Micropropagation of Achillea filipendulina cv. ‘Parker’

Achillea filipendulina (family Asteraceae) is widespread throughout temperate North America. In order to clean stock plants from endemic fungal and bacterial contaminations a method for large-scale propagation of A. filipendulina through meristem culture was sought and found and is described in this paper. The best conditions for propagating A. filipendulina was found to be MS (Murashige and Skoog) salt medium supplemented with 3% sucrose and 1 mg l^–1 IAA (indole-3-acetic acid) plus 2 mg l^–1 BA (6-benzyladenine) under 16 h of cool fluorescent light. Rooted plants were successfully acclimatized within a short time after propagating on this medium. The propagation via tissue culture did not affect plant's presentation. The use of clean stock plants made it possible to increased Israeli production of Achillea from about 150,000 stems a year to about 1,300,000 stems a year.     

Micropropagation of Calluna vulgaris cv. ‘H.E. Beale’

Axillary shoot producing cultures were obtained from microcuttings and shoot tips of Calluna vulgaris cv. H.E. Beale. For cultures derived from microcuttings the highest multiplication rate of 38 shoots (5 mm or longer) was obtained on a reduced salt medium with the addition of 0.5 mgl^-1 2-isopentenyladenine (2iP) during an 8 week subculture. For shoot tip derived cultures 0.2 mgl^-1 6-benzyladenine (BA) was the best cytokinin and led to a multiplication rate of 26 for a 6 week subculture. The addition of 1 g/l casein hydrolysate to a multiplication medium enhanced shoot proliferation in presence of 0.5 mgl^-1 BA.Despite various auxin treatments shoots formed no roots in vitro but rooted readily if transferred to a peat substrate ex vitro. A high rooting percentage (80%) was also obtained with shoots taken from the end of a multiplication phase and rooted directly. An additional subculture on low auxin containing media before transfer to peat substrate is recommended because the shoot condition can be improved in this way. A high number of rooted plantlets was produced, so the methods described will allow mass propagation.     

Micropropagation of fig (Ficus carica L.) ‘Roxo de Valinhos’ plants

Summary An in vitro protocol for Ficus carica cv. ‘Roxo de Valinhos’ was optimized. Nodal explants containing two buds were excised from field-grown mature plants, and transferred to different proliferation media consisting of combinations of distinct concentrations of activated charcoal with benzyladenine (BA), kinetin with gibberellic acid (GA₃), and WPM (woody plant medium) with kinetin. The regular strength of WPM in combination with 0.5 mgl⁻¹ kinetin was the best condition for shoot proliferation of Ficus carica ‘Roxo de Valinhos’ plants. The addition of activated charcoal in the medium completely inhibited shoot proliferation. The inclusion of BA in the medium induced excessive callus formation as well as small and vitrified shoots, while GA₃ induced excessive elongation associated with vitrification, chlorosis, and tip-burned shoots.     

Micropropagation of seed-derived plants ofCynara scolymus L., cv. ‘Green Globe’

Growth of Cymbidium kanran rhizome was enhanced by higher NAA:BAP ratios in modified Murashige & Skoog (MS) media. Only vegetative shoots resulted from rhizomes cultured in vitro when lower NAA:BAP ratios were used. The rhizomes were induced from the axils of leaves when shoots were explanted to medium containing higher concentrations of NAA. Root formation of C. kanran was inhibited by the addition of either auxin or cytokinin to the culture media. Differentiation of the rhizomes into plantlets occurred when the concentrations of ammonium nitrate and potassium nitrate in MS medium wewe reduced. The modified MS medium containing lesser amounts of potassium nitrate and ammonium nitrate than those of the original MS media, and was optimal for the production of plantlets from rhizomes of C. kanran without addition of auxin and cytokinin.Abbreviations NAA -naphthaleneacetic acid - BAP N⁶-benzylaminopurine - MS medium Murashige & S Skoog medium     

