Improved yield of apple leaf protoplasts from in vitro cultured shoots by using very young leaves and adding L-methionine to the shoot medium

Protoplast isolation from apple leaf tissue of in-vitro cultured shoots is possible if very young leaves from buds are used. Digestible leaves are found in apple shoots of the subculture kerö 14 days after transferring the shoot and of M26 some days later.The yield of protoplasts is considerably improved if the apple shoots are cultured in a medium containing L-methionine (0.5 mM).     

An efficient procedure for isolation of nuclei from plant protoplasts

Summary The present communication describes an easy, efficient and rapid method for isolation of nuclei from plant protoplasts. Release of nuclei is accomplished by disruption of protoplasts in an appropriate buffer containing a very low concentration (0.01%) of the detergent Triton X-100. The pH of the nuclei isolation buffer (5.3) played a critical role in the recovery of stable nuclei in large numbers. Supplementation of buffer (10 mM MES) with spermine (0.1 mM), dithiothreitol (2.5 mM), ethylenediaminetetraacetic acid (2.5 mM) and Nad and KCl (10 mM each) improved nuclear yield and quality. With the method developed it is possible to routinely recover 95% nuclei from the protoplasts within 30 minutes. The nuclear preparations are of high purity with little detectable cytoplasmic contamination and no clumping of the nuclei. The structural integrity of the nuclei has been assessed and confirmed by Nomarski differential interference contrast optics and ultrastructural observations.     

Isolation and culture of soybean hypocotyl protoplasts

Summary Protoplasts were isolated seedling hypocotyls of soybean (Glycine max), and cultured in both liquid and agarose-solidified, modified K8P medium. Nuclear staining revealed that only 2% of protoplasts lacked a nucleus, 93% contained a single nucleus, and 5% contained more than one. Maximum protoplast yields and subsequent division frequencies, in liquid medium, were obtained from 5 days-old seedlings. Maximum division frequencies (54%) were obtained from hypocotyl protoplasts plated at a density of 5×10⁴ ml⁻¹. Using different osmolality reduction régimes for liquid cultures, hypocotyl protoplasts developed into green, nodular callus, similar to that which has previously given rise to shoot buds in perennialGlycine species. This tissue, however, did not produce shoot buds in soybean. N. H. was supported by a SERC CASE studentship and a postdoctoral fellowship from Shell Research Ltd., Sittingbourne, Kent, UK.     

Isolation and selection of cellulolytic fungi from palm oil mill effluent

Cellulolytic fungi, 34 strains, were isolated from samples taken from palm oil mill residues and effluent, and high cellulase producers selected in comparison with nine known reference strains. Although 13 isolates showed good filter paper distintegration within 14 days, only eight isolates exhibited clearing zones around their colonies on carboxymethylcellulose (CMC) agar medium. Quantitative cellulase activity measurements, using CMC as carbon source, selected three of the eight isolates as potential cellulase producers. Using dried palm oil mill condensate as carbon source, only one of the isolates (F 11) showed similar results on both carbon sources. During media optimization for CMCase production, a four-fold increase from 0.058 to 0.275 U/ml was obtained using a medium, containing 0.1% (v/v) Tween 80 0.02% (w/v) NH₄NO₃, 0.025% (w/v) proteose-peptone and 0.1% (w/v) CMC dissolved in undiluted condensate from the sterilization of oil palm bunches, with an initial pH of 5.5.     

肿瘤标记物的分类、筛选及其在肿瘤病理诊断中的应用

随着免疫组织化学研究的进展,各种新的肿瘤标记物的发现对肿瘤的来源和罕见肿瘤的诊断、以及转移瘤原发部位的确定,提供了客观而可靠的依据。但是免疫组化还面临不少问题。越来越多的肿瘤标记物的相对特异性和交叉反应,未分化或低分化肿瘤不表达有关抗原,给诊断带来困难。尤其是软组织肿瘤,由于组织起源的多样性和多向分化的潜能,瘤细     

The isolation and identification of the prosthetic group released from a bound form of abscisic acid

A bound form of abscisic acid, herein called adduct, has been isolated from peas and barley and has been shown to release methyl abscisate upon mild acid hydrolysis. Mass spectrometry of the methyl abscisate released by ²H₂SO₄ in ²H₂O showed that the remainder of adduct was linked as an enolate of the ketone of the moiety that releases methyl abscisate. Some evidence is adduced to suggest that adduct is a metabolic precursor of abscisic acid. The implications of the distribution of adduct between roots and shoots for the plant's response to stress is discussed.     

