Changes in pectins and MAPKs related to cell development during early microspore embryogenesis in Quercus suber L

The occurrence and significance of changes in cell wall components and signalling molecules has been investigated during early microspore embryogenesis in cork oak (Quercus suber L.) in relation to cell proliferation and cell differentiation. Microspore embryogenesis has been induced in in vitro anther cultures of Q. suber by the application of a stress treatment of 33 degrees C. After the treatment, microspores at the responsive developmental stage of vacuolate microspore switched towards proliferation and the embryogenesis pathway to further produce haploid plantlets. Ultrastructural and immunocytochemical analysis revealed changes in cell organisation after induction at different developmental stages, the cellular features displayed being in relation to the activation of proliferative activity and the beginning of differentiation in young and late proembryos. Immunogold labelling with JIM5 and JIM7 antibodies showed a different presence of pectin and level of its esterification in cell walls at different developmental stages. Non-esterified pectins were found in higher proportions in cells of late proembryos, suggesting that pectin de-esterification could be related to the beginning of differentiation. The presence and subcellular distribution of Erk 1/2 MAPK homologues have been investigated by immunoblotting, immunofluorescence and immunogold labelling. The results showed an increase in the expression of these proteins with a high presence in the nucleus, during early microspore proembryos development. The reported changes during early microspore embryogenesis are modulated in relation to proliferation and differentiation events. These findings provided new evidences for a role of MAPK signalling pathways in early microspore embryogenesis, specifically in proliferation, and would confer information for the cell fate and the direction of the cell development.     

Pro-embryos induction from <Emphasis Type="Italic">Olea europaea</Emphasis> L. isolated microspore culture

Studies were undertaken with one olive (Olea europaea L.) cultivar to identify buds with microspores competent to embryogenesis in vitro. Isolated microspore cultures were performed for the induction of gametic embryogenesis. Different pollen development stages and stress conditions (heat or cold shock) were evaluated. The correlation of inflorescence, anther morphology and the suitable stage of microspore development were analysed. The morphology of responsive buds was identified which corresponded with microspores from the late uni-nucleate to early bi-nucleate pollen stages. Symmetrical divisions of microspores as well as resulting multinucleate structures and pro-embryos were observed. In this paper, a new method of isolated microspore culture that leads to cell division and pro-embryos in olive, is reported.     

Carrageenan oligosaccharides enhance stress-induced microspore embryogenesis in Brassica oleracea var. italica

Embryogenic induction in cultures of isolated microspores is a stress-dependent process, which can be triggered by heat shock, sucrose or nitrogen starvation or by anti-microtubular drugs. As they are known to mimic biotic stress, oligosaccharides were tested as an alternative source of compounds to induce microspore embryogenesis in Brassica oleracea var. italica. Among the eight oligosaccharide series that were investigated and the corresponding polysaccharides, namely pectin, alginate, fucan, laminarin, agar and κ-, ι-, and λ-carrageenans, only the carrageenan oligomers displayed significant effects on microspore induction. When supplied in combination to heat stress, they markedly enhanced the final yields of microspore-derived embryos, with a two-fold increase in the most reactive treatment, i.e. in the presence of λ-carrageenan oligosaccharides. A 30 min treatment was enough to stimulate embryogenesis, and two optimal concentrations were observed, 170 nM and 34 μM.     

Microspore-derived embryogenesis in pepper (Capsicum annuum L.): subcellular rearrangements through development

Background information. In vitro-cultured microspores, after an appropriate stress treatment, can switch towards an embryogenic pathway. This process, known as microspore embryogenesis, is an important tool in plant breeding. Basic studies on this process in economically interesting crops, especially in recalcitrant plants, are very limited and the sequence of events is poorly understood. In situ studies are very convenient for an appropriate dissection of microspore embryogenesis, a process in which a mixture of different cell populations (induced and non-induced) develop asynchronically.Results. In the present study, the occurrence of defined subcellular rearrangements has been investigated during early microspore embryogenesis in pepper, an horticultural crop of agronomic interest, in relation to proliferation and differentiation events. Haploid plants of Capsicum annuum L. (var. Yolo Wonder B) have been regenerated from in vitro anther cultures by a heat treatment at 35 degrees C for 8 days. Morphogenesis of microspore-derived embryos has been analysed, at both light and electron microscopy levels, using low-temperature-processed, well-preserved specimens. The comparison with the normal gametophytic development revealed changes in cell organization after embryogenesis induction, and permitted the characterization of the time sequence of a set of structural events, not previously defined in pepper, related to the activation of proliferative activity and differentiation. These changes mainly affected the plastids, the vacuolar compartment, the cell wall and the nucleus. Further differentiation processes mimicked that of the zygotic development.Conclusions. The reported changes can be considered as markers of the microspore embryogenesis. They have increased the understanding of the mechanisms controlling the switch and progression of the microspore embryogenesis, which could help to improve its efficiency and to direct strategies, especially in agronomically interesting crops.     

