Micro-bioreactor arrays for controlling cellular environments: design principles for human embryonic stem cell applications

We discuss the utilization of micro-bioreactor arrays for controlling cellular environments in studies of factors that regulate the differentiation of human embryonic stem cells. To this end, we have designed a simple and practical system that couples a microfluidic platform with an array of micro-bioreactors, and has the size of a microscope slide [E. Figallo, C. Cannizzaro, S. Gerecht, J.A. Burdick, R. Langer, N. Elvassore, G. Vunjak-Novakovic, Lab Chip 7 (2007) 710-719]. The system allows quantitative studies of cells cultured in monolayers or encapsulated in three-dimensional hydrogels. We review the operating requirements for studies of human embryonic stem cells (hESCs) under steady-state and dynamic conditions, and the related control of the mass transport and hydrodynamic shear. We describe the design and fabrication of the individual bioreactor components, and the criteria for selecting the bioreactor configuration and operating parameters, based on the analysis of the characteristic times and scales of reaction, convection and diffusion. To illustrate the utility of the bioreactor, we present a "case study" of hESC cultivation with detailed experimental methods and representative biological readouts.     

Blätteralkohol (XVI)Blätteralkohol (XVII)

2-Methyl-4, 6-cyclohexadienaldehyde and n-butyraldehyde were treated with sodium in p-xylene to yield the aromatized “leaf alcohol reaction” product, 2-methyl-benzylalcohol, in a better yield than that with the cyclohexadienaldehyde alone. n-Butyric acid isolated from the reaction mixture unequivocally showed the operation of the “crossed Cannizzaro disproportionation” in this reaction, aliphatic aldehyde serving as the hydride donor. 2-Propyl-5-ethyl-4, 6-cyclohexadienaldehyde was obtained by the NaOH/H₂O-EtOH Michael-Aldol condensation of leaf aldehyde, gave 2-propyl-5-ethyl-benzylalcohol along with caproic acid.

On the basis of “leaf alcohol-reaction” mechanism, it was obtained following benzyl-alkohols; 2-methyl-, 2-propyl-, 2-methyl-5-ethyl-, 2-propyl-5-ethyl-benzylalcohol, from leaf alcohol and crotylalcohol.     

Synthesis and evaluation of glycosidase inhibitory activity of octahydro-2H-pyrido[1,2-a]pyrimidine and octahydro-imidazo[1,2-a]pyridine bicyclic diazasugars

An efficient chiron approach for the synthesis of bicyclic diazasugars 4a and 4b having both -CH(2)OH and -OH functionality at the same carbon atom (C-6) is reported. Thus, easily available alpha-D-xylo-pentodialdo-1,4-furanose 5, obtained from D-glucose, on aldol-crossed Cannizzaro reaction followed by hydrogenolysis afforded 7. The regio-selective beta- and alpha-sulfonylation of hydroxymethyl groups in 7 afforded 8a (beta-sulfonylation) and 11 (alpha-sulfonylation) in good yields. The cleavage of the 1,2-acetonide functionality, individually in 8a and 11, followed by reaction with ethylenediamine gave in situ formation of sugar aminals that undergo concomitant nucleophilic displacement of the sulfonyloxy group, by amino functionality, to give hitherto unknown bicyclic diazasugars 4a and 4b, respectively. The inhibitory potency of the earlier reported bicyclic diazasugars 3a,b and 4a,b was evaluated against alpha- and beta-glycosidases and they were found to be potent and specific against the beta-glycosidases with IC(50) and K(1) values in the micro molar range.     

Adaptation and the origin of species

As reflected in the title of his masterwork On the Origin of Species, Darwin proposed that adaptation is the primary mechanism of speciation. On this, Darwin was criticized for his neglect of reproductive isolation, his lack of appreciation for the role of geographic barriers, his failure to distinguish varieties from species, and his typological species concept. Two developments since Darwin, the biological species concept of Ernst Mayr and the methods of Coyne and Orr for estimating the contribution of different barriers to the total reproductive isolation, provide a framework for reconciling Darwin's view on the primacy of adaptation in speciation with later proposals that emphasize reproductive isolation. A review of the few studies that have estimated the contributions of multiple isolating barriers suggests that habitat isolation and other barriers that operate before hybrid formation are much stronger than intrinsic postzygotic isolation. In light of these data, I suggest that Darwin's focus on adaptation in the origin of species was essentially correct, a conclusion that calls for future studies that explore the links between adaptation and speciation, in particular, ecogeographic isolating barriers that result from adaptive divergence in habitat use. The recent revival in thinking about ecological factors and adaptive divergence in the origin of species echoes Darwin's much-criticized "principle of divergence" and suggests that the emerging views from today's naturalists are not so different from those espoused by Darwin some 150 years ago.     

