Tissue Culture Laboratory Research Articles

Enraizamento in vitro do morangueiro (Fragaria x ananassa Duchesne) em diferentes concentrações do meio MS

O presente trabalho foi realizado no Laboratório de Cultura de Tecidos da EMBRAPA/CPACT, Pelotas-RS, com o objetivo de verificar o comportamento in vitro de duas cultivares de morangueiro em diferentes concentrações do meio MS. Explantes de morangueiro, cultivares Hofla e Tangi provenientes do cultivo in vitro foram cultivados em meio MS, na concentração plena, 3/4 e 1/2 adicionado de sacarose a 30g/l, mio-inositol a 100mg/l e benzilaminopurina (BAP) a 0,005mg/l. Utilizaram-se frascos com capacidade de 250ml, que continham 40ml de meio de cultura, nos quais inocularam-se cinco explantes. O cultivo foi mantido em sala de cultura, onde permaneceu por 32 dias sob fotoperíodo de 16 horas, luminância de 1500 lux e temperatura de 25°C. Os resultados indicaram que a redução na concentração dos sais MS incrementa o percentual de explantes enraizados, aumentando o número de raízes da cultivar Tangi. O maior comprimento médio de raízes foi obtido com a cultivar Hofla na concentração 3/4 e 1/2 do meio MS. Não se observaram diferenças no desenvolvimento das plântulas, entre as cultivares, com a redução das concentrações do meio MS.

Uso de ágar, amido e ácido indolbutírico no enraizamento in vitro do porta-enxerto de macieira MM 111

O ágar é um dos componentes mais caros do meio de propagação in vitro. Visando a reduzir custos, os estudos foram desenvolvidos no Laboratório de Cultura de Tecidos da EMBRAPA/CPACT, Pelotas RS, objetivando-se testar a ação do ágar e da sua substitução parcial pelo amido de mandioca na composição do agente solidificante (AS) do meio MS, associados às concentrações de 0; 2,5; 5,0; 7,5 e 10µM de ácido indolbutírico (AIB). Os dois AS de meio de cultivo testados: AS1 - 6g/l de ágar e; AS2 - combinação de 3g/l de ágar mais 50g/l de amido de mandioca (fécula), tiveram redução de Vi dos sais de MS e acréscimos de 100mg/l de mio-inositol e 30g/l de sacarose, juntamente com pH ajustado em 5,9 antes da autoclavagem. Os explantes foram mantidos por 30 dias em sala de crescimento sob condições de 25±2°C, 16 horas de fotoperíodo e ±2000lux de intensidade luminosa. O AS2, além de apresentar o maior número de raízes, e maximizá-las na presença de 5µM de AIB, antecipou em cerca de 10 dias o surgimento destas e apresentou consistência semelhante ao AS1, sendo apenas menos transparente.

BIOTECHNOLOGY IN THE SOUTH PACIFIC ISLAND REGION

Biotechnology in the South Pacific region is limited to the use of tissue culture for propagation and conservation purposes. Regional and national tissue culture laboratories have been established in several of the Pacific Island countries; their main priorities are the provision of planting material for growers and in vitro conservation. Root and tuber crops have been the main focus of tissue culture technology in the region, but with the increase in the number of national tissue culture laboratories, and the need to ensure that these laboratories can generate some income, attention has turned to other more high value plants such as orchids, anthruriums, strawberries, etc. Micropropagation techniques for crops such as taro have been optimised, and with crops such as Abelmoschus manihot, (bele), tissue culture methodology has been developed. In recent years significant losses have occurred in the field collections of the root and tuber crops maintained in many of the Pacific Island countries. The extended use of in vitro conservation is now being considered as a more secure strategy for safeguarding these genetic resources. Experiments are underway to evaluate the use of certain slow-growth methods, and the feasibility of using cryopreservation for some crops in the region. The region has access to the other areas of biotechnology important to agriculture such as plant transformation, marker technology, through collaboration with institutions outside of the Pacific Island countries. This paper reviews the progress made by tissue culture in the South Pacific Island region over the last decade and considers what influence biotechnology might have in the future.