毛白杨优良无性系‘LM50’花药培养植株再生体系建立*

毛白杨‘LM50’具有速生、抗逆、不飞絮等优良特性,是木本植物进行遗传转化的理想材料。长期无性繁殖会导致优良性状衰退,在进行组织培养时,往往出现外植体不定芽分化和生根困难等问题。通过花药培养可以在较短时间内使毛白杨复幼,从而消除因外植体老化带来的负面影响,为转化研究提供理想的材料。与此同时,期望通过花药诱导创制单倍体植株,为基因组学研究和倍性育种提供材料。以山东冠县毛白杨基因库的‘LM50’为试验材料,对其花药发育时期与花芽形态的关系进行鉴定,选择单核靠边期的花药进行试验,探究了生长素和细胞分裂素在花药愈伤组织形成、不定芽分化及不定芽生根中的作用,建立了毛白杨花药离体再生体系,采用流式细胞仪和染色体压片计数法对诱导获得的再生植株进行了倍性鉴定。进一步利用花药培养再生植株的叶片建立了分化率高、生根率高的植物再生体系。小孢子发育时期与花芽外部形态特征对比表明,花芽长度为(1.98 ± 0.06) cm,1/4花序露出芽鳞的花芽,此时小孢子大部分处于单核靠边期;选择处于此时期的花药诱导形成愈伤组织,愈伤组织诱导率最高的培养基为H + 1.00 mg/L NAA + 1.00 mg/L BA,诱导率约为28.89%;愈伤组织进一步分化为不定芽,最佳分化培养基为MS + 0.05 mg/L NAA + 0.50 mg/L BA,分化率约为22.23%;不定芽接种至生根培养基,最佳生根培养基为1/2 MS + 0.30 mg/L IBA,生根率约为93.30%;利用流式细胞仪和染色体压片法对花药培养的27株再生植株进行倍性鉴定,鉴定植物均为二倍体;再生植株叶片分化成芽的最佳培养基为MS + 0.10 mg/L TDZ + 0.10 mg/L NAA + 0.50 mg/L BA,分化率高达92.23%。该叶片分化产生的不定芽的生根培养基与愈伤组织诱导不定芽生根的培养基相同,生根率一致。研究获得了毛白杨‘LM50’花药诱导再生植株,并建立了再生植株的叶片培养体系,可用于该优良无性系的快速繁育和毛白杨的遗传转化研究,为毛白杨的分子设计育种奠定了基础。     

First stages of microspore reprogramming to embryogenesis through anther culture in Prunus armeniaca L.

Prunus armeniaca L. is a worldwide known species, very important particularly in the Mediterranean basin. Microspore embryogenesis through in vitro anther culture is a widely used method to obtain haploid and doubled haploid (DHs) plants which are being routinely used in breeding programmes for new superior cultivar development in many crops. Haploid-diploidization through gametic embryogenesis allows single-step development of complete homozygous lines from heterozygous parents. In the case of fruit crops, with long reproductive cycle, a high degree of heterozygosity, large size, and, often, self-incompatibility, there is no way to obtain haploidization through conventional methods. Induction of microspore embryogenesis in vitro is switched by a stress treatment. In many species, heat or cold stress has been reported to trigger pollen embryogenesis, the response being genotype dependent. In the present work we analyzed whether microspore reprogramming could be induced in apricot cultivars by cold stress through anther culture. We report the development of an in vitro anther culture protocol in P. armeniaca L. and analyse the response of several cultivars to stress treatments and culture media for inducing pollen embryogenesis. Results showed the formation of multicellular pollen and proembryos. The effect of two culture media in the embryogenic response was also analyzed, being the responses genotype-dependent. Monitoring of the cellular changes on the microspores was performed by structural and confocal microscopy analyses. Results indicated that the reprogramming of the microspore and the first steps of the embryogenic pathway have been achieved in different varieties of P. armeniaca, which constitutes a crucial step in the design of protocols for the regeneration of microspore-derived embryos and DH plants, for future potential applications in breeding programmes of this economically important fruit tree.     

