Induction of freezing tolerance in alfalfa cell suspension cultures

The effects of ABA, 2,4-D, kinetin and cold exposure on the cold hardiness of Medicago sativa L. cell suspensions were investigated. Cultures treated with 5×10^–5 M ABA at 2°C for 4 weeks in the absence of kinetin showed a 50% survival after freezing to –12.5°C, whereas cultures grown at 25°C under normal conditions tolerated freezing to only –3°C. The optimum ABA treatment of 5×10^–5 M for 4 weeks was effective only in combination with cold exposure. Of six cell lines tested, all showed different degrees of induced cold hardiness. The results suggest that ABA alone cannot induce freezing tolerance on alfalfa cell suspension cultures and that the deletion of kinetin and combination of low temperature and ABA is critical for the induction of cold hardiness in alfalfa cell suspension cultures.Abbreviations ABA abscisic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - LT₅₀ 50% killing temperature     

Cryopreservation of periwinkle,Catharanthus roseus cells cultured in vitro

A procedure for prolonged cryogenic storage of periwinkle cell cultures is described. Cells derived from periwinkle, Catharanthus roseus (L.) G. Don, and subcultured as suspension in 1-B5C nutrient medium have been frozen, stored in liquid nitrogen (–196°C) for 11 weeks, thawed and recultured. Maximal survival was achieved when 3–4 day-old cells precultured for 24 h in nutrient medium with 5% DMSO were frozen at slow cooling rates of 0.5 or 1°C/min prior to storage in liquid nitrogen. The only loss in viability of cells occurred subsequent to treatment with DMSO. Abbreviations: DMSO, dimethylsulfoxide; 2,4-D, 2,4-dichlorophenoxyacetic acid; TTC, triphenyltetrazolium chloride.NRCC No. 20082     

A cellulase-free xylanase from alkali-tolerantAspergillus fischeri Fxn1

Summary An alkali-tolerant fungusAsperqillus fischeri Fxn1 isolated from xylan enrichment grew in the pH range 5–10 and secreted an extracellular cellulase-free xylanase. Arabinose, lactose, maltose, cellobiose and glucose induced low levels of xylanase (1.8–9.0 IU/ml), whereas xylose, xylan and wheat bran induced higher level (34–45 IU/ml).CMcellulose and FPcellulose did not support growth. The optimum pH of xylanase was 6.0–6.5 and it was stable in a wide range of pH 5–9.5. The optimum temperature was 60°C and it was stable upto 55°C. The half-lives at 50 and 55 °C were 240 and 40 min. respectively. This enzyme released reducing sugars from pulp at pH 9.0 and 40°C.     

Production of dextranase byLipomyces starkeyi

Summary The optimal growth rate ofLipomyces starkeyi, with dextran as sole carbon source, was found within the pH range 2.5–4.0, and temperature between 25–30°C. This yeast was unable to grow above 33°C. Dextranase production optima paralleled growth optima, except at pH 2.5. Decrease in enzyme yield at this pH could not be attributed to poor yeast growth or enzyme stability.     

In vitro vegetative propagation of Leucaena leucocephala (Lam.) de Wit

A method for in vitro clonal multiplication of Leucaena leucocephala cv K-8 is described. On MS with BAP (3×10^–6M), at the optimum temperature of 30°C, shoots from seedling and adult trees multiplied at a rate of 6–7 fold every three weeks. The addition of adenine or glutamine reduced precocious leaf drop. All shoots rooted on MS with IAA (5×10^–6M). Micropropagated plants have been successfully transferred to soil.     

Ethanol production by Zymomonas mobilis CP4 from sugar cane chips

Summary The fermentation of large sugar cane chips (1.0–1.5 in) to ethanol by Zymomonas mobilis CP4 (Z. mobilis) was studied in two glass fermentors operating with culture circulation for agitation (the EX-FERM type): a. A laboratory scale(2.5 liter) cylindrical vessel; b. A bench scale (8 liter) wide vessel. Z. mobilis cultures consumed 89–96% of the cane sucrose, converting it to ethanol by 90–97% of the theoretical yield in the laboratory scale fermentor and by 83–90% in the bench scale fermentor culture. Comparative Saccharomyces spp. cultures in laboratory fermentor consumed 96–98% of the cane sucrose, with ethanol conversion of only 75–79% of the theoretical yield.These preliminary results indicated that sucrose in agricultural size sugar cane chips was ethanol fermentable as compared to small size sugar cane chips or to sugar cane juice. Z. mobilis CP4 cultures converted sucrose more efficiently to ethanol than Saccharomyces spp. as shown in the laboratory scale fermentor studies.The ethanol yields in a wide bench scale fermentor cultures were slightly lower than in a laboratory fermentor.     

