Richard Fenwick Micropropagation Explained
ŠRichard Fenwick
Horticulture Course Manager & Lecturer at
Richard Fenwick Course Manager/Instructor in Horticulture Bishop Burton College Modules currently tutoring Level 3 Extended Diploma in Horticulture Understand the principles of organic crop production Horticultural production techniques outdoors & indoors Principles of plant health & protection Investigative project in the horticultural sector Business management in the land based sector Level 3 Extended Diploma Fish Management Business management in the land based sector Level 3 Extended Diploma in Agriculture Principles of plant science Business management in the land based sector
Horticultural Advisor/Writer Garden Tripod Magazine www.gardentripod.com
First Published August 2013 All The Materials Contained May Not Be Reproduced, Copied, Edited, Published, Transmitted Or Uploaded In Any Way Without the artist/photographers Permission. These Images/writings Do Not Belong To The Public Domain. All images and information within the Garden Tripod magazine are the responsibility of the owner/artist/writer/photographer & not the Garden Tripod magazine 2012-2013
Micropropagation
Explained
Micropropagation
[Definition] High yield plant production, grown in a nutrient rich gel under sterile lab conditions.
Advantages If you have the following propagation problems then Micropropagation may be the answer
Seed growth lacks uniformity. Seed growth is not true to type. Seeds take too long to mature. Seeds difficult to handle. Seeds not available. Low germination rate with seeds. Cuttings grow too slowly. Seed/cuttings require too much care. Only 1 virus free plant. Only 1 hybrid. Only 1 desirable mutant / sport e.g. Hosta 'George Smith' Cuttings have a poor survival rate e.g. Clematis armandii Vulnerable to pests and diseases. Shortage of stock plants. Not cost effective to keep/maintain stock plants e.g. insufficient room
Or would like the following High number of plants in a short period of time infinite number of plants. Clean material for genetic engineering (GM), hybrid development, pesticide testing etc
Micropropagation has also been responsible for Saving plants that are in danger of extinction. E.g. Musa acuminata [Banana] A 'type' of Micropropagation is used in the field on long excursions into unexplored rain forests. Exporting plants in test tubes often requires little to no inspection or plant passports.
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Disadvantages Staff training costs. High start up and chemical costs. If there is a defect from chemical imbalance or mutation while in culture. This may not be detected for a number of years. Distorted plants can develop if high levels of hormones are used over a long period of time. Tissue proliferation Plant introduction [New Start] may take up to a year before multiplication starts. Thrip infestations can damage plant material, contaminate/spread infections and destroy cultures. Long term bacterial contamination from New Start or Multiplication stage can result in poor growth/multiplication and/or rooting.
Nutrient gel First we need to discuss the nutrient rich gel; this is used throughout the 3 stage process of Micropropagation Key points are- what the gel contains, how the gel helps explants grow/develop and the preparation method. Each variety of plant requires a unique gel recipe. It’s very unusual for plants to grow and develop in an ‘off the shelf mix’
A general mix would contain Distilled water Fertilizer or salts Vitamins - essential for plant metabolism and growth Cytokinin -for growth, branching, cell elongation, bud formation Auxin - very important to plant growth and to balance levels of cytokinin Gibberellic acid - growth regulator Amino acids - building blocks of proteins Sucrose – supplemented food source as the artificial lights in the growing room are not as strong as sunlight
Note pH adjustment - just like soil this needs to be correct Agar – a semi-solid to support the explants and allow water/nutrient uptake
Preparation Method This is a bit like making a cake, follow the recipe by Adding all the required stock solutions to the water and then mixing in the dry ingredients sucrose /agar. Just like making a fruit jelly, the agar needs to be heated to just below boiling point. [90◦C] The liquid should go from a cloudy to a clear solution; it is then ready to be poured into clean/dry test tubes, jars or pots. A 1L flask should give you approx 20 jars. Due to the sucrose content, the jars containing the medium need to be sterilized otherwise you would just grow mould. This is normally done in an Autoclave [big pressure cooker] to a temperature of 121◦C and 2 -3 bars of pressure. Once cold, store in a dark fridge/cold store for up to 6 months. Before use, bring back up to room temperature for 7 -14 days and inspect for any mould growth.
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New start Initiation of a new variety into culture [New start] Similar to any propagation method, you should start with a healthy plant that is disease, virus*, and pest free. I also recommend the plant should also be under watered, herbicide, pesticide, dust free for at least 6 weeks *A quantity of viruses have advantages or traits The plant should have little to no stress; however that can be an advantage in some varieties E.g. Clematis in low light, stressing shoots to elongate This is due to the elongation of the shoots there is a higher concentration of natural auxin in the shoots that can lead to a more vigorous culture. A small section can be taken from a stem, root, leaf, bud or single cell; often from meristematic tissue this is then called an explant. Due to the presence of sucrose in the gel, all traces of dirt, bacteria, sources of contaminate need to be removed before placing the explants in the medium. Surface sterilize the explants – Askham Bryan College Method One 5% Tween 20 for 5 min [Optional 0.1% Hg2Cl2 [Mercury chloride] for up to 10 min] 10% Bleach for 10 - 15 min [Sodium hypochlorite] [Hydrogen peroxide] 3 X wash distilled autoclaved water Remove any damaged tissue Place in the gel and store in the growth room, growth cabinet or on the trolley. The environment is air-conditioned @ 25°C - 27°C depending on variety and the explants are grown under white light (normally) with a Lux level of 1200. New starts need to be inspected daily for early bacterial contamination.