Donizetti and the music of mental derangement: Anna Bolena, Lucia di Lammermoor, and the composer's neurobiological illness.

The composer Gaetano Donizetti, who died in a state of mental derangement due to neurosyphilis, created some of opera's greatest scenes of psychosis. His letters reveal the clinical progression of his neurobiological illness, which was confirmed by autopsy. One can hypothesize that the composer's brain disease, which led to his psychosis and death, may have had an influence on his ability to create the powerful and unforgettable scenes of psychosis in his operas. In Anna Bolena, he captured in musical and dramatic terms Anne Boleyn's historically corroborated mental disorder during her imprisonment in the Tower of London. Sixteen years after having composed Anna Bolena, Donizetti himself would be locked up, against his will, in a mental institution. In Lucia di Lammermoor, Donizetti portrayed a girl given to hallucinations who, in her unforgettable "mad" scene, comes on stage, a pathetic embodiment of a human being in the throes of psychosis. Thirteen years after Lucia's première, Donizetti would die, psychotic and paralyzed, of untreated neurosyphilis. Studying Donizetti's neurosyphilis and the portrayals of psychosis in his operas can help one to appreciate the pain of human beings trapped in the prison of a brain subjected to the devastation of mental derangement.     

Measurement of gas free body volume by a diving device.

A device is presented for determination of gas free body volume, by which total body volume as well as lung volume together with intestinal gas can be measured. The measurement is performed under water. The subject to be measured keeps his head in an airfilled cylinder which allows free breathing during the measurement procedure under water. The subject sits on a seat which is connected with a vertical "probe" that is submerged in a water basin. This "body density probe" carries several measuring rods of different diameter on its upper end, which lead to an increase of buoyancy when sinking deeper into the water after additional weights have been put on the device. These rods lead to new equilibrium states which are also dependent on the compressable gas volume attached to the probe. Three equilibrium states with different added weights give a physical definition of gas free body volume, which can be calculated from a number of accurately measurable single items.     

J.G. Mendels „Versuche über Pflanzen-Hybriden“ und ihre Würdigung in der Zeit bis zu ihrer Wiederentdeckung

Summary During the centenary of the publication of Mendel's paper Versuche über Pflanzen-Hybriden the significant and continuous effect of his work directs our attention, in the light of new historical findings, to Mendel's personality and the evaluation which his paper received up to the date of the rediscovery in 1900. Mendel was prepared for his experiments by a two-years-study of mathematical and natural sciences at the University of Vienna. About the reception of his famous lecture there exist four different versions. We try to document the reaction to his paper in both German speaking and foreign countries, and analyze the different references made to Mendel's paper during those years. We also consider Mendel's own reaction. Mendel's paper was not understood because his contemporaries were unable to follow the biological hypotheses of his experiments at a time when the development of cytology was yet to come. Moreover, they did not understand Mendel's method which consisted of a systematic and intuitively correct application of statistical principles to the questions he posed.

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Erweiterte Fassung der geschichtlichen Notizen eines am 9. 12. 1965 im Colloquium des Institutes für angewandte Genetik der Technischen Hochschule Hannover gehaltenen Vortrages.     

Osservazioni sulla secrezione delle ghiandole sottomandibolare e sottolinguale di embrione di topo,in condizioni normali e in cultura in vitro

