The types of bioreactors used for shoots and embryos

Plant regenerated organs such as shoots, bulbs, microtubers, corms, embryos, etc. have been successfully proliferated in the bioreactor. The use of a bioreactor leads to the development of technology suitable for large scale plant propagation. The basic construction and characteristics of various types of bioreactor systems are reviewed in relation to shoot and embryo cultures. A pilot scale 500 liter bioreactor system was applied to the production of large scale Stevia rebaudiana shoots.Abbreviations DW dry weight - EC electrical conductivity - FW fresh weight - ORP oxidation-reduction potential     

Factors affecting variability in anther culture and in conversion of androgenic embryos ofSolanum phureja

The variation for embryo production in anther ofSolanum phureja was examined as a function of maximum greenhouse temperature prior to bud harvest and innate responsiveness among anthers within a bud. Four clones ofS. phuyreja were grown in a greenhouse under a 16-h photoperiod. The temperature was monitored continuously. Buds (60 per day on 10 days) were collected and the anthers cultured in two groups of five flasks (30 anthers per flask). In the first group, each flask contained the 30 anthers from six buds; in the second group, each flask contained one anther from each of 30 buds. Significantly smaller coefficients of variation were observed for the second group, suggesting that variation for embryogenic capcity among buds was greater than that among anthers within a bud. Variation in embryo yield as a function of greenhouse temperature was examined by stepwise regression analysis. Embryogenic capacity of one clone was adversely affected by high temperatures (31–37°C) that occurred two and seven days before bud harvest. However, similarly high temperatures appeared to enhance the androgenic response of another clone. Conversion of anther-derived embryos over three subcultures to fresh regeneration medium was examined as a function of anther donor or clone, cold pretreatment of embryos, and morphological classification of embryos. Only clonal origin significantly affected conversion rate which ranged from 12.5% to 46.0%. Conversion rate declined on each serial subculture.Abbreviations BA N⁶-benzyladenine - GA₃ gibberellic acid, IAA-indole-3-acetic acid     

Gibberellins in suspensor,embryo and integument from very young seeds of Phaseolus coccineus L.

Endogenous gibberellins (GAs) were extracted from suspensor, embryo and integument of very young seeds of Phaseolus coccineus L. and detected by combined gas chromatography-mass spectrometry (GC-MS). Results show the presence of one C₂₀-GA, GA₄₄ and five C₁₉-GAs in the suspensor: GA₁, GA₄, GA₅, GA₆ and GA₈, and four C₁₉-GAs in the integument: GA₁, GA₅, GA₆ and GA₈. Only traces of GA₁ and GA₅ were identified in the embryo. A compound structurally related to GAs was identified as tetrahydroxy-Kauranoic acid in suspensor, integument and, only in trace amounts, in the embryo.     

Acquisition of desiccation tolerance by isolated maize embryos exposed to different conditions: the questionable role of endogenous abscisic acid

The effect of exogenous ABA on acquisition of desiccation tolerance has been well documented for the embryos of several species. including maize ( Zea mays L.). It has also been suggested that endogenous ABA plays a role in regulating the same phenomena. To test this hypothesis, endogenous ABA was quantified by radioimmunoassay. Our results show that: (1) during embryogenesis in maize, endogenous ABA increase-concomitantly with the acquisition of desiccation tolerance: (2) ABA deficient embryos of the vp 5 mutant are desiccation intolerant, but tolerance can he induced by exogenous ABA: and (3) desiccation tolerance is acquired if desiccation sensitive embryos undergo a slow drying treatment, during which ABA increases. However, when embryos were preincubated in fluridone to prevent ABA accumulation during slow drying, desiccation tolerance was induced in spite of the low level of endogenous ABA in the embryo. Our results cast doubts on an exclusive role of ABA in the acquisition of desiccation tolerance in maize embryo.     

Presence and role of jasmonate in apple embryos

(-)Jasmonic acid (JA) was identified in extracts front embryos of apple (Mulus domestica) by combined gas chromatography-mass spectromelry. Quantification of JA in embryos isolated from seeds at different perexts of stratification by gas chromalography combined with mass spectrometry/selective ion monitoring indicated a sharp peak at day 30. At the same time the maximal ratio of conjugated to free JA was found by enzyme-linked imrnunosorbent assay (ELISA). Germination of embryo.s was stimulated by added JA and inhibited by salieylhydroxamic acid (SHAM, an inhibitor of lipoKygenase). Both stimulation and inhibition disappeared in embryos stratified for more than 30 days. Methyl jasmonate was more effective in stimulation of embryo germination than free JA. while JA-isoleucine inhibited germination. The possible mechanism responsible for changes in JA level as wel! as the role of JA and its conjugates in removal of dormancy in apple seeds are discussed.     

