Plant regeneration via somatic embryogenesis from protoplasts of Uganda cherry orange (Citropsis schweinfurthii)

Protoplasts isolated from embryogenic callus of Citropsis schweinfurthii (Engl.) Swing. & M. Kell were cultured in MT (Murashige and Tucker 1969) basal medium containing 5% sucrose supplemented with 0.0, 0.001, 0.01, 0.1 or 1.0 mg l^–1 BA, 0, 300, 600 or 900 mg l^–1 malt extract and 0.6 M sorbitol. The highest plating efficiency was obtained on MT basal medium containing 5% sucrose supplemented with 0.01 mg l^–1 BA and 600 mg l^–1 malt extract. MT basal medium containing 5% sucrose and supplemented with 0.01 mg l^–1 kinetin was found to be a medium suitable for the development of globular somatic embryos derived from protoplasts into heart-shaped somatic embryos with cotyledon-like structures. The highest percentage of shoot formation was obtained using 0.1 mg l^–1 GA₃. A complete protoplast to-plant system was developed for C. schweinfurthii, which could facilitate the transfer of nuclear and cytoplasmic genes from this species into cultivated Citrus through protoplast fusion.Abbreviations BA N⁶-benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - FDA fluorescein diacetate - GA₃ gibberellin A₃ - ME malt extract     

Evaluation ofMicrothrix inconspicuella, [Lepidoptera: Pyralidae], a potential biological control agent forEmex australis in Australia,carried out in apple orchards in South Africa

Host specificity tests carried out in the laboratory in Australia during 1977, showed thatMicrothrix inconspicuella Ragonot could develop on young apple leaves (Harley et al., 1979). Field studies in unsprayed apple orchards in South Africa showed that some feeding occurred, but fewer than 40% of late instar larvae developed to adults when confined in sleeves on apple tree branches. No feeding or survival occurred in large field cages or in the open. Adults which developed from apple fed larvae were smaller, deformed, occasionally mated and laid fertile eggs but their progeny did not feed or develop on apple fruit or leaves. In conclusion,M. inconspicuella larvae did not develop on apple fruit or leaves in the field, damage was mainly limited to apples already injured and feeding on leaves was minimal. Under normal pest control practicesM. inconspicuella populations did not survive on any part of the apple tree or onE. australis growing under the trees.      

Different nitrogen sources and growth responses of Spirulina platensis in microenvironments

Spirulina platensis was cultivated, in comparative studies, using several sources of nitrogen. The standard source used (sodium nitrate) was the same as that used in the synthetic medium Zarrouk, whereas the alternative nitrogen sources consisted of ammonium nitrate, urea, ammonium chloride, ammonium sulphate or acid ammonium phosphate. The initial nitrogen concentrations tested were 0.01, 0.03 and 0.05 M in an aerated photobioreactor at 30 °C, with an illuminance of 1900 lux, and 12 h-light/12 h-dark photoperiod over a period of 672 h. Maximum biomass was produced in medium containing sodium nitrate (0.01–0.03–0.05 M), followed by ammonium nitrate (0.01 M) and urea (0.01 M). The final biomass concentrations were 1.992 g l^–1 (0.03 M sodium nitrate), 1.628 g l^–1 (0.05 M sodium nitrate), 1.559 g l^–1 (0.01 M sodium nitrate), 0.993 g l^–1 (0.01 M ammonium nitrate) and 0.910 g l^–1 (0.01 M urea). This suggested that it is possible to utilize nitrogen sources other than sodium nitrate for growing S. platensis, in order to decrease the production costs of scaled up projects.     

Effects of growth regulators on inflorescence proliferation of Bambusa edulis

The effects of various growth regulators in Bambusa edulis inflorescence proliferation were studied. Cytokinin is essential for inflorescence proliferation. Thidiazuron (TDZ) had been the most efficient cytokinin to induce inflorescence proliferation. The optimal TDZ concentration was 0.01–0.1 mg l^–1. Inflorescences did not proliferate in media containing auxin, gibberellic acid (GA₃), abscisic acid (ABA), or 1-amino- cyclopropane-1-carboxylic acid (ACC) alone. In TDZ-containing medium, the proliferation ratio decreased when the naphthaleneacetic acid (NAA) concentration higher than 5 mg l^–1.     

