薄层培养的应用现状与前景

植物薄层培养 (TCL)实验系统是 70年代初发展起来的一个新颖精致的实验系统。由于薄层培养具有组织结构简单、对调控因子敏感、培养易成功等优点 ,目前已经在植物细胞的分裂、分化及植物生长、发育的调控机制与分子机理等方面的研究得到了广泛的应用。本文综述了薄层培养在形态发生、细胞学、分子生物学等研究领域的应用现状与进展 ,并对当前薄层培养存在的问题作了评述。     

细胞薄层培养在植物激素调节形态发生研究中的应用

从外源激素对形态发生的调节、植物激素的作用机制、形态发生的内源激素动态等方面概述了细胞薄层培养在植物激素诱导与调节形态发生研究中的应用进展,并对其在植物信号转导、玻璃化现象的发生机制、离体再生障碍的克服等研究领域的发展前景和尚存在的问题作了讨论。     

植物维管系统形成的调节机制

植物维管系统形成与分化包含着贯穿植物生长发育全过程的一系列细胞分裂与分化事件。这些事件的发生和发展过程受到很多遗传和其他内源因子的调控。对维管系统形成及其调控的认识近年来获得了较大的进展。研究表明植物激素、转录因子、短肽信号分子和microRNA等在植物维管系统建成中发挥重要调控作用。     

植物内生菌在食用农产品质量安全与营养品质调控中的研究进展

食用农产品是食品的基础原料,直接关乎人类日常生活和健康,对其质量安全和营养品质调控是农业和食品领域研究的重大课题。植物内生菌是健康植物体内的共生菌,菌体及其代谢产物能对调控植株生长发育以及环境胁迫适应等方面产生重要影响。目前对植物内生菌的研究已渗透到农业生产过程中的各个领域,其应用价值逐渐崭露头角,成为助推农业生产和提升农产品品质的潜在辅助手段,特别在食用农产品质量安全与营养品质调控上具有独特优势。本文就植物内生菌在食用农产品生产过程中产地环境危害物、农药投入品危害物、病虫害以及营养品质指标等调控方面的最新研究状况进行系统综述,并就植物内生菌在食用农产品质量安全与营养品质指标调控研究上的技术瓶颈以及未来可拓展的研究和应用展开探讨,为食用农产品质量安全与营养品质调控领域研究提供最新的有参考价值的文献资料和潜在技术创新思路。     

重金属污染土壤植物修复中细胞分裂素的作用与机制

植物修复因投资成本低、环境扰动少、二次污染易控制、美化环境等优点成为重金属污染土壤修复重要的治理技术。植物内源细胞分裂素调控植物生理活动,外源细胞分裂素对植物生理生态特征产生显著影响,且在植物修复中逐渐受到研究人员的关注。细胞分裂素能够调控植物根茎发育、叶片衰老、激素传递等过程,同时在重金属胁迫下也参与蒸腾、光合、抗性、解毒等系统的运转。以细胞分裂素对植物生理活动的调控作用研究为基础,阐述了细胞分裂素在植物修复中的作用机制。主要包括：增强光合作用,延缓叶片衰老,提升植物抗性能力;调控根茎叶发育,增加植物生物量,强化植物富集效果;增强转运蛋白表达,提高叶面蒸腾作用,促进重金属吸收转运;参与解毒过程,降低重金属毒性,调控重金属体内转化。最后提出了细胞分裂素在重金属污染土壤植物修复中的研究方向,这对促进细胞分裂素在植物修复中的实际应用具有重要意义。     

氢气调节植物生长发育和提高植物抗逆性的研究进展

氢气作为新发现的活性气体被广泛研究。在植物生长发育方面,氢气具有促进种子发芽、幼苗发育、不定根生长等作用;在植物遭受逆境胁迫过程中,氢气通过调控抗氧化酶活性、抗氧化物质的生成及其相应的转录本来应对胁迫带来的氧化损伤,提高植物对干旱、盐胁迫、重金属胁迫、除草剂、紫外照射等胁迫的抗性,同时氢气还可以调控与抗病虫害等胁迫相关基因的表达。该文对国内外有关氢气在促进植物生长发育和提高植物抗性方面的作用,以及逆境胁迫下氢气作为信号分子通过调控抗氧化防御系统提高植物抗逆性的机制进行综述,以期更好地了解和促进氢气在农业科学上的研究与应用。     

