35-37 - Aseptic technique o Stirring of the Tissues
Also facilitates good surface contact
Sources of contamination: o Ultrasonic bath o Microorganisms in nutrient medium proper Ensure good surface contact din plugging and autoclave! 120C, 20min, 15psi Like those used to clean dentures o In explant surface-sterilize for 30 mins o After surface sterilization o During transfer of plant sterilize inoculation Minimum 3 sequential rinses with chamber by UV radiations sterile dH2O to remove remaining Adjust medium to pH 5.6-6.0 before autoclaving chemical sterilizing agent (extreme pH affects nutrient uptake) Agar 0.8% - 1.0% concentration (hard medium 40 - in vitro environment decreases nutrient uptake) Explant plant tissue removed from original site and 37 - Sterilization of plant tissues transferred to artificial culture media Table na nasa postlab ni sir Wash in weak detergent solution and rinse several times with dh2o prior to sterilization 40-42 - pre-treatment of explant tissues prior to culture Immerse briefly in 70% EtOH (spreads over tissues more effectively than higher concentration OH) - cleans Woody tissue explants produce phenolics into culture woody tissues, buds, and twigs medium browning of explant material Sodium hypochlorite (NaOCl) - most commonly used o Antioxidants such as L-ascorbic acid, citric acid, (0.025-0.25% NaOCl) and L-cysteine are commonly used for checking o Diluted household bleach can also be used browning problem ? (best results: freshly (5.25% NaOCl) less expensive prepared antioxidants) Calcium hypochlorite (CaOCl) substitute for NaOCl o Browning pigments are toxic to plant growth in o Less damage to plant tissues but tends to cultures precipitate out o Prepare mixture of L-ascorbic acid (100mg/l) o To avoid CaOCl accumulation on tissue and citric acid (150mg/l) in double dh2o?? surfaces, sterilization solutions should be o Filter sterilize through 0.22micrometer filter unit filtered or decanted prior to use o Store explant in cold antioxidant mixture H2O2 (3-10%) o Incubate explants in ref at 0 degrees for 5-30 o Much easier to remove than NaOCl and CaOCl mins to allow soaking of antioxidant solution Other substances o Commercial bleach contains about 5% sodium o Bromine water (1-2%) hypochlorite and thus may be used at a o Silver nitrate (1%) concentration at 10-20%, which is equivalent to o Mercuric chloride (HgCl2 0.1-1%) 0.5%-1% sodium hypochlorite o Table (KAILANGAN PA BA YON PUTEK) 38 - Sterilization, surfactants Sterilization procedures may be enhanced by the following method Autoclave glasswares and instruments at 121C for 1 hr o Place material in 70% EtOH before treating with Dry-heat: metal instruments 140-160 degrees 2 hrs another disinfectant solution. 2 step/source Filter sterilization for heat labile amino acids, vitamins, sterilization procedure is beneficial growth regulators, antibiotics, natural complexes by o Use wetting agent (Tween 20 or 80) Millipore filtration assembly Reduce surface tension o Filter membrane 0.45 or 0.22 micrometer Better surface contact porosity o Conduct sterilization under vacuum o Plug receiver flask with cotton Removes air bubbles o Wrap filtration unit with paper or foil Effective sterilization process o Autoclave both 121 degrees 1 hr Procedure o Attach filtration unit with receiver flask with a o Wash explants in mild detergent before vacuum pump in laminar flow bench?? disinfecting (exclude herbaceous materials o Pour solution to be sterilized into filtration unit which may not need this treatment) o Apply slight air pressure to start filtration o Rinse thoroughly under running tap water 10-30 o Transfer desired volume to sterile flasks under mins laminar airflow bench o Submerge into disinfectant solution o Use sterile pipette for drawing filter sterilized o Under sterile conditions, decant and rinse solution to autoclaved medium several times with sterile dh2o Surfactants o Explants from adult woody species o Tween 20 or Triton X-100 contaminated!!! enhance sterilization! 0.05% (low concentration Aeration of cultures procedure Ensures that sterilizing agent comes in o Semi-solid medium di na kailangan ng special contact with entire tissue surface device for aeration o Liquid medium: shake with gyratory shaker o Prepare stocks with distilled or demineralized h2o 43 - isolation of plant material o Reagent grade chemicals should be used to ensure maximum purity Explant - 2-4 mm3 sterile segment o Nitrate stocks precipitates out, must be heated Stock plant until crystals are completely dissolved Age of stock plant and location on the stem from which o Discard stock with cloudy or microbial growth explants are removed greatly affect callus establishment o Do not combine stock to other stocks unless they are stable and compatible 43-44 - age of plant tissue Plant growth regulators o Auxin IAA, NAA, 2,4-D, IBA Stem apex and seeds commonly used to raise callus Induce cell division and root initiation Injured tissues during explant excision release 2,4-D callus induction; others for root compounds that are air oxidized induction o Turn brown (lethal!!!) Dissolve crystals in EtOH, 1N NaOH, o USE SHARP SCALPEL TO EXCISE EXPLANT or 1N KOH; rapidly add dh2o to 100 FROM STOCK PLANT HAHAHA ml IAA destroyed by low pH, light, 46 - procedure of removing plant parts (micropropagation) oxygen and peroxidase NAA and 2,4-D most stable form Selection of an elite mother plant o Cytokinins KN, BAP, 2iP, Zeatin Explant pieces Adenine