Sex pheromones of Spilonota ocellana and Spilonota laricana

The sex pheromones of Spilonota ocellana D. & S. and Spilonota laricana Hein. (Lepidoptera: Tortricidae) were identified by chemical analysis and field trapping. Female moths of the two species produce (Z)-8-tetradecenyl acetate, (Z)-8-tetradecen-1-ol and dodecyl acetate in almost the same proportions (98:1:1 and 97:3:1). Males of both species were best attracted to a blend of 10:1 to 1:1 Z8-14Ac:Z8-14OH. This indicates that mating barriers other than sex pheromones exist between sympatric populations.     

Isolation of rat hepatocytes with EDTA and their metabolic functions in primary culture

Summary Isolated hepatocytes from adult rat liver were prepared after dissociation of the liver with EDTA. The morphological appearance, viability (94.5%) and yield (1.76.10⁷ cells/g liver) compare well with those of previously described methods using collagenase. Differentiated functions of the hepatocytes in primary culture such as albumin secretion (10.9 μg/mg cell protein/d) and triglyceride synthesis and secretion are maintained. Induction of triglyceride synthesis and secretion by oleic acid takes place to an extent similar to that observed in vivo and liver perfusion. Particles with a lipid composition resembling circulating very low density lipoproteins are secreted into the medium. These characteristics demonstrate the ability of hepatocytes isolated with EDTA and subsequently used in primary culture to retain complex and highly differentiated functions of the intact liver.     

New ecdysteroid conjugate: Isolation and identification of 26-hydroxyecdysone 26-phosphate from eggs of the tobacco hornworm,Manduca sexta (L.)

The major ecdysteroid conjugate present in eggs (48–64 h old) of the tobacco hornworm has been purified by XAD-2 chromatography, C₁₈ SEP-PAK separations, and ion suppression reversed-phase high-performance liquid chromatography. Enzymatic hydrolysis of the conjugate with acid phosphatase from human seminal fluid gave 26-hydroxyecdysone. The conjugate was identified as 26-hydroxyecdysone 26-phosphate by nuclear magnetic resonance and fast atom bombardment mass spectrometry. This compound is also the major conjugate of newly laid eggs (0–1 h old) of the tobacco hornworm. The role for ecdysteroid conjugates is discussed.     

Is population genetic structure of vascular plants shaped more by ecological or geographic factors? A study case on the Mediterranean endemic Centaurea filiformis (Asteraceae)

• All known populations of the Sardinian endemic Centaurea filiformis Viv. (Asteraceae) were studied in order to understand the impact of both geographic and ecological factors on the genetic structuring of this species.
• Fourteen populations and 234 individuals were sampled. The demographic structure of the populations and the reproductive ecology were estimated in 28 plots. Population genetic analyses were based on SSR markers. Genetic structure was investigated by spatial Bayesian methods.
• Average densities of 0.51 individuals m^?2 were detected, with a prevalence of adults. Ten species of pollinators were identified; C. filiformis ability to self‐pollinate and myrmecochory were demonstrated experimentally. The populations displayed an average heterozygosity value of H _e = 0.576 and high genetic differentiation (overall F _ST = 0.218). Bayesian analysis suggests that five is the most probable number of gene pools of origin. A strong correlation between geographic distances and genetic distances among populations was highlighted.
• The demographic population structure of C. filiformis is dominated by adults, suggesting that it is a stable‐regressive or senile species, investing more in local persistence than colonisation ability. Despite the scattered distribution, the populations studied do not present evidence of genetic erosion. The analysis of genetic differentiation reveals very high differentiation levels among populations, thus indicating that effective barriers exist against gene flow. A general conclusion is that population distribution results in a clear genetic structure for the populations studied, and that geography and not ecology is shaping the present distribution of this species.