Frankenstein Hosta - Tissue Culture and Micropropagation
PRELIMINARY - NOT EDITED OR READY FOR PUBLICATION
this article is still being edited and as such, is not really ready for publication. it's just here now to remind me to finish writing it.
Plant tissue culture, or micropropagation, is a process which is used to reproduce genetically-identical plants from a small piece of a donor plant. This technique produces many more plants than propagation by crown division. Usually a stem tip, node, meristem, or embryo is prepared and placed it in a sterile nutrient medium so that it can multiply. The type of growth is determined by the nutrient medium - for example if you want to create more undifferentiated callus tissue, multiply the number of plantlets, grow roots, or multiply embryos for artificial seed, you would use different nutrients to stimulate the different types of cells to grow and multiply.
When micropropagating hostas, two different source tissues are usually used: the meristem, and the adventitious scape shoot. Each type of tissue donor reproduces different characteristics. For example, when using the shoot tip, or meristem, all properties of the donor plant will be reproduced, however, when using the adventitious shoots from young flower stalks (scapes), only solid green plants will result (no stripes or variegation).
While it is true that bacteria, mold spores, and other contaminants will definitely overrun a culture if you aren't careful in your sterile procedures, disinfection of implements, surface and nearby areas helps minimize contaminants and gives you a better chance at reproducing the plant you intended, rather than mold.. The simplest type of transfer area suitable for tissue culture work is an enclosed box ,commonly called a glove box. This type of culture hood can be sterilized by an ultraviolet light and wiped down periodically with 70% alcohol. You can make your own using any non-permeable material, such as glass, or plastic, which has been thoroughly disinfected. The 'hood' or 'glove box' need not be elaborate, just sterile, so using a large fish tank turned on its side or the inside of a 55 gallon drum, held up with rods to form a 'tent' would work if you are careful and thorough with your sterlization protocols.
Three stages of tissue culturing:
- Initial - Micropropagation is initiated from the donor plant tissue (explant) and cell multiplication begins. Culture medium used: Hosta Initiation/ Multiplication Medium.
- Multiplication - During this mid-stage, the explant multiplies to form many new shoots branching out from the original donor tissue. Culture medium used: Hosta Multiplication Medium
- Rooting - Once enough base plant material has been grown, shoots are stimulated to form roots. Culture medium used: Hosta Rooting Medium
Initial Stage - Establishing Hosta Cultures from Isolated Shoot Tips
- Wipe down all surfaces of your work area with 70% isopropyl alcohol. Take your time and do a good job since even a few fungus or mold spores can ruin your entire tissue culture batch.
- If you are working under a hood, allow the hood to run for 20 minutes or so before you start using it.
- Sterilize all of your equipment by either autoclaving it, or wiping it down with alcohol, bleach, and then a final round with Lysol. Be sure to dry all tools so that no traces of disinfectant remain.
- Place your sterile tools and fresh culture medium inside your work area (either your laminar flow hood, or your glove box).
- At this time also place about 100ml of 70% alcohol in a sterile 250-ml beaker inside your hood or box.
- Prepare the tissue sample(s):
- Remove the crown(s) from the soil when the plant(s) start to break dormancy and new shoots are 2-8 cm tall (before the first leaves unfold).
- Gently wash off all soil.Again, take your time with this and remove all traces of soil and 'outside' debris. You may wish to use a very soft brush, like a painter's acrylic bristle brush, to gently dislodge any scum or 'film' which may be on the plant from being outdoors.
- To isolate the meristem (growing point) cut off the shoot and a small portion of the basal stem (crown) tissue.
- remove the layers of petioles and leaves until only a small point, the shoot tip, about 1 – 1.5 cm long remains.
- Cut the basal stem away until about 0.5 cm thickness remains. Remove all discolored tissue since these could be signs of bacterial or fungal contamination. In general, the smaller the amount of tissue remaining, the less chance there is for endogenous (internal) contaminants.
- Repeat this process until the desired number of shoot tips has been isolated.
Multiplication and Rooting Stages
All work should be performed under sterile conditions in a laminar flow hood as previously outlined for culture establishment. Wipe the outside of the stage II culture container with 70% isopropyl alcohol and place it in the hood along with fresh media, sterile Petri dishes, and a sterile forceps and scalpel.
- Remove the Hosta shoot mass from the medium and place it on a sterile Petri dish.
- Using the forceps and scalpel, cut or break apart the base of the mass into individual shoots.
- Place these shoots onto fresh medium; 2-4 shoots can be placed in each container of medium.
- Replace the lid of the container(s) and put the culture(s) under fluorescent light as previously indicated for new cultures. .
- Subculture (break shoots apart and transfer to fresh medium) as desired. This is typically done at 30 – 60 day intervals to maintain actively growing cultures.