Mould
Mould
Mould
Mould
Shoot blindness = add gibberellin to the mix & lower light Ethylene gas build up = sunfim plus multiplication quicker & reduce temperature in the growth room or move jar to a lower level & add charcoal to the mix. Â Contamination = don't work on the same variety on same day & use a good micro technique. Try to catch contamination in the early stages and use Sodium hypochlorite
Always keep a record of crop numbers, changes in mediums or technique..
Ethylene gas build up
Bleeding 8
Contamination Bacterial contamination can be the result of New start plant material not being completely clean Cross contamination from another culture The human element infecting the plant. E.g. skin cells Loss of power to the Laminar Flow Cabinet Infection / virus in the stock plant leaching into the culture If this occurs the explant will require a further dip in Sodium hypochlorite and a new jar of medium.
Problems that can arise throughout the life of the culture Explant dies = Dom strength or Medium to strong or wrong time of year Explant blackens / dies = Contamination, bleeding, wrong mix, GR to hot or cold Growth slow = wrong mix, dormant period, virus, unseen contamination, old culture or Ethylene gas build up stunting / killing the growth Bleeding = phenolic exudates [organic metabolic by-products transported by the Phloem] from the culture that colours the medium inky purplish black and is often toxic = dull knives, old cultures or too liquid a medium Shoots long and spindly = add more cytokinin Shoots too short = reduce cytokinin Fat stems but small leaves = reduce cytokinin Unwanted callus = wrong hormones try reducing cytokinin or a new mix/gel Red stems = stress change temp, light, reduce sugar & increase nitrate Vitrification = lower cytokinin or/and nitrate + increase agar + sunfilm to reduce water or it’s an old culture therefore start thinking 'New Start'. Desiccant tubes can also be used to reduce vitrification however this can also be a weaning method
Multiplication Multiplication is normally on a 4-6 week rotation, depending on health and variety of the plant. Daily observations should be made for any contamination, gas build up or early signs of vitrification/hyperhydracity. Generally the culture has no roots at this stage just top growth/callus in a multiplication mix. Before opening a jar to multiply, always look for any Bacterial contamination Phenolic exudates Callus type/ differentiation Plant health – vitrification & pest/diseases Ethylene gas build up Always wash your hands before working and spray with 10% ethanol in the laminar flow cabinet. You should also have two lab coats, one for working in the growth room and one for working in the cabinets. These should be washed on a daily basis and will reduce plant culture problems.
All equipment should be sterilized before use. Procedure while working in the laminar flow cabinet Take plants out of the jar from the left Work off a white tile and multiply plant material Re-flame scalpel and tweezers between plants Place these new sections into fresh medium in the right Use autoclaved paper over the tile before starting a new jar, this will reduce any cross contamination Label jars with a plant code, date, gel code and your initials Enter jar numbers into a diary plus any additional info; gel type, Sun Film use and any other observations Plant numbers are increased over a period of time, typically 200+ jars before the rooting stage Environmental conditions within the jar - 90% - 100% Humidity There is enough O2 and CO2 mix for the plants to grow/multiply until the plants are multiplied again. If a solid cap is used there is little gas exchange however Sun Film provides gas exchange through a permeable layer.
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Rooting The rooting mix is often completely different to the multiplication mix and will require research and development. Once this has been developed, large number of plants can be transferred and rooted. Rooting time scale can be 4-6 weeks with daily monitoring; once 80% of the jar is rooted the plants can be weaned. The last 20% should root once weaned and in compost.
Weaning & Aftercare Once your culture has achieved a rooting percentage of 80%+ it’s time for weaning/hardening off.
It’s important to remember Cultures can be many years old, have been growing in 90% to 100% humidity and have lost the ability to open and close their stomata. The leaves have abnormally high number of stomata per leaf and little to no cuticle. In some cases, plants have thinner roots, stems, leaves and less chlorophyll in the leaf. Without this important weaning stage any micropropagated plants left in the open would be dead in approx 20 minutes as water escapes through all pores in the leaves. To help this process while the plants are still in culture the following can be used to help the weaning process Add calcium or reduce sugar content in multiplication/rooting mix this can shorten weaning time and reduce fatalities. Small bags of silica gel can be hung in the jars to lower the humidity however the culture must not be allowed to dry out. In the growing room or a clean room, jar lids can be loosened to allow more water vapour to escape. Increase agar content to reduce available moisture [= a firmer gel] however this will make it more difficult to separate roots from agar.
