Sago: An Alternative Cheap Gelling Agent for Potato In Vitro Culture

Sago, a processed (gelatinized) edible starch, was successfully used as a gelling agent in culture medium. The efficacy of sago-gelled (80 g dm^–3) medium was studied in ten potato (Solanum tuberosum L.) genotypes during micropropagation and minimal growth conservation. Sago starch provided a firm gelling surface throughout the entire culture period, and fostered optimum plantlet growth in terms of shoot height, number of nodes per plant, number of leaves and fresh mass. No softening of the sago-gelled medium occurred over prolonged (six months) storage. The study showed that sago starch could be used as a substitute to agar in culture medium to substantially reduce the medium cost.     

Direct organogenesis in hop - a prerequisite for an application ofA. tumefaciens-mediated transformation

The regeneration ability of primary explants derived from mericlones of two commercial Bohemian hops was investigated. It was found that these hops are able to regenerate shoots by direct organogenesis on media containing BAP or zeatin at concentrations 0.5–2 mg dm⁻³. The highest regeneration of shoots was achieved from either petioles or internodes at frequencies 21% and 52%, respectively, on the medium containing zeatin (2 mg dm⁻³), while relatively low amount of regenerated shoots (1.3%) was observed for leaf blade explants. On the other hand, more efficient rooting occurred on the leaf blades then on other explants. A similar pattern of regeneration we observed for HLVd-infected mericlones of clone Osvald 31 even though viroid concentration inin vitro cultures was about 8-fold higher than in field-grown plants and was 31.1 pg mg⁻¹ of fresh mass in the average. These results suggest that HLVd infection did not impair organogenesis. We found that high 2,4-D concentration pretreatment (11 mg dm⁻³) did not promote somatic embryogenesis. Although this treatment suppressed direct organogenesis, the inhibition was not complete and in low frequency the shoot regeneration was seen. Sensitivity of hop explants to antibiotics commonly used inAgrobacterium-mediated transformation was assayed. It was found that kanamycin (100–200 mg dm⁻³) suppressed efficiently callogenesis, root formation and shoot proliferation. An estimation of effect of kanamycin (200 mg dm⁻³) and ticarcillin (500 mg dm⁻³) on morphogenesis was performed using regeneration medium. The inhibitory effects observed suggest that these conditions could be used inAgrobacterium transformation/selection system. Communicated by J. TUPY     

Factors Affecting In Vitro Multiplication of Date Palm

Rapid method of in vitro multiplication of date palm was developed. Shoot tips were cultured on Murashige and Skoog (MS) medium supplemented with 2 mg dm^–3 dimethylaminopurine (2iP) + 1 mg dm^–3 naphthalene acetic acid (NAA). Shoot buds were proliferated from white nodular cultures on hormone free medium. Shoot bud proliferation strongly enhanced when cultured on MS-medium contained 3 mg dm^–3 2iP + 0.5 mg dm^–3 NAA. Culturing on full-strength MS medium showed higher multiplication rate compared with half-strength MS medium. Among four concentrations of sucrose used, 30 g dm^–3 speeded up the bud proliferation more than 10, 20 and 40 g dm^–3. However, the largest shoot buds were observed with 40 g dm^–3 sucrose. Solidification of culture media by 1.75 g dm^–3 Phytagel showed the highest proliferation rate, but the largest buds were observed with 1 g dm^–3 Phytagel.     

Wood biodeterioration control potential of Acalypha hispida leaf phenolic extract in combination with Trichoderma viride culture filtrate

The phenolic extract of Acalypha leaves inhibited growth of Gloeophyllum sepiarium and Pleurotus sp. (test wood-rot fungi) in potato dextrose agar, starch agar, starch glucose agar, carboxyl methyl cellulose agar and carboxyl methyl cellulose glucose agar. Fungicidal or fungistatic concentration of the extract (10–14 mg/ml) depended on the medium. However a lower concentration of the extract (8–10 mg/ml) in combination with Trichoderma viride culture filtrate caused a similar inhibitory pattern. Degradation of obeche (Triplochiton scleroxylon), mahogany (Khaya ivorensis) and walnut (Lovoa trichilioides) by the test fungi was limited or prevented by extract treatment of 8–10 mg/g wood. A similar inhibitory effect again occurred when a combination of T. viride filtrate and lower extract concentration (6–8 mg extract per gram of wood) was used. On-going wood decay was limited or halted by a combined treatment involving 8–12 mg extract per gram of wood depending on the fungal residence period. Treated stakes exposed to 6 months of tropical wet season retained resistance to fungal attack including soft rot. The phenolic extract of A. hispida may prove useful in an integrated chemical and biological approach to wood treatment.     

