Effects of pre-storage conditions on storage of in vitro cultures of Nephrolepis exaltata (L.) Schott and Cordyline fruticosa (L.) A. Chev.

In vitro cultures of Nephrolepis exaltata and Cordyline fruticosa were stored at 5°, 9° or 13°C, at a low irradiance (3–5 mol m^–2 s^–1) or in darkness. Prior to storage the cultures were subjected to 18°, 21°, 24° or 27°C and 15, 30 or 45 mol m^–2 s^–1 in a factorial combination.The optimal storage conditions for Nephrolepis were 9°C in complete darkness. These cultures were still transferable to a peat/perlite mixture at the end of the experimental period of 36 months.The optimal storage conditions for Cordyline were 13°C and a low light level (±3–5 mol m^-2 s^-1). When the pre-storage conditions were normal growth room conditions (24°C and 30 mol m^-2 s^-1), in vitro cultures could be stored for 18 months. With the most favourable pre-storage treatment (18°C and 15 mol m^-2 s^-1) some cultures still had green shoots after 36 months of storage, but did not survive transfer to peat/perlite.Pre-conditioning before storage was most favourable for Nephrolepis, and not that important, but still favourable, for Cordyline. There was an interaction between pre-storage temperature and pre-storage irradiance. For both species a high irradiance level was less favourable than a low irradiance level when combined with high growth room temperatures.Abbreviations BA 6-benzyladenine - IAA indole-3-acetic acid - NOA 2-naphthoxyacetic acid     

Survival of somatic embryos and recovery of plants of sweet orange (Citrus sinensis (L.) Osb.) after immersion in liquid nitrogen

Somatic embryos of Washington Navel sweet orange (Citrus sinensis (L.) Osb.) derived from in vitro cultured ovules excised from immature fruits, were frozen to the temperature of liquid nitrogen. A method of slow cooling at a rate of 0.5°C min^-1 down to –42°C followed by storage in liquid nitrogen was used. Thawing was achieved by keeping the specimens at room temperature for 15 min. A small number of frozen embryos survived and developed into proliferating cultures that produced whole plants. The plants obtained from frozen cultures were transferred to soil and are growing successfully.     

Plant regeneration from grape callus stored under a combination of low temperature and silicone treatment

Effects of the combination of low temperature and silicone treatment on the storage of grape callus (Vitis vinifera L. x V. labrusca L. cv. Kyoho; V. vinifera L. cv. Koshusanjaku) were examined. In Kyoho, the calli were stored at 10°C successfully for up to 360 days. Embryogenic calli of Koshusanjaku stored at 10°C retained the ability of embryogenesis after 360 days of storage. However, the color of both calli became brownish. This was improved by the combination of low temperature and silicone treatment. The calli of Kyoho survived by the storage under the combination of 15°C and silicone. Embryogenic calli stored at 10 and 15°C in combination with silicone survived for 360 days, and regenerated only after transfer onto a regeneration medium. Thus the combination of low temperature and silicone affects the longevity of the grape callus.     

Photosynthetic activities of a membrane preparation of the thermophilic cyanobacterium Mastigocladus laminosus

Photosynthetically active membranes have been prepared from the thermophilic cyanobacterium Mastigogladus laminosus by treatment with lysozyme. The membranes were active in electron transport through photosystem I and II as well as in photophosphorylation and proton uptake. Cells were grown at 40°, 45° and 55°C respectively. The temperature optimum of oxygen evolution of whole cells was about 10°C higher than the growth temperature. In isolated membranes the temperature optimum for cyclic photophosphorylation was identical to the growth temperature of the cells whereas the optimum for photosystem II electron transport never exceeded 40°C. Photophosphorylation was inhibited by N, N-dicyclohexyl carbodiimide (DCCD), carbonyl-cyanide-m-chlorophenylhydrazone and NH₄Cl, whereas proton uptake was enhanced by DCCD. Electron transport was slightly inhibited by these treatments. The membranes could be stored for several weeks at-20°C in 50% glycerol without any loss in the activities.Abbreviations DPIP 2,6-dichlorophenolindophenol - CCCP Carbonyl-cyanide-m-chlorophenylhydrazone - DCCD N,N-dicyclohexyl carbodiimide - PMS N-methylphenazonium methosulfate - DCMU 3-(3-4-dichlorophenyl)-1,1-dimethylurea - TMP 20 mM Tris-HCl buffer pH 7.8, 10 mM MgCl₂, 5 mM phosphate buffer pH 7.8     