Riassunto E' stata studiata l'istogenesi e l'istochimica della ghiandola sottomandibolare e sottolinguale di embrione di topo, in condizioni di sviluppo normale e in cultura organotipica in vitro. In condizioni normali l'attività secretrice diviene dimostrabile con mezzi istochimici al 16 giorno di gestazione, come materiale intracellulare. La secrezione presenta fino dall'inizio reazione positiva ai metodi del PAS e di Hale, ed è colorabile col blu alcian. Al 17 giorno compaiono fra le due ghiandole differenze nell'aspetto morfologico del secreto, nell'intensità delle suddette reazioni istochimiche, e nel comportamento rispetto al blu di toluidina che dopo solfatazione dà colorazione metacromatica nella sottolinguale, ma non nella sottomandibolare. Ciò indica che il componente polisaccaridico del secreto ha caratteristiche un po' diverse nelle due ghiandole.Sono state eseguite culture in vitro di ghiandole al 13 giorno di gestazione, su un coagulo di plasma e di estratto embrionale di pollo. Al momento dell'espianto, esse sono all'inizio della loro differenziazione, e sono formate ciascuna da un cordoncino epiteliale e da una gemma solida, entro una comune capsula mesenchimale. Il processo di ramificazione si svolge più lentamente che in vivo, ma la secrezione compare alla stessa età totale. Le ghiandole si sono sviluppate in vitro per 10 giorni e le loro caratteristiche istochimiche sono apparse eguali a quelle che si osservano in condizioni normali di sviluppo.

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Summary Histogenesis and histochemistry of submandibular and sublingual glands of mouse embryo were studied both during normal development and in vitro. In normal conditions secretory activity becomes detectable by histochemical methods at the 16th day of gestation as intracellular material. From the start, secretion shows a positive reaction to the PAS and Hale's method, and stains with alcian blue. At the 17th day differences may be shown between the two glands in the morphological features of the secretion, in the intensity of the above said histochemical reactions, and in the behaviour towards toluidine blue, which after sulfation gives metachromatic staining in the sublingual, but not in the submandibular gland. This indicates that the polysaccharidic component of the secretion has different characters in the two glands.Cultures in vitro were made of glands at the 13th day of gestation, on a clot of chicken plasma and embryo extract. At explantation time, they are just beginning their differentiation, and both consist of an epithelial cord and a solid bud, contained in a common mesenchymal capsule. The ramification process goes on at a slower rate in vitro than in vivo, but secretion appears at the same total age. The glands developed in vitro for 10 days and their histochemical features appeared to be comparable to those which were observed in normal developmental conditions.

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Lavoro eseguito coi mezzi forniti da contratti di ricerca dell'U.S.A.E.C. (NYO-3355-10) e dell'Euratom (043-65-1 BIO I) e con un contributo del C.N.R.     

Ultrastructural features of cytofilaments within mammalian endothelial cells

Summary A submicroscopic study of the endothelium lining the blood capillaries and arterioles contained in the Cyon's nerve was made. The cytoplasm of some endothelial cells was found containing bundles of thin filaments. These measure about 60 Å in diameter, and do not show any cross striation, nor contacts with other cytoplasmic components. They are oriented parallel to each other and to the cell surface. No attachment plate of cytofilaments to the plasma membrane was seen. The filamentous structures were mostly found within the supranuclear cytoplasm. The endothelial cells in question never showed contacts with axons.In the light of these findings it can be advanced the view that the cytofilaments present in endothelial cells are supportive in function; namely they may confer a higher elasticity to these cells, subject to continuous pressure and morphological variations.

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Riassunto Lo studio ultrastrutturale dell'endotelio di piccoli vasi sanguigni contenuti nel nervo cardio-aortico depressore di Cyon del coniglio ha consentito di rilevare la presenza in alcune cellule endoteliali di esili filamenti raggruppati in fasci più o meno numerosi. Detti citofilamenti presentano uno spessore di circa 60 Å, occupano per lo più il citoplasma sopranucleare e sono orientati parallelamente tra loro e rispetto alla superficie cellulare. Essi non mostrano struttura periodica né rapporto alcuno, se non di semplice contiguità, con altre componenti citoplasmatiche. Non presentano, inoltre, punti di attacco sulla membrana cellulare, né connessioni con fibre nervose. Numerose le vescicole pinocitotiche osservate lungo i bordi superficiale e basale delle cellule endoteliali in oggetto.In base a tali reperti si avanza l'ipotesi che i citofilamenti endoteliali svolgano una funzione di sostegno nell'ambito del citoplasma, e siano capaci di conferire un più elevato grado di elasticità all'endotelio vasale, soggetto a continue variazioni di pressione e di forma.

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Dedicated to Prof. Wolfgang Bargmann on his 60th birthday.—This investigation was supported in part by a grant from the Italian C.N.R.

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The author wishes to thank Dr. Pasquale Romeo for his kind help in the preparation of the paper, and Mr. Ciro Paesano for making the photographic prints.     