我国体外诊断行业发展现状与对策建议*

体外诊断在疾病诊疗过程中扮演着非常重要的角色,素有“医生的眼睛”之称。新冠肺炎疫情让人们对生命健康的关注程度空前提高,在此背景下,体外诊断作为大健康产业的重要一环,迎来爆发式增长。概述全球和中国体外诊断行业发展现状,重点分析市场规模、竞争格局和国产化情况,分析现阶段我国体外诊断行业遇到的问题,并从突破原始性创新、加强资金保障、构建产业链生态、加大人才培养力度等方面提出建议和对策。     

Successful cocultivation of Brassica napus microspores and proembryos with Agrobacterium

Brassica napus microspores and microspore-derived proembryos were cocultivated with Agrobacterium tumefaciens harbouring a binary vector. The vector contained selectable genes for kanamycin and hygromycin antibiotic resistance. Microspores and proembryos survived the cocultivation procedure and subsequent antibiotic selection. Thousands of plantlets can be regenerated from a single experiment. Biochemical analysis indicated up to 7.3% of plants exhibited neomycin phosphotransferase II enzyme activity. Success of the cocultivation procedure depended largely on choosing the proper coculture conditions while allowing microspore embryogenesis to proceed.Abbreviations Hm hygromycin - Km kanamycin     

甘蓝型油菜小孢子胚状体发生的细胞学观察

应用甘蓝型油菜ＤＨ系保６０４为材料研究小孢子胚发生过程,结果表明,在小孢子离体培养１～５ｄ内,随培养天数增加,小孢子的存活率迅速下降,部分小孢子培养后出现细胞膨大和分裂,并沿２－细胞。“ｆ”形３细胞,多细胞原体,胚柄球形胚,心形胚最终发育成鱼雷形胚,一般在心形胚阶段,胚柄脱离胚主体部分游离到培养基中,大多数膨大的细胞不能分裂或分裂后停止发育或发育异常。     

Young microspore-derived maize embryos show two domains with defined features also present in zygotic embryogenesis

In this work we report the existence of two domains in early microspore maize proembryos displaying similar features of zygotic embryogenesis. The large or so-called endosperm-like domain exhibits specific features: coenocytic organisation, synchronous mitosis, vacuolated cytoplasm, starch granules, incomplete walls containing callose and differential tubuline organisation. The small or embryo-like domain displays small polygonal uninucleate cells with typical organisation of proliferating cells. The structural organisation and the subcellular localization of specific cytoplasmic and cell wall components (starch, tubuline and callose) in both proembryo domains have been determined by using specific cytochemical and immunocytochemical methods. Four morphological types of proembryos containing both domains have been characterised. Taking into account their relative size, the high asynchrony of the culture and the homologies between structural features of endosperm-like domain and zygotic endosperm development, they could represent different stages in microspore embryogenesis development. The ZmAE1 and ZmAE3 genes expressed in the Embryo Surrounding Region of the endosperm during zygotic embryogenesis (Magnard et al., 2000), were revealed to be expressed in early microspore proembryos by in situ hybridization at light and electron microscopy levels. This data supports the existence of an endosperm-like function during early microspore embryogenesis and provides new insights into the onset of microspore embryogenesis in maize, and its parallelism with zygotic embryogenesis.     

The metabolism of dihydrotachysterols: Renal side chain and non-renal nuclear hydroxylations in vivo and in vitro