Somatic embryogenesis in callus cultures of Rosa hybrida L. cv. Landora

Callus was initiated from immature leaf and stem segments of rose (Rosa hybrida cv. Landora) and subcultured every four weeks on a basal medium of half-strength Murashige & Skoog (1962) salts plus 30 g l^-1 sucrose (1/2 MS) and supplemented with 2.2 M BA, 5.4 M NAA and 2.2–9.0 M 2,4-D. Embryogenic callus and subsequently somatic embryos were obtained from 8-week-old callus culture on 1/2 MS+2.2 M BA+0.05 M NAA+0.3 M GA₃+200–800 mg l^-1 L-proline. Long-term cultures were established and maintained for up to 16 months by repeated subculture of embryogenic callus on L-proline deficient medium. About 12% of cotyledonary stage embryos taken from cultures cold-stored at 8±1°C for 4 days germinated on 1/2 MS+2.2 M BA+0.3 M GA₃+24.7 M adenine sulphate.Abbreviations BA benzyladenine - NAA -naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid     

Characterization of groups of the zygomycete genusRhizopus

Rhizopus is a zygomycetous genus. Several species of this taxon may infect humans and lower animals. Seventeen isolates ofRhizopus species in three distinct morphological groups were studied: the stolonifer group (sporangiophores greater than 1 mm in height, sporangial diameters of 100–275 µm, branched rhizoids); the arrhizus group (sporangiophores greater than 1 mm in height, branched rhizoids, sporangial diameters of 100–240 µm); and the microsporus group (sporangiophores less than 0.8 mm in height, sporangial diameters less than 100 µm, simple rhizoids). Maximal growth temperatures were characteristic: the stolonifer group grew at 30°C, the arrhizus group grew at 36°C, and the microsporus group grew at 45°C. The DNA mol% G + C base composition of all isolates ranged from 34.9 to 40.2% Species within the three groups were grouped by DNA differences. The arrhizus group was most distinctive with a value of 34.9–36.3%; the stolonifer and microsporus groups had G + C values of 37.0–39.3% and 37.8–40.2%, respectively. Our research clarifies and defines the G-C values of the three important groups ofRhizopus species.     

Soybean seedling emergence at high temperatures

Bragg and Cobb soybean [Glycine max (L.) Merr.] seeds were germinated in sand at temperatures ranging from 25 to 40°C. Emergence decreased with increasing temperature above 37°C, with virtually no emergence at 40°C. Emergence of 12 other cultivars at 38°C ranged from 25 to 95%. Foster and Coker 338 were more sensitive to high temperature than the other cultivars.     

Sucrose in storage media and cultivar affects post-storage regrowth of <Emphasis Type="Italic">in vitro</Emphasis> Hosta propagules

The goal of this research was to investigate if culturing in high sucrose (5%) liquid media during multiplication phase (stage II) would enhance endogenous sugar levels and dry matter sufficiently to allow storage of in vitro plants in sugar free media without adversely affecting post-storage recovery. Hosta tokudama Newberry Gold (NBG) and Hosta Striptease were cultured in Murashige and Skoog (MS) media containing 5% sucrose during stage II and transferred to rooting phase (stage III) in MS medium without (0%) sucrose or with 3% sucrose for 4weeks. At the end of stage III, cultures were stored, with the remaining media, at 10°C with 5molm^–2s^–1 photosynthetic photon flux (PPF) from cool white fluorescent lamps for 7 or 14weeks with or without a 2-week dark period prior to removal from storage. In both cultivars, stage III plants cultured in 3% sucrose media had higher soluble sugar levels and greater shoot and root biomass than those cultured in 0% sucrose media. Shoot and root soluble sugars decreased during storage. Shoot growth ceased during storage in both media. Root dry matter continued to increase in plants stored in 3% sucrose media but did not change in 0% sucrose media. Plants cultured in 3% sucrose media had less leaf chlorosis and less mortality after 7 or 14weeks of low temperature storage than the plantlets from sugar free media. Extending the storage period from 7 to 14weeks or introduction of 2-week dark period at the end of storage did not affect leaf chlorosis or plant mortality during acclimatization. Post-storage growth varied with the cultivar. Benefit of having sucrose in storage media was to develop a strong root system that aided the acclimatization and post-storage growth following 7 or 14week storage. Sucrose loading by culturing plants in liquid media containing 5% sucrose did not allow storage in sugar free media without adversely affecting post-storage growth in both cultivars.     