A simple culture method for Brassica hypototyl protoplasts

Hypocotyl protoplasts from oil rape, Brassica napus L. cv. Isuzu were cultured in the dark at 25°C in a modified Nitsch and Nitsch medium containing 13% sucrose, 5 g/l Ficoll, 0.5 mg/l BAP, 1 mg/l NAA and 0.5 mg/l 2–4 D. Protoplasts floated on the surface of the medium and developed into microcolonies 0.5 mm in diameter in 4–6 weeks. The microcolonies also remained on the surface of the medium. Transfer to MS medium supplemented with 200 mg/l casein hydrolysate, 5mg/l BAP, 0.5 mg/l NAA and solidified with 0.6% agarose induced shoot regeneration in 3–4 weeks.Abbreviations BAP 6-benzylaminopurine - NAA 1-naphthaleneacetic acid - 2,4-D 2,4 — dichlorophenoxyacetic acid     

Somatic embryogenesis and organogenesis in callus cultures of a tree legume — Albizia richardiana King

Hypocotyl explants of 1 and 10 mm lengths were excised from 12-day-old in vitro-grown seedlings of Albizia richardiana. The larger pieces, after 40 days of culture, developed shoots along with green calli on B₅ + BAP (10^–7–10^–5M), while the smaller segments produced only green calli on B₅+BAP (10^–7–10^–4M) medium. Some of the green calli turned morphogenic and started producing somatic embryos with the 2nd sub-culture and shoots from 7th sub-culture onwards. Calli retained the morphogenic potential even after repeated sub-culturing for over two years. The number of embryos in an embryogenic culture varied from 2 to 20 per callus mass of 5–6.5 cm³. Sucrose at the 2% level in MS medium was optimal for embryogenesis while 4% was optimal for shoot bud differentiation. Higher levels of sucrose (6–10%) caused browning of green calli and also inhibited differentiation into embryos and shoot buds. By selective sub-culturing of 0.1 cm³ pieces of embryogenic calli on MS+10^–5M BAP, 46% of the cultures produced somatic embryos. The latter germinated into plantlets on Knop's medium.Abbreviations BAP 6-benzylaminopurine - B₅ Gamborg et al., 1968 medium - IAA Indole-3-acetic acid - MS Murashige and Skoog's (1962) medium     

Optimum tissue culture conditions for selection of resistance to Phytophtora cinnamomi in pine callus tissue

The present experimentation compared the best nutrient medium, temperature, and growth hormones for callus induction and growth of various pine species from different seed sources with their effect on growth of Phytophthora cinnamomi. Callus tissues maintained on a modified Murashige and Skoog medium with 10^–5M 2,4-D at 26°C in the dark optimized the expression of differential resistance when inoculated with hyphae of P. cinnamomi. High concentration of 2,4-D (5×10^–5M) inhibited growth of P. cinnamomi.Abbreviations AL loblolly pine-Alabama - PL South Carolina - AS shortleaf pine-Alabama - CS Georgia - AV Virginia pine-Alabama     

Biological studies upon Enchytraeus variatus Bouguenec & Giani 1987 in breeding cultures

The life history of an enchytraeid worm, Enchytraeus variatus, was studied under laboratory conditions at 18–22 °C. This species can reproduce simultaneously by asexual (architomy) or sexual reproduction. The number of ova per cocoon varies from 5 to 20 (x = 10.9). The generation period (from cocoon to next cocoon) varies from 14 to 39 days (x = 26.1) according to the period of the year. The number of generations per year is between 7.3 and 26.1 (x = 14). A mature worm can lay between 23.7 and 25.8 cocoons during its life (254 days as maximum observed) at a mean rate of 0.12 cocoon worm^–1 day^–1. Experimental cultures were carried out to determine the structure, density and biomass of the populations. A maximal density of 1 396 314 worms was recorded after 85 days of culture. Net production reached 21.48 g m^–2 day^–1 after 26 days in a culture initiated from cocoons.     