Shoot Regeneration from Immature Cotyledons of Cicer Arietinum

Shoot regeneration was achieved from immature cotyledons of five chickpea (Cicer arietinum L.) genotypes: C235, ICC4971, ICC11531, ICC12257 and ICC12873. The cotyledons cultured on Murashige and Skoog (MS) medium supplemented with 3 or 5 mg dm^–3 zeatin with or without 0.04 mg dm^–3 indole acetic acid (IAA) showed formation of cotyledon like structures (CLS) at their proximal ends. Subsequently, shoot regeneration took place in some of the CLS forming explants. CLS were also formed in cotyledons cultured on MS + 0.2 – 1 mg dm^–3 thidiazuron (TDZ); direct shoot regeneration was observed in cotyledons cultured on 1 mg dm^–3 TDZ. The shoot buds elongated on media containing indole butyric acid (IBA), benzylaminopurine (BAP) and gibberellic acid (GA₃). Complete plantlets were obtained by rooting of shoots following pulse treatment with 200 mg dm^–3 IBA for 5 min and culture on growth regulator free half-strength MS medium.     

Use of phosphomannose isomerase-based selection system for <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of tomato and potato

Two selection systems for Agrobacterium tumefaciens mediated transformation of tomato and potato were compared. In the tomato (Lycopersicon esculentum cv. Moneymaker), the highest transformation rate, 4.2 %, of cotyledon explants on mannose-selection medium was obtained when mannose/sucrose concentration in the regeneration medium was 5/15 g dm⁻³. The best transformation efficacy with the commonly used concentration of 100 mg dm⁻³ kanamycin as a selection agent was 9 %. In the potato (Solanum tuberosum cv. Bintje), the highest transformation frequency was 53.3 % when mannose concentration in the regeneration medium was 5 g dm⁻³ during the first 3 weeks after transformation and 10 g dm⁻³ afterwards. The optimum concentration of sucrose was 20 g dm⁻³. The transformation efficiency using kanamycin as a selection agent at a concentration 100 mg dm⁻³ was 33.3 % with potato. Our results demonstrate that the transformation efficiency using mannose selection is 1.6-fold higher for potato and about 2 times lower for tomato comparing with the ordinary protocol using kanamycin.     

Efficient regeneration of <Emphasis Type="Italic">Brassica napus</Emphasis> L. hypocotyls and genetic transformation by <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>

An efficient system for shoot regeneration and Agrobacterium-mediated gene transfer into Brassica napus was developed through the modification of the culture conditions. Different concentrations of benzyladenine (1.5, 3.0 and 4.5 mg dm^–3) and thidiazuron (0.0, 0.15 and 0.30 mg dm^–3) were evaluated for shoot regeneration of 7, 14 and 21-d-old hypocotyl explants. Maximum shoot regeneration frequency was obtained in 21-d-old explants using 4.5 mg dm^–3 benzyladenine and 0.3 mg dm^–3 thidiazuron. Under above culture condition, the highest percentage of shoot regeneration frequency was 200 %. Agrobacterium-infected explants grown on the selection medium gave rise to transgenic shoots at a frequency of 11.8 %. Transformed shoots rooted when cultured on a medium supplemented with 2 mg dm^–3 of indolebutyric acid and 10 mg dm^–3 kanamycin. The rooted plantlets were successfully established in the soil and developed fertile flowers and viable seeds. Evidences for transformation were confirmed by GUS assay and PCR analysis.     

Micropropagation of an Endangered Orchid Anoectochilus formosanus

A rapid and efficient procedure is outlined for in vitro clonal propagation of an elite cultivar of jewel orchid (Anoectochilus formosanus). Multiple shoot proliferation was induced in shoot tip explants on Hyponex (H3) media supplemented with 1 mg dm^–3 benzyladenine or 1 – 2 mg dm^–3 thidiazuron (TDZ). Addition of activated charcoal (1 g dm^–3) to the TDZ containing medium promoted multiple shoot formation (11.1 shoots per explant). However, the regenerated shoots had slow growth rate and failed to elongate. This problem was overcome by transferring the shoot clumps to a hormone free H3 medium supplemented with 2 % sucrose and 0.5 g dm^–3 activated charcoal. Rooting was induced in 100 % of the regenerated shoots in the same media. The plantlets were acclimatized and established in greenhouse.     