Plant regeneration from protoplasts of Gentiana by embedding protoplasts in gellan gum

A protoplast-to-plant system was developed in Gentiana using a gellan gum-embedding culture. Viable protoplasts could be routinely isolated from in vitro-grown plantlets, and they were embedded in 0.2% gellan gum-solidified B5 medium containing 2 mg l^-1 NAA, 0.1 mg l^-1 TDZ, 0.1 M sucrose and 0.4 M mannitol. Weekly addition of fresh liquid medium was essential for preventing cell browning. Colony growth was promoted by lowering mannitol concentration of the culture media after one month, and visible colonies were produced after 2 months of culture. Shoot regeneration from protoplast-derived calluses was stimulated by 1 to 10 mg l^-1 TDZ in combination with 0.1 mg l^-1 NAA. Protoplast-derived plants were recovered following rooting of the shoots in plant growth regulator-free medium and they were successfully transferred to soil.Abbreviations BA benzylaminopurine - FDA fluorescein diacetate - FW fresh weight - MES 2-N-morpholinoethane sulfonic acid - NAA -naphthaleneacetic acid - TDZ N-1,2,3-thiadiazol-5-yl-N-phenylurea (also called thidiazuron)     

Stimulation of menthol production in <Emphasis Type="Italic">Mentha piperita</Emphasis> cell culture

Plant cell culture provides an alternative means for producing secondary metabolites. In this study, experiments were carried out to study the impact of several parameters, independently and in combination, on the stimulation of menthol production in the cell suspension culture of Mentha piperita. Callus was obtained from leaf segments of in vitro grown plantlets on Murashige and Skoog (MS) medium supplemented with 0.2 mg l⁻¹of 2,4-dichlorophenoxy acetic acid to initiate cell suspension culture. This culture was maintained in half-strength MS medium supplemented with 0.2 mg l⁻¹of 2,4-dichlorophenoxy acetic acid at 15 d interval and used for further studies. Precursor feeding alone, i.e., menthone, at 35 μM concentration showed slightly improved productivity. γ-Cyclodextrin alone at 60 μM concentration and in combination with menthone feeding at 35 μM increased menthol yield up to 92 and 110 mg l⁻¹ in comparison to 77 mg l⁻¹ of control culture. Synergistic potentiation effect of menthone feeding at 35 μM and γ-cyclodextrin at 60 μM treatment followed by in situ adsorption with RP-8 also showed potential stimulation of menthol production in M. piperita cell culture. Fungal elicitor treatment showed enhanced production level up to 140.8 mg l⁻¹ in comparison to that of control. Further studies were carried out with the establishment of Agrobacterium tumefaciens (Ach5) gall-mediated calli, and consequently, cell suspension culture and results showed the significant enhancement of menthol yield up to 278 mg l⁻¹. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Micropropagation of adult Swainsona formosa (Leguminosae: Papilionoideae: Galegeae)

Summary Explants of axillary buds excised from mature adult stems of Swainsona formosa (G. Don) J. Thompson (syn. Clianthus formosus) were cultured on Murashige and Skoog medium supplemented with a range of auxins, cytokinins, and sucrose concentrations. Auxins did not increase shoot or bud numbers above controls, and 2,4-dichlorophenoxyacetic acid was the only auxin to significantly increase callus production. Benzyladenine or thidiazuron incorporated into the medium at 0.1 μM stimulated shoot and bud production, and shoot growth occurred following removal of cytokinins from the medium after 4 wk. Shoot number increased linearly with sucrose concentration up to 40 g l⁻¹, but shoot height and the number of cytokinin-induced buds were optimal at sucrose levels of 20–30 g l⁻¹. Roots were initiated in vitro following treatment of cuttings with 0.1% indole-3-butyric acid and 0.1% α-naphthaleneactic acid. Plantlets were successfully established in soil but were plagiotropic and exhibited distichous phyllotaxy.     