The metabolism of dihydrotachysterol (DHT), a hydrogenated analogue of vitamin D, has been studied in vivo using man and rat and in vitro using the perfused rat kidney, and hepatoma (3B) and osteosarcoma (UMR-106) cell lines. In vivo a large number of metabolites appeared in the plasma of rats given DHT₂ and DHT₃. Of particular interest was a compound more polar than 25-hydroxy-DHT, which has been designated compound H. Further study of this compound showed that it was composed of two components, one (Ha) being in much lower concentration than the other (Hb). The production of T2/H (peak H from DHT₂) was demonstrated in human plasma after administration of oral DHT₂. Comparison of the metabolites formed in vivo with those isolated from the rat kidney perfused with 25-hydroxy-DHT₃ in vitro showed that 25-hydroxy-DHT₃ was metabolized along two metabolic pathways previously described for vitamin D, culminating in the production of 25-hydroxy-DHT₃-23,26-lactone and 23,25-dihydroxy-24-oxo-DHT₃. The osteosarcoma cell line metabolized 25-OH-DHT₃ in vitro along the same two metabolic pathways already demonstrated in the perfused rat kidney. More polar metabolites than compound H seen in rat plasma in vivo were shown to be metabolites of compound H and similar metabolites were also produced in the osteosarcoma cell line from chemically synthesized 1,25-dihydroxy-DHT₃. The hepatoma cell line 25-hydroxylated DHT and no feed-back inhibition was observed. Use of the hepatoma cell to 25-hydroxylate a number of chemically synthesized 1-hydroxy-DHTs indicated that compound Ha was indistinguishable from 1,25-dihydroxy-DHT whereas compound Hb is possibly 1β,25-dihydroxy-DHT. Studies with the VDR in both chick gut and calf thymus indicated that 1,25-dihydroxy-DHT is very effective in displacing radiolabelled 1,25-dihydroxyvitamin-D₃ and is thus most likely to be the calcaemic metabolite of DHT.     

Platelet activity in immune lysis of Trypanosoma musculi

It was observed that, when blood from CBA/J mice recovering from T. musculi infection was added to parasites in vitro, platelets adhered to the trypanosomes which subsequently died. This platelet adherence trypanosomal lysis (PATL) activity of the blood appeared late in infection and persisted long after parasites had been eliminated from the blood, although showing a cyclical pattern of activity. Trypanosomal lysis in vitro by fresh (non-heat-inactivated) immune plasma free of platelets could also be demonstrated.     

A mechanism for heterogeneous endothelial responses to flow in vivo and in vitro

Exposure of endothelium to a nominally uniform flow field in vivo and in vitrofrequently results in a heterogeneous distribution of individual cell responses. Extremes in response levels are often noted in neighboring cells. Such variations are important for the spatial interpretation of vascular responses to flow and for an understanding of mechanotransduction mechanisms at the level of single cells. We propose that variations of local forces defined by the cell surface geometry contribute to these differences. Atomic force microscopy measurements of cell surface topography in living endothelium both in vitro and in situ combined with computational fluid dynamics demonstrated large cell-to-cell variations in the distribution of flow-generated shear stresses at the endothelial luminal surface. The distribution of forces throughout the surface of individual cells of the monolayer was also found to vary considerably and to be defined by the surface geometry. We conclude that the endothelial three-dimensional surface geometry defines the detailed distribution of shear stresses and gradients at the single cell level, and that there are large variations in force magnitude and distribution between neighboring cells. The measurements support a topographic basis for differential endothelial responses to flow observed in vivo and in vitro. Included in these studies are the first preliminary measurements of the living endothelial cell surface in an intact artery.     

RU 58668: Further in vitro and in vivo pharmacological data related to its antitumoral activity

Previous studies with the pure antiestrogen RU 58668 showed that this compound proved to be highly antiproliferative in vitro, and to be the only antiestrogenic compound so far known to induce long-term regression of MCF-7 tumours implanted into nude mice. In order to obtain more insight into the therapeutic potential of this molecule, we performed a new set of experiments in vitro and in vivo in comparison with tamoxifen and/or ICI 182,780. In vitro, 1 nM RU 58668 induced an accumulation of MCF-7 cells in G0/G1 phases of the cell cycle within 48 h and, in contrast to trans-4-hydroxy-tamoxifen, blocked the invasiveness of ras-transfected MCF-7 cells into the chick embryo heart during the three weeks of co-culture. An in vivo dose-effect relationship study showed that RU 58668 induced a regression of MCF-7 tumour with as low a dose as 10 mg/kg/week, and that such an effect can not be obtained either with a sublethal dose of adriamycin or with ICI 182,780, (2–250 mg/kg/week). This reduction in the tumour volumes accords with histological modifications of the tumours, which showed a decrease in the ratio of epithelial cells over the tumoral mass, and with a concomitant decrease in their regrowth potential when reimplanted into naive nude mice. Taken together, these results suggest a promising usefulness for RU 58668 in the treatment of metastatic breast cancer in women.     