Production of an amylase-degrading raw starch by Gibberella pulicaris

An endophytic fungus, Gibberella pulicaris, produced an amylase which degraded raw starches from cereals and other crops including raw potato, sago, tapioca, corn, wheat and rice starch. In each case, glucose was the main product. Among the raw starches used, raw potato starch gave the highest enzyme activity (85 units mg^–1 protein) and raw wheat starch the lowest (49 units mg^–1 protein). The highest amylase production (260 units mg^–1 protein) was achieved when the concentration of raw potato starch was increased to 60 g l^–1. Optimum hydrolysis was at 40°C and pH 5.5.     

Growth kinetics ofSaccharomyces cerevisiae in batch and fedbatch cultivation using sugarcane molasses and glucose syrup from cassava starch

Growth kinetics ofSaccharomyces cerevisiae in glucose syrup from cassava starch and sugarcane molasses were studied using batch and fed-batch cultivation. The optimum temperature and pH required for growth were 30°C and pH 5.5, respectively. In batch culture the productivity and overall cell yield were 0.31 g L^–1 h^–1 and 0.23 g cells g^–1 sugar, respectively, on glucose syrup and 0.22 g L^–1 h^–1 and 0.18 g cells g^–1 sugar, respectively, on molasses. In fed-batch cultivation, a productivity of 3.12 g L^–1 h^–1 and an overall cell yield of 0.52 g cells g^–1 sugar in glucose syrup cultivation and a productivity of 2.33 g L^–1 h^–1 and an overall cell yield of 0.46 g cells g^–1 sugar were achieved in molasses cultivation by controlling the reducing sugar concentration at its optimum level obtained from the fermentation model. By using an on-line ethanol sensor combined with a porous Teflon^® tubing method in automating the feeding of substrate in the fed-batch culture, a productivity of 2.15 g L^–1 h^–1 with a yield of 0.47 g cells g^–1 sugar was achieved using glucose syrup as substrate when ethanol concentration was kept at a constant level by automatic control.     

Adventitious Shoot Regeneration and Micropropagation in Calendula officinalis L.

Hypocotyl, cotyledon and cotyledonary node explants of Calendula officinalis L were cultured on Murashige and Skoog (MS) media supplemented with various concentrations of thidiazuron (TDZ), kinetin (KIN), -naphthaleneacetic acid (NAA) and indole-3-butyric acid (IBA) to induce adventitious shoot regeneration and micropropagation. The highest frequency of adventitious shoot regeneration was achieved from hypocotyl and cotyledon explants on MS media supplemented with 0.75 mg dm^–3 TDZ and either 0.25 or 0.50 mg dm^–3 IBA. Efficient in vitro clonal propagation was also induced from cotyledonary nodes on a range of media supplemented with 0.75 mg dm^–3 TDZ and 0.05 mg dm^–3 NAA or 2 mg dm^–3 KIN and 1 mg dm^–3 NAA. Regenerated shoots were excised and rooted in MS medium supplemented with 1 mg dm^–3 NAA. The rooted plantlets were finally transferred to pots.     

Solanum Nigrum is a Model System in Plant Tissue and Protoplast Cultures

Solanum nigrum is a model system especially for newcomer to the subject of plant tissue culture. Shoot culture has been easily established from shoot cutting of germinated seeds on Gamborg (B5), or Murashige and Skoog (MS) medium without phytohormones. Direct regeneration was possible using basal media B5, B5C (B5 supplemented with 5 % coconut endosperm milk), Schenk and Hildebrandt (SH), and MS, leaf, stem, shoot tip as explants, cytokinins benzylaminopurine (BAP) or kinetin (KIN) at concentrations from 0.25 to 2 mg dm^–3, and different light treatments (dark, dim and normal light). The best culture condition for shoot formation was the culture of stem internode segments on B5 medium supplemented with 0.5 mg dm^–3 BAP at 16-h photoperiod (irradiance of 100 µmol m^–2 s^–1). Also, root formation was possible under different culture conditions. The best culture condition was the culture of microshoot segments on half strength MS medium supplemented with 1 mg dm^–3 isobutyric acid. Induction of callus formation from young and mature tissues on MS medium supplemented with 0.5 mg dm^–3 BAP, 0.1 mg dm^–3 2,4-dichlorophenoxyacetic acid and 1 mg dm^–3 naphthalene acetic acid, and subsequent plant regeneration on B5C medium supplemented with 0.5 mg dm^–3 BAP was easy. Regeneration of protoplasts isolated from shoot tips and fully expanded leaves was also simple. Finally, the transfer of rooted plantlets to the soil was successful.     

