屋顶长生草叶的解剖结构及其离体培养形态发生的研究

对屋顶长生草叶的解剖结构及其在离体培养条件下形态发生过程进行了研究。结果表明,屋顶长生草的叶具有肉质旱生植物叶的特点,表皮细胞外有角质层,叶有较密的腺毛分布,气孔器由两个肾形的保卫细胞和两个镰刀形的护卫细胞组成;叶肉细胞没有栅栏组织与海绵组织之分,细胞比较大,有贮水作用;维管束平行排列,导管和筛管分子都很小,为一圈维管束鞘所包围。屋顶长生草叶片离体培养形态发生途径主要有两种:一种是由外植体直接产生不定芽(器官型)途径;另一种是叶肉细胞脱分化成胚性细胞,经胚性细胞团形成愈伤组织,再分化产生芽、根等器官(器官发生型),芽分化为内起源。     

杨树叶薄层培养中不定芽形态发生的细胞组织学研究

将杂种杨树(Populus nigra var.betulifolia×P.trichocarpe)NE299叶主脉用振动切片机横切成400μm或800μm的薄切片,培养在附加0.2mg/L BA和0.01mg/L NAA的木本植物培养基上。培养后,位于主脉维管束两侧中上部的维管束鞘薄壁细胞首先启动分裂。几乎同时,与其邻接的一些栅栏组织细胞也分裂,并很快形成胚性分生细胞团。主脉的愈伤组织主要由维管束鞘薄壁细胞,以及与其邻接的一些栅栏组织细胞和韧皮部的薄壁细胞分裂而来。不定芽通常发生在愈伤组织的周边区,也可以起源于维管组织结节(vascular nodules)周围的形成层状细胞。侧脉的维管束鞘细胞分裂活动很强,可不经愈伤组织直接长成不定芽。杨树叶主脉处的维管束鞘薄壁细胞在与叶肉组织相邻接的细胞中,通常含有少量较小的叶绿体,而位于背腹面的细胞中含有贮藏的淀粉。对形态发生的特定部位及其细胞进行了讨论。     

Production of<Emphasis Type="Italic"> Hevea brasiliensis</Emphasis> transgenic embryogenic callus lines by<Emphasis Type="Italic"> Agrobacterium tumefaciens</Emphasis>: roles of calcium

A procedure has been established for Agrobacterium tumefaciens-mediated genetic transformation of Hevea brasiliensis embryogenic friable calli. Precultivation of tissues on a CaCl(2)-free maintenance medium dramatically enhanced the transient activity of the reporter gene, gusA encoding beta-glucuronidase (GUS). The increase was first noticed in highly active cells (undifferentiated or/and embryogenic), in tissues precultured for 2-8 weeks. Beyond 8 weeks of preculture, GUS activity increased again, but this time in tissues consisting of differentiated cells accumulating polyphenols. Out of five Agrobacterium strains cocultivated with CaCl(2)-free precultured tissues, only inoculation with EHA105pC2301 led to high transient GUS activity. Paromomycin proved more effective than kanamycin for the selection of transformed cells, as it inhibits the growth of non-transformed cells more radically. Five paromomycin-resistant callus lines were established. The presence of gusA and neomycin phosphotransferase ( nptII) genes in the plant genome was confirmed by DNA amplification, and by Southern hybridization. These results confirmed that A. tumefaciens is an effective system for mediating stable transformation of rubber tree calli with a low copy number of transgenes. Transgenic callus lines constitute a useful tool for studying genes of interest on a cellular level and for regenerating transgenic rubber trees.     

Hepatitis C virus ultrastructure and morphogenesis

Details of the ultrastructure of hepatitis C virus (HVC) virion remain unclear because it has proved extremely difficult to visualise virus particles from infected serum and tissues directly. In addition, although much is known about the viral genome, first cloned in 1989, little is known about HCV morphogenesis, due to the lack of an efficient in vitro culture system for HCV propagation. Virus-like particles (VLPs) obtained by expressing genes encoding the HCV structural proteins in mammalian cells can be used as an alternative model for studying HCV morphogenesis. In particular, this HCV-LP model has made it possible to demonstrate that HCV budding occurs at the ER membrane and that the core protein drives this process. The HCV-LP model opens up new possibilities for the investigation of viral morphogenesis and virus-host cell interactions, which may make it possible to establish the long-awaited in vitro culture system for HCV.     