The respiratory chain of the facultative thermophile,Bacillus coagulans

Two oxidases were found to be present in membranes from the facultative thermophile Bacillus coagulans grown at 55°C, compared to one in cells grown at 37°C. Cytochrome spectra and inhibitors of the respiratory chain identified them as cytochrome oxidases aa ₃ and d. Both were present in membranes from 55°C grown cells, but only cytochrome oxidase aa ₃ was found in membranes from 37°C grown cells. The presence of cytochrome d in 55°C grown cultures was found to be due to decreased oxygen tension and not to the high growth temperature. This was confirmed by (a) induction of cytochrome d at 37°C under conditions of oxygen limitation and (b) its repression at 55°C under conditions of high aeration and its subsequent induction on lowering the dissolved oxygen concentration in chemostat cultures. Two cytochromes b (_max 558 and _max 562) were present in both 37°C and 55°C grown cells. Results from the inhibition of substrate oxidation by membranes suggested different pathways of electron transport by the respiratory chain.     

Characterization of a new Bacillus stearothermophilus isolate: a highly thermostable α-amylase-producing strain

A novel strain of Bacillus stearothermophilus was isolated from samples of a potato-processing industry. Compared to known -amylases from other B. stearothermophilus strains, the isolate was found to produce a highly thermostable -amylase. The half-time of inactivation of this -amylase was 5.1 h at 80°C and 2.4 h at 90°C. The temperature optimum for activity of the -amylase was 70°C; the pH optimum for activity was relatively low, in the range 5.5–6.0. -Amylase synthesis was regulated by induction and repression mechanisms. An inverse relationship was found between growth rate and -amylase production. Low starch concentrations and low growth temperatures were favourable for enzyme production by the organism. At the optimal temperature for growth, 65°C, the -amylase was a growth-associated enzyme. The optimal temperature for -amylase production, however, was 40°C, with -amylase increasing from 3.9 units (U)/ml to 143 U/ml when lowering the growth temperature from 65°C to 40°C. Maximal -amylase production in a batch fermentor run at 65°C was 102 U/ml, which was 26-fold higher than in erlenmeyer flasks at 65°C. The dissolved O₂ concentration was found to be a critical factor in production of the -amylase.     

Response of effective quantum yield of photosystem 2 to in situ temperature in three alpine plants

The response of effective quantum yield of photosystem 2 (F/F_m) to temperature was investigated under field conditions (1 950 m a.s.l.) in three alpine plant species with contrasting leaf temperature climates. The in situ temperature response did not follow an optimum curve but under saturating irradiances [PPFD >800 µìmol(photon) m^–2s^–1] highest F/F_m occurred at leaf temperatures below 10°C. This was comparable to the temperature response of antarctic vascular plants. Leaf temperatures between 0 and 15°C were the most frequently (41 to 56%) experienced by the investigated species. At these temperatures, F/F_m was highest in all species (data from all irradiation classes included) but the species differed in the temperature at which F/F_m dropped below 50% (Soldanella pusilla >20°C, Loiseleuria procumbens >25°C, and Saxifraga paniculata >40°C). The in situ response of F/F_m showed significantly higher F/F_m values at saturating PPFD for the species growing in full sunlight (S. paniculata and L. procumbens) than for S. pusilla growing under more moderate PPFD. The effect of increasing PPFD on F/F_m, for a given leaf temperature, was most pronounced in S. pusilla. Despite the broad diurnal leaf temperature amplitude of alpine environments, only in S. paniculata did saturating PPFD occur over a broad range of leaf temperatures (43 K). In the other two species it was half of that (around 20 K). This indicates that the setting of environmental scenarios (leaf temperature×PPFD) in laboratory experiments often likely exceeds the actual environmental demand in the field.This revised version was published online in March 2005 with corrections to the page numbers.     