脱落酸在植物花发育过程中的作用

植物激素脱落酸(ABA)对植物的生长发育具有多方面的调节作用,比如种子休眠、萌发,营养生长,环境胁迫反应等。大量研究显示,ABA也参与了植物的成花调控。影响植物成花调控的环境因子,包括光周期变化、春化作用、干旱等均会导致植物体内ABA代谢的变化。本文从调控植物开花的4条主要途径与植物体内ABA代谢变化之间的相互关系,花芽分化时期ABA在植物叶芽和花芽中的动态分布以及离体培养条件下ABA对花芽分化的影响等方面总结了ABA与植物花发育这一领域的最新研究进展。对ABA在植物成花诱导和花发育中的作用进行了综合分析。     

CRISPR/Cas9系统在培育抗病毒植物新种质中的应用

随着CRISPR/Cas9系统在基因组编辑技术上的开发和完善,CRISPR/Cas9系统在应用于动物病毒感染性疾病防治并取得相当成效的同时,也逐步被应用到对植物病毒基因组进行高效靶向修饰的研究中。CRISPR/Cas9系统对基因组靶向修饰作用不仅实现了对植物DNA病毒基因组序列的编辑,还展示了其有效作用于植物RNA病毒基因组的潜力,同时CRISPR/Cas9系统还能在基因转录和转录后调控水平发挥作用,说明该系统具有通过多种途径调控植物病毒复制的潜能。相对其他植物病毒病防治策略,该系统对病毒基因组的编辑更精准、对基因表达的调控更稳定,对病毒病的抗性也更为广谱。本文将CRISPR/Cas9系统与其他植物病毒病防治策略进行了比较,概述了该系统在培育植物抗病毒病新种质中的优势,分析了其具体应用在该领域中面临的主要问题,讨论了该系统在培育抗病毒植物新种质应用中的发展趋势。     

分室培养装置在丛枝菌根真菌研究中的应用及其发展

重点围绕玻璃珠分室培养系统、H形分室培养系统、根排斥室培养系统、供体自养植物的双分室体外培养系统、丛枝菌根(AM)真菌与普通植物根器官的双重培养系统、AM真菌与Ri T-DNA转型根的双重单胞无菌培养系统、AM真菌与Ri T-DNA转型根双重培养的改良分室单胞培养系统等7个不同的分室培养装置, 对AM真菌的培养类型及其应用进行了系统的评述。其中, 采用玻璃珠分室培养装置易于将AM真菌与培养基质分开, 能获得大量纯净的AM真菌繁殖体, 用于研究AM真菌对矿质元素和微量元素的吸收, 具有不可替代的作用。H形分室培养系统和根排斥室(RECs)培养系统均能够获得连续的、可切断的共生菌根网络(CMNs), 可用于研究植物-植物、植物-昆虫之间化感作用产生的信息交流。供体自养植物的双分室培养系统有益于研究AM真菌对宿主植物在单作和混作条件下生长效应的影响。AM真菌与植物根器官的双重培养系统为研究AM真菌的侵染过程及生理、生化特性提供了极大的方便, 同时为纯培养研究提供了重要的理论依据。AM真菌与Ri T-DNA转型根的双重单胞无菌培养体系可以获得AM真菌纯净菌体, 是研究AM真菌遗传、生理、生化等特性的理想方法。以AM真菌与Ri T-DNA转型根的双重单胞无菌培养系统为基础, 可以在菌丝生长室置换培养基、在根室中补充适量碳源, 并多次收获AM真菌繁殖体。转型根改良双重培养系统是提高AM真菌孢子接种剂产量的有效方法。综上所述, AM真菌的分室培养系统已经取得显著进展, 为开展个体、种群、群落等不同层次的菌根生态学研究提供了依据。     

四环素诱导调控表达系统的研究与应用

四环素(Tet)诱导调控表达系统是在大肠杆菌Tn10转座子中特异的Tet抗性操纵子基础上建立起来的一种用于诱导基因表达的调控系统。经过近几年来的发展,衍生出了Tet-on、Tet-off两套调控系统;随着对Tet诱导调控表达系统的改进,其对基因表达调控的严谨性、诱导效率及安全性等方面逐步得到改善,并被广泛应用于基础研究及体内外的实验治疗研究。此外Tet诱导调控系统与其他诱导调控体系及新兴技术的联合应用,为疾病的基因治疗研究提供有力的手段。     

