glycine max) cultivars

The effects of eight germination temperatures from 10°C to 35°C on germination and dehydrogenase activities of two soybean (Glycine max [L.] Merr.) cultivars were investigated after 48 h of seedling growth. Axis fresh weights of cv. Chippewa increased as germination temperature increased from 10°C to 35°C. In contrast, axis fresh weights for the cv. Wells increased more slowly with increasing temperature and reached a maximum at c. 25°C. In general, in vitro activities of glutamate dehydrogenase (GDH), NADP-isocitrate dehydrogenase (NADP-ICDH), and malate dehydrogenase (MDH) from the axes of cv. Chippewa correlated well with increases in axis fresh weights. GDH and MDH activities from axes of the cv. Wells also reflected increases in axis fresh weights although the correlation was not as evident as for the cv. Chippewa. NADP-ICDH activity from ‘Wells’ axes was highest at 35°C even though germination was poor at this temperature. GDH and MDH activities from cotyledons of both cultivars were not correlated with axis weight increases. No GDH activity was detected in ‘Wells’ cotyledons from seeds germinated at 35°C.     

Hybrid Weakness in Phaseolus vulgaris L. I. Disruption of Development and Hormonal Allocation

A reduced concentration of cytokinins may cause the abnormal growth and development found in F1 hybrids between Andean and Mesoamerican races of Phaseolus vulgaris L. In this study, concentrations of the transportable cytokinin zeatin riboside (ZR) were measured by ELISA for ZR (cross reactivities dihydrozeatin, 14%, zeatin 7.6%) in roots, stems, and leaves of a Phaseolus Mesoamerican landrace (P. vulgaris L. cv. Redkloud), an Andean landrace (P. vulgaris L. cv. Batt), and their F1 hybrids. Concentrations of ZR in roots and leaves of F1 hybrids were significantly less than that found in roots and leaves of parental cultivars. Approximately 90% of the ZR found in F1 hybrids was found sequestered in the stems, whereas cytokinins of the parental cultivars were distributed throughout the plant (roots: Batt 37%, Redkloud, 44%; stems: Batt 35%, Redkloud 42%; leaves: Batt 28%, Redkloud 14%). These results suggest that abnormal growth and development of F1 hybrids may involve interruption of the regulation of cytokinin allocation, thereby disrupting the root-shoot feedback loop between root-sourced cytokinins and putative shoot-produced factors. Received October 15, 1998; accepted May 12, 1999     

Impact of temperature on olive (Olea europaea L.) pollen performance in relation to relative humidity and genotype

Olive varieties ‘Koroneiki’, ‘Kalamata’, ‘Mastoidis’ and ‘Amigdalolia’ were employed in two experiments for 3 years to assess the effect of temperature on olive pollen germination and tube growth in relation to relative humidity and genotype. Pollen samples were subjected to pre-incubation at 10, 20, 30 or 40 °C in combination with decreased air relative humidity – 80, 40, 30 or 20%, respectively – for 24 h to simulate temperature stress that is observed during pollen dispersal; and subsequently in vitro cultured. In the second experiment, pollen was exposed at 15, 20, 25 and 30 °C for 24 h in vitro to evaluate pollen response in conditions of water and nutrients availability and to determine the optimum pollen germination and tube growth temperatures for each cultivar. The highest pre-incubation temperature treatment (40 °C) prevented pollen germination in ‘Koroneiki’ and ‘Mastoidis’, with the less affected varieties (‘Amigdalolia’ and ‘Kalamata’) having average germination percentages of only 7.6 and 2%, respectively. Pre-incubation at 30 °C had a negative impact on pollen germination in ‘Koroneiki’ (?65%), ‘Kalamata’ (?20%) and ‘Amigdalolia’ (?72%) compared to the control (20 °C). Pollen pre-incubation at 40 °C decreased significantly the pollen tube length in ‘Kalamata’ (?50%) and ‘Amigdalolia’ (?52%). In the second experiment, in vitro pollen germination increased after incubation at 25 °C for ‘Koroneiki’ (+6%), ‘Mastoidis’ (+52%), ‘Kalamata’ (+10%) and ‘Amigdalolia’ (+10%) compared to the control (20 °C). At 30 °C germination percentages for ‘Mastoidis’, ‘Kalamata’ and ‘Amigdalolia’ were 8, 6 and 14% higher, respectively, compared to the control (20 °C). Pollen tube length also increased with incubation temperature for all of the studied cultivars. Based on the cumulative stress response index (CSRI) that was calculated for high temperature stress the varieties were classified: ‘Mastoidis’ and ‘Kalamata’ as tolerant and ‘Koroneiki’ and ‘Amigdalolia’ as intermediate at 30 °C while all studied cultivars were sensitive at 40 °C. The observed strong genotype-differentiated response in high and low temperature stress could be exploited by plant breeders towards producing new tolerant olive varieties.     