Overlap of laccases/cellobiose dehydrogenase activities during the decolourisation of anthraquinonic dyes with close chemical structures by Pycnoporus strains

Pycnoporus strains were used as model to understand the role of laccases in the in vivo decolourisation of three anthraquinonic dyes. The decolourisation capability of Pycnoporus sanguineus MUCL 41582 (PS7), which produces laccases as the main oxidative enzyme, was assayed and compared with the decolourisation capability of a control strain, Pycnoporus cinnabarinus MUCL 39533 (PC330) described as laccase-deficient strain. In absence of dye, laccase activity was observed during the trophophase and the idiophase with PS7, while no laccase activity was observed with PC330. Acid Blue 62 (ABu62), Acid Blue 281 (ABu281) and Reactive Blue 19 (RBu19) caused an increase in laccase activity and surprisingly laccase activity was detected with PC330. In vitro, oxidation of all three anthraquinones by a laccase preparation was obtained to a lesser extent than the whole cell process; suggesting that other factor(s) could be required for a complete decolourisation. As the time space of laccase production in the tested fungi was not perfectly coincidental with the decolourisation process, the activity of cellobiose dehydrogenase (CDH) was monitored. Present early in the broth during the growth of the fungi, CDH displayed in vitro a synergism with laccases in the decolourisation of ABu62, and an antagonism with laccases in the decolourisation of ABu281 and RBu19.     

In vivo strain gage implantation in rats

A technique for the fabrication of encapsulated micro-miniature rosette strain gages for in vivo implantation is described. The gage units have an overall area of ten square millimeters (2.5 mm × 4.0 mm), and hence can be installed in very small experimental animals, particularly rodents. Using a rat model, strain data for up to 12 days have been obtained and in vitro studies have validified the in vivo strain recordings.     

Specific effect of manganese on the allantoinase of Vigna Radiata

Vigna radiata seedlings germinated in the presence of Mn²⁺ show an unusual increase in allantoinase activity which is proportional to Mn²⁺ concentration up to 5 mM. Though Mn²⁺ is not an activator for V. radiata allantoinase, it specifically protects allantoinase against thermal as well as papain-catalysed inactivation. Evidence is presented to show that the primary effect of Mn²⁺ is a protective one, both in vitro and in vivo, and that this is reflected in the observed enhancement of allantoinase activity in Mn²⁺ grown seedlings. That this unusual effect of Mn²⁺ is a specific one is indicated by the lack of a similar effect with Mg²⁺. Cu²⁺ is shown to destabilize V. radiata allantoinase in vitro as well as in vivo.     

Ecdysterone induced sexual reproduction in Opalina sudafricana parasitic in Bufo regularis

Ecdysterone induced sexual reproduction in Opalina sudafricana parasitic in Bufo regularis. International Journal for Parasitology 3: 863–868. Ecdysterone injections (0·5 mg/ml) induced sexual reproduction in Opalina sudafricana parasitic in male or female Bufo regularis after 12 days. Hypophysectomy or gonadectomy did not prevent the hormone from producing its effect on the parasites. Ecdysterone did not induce sexual reproduction in the parasites in vitro. Urine of toads injected with ecdysterone induced the opalinids to encyst in vitro.     

In vitro and in vivo interactions between nuclear receptors at estrogen response elements

To study mechanisms involved in the antiestrogenic effect of retinoic acid (RA), previously described in mammalian cells, we used in vitro and in vivo approaches. One hypothesis was direct competition between nuclear receptors (ER, RAR and RXR) at the DNA level. We first showed in vitro that the RAR/RXR heterodimer could weakly bind an ERE and that retinoid receptors reduced binding of ER to an ERE. We next checked whether, in yeast, direct competition between receptors that recognize the same responsive element could be monitored in a reconstituted heterologous estrogen-responsive system, by determining the expression of a reporter gene. We then co-transformed RAR and RXR in an estrogenic responsive strain. This model demonstrated that, even though RAR/RXR was able to bind an ERE, the addition of retinoic acid had no inhibitory effect on estrogen-induced responses in this yeast system, unlike in mammalian cells. Interference between these receptors should require other factors than interactions at the ERE level. This model could be used to identify mammalian factors interacting with estrogen and retinoic acid receptors which could play a role in crosstalk between these receptors.