Production of thermotolerant β-amylase byBacillus circulans

An isolate from a Hong Kong soil sample which produced -amylase was identified as a thermotolerant strain ofBacillus circulans with a growth range of 35 to 55C. The -amylase was stable at 45°C for 30 min but lost half of its activity after 30 min at 50°C. Maximum specific activity of -amylase (36.2 units/mg protein) in the culture broth was detected after 36 h of cultivation at 45°C in a medium containing soluble starch, beef extract, coconut water and inorganic salts.     

Temperature effects on self incompatibility in Lilium longiflorum

Summary Detached pistils of the clonal variety, Lilium longiflorum Aral No. 5, were submerged before pollination in 50°C water for 0, 1, 2, 3, 4, 5, 6 or 7 min and then immediately compatibly and incompatibly pollinated. Incompatibility, as indicated by pollen tube length after 48 h at 23.5°C, was eliminated by a 1–2 min submersion while compatibility was removed by a 4–5 min one. The window of incubation temperatures at which incompatible and compatible pollen tubes are clearly differentiated occurred between 15 and 30°C.     

Initiation and culture of potato tuber callus tissue with picloram

Callus cultures of 7 potato cultivars were initiated from tuber tissue and maintained on Gelrite-solidified media with 1–20 M picloram as the only PGR. Ten M picloram was the optimal concentration for callus induction. By 4–6 weeks after explanting, there was sufficient callus produced for subculture to maintenance media which contained 1–20 M picloram as the only PGR. When grown in the dark at 25°C, subcultured callus typically increased 10-fold in wet weight in 4–5 weeks. The callus produced was friable and a light grey to cream color. Callus cultures were used to establish cell suspension cultures. Callus and cell suspension cultures have been maintained for over 2 years on the picloram containing media.Abbreviations BA benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - MS Murashige-Skoog - NAA naphthaleneacetic acid - PGR plant growth regulator Research paper #9053 of the Idaho Agricultural Experiment Station.     

Cold storage of in vitro cultures of hybrid poplar shoots (Populus alba L. x P. grandidentata Michx.)

Shoot cultures of P. alba x P. grandidentata Crandon were maintained for more than 5 years at 4°C with minimal growth. The highest survival after 2 years and 5 years of cold storage were 70% and 25% respectively using 1-month of pre-storage culture on MS medium containing 1.33 M BA. When 5-year-old cold-stored shoot cultures were transferred to the greenhouse, color variations were observed. The frequencies of albino and red pigmented plants were 0.25% and 12.8%, respectively. A rosette type growth pattern was also observed on 0.3% of the long-term cold-stored plants. During long-term cold storage there were local disruption of cambium connections and the accumulations of chemicals in some cells, as observed by light microscopy.     

Rooting apple cultivars in vitro: Interactions among light,temperature, phloroglucinol and auxin

Delicious apple (Malus domestica Borkh.) and several of its strains, which have been difficult to root in vitro, were successfully propagated with rooting percentages up to 100%. The combination of treatments used to achieve this result included placing the shoots on rooting medium in the dark at 30°C for the first week of the rooting stage, then moving them to a regime of 16 hr light-8 hr dark at 25°C. The rooting medium contained half strength Murashige and Skoog salts plus 1.2 M thiamine HCl, 0.56 mM myo-inositol, 1 mM phloroglucinol (PG), 1.4 M indolebutyric acid (IBA), 1.3 M gibberellic acid (GA₃), 87.6 mM sucrose, and 7 g l^–1 Difco Bacto agar. Dark treatment applied during the proliferation stage (etiolation) was less effective than one applied at the beginning of the rooting stage. The optimum length of dark treatment during rooting was 4 to 7 days. Increasing the temperature from 25°C to 30°C improved rooting of Delicious, Royal Red Delicious, and Vermont Spur Delicious in the absence of PG but generally had less effect in the presence of PG. Further increase in temperature to 35°C stimulated rooting of Royal Red Delicious but reduced rooting of Vermont Spur Delicious. Transfer of the cuttings to auxin-free medium after 1 week had no effect on percentage rooting and increased the number of roots per cutting for only 1 of 4 cultivars tested and then only in the presence of PG. In general PG stimulated rooting of Delicious and its strains, but had no effect on Golden Delicious.     

