2Terminology Aseptic procedure

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Plant Biotechnology Laboratory CCS HAU Regional Research Station, Karnal Dr. Ashok K. Dhawan

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Tissue Culture: Laboratory, Terminology, Aseptic Procedures

PTC Requirements:

PTC Requirements Media Preparation Sterile work environment laminar flow benches or glove boxes autoclave or pressure cooker sterilizing solutions - bleach, ethanol, H 2 O 2 Culture facilities Protocol most must be developed independently

Location of a PTC Laboratory:

Location of a PTC Laboratory Avoid Locating the Lab near: - Labs that handle microbes/ Insects etc. - Facilities that store seeds, other plant materials -Areas of High Foot traffic: invites dust which can be a source of contamination Air Vents can be a source of contamination Potted plants may hide mites and other contamination sources

Tissue Culture Laboratory:

Tissue Culture Laboratory A PTC Laboratory should be like a Production line: - Washing areas: Includes autoclaves -Media room: Balance, pH meter, refrigerator, hot-plate -Sterilization: Oven, autoclaves, ultra-filtration sets -Media storage -Aseptic procedures -Culture rooms Aseptic area must free from dust and isolated. Culture area should have temp control (25-27 0 C) and control of light intensity, quality and duration (Lamps + Tubes). Green house, poly-houses, glass house, screen house, net house and Fields

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Explant: Plant part used as a starting material in TC. Media: Mixture of chemicals used to grow explants e.g. MS-media is Murashige and skoog media. Sub culture: Aseptic transfer of a part of culture to fresh medium. Important Terms

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Inoculation: To transfer an explant to a media Sterilization: To kill all living micro- organisms, spores and cells Important Terms

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Callus: Tumour like mass of cells . Friable = Soft, cells are easily separable; Compact = Hard callus, has lesser regeneration Micro-propagation: Commercial multiplication of plants using tissue culture. Important Terms

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Hardening or acclimatization : a process before transfer to the field Adventitious : strictures from a place other than its usual location e.g. roots on leaves. Axenic: with no contamination ; sterile. Important Terms

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In vivo: “ in living ” processes in the intact living organism. In vitro: “ in glass ” processes in artificial conditions. Meristem culture: culture of apical meristem- explant of only apical dome tissue distal to youngest leaf primordia. Plantlet: miniature plants with roots and shoots made in tissue culture. Rhizo genesis: initiation of adventitious root primordia . Important Terms

Asceptic Procedures :

Asceptic Procedures The key to success in Tissue Cultures

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Common Terms Antimicrobial - An agent that kills or inhibits the growth of micro-organisms. Antiseptic - A chemical applied topically to inhibit the growth of micro-organisms. Asepsis - Prevention of microbial contamination of tissues or sterile materials by excluding, removing or killing micro-organisms.

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Autoclave - A steam sterilizer . Early models were termed "autoclaves" because they were fitted with a self-closing door . Contamination - Introduction of micro-organisms to sterile materials. Disinfectant - An agent that is kills or removes microbes, with exception of bacterial spores . Pasteurization - A process that kills nonspore -forming micro-organisms by hot water or steam at 65-100oC . Common Terms

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Disinfestation – To remove surface organisms from an explant. Sanitization - A process that reduces microbial contamination to a low level by the use of cleaning solutions , hot water or chemical disinfectants . Sterilization - The complete destruction of micro-organisms. Tydallization- Repeated boiling for 20 min on 3 consecutive days; Keep at 37 C Common Terms

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Aseptic areas to be located in a manner that there are no air currents . These carry contaminating spores . Laminar air flow is the most common equipment used. These are cabinets with gentle flow of ultra filtered air . Also contain UV lamps with emission of 253.7 nm which is germicidal . 1. Aseptic Areas

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Dirty hands and fingernails can be sources of contamination in laminar flow cabinets. Caution

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Glassware, metal instruments and materials not charred by high temperature Use laboratory ovens . Complete sterilization can be obtained by: -Heating for 45 min at 160 0C -Heating for 18 min at 170 0C -Heating for 7.5 min at 180 0C -Heating for 1.5 min at 190 0C 2. Dry Heat Methods:

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Employs an autoclave or a home pressure cooker . Pressure of 15 lbs/sq inch (103.4 kPa) at a temp of 121 0C. Paper products, glassware , instruments and liquid volumes up to 50 ml/container require 15 min . 3. Wet heat

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Bacillum macerans and Clavibacter species are viable on forceps stored in alcohol Also survive alcohol flamming Must autoclave the forceps Heat Resistant Bacteria

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Components that are unstable at high temp . Ultra filters made up of polyethylene film , cellulose esters, teflon or fluorocarbon polymers having pore size of less than 0.4 µ are employed, even though 0.22 µ is more preferable. 4. Ultra filtration:

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70% ethanol or isopropanol : 80-90% more inflammable; is less effective as it causes quick dehydration and lessens chances of killing 0.5% sodium hypochlorite or calcium hypochlorite for 10 min ( CLOROX : common bleach is 5% hypochlorite). Mercuric chloride (0.1%) is also used, but is toxic. Hydrogen peroxide 3-10% is effective Chlorine Gas: For seed kept in a dessicator and chlorine released by adding HCl to clorox NaDCC (Sodium dichloro isocyanurate) is less toxic to plants and does not need rinsing 5. Chemicals:

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Addition of Teepol or Tween-20 or Triton as wetting agent for plant materials containing cutin, suberin . These are surfactants . Two-stage disinfection e.g. alcohol + sodium hypochlorite is employed. Extremely Helpful!