The importance of the explant on regeneration in thin cell layer technology

Summary The basic factor underlying the success of the tissue culture, large-scale micropropagation and genetic transformation of any plant species is regeneration. This has been achieved over the years through the use of various-sized explants ranging from protoplasts (small scale) to entire organs (large scale). Inherent problems underlie the use of either extreme, leading to both nonspecific morphogenic reactions in the latter, or to undesired necrosis in the former. This review investigates the importance of different aspects of a thin cell layer (TCL) explant, from its source to its size. TCLs, as a results of their size and origin, in combination with other controllable factors such as media and environmental conditions, have shown this system to be superior to the use of conventional explants. Numerous species that were previously unsuccessfully tissue-cultured have, with the use of TCL technology, resulted in their successful micropropagation and regeneration. These successes, based on the inherent qualities of the TCL explant—specific for a given species—are also examined.     

Somatic embryogenesis in peach palm using the thin cell layer technique: induction, morpho-histological aspects and AFLP analysis of somaclonal variation

BACKGROUND AND AIMS: The thin cell layer (TCL) technique is based on the use of very small explants and has allowed enhanced in vitro morphogenesis in several plant species. The present study evaluated the TCL technique as a procedure for somatic embryo production and plantlet regeneration of peach palm. METHODS: TCL explants from different positions in the shoot apex and leaf sheath of peach palm were cultivated in MS culture medium supplemented with 0-600 microM Picloram in the presence of activated charcoal. The production of primary calli and embryogenic calli was evaluated in these different conditions. Histological and amplified fragment length polymorphism (AFLP) analyses were conducted to study in vitro morphogenetic responses and genetic stability, respectively, of the regenerated plantlets. KEY RESULTS: Abundant primary callus induction was observed from TCLs of the shoot meristem in culture media supplemented with 150-600 microM Picloram (83-97%, respectively). The production of embryogenic calli depends on Picloram concentration and explant position. The best response observed was 43% embryogenic callus production from shoot meristem TCL on 300 microM Picloram. In maturation conditions, 34+/-4 somatic embryos per embryogenic callus were obtained, and 45.0+/-3.4% of these fully developed somatic embryos were converted, resulting in plantlets ready for acclimatization, of which 80% survived. Histological studies revealed that the first cellular division events occurred in cells adjacent to vascular tissue, resulting in primary calli, whose growth was ensured by a meristematic zone. A multicellular origin of the resulting somatic embryos arising from the meristematic zone is suggested. During maturation, histological analyses revealed bipolarization of the somatic embryos, as well as the development of new somatic embryos. AFLP analyses revealed that 92% of the regenerated plantlets were true to type. The use of TCL explants considerably improves the number of calli and somatic embryos produced in comparison with previously described protocols for in vitro regeneration of peach palm. CONCLUSIONS: The present study suggests that the TCL somatic embryogenesis protocol developed is feasible, although it still requires further optimization for in vitro multiplication of peach palm, especially the use of similar explants obtained from adult palm trees.     

A modeled hydrophobic domain on the TCL1 oncoprotein mediates association with AKT at the cytoplasmic membrane

AKT has a critical role in relaying cell survival and proliferation signals initiated by ligand binding to surface receptors in mammalian cells. Induction of AKT serine/threonine kinase activity is augmented by the T-cell leukemia-1 (TCL1) oncoprotein through a physical association requiring the AKT pleckstrin homology domain. Here, we used molecular modeling and identified an exposed hydrophobic patch composed of two discontinuous amino acid stretches near one end of the TCL1 beta-barrel that was required for a TCL1-AKT association. Site-directed mutations of this region did not affect TCL1 secondary structure, yet they disrupted interactions with AKT. This region was found in other members of the TCL1 oncoprotein family, such as TCL1b and MTCP1, and suggested a conserved, novel AKT binding domain. Interestingly, TCL1 and AKT co-localize in multiple cell compartments, but only extracts from the plasma membrane stimulate optimal complex formation in vitro. Identification of an AKT binding domain on TCL1 is an important step in deciphering the complex interactions that regulate AKT kinase activity in lymphocyte development and neoplasia within the immune system.     