Influence of antibiotic cefotaxime on somatic embryogenesis and plant regeneration in indica rice

An antibiotic, cefotaxime (Omnatax) has been found to promote somatic embryogenesis and subsequent plant regeneration in vitro in indica-type basmati rice cultures. Response was highly genotype specific. The number, mass and morphology of the calli formed on the scutellar tissues were dependent on the growth medium (with or without cefotaxime). The embryogenic nature of nodular calli was confirmed through histological analysis and their plant regeneration ability. The calli of variety Pusa basmati 1 grown on medium supplemented with cefotaxime (100 mg/L) exhibited up to 70.5% plant regeneration as compared to control (51.51%). Plants regenerated from emryogenic calli were phenotypically normal and identical to seed-derived plants and exhibited normal fertility. A limited humidity and an optimal aeration of the culture tubes further enhanced the frequency of somatic embryogenesis and plant regeneration.     

The effect of cytokinins on in vitro cultured inbred lines of Raphanus sativus var. radicula Pers. with genetically determined tumorigenesis

The higher plant tumors are convenient models for studying the genetic control mechanism of plant cell division. There are two types of tumors: induced by the pathogenic factor and genetically determined. The development of both tumor types was related to the changes in cytokinin metabolism and/or signal transduction. In this work, the effect of synthetic cytokinins on the in vitro morphogenesis of cotyledon explants and isolated apices of radish seedlings was studied in several inbred radish lines (Raphanus sativus var. radicula Pers.) that differed in their in vivo tumorigenic properties. It was noted that root formation was stronger affected by kinetin while the treatment with thidiazuron tended to induce active callus formation in cotyledon explants of all inbred lines, except IIa. Growing with benzyladenine produced an intermediate effect as regards all morphogenetic responses. Cytokinin treatment of tumorigenic lines enhanced necrotic development in cotyledon explants. Culturing isolated apices of regenerated plants produced tumors anatomically and morphologically similar to those developing in vivo. Some of the lines nontumorigenic in vivo with enhanced formation of calli on cotyledon explants also developed tumors on apical explants in vitro when treated with cytokinins. These data suggest that different mechanisms for tumor formation operate in various radish lines. The radish lines are classified into three types: (1) necrotic lines with tumor formation putatively related to endogenous cytokinin level, (2) callus-forming lines with cell division enhanced in response to cytokinins, and (3) necrosis-and callus-forming lines with both mechanisms of tumor formation involved.     

Extracellular matrix as the early structural marker for <Emphasis Type="Italic">Centella asiatica</Emphasis> embryogenic tissues

Embryogenic and non-embryogenic calli were induced from the Centella asiatica leaf explants on Murashige and Skoog medium supplemented with kinetin and 2,4-dichlorophenophenoxyacetic acid. The extracellular matrix (ECM) layer was seen on the surface of embryogenic cells but not on the non-embryogenic cells. The ECM formed bridges with net-like material between the embryogenic cells. This network like structure was believed to play an important role in plant morphogenesis and can serve as an early structural marker of embryogenic competence in Centella asiatica calli culture.     

Genetic control of totipotency of plant cells in vitro

The main approaches have been considered to studying the genetic control of plant cell totipotency in an in vitro culture. The capacity of cultured plants for callusogenesis, organ formation, and somatic embryogenesis depends on the activity of genes that determine and maintain the meristematic state of cells, level of hormones in the cells, and sensitivity to hormones, as well as on the activity other genes that control different stages of plant morphogenesis.     

Trichome morphogenesis in Arabidopsis

Trichomes (plant hairs) in Arabidopsis thaliana are large non-secreting epidermal cells with a characteristic three-dimensional architecture. Because trichomes are easily accessible to a combination of genetic, cell biological and molecular methods they have become an ideal model system to study various aspects of plant cell morphogenesis. In this review we will summarize recent progress in the understanding of trichome morphogenesis.     