Dicentric Y chromosome in a patient with gonadal dysgenesis and seminoma

Summary A male patient with ambiguous external genitalia developed a seminoma in the left inguinal region; his internal genitalia included a streak gonad on the right and a small uterus.Cytogenetic studies demonstrated a dicentric Y chromosome with unstable behavior during cell division, which resulted in 45,X/46,X,dic(Y)/47,X,dic(Y),dic(Y) mosaicism.Immunogenetic studies allowed the identification of the male-determining H-Y antigen on both leukocytes and red cells of the patient.The significance of these results is discussed with respect to recent data on the genetic control of H-Y antigen.This work was supported in part by CNR Centro di studio per l'Immunogenetica e l'Istocompatibilità     

Accrescimento e rinnovamento strutturale della compatta in ossa sottratte alle sollecitazioni meccaniche

In questa Nota I sono compresi i capitoli relativi alla letteratura sull'argomento, al piano delle ricerche, al materiale e alla tecnica impiegata, ai mutamenti di peso e di forma delle ossa inerti. Nella Nota II saranno esposte le modificazioni di struttura di 2 e di 3 ordine delle ossa immobilizzate e le conclusioni generali.     

Biotin-c10-AppCH2ppA is an effective new chemical proteomics probe for diadenosine polyphosphate binding proteins

Here we report on the synthesis of a synthetic, stable biotin-c10-AppCH₂ppA conjugate involving an unusual Cannizzaro reaction step. This conjugate is used to bind prospective Ap₄A binding proteins from Escherichia coli bacterial cell lyzates. Following binding, identities of these proteins are then determined smoothly by a process of magnetic bio-panning and electrospray mass spectrometry. Protein hits appear to be a definitive set of stress protein related targets. While this hit list may not be exclusive, and may vary with the nature of sampling conditions and organism status, nevertheless hits do appear to correspond with bona fide Ap₄A-binding proteins. Therefore these hits represent a sound basis on which to construct new hypotheses concerning the cellular importance of Ap₄A to bacterial cells and the potential biological significance of Ap₄A-protein binding interactions.     

Synthesis and biologically relevant properties of polyphosphazene polyacids

Polyphosphazene polyacids show potential as immunostimulating compounds and materials for microencapsulation. Their synthesis requires multistep chemical transition from a hydrolytically unstable macromolecular precursor, poly(dichlorophosphazene), to a water-soluble polyelectrolyte. Insufficient synthetic control in these reactions can lead to molecular weight variations and formation of macromolecules with "structural defects" resulting in significant variations in polymer performance. Simple and reproducible "one pot-one solvent" method is reported for the preparation of polyphosphazene polyacids-poly[di(carboxylatophenoxy)phosphazene] and its copolymers. Molecular weight characteristics and polymer compositions were studied as a function of reaction parameters. Macromolecular byproducts, incompletely substituted polymers containing hydroxyl groups and partially deprotected polymers containing propyl ester functionalities, were synthesized and characterized. It was demonstrated, that the presence of such groups can affect polymer characteristics, such as hydrolytic degradation profiles, immunostimulating activity, and microsphere forming properties. In vivo studies showed that the immunostimulating activity of polyphosphazene polyacids correlates with the content of acid functionalities in the polymer.     

Trigonia and the origin of species

Conclusion While the Trigonia story is a microcosmic representation of nineteenth-century evolutionary debates, it also serves as a model for assessing the impact of new empirical material upon a controversial issue potentially explained by several internally consistent but contradictory theories; for there can be no fact quite so pristine as a discovery anticipated by no one. The reaction to modern trigonians was, I suspect, completely typical; all parties to the dispute managed to incorporate the new datum into their systems. Evolutionists emphasized the morphological differences between Mesozoic and modern forms and assumed that the disjunct distribution was an artifact of an imperfect record. Agassiz cited the known distribution in support of special creation, but announced that the discovery of a Tertiary species would discredit none of his ideas. Parkinson could not readily encompass the difference without evident improvement in his progressionist synthesis, but invoked almighty wisdom in his ignorance.I do not doublt that all these naturalists proceeded properly in refusing to yield to the anomalies of a single fact which destroyed no deductive sequence in any of their theories. When one considers the stupendous amount of misinformation current in early nineteenth-century scientific circles,⁴⁹ it is easy to appreciate the salutary aspects of stubbornness in the face of inevitable contradictory citations.De Beer⁵⁰ has marveled at Darwin's ability, in the 1844 sketch, to work his way through a mire of misinformation: It is a matter for wonder that with the meagre materials at his disposal he was able to steer a straight course across a largely uncharted ocean of ignorance, with rocks of falsehood right across his path. Yet Darwin approached these rocks with the idea of natural selection already firmly in mind. Any pure empiricist would have surrendered to confusion long before 1859.     