Computer-controlled laser ablation: a convenient and versatile tool for micropatterning biofunctional synthetic surfaces for applications in biosensing and tissue engineering.

This paper describes laser-based methods for preparing micropatterns of bioactive molecular species in self-assembled monolayers (SAMs) and micropatterns of proteins and other biological molecules immobilized on solid substrates. Applications of these micropatterned surfaces in multianalyte biosensing and tissue engineering are emphasized. The focus of the paper is on the use of a computer-controlled laser ablation system comprising a research-grade inverted optical microscope, a pulsed nitrogen-pumped dye laser emitting at 390 nm, a programmable sample stage, and the computerized control system. The laser system can be implemented in a typical biosensor or tissue culture laboratory to enable the facile and reproducible fabrication of micropatterned surfaces by several methods. Various methods for patterning are discussed with examples given and emphasis placed on (1) laser ablation in the fabrication of photolithography masks, (2) electrochemical patterning of SAMs, and (3) laser desorption of SAMs. The relative merits of each technique are discussed with respect to application in fabrication of active surfaces for biosensing and tissue culture applications.

In Vitro Propagation of Garlic (Allium sativum L.)

This study was carried out in the Tissue Culture Laboratory and the Experiment Field of the Dept. of Horticulture, Assiut Univ. Two garlic cultivars i.e., El-Balady (local cultivar) and the Chinese were used. Two media, MS and B5, were supplemented with 4%, 8%, 12% and 16% sucrose. The 8% sucrose gave best number of roots, root length and leaf length as compared to the other sucrose concentrations used in this study. Four percent or 16% sucrose gave the lowest values of these characters. The MS gave higher values in most of the studied characters than that of the B5. In another experiment three growth regulators i.e. IBA, BA and GA3 or used at 0, 5, 10, 20 ppm in two media, MS and B5 supplemented with 3% sucrose. All the studied growth regulators gave higher values than the control in respect of the all studied characters. The IBA was better than BA and GA3 in root formation, while GA3 was better than BA and IBA in leaf formation. It is found that 5 ppm of IBA formed the highest number of roots, root length and percentage of root formation. On the other hand, the 20 ppm of GA3 gave the highest number of leaves and leaf length. The garlic plantlets were successfully acclimatized in the laboratory giving 67.8% survival for the Balady cv. and about 62.6% for Chinese cv. The survival rate of these plantlets were 74.5% for the Balady cv. and 78.5% for the Chinese cv. on transfer to the open-field. No chromosomal aberrations was noticed.

Laboratory contamination management: the requirement for microbiological quality assurance

Microbial contamination is the single most important cause of losses in commercial and scientific plant tissue culture laboratories [1,2]. However, even in commercial companies the severity and implications of the problem are not recognised or admitted. Many scientific laboratories fail to record contamination losses, and the micropropagation industry often only recognises the sources of contamination after severe losses have occurred. Following a rapid increase in the production of micropropagated plants in the 1980s [3], there has been a steady decline in the number of micropropagation laboratories in the 1990s, which was at least partially caused by the inability of laboratories to reduce contamination losses to a level which allows a predictable production output and quality of micropropagated plants.

Basic cell culture.

This article will describe the basic techniques required for successful cell culture. It will also act to introduce some of the other chapters in this volume. It is not intended, as this volume is not, to describe the establishment of a tissue culture laboratory, nor to provide a historical or theoretical survey of cell culture. There are several books that adequately cover these areas, including the now somewhat dated but still valuable volume by Paul (1), the multi-authored Methods in Enzymology volume edited by Jakoby and Pastan (2), and the new edition of Freshney (3). Instead, this chapter's focus will be on the techniques for establishing primary rodent cell cultures from embryos and adult skin, maintaining and subculturing these fibro-blasts and their transformed derivatives, and the isolation of genetically pure strains. The cells described are all derived from Chinese hamsters since, to date, these cells, have proved to be the most useful for somatic cell genetics (4,5). The techniques, however, are generally applicable to most fibroblastic cell types.