Morphogenetic effect of glycerol on tissue cultures of the red seaweed Grateloupia doryphora

Explants of Grateloupia doryphora were cultivated in Provasoli Enriched Seawater culture medium (PES) supplemented with glycerol (0.1, 0.3, 0.5 or 0.8 mol 1^–1) or carbohydrates (0.1 or 0.3 mol 1^–1 mannose, glucose and galactose) and agar (3, 8, 15 g 1^–1 ). The osmolality of the medium was adjusted by dilution of the seawater (70 or 100%, v/v). The increase in fresh weight of explants cultivated in liquid medium with glycerol (0.3 mol 1^–1) and without glycerol was compared. All experiments were carried out in the light, except for one assay in which the explants were cultivated in the dark. Glycerol was an effective carbon source for the vegetative propagation of G. doryphora in solid and liquid media. Mannose, glucose and galactose all had no effect on growth or morphogenesis of the explants. In solid media the main effect of glycerol was as a morphogenetic inductor, with PES70 (70% seawater) + 0.1 or 0.3 mol 1^–1 glycerol + 3 or 8 g 1^–1 agar the best formulation. An increase in the concentration of agar in glycerol-containing medium reduced the morphogenetic capacity of the explants, which developed into compact cell masses. The effects of glycerol were observed only in explants cultivated under light.     

Micropropagation of Endangered Species Daphne cneorum

A new protocol for micropropagation of endangered Daphne cneorum through multiple shoot formation has been developed. Two different types of explants (dormant apical buds and in vitro seed-derived young seedlings) from plants in two different localities were used for the initiation of multiple shoots on agar woody plant medium (WPM) with 0.2 mg dm^–3 benzylaminopurine (BAP), 0.1 mg dm^–3-indolebutyric acid (IBA), 200 mg dm^–3 glutamine, and 200 mg dm^–3 casein hydrolysate. From 10 seeds only one germinated and the multi-apex culture bearing 12 shoots sprouted out from in vitro seed-derived young seedling. After 6-month cultivation 35 multi-apex cultures were achieved from in vitro seed-derived young seedling. On 1/3 strength WPM medium supplemented with 2.83 mg dm^–3 IBA 50 % of cultures (clusters of 3 – 5 shoots) rooted but no rooting occurred in the presence of -naphthaleneacetic acid (NAA). The rooted plantlets were acclimatized for 4 weeks in the greenhouse and then transferred into natural conditions. The plants successfully survived the winter and flowered.     

Callus Induction and in vitro Regeneration from Barley Mature Embryos

We have assayed different combinations of nutrient media and growth regulators to induce callus and plant regeneration from explants of root, shoot and leaf, complete seed, and isolated mature embryo of barley (Hordeum vulgare L. cv. Hassan). The best results were obtained with mature embryo in J25-8 medium supplemented with 2.0 mg dm^–3 2,4-dichlorophenoxyacetic acid where about 75 % developed friable calli. Some 80 – 85 % of these calli regenerated barley plants in the same J25-8 medium supplemented with 1.0 mg dm^–3 indole-3-butyric acid and 0.1 mg dm^–3 kinetin.     

Growth of Fusarium moniliforme on n-Alkanes: Comparison of an immobilization method with conventional processes

Summary In submerged culture there was negligible growth of Fusarium moniliforme with either n-tetradecane or gasoil (C₁₃–C₁₉) as the only carbon and energy source. In surface culture the cell yield was about 0.25 g dm^–3 dry weight after four weeks incubation. Some oxidation products, mainly isomeric tetradecanones (4-one, 5-one, 6-one and 7-one), could be identified. However the cell yield in a trickle-flow column was about 3 g dm^–3 dry weight after 7 days. Only traces of oxidation products could be detected. In a fixed-bed reactor, filled with glass rings, cell yields were similar to those in the trickle-flow column and depended on the medium flow rate.After termination of growth in the fixed-bed reactor, similar amounts of gibberellic acid were produced in a nitrogen-free medium with either gasoil or glucose.     

