Transformation of Nicotiana tabacum,N. debneyi,and N. rustica: Inheritance and protoplast expression of antibiotic resistance

Agrobacterium tumefaciens strains harbouring plasmid vectors pBCAT1, pVU1011 or pMON806 were used to transform leaf explants of Nicotiana tabacum cultivars Delgold and Candel, N. debneyi, and N. rustica var. NRT. Transgenic plants resistant to the selective agents kanamycin, hygromycin or methotrexate were regenerated and used as sources of leaf mesophyll protoplasts. Protoplasts divided and regenerated plants in the presence of selective agents at levels inhibitory to protoplasts of non-transformed plants. Cross-resistance of protoplasts to more than one selective agent was not observed in this study which suggests that this approach may lead to an efficient interspecific somatic hybrid selection system.     

Multidrug resistance evaluation by confocal microscopy in primary urothelial cancer explant colonies

Assessing functional multidrug resistance (MDR) status in clinical biopsy material using drug autofluorescence has potential applications to clinical management. The small size of many cystoscopy specimens has led us to develop, as an alternative to flow cytometry, a protocol for studying epirubicin accumulation in adherent colonies of primary bladder cancer cells viewed live andin situ by confocal microscopy. The limitations to quantitation inherent in this technique are compensated for by preservation of cellular organisation and the elimination of non-malignant cells. Biopsy material is disaggregated and explanted into culture-grade petri dishes. After incubation for three to seven days plaques of epithelial cells have developed. Classical patterns of sensitive and resistant drug distribution are observed. Cells of the rolled edges of the colony accumulate more drug than those of the inner epithelial monolayer. Some central areas of larger colonies give the appearance of drug arrested at the intercellular junctions to give a fenestrated pattern. These observations contribute to the understanding of mechanisms in MDR as well as forming the basis for a clinical urological MDR evaluation protocol.     

几种细胞分裂素诱导石竹外植体直接再分化成花芽的机理

用ＭＳ＋２,４－Ｄ０．１ｍｇ／Ｌ为基本培养基,分别附加不同浓度的ＺＴ、ＢＡ和ＫＴ,对石竹的叶片、茎和花等外植体直接再分化成花芽的试验表明：叶片的外植体在有ＢＡ时可再分化出只有红色花瓣的不正常花芽;在含ＺＴ的培养基中再分化出具有１～２片叶或无叶的花芽,其花有两性花,雌花和仅单个雌蕊的不正常花,两性花可发育成结籽的果实;ＫＴ未能诱导花芽分化。茎外植体,３种ＣＴＫ都不能诱导出花芽。花等外植体仅在ＫＴ３ｍｇ／Ｌ的培养中再分化出无叶的不正常花芽,ＺＴ和ＢＡ的处理均不能诱导出花芽。     

不同激素及其不同配比对绞股兰愈伤组织诱导的统计学分析

绞股兰的茎段和叶碎片外植体可在合适的激素调节诱导下形成愈伤组织．通过含有不同水平激素的MS培养基对绞股兰愈伤组织诱导试验,经统计学分析,可以找出对绞股兰脱分化形成愈伤组织细胞影响显著的激素及其适宜的激素水平．     

The root: a potential new source of competent cells for high-frequency regeneration in Tylophora indica

The purpose of this study was to develop a new micropropagation system for Tylophora indica, an important medicinal plant in India, using root explants as starting material. Root explants cultured on MS medium supplemented with 6-benzyladenine (BA) or 2-isopentyladenine (2iP) produced organogenic nodular meristemoids (NMs) within 4 weeks. NMs induced from the cut ends of root segments showed two types of organogenic response—direct shoot bud formation and somatic embryogenesis—when maintained on induction medium. In 42% of the explants, NMs developed shoot buds directly in the presence of 10.72–26.80 M BA. On average, 18.5±0.7 shoots per gram of NM tissue were obtained after each 4-week subculture. Elongation of microshoots and root initiation were correlated with the auxin used, with the optimal response occurring in the presence of 28.54 M indole-3-acetic acid. In 39% of the explants, NMs dedifferentiated into friable embryogenic callus (FEC) in the presence of BA or 2iP after 12 weeks of culture. Of the different treatments, MS medium supplemented with 10.72 M BA was the most effective in inducing FEC and somatic embryogenesis: at this concentration 64% of the cultured NMs developed FEC and, on the same medium, 89% of the FEC produced globular somatic embryos (SEs). FEC biomass increased nearly five-fold with every 4-week subculture, and about 30 SEs were recovered per gram of FEC during this period. The best conversion of mature SEs to complete plantlets was obtained on basal MS medium–42%. Plantlets derived via somatic embryogenesis and shoot organogenesis were successfully hardened (88–96%) and transferred to the field.Abbreviations BA: 6-Benzyladenine - 2,4-D: 2,4-Dichlorophenoxyacetic acid - FEC: Friable embryogenic callus - GRS: Green root segment - IAA: Indole-3-acetic acid - IBA: Indole-3-butyric acid - 2ip: 2-Isopentyladenine - Kin: Kinetin - NAA: -Naphthaleneacetic acid - NM: Nodular meristemoid - SE: Somatic embryo - WRS: White root segmentCommunicated by P. Lakshmanan     