Influência da benzilaminopurina (BAP) NA multiplicação in vitro de kiwi (Actinidia deliciosa)

Este experimento foi conduzido no Laboratório de Cultura de Tecidos da EMBRAPA/CPACT, Pelotas, RS, com o objetivo de estudar a influência de diferentes concentrações de 6-benzilaminopurina (BAP) na multiplicação in vitro de kiwi (Actinidia deliciosa), cv. Tomun. Utilizou-se o meio de cultura MS, acrescido de sacarose, mio-inositol, ágar e das seguintes concentrações de BAP: 0,0; 0,5; 1,0; 1,5; 2,0 e 2,5 mg/l. Os explantes foram constituídos de microestacas provenientes da coleção in vitro do Laboratório, com aproximadamente 10mm de comprimento. Verificou-se que, para o número e comprimento de brotações, número de gemas e folhas a concentração em torno de 1,5mg/l de BAP proporcionou os memores resultados. Nas condições em que o trabalho foi realizado, pode-se concluir que o BAP foi eficiente na multiplicação in vitro de kiwi, cv. Tomuri.

Observations and Suggestions for Improving Somatic Hybridization by Plant Protoplast Isolation, Fusion, and Culture

De (Triticum aestivum): In vitro production and utilization of doubled haploids, p. 101–124. In: Y.P.S. Bajaj (ed.). Biotechnology in agriculture and forestry. vol. 12: Haploids in crop improvement I. Springer-Verlag, Berlin. Powell, W., P.D.S. Caligari, and J.M. Dunwell. 1986. Field performance of lines derived from haploid and diploid tissues of Hordeum vulgare. Theor. Applied Genet. 72:458–465. Reed, S.M. and E.A. Wernsman. 1989. DNA amplification among antherderived doubled haploid lines of tobacco and its relationship to agronomic performance. Crop Sci. 29:1072–1076. 401 Research Group, Laboratory of Plant Cell and Tissue Culture, Institute of Genetics, Academia Sinica. 1975. Primary study on induction of pollen plants of Zea mays. Acta Genet. Sin. 2:143. Research Laboratory of Breeding. 1981. A preliminary study on the heredity and vitality of the progenies of tobacco pollen plants, p. 223–225. In: H. Hul (ed.). Plant tissue culture. Proc. Symp. Plant Tissue Culture, Peking, 1978. Pitman Publishing, London. Rufty, R.C., E.A. Wernsman, C.E. Main, and G.V. Gooding, Jr. 1990. Registration of NC-BMR 42 and NC-BMR 90 germplasm lines of tobacco. Crop Sci. 30:241–242. Schaeffer, G.W., F.T. Sharpe, Jr., and P.B. Cregan. 1984. Variation for improved protein and yield from rice anther culture. Theor. Applied Genet. 67:383–389. Simmonds, N.W. 1979. Principles of crop improvement. Longman, London. Thomas, E., F. Hoffman, and G. Wenzel. 1975. Haploid plantlets from microspores of rye. Z. Pflanzenzuchtg. 75:106–113. Wang, Y., C. Sun, C. Wang, and N. Chien. 1973. The induction of the pollen plantlets of Triticale and Capsicum annuum from anther culture. Sci. Sinica 16:147–151. Wenzel, G., F. Hoffmann, and E. Thomas. 1977. Anther culture as a breeding tool in rape. I. Ploidy level and phenotype of androgenetic plants. Z. Pflanzenzuchtg. 78:149–155. Wenzel, G., O. Schieder, T. Przewozny, S.K. Sopory, and G. Melchers. 1979. Comparison of single cell culture derived Solanum tuberosum L. plants and a model for their application in breeding programs. Theor. Applied Genet. 55:49–55. Winzeler, H., J. Schmid, and P.M. Fried. 1987. Field performance of androgenetic, doubled haploid spring wheat lines in comparison with lines selected by the pedigree system. Plant Breeding 99:41–48. Wu, J. 1986. Breeding haploid corn by anther culture, p. 149–161. In: H. Hu and H. Yang (eds.). Haploids of higher plants in vitro. Springer-Verlag, Berlin. Yang, X. and H. Fu. 1989. Hua-03—A high protein indica rice. Intl. Rice Res. Nwsl. 14(3):14–15. Zhu, D. and X. Pan. 1990. Rice (Oryza sativa L.): Guan 18—An improved variety through anther culture, p. 204–211. In: Y.P.S. Bajaj (ed.). Biotechnology in agriculture and forestry 2: Haploids in crop improvement I. Springer-Verlag, Berlin. Zhao, Y., X. He, J. Wang, and W. Liu. 1990. Anther culture 28—A new diseaseresistant and high-yielding variety of winter wheat, p. 353–362. In: Y.P.S. Bajaj (ed.). Biotechnology in agriculture and forestry 13: Wheat. SpringerVerlag, Berlin.

