Tissue Culture Problems

Question 1

Activated Charcoal

Answer 1

• an absorbant material
• used to absorb excess phenolic exudates from explants in culture
  
  • so that the phenolic compounds dont oxidize to form a brown material in the medium and inhibit shoot formation

Question 2

Ascorbic Acid

Answer 2

• an antioxidant used to minimize excess phenolic exudates from woody perennial explants in culture
• citric acid also used
• if phenolic exudates not minimized, then they oxidize to form a brown material in the medium and inhibit shoot formation

Question 3

Chimeral Breakdown

Answer 3

• a revision toward the inner tissue genotype, particularly during tissue culture propagation
• can result from adventitious shoot initiation
  
  • that is why chimeras can only be reliably propagated from shoot tip and axillary shoot cultures

Question 4

Epigenetic Variation

Answer 4

• variations due to epigenetic effects primarily involve phase changes involved with the juvenile-mature cycle.
• important in:

1. rejuvenation
   * cuttings that fail to root from mature stock plants often can be rooted from microcuttings from tissue culture
2. flowering
   * ones from tissue culture flower much sooner than just seeds

Question 5

Genetic Variation

Answer 5

• same thing as somaclonal variation
• also called chimera variation
• genetic changes prior to , or during, micropropagation are the most serious types of aberration b/c they are permanent and may be difficult to detect during the operation
• occurances of variation can be reduced by:
  
  • avoid long term cultures
  • use axillary shoot induction systems where possible (not adventitious shoots)
  • propagate chimeras by other clonal systems

Question 6

Habituation

Answer 6

• a problem encountered in tissue culture
• when a culture continues to develop in the absence of auxin or cytokinin
• shoots will not develop without cytokinin but in habituation, even after cytokinin is removed, still makes shoots like crazy.

Question 7

Hyperhydricity

(vitrification)

Answer 7

• a problem encountered in tissue culture
• when tissues becoe water-soaked and translucent
• caused by excessive water uptake (“waterlogging”)
• they then fail to regenerate
• usually controlled by changing agar concentration or source

Question 8

Internal Pathogens

Answer 8

• a problem encountered in tissue culture
• can be either virus or bacteria from the initial explant
  
  • internal - so cant wash off or kill with chlorine
• antibacteria are used in some difficult to eliminate bacteria cases
• a VERY BIG PROBLEM

Question 9

Phenolic Exudation

Answer 9

• a problem encountered in tissue culture
• problem found in established explants in culture (esp. woody perennials)
• these compounds oxidize to form a brown material in the medium and inhibit shoot formation
• treatments to minimize exudation include:
  
  • add antioxidant like ascorbic or citric acid
  • add absorbent material like activated charcoal
  • frequent transfers to new media

Question 10

Rejuvenation

Answer 10

• .the shift from the adult phase to the juvenile of transitional phase
• micropropagation has resulted in the reversion of plant shoots to the juvenile phase
• repetitive subculturing of mature walnut shoot tip culture lef to rejuvenation of mature clones and enhanced rooting of micro-cuttings

Question 11

Shoot-Tip Necrosis

Answer 11

• a problem encountered in tissue culture
• occurs in some woody perennial tissue cultures
• when actively growing shoot tips develop die-back
• usually cause by calcium deficiency in medium
  
  • to fix usually just add more calcium to the medium

Question 12

Somaclonal Variation

Answer 12

• geneti mutations that occur in tissue culture
• typical types of mutations include changes in plant form:
  
  • chimera varigation
  • change in leaf size
  • various forms of fasciation
• usually undesirable but can lead to new cultivars
• occurance of somaclonal variation can be reduced by:
  
  • avoid long term cultures
  • use axillary shoot induction systems where possible (rather than adventitious)
  • propagate chimeras by other clonal systems

Question 13

Tissue Proliferation

Answer 13

• a problem encountered in tissue culture
• formation of a gall-like growths on the stem of some micropropagated plants
  
  • especially ericaceous plants (heath family)
• cause is unknown
  
  • but not pathogen related
• similar gall-like structures have been seen naturally on Rhodendendron and it has been suggested that the tissue culture environment may promote the expression of TP in species or cultivars having this tendency naturally.

Question 14

Transient Phenotypic Expression

Answer 14

• a type of variation in micropropagation plants
• increases vigor
• changes in developmental stage
• increased axillary branching (plant will grow out of it, but initially get nice branched plant)
• these effects are temporary and disappear as the plants continued to grow and/orwere re-propagated

Question 15

Problems encountered in tissue culture:

Answer 15

1. hyperhydricity
2. internal pathogens
3. phenolic exudation
4. shoot-tip necrosis
5. cytokinin carryover
6. habituation
7. tissue roliferation
8. somaclonal variation