Life history characteristics of the western flower thrips,Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae), under fluctuating conditions of temperature or relative humidity

Frankliniella occidentalis (Pergande) is a major insect pest of greenhouse crops such as leaf vegetables, flowers and vegetable fruits worldwide. The life history characteristics of F. occidentalis were investigated at control temperature and humidity (27.3 ± 0.54 °C, 79.9 ± 2.79% RH) (mean ± SD), a 10 °C-range fluctuation in temperature (27.1 ± 5.28 °C, 81.5 ± 4.03% RH), a 20 °C-range fluctuation in temperature (26.5 ± 10.09 °C, 80.4 ± 5.76% RH), a 20%-range fluctuation in humidity (26.8 ± 0.37 °C, 80.7 ± 9.55% RH) and a 30%-range fluctuation in humidity (27.3 ± 0.41 °C, 76.3 ± 15.28% RH). Overall, the life history traits of F. occidentalis were more negatively affected by fluctuating environmental conditions. The impact of temperature fluctuation was more severe than that of humidity fluctuation. Additionally, the degree of impact increased as the fluctuation range of the temperature increased, while the reverse trend was observed with humidity fluctuations. With the 20 °C-range fluctuation in temperature, F. occidentalis died at the 1st instar larval stage. The offspring’s sex ratio was significantly higher at the 20%- and 30%-range fluctuations in humidity (0.47 and 0.49, respectively) compared to the control (0.35) and at the 10 °C-range fluctuation in temperature (0.33). From the fertility life table analysis, the intrinsic rate of increase (r) was higher at the 30%-range fluctuation in humidity and control conditions as 0.218 and 0.205, respectively. At the 10 °C-range fluctuation in temperature conditions, r was significantly lower as 0.169. High fluctuations in temperature and low fluctuations in humidity appear to be the best conditions for controlling F. occidentalis populations in greenhouses.     

Photosynthetic pigments,morphology and leaf gas exchange during ex vitro acclimatization of micropropagated CAM Doritaenopsis plantlets under relative humidity and air temperature

The effect of relative humidity (RH) and temperature on CO₂ assimilation (An), stomatal conductance (Sc), transpiration rate (Tr), chlorophyll content, fresh and dry weight, leaf length, leaf area, leaf width, formation of new root and survival rate have been assayed in Doritaenopsis in growth chamber after 1 month of acclimatization. Reduced growth was observed at below and above 25 °C whereas it was increased with increasing humidity. Relative water content (RWC) was decreased at 50% and 70% humidity after second day of transfer and recovered completely with the progression of acclimatization. RWC also reduced at high temperature but recovered slowly and a gradual decrease of RWC was observed at 15 °C. A visual symptom of severe leaf tip burn was observed at 50–70% humidity and at 35 °C during acclimatization. At 15 °C and 50% humidity sudden decrease of photosynthetic efficiency (F_v/F_m) was observed, which could not recover in temperature treated plantlets during acclimatization period. Chlorophyll content increased with increasing humidity and at 15 and 35 °C chlorophyll content was decreased compared to 25 °C. Chlorophyll a/b ratio was unchanged while total chlorophyll/carotenoids ratio was increased from low to high temperature. Exposure of plantlets to high temperature led to a noticeable decrease in An, Sc and Tr, and at 15 °C they were more decreased whereas significant differences were not observed in the parameters tested under humidity after 25 days of acclimatization. During daytime at 15 °C, increase in An, Sc and Tr indicates the plantlets adaptability in the new environment. The peroxidase activity remained unaffected in all humidity stress whereas low temperature increased the peroxidase activity compared to high temperature. These finding suggests that photosynthetic properties was greatly affected by air temperature conditions with a reduction of An, Sc and Tr at 15 and 35 °C compared to humidity stress that played a greater role in limiting photosynthesis.     