Automated, in vitro harvest of somatic embryos

This work has demonstrated the aseptic, automated harvest of somatic embryos from a bioreactor suspension culture. Machine vision, emulating the selection criteria of an experienced biologist, classified embryos as harvestable or non-harvestable as they flowed through a 3 mm glass conduit. Embryos classified as harvestable were separated in a sealed harvest chamber. The system harvested 60% of the embryos at a rate of 2.4 embryos/h and incorrectly harvested less than 1% of the non-harvest objects. The low harvest rate precludes the applicability of this technique to research and commercial tissue culture laboratories. The suspension feed-rate, culture population density and culture homogeneity were identified as the most important factors influencing embryo harvest rate. The performance of this technique on more densely populated cultures was projected using anticipated improvements in suspension feedrate. It was concluded that, under the conditions of this analysis, the harvester would be of limited value in a commercial propagation environment but could be beneficial to many research labs working with plant somatic embryos.

Laboratory Exercises Illustrating Organogenesis and Transformation using Chrysanthemum Cultivars

A tissue culture laboratory exercise illustrating regeneration of whole plants from leaf segments of chrysanthemum by organogenesis is described. Using simple, common media, shoots can be generated in 5 weeks and rooted after an additional 3 weeks. Acclimatization of plants can be accomplished in a simple mistbed in the greenhouse. The exercise is adaptable to depict genotype differences among cultivars, optimization of shoot induction, effects of growth regulators, and experimental design. Callus is typically not formed during shoot formation; however, co-cultivation of leaf segments with a virulent strain of Agrobacterium tumefaciens produces callus with a strain of disarmed A. tumefaciens harboring NPTII construct affects regeneration of plants resistant to kanamycin.

Incidence of cymbidium mosaic and odontoglossum ringspot viruses and their significance in orchid cultivation in Singapore

A survey of cymbidium mosaic virus (CyMV) and odontoglossum ringspot virus (ORSV) in orchids cultivated in Singapore was conducted during 1988–1991. Of the 12 genera from four major commercial orchid farms, 54.6% of the plants were infected with CyMV, 4.0% with ORSV, and 14.2% with both CyMV and ORSV. Among the 29 genera from the orchid nursery of Botanic Gardens, 34.5% were infected with CyMV, 0.3% with ORSV, and 8.3% with both CyMV and ORSV. From the 13 orchid genera cultivated in tissue-culture flasks in the tissue culture laboratory of the Botanic Gardens, 50.5% were infected with CyMV, none with ORSV alone, and 0.5% with both CyMV and ORSV. These findings emphasize the need to implement a virus-free certification programme that would greatly enhance the management of these two prevalent viruses in the Singapore orchid industry.