Abscisic acid biosynthesis during corn embryo development

In this study we examined the biosynthesis of abscisic acid (ABA) by developing corn (Zea mays L.) embryos. Three comparisons were made: ABA biosynthesis in embryos isolated from kernels grown in vitro with those grown in the field; the developmental profile of ABA content with that of biosynthesis; and ABA biosynthesis in corn embryos lacking carotenoid precursors with ABA biosynthesis in normal embryos. Embryos were harvested at various times during seed development and divided into two groups. Endogenous levels of ABA were measured in one group of embryos and ABA biosynthetic capacity was measured in the other group. The ABA biosynthetic capacity was measured with and without tetcyclacis (an inhibitor of ABA degradation) in embryos from both field-grown and in-vitro-grown corn kernels. Reduced-carotenoid (either fluridone-treated or genetically viviparous) embryos were also included in the study. Corn kernels developing under field and in-vitro conditions differed from each other in their responses to tetcyclacis and in their profiles of ABA biosynthesis during development. Therefore, in-vitro kernel culture may not be an appropriate substitute for field conditions for studies of embryo development. The developmental profiles of endogenous ABA content differed from those of ABA biosynthesis in isolated embryos of both in-vitro-and field-grown kernels. This indicated that ABA levels in the developing embryos were determined by import from the maternal tissues available to the embryos rather than by in-situ biosynthesis. In embryos with reduced levels of carotenoids, either fluridone-treated or genetically viviparous embryos, ABA biosynthesis was low or nonexistent. This result is expected for the presence of an indirect pathway of ABA biosynthesis and in the absence of ABA precursors.Abbreviations ABA abscisic acid - DAP days after pollination     

Genetic analysis of populations of threatened snake species using RAPD markers

Snakes are a particularly threatened vertebrate taxon, with distributions of many species and populations becoming increasingly fragmented. At present, little is known about the degree of genetic differentiation that exists between isolated populations even though such information may be critical to their survival and conservation. As an example of how recently developed RAPD genetic markers can be used in conservation genetics, we present preliminary results from a study which used these DNA-based markers to assess population divergence in two threatened Canadian snakes, the black rat snake ( Elaphe o. obsoleta ) and the eastern massasauga rattlesnake ( Sistrurus c. catenatus ). We present information on the levels of variation and reliability of amplification for fragments generated from five primers. We then use a recently developed analytical technique to estimate levels of nucleotide diversity within populations and sequence divergence between populations. Our results show that intrapopulation levels of divergence as estimated by the methods of Clark & Lanigan ( Molecular Biology and Evolution 1993, 10 , 1096–1111) approximate those found for mtDNA in vertebrates and that diversity between snake populations is small and non-significant when tested using randomization procedures. Thus, our study provides an example of how RAPDs can be applied to conservation genetic studies of vertebrates and suggest that the snake populations we examined have only recently become isolated and maybe considered genetically equivalent from a conservation perspective, although this conclusion needs to be confirmed with other DNA-based markers.     

小麦体细胞胚发生的超微结构研究

对小麦（Ｔｒｉｔｉｃｕｍ ａｅｓｔｉｖｕｍ Ｌ．）幼胚培养中体细胞胚发生过程的超微结构变化进行了研究,结果如下：（１）原体细胞中大液泡消失,存在大量小液泡,细胞质的电子密度加强,核大,核仁明显,出现多核仁;（２）细胞器数量和种类,如质体、核糖体和线粒体增加;（３）细胞壁加厚,胞间连丝逐渐消失,细胞器数量丰富,胚性细胞中积累淀粉;（４）细胞壁加厚的胚性细胞中存在核仁液泡、自体吞噬泡和分泌泡;（５）多细胞原胚、球形胚和梨形胚被一层外壁包围,但胚体内细胞间广泛存在胞间连丝;（６）成熟胚生长点部位的细胞内质体中出现膜系结构,已向叶绿体转变,类囊体已形成     