The influence of some inhibitors on the formation of caffeic acid in cultures ofPerilla cell suspensions

A cell suspension culture, prepared fromPerilla frutescens var.crispa callus induced by Murashige and Skoog (1962) medium containing 2,4-dichlorophenoxyacetic acid (2,4-D, 1.0 ml/l) and kinetin (0.1 mg/l), contained caffeic acid derivatives as the phenolic components. Fresh and dry weights of the cells increased exponentially for about 11 days after transfer to a fresh medium. The contents of caffeic acid and protein also reached a maximum on the 11th day, but α-amino nitrogen phenylalanine and tyrosine continued to increase in amount until the 20th to 23rd day. Caffeic acid formation in the cells was increased by lowering the concentration of 2,4-D. The administration ofl-2-aminooxy-3-phenylpropionic acid (l-AOPP), 2-aminooxyacetic acid (AOA) andN-(phosphonomethyl)glycine (glyphosate) to the cells inhibited caffeic acid formation to a large extent. An 80% inhibition of caffeic acid formation was caused by 10⁻⁴Ml-AOPP whereas phenylalanine and tyrosine contents of the cells became 7.5 and 2.3 times higher at thisl-AOPP concentration than those in the control. An 85% inhibition of caffeic acid formation was achieved at 10⁻³M glyphosate concentration, while 10⁻³M AOA inhibited caffeic acid formation by 95% and also growth rate by 80%. The influence of inhibitors on caffeic acid formation is discussed in relation to the level of α-amino nitrogen, particularly aromatic amino acids, in the cell suspension cultures.     

Organogenesis and embryogenesis from callus cultures of Azadirachta excelsa

ABSTRACT

Callus production from leaf explants of Azadirachta excelsa (Jack) Jacobs was favoured by Murashige and Skoog medium supplemented with 4 mg l^-1 indole-3-butyric acid (IBA) and 1 mg l^-1 6-benzyladenine (BAP). Increasing the concentration of BAP to 2 mg l^-1 induced shoot regeneration. Adding polyvinylpyrrolidone (PVP) to the medium resulted in a significantly increased number of shoots. Transfer onto medium containing 0.5 mg l^-1 BAP, 0.4 mg l^-1 gibberellic acid (GA₃) and 2% sucrose stimulated elongation of the internodes; subsequent transfer onto medium containing 1 mg l^-1 IBA induced root formation. The histological analysis demonstrated that organogenesis and embryogenesis occurred in the same callus. However, shoots originated inside the callus mass, whereas the embryos originated on the surface. Given that the embryos did not develop beyond the globular or heart-shaped stage, we concluded that the plants regenerated from callus were derived only from organogenesis.     

Plant regeneration from cell suspension-derived protoplasts of Saintpaulia ionantha Wendl

Summary Friable calli were induced on leaf segments of Saintpaulia ionantha Wendl. on B5 medium containing 1 mg l^–1 2,4-D and 2 g l^–1 casein hydrolysate. Cell suspension cultures were readily established from these friable calli and protoplasts could be isolated from the cells with yields of 1–3×10⁷/g f. wt.. By culturing in 0.1 % gellan gum-solidified B5 medium supplemented with 1 mg l^–1 2,4-D and 0.1 M each of sucrose and mannitol at a density of 1×10⁵/ml, the protoplasts divided within 6 days and formed macro-colonies after 2 months of culture. Shoot regeneration from protoplast-derived calli was obtained by sequential treatment of the calli with plant growth regulators: initially with 1 mg l^–1 each of NAA and BA for 2 months followed by 0.01 mg l^–1 NAA and 5 mg l^–1 BA for 4 months. Regenerated plants were established after rooting of the shoots on half-strength MS medium, and successfully transferred to the greenhouse. The regenerated plants grew into flowering stage and showed the same phenotype as the parent plant.Abbreviations BA benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - FDA fluorescein diacetate - f. wt fresh weight - MES 2-(N-morpholino)-ethanesulfonic acid - MS Murashige and Skoog (1962) - NAA -naphthaleneacetic acid - PE plating efficiency     

The suitability of the free-living nematode Panagrellus redivivus as live food for first-feeding fish larvae