LABORATORY EXERCISES ILLUSTRATING ORGANOGENESIS AND TRANSFORMATION USING CHRYSANTHEMUM CULTIVARS

A tissue culture laboratory exercise illustrating regeneration of whole plants from leaf segments of Chrysanthemum by organogenesis will be described. Using simple, common media, shoots can be generated in five weeks and rooted after an additional three weeks. Acclimatization of plants can be accomplished in a simple mistbed in the greenhouse. The exercise is adaptable to depict genotype differences among cultivars, optimization of shoot induction, effects of growth regulators, and experimental design. Callus is typically not formed during shoot formation; however, co-cultivation of leaf segments with a virulent strain of Agrobacterium tumefaciens produces callus autotrophic for growth regulators. Co-cultivation with a strain of disarmed A. tumefaciens harboring a NPTII construct affects regeneration of plants resistant to kanamycin.

SELECTION AND INTRODUCTION OF WOODY LANDSCAPE ORNAMENTALS AT THE UNIVERSITY OF GEORGIA

The Department of Horticulture has introduced over 20 new plants into the Georgia and southern nursery trades. The program serves as a conduit for evaluating and distributing plants from the U.S. National Arboretum, Arnold Arboretum, individuals and nurseries. Propagation requirements and cold hardiness estimates are determined for most of the potential introductions. Cooperating nurseries provide real-world assessments of container and field cultural criteria. Further, plants are tested under field conditions at the Department's research farm. Plants are promoted through lectures, articles, and distribution to any interested Georgia nursery. Numerous trade, popular, and refereed articles were spawned from the program. Selected plants have been provided to commercial tissue culture labs for stock increase.To date, the University has realized no financial return. A genuine need exists to generate income from royalties to sustain and enlarge the program.A list of the Georgia introductions will be available to all participants and several of the more notable introductions will be discussed.

BREEDING STRATEGIES AND METHODS FOR FOLIAGE AND FLOWERING ORNAMENTALS

Tissue culture labs based in countries with high labor costs are becoming more dependent on proprietary plants. This has increased the necessity of high profile plant breeding programs. Foliage and flowering plant breeding programs have evolved rapidly to take advantage of the benefits associated with tissue culture labs. Breeding strategies and methods will be discussed on existing flowering and foliage programs for Anthuriums, Euphorbia, Aloe, Spathiphyllum, Homalomena, and Dieffenbachia. Embryo rescue in the lab has increased the survival of wide crosses from different species within a genera. Rapid multiplication of selected clones has increased the efficiency of screening for disease and insect resistance in the selection of new cultivars. Marketing, along with improved horticultural characteristics, determine the success of new releases.

Characterization of human purified epithelial and stromal cells from endometrium and endometriosis in tissue culture

To initiate in vitro cultures of separate stromal and epithelial elements from endometriotic tissue and to compare the characteristics of these cells with those of cultured endometrial cells. The study involved testing the viability of a culture system for endometriotic tissue and examination of the phenotype of the cells. Fresh tissue samples were collected from the operating theater and transferred to the tissue culture laboratory. Twenty patients undergoing laparotomy for endometriosis and patients undergoing surgery for benign conditions were recruited. Endometrium and endometriotic tissue were separated, cultured in vitro, and labeled by indirect immunofluorescence with monoclonal antibodies against cytoskeletal components and epithelial mucins. Endometriotic cells have been maintained in vitro and found to resemble endometrial cells closely. With respect to the staining patterns for cytokeratins 18 and 19, vimentin, and three different epithelial mucins, cultured cells from both endometrium and endometriotic tissue had similar properties. Cytokeratins were located in epithelial cells, and vimentin was expressed in both stromal and epithelial cells. The antimucin antibodies all gave distinct patterns of intracellular staining of epithelial cells. Our results indicate a close similarity between cultured stromal and epithelial cells from endometrium and endometriotic deposits. Culture of these cell populations will permit study of their properties and interactions and may provide some insight into the cause of endometriosis.

Conservation of rare and endangered plants using in vitro methods

Many botanic gardens now have tissue culture laboratories for the micropropagation of plants that are difficult to propagate by conventional horticultural techniques. In many cases the work centers on rare and endangered species. Examples of the use of different techniques including micropropagation, in vitro seed germination, dual culture with symbiotic fungi, and regeneration from callus are discussed with reference to their application to plant germplasm conservation.