Expression and localization of angiogenin in placenta: enhanced levels at term over first trimester villi

Human angiogenin, a 14-kDa non-glycosylated polypeptide with both angiogenic and ribonucleolytic activities, is implicated in angiogenesis, a complex process of proliferation and formation of new capillary blood vessels from existing blood vessels. Placental growth requires extensive angiogenesis, which develops its vascular structure in both fetal chorionic villi and maternal deciduas. In this study, we investigated the expression profiles of angiogenin in placental villi from early and late gestation at both mRNA and protein levels using explant cultures in vitro followed by RT-PCR, immunoblot, and immunohistochemical analyses. From functionally active placental explants, angiogenin was detected in conditioned media of all the samples from first trimester and term group. The mean levels of angiogenin produced by term villi were found to be 2.6-, 2.1-, and 2.2-fold higher (P < 0.01) than first trimester villi at 24, 48, and 72 hr of culture, respectively. Expression profiles of angiogenin from term and first trimester villi seem to agree with its mRNA levels and immunoblot analysis; the expression in term villi was twice that in first trimester villi. The presence of angiogenin in placental villi and upregulation of its production towards term indicate that angiogenin production by the placenta is specific to the developmental stage. In conclusion, the observed changes in the localization and mRNA expression of angiogenin during placental development raise the possibility that it is involved in morphological and angiogenic changes in this endocrine organ vital to the successful fetal outcome during pregnancy.     

Histological and ultrastructural regulation in rabbit endometrial explants by estrogen in serum-free culture

A repertoire of hormonal signals including estrogen regulate the growth, differentiation, and functioning of diverse target tissues, including the ovary, the mammary gland, and skeletal tissue. A serum-free culture system derived from rabbit endometrium explants has been devised and is reported here to explore estrogen action in vitro. The system involves aseptically harvesting the uterus from a virgin rabbit, dissecting the endometrium, explanting it into 1- to 2-mm(3) pieces weighing approximately 1-2 mg each, and incubating these pieces in serum-free Medium-199. The culture is carried out for a period of 4 d in a humidified CO(2) incubator at 37 degrees C with 5% CO(2). The effect of extraneously added estrogen (1 microg/ml) was investigated by histological and ultrastructural procedures. It was observed that estrogen could induce specific changes, such as abundant mitochondria, rough endoplasmic reticulum, golgi complex, and intracellular collagen deposition, in both the epithelial and the fibroblast cell components of the explanted tissue. The study, therefore, indicates that the proposed system is an ideal tool for exploring and demonstrating estrogen responsiveness under in vitro conditions.     

Methods for Culturing Human Femur Tissue Explants to Study Breast Cancer Cell Colonization of the Metastatic Niche

Bone is the most common site of breast cancer metastasis. Although it is widely accepted that the microenvironment influences cancer cell behavior, little is known about breast cancer cell properties and behaviors within the native microenvironment of human bone tissue.We have developed approaches to track, quantify and modulate human breast cancer cells within the microenvironment of cultured human bone tissue fragments isolated from discarded femoral heads following total hip replacement surgeries. Using breast cancer cells engineered for luciferase and enhanced green fluorescent protein (EGFP) expression, we are able to reproducibly quantitate migration and proliferation patterns using bioluminescence imaging (BLI), track cell interactions within the bone fragments using fluorescence microscopy, and evaluate breast cells after colonization with flow cytometry. The key advantages of this model include: 1) a native, architecturally intact tissue microenvironment that includes relevant human cell types, and 2) direct access to the microenvironment, which facilitates rapid quantitative and qualitative monitoring and perturbation of breast and bone cell properties, behaviors and interactions. A primary limitation, at present, is the finite viability of the tissue fragments, which confines the window of study to short-term culture. Applications of the model system include studying the basic biology of breast cancer and other bone-seeking malignancies within the metastatic niche, and developing therapeutic strategies to effectively target breast cancer cells in bone tissues.