The free‐living nematode Panagrellus redivivus has been recommended as a suitable food source for first‐feeding fish. A new technology for mass production of P. redivivus enables fish hatchery operators to rely on an inexpensive, standardized and permanently available live food for first‐feeding fish larvae. The proximate composition, and the fatty acid and amino acid profiles of nematodes mass produced on oat‐based and purified ingredient media were determined. The quality of nematodes was significantly influenced by the culture medium used. The lipid content and fatty acid composition of nematodes could be modified by using lipid‐enriched media. Mass‐produced nematodes were tested on first‐feeding common carp (Cyprinus carpio L.) and whitefish (Coregonus lavaretus) larvae. Carp larvae, grown on nematodes cultured on oat medium enriched with sunflower oil, showed a higher survival rate (87.1%) than the control group fed frozen zooplankton (82.9%) at the end of the 1‐week feeding experiment. Differences in larval mass between the treatments disappeared after subsequent feeding of a dry diet for 2 weeks. Whitefish larvae can be reared exclusively on a dry diet; here, the initial feeding of nematodes had no effect on final biomass and survival of larvae.     

Establishment of Aster sedifolius and Aster caucasicus callus cultures as a potential source of antioxidants

Abstract

Callus cultures were established for Aster sedifolius and Aster caucasicus, two Aster species used in natural medicine for their anticancer, antibacterial and antiviral activities attributed to the high content of antioxidant compounds such as polyphenols and ascorbate. The effects of growth medium and light condition on the induction and growth rate of callus from leaf, petiole and root explants are reported. Callus induction and proliferation depended on the genotype and the experimental conditions. In particular, a profuse callus culture was obtained from leaf explants grown in the light on medium supplemented with 2,4-D (0.1 mg l^?1) for A. caucasicus and on medium supplemented with 2,4-D (0.44 mg l^?1) plus 6-benzil-ammino-purine (BAP) (0.22 mg l^?1) for A. sedifolius. The content of total polyphenol and ascorbic acid was estimated in leaf and petiole explants of in vivo plants and in the relative derived calli. In calli, polyphenol content was lower than in the corresponding in vivo organs. Furthermore, the total ascorbic acid content decreased in calli while the reduced ascorbic acid pool increased. These findings demonstrate that Aster callus cultures produce antioxidant compounds and as such might be a model system to investigate the regulation and production of these important metabolites.     

Plant regeneration via somatic embryogenesis from protoplasts of six plant species related to Citrus

Protoplasts isolated from embryogenic callus of Fortunella polyandra (Ridl.), Atalantia bilocularis (Pieree ex Guill.), Hesperethusa crenulata (Roxb.), Glycosmis pentaphylla (Retz.) Corr., Triphasia trifolia (Burm. f.) P. Wils. and Murraya koenigii (L.) Spreng. were cultured in MT (Murashige and Tucker 1969) basal medium containing 5% sucrose supplemented with 0.0, 0.001, 0.01, 0.1 or 1.0 mg l^–1 BA and 0.6 M sorbitol. The highest plating efficiencies for all species were obtained on MT basal medium containing 5% sucrose supplemented with 0.001 mg l^–1 BA. F. polyandra produced higher percentages of globular somatic embryo development, while A. bilocularis consistently showed a lower percentage of globular somatic embryo development in all 5 concentrations of BA. MT basal medium containing 5% sucrose and supplemented with 0.001 mg l^–1 BA was found to be a suitable medium for development of globular somatic embryos derived from protoplasts to form heart-shaped somatic embryos with cotyledon-like structures. The highest percentages of shoot formation for all 6 species were obtained using 0.1 mg l^–1 GA₃. A complete protoplast-to-plant system was developed for F. polyandra, A. bilocularis and T. trifolia, which could facilitate the transfer of nuclear and cytoplasmic genes from these species into cultivated Citrus through protoplast fusion.Abbreviations BA N⁶-benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - FDA fluorescein diacetate - GA₃ gibberellin A₃     