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Once the plants are removed from the jar – Wash off all traces of agar, this will reduce moulds, yeast's bacteria formation and the need to use fungicides. Transfer plantlets to compost and store in a fog or mist tunnel/bench for 2 - 4 weeks reducing the mist gradually. Monitor and if required us fungicides, this can increase the survival rate. A fully weaned plant is free from pest/diseases, is often stronger and has better root system compared to normal propagation techniques. E.g. Soft or semi-ripe cutting.  At this stage the plant is able to open and close the stomata according to turgidity.Â
Equipment Basic Micropropagation can be performed at home, in a kitchen or spare room this can give good results. Its best to avoid a carpeted room as any movement often dissipates dust and bacteria into the air. That’s the advantage of a tiled, wooden or vinyl floor; it can easily be sterilized using a household cleaner.
Equipment required A sterile still air cabinet used to transfer plants. A fish tank on its side makes an ideal transfer cabinet. Any perspex or glass chamber with dimensions of 50 cm (length), 40 cm (height) and 40 cm (depth) could easily be made into a transfer cabinet. A pressure cooker or microwave. [DO NOT use Aluminum Foil in the Microwave] For sterilization of media, instruments, water, paper etc. [Once sterile don’t open until you are ready to use the equipment in the transfer cabinet] Glass jars (baby food jars are excellent) and take away food containers with lids which can withstand the heat inside a pressure cooker are ideal vessels to use. Scalpel and forceps. White Tile + A4 white copy paper, cut to size, can be sterilized and used for a sterile cutting surface. spirit lamp containing Mineralised Methylated spirit [Purple or Clear] for flaming the instruments. [DO NOT use Methanol as it is toxic!]. Hand held spray bottle containing 70% alcohol solution e.g. Mineralised Methylated spirit to spray the transfer chamber, jars and other surfaces. Dilute chlorine solution for a New Start e.g. 1/4 dilution of the household bleach for use in surface sterilization of plant material.
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required for home tissue culture
Preparation
Media Preparation
Most of the ingredients indicated below can be purchased using the local super market or a health food shop. [With this recipe there is no need to buy expensive weighing scales] Two cups of rain water. A quarter cup of sugar. Fertiliser stock: 1/2 tablespoon all purpose 10:10:10 (N.P.K.) water-soluble fertiliser in 1L of water: use one cup of stock for this recipe. Inositol tablets (500 mg): 1/2 tablet. Vitamin tablet with thiamine: 1/2 tablet- Any multivitamin tablet may be used. Agar flakes: 4 tablespoons.
Preparation of Multiplication and Rooting media Add 1/2 cup of fresh or tinned coconut milk. Replacing the coconut milk with 1/2 cup of green tomato puree or adding and 1/2 teaspoon of malt or 1/2 cup of freshly squeezed orange juice may produce different responses. Ensure that the pH of medium is always between 5 and 6 using narrow range pH indicator tape. Adjust pH if necessary, with acid. E.g. Citric acid or base e.g. bicarb soda. Mix the ingredients in a saucepan and gently boil until the agar has dissolved, stirring continuously to stop the agar sticking and subsequently burning at the bottom of the pan. Dispense into empty glass jars, using a ladle, so that the medium is about 2 cm deep. Cover and process in a pressure cooker. Cook for 15 minutes after the pressure is reached.
Please note: Always take great care when embarking on any home Micropropagation. Comply with all advice given in MSDA Data Sheets for the safe use and handling of products / chemicals. Good practice should always be upheld while working in a Micropropagation Lab.
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The projected
Future of Micropropagation Within the next 10 years Increased number of Micropropagation labs, based at large commercial Nurseries but networking with surrounding small nurseries/garden centers making it more cost effective. Robotics to increase production and reduce labour costs. Development of UV rooms [with an automotive robotic workforce] to eliminate contamination to between 80 - 100%. Replacing the scalpel blade with laser cutting beam in an automated device. This reduces cross contamination/damage to neighboring cells and has been found to break bud dormancy of in woody plants. E.g. Avocado [Persea americana] Strawberry Tree [Arbutus unedo] Carbohydrate/Sucrose free cultures reducing bacterial contamination to workable conditions. This could allow cultures to be grown outside under sunlight with or without CO² enrichment.
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INDEX 4 Advantages 5 Disadvantages 6 Nutrient gel 7 New start 8 Contamination 10 Multiplication 11 Rooting 12 Weaning & Aftercare 14 Equipment 16 Preparation 18 Future of Micropropagation
Garden Tripod Magazine
All The Materials Contained May Not Be Reproduced, Copied, Edited, Published, Transmitted Or Uploaded In Any Way Without the artist/photographers Permission. These Images/writings Do Not Belong To The Public Domain. All images and information within the Garden Tripod magazine are the responsibility of the owner/ artist/writer/photographer & not the Garden Tripod magazine 2012-2013