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Goal of surface sterilization is to remove all microbes with minimum damage to plant tissue . These chemicals are for surface sterilization and microbes inside the plant tissue are a special problem.

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Antibiotics not a substitute for strict adherence to proper sterilization No known antibiotic effective against all microbes that cause contamination. Antibiotics affect plant growth . Only arrest growth , microbes tend to reappear Cause resistance among microbes Bavastin, Getamycin and mycostatin, ampicillin are common. 6. Antibiotics:

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UV lamp emission of 253.7 nm is germicidal. Brief exposure of UV lamp (2-5 min) is sufficient to sterilize working areas. UV burns the eyes ( actinic keratitis ). This is very painful though not lasting. Long hours of UV may change O2 to O3 which may cause explosion. 7. UV:

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Sterilization of Explants -Microbes present on the surface, in small cervices, between the layers, developing leaves of buds etc -Surfaces covered with wax have spores sticking and epidermal hairs trap microbes -Also contamination within the vascular tissue and inside the tissues

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Source of Explants is Important -Select plant materials in active state of growth e.g. spring flush shoots -Plant parts near on inside the soil more contaminated

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When Explants Harbour Internal Microbes Rapidly growing shoot tips, ovules, immature flower parts, tips of runners etc may be better Seeds can be asceptically grown Transfer plants to a pot in glass house for 1-2 months before collection of explants Grow plants ascetically (S-0)

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When Explants Harbour Internal Microbes Antibiotics suppress microbes , even though they tend to re-appear Heat treatment can be effective for some bacteria, viruses or fungi: in water bath at 550C for 1 h or growth chamber at 400C for longer periods Fungicide treatment prior to cultures helps

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THANKS

PTC Applications:

PTC Applications Micropropagation $$$$$$$$$ Artificial seeds and forestry In vitro breeding interspecific crosses haploid breeding Somaclonal variation and cell selection Cell culture (similar to fermentation)

Micropropagation:

Micropropagation Large scale multiplication of high value plant material Normally use meristem as explant meristem is the growing point of a plant consists of cells which are undifferentiated All regenerated material are clones

Micropropagation:

Micropropagation Allows for continuous production Permits seasonal production Allows for long term preservation of plants Small space requirements Because the meristem is starting material, products are virus and disease free seed potato industry

Seeds:

Seeds Seeds are the reproductive product of higher plants Consist of a seed coat (protective layer), endosperm or cotyledon (nutrient storage) and an embryo Seeds are the product of fertilization (2 haploid gametes fuse to form new embryo)

Somatic Embryos:

Somatic Embryos Tissue culture maintains the genetic of the cell or tissue used as an explant Tissue culture conditions can be modified to cause to somatic cells to reprogram into a bipolar structure These bipolar structures behave like a true embryo - called somatic embryos

Artificial Seeds and Forestry:

Artificial Seeds and Forestry U of S patent Production of artificial seeds for conifers Can produce 1,000,000s of genetically identically clones Company in Victoria involved in reforestation MONOCULTURE to the n th degree

In Vitro Breeding:

In Vitro Breeding Plant breeders use TC to transfer genes from one species into another with the use of genetic engineering. Two methods have been successful - protoplast fusion and embryo rescue Protoplasts are plant cells without cell walls Can be produced in a culture dish with enzymes

Protoplast fusion:

Protoplast fusion Protoplasts are made from two species that you want to cross The membranes are made to fuse osmotic shock, electrical current, virus Regenerate the hybrid fusion product Contain genome from both organisms Very, very difficult

Embryo Rescue:

Embryo Rescue Place pollen from one species on the emasculated flower of the second plant After a few days dissect out the developing seed and place into tissue culture Regenerate the hybrid Triticale - developed by crossing wheat and rye

Haploid Culture:

Haploid Culture Culture of the plant gametes - microspore and ovule (egg cells) Cells are haploid (1 set of chromosomes) Regenerated plants are haploid Regenerated plants are sterile Can’t undergo meiosis

Haploid Culture:

Haploid Culture Chromosomes need to be doubled Use mitotic inhibitors - colchicine, fluoralin Recovered plants are diploid Recovered plants are 100% homozygous “Fixes” traits, decreased breeding time Most canola varieties are doubled haploid

Somaclonal Variation:

Somaclonal Variation Although PTC is assumed to preserve genetic identity, genetic variation does occur This variation can be selected for and has been termed somaclonal variation Problem is that it has proven to be unstable in most cases - epigenetic inheritance

Somaclonal Variation:

Somaclonal Variation True SV has had little success (flax, sugarcane, potato), cell selection improves outcome Selection is completed in presence of a selection agent and often a mutagen is used Herbicide resistant canolas, virus and disease resistant potatoes

Cell Culture :

Cell Culture Similar to fermentation Culture of cells mainly for plant secondary metabolites Many, many problems $$$Billions$$$ invested with little payoff