Dissecting the Concept of the Thin Cell Layer: Theoretical Basis and Practical Application of the Plant Growth Correction Factor to Apple,Cymbidium and Chrysanthemum

A thin cell layer (TCL) is a thin layer of plant cells. TCLs have served as a simple, but important biotechnological tool in plant science, with several dozen crop species having had tissue culture regeneration protocols developed using TCLs generated from multiple explant sources. There are two types of TCLs, transverse TCLs, or tTCLs and longitudinal TCLs, or lTCLs. The former is the most common, ranging from 100 μm to 1–2 mm in thickness, usually cutting through several tissue types. In contrast, the latter usually targets a very specific layer of cells or tissues, and may vary in length but is as thick as a tTCL. The developmental question that needs to be addressed will determine the choice between one or the other and its use in plant tissue culture. The often unappreciated beauty of the TCL is not so much in its actual regeneration capacity, but rather in its potential regeneration capacity. Herein, we use data from three model species, a woody temperate fruit tree, Malus sp. (apple; Rosaceae), and two herbaceous ornamentals, Cymbidium (orchid; Orchidaceae) and Dendranthema (chrysanthemum; Asteraceae), to demonstrate the theory and functionality of TCLs. Moreover, using a new concept, the plant growth correction factor, or GCF, the ability to theoretically predicts the organogenic outcome in vitro is presented through mathematical models based on the geometric analysis of explant size and shape. A new factor, the geometric factor, or GF, was also determined for all three plants to compare regeneration from different explant types with different shapes. The GF, which is calculated, is independent of plant species or any in vitro conditions, but depends only on the size and shape of the explant and on tissue that is capable of regeneration. The GF and GCF would, in theory, allow for the direct comparison of plant in vitro studies in different laboratories provided that explant size is known, and to predict the theoretical outcome of a regeneration protocol if different explants were to be used.     

Adaptation of the Transverse Carpal Ligament Associated with Repetitive Hand Use in Pianists

The transverse carpal ligament (TCL) plays a critical role in carpal tunnel biomechanics through interactions with its surrounding tissues. The purpose of this study was to investigate the in vivo adaptations of the TCL’s mechanical properties in response to repetitive hand use in pianists using acoustic radiation force impulse (ARFI) imaging. It was hypothesized that pianists, in comparison to non-pianists, would have a stiffer TCL as indicated by an increased acoustic shear wave velocity (SWV). ARFI imagining was performed for 10 female pianists and 10 female non-pianists. The median SWV values of the TCL were determined for the entire TCL, as well as for its radial and ulnar portions, rTCL and uTCL, respectively. The TCL SWV was significantly increased in pianists relative to non-pianists (p < 0.05). Additionally, the increased SWV was location dependent for both pianist and non-pianist groups (p < 0.05), with the rTCL having a significantly greater SWV than the uTCL. Between groups, the rTCL SWV of pianists was 22.2% greater than that of the non-pianists (p < 0.001). This localized increase of TCL SWV, i.e. stiffening, may be primarily attributable to focal biomechanical interactions that occur at the radial TCL aspect where the thenar muscles are anchored. Progressive stiffening of the TCL may become constraining to the carpal tunnel, leading to median nerve compression in the tunnel. TCL maladaptation helps explain why populations who repeatedly use their hands are at an increased risk of developing musculoskeletal pathologies, e.g. carpal tunnel syndrome.     

Characterization of TCL, a new GTPase of the rho family related to TC10 andCcdc42

GTPases of the Rho family control a wide variety of cellular processes such as cell morphology, motility, proliferation, differentiation, and apoptosis. We report here the characterization of a new Rho member, which shares 85% and 78% amino acid similarity to TC10 and Cdc42, respectively. This GTPase, termed as TC10-like (TCL) is encoded by an unexpectedly large locus, made of five exons spanning over 85 kilobases on human chromosome 14. TCL mRNA is 2.5 kilobases long and is mainly expressed in heart. In vitro, TCL shows rapid GDP/GTP exchange and displays higher GTP dissociation and hydolysis rates than TC10. Using the yeast two-hybrid system and GST pull-down assays, we show that GTP-bound but not GDP-bound TCL protein directly interacts with Cdc42/Rac interacting binding domains, such as those found in PAK and WASP. Despite its overall similarity to TC10 and Cdc42, the constitutively active TCL mutant displays distinct morphogenic activity in REF-52 fibroblasts, producing large and dynamic F-actin-rich ruffles on the dorsal cell membrane. Interestingly, TCL morphogenic activity is blocked by dominant negative Rac1 and Cdc42 mutants, suggesting a cross-talk between these three Rho GTPases.     