Root hair protoplasts ofLotus corniculatus L. (birdsfoot trefoil) express their totipotency

Treatment of the roots of 24–48 h old seedlings of the forage legumeLotus corniculatus with 1.0% Cellulase YC, and 0.1% Pectolyase Y-23 in 4.2% mannitol solution released protoplasts from the tips of root hairs within 30–40 sec of enzyme incubation. Roots from approximately 1000 seedlings yielded 1.7×10⁵ protoplasts. Ten percent of protoplasts divided to form cell colonies when cultured at 1.0×10⁵ ml^–1 in droplets of KM8P medium with 0.6% Sea Plaque agarose. Colonies formed callus on UM agar medium; protoplast-derived tissues produced shoots on B5 medium containing 0.05 mg 1^–1 of BAP. Regenerated plants were phenotypically and cytologically normal (2n=2x=24±2), and produced nitrogen fixing root nodules following inoculation withRhizobium. These results confirm the totipotency of protoplasts isolated from specialised epidermal cells of seedling roots ofLotus corniculatus.     

Effect of temperature on sucrose to ethanol conversion by Zymomonas mobilis strains

Summary Two strains of Zymomonas mobilis were tested for their ability to ferment sucrose to ethanol at elevated temperatures (30–42.5°C). The optimal temperature for efficient sucrose to ethanol conversion was 35°C with 22–27 h fermentation time and 75% conversion efficiency. Increases in magnesium concentration improved one of the strains at 40°C from 38 to 76% ethanol yield efficiency.     

Removal of NH3-N from domestic waste water by fungi

Summary Nine species of fungi viz.,Aspergillus niger,A. flavus,A. terreus,Fusarium solani,Mucor sp.,Neurospora crassa,Penicillium janthinellum,Trichoderma harzianum andTrichothecium roseum were evaluated for their potential to remove NH₃–N from domestic waste water. Of the fungi tested,A. flavus was found to be the most effective in the removal of NH₃–N. Maximum reduction (92%) of NH₃–N by this organism was observed at pH 8.0 at 20°C.     

Nutritional and hormonal requirements of Ginkgo biloba embryo-derived callus and suspension cell culture

Calli were obtained from Ginkgo biloba embryos grown on Murashige and Skoog (MS) medium. The G. biloba cells could grow on either MS or Gamborg B5 mineral salt medium supplemented with sucrose (3% and 2%, respectively) and naphthaleneacetic acid (NAA) and kinetin (K) in concentrations ranging from 0.1 to 2.0 mg·L^–1. Best growth and maintenance of callus cultures were achieved using MS medium supplemented with 2 mg·L^–1 NAA and 1 mg·L^–1 K (N₂K₁MS). Light was required to maintain healthy growth of the callus tissue.In both MS and B5 based media, sucrose was hydrolyzed extracellularly before being taken up by Ginkgo cell suspension cultures. Specific growth rates of 0.13 d^–1 and 0.08 d^–1 were obtained in MS medium supplemented with 1 mg·L^–1 NAA, 0.1 mg·L^–1 K and 30 g·L^–1 sucrose (N₁K_0.1MS) and B5 medium supplemented with the same growth regulator regime and 20 g·L^–1 sucrose (N₁K_0.1B5) respectively. Complete phosphate and ammonium uptake was observed in 11 days when cultured in MS medium and 10 days and 4 days respectively when cultured in B5 medium. During the culture, G. biloba cells consumed only 64% and 29% of the nitrate content of N₁K_0.1MS and N₁K_0.1B5 media respectively. Maximum dry biomass concentrations were 13.4 g·L^–1 and 7.9 g·L^–1, and yields on carbohydrate were 0.39 and 0.45 in N₁K_0.1MS and N₁K_0.1B5 media respectively. The better performance of MS cultures came from the higher sucrose and nitrogen salts concentrations of this medium.Abbreviations B5 Gamborg mineral salt medium - d.w. Dry weight - K Kinetin - MS Murashige and Skoog mineral salt medium - N or NAA Naphthaleneacetic acid - N_iK_jMS i and j are the respective concentrations (mg·L^–1) of NAA and K - n Number of experimental points - r Linear regression correlation coefficient     

Effluent treatment by anAzotobacter sp.

