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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

Explant 3mm x 5mm x 5mm

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