In vitro Reaction of Sunflower (Helianthus annuus L.) to the Toxin(s) Produced by Alternaria alternata, the Casual Agent of Brown Leaf Spot

A bioassay system was developed for studying the in vitro reaction of sunflower ( Helianthus annuus L. cv. 'Nanus') against the toxin produced by the virulent pathotype IMI 366417 (1) of the pathogenic fungus Alternaria alternata. Cotyledons from 2-week-old seedlings were cultured on a MS (Murashige and Skoog) medium supplemented with 0.3 μM NAA (α-napthylacetic acid) and 1.3 μM BA (6-benzyladenine). Exponentially growing calli were transferred to selective media containing toxin solutions at various concentrations. The fresh weight of the cultured calli was reduced as the toxin concentration increased, although the viability of the cells, expressed as callus dehydrogenase activity, increased. Selection for toxinresistant genotypes was attempted at 30% toxin concentration, which causes a 90% reduction in callus growth. After one month in culture, 18% of the calli demonstrated resistance to the toxin. However, no plants could be regenerated from those calli after transfer onto a MS medium supplemented with 5.4 μM NAA and 4.4 μM BA. The effect of the toxin purification method on toxin yield and biological activity, as well as its possible mode of cellular action are discussed. The results of these experiments may contribute to a better understanding of the disease mechanism and help establish an efficient selection method of resistant sunflower genotypes.     

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.     

Extracellular metalloproteinases in neural crest development and craniofacial morphogenesis

Abstract

The neural crest (NC) is a population of migratory stem/progenitor cells that is found in early vertebrate embryos. NC cells are induced during gastrulation, and later migrate to multiple destinations and contribute to many types of cells and tissues, such as craniofacial structures, cardiac tissues, pigment cells and the peripheral nervous system. Recently, accumulating evidence suggests that many extracellular metalloproteinases, including matrix metalloproteinases (MMPs), a disintegrin and metalloproteinases (ADAMs), and ADAMs with thrombospondin motifs (ADAMTSs), play important roles in various stages of NC development. Interference with metalloproteinase functions often causes defects in craniofacial structures, as well as in other cells and tissues that are contributed by NC cells, in humans and other vertebrates. In this review, we summarize the current state of the field concerning the roles of these three families of metalloproteinases in NC development and related tissue morphogenesis, with a special emphasis on craniofacial morphogenesis.     

Flow cytometric analysis of polysomaty and in vitro genetic instability in potato

The analysis of nuclear DNA contents in various tissues of potato genotypes showed that flow cytometry is a rapid method to characterize large populations of cells for polysomaty, that is, the occurrence of cells with normal DNA levels together with cells containing endoreduplicated nuclei. The proportion of endoreduplicated nuclei varied in different tissues and genotypes of potato. The analysis of callus and cell cultures showed that the temporal changes in nuclear DNA contents during in vitro growth can be followed and the degree of polyploidization quantified. It is concluded that flow cytometry is a highly suitable method to detect ploidy changes in differentiated plant tissues and calli which are often not amenable for chromosome number determination.     

Confocal microscopy of cells implanted into tissue blocks: Cell migration in long-term histocultures

Summary In three-dimensional tissues in vivo, cells find themselves in a unique, heterogeneous microenvironment among various cellular and noncellular elements. Cells are greatly affected by and contribute to their physical and chemical microenvironments. However, live cells are currently studied predominantly in homogeneous monolayer cultures where newly established contacts might be fundamentally different from contacts in vivo. Several systems have been suggested to simulate the three-dimensional environment of real tissue. In this report, we describe a new system for studying cell behavior inside real tissues in vitro. By fluorescently labeling mouse tumor cells, then implanting them into cultured tissue blocks (histocultures), we have observed cellular location and followed their locomotion, within tissues in vitro for days. We discuss the potential of the described system for studying different aspects of cell behavior in a nativelike microenvironment.     

Liposome-mediated gene delivery into plant cells

Liposomes may offer several advantages as vectors for gene delivery into plant cells: (1) enhanced delivery of encapsulated DNA by membrane fusion, (2) protection of nucleic acids from nuclease activity, (3) targeting to specific cells, (4) delivery into a variety of cell types besides protoplasts by entry through plasmodesmata, (5) delivery of intact small organelles. Realization of these advantages calls for the construction of efficient liposomes, for appropriate fusion conditions and for an understanding of the nature of liposome-cell interactions. Various characteristics and techniques of the liposome-cell system are described (mode of delivery, liposome types and composition, and means of promoting delivery of liposome contents). Data of liposome-mediated delivery of various macromolecules into plant cells, with special reference to protoplasts, calli and pollen are reviewed. This includes data obtained by the use of fluorescent probes, radioactive-labelled DNA, viral nucleic acids and expression of plasmid-DNA. Structure and characteristics of plant surfaces and plasmodesmata are discussed with respect to DNA entry. It is suggested that liposome-mediated gene delivery into plant cells, and not only protoplasts, will be advantageous in certain specific tissues and situations.     