- Day 0 - Isolation of fresh explants
- Day 60 - 90 - First subculture of explant-derived shoots
- Day 91 - 120 - Transfer to soil when plantlets are large enough to handle
One of the most important factors governing the growth and morphogenesis of plant tissues in culture is the composition of the culture medium. The basic nutrient requirements of cultured plant cells are very similar to those of whole plants. Plant tissue and cell culture media are generally made up of some or all of the following components: macronutrients, micronutrients, vitamins, amino acids or other nitrogen supplements, sugar(s), other undefined organic supplements, solidifying agents or support systems, and growth regulators. Several media formulations are commonly used for the majority of all cell and tissue culture work. These media formulations include those described by White, Murashige and Skoog, Gamborg et. al., Schenk and Hilderbrandt, Nitsch and Nitsch, and Lloyd and McCown. Murashige and Skoog’s MS medium, Schenk and Hildebrand’s SH medium, and Gamborg’s B-5 medium are all high in macronutrients, while the other media formulations contain considerably less of the macronutrients.
The optimum concentration of each macronutrient for achieving maximum growth varies a great deal depending on which plant is being cultured. In general the macronutrients provide six major elements required for plant cell or tissue growth:
- nitrogen (N) - Culture media should contain at least 25-60 mM of inorganic nitrogen for adequate plant cell growth. Plant cells may grow on nitrates alone, but considerably better results are obtained when the medium contains both a nitrate and ammonium nitrogen source. Certain species require ammonium or another source of reduced nitrogen for cell growth to occur. Nitrates are usually supplied in the range of 25-20 mM; typical ammonium concentrations range between 2 and 20 mM. However, ammonium concentrations in excess of 8 mM may be deleterious to cell growth of certain species. Cells can grow on a culture medium containing ammonium as the sole nitrogen source if one or more of the TCA cycle acids (e.g., citrate, succinate, or malate) are also included in the culture medium at concentrations of approximately 10 mM. When nitrate and ammonium sources of nitrogen are utilized together in the culture medium, the ammonium ions will be utilized together in the culture medium, the ammonium ions will be utilized more rapidly and before the nitrate ions.
- potassium (K) - Potassium is required for cell growth of most plant species. Most media contain K, in the nitrate or chloride form, at concentrations of 20-30 mM.
- phosphorus (P) - The optimum concentration of phosphorous can range from 1 to 3 mM when all other requirements for cell growth are satisfied, however higher concentrations may be required if deficiencies in other nutrients exist.
- calcium (Ca) - The optimum concentration of calcium can range from 1 to 3 mM when all other requirements for cell growth are satisfied, however higher concentrations may be required if deficiencies in other nutrients exist.
- magnesium (Mg) - The optimum concentration of magnesium can range from 1 to 3 mM when all other requirements for cell growth are satisfied, however higher concentrations may be required if deficiencies in other nutrients exist.
- sulfur (S) - The optimum concentration of sulfur can range from 1 to 3 mM when
all other requirements for cell growth are satisfied, however higher
concentrations may be required if deficiencies in
other nutrients exist.
Micronutrients for plant cell and tissue growth include:
- iron (Fe) - Iron may be the most critical of all the micronutrients. Iron citrate and tartrate may be used in culture media, but these compounds are difficult to dissolve and frequently precipitate after media are prepared. Murashige and Skoog used an ethylene diaminetetraacetic acid (EDTA)-iron chelate to bypass this problem, which is why chelated forms of iron are commonly used in culture media.
- manganese (Mn), molybdenum (Mo) and boron (B) - Are included in most culture media.
- zinc (Zn) - Chelated forms of zinc are commonly used in culture media.
- copper(Cu), cobalt (Co) and iodine (I) - Sometimes added, but definitive requirements have not been established.
- sodium (Na) and chlorine (Cl) - Also used in some media, but are not essential for cell growth.
Carbon and Energy Source
Carbohydrates must be supplied to the culture medium because few plant cell lines have been isolated that are fully autotropic, e.g., capable of supplying their own carbohydrate needs by CO2 assimilation during photosynthesis. The preferred carbohydrate in plant cell culture media is sucrose.
Glucose and fructose may be substituted in some cases, glucose being as effective as sucrose and fructose being somewhat less effective. Other carbohydrates that have been tested include lactose, galactose, rafinose, maltose, and starch. Sucrose concentrations of culture media normally range between 2 and 3 percent. Use of autoclaved fructose can be detrimental to cell growth.Vitamins
Vitamins are the catalysts in various metabolic processes. Plants usually synthesize the vitamins that they require. When plant cells and tissues are grown artificially as with tissue culture, some vitamins may become the limiting factors for cell growth. This is why the addition of vitamins has proven beneficial when cutluring plant media. The vitamins most frequently used in cell and tissue culture media include:
- thiamin (B1) - Thiamin is the one vitamin that is required by all cells for growth. Thiamin is normally used at concentrations ranging from 0.1 to 10.0 mg/liter.