Tissue culture inHaworthia

Effects of three different auxins and kinetin in various combinations on greening and redifferentiation of the callus ofHaworthia setata were investigated. All auxins at the concentration of 50 mg/l inhibited callus greening. NAA in combination with kinetin promoted both callus greening and production of redifferentiated shoots. Low concentrations of IAA without kinetin promoted redifferentiation of shoots, but not callus greening. Addition of 2,4-D completely inhibited both greening and redifferentiation regardless of the level of kinetin except for the effects on shoot formation in the medium with 0.1 mg/l 2,4-D added. The calluses with the highest chlorophyll content were observed in the medium containing 2.0 mg/l kinetin without any auxins or with 0.1 mg/l NAA added. Most frequent shoot redifferentiation was observed in the medium containing 0.1 mg/l IAA without kinetin (redifferentiation rate; 67%), followed by the medium containing 10 mg/l NAA with 2.0 mg/l kinetin (44%), and 0.1 mg/l 2,4-D with 0.2 mg/l kinetin (33%). Generally, higher degrees of greening were associated with better growth. However, the auxins (IAA, NAA and 2,4-D) given at concentrations optimal for growth did not exhibit the highest degree of callus greening. Differences of the three auxins in their actions and interaction with kinetin were disclosed. Contribution from the Laboratory of Genetics, Faculty of Agriculture, Kyoto University, Japan, No. 423     

Effect of root extracts of resistant pasture plants on the feeding and survival of black beetle larvae,Heteronychus arator (Scarabaeidae)

Abstract

Crude root extracts of the black beetle-resistant legumes red clover, white clover, lupin, Lotus pedunculatus, and lucerne significantly reduced feeding by 3rd-instar black beetle larvae when incorporated in an artificial medium containing a strong feeding stimulant. The same extracts were toxic when administered orally. Lucerne and L. pedunculatus contain particularly active feeding deterrents and toxins. The root of the black beetle-resistant grass Phalaris aquatica (= P. tuberosa), like that of the susceptible perennial ryegrass, had no effect on larval feeding or survival. Lotus pedunculatus was very much more active against black beetle larvae than L. corniculatus or L. corniculatus × pedunculatus.     

Fungal biotransformation of p-coumaric acid into caffeic acid by Pycnoporus cinnabarinus: an alternative for producing a strong natural antioxidant

Fungal biotransformation of p-coumaric acid into caffeic acid, potentially a strong antioxidant, was evidenced in Pycnoporus cinnabarinus cultures grown with high feeding of p-coumaric acid. Preliminary experiments showed no toxicity of both p-coumaric and caffeic acids at concentrations ranging from 0 to 500 mg l^–1. Feeding 450 mg p-coumaric acid l^–1 into P. cinnabarinus cultures grown on 20 g l^–1 glucose medium resulted in the production of 257 mg caffeic acid l^–1with a molar yield of 21%.     

Induction of haploids in Populus maximowiczii via embryogenic callus

Anthers of Populus maximowiczii with microspores at the mononucleate stage were cultured at 20°C in the dark on agar-solidified Murashige and Skoog medium after 4 days of cold treatment (4°C). After 4 to 8 weeks anthers on medium supplemented with 0.5, 1.0 or 2.0 mg l^-1 2,4-D in combination with 0.1 mg l^-1 kinetin developed calli that were characterized by smooth surface and gel-like consistency. These calli were comprised of expanding microspores surrounded by a mucilaginous matrix. After transfer of anthers with embryogenic calli to MS medium with low hormone levels (NAA at 0, 0.1 and 0.1 mg l^-1 and BA at 0, 0.1 and 1.0 mg l^-1) microspores started to divide and initiated independent meristematic nests, which developed into embryoidal structures, resembling globular to bi-polar heart-shaped embryoids. The embryoids germinated precociously without developing cotyledons. After transfer to medium with a range of levels of BA (1.0, 2.5 and 5.0 mg l^-1), adventitious shoots developed mainly from the roots. Shoots were rooted in half strength MS medium supplemented with 0.025 mg l^-1 NAA. Via this pathway anther response in the best treatment combination was 10%.Abbreviations BA benzyladenine - MS Murashige & Skoog - NAA naphthaleneacetic acid - 2,4-D-2,4 dichlorophenoxyacetic acid     

Isolation of <Emphasis Type="Italic">Aspergillus oryzae</Emphasis> mutants for heterologous protein production from a double proteinase gene disruptant