derivatives Trimming Cell division, shoot proliferation, Surface sterilization organogenesis, somatic Washing embryogenesis Establishment of cultures on appropriate growth medium Dissolve crystals in 1N HCl and water, Transfer on multiplication medium + gentle heat, add double dh2o Rapid shoot or embryo formation Thermostable in autoclave Embryo shoots or plantlets transferred to sterilized soil o Gibberellins inhibit callus growth or artificial medium by different gradual weaning Used in plant regeneration and processes (gradual reduction in humidity and nutrient elongation after shoot primordial levels) formation Dissolve in water, adjust pH to 5.7 47 - callus tissue and organogenesis Not thermostable, should be filter sterilized Callus amorphous aggregate of loose parenchyma o Abscisic acid embryo culture and somatic cells embryogenesis o No organized meristem Double dh2o, colored bottle o May be hard due to lignification of CW or brittle Heat stable but light sensitive and sometimes soft Vitamins After subcultures, soft callus can be inoculated into liquid o Nicotinic acid B3 medium suspension culture o Thiamine B1 Clone: plate suspension on agar o Pyridoxine B6 Organogenesis adventitious organs or primordial o Add before autoclaving o Stocks at 100x or 1000x (10ml per 1000ml or 1 (embryoid) from callus o High auxin:cytokinin root ml per 1000 ml) o Low shoot Carbon source Caulogenesis type of organogenesis na only o Low levels of carbs for protoplast culture; high adventitious shoot bud initiation takes place for embryo or anther culture o Sugars 50-54 - media components Caramelization if autoclaving prolonged Inorganic salt React with amino acid compounds o MS formulation widely used Degraded, form melanoidin o MS high nitrate, potassium, ammonium Brown, high MW, inhibit cell o Stocks prepared at 100x the final medium growth concentration o Hexitols o Each stock added at 10 ml per 1000 ml of Myo-inositol important, growth medium prepared promoter, carbon source and vitamin- o Na-Fe EDTA stock amber or wrap in Al foil like (protect from light) Mannitol or sorbitol good osmotica for protoplast isolation Water soluble, 100x Broccoli : Setup A 11.62 microM Kinetin; Setup B - + Gelljng agent 45.67microM IAA o TC grade ? or Difco-bacto agar o KEEP AGAR IN MOTION WHILE Transfer to hypochlorite solution DISSOLVING, OTHERWISE IT WILL BURN Shake 5 sec every minute for 10 mins THE BOTTOM OF THE FLASK Pour to waste beaker o 15 min 121 degrees autoclave Wash 4x Amino acids and amides o 100 ml sterile h2o, shake 5 secs, pour o L forms of amino acids Using sterile forceps cooled in wash water, transfer 6 L-tyrosine shoot initiation explants per bottle L-arginine rooting o Flame sterilize and cool forceps after each dish L-serine haploid embryo induction in is complete microspore cultures Cap bottle and seal with parafilm to reduce dehydration L-cysteine control phenol leaching Incubate in light at 20-28 degrees o Amides L-glutamine, L-asparagine When shoots = 1-2cm Induce somatic embryogenesis Antibiotics Cut from the mass larger shoots and connected material o Fungicides and bactericides in case explants at bases are excessively contaminated Transfer 4 shoots to bottle with hormone-free medium o Toxic to contaminant AND explant materiels so Seal, incubate restricted use for additions to culture medium After 2-3 weeks, roots! Transfer ulit to bottles with o Freshly made, filter sterilization hormone-free medium or to pots of sterile compost Natural complexes o Coconut (endosperm) milk (CM), Yeast extract Carrot: Setup C 0.45microM 2,4-D (YE), Malt extract (ME), Tomato juice, Potato extract, Casein hydrolysate, Fish emulsion Fresh undamaged carrots which sink in water should be used Antioxidants (without internal air spaces para sterile root interior) o Citric acid, ascorbic acid, pyrogallol, phloroglucinol, L-cysteine reduce excessive Surface sterilize with bleach and wetting agent browning (detergent) o Adsorbents: PVP, activated charcoal also used Shake 5 secs every 5 mins for 20 mins to check excessive browning Pour to waste MS medium = Murashige and Skoog Wash 4x o Sterile h2o, shake 5sec, pour 55 - sterilization of media Transfer to sterile petri dish, cut 1 cm from each end (discard) Tissue culture media generally sterilized by autoclaving Insert sterile scalpel to core at broad/shoot end to hold at 121C at 15-20psi Cut 1-3mm thick transverse section, shoot-pole Time depends on volume of medium in vessel uppermost, transfer to sterile petri dish Dispense medium in small aliquots as many components Cut smaller sections approx. 5 mm squared (w phloem, are broken down on prolonged heat exposure poor xylem, and cambium) by cutting across cambium cell growth Transfer each explant 2 per bottle root-pole downwards Minimum autoclaving itme to sterile agar with 2,4-D o Includes time required for liquid volume to Seal, incubate in dark at 25C reach sterilizing temperature (121C) + 16 mins at 121C After 4-5 weeks, callus! Discard dark necrotic tissue. o Several components are thermolabile, should not be autoclaved Use sterile scalpel to cut off healthy (yellow/cream- o Stock solns of heat labile components are filter colored) pieces of callus, approx. 5 mm cubes sterilized through 0.22mm filter to sterile Transfer to petri dish ? w fresh growth medium container then aseptically added medium (wc Seal, incubate, blah has been autoclaved and cooled to 35-45C) Kinetin, 2,4-D: 1N NaOH PROTOCOL IAA, NAA: ethanol