Changes in the proliferative response of human lymphocytes in vitro on exposure to subcellular components of Bordetella pertussis]

Different components of B. pertussis were found to have a similar inhibitory effect on thymidine-3H incorporation caused by phytohemagglutinin (PHA) in the culture of lymphocytes taken from donors immunized with tetanus toxoid. However, the same fractions of B. pertussis produced differential effects on the reaction of lymphocyte proliferation in response to tetanus toxoid: murein-"ontaining membranes enhanced thymidine-3H incorporation, RNA-containing fractions reduced it and water-soluble components of disintegrated B. pertussis produced no effect on lymphocyte proliferation.     

PSYCHOLOGICAL ASPECTS OF PAIN IN PATIENTS WITH TERMINAL CANCER

The dying patient reacts emotionally to the problems encountered in the terminal period according to his established pattern of response to stress. The nature of this pattern will play a part in his experience of pain. Some of the types of reaction include the bizarre misinterpretation of bodily sensation of the psychotic, the development of conversion symptoms, the increase in pain through muscle tension in the anxious but overcontrolled person, and the stoical acceptance by guilt-ridden patients. Physicians are sometimes reluctant to devote full attention to the care of the terminally ill for a number of reasons, including the attitude that "curing" is the only worthwhile activity of a doctor of medicine. Observers have found that the physician's attention to the day to day anxieties of the patient in a terminal stage may contribute substantially to his comfort.     

Type specimens of Pectinidae (Mollusca: Bivalvia) described by Linnaeus (1758–1771)

Linnaeus listed and described (as Ostrea species) 20 recent pectinid species in the 10th edition of his Systema Naturae and one pectinid species in his Mantissa. These are now placed in 17 genera of the family Pectinidae. Nine species are cited to Museum Ludovicae Ulricae. Ten primary types are present in the Linnéan Society of London, and the same number in die Zoological Museum of die Uppsala University. Two lectotypes were designated recendy by Smith and Waller; 18 lectotypes and one neotype are selected herein. Nine of the species are type species of currently accepted pectinid genera. Seven new type localities are also designated for species which had unknown or erroneous type localities, and six are more restricted. Potential type material of eight species is also traced in the Gualtieri collection of the Museo di Storia Naturale e del Territorio at Certosi di Calci (Italy).     

Colture in vitro di cellule spleniche di topo

Riassunto Colture di cellule spleniche provenienti da topo immunizzato e controllo sono state seguite nella loro evoluzione morfologica, con particolare riguardo alla trasformazione in elementi di tipo blastoide e in elementi binucleati. Con la tecnica di Jerne si è testata la capacità anticorpopoietica delle culture di immunizzato a intervalli vari di tempo e si è fissata a 15 giorni la durata della competenza immunologica. Si è poi studiata, mediante autoradiografia l'andamento in vitro della sintesi del DNA e dell'RNA, articolata quest'ultima attraverso l'entrata in azione dei tre pools, e dal confronto costante dei due tipi di colture si è concluso che in esse qualitativamente non sono apprezzabili differenze nella dinamica di sintesi.

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In vitro cell cultures of the spleen of the mouseAntibody response and synthesis of nucleic acids

Summary Spleen cell cultures from immunized and control mice have been followed in their morphological evolution, especially towards blastoid and binucleated cells. The immunological activity of cultures from immunized mice has been tested at different ages of culture by the Jerne antibody plaque essay. The ability of spleen cells to synthetize antibody was maintained in vitro for 15 days. DNA and RNA synthesis have been studied by radioautography using thymidine and uridine as precursors, respectively. No differences in the kinetic of synthesis appeared by relating the two types of cultures.

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Il presente lavoro è basato in parte sopra una tesi di laurea discussa davanti alla Facoltà di Medicina dell'Universita di Pavia il 28 ottobre 1968.     