Identification of Akt association and oligomerization domains of the Akt kinase coactivator TCL1

Serine/threonine kinase Akt/protein kinase B, the cellular homologue of the transforming viral oncogene v-Akt, plays a central role in the regulation of cell survival and proliferation. We have previously demonstrated that the proto-oncogene TCL1 is an Akt kinase coactivator. TCL1 binds to Akt and mediates the formation of oligomeric TCL1-Akt high-molecular-weight protein complexes in vivo. Within these protein complexes, Akt is preferentially phosphorylated and activated. The MTCP1/TCL1/TCL1b oncogene activation is the hallmark of human T-cell prolymphocytic leukemia (T-PLL), a form of adult leukemia. In the present study, using a PCR-generated random TCL1 library combined with a yeast two-hybrid screening detecting loss of interaction, we identified D16 and I74 as amino acid residues mediating the association of TCL1 with Akt. Based on molecular modeling, we determined that the beta C-sheet of TCL1 is essential for TCL1 homodimerization. Studies with mammalian overexpression systems demonstrated that both Akt association and oligomerization domains of TCL1 are distinct functional domains. In vitro kinase assays and overexpression experiments in mammalian cells demonstrated that both TCL1-Akt interaction and oligomerization of TCL1 were required for TCL1-induced Akt activation and substrate phosphorylation. Assays for mitochondrial permeability transition, nuclear translocation, and cell recovery demonstrated that both Akt association and homodimerization of TCL1 are similarly needed for the full function of TCL1 as an Akt kinase coactivator in vivo. The results demonstrate the structural basis of TCL1-induced activation of Akt, which causes human T-PLL.     

Luminescent radio frequency radiation dosimetry

Thermoluminescent dosimetry has been the industry standard for ionizing radiation dosimetry because it is inexpensive, sensitive, and accurate. No such system exists for radio frequency radiation. This paper describes the state of the art of efforts toward developing such a system. Thermochemiluminescent (TCL) dosimetry, first reported in 1991, is a first step toward achieving this goal. However, it has had problems in the production of TCL materials and in conversion of the luminescent signal into specific absorption rate (SAR). The former problem has been solved by the development of a genetically engineered Escherichia coli bacterium (JM 109/plC20RNR_1.1), described herein, that produces the TCL material in a fermentation process. The latter problem stems from the difficulty in determining the structure of the currently best TCL material diazoluminomelanin. A theoretical approach for the solution of this problem has been achieved by combining equations for delayed fluorescence, temperature determination by TCL, and the free energy equation for equilibrium reactions. It has led to an explanation for the stable display of steady‐state energy disposition, illustrated by TCL, in phantoms without the expected disruption by thermal conduction or convection, at frequencies ranging from 2.06 GHz to 35 GHz. Bioelectromagnetics 20:46–51, 1999. Published 1999 Wiley‐Liss, Inc.     

Plant Thin Cell Layers: A 40-Year Celebration

The concept of a thin cell layer (TCL) was initially presented by Tran Thanh Van in two key papers exactly 40 years ago. At that time, Nicotiana tabacum was the model plant used to establish three main pathways for de novo organogenesis by developing a flower, vegetative bud, and root “programme” from pedicel tissue. Over the last 40 years, a wealth of research in plant tissue culture based on TCLs has emerged, fortifying the importance of this very simple technique, and highlighting its fundamental importance as a key tool in plant cell and tissue differentiation as well as organ development. This review not only highlights the achievements made using TCLs in the plant kingdom over these 40 years, it also reports on the success of this technique in ornamentals, fruit and forestry species, vegetables, and medicinal plants. There is overwhelming evidence of the importance of this technique for plant biotechnology, and it provides one solution for the mass clonal propagation of plants, use in bioreactors, genetic transformation, or micropropagation.