Quinidine-sensitive K+ channels in the basolateral membrane of embryonic coprodeum epithelium: regulation by aldosterone and thyroxine

Basolateral K⁺ channels and their regulation during aldosterone- and thyroxine-stimulated Na⁺ transport were studied in the lower intestinal epithelium (coprodeum) of embryonic chicken in vitro. Isolated tissues of the coprodeum were mounted in Ussing chambers and investigated under voltage-clamped conditions. Simultaneous stimulation with aldosterone (1 mol·l^-1) and thyroxine (1 mol·l^-1) raised short-circuit current after a 1- to 2-h latent period. Maximal values were reached after 6–7 h of hormonal treatment, at which time transepithelial Na⁺ absorption was more than tripled (77±11 A·cm^-2) compared to control (24±8 A·cm^-2). K⁺ currents across the basolateral membrane with the pore-forming antibiotic amphotericin B and application of a mucosal-to-serosal K⁺ gradient. This K⁺ current could be dose dependently depressed by the K⁺ channel blocker quinidine. Fluctuation analysis of the short-circuit current revealed a spontaneous and a blocker-induced Lorentzian noise component in the power density spectra. The Lorentzian corner frequencies increased linearly with the applied blocker concentration. This enabled the calculation of single K⁺ channel current and K⁺ channel density. Single K⁺ channel current was not affected by stimulation, whereas the number of quinidine-sensitive K⁺ channels in the basolateral membrane increased from 11 to 26·10⁶·cm^-2 in parallel to the hormonal stimulation transepithelial Na⁺ transport. This suggests that the basolateral membrane is a physiological target during synergistic aldosterone and thyroxine regulation of transepithelial Na⁺ transport for maintaining intracellular K⁺ homeostasis.Abbreviations f frequency - f _c Lorentzian corner frequency - g _K single K⁺ channel conductance - HEPES N-2-hydroxyethylpiperazin-N'-2-ethansulfonic acid - i _K single K⁺ channel current - I_Ampho amphotericin B induced K⁺ current - I _sc short-circuit current - I _K quinidine blockable K⁺ current - I _max maximally blocked current by quinidine - IC ₅₀ half-maximal blocker concentration - k _on, k _off on- and off-rate coefficients of reversible single channel block by quinidine - M _K number of conducting K⁺ channels - [Q] quinidine concentration - R _t transepithelial resistance - S spectral density - S _o Lorentzian plateau - TBM cells toad urinary bladder cell line Present address: University of California at Berkeley, Dept. of Molecular and Cell Biology Berkeley, CA 94720, USA     

Structural and viral association comparisons of bovine zonae pellucidae from follicular oocytes, Day-7 embryos and Day-7 degenerated ova

Bovine zonae pellucidae (ZP) from follicular oocytes and from embryos and degenerated ova collected on Day 7 from superovulated cows were examined by scanning electron microscopy, by dimensional measurement, and by total protein determination. The number of plaque-forming units (PFU) of infectious bovine rhinotracheitis virus (IBRV) that were associated with ZP-intact embryos/ova from each of the 3 sources after in vitro exposure was also determined.

Scanning electron microscopy revealed that the surfaces of Day-7 embryos and degenerated ova were smoother than those of follicular oocytes. Mean dimensional measurements of the diameter/thickness of the ZP from follicular oocytes, Day-7 embryos, and degenerated Day-7 ova were 156.7 μm/12.3μm, 161.3μm/12.6μm, and 158.9μm/12.8μm, respectively. The mean total protein per ZP of follicular oocytes, embryos, and degenerated ova was 0.331 μg, 0.349 μg, and 0.254 μg, respectively. Considerable variability existed within groups, but significantly greater quantities of IBRV were associated with follicular oocytes (mean PFU/oocyte = 68.1) than with Day-7 embryos (mean PFU/embryo = 43.0; P<0.05) or with Day-7 ova (mean PFU/ovum = 31.9; P<0.01).

The reliability of using an assay for IBRV associated with nontransferable ova/embryos as an indicator of the presence or absence of the virus in transferable embryos from the same collection (Day 7) was supported. Although structural differences between the ZPs of follicular oocytes and Day-7 embryos were observed in this study, further investigation is needed to determine if there are differences in the protective function of the respective ZPs.