Ultrastruttura della Pars distalis dell'ipofisi di Lacerta s. sicula Raf., nei maschi in attività sessuale ed in quelli castrati

Riassunto Facendo seguito ad uno studio in microscopia ottica sull'ipofisi della Lacerta s. sicula Raf. durante vari periodi dell'anno, gli AA. espongono, per la stessa specie, i dati ultrastrutturali per le cellule adenoipofisarie, di maschi ancora in periodo di attiva spermatogenesi e di accoppiamento, e di altri castrati e sacrificati dopo 3–4 mesi. In base alla localizzazione nella ghiandola ed alle variazioni dopo castrazione, si distinguono cellule del 1 tipo (STH) con granuli rotondeggianti ed uniformi (circa 310 m ), cellule del 2 tipo (gonadotrope) a granuli polimorfi, cellule del 3 tipo (pure gonadotrope) a granuli tondi e piccoli (150–270 m) e a globuli di 700–800 m, e cellule del 4 tipo a granuli piccoli ed uniformi (100–200 m), di dubbio significato. Presentano dati a favore dell'ipotesi che la condensazione del secreto in forma di granuli avvenga nelle cisterne del Golgi. Osservano fenomeni di degenerazioni cellulari ed aspetti di distruzione citoplasmatica con liberazione di granuli negli spazi tra le cellule. Negli animali castrati notano degranulazione e segni di intensa attività delle cellule del 2 e del 3 tipo, oltre alla comparsa di cellule vacuolizzate, per dilatazione del reticolo endoplasmico ruvido.

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The ultrastructure of the pars distalis of the hypophysis in sexually active and castrated males of Lacerta s. sicula Raf

Summary In addition to light microscopic studies on the hypophysis of the lizard, Lacerta s. sicula Raf., during different periods of the year, the authors of this paper explain the ultrastructure of the adenohypophysial cells of normal sexually active male lizards and of castrated lizards. On the basis of the location and the structural variations after castration several hypophysial cell types can be distinguished: Type 1 (STH) with spheroid granules (about 310 m diameter); type 2 with polymorphous glycoprotein granules; type 3 with small round granules (150–270 m diameter) and globules of about 700–800 m diameter (both gonadotropes). A fourth cell type with small uniform granules (diameter about 100–200 m has also been observed; its function is uncertain. A condensation of secretory granules in the Golgi complex and phenomena of cellular degeneration and cytolysis followed by a release of granules into the cellular interstices have been observed. In castrated animals degranulation and signs of an increased activity in the cell types 2 and 3 have been shown. In these animals the cisternae of endoplasmic reticulum become greatly distended.

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Lavoro eseguito con il contributo del Consiglio Nazionale delle Ricerche, Impresa Endocrinologia, e svolto, per le osservazioni, presso il Centro di Studio di Microscopia Elettronica della Facoltà di Scienze dell'Università di Napoli; ringraziamo il Prof. Gianfranco Ghiara per la continua critica costruttiva e il dott. Carlo Taddei per i suggerimenti nelle tecniche di microscopia elettronica.     

Agrobacterium-mediated transformation of commercial melon (Cucumis melo L., cv. Amarillo Oro)

Cotyledon explants of muskmelon (Cucumis melo L., cv. Amarillo Oro) seedlings were co-cultivated with disarmed Agrobacterium tumefaciens strain LBA4404 that contained the binary vector plasmid pBI121.1. The T-DNA region of this binary vector contains the Nopaline synthase/neomycin phosphotransferase II (NPTII) chimeric gene for kanamycin resistance and the Cauliflower Mosaic Virus 35S/-glucuronidase (GUS) chimeric gene. After infection, the cotyledon pieces were placed in induction medium containing 100 mg/l kanamycin. Putative transformed shoots were obtained, followed by the development of morphologically normal plantlets. The transgenic nature of regenerants was demonstrated by polymerase chain reaction, Southern blot analysis, plant growth on medium selective for the transgene (NPTII) and expression of the co-transformed GUS gene. Factors affecting the transformation procedure are discussed.Abbreviations CaMV Cauliflower Mosaic Virus - Cf Cefotaxime - GUS -glucuronidase - Km Kanamycin - MS Murashige and Skoog - NOS nopaline synthase - NPTII neomycin phosphotransferase II - PCR polymerase chain reaction