Adaptive response to cold temperatures and characterization of cspA in Salmonella typhimurium LT2

Salmonella typhimurium is a major foodborne microbial pathogen which primarily contaminates poultry products causing salmonellosis in humans. S. typhimurium LT2 cultures, when transferred from 37 °C to 5 °C or 10 °C, showed an initial lag period in growth with an approximate generation time of 10–25 h. Western blot assay using E. coli CS7.4 antibody and analysis of radiolabeled total cellular proteins from S. typhimurium cultures after exposure to 10 °C or 5 °C showed elevated expression of a major cold shock protein, CS7.4. Identification of a decreased level of CS7.4 at 37 °C suggests that the expression of this protein may require a large temperature downshift. Putative regulatory protein binding segment on the 5-untranslated region referred as Fragment 7 in S. typhimurium exhibited a 90.6% and a 56.25% nucleotide sequence identity when compared with the Fragment 7 of E. coli and S. enteritidis, respectively. The differences in the nucleotide sequence within the Fragment 7 between S. typhimurium and S. enteritidis may explain the differential expression of CspA at 37 °C. The nucleotide sequence of the open reading frame of S. typhimurium cspA gene showed a single base difference at 816 bp position from a G to a C which altered the amino acid residue from a glycine to an alanine. In addition to CspA, an elevated expression of a 105 kDa, and decreased expression of 6 proteins were evidenced when cultures of S. typhimurium were exposed to 10 °C or 5 °C. Differential expression of the CspA and other proteins in S. typhimurium following exposure to cold temperatures suggest that adaptation and continued growth and survival at cold temperatures in this pathogen may be aided by these cold-responsive proteins.     

Slow growth in vitro conservation of coffee (Coffea spp.)

The effects of reduced sucrose concentrations and low temperature on a collection of coffee microcuttings have been examined. Sucrose concentrations of 0.5 g l^-1 and 20 g l^-1 and temperatures of 20°C and 27°C were compared in three accessions: the Arabusta (interspecific hybrid) and Coffea arabica L. cv. Caturra amarillo and cv. Mokka de Tahiti. After six months, low sucrose concentrations reduced microcutting growth, rooting and survival rate. At 20°C, microcutting growth was also reduced, but leaf loss and survival rate were promoted. The genotypic differences at six months were minor. After one year without subculture, survival rate was influenced by sucrose concentration and by genotype. These two species can be cold-stored six months at 20°C on a medium containing at least 20 g l^-1 sucrose.Abbreviations BA 6-benzylaminopurine - MS Murashige & Skoog     

A new technique for storage of meristem-tip cultures of ‘Cavendish’ banana

Meristem-tip tissues of Cavendish banana were incubated at 17°C under light on cheesecloth over cotton saturated with 3% solution of various kinds of sugars. All the sugars tested were found to be capable of extending the survival time of banana tissues and ribose was the best followed by sucrose, glucose, fructose and lactose. After 21 months, 33% of banana tissues incubated with ribose solution remained viable and resumed growth within two weeks after being transferred to fresh SM medium. About 99% of plantlets developed from these tissues were normal. In a separate experiment, 67% of banana tissues incubated with 3% ribose solution remained viable after 24 months. Ribose solutions at 0.3% and 0.03% were not as effective and over saturation of cotton with 3% ribose solution was not beneficial. The new technique may be useful for storage of tissue cultures of other species and cultivars.     

In vitro propagation of Alstroemeria ‘Alsaan’

Plantlets were regenerated from Alstroemeria Alsaan rhizome tips cultured in vitro on solid and liquid media based on Murashige and Skoog salt formulation. The quality of the cultures was superior when intact rather than longitudinally sliced rhizome tips were used as explants and when a temperature of 8°C rather than 22°C was used at the initiation stage. More roots were produced on rhizome tips containing a rhizome apical meristem than on rhizome sections lacking such a meristem. Most (90%) of the rooted plantlets were successfully acclimatized and developed into true-to-type flowering plants.