- nicotinic acid and pyridoxine (B6) - Nicotinic acid and pyridoxine are often added to culture media but are not essential for cell growth in many species. Nicotinic acid is normally used at concentrations of 0.1-5.0 mg/liter; pyridoxine is used at 0.1-10.0 mg/liter.
- myo-inositol - Myo-inositol is commonly included in many vitamin stock solutions. Although it is a carbohydrate and not technically a vitamin, it has been shown to stimulate growth in certain cell cultures. Its presence in the culture medium is not essential, but in small quantities myo-inositol is thought to stimulate cell growth in plants. Myo-inositol is generally used in plant cell and tissue culture media at concentrations of 50-5000 mg/liter.
Hosta Multiplication Medium
explain it here
Hosta Rooting Medium
explain it here
All types of tissue cultures should be incubated under conditions of well-controlled temperature, humidity, air circulation, and light quality and duration. Environmental factors may influence the growth and differentiation process directly during culture or indirectly by affecting their response in subsequent generations. Protoplast cultures, low-density cell suspension cultures, and anther cultures are particularly sensitive to environmental condition. Typically, the culture room in a lab should have a temperature between 15° and 30° C, with a temperature fluctuation of less than ±0.5° C.
Cleaning glassware used for tissue culture:
The conventional laboratory method of washing glassware requires that the glass be soaked in a chromic acid-sulfuric acid bath, followed by water rinses, distilled water rinses, and finally double-distilled water rinses, in that order. Due to the corrosive nature of chromic acid, the use of this procedure has been eliminated except for highly contaminated or soiled glassware. Adequate cleaning of most glassware for tissue culture purposes can be achieved by washing in hot water (70°C+) with commercial detergents, rinsing with hot tap water (70°C+), and finally rinsing with distilled and double-distilled water. However, highly contaminated glassware should be cleaned in a chromic acid-sulfuric acid bath or by some other proven method such as (1) ultrasonic cleaning, (2) washing with sodium pyrophosphate, or (3) boiling in meta-phosphate (Alconox), rinsing then boiling in a dilute hydrochloric acid solution, and then finally re-rinsing. Cleaned glassware should be inspected, dried at 150°C in a drying oven, capped with aluminum foil, and stored in a closed cabinet.
- Autoclave all glassware with media and cultures still in it. This kills any contaminating microorganisms that may be presents.
- After the autoclaved media has cooled, but while it is still in a liquid state, pour it into biohazard plastic bags or thick plastic bags, seal, then discard.
- Wash all glassware in hot soapy water using a suitable bottle brush to clean the internal parts of the glassware.
- Any glassware that is stained should be soaked in a concentrated sulfuric acid-potassium dichromate acid bath for 4 hr, then rinsed 10 times before washing it with soapy water.
- All glassware should be rinsed three times in tap water, three times in deionized water, three times in double-distilled water, dried, and stored in a clean place.
- Wash all instruments and new glassware in a similar manner.
Further Reading and Reference:
- Debergh, P.C. and R.H. Zimmerman, eds. 1991. Micropropagation, Technology and Application.Kluwer Academic Publishers. Lab design, info on labs worldwide, in depth discussions of problems. Not for the beginner.
- Donnelly, D.J., and W.E.Vidaver, 1988. Glossary of Plant Tissue Culture, Portland, OR. Timber Press, Good definitions of tissue culture terms.
- Kyte, Lydiane and J. Kleyn, 1996. Plants from Test Tubes: An Introduction to Micropropagation, 3rd ed., Timber Press, 1996 Good basics for the beginning amateur or grower.
- Smith, Roberta H., 1992. Plant Tissue Culture-Techniques and Experiments. Academic Press. Good introduction and broad base for college course.
- Trigiano, Robert N, and Dennis J. Gray, eds.1996, Plant Tissue Culture Concepts and Laboratory Exercises. CRC Press. Advanced
Tissue Culture Supplies:
- Carolina Biological
- Edmond Scientific
- PhytoTechnology Laboratories This company specializes in plant tissue culture supplies. Downloadable documents (choose MS Word or PDF format) on Media Preparation, Setting Up a Tissue Culture Lab, Basic Laboratory Procedures, and more are available in the "Technical" section of the web site.
- Sigma, "Plant Tissue Culture Catalog". In addition to media ingredients, premixes, equipment and supplies, this catalog contains a media comparison chart, procedures for media preparations.references and other valuable data.