Application of the principles of good manufacturing practices (GMP) to the design and operation of a tissue culture laboratory

Recommendations are presented for the design and operation of a tissue culture laboratory to reduce microbial contamination of cultures. These recommendations are based on practices currently used in the manufacturing of sterile drug products, certain medical devices, and in vitro diagnostic products. The recommendations depend on qualification and validation of equipment, systems, and procedures and on the control and monitoring of the laboratory environment and procedures.

Aspergillus fumigatus contamination of lymphokine-activated killer cells infused into cancer patients

Lymphokine-activated killer (LAK) cells, prepared by incubating autologous lymphocytes in cell culture medium with interleukin-2, selectively lyse tumor cells and are effective immunotherapy of some cancers. During a 3-month period, two patients at our center were infused with LAK cells subsequently found to have been contaminated by Aspergillus fumigatus. Each case was investigated by obtaining environmental cultures and assessing aseptic practices during LAK cell preparation. Investigation of the first case demonstrated a malfunction of the laminar air flow hood, under which interleukin-2 and the patient's lymphocytes had been added to cell culture medium, and showed heavy A. fumigatus contamination of the hood, adjacent countertop, and cell culture incubator. Despite repair of the laminar air flow hood and cleaning of the laboratory, a second case occurred, and cultures at that time implicated the humidified cell culture incubators as the source of A. fumigatus. Following incubator sterilization and removal of the humidification apparatus from the incubators, weekly environmental cultures in the LAK cell laboratory were negative, and none of the LAK cell cultures from the 20 patients treated during the ensuing 15 months grew A. fumigatus. Our findings show that growth of fungi in humidified incubators, which previously has caused contamination problems in tissue culture and clinical microbiology laboratories, can result in patient infections when humidified incubators are used to prepare cells for reinfusion.

Tropical forest genetics at the Oxford Forestry Institute : Exploration, evaluation, utilization and conservation of genetic resources

Research and development of tropical tree genetic resources began at the Oxford Forestry Institute (OFI) in 1963 with Pinus caribaea. The sequence started with exploration of the natural distribution, a study of variation through classical taxonomic procedures backed up by chemotaxonomy and isozyme analysis, and observations on general morphology. Seed was collected to cover the range of variation, then processed, tested and stored in England, and distributed for trial at representative sites and in recommended designs. Standardized assessment techniques were developed and evaluation of the field trials was conducted by OFI staff for a selection of sites and by national staff in the other locations. Following analysis, interpretation and publication of the data, second-stage seed collections were made from the most promising natural populations and scions were collected from selected phenotypes in the provenance trials themselves; these scions were shipped to Oxford and bulked through grafting, and will eventually be transferred to field sites as packaged seed-orchards and breeding populations through scions and micropropagules. The second sampling was structured to gain a better understanding of variation in the promising provenances. Appropriate breeding strategies were evolved and practically implemented through direct advice to participating countries, and a philosophy was developed for the conservation of genetic diversity, both in situ and ex situ. This work with P. caribaea created the capability in terms of specialist staff and facilities for the full genetic development and conservation of a species. As current needs have been indicated by world forestry forums, more species have been brought into the programme including other tropical pines (for example P. oocarpa, P. tecunumanii), various tropical and sub-tropical timber hardwoods (e.g. Cedrela and Cordia species) and dry-zone species for multipurpose use in agroforestry and silvo-pastoral systems (including Gliricidia, Leucaena and Parkinsonia species from central America and Acacia species from eastern Africa). During the past 25 years, the OFI has distributed over 22 000 separate seedlots of 108 species to 122 countries. Many overseas graduate students have used material from these trials for their research degrees. All records of origin, distribution, assessment and literature for each seedlot are maintained in a computerized database. Expansion of the OFI's gel-electrophoresis and tissue-culture laboratories are seen as important steps to increase the efficacy and speed with which tree species can be conserved and bred in the future.