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.     

Modeling seed germination of unprimed and primed seeds of sweet sorghum under PEG-induced water stress through the hydrotime analysis

The effects of reduced water potential (ψ) on seed germination at 25 and 15 °C in unprimed (UP) and primed (P) seeds of two cultivars of sweet sorghum (cv. Keller and cv. Makueni local), were analyzed through the hydrotime model. Six ψ (from 0 to ?1.0 MPa) in polyethylene glycol 6000 (PEG) solutions were used for the tests. Seeds were primed in 250 g/L PEG solution at 15 °C for 48 h. Decreasing ψ of imbibition solution reduced and delayed germination. At 15 °C seeds germinated less and slower than at 25 °C at any ψ. Seeds of cv. Makueni local exhibited a greater sensitivity to water stress in terms of germination percentage, than seeds of cv. Keller, but they were faster in germination. Osmopriming was beneficial for seed germination, both in terms of final percentage and rate, at any temperature and ψ. The hydrotime analysis revealed that predicted θ _H constant was increased when temperature was reduced to 15 °C and at this temperature median base water potential [ψ _b(50)] for germination was higher (less negative) than at 25 °C. Seed priming shifted ψ _b(50) towards more negative values and reduced θ _H requirements for germination. At 25 °C the two cultivars behaved similarly while at 15 °C cv. Keller exhibited a ψ _b more negative but required a greater θ _H to germinate, indicating a greater water-stress tolerance but a slower germination, than cv. Makueni local. The application of the model allows to identify water stress tolerant cultivars during germination, to include into breeding programs for the selection of well-performing cultivars under stress conditions.     

The Influence of Temperature on Nitrate Reductase Activity of Agrostis palustris and Cynodon dactylon

The influence of temperature was studied in relation to nitrate reductase activity of creeping bentgrass (Agrostis palustris Huds. cv. ‘Toronto’) a cool season grass and bermudagrass (Cynodon dactylon L. cv. ‘Tifgreen’) a warm season grass. Maximum nitrate reductase activity of both species occurred at 20°C. The nitrate reductase level in bentgrass leaves was reduced when grown at 35°C while bermudagrass leaves were relatively unaffected. The activity per se of the bentgrass enzyme preparation was inhibited rather than synthesis of the enzyme.     

Predicting military working dog core temperature during exertional heat strain: Validation of a Canine Thermal Model

Military working dogs (MWDs) operate under a wide range of conditions, including hot environments. Predicting how long a MWD can safely work without overheating is important for both health and performance. A Canine Thermal Model (CTM) was developed to predict core temperature (Tc) of MWDs. The CTM calculates heat storage from the balance of heat production from metabolism and heat exchange with the environment. Inputs to the CTM are: meteorological conditions (ambient temperature, relative humidity, solar radiation and wind speed), physical characteristics of the dog (mass, length), and metabolic activity (MET level, estimated from accelerometer data). The CTM was validated against Tc measured in 23 MWDs during training sessions (11.6 ± 5.0 min (mean ± standard deviation), range 4–26 min) in October (24 °C, 52% RH), March (14 °C, 74% RH), or August (28 °C, 64% RH), and 24 kennel MWDs during a standard exercise walk (11.4 ± 3.3 min, range 5.6–18 min) in July (26 °C, 77% RH). The CTM was considered acceptable if predicted Tc was within ±0.5 °C of measured Tc at the end of exercise. Compared to Tc at the end of training sessions (39.8 ± 0.6 °C, range 38.4–41.1 °C) and exercise walks (40.0 ± 0.7 °C, range 38.9–41.4 °C), the CTM-predicted Tc was within ±0.5 °C for 71 of 84 cases (85%) and 19 of 24 cases (79%), respectively. The mean difference between CTM-predicted and measured final Tc during training was -0.04 ± 0.43 °C, with 80 of 84 cases (95%) within the range of ±2 SD (Bland Altman comparison). During exercise walks the mean difference was -0.15 °C ± 0.57, with 23 of 24 cases (96%) within ±2 SD. These results support the use of the CTM to predict Tc of MWDs for the types of physical activities described above.     