Micropropagation of juvenile tissue of Eucalyptus erythronema × eucalyptus stricklandii cv. ‘urrbrae gem’

Summary Micropropagation via enhanced axillary shoot proliferation was investigated in the ornamental Eucalyptus cv. ‘Urrbrae Gem’ using in vitro germinated seedlings and was successfully achieved using woody plant medium (WPM) supplemented with 2.2 μM benzylaminopurine, 1.0 μM α-naphthaleneacetic acid, and 1.5 μM gibberellic acid (GA₃), gelled with 5 g l⁻¹ Phytagel^?. Shoot proliferation was greater on WPM and QL media with GA₃ compared to B5, AP, and TK media with or without GA₃. GA₃ was required for shoot elongation as the internodes were otherwise very short and unsuitable for multiplication or root initiation. Root initiation was improved using (1/2) WPM supplemented with 20 μM indole-3-butyric acid (IBA) over a 7 d pulse, followed by subculture to IBA-free medium, compared to placing shoots on low levels of IBA for 4–6 wk. Plantlets were successfully hardened off to the natural environment via a fogger at 67% relative, humidity at 21°C for 3 d and continued to thrive as potted plants. This is the first report of successful, micropropagation in an ornamental eucalypt (subgenus Symphyomyrtus) from seedling explants.     

Micropropagation of ‘Yamatoimo’ Chinese yam (Dioscorea opposita) from immature leaves

A micropropagation system for Yamatoimo Chinese yam (Dioscorea opposita Thunb.) was developed. Immature leaves collected from virus-free plants growing in the greenhouse were cultured on MS medium supplemented with 8.9 M benzyladenine (BA), 3% (w/v) sucrose and 0.8% (w/v) agar. After 2–3 months, multiple buds that were clumps of green-colored bulbous structures including adventitious buds and meristematic regions 2–3 mm in diameter were formed on immature leaves. Transplanting clusters of multiple buds to fresh MS medium supplemented with 0.11 M -naphthaleneacetic acid (NAA), 0.89 M BA and 6% (w/v) sucrose was effective for inducing shoot formation, leading to plantlet formation. After 6 months, a large number of microtubers, about 3–7 mm diameter, were obtained.Abbreviations NAA -naphthaleneacetic acid - BA benzyladenine - MS Murashige and Skoog     

Micropropagation of ‘Holy Basil’ (Ocimum sanctum Linn.) from young inflorescences of mature plants

In vitro micropropagation of holy basil (Ocimum sanctum L.), an Indian medicinal herb, has been accomplished on Murashige and Skoog (MS) medium utilizing young inflorescence explants. MS supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) or thidiazuron (TDZ) produced only non-morphogenetic callus. Direct multiple shoots differentiated within 2--3 weeks when explants were cultured on MS containing 6-benzyl aminopurine (BAP). Of the various levels of BAP tested, MS + BAP (1.0 mgl^–1) produced the maximum number of shoots. Incorporation of indole-3-acetic acid (IAA) (0.05 mgl^–1) along with BAP (1.0 mgl^–1) in the culture medium showed a marked increase in the number of shoots. About 92% of the in vitro regenerated shoots rooted on MS hormone-free medium within 2--3 weeks of culture and 85% of the micropropagated plantlets could be successfully established in soil, where they grew normally.     

Species-specific ‘fingerprints’ of deer in China

State governments in China have laid down laws for protecting rare and endangered species, but the effective protection depends on accurate species identification. Although DNA fingerprinting has appeared in court as legal evidence for many years, it is unsuitable for species identification as previous multilocus probes produce individual-specific fingerprints. This study outlines the development of a new species-specific probe – the pta₂ probe. Using this oligonucleotide probe, the identification of all deer species in China is made possible by species-specific fingerprints. Material evidence can then be provided to any judicial organization, which will have a profound effect on the criminal activities of poachers, thereby helping to protect wild animals and conserve biodiversity. Interestingly, the pta₂ probe can also reveal the gender of detected deer species.     

