1. Other Plant Tissue Culture Topics

2. Somaclonal Variation
Somaclonal variation is a general phenomenon of all plant regeneration systems that involve a callus phase
There are two general types:
Heritable, genetic changes (alter the DNA)
Stable, but non-heritable changes (alter gene expression, epigenetic)
With or without mutagen

3. Somaclonal/Mutation Breeding
Advantages:
Screen very high populations (cell based)
Can apply selection to single cells
Disadvantages:
Many mutations are non-heritable
Requires dominant mutation (or double recessive mutation); most mutations are recessive
Can avoid this constraint by not applying selection pressure in culture, but you lose the advantage of high through-put screening –have to grow out all regenerated plants, produce seed, and evaluate the M2

4. Somaclonal Breeding Procedures
Use plant cultures as starting material
Idea is to target single cells in multi-cellular culture
Usually suspension culture, but callus culture can work
Optional: apply physical or chemical mutagen
Apply selection pressure to culture?
Target: very high kill rate, you want very few cells to survive, so long as selection is effective
Regenerate whole plants from surviving cells

5. Requirements for Somaclonal Breeding
Effective screening procedure
Most mutations are deleterious
Most mutations are recessive
Must screen M2 or later generations
Consider using heterozygous plants?
Haploid plants seem a reasonable alternative if possible
Very large populations are required to identify desired mutation:
Can you afford to identify marginal traits with replicates & statistics? Estimate: ~10,000 plants for single gene mutant
Clear Objective
Can’t expect to just plant things out and see what happens; relates to having an effective screen
This may be why so many early experiments failed

6. Introduction into suspension
Sieve out lumps
1 2
Initial high
density +
Subculture
and sieving
Plate out

7. Synchronization Cold treatment: 4oC
Starvation: deprivation of an essential growth compound, e.g. N →accumulation in G1
Use of DNA synthesis inhibitors: thymidine, 5-fluorodeoxyuridine, hydroxyurea
Colchicine method: arresting the cells in metaphase stage, measured in terms of mitotic index (% cells in the mitotic phase)

8. Selection Select at the level of the intact plant Select in culture
single cell is selection unit
possible to plate up to 1,000,000 cells on a Petri-dish.
progressive selection over a number of phases

9. Selection Strategies
Positive
Negative
Visual
Analytical Screening

10. Positive selection
Add into medium a toxic compound e.g. hydroxy proline, kanamycin
Only those cells able to grow in the presence of the selective agent give colonies
Plate out and pick off growing colonies.
Possible to select one colony from millions of plated cells in a days work.
Need a strong selection pressure - get escapes

11. Negative selection
Add in an agent that kills dividing cells e.g. chlorate / BUdR.
Plate out leave for a suitable time, wash out agent then put on growth medium.
All cells growing on selective agent will die leaving only non-growing cells to now grow.
Useful for selecting auxotrophs.

12. Visual selection
Only useful for colored or fluorescent compounds e.g. shikonin, berberine, some alkaloids
Plate out at about 50,000 cells per plate
Pick off colored / fluorescent-expressing compounds (cell compounds?)
Possible to screen about 1,000,000 cells in a days work.

13. Analytical Screening Cut each piece of callus in half
One half subcultured
Other half extracted and amount of compound determined analytically (HPLC/ GCMS/ ELISA)

14. Targets for Somaclonal Variation
Herbicide resistance and tolerance
Specific amino acid accumulators
Screen for specific amino acid production
e.g.Lysine in cereals
Abiotic stress tolerance
Add or subject cultures to selection agent–e.g.: salt, temperature stress
Disease resistance
Add toxin or culture filtrate to growth media

15. T-DNA Tagging

17. Embryo Culture

18. Embryo Culture Uses Rescue F1 hybrids from wide crosses
Overcome seed dormancy, usually with addition of hormone (GA) to medium
To overcome immaturity in seeds
To speed generations in a breeding program
To rescue a cross or self (valuable genotype)

20. Haploid Plant Production
Embryo rescue of interspecific crosses
Bulbosum method
Anther culture/Microspore culture
Culturing of anthers or pollen grains (microspores)
Derive a mature plant from a single microspore
Ovule culture
Culturing of unfertilized ovules (macrospores)