Changes in cytokinin levels of Phalaenopsis leaves at high temperature

The effect of high temperatures on cytokinin levels in Phalaenopsis hybrida leaves was investigated. Endogenous cytokinins were identified and quantified in Phalaenopsis leaves grown under high temperature conditions (30/25 °C day/night) using high performance liquid chromatography, bioassay and gas chromatography-selected ion monitoring-mass spectrometry. After 5 and 20 d of low temperature (25/20 °C day/night), zeatin, zeatin riboside and dihydrozeatin levels in the leaves were higher than that in leaves subjected to high temperature treatments. When Phalaenopsis leaves were exposed to low temperatures, about 76 % of the free cytokinins detected were of the zeatin-type. Glucoside cytokinins in the leaves increased significantly 5 d following high temperatures, and the rate of increase in glucoside cytokinins corresponded to the duration of high temperatures. At the same time, zeatin riboside and dihydrozeatin declined significantly following high temperature application. A significant accumulation of glucoside cytokinins, zeatin-9-glucoside, zeatin-O-glucoside, zeatin riboside-O-glucoside, and dihydrozeatin-O-glucoside was observed 20 d following high temperatures. These results suggest that high temperatures lead to an accumulation of glucoside cytokinins and a reduction of free base and riboside cytokinins.     

Life history ofAphis gossypii on cucumber: influence of temperature, host plant and parasitism

Life table data forAphis gossypii Glover (Homoptera: Aphididae), an important pest in glasshouse cucumber crops, were studied at 20, 25 and 30°C on two cucumber cultivars (Cucumis sativus L.) in controlled climate cabinets. The development time on the cucumber cv. ‘Sporu’ ranged from 4.8 days at 20°C to 3.2 days at 30°C. Immature mortality was approximately 20% and did not differ between temperatures. Most mortality occurred during the first instar. Reproduction periods did not differ among temperatures, but at 25 and 30°C more nymphs were produced (65.9 and 69.8 nymphs/♀, respectively) than at 20°C (59,9 nymphs/♀) because of a higher daily reproduction. Intrinsic rate of increase was greatest at 25°C (r _m =0.556 day⁻¹). At 20 and 30°C the intrinsic rate of increase was 0.426 and 0.510, respectively. On cv. ‘Aramon’, the development time ofA. gossypii was approximately 20% longer at all temperatures. Immature mortality did not differ between the two cultivars. The intrinsic rate of increase on cv. ‘Aramon’ was 15% smaller than on cv. ‘Sporu’. The use of cucumber cultivars partially resistant to aphids is discussed in relation to biological control of cotton aphid in glasshouses. Development time and immature mortality on leaves of the middle and upper leaf layer of glasshouse grown cucumber plants (cv. ‘Aramon’) were comparable to development in the controlled climate cabinets. On the lower leaves immature mortality was much higher (approximately 82%) than on leaves of the middle (24.0%) and upper leaf layer (24.5%). Reproduction was less on the lower leaf layer (45.9, 70.5 and 70.1 nymphs/♀ on leaves of the lower, middle and upper leaf layer, respectively). Aphids, successfully parasitized byAphidius colemani Viereck (Hymenoptera: Braconidae) only reproduced when they were parasitized after the third instar. Fecundity was 0.1 to 0.9 and 10.5 to 13.3 nymphs/♀ for aphids parasitized in the fourth instar or as adults, respectively. Reproduction of aphids that were stung but survived the attack was lower than for aphids not stung. Average longevity of these aphids was equal to the longevity of aphids not stung byA. colemani.     

Bacterial Rot of Dieffenbachia maculata (Lodd.) G. Don. Caused by Erwinia chrysanthemi

Five bacterial isolates were obtained in a nursery near Gent (Belgium) from diseased Dieffenbachia maculata (Lodd.) G., Don. cv. Compacta, cv. Camillo and cv. Veerle plants. They were identified by API 20E, API 50 CHE and API ZYM systems as Erwinia chrysanthemi. These strains and seven collection strains were pathogenic to all three D. maculata cultivars tested: cv. Camillo, cv. Compacta and cv. Tropic Snow. Inoculation in the stem or petiole was the only effective method for obtaining systemic infection. The petiole appeared to be the part of the plant in which disease developed most readily. Wounding was required for the induction of the disease. Temperatures between 25° and 30° C, a relative air humidity of 75 % or more and low light intensity (0.5 ft-c) favoured the disease, whereas high inoculum sizes (10⁶ or more cells) accelerated and increased it. Histological studies showed tissue degradation in infected areas. Petioles were most severely affected. Transport of bacteria seemed to occur after vessel infection in stem or petiole tissues.     

