Tissue Culture General & Orchids

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Features to consider for suspension cell culture
There are two basic ways to grow cells in culture: as monolayers stuck to a surface or artificial substrate (adherent culture), or floating free in culture medium (suspension culture). When it comes to suspension cell culture, constant agitation ensures that the cells are aerated and nutrients are available to help the cells grow uniformly. The shaking “prevents the bacteria or other cell types from settling at the bottom of the flask, which would result in cell death from the lack of nutrient and oxygen availability,” says Jayne Bates, technical support manager at Cole-Parmer (Vernon Hills, IL). She adds that shaking also keeps clumps from forming.

To accomplish these vital functions in suspension cell culture, one requires a shaker that meets certain requirements.

The shaking motion needed when using an Erlenmeyer flask is a “nice swirl,” says Sara Livingston, global product manager for orbital shakers at Thermo Fisher Scientific (Waltham, MA), and there should be no sloshing. This circular (aka orbital) motion will increase the surface area of the top of the liquid to maximize gas exchange between the liquid and the air. Single-use bags, however, perform best on a two-dimensional rocking platform, says Brian Canna, vice president of marketing and business development at Boekel Scientific (Feasterville, PA). Bates notes that cultures in microtitre plates require rapid mixing, so more of a vibration mixing action is typically employed.

Variable speeds are needed to accommodate different culture vessel sizes, volumes, and numbers and types of cells. All these factors explain why determining the optimal speed for a particular application is such a common challenge encountered in suspension cell culture. “Some cells can be shaken faster than others,” says Livingston. For example, Bates explains that bacterial cells such as E. coli require vigorous shaking, whereas insect cell cultures should be shaken at much lower speeds. It is therefore important to use a shaker that has adjustable speeds, says Canna.

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Am also reposting this image on this page.



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Ray's comment.

When trying to get multiple plants from the meristem, the first thing done is exacting it and getting it to grow undifferentiated. That is often done by rotating or oscillating the growing chamber.

Once sufficient undifferentiated cells have grown, the material is divided to provide many such clusters, and when left static, they will differentiate, ultimately becoming individual plants.

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Plant Tissue Culture Techniques: 6 Methods & Protocols

Plant Genotype
All species of the plants don’t equally respond in tissue culture. Large differences exist between different genotypes, species, or cultivars, making some easily grown and some non-responsive or recalcitrant.

Explant culture
In biology, explant culture is a technique to organotypically culture cells from a piece or pieces of tissue or organ removed from a plant or animal. The term explant can be applied to samples obtained from any part of the organism. The extraction process is extensively sterilized, and the culture can be typically used for two to three weeks.

Types of explants used in tissue culture.
All parts of plants can be used as explants for tissue culture purposes: leaves, stems, a portion of shoots, flowers, anthers, ovary, single undifferentiated cells, or mature tissues. However, the cells should be capable of de-differentiating and resume cell division and have cytoplasm.

Some parts, like the root tip, are rarely used as explants for tissue culture processes. Root tips are difficult to isolate and contain microbes in their tissues, forming a strong association with them. Also, soil particles are attached to roots which are difficult to remove without damaging some tissues.

The age of the explant is an important factor. Young tissues are newly generated, they are better suited for the rough process of surface sterilization.

Different seasons have different effects on the explants. Explants collected during the spring while in their flush state are good examples & in winter their dormant state stop them from growing as explants.

Smaller explants are difficult to culture and are less responsive than the larger size explants. Smaller explants require additional components to sustain the culture.[COLOR="Silver"]

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