Genetic engineering of cytokinin metabolism: Prospective way to improve agricultural traits of crop plants

Cytokinins (CKs) are ubiquitous phytohormones that participate in development, morphogenesis and many physiological processes throughout plant kingdom. In higher plants, mutants and transgenic cells and tissues with altered activity of CK metabolic enzymes or perception machinery, have highlighted their crucial involvement in different agriculturally important traits, such as productivity, increased tolerance to various stresses and overall plant morphology. Furthermore, recent precise metabolomic analyses have elucidated the specific occurrence and distinct functions of different CK types in various plant species. Thus, smooth manipulation of active CK levels in a spatial and temporal way could be a very potent tool for plant biotechnology in the future. This review summarises recent advances in cytokinin research ranging from transgenic alteration of CK biosynthetic, degradation and glucosylation activities and CK perception to detailed elucidation of molecular processes, in which CKs work as a trigger in model plants. The first attempts to improve the quality of crop plants, focused on cereals are discussed, together with proposed mechanism of action of the responses involved.     

Stimulation of in vitro morphogenesis,antioxidant activity and over expression of kaurenoic acid 13-hydroxylase gene in Stevia rebaudiana Bertoni by chlorocholine chloride

Phytoconstituents from medicinal plants are considered as important source of raw materials of drugs for pharmaceutical industries. Biotechnology has become an inevitable approach in the area of research and development of medicinal plants for many decades. The present work has been carried out to ascertain the role of chlorocholine chloride (CCC) on in vitro morphogenesis, antioxidant activity and expression level of kaurenoic acid 13-hydroxylase (KA13H) gene in Stevia rebaudiana. To fulfill these purposes chlorocholine chloride was applied in the Murashige and Skoog (Physiol Plant 15(3):473–497, 1962) medium in combination with other plant growth regulators such as 1-naphthalene acetic acid, kinetin and thidiazuron. Chlorocholine chloride was found to contribute significant role on in vitro morphogenesis of S. rebaudiana as evidenced by the formation of embryogenic calli and increase in callusing and microshooting efficiency of explant, i.e., cotyledonary leaf. Moreover, antioxidant enzyme activity as well as ascorbic acid content of the calli and leaves was also stimulated after application of chlorocholine chloride. Q-PCR amplification using gene-specific primers revealed that CCC also promoted the expression level of KA13H gene in S. rebaudiana leaves. The overall study highlighted the promising role of chlorocholine chloride on regeneration efficiency of cotyledonary leaf, significant promotion in antioxidant potential and expression of KA13H gene in S. rebaudiana.     

Single primary fetal lung cells generate alveolar structures in vitro

Organ morphogenesis, including lung morphogenesis, involves a series of cellular behaviors that are difficult to observe and document in vivo due to current limitations in imaging techniques. Therefore, in vitro models are necessary to study these cellular behaviors as well as basic developmental processes relevant to in vivo morphogenesis. Here, we describe a novel in vitro three-dimensional (3D) culture system for assessing mouse lung alveolar morphogenesis using primary fetal mouse lung cells cultured in Matrigel supplemented with fibroblast growth factor 10 and hepatocyte growth factor. In our in vitro 3D culture system, single primary mouse fetal lung cells successfully grew, developed lumen, and formed multivesicular epithelial structures, resulting in a morphology that was highly similar to that of lung alveoli. This culture system is a useful tool for investigating the cellular and molecular mechanisms involved in lung alveolar morphogenesis.     

胚珠发育的分子机理

胚珠是研究器官形态发生和模式建成遗传分子机理的一个理想系统。近年来, 关于胚珠特征的决定、模式建成、珠被形态建成和胚囊形成等发育事件分子机理的研究取得了重要进展, 初步建立了胚珠发育的基因调控模型。同时, 离体花器官再生系统为研究激素调控胚珠发育的机理提供了有效途径。本文对拟南芥(Arabidopsis thaliana)胚珠发育的分子调控机制进行了综述。     

胚珠发育的分子机理

胚珠是研究器官形态发生和模式建成遗传分子机理的一个理想系统.近年来,关于胚珠特征的决定、模式建成、珠被形态建成和胚囊形成等发育事件分子机理的研究取得了重要进展,初步建立了胚珠发育的基因调控模型.同时,离体花器官再生系统为研究激素调控胚珠发育的机理提供了有效途径.本文对拟南芥(Arabidopsis thaliana)胚珠发育的分子调控机制进行了综述.