Summary The effect of pH, temperature and carbon source on the specific growth rate of anAzobacter sp. was studied in nitrogen-deficient media. The optimum pH and temperature were 7.0 and 30°C respectively, with COD removal 27–85%. The strain was also used for treating effluents of a pharmaceutical industry, with 37–45% COD removal.     

Silver toxicity to ferrous iron and pyrite oxidation and its alleviation by yeast extract in cultures ofThiobacillus ferrooxidans

Summary Ferrous-ion oxidation byThiobacillus ferrooxidans was inhibited by 10^–6 M Ag⁺ while a slight inhibition of growth was apparent with 10^–7 M Ag⁺. The threshold toxic concentration was the seme for four different test strains. While prolonged lag phases resulted from culture exposure to Ag⁺, Fe²⁺ oxidation rates after the onset of growth showed little variation under these conditions. Yeast extract (0.02%) partially alleviated the toxicity of silver-ion by reducing the lag periods. Pyrite oxidation byT. ferrooxidans and mixed cultures of acidophiles was tested at 8.3×10^–7 to 8.3×10^–5 M Ag⁺. Strong inhibition was apparent at 8.3×10^–5 M Ag⁺ and little to no inhibition was observed at 8.3×10^–7 M Ag⁺.     

Chrysonila sitophila (TFB-27441): A hyperlignolytic strain

Summary C. sitophila strain TFB-27441 showed 2–3 times higher lignolytic activity thanPhanerochaete chrysosporium (BKM-F-1767 strain). Lignin had a marked effect on the ligninase activity indicating that some induction or activation mechanism is involved in lignin degradation byC. sitophila.     

Halophilic n-alkane utilizing yeast from the Arabian gulf

Summary A facultative halophilic yeastTorulopsis candida (Saito) Lodder, isolated from the Arabian Gulf, was oua to grow on n-alkanes (C₁₃–C₁₉) at 37°C and pH 3–7 in media containing 1.4- 3M NaCl or 30–90% seawater. The influence of pH, temperature, growth factors, salt and pure n-alkanes on its growth were studied. Its amino acid profile and crude protein were compatible with those of commercial n-alkane yeast products.     

Induction of freezing tolerance in microspore-derived embryos of winter Brassica napus

Freezing tolerance was induced in microspore derived embryos of winter Brassica napus cv. Jet neuf by the addition of ABA or mefluidide to the culture media during embryogenesis. Survival after freezing was estimated by culture of frozen-thawed embryos to plantlets. A higher freezing tolerance (50% survival at –15°C) was induced when 50 M ABA or 3.2 M mefluidide was incorporated initially into the medium during embryogenesis at 25°C followed by culture at 2°C for 3 weeks. When embryos were induced in the absence of ABA or mefluidide and maintained at 2°C for even as long as 12 weeks a lower degree of freezing tolerance (10% survival at –15°C) was obtained. Plants regenerated from embryos hardened maximally by a combination of either ABA or MFD with low temperature did not require further vernalization for flowering.Abbreviations ABA abscisic acid - MFD mefluidide - 2,4-D 2,4-dichlorophenoxyacetic acid - LT₅₀ killing temperature for 50% of the embryos     

Solid state fermentation for production of higher titres of thermostable alpha-amylase with two peaks for pH optima byBacillus licheniformis M27

Summary Bacillus licheniformis M27 produced 21, 000 units of alpha-amylase/g dry bacterial bran under solid state fermentation in wheat bran medium enriched with 3.3% di-ammonium hydrogen phosphate. The crude enzyme, with temperature optimum at 90°C in 0.5% starch solution, showed pH optima at 6.5–7.0 and 9.5 and over 75% activity over the pH range 6.0–10.5.