Soy-based medium for ethanol production byEscherichia coli KO11

An optimized soy-based medium was developed for ethanol production byEscherichia coli KO11. The medium consists of mineral salts, vitamins, crude enzymatic hydrolysate of soy and fermentable sugar. Ethanol produced after 24 h was used as an endpoint in bioassays to optimize hydrolysate preparation. Although longer fermentation times were required with soy medium than with LB medium, similar final ethanol concentrations were achieved (44–45 g ethanol L^–1 from 100 g glucose L^–1). The cost of materials for soy medium (excluding sugar) was estimated to be $0.003 L^–1 broth, $0.006 L^–1 ethanol.     

The effects of the physical characteristics of the culture medium on the development of red seaweeds in tissue culture

Explants of Gelidium versicolor, Grateloupia doryphora and Laurencia sp. were cultivated in Provasoli enriched seawater culture medium (PES) adjusted to several osmolalities (0.5, 0.7, 1.0 and 1.5 Os kg^–1) and solidities (agar concentration = 3, 8 and 15 g L^–1). Osmolality was adjusted by dilution of seawater with distilled water (50, 70 and 100% seawater) and by NaCl addition. Explants of Laurencia sp. and Grateloupia doryphora showed bud regeneration and callus formation. Explants of Gelidium versicolor only showed bud regeneration. Osmolalities of 0.5 and 1.05 Os kg^–1. inhibited or drastically reduced bud regeneration and callus formation. The highest callus formation and bud regeneration were observed at 0.7 to 1.0 Os kg^–1. An increase in the agar concentration of the culture medium was positively correlated with callus formation and negatively correlated with bud regeneration. An increase in the percentage of seawater increased the solidity of the culture medium and was positively correlated with callus formation. Glycerol was an effective carbon source for the vegetative propagation of axenic explants of Grateloupia doryphora, promoting growth and bud regeneration. An increase in glycerol concentration in the culture medium increased its osmolality, inhibiting the growth of the explants and their morphogenetic development.     

High Frequency Multiple Shoot Regeneration from Decapitated Embryo Axes of Chickpea and Establishment of Plantlets in the Open Environment

Multiple shoot regeneration from the cut plumular ends of embryo axes of chickpea (Cicer arietinum L.) was evaluated on Murashige and Skoog medium having different concentrations of thidiazuron (TDZ) (0.1 to 10.0 mg dm^–3) 6-benzylaminopurine (BAP) (0.5 and 1.0 mg dm^–3), kinetin (0.5 and 1.0 mg dm^–3) or zeatin (2.0 and 4.0 mg dm^–3). TDZ (0.2 mg dm^–3) was found to be the most effective cytokinin as it produced multiple shoots in 100 % of the explants from genotypes C235, ICC5166, ICC12269, ICC4951, ICC11531, BG256 and a local cultivar. Shoots were elongated on growth regulator-free medium, and rooted on growth regulator-free medium containing 1/4 MS salts + full vitamins + 3 % sucrose. Plantlets formed were acclimatized for 12 – 15 d in MS medium with a gradual reduction in sucrose concentration and transferred into pots filled with soil and kept in the field; this resulted in more than 70 % survival. The plants developed normally and produced fertile flowers and set seeds. Low temperatures, maximum 19.0 °C, and minimum 8.2 °C, during the first 15 d of transfer favoured survival on transfer to pots.     

Multiple Shoot Induction from Cotyledonary Node Explants of Terminalia chebula

A protocol for multiple shoot induction from cotyledonary node explants of Terminalia chebula Retz. has been developed. Germination frequency of embryos (up to 100 %) was obtained on Murashige and Skoog (MS) medium supplemented with 0.5 mg dm^–3 gibberellic acid (GA₃). Maximum number of shoots (6.4 shoots per cotyledonary node) was obtained on half-strength MS + 0.3 mg dm^–3 GA₃+ 1.0 mg dm^–3 indole-3-butyric acid (IBA) + 10.0 mg dm^–3 benzylaminopurine (BAP) after 4 weeks of culture. When the cotyledonary nodes along with the axillary shoot buds were allowed to grow in the same medium upto 19.2 shoots were obtained after 8 – 9 weeks. Best rooting (100 %, 5.5 roots per shoot) was observed when shoots were excised and transferred to half-strength MS medium containing 1.0 mg dm^–3 IBA + 1 % mannitol and 1.5 % sucrose. Survival of rooted plants in vivo was low (35 – 40 %) when they were directly transferred to soil in glasshouse. However, transfer to soil with MS nutrients and 1.0 mg dm^–3 IBA in culture room for a minimum duration of 2 weeks increased the survival percentage of plants to 100 %.