Kinetics of degradation of ‘short-’ and ‘long-lived’ proteins in cultured mammalian cells

Two distinct classes of protein referred to as short- and long-lived (Poole and Wibo, 1973) were found in pulse-chased HeLa S-3 and BHK 21/C13 cells. From experiments with pulse times ranging from 1 min to 20 h, a clear inverse correlation emerged between the pulse length and the percentage of protein which was hydrolysed intracellularly in the first h of chase. Using a 5 min pulse labelling with ³H-leucine, cells were either harvested immediately or after a 2 h chase. Approximately 35% of the label incorporated by cells was lost in a 2h chase; however, electrophoretic separation of cytosol and residual cytoplasmic fractions revealed no significant alteration in their protein profiles. The technique of selectively labelling ‘short’ and ‘long-lived’ proteins, which implies qualitative differences between them, is more readily interpreted as an artificial polarisation of a declining statistical probability curve of proteolysis with time which is similar for all nascent proteins.     

Effects of the torso boundary and internal conductivity interfaces in electrocardiography: An evaluation of the ‘Infinite medium’ approximation

The analytic, eccentric spheres model of the torso was used to examine the validity of approximating the ‘infinite medium’ potential by integrating ‘finite medium potentials’ over the torso surface. Although idealized, the analytic model is sophisticated enough for all important torso conductivity and geometry parameters to be preserved in the formulation. The model generates both ‘finite medium’ potentials (for which the torso is surrounded by air) and also ‘infinite medium’ potentials (for which the outermost layer of the torso extends outward to infinity). The finite medium torso potentials were integrated over the torso surface in accordance with the approximation used by many investigators in an effort to make the surface distribution more representative of the primary cardiac sources. The resulting potential distribution was compared with the true infinite medium potential, in which the effects of internal inhomogeneities (secondary sources) were taken into account. The difference between the two representations was found to be significant, and caution should be used when interpreting such data.     

Micropropagation of ‘Ãkia (Wikstroemia uva-ursi A. Gray)

A protocol for the micropropagation of Wikstroemia uva-ursi A. Gray ('Ãkia) was developed by the establishment of axenic shoot cultures from field-grown plants, induction of shoot proliferation, and rooting in vitro. The best shoot proliferation was obtained from the distal half of stem explants in 8.8 μM 6-benzyladenine. High frequencies of rooting were obtained. The best rooting occurred for shoots grown in half-strength MS plus 2.46 μM indole-3-butyric acid for two weeks.     

‘Conservative’ and ‘variable’ clusters of Alu-family DNA repeats in human chromosomes

The distribution of Alu-family DNA repeats (AFRs) in chromosomes of phytohaemagglutinin-stimulated peripheral blood lymphocytes of four normal donors and non-stimulated bone marrow cells of four patients with acute leukemia (ALL and ANLL) was studied by in situ hybridization using DNA of recombinant phage lambda containing multiple inserts of AFR as a probe. Over some chromosome bands (14cen, 16p13, 16cen) from normal donors and from leukemic patients clusters of silver grains were detected. Over other three bands (3q26, 8p11-p12 and 14q24) the clusters were found only in chromosomes from the four acute leukemia patients, and were absent from chromosomes of healthy donors. The results suggest non-random long-range distribution of AFRs in human chromosomes, and somatic variations in the distribution of the repeats.     

Problems in application of the terms ‘blastic’ and ‘thallic’ to modes of conidiogenesis in some onygenalean fungi

A woman suffering from acute tubulo-interstitial nephritis was admitted to the hospital ten days after deliberate intoxication by ingestion of Cortinarius orellanus. Orellanine, the main toxin responsible for orellanine poisoning, was detected in biological fluids and renal biopsies. It was assayed by direct spectrofluorimetry on two-dimensional thin-layer chromatograms after specific photodecomposition into orelline. The orellanine concentration was 6.12 mg/l in the plasma (10 days after ingestion). Orellanine levels in renal biopsies were 7 g per 25 mm³ of the first biopsy (13 days after ingestion) and 24 g per 8 mm³ of the second biopsy (6 months later).Taken in part from the doctorate thesis of S. Rapior.     