A low-temperature method for maintaining plant regeneration activity in embryogenic callus of rice (Oryza sativa L.)

 A method was developed to maintain plant regeneration activity of rice cells (Oryza sativa L.) using embryogenic callus. Calluses were cultured in suspension, then on solid medium, to form compact globular callus resistant to low-temperature stress and with high plant regeneration activity. Callus preserved at 5  °C for 5 months regenerated plants from protoplasts at a frequency higher than from non-preserved callus from cv. Nipponbare, and cv. Koshihikari, but at lower rates from cv. Akitakomachi. Similar results were obtained from protoplasts of the three cultivars. Callus preserved at 5  °C for 8 months incurred cell damage, yet some surviving cells divided in suspension culture and eventually regenerated whole plants. Preserved and non-preserved regenerated plants showed similar levels of somaclonal variation. Received: 7 January 1999 / Revision received: 28 April 1999 / Accepted: 26 May 1999     

Endogenous auxin level is a critical determinant for in vitro adventitious shoot regeneration in potato (Solanum tuberosum L.)

Endogenous levels of indole-3-acetic acid (IAA) were determined in the internodal explants of Indian tetraploid potato cultivars (cvs) viz., Kufri Sutlej (K.Sutlej) and Kufri Giriraj (K.Giriraj). Seven fold higher level of endogenous IAA was recorded for cv K.Sutlej over cv K.Giriraj. As a result, perhaps there was a callusing response from the cut end of the internodal explants of both the cvs in the MS basal medium. The extent of callusing was relatively higher in cv K.Sutlej when compared to that in K.Giriraj. The callusing response was inhibitory to shoot morphogenesis. Inclusion of an established anti-auxin, 2,3,5-tri-iodobenzoic acid (TIBA) in the regeneration medium facilitated a high frequency adventitious shoot regeneration response with lower cytokinin levels. Murashige and Skoog (MS) medium containing TIBA at 2.5?mg?l^?1 and 0.25?mg?l^?1 zeatin evoked a 100% regeneration response (4.5 shoot buds per explant) in cv K.Sutlej within 20?C25?days. However, in cv K.Giriraj, which had lower levels of endogenous IAA, 80% regeneration response (1.4 shoot buds per explant) was recorded in an extended period of 40?C45?days on a medium containing 0.5?mg?l^?1 TIBA and 0.1?mg?l^?1 zeatin. Although, TIBA and zeatin induced shoot bud formation, it failed to support sustained growth of the regenerated shoots in cv K.Giriraj. Hence, 0.01?mg?l^?1 ??-naphthaleneacetic acid (NAA) with a relatively higher concentration of zeatin (1.0?mg?l^?1) were used for sustained shoot regeneration (3.3 shoot buds per explant) within 25?days. From our results, it is evident that there was a difference in the requirement of exogenous zeatin levels required to induce a regeneration response in two cultivars of potato and this is attributed to the variable levels of endogenous IAA.     

Characterization of recombinant human granulocyte colony-stimulating factor expression by FT-IR spectroscopy: Studies on thermal induction and media formulation on the stability of the protein secondary structure

The Fourier-transform infrared (FT-IR) spectroscopic approach has been employed to understand the recombinant human G-CSF (rhG-CSF) protein accumulation, secondary structure, and thermal stability in Escherichia coli grown under a temperature shift strategy (37 and 28°C) in various media formulations. The choline?+?sodium pyruvate (37°C) and sodium pyruvate (28°C) formulations have shown the highest inclusion body (IB) accumulation of 0.41 and 0.46?mg/mL, respectively. Furthermore, insights on the structure of the rhG-CSF within IBs and intact cells have been investigated through secondary structure analysis. Thermal stability experiments were also carried out to explain the pattern of the second derivative structure of rhG-CSF. The studies showed that choline?+?sodium pyruvate formulation has preserved the protein secondary structure even at 82°C. Overall, the FT-IR spectroscopic technique can also be adopted to accelerate the characterization of other recombinant therapeutic proteins of E. coli origin.