The recombinant Pichia pastoris harboring an improved methionine adenosyltransferase (MAT) shuffled gene was employed to biosynthesize S-adenosyl-l-methionine (SAM). Two l-methionine (l-Met) addition strategies were used to supply the precursor: the batch addition strategy (l-Met was added separately at three time points) and the continuous feeding strategies (l-Met was fed continuously at the rate of 0.1, 0.2, and 0.5 g l⁻¹ h⁻¹, respectively). SAM accumulation, l-Met conversion rate, and SAM productivity with the continuous feeding strategies were all improved over the batch addition strategy, which reached 8.46 ± 0.31 g l⁻¹, 41.7 ± 1.4%, and 0.18 ± 0.01 g l⁻¹ h⁻¹ with the best continuous feeding strategy (0.2 g l⁻¹ h⁻¹), respectively. The bottleneck for SAM production with the low l-Met feeding rate (0.1 g L⁻¹ h⁻¹) was the insufficient l-Met supply. The analysis of the key enzyme activities indicated that the tricarboxylic acid cycle and glycolytic pathway were reduced with the increasing l-Met feeding rate, which decreased the adenosine triphosphate (ATP) synthesis. The MAT activity also decreased as the l-Met feeding rate rose. The reduced ATP synthesis and MAT activity were probably the reason for the low SAM accumulation when the l-Met feeding rate reached 0.5 g l⁻¹ h⁻¹.     

Media optimization for maximum biomass production in cell cultures of pacific yew

Three cell lines of Taxus brevifolia Nutt. with differing growth rates were used to assess the effects of basal salt mixtures, carbohydrates, organic nitrogen additives, vitamin formulations, and plant growth regulators on callus growth. Gamborg's B5 major salts provided significantly better growth than all other salt formulations tested. The greatest biomass was obtained with 1% total carbohydrate. The best carbohydrate combination, 0.5% fructose + 0.5% sucrose, was significantly better than all other combinations of carbohydrates tested. A complex vitamin mixture was significantly better than any one previously published vitamin formulation. Greatest rates of callus growth were obtained with 4.14 M (1 mg l^-1 picloram, 0.46 M (0.1 mg l^-1 kinetin, and 0.38 M (0.1 mg l^-1) abscisic acid or 0.29 M (0.1 mg l^-1 gibberellic acid. Our final medium, TM5, is superior to published methods for the general callus culture of T. brevifolia. This medium has improved growth in three tested cell lines to provide doubling times of 3.5 to 5.6 days, an average 5.3-fold increase over our previously published medium.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - 2,4,5-T 2,4,5-trichlorophenoxyacetic acid, 2ip-6-(,-dimethylamino)-purine - ABA abscisic acid - BA 6-benzyladenine - GA₃ gibberellic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - kinetin 6-furfurylaminopurine - NAA napthaleneacetic acid - picloram 4-amino-3,5,6-trichloropicolinic acid     

Plant regeneration of the mining ecotype <Emphasis Type="Italic">Sedum alfredii</Emphasis> and cadmium hyperaccumulation in regenerated plants

An efficient micropropagation system for mining ecotype Sedum alfredii Hance, a newly identified Zn/Cd hyperaccumulator, was developed. Frequency of callus induction reached up to 70% from leaves incubated on Murashige and Skoog (MS) medium supplemented with 1.0 mg l⁻¹ 2,4-dichlorophenoxy acetic acid (2,4-D) and 0.5 mg l⁻¹ 6-benzyladenine (BA), and 83% from internodal stem segments grown on MS medium with 0.1 mg l⁻¹ 2,4-D and 0.1 mg l⁻¹ BA. Callus proliferated rapidly on MS medium containing 0.2 mg l⁻¹ 2,4-D and 0.05 mg l⁻¹ thidiazuron. The highest number of adventitious buds per callus (17.3) and frequency of shoot regeneration (93%) were obtained when calli were grown on MS medium supplemented with 2.0 mg l⁻¹ BA and 0.3 mg l⁻¹ α-naphthalene acetic acid (NAA). Elongation of shoots was achieved when these were incubated on MS medium containing 3.0 mg l⁻¹ gibberellic acid. Induction of roots was highest (21.4 roots per shoot) when shoots were transferred to MS medium containing 2.0 mg l⁻¹ indole 3-butyric acid rather than either indole 3-acetic acid or NAA. When these in vitro plants were acclimatized and transferred to the greenhouse, and grown in hydroponic solutions containing 200 μM cadmium (Cd), they exhibited high efficiency of Cd transport, from roots to shoots, and hyperaccumulation of Cd.