Improvement of in vitro propagation of apricot cultivar ‘Bebecou’

Factors affecting successful establishment in vitro, rapid proliferation and rooting of apricot cultivar ‘Bebecou’ were studied. Ethanol and NaOCl were applied in several combinations for disinfection; chilling, plant growth regulators BA, IAA and GA₃, antibiotics, different culture vessels and systems of subculture were evaluated for the optimization of shoot proliferation and the auxins NAA and IBA were assessed for root induction. The highest number of new microshoots/explant (18.7) was obtained in a culture medium supplemented with 2.2 μM BA+0.57 μM IAA after 300 h of chilling. The effect of GA₃ (11.4 μM) on shoot proliferation was positive in combination with 4.4 or 8.9 μM BA. Shoot length and productivity were highest at 2.2 μM BA+11.4 μM GA₃+0.57 μM IAA and at 2.2 μM BA+0.57 μM IAA, respectively and decreased as cytokinin concentration increased. The antibiotic ‘Na-cefotaxime’ had a minimal impact on shoot growth when used at the lowest concentration (250 mg l⁻¹). Subculture every 2 weeks in a medium supplemented with 2.2 μM BA and 0.57 μM IAA was more efficient for shoot induction than alternation of 20 days culture in a propagation medium supplemented with 2.2 μM BA and 10 days culture in an elongation medium supplemented with 1.1 μM BA and 5.71 μM IAA. The highest number of roots/shoot (8.1) was recorded at 19.6 μM IBA.     

Organogenesis from ‘Passe Crassane’ and ‘Old Home’ pear (Pyrus communis L.) protoplasts and isoenzymatic trueness-to-type of the regenerated plants

Summary Large numbers of highly viable mesophyll protoplasts were isolated from shoot cultures of the scion cv Passe Crassane and the rootstock genotype Old Home of common pear (Pyrus communis L.). Protoplasts were cultured for both genotypes either as liquid layers or as liquid-over-agar cultures, in ammonium-free MS medium with 0.5 M mannitol, 50 mg/l casein enzymatic hydrolysate (CEH), 2.0 mg/l NAA and 1.0 mg/l BAP, plus either 0.5 mg/l IAA (for Old Home) or 2.0 mg/l IAA (for Passe Crassane). Protoplast microcalli, obtained by day 60 (Passe Crassane) or day 80 (Old Home), were transferred for further growth to ammonium-free MS medium with 2.0 mg/l NAA and 1.0 mg/l BAP. Shoot bud regeneration from the protoplastderived callus was first attempted between 100 (Passe Crassane) and 120 (Old Home) days after protoplast isolation. For Passe Crassane, shoot buds were regenerated (day 130) on a half-strength MS medium with 0.1 mg/l IBA, 0.5 mg/l BAP, 50 mg/l CEH and 20 mg/l Ca-panthotenate. For Old Home, shoot but regeneration only occurred 30 days later and on the same medium as above, which was additionally supplemented with double the concentration of the group B vitamins found in the original MS formulation and 0.05 mg/l GA₃. Following micropropagation and in vitro rooting of shoots, the plants were transferred to soil following standard procedures. Trueness-to-type of the regenerated plants was assessed by analysing their leaf isozyme banding profiles (for EST, AP, PRX, SOD, ENP, LAP, PGI, AAT, ADH, MDH and PGM) and comparing them to those corresponding to the original shoots that provided the protoplasts. No differences between the mother shoots and the protoclones were observed for any one of the 11 isozyme systems studied.