21. Androgenesis
History
1964, 1966 Datura innoxia (Guha and Maheshwari)
1967 Nicotiana tabacum (Nitsch)
Critical factor - change in developmental pattern from mature pollen to embryogenesis

22. Factors influencing androgenesis
Genotype of donor plants
Anther wall factors
Culture medium and culture density
Stage of microspore or pollen development
Effect of temperature and/or light
Physiological status of donor plant

27. H. Bulbosum chromosomes eliminated
Bulbosum Method
Hordeum bulbosum
Wild relative
2n = 2X = 14
Hordeum vulgare
Barley
2n = 2X = 14 X ↓
Embryo Rescue
Haploid Barley
2n = X = 7
H. Bulbosum chromosomes eliminated
This was once more efficient than microspore culture in creating haploid barley
Now, with an improved culture media (sucrose replaced by maltose), microspore culture is much more efficient (~2000 plants per 100 anthers)

28. Ovule Culture Haploids can be induced from ovules
The number of ovules is less and thus is used less than anther culture
May be by organogenesis or embryogenesis
Used in plant families that do not respond to androgenesis
Liliaceae
Compositae

29. Haploids Typically weak, sterile plant
Usually want to double the chromosomes, creating a dihaploid plant with normal growth & fertility
Chromosomes can be doubled by
Colchicine treatment
Spontaneous doubling

30. Protoplasts Created by degrading the cell wall using enzymes
Very fragile

31. Protoplasts can be induced to fuse with one another:
Electrofusion: A high frequency AC field is applied between 2 electrodes immersed in the suspension of protoplasts- this induces charges on the protoplasts and causes them to arrange themselves in lines between the electrodes. They are then subject to a high voltage discharge which causes their membranes to fuse where they are in contact.
Polyethylene glycol (PEG): causes agglutination of many types of small particles, including protoplasts which fuse when centrifuged in its presence
Addition of calcium ions at high pH values

32. Uses for Protoplast Fusion
Combine two complete genomes
Another way to create allopolyploids
Partial genome transfer
Exchange single or few traits between species
May or may not require ionizing radiation
Genetic engineering
Micro-injection, electroporation, Agrobacterium
Transfer of organelles
Unique to protoplast fusion
The transfer of mitochondria and/or chloroplasts between species

33. = chloroplast
= mitochondria
Fusion
= nucleus
heterokaryon
cybrid
hybrid
cybrid
hybrid

34. Identifying Desired Fusions
Complementation selection
Can be done if each parent has a different selectable marker (e.g. antibiotic or herbicide resistance), then the fusion product should have both markers
Fluorescence-activated cell sorters
First label cells with different fluorescent markers; fusion product should have both markers
Mechanical isolation
Tedious, but often works when you start with different cell types
Mass culture
Basically, no selection; just regenerate everything and then screen for desired traits

35. Germplasm Preservation
Extension of micropropagation techniques:
Two methods:
1. Slow growth techniques
↓Temp., ↓Light, media supplements (osmotic inhibitors, growth retardants), tissue dehydration, etc
Medium-term storage (1 to 4 years)
2. Cryopreservation
Ultra low temperatures. Stops cell division & metabolic processes
Very long-term (indefinite?)

36. Why not seeds? Some crops do not produce viable seeds.
Some seeds remain viable for a limited duration only and are recalcitrant to storage.
Seeds of certain species deteriorate rapidly due to seed borne pathogens.
Some seeds are very heterozygous not suitable for maintaining true to type genotypes.
Effective approach to circumvent the above problems may be application of cryopreservation technology.

37. Cryogenic explants: Undifferentiated plant cells Embryonic suspension
Callus
Pollen
Seeds
Somatic embryos
Shoot apices

38. Cryopreservation
Requirements: •Preculturing–Usually a rapid growth rate to create cells with small vacuoles and low water content •Cryoprotection–Glycerol, DMSO, PEG, etc…, to protect against ice damage and alter the form of ice crystals •Freezing–The most critical phase; one of two methods: •Slow freezing allows for cytoplasmic dehydration •Quick freezing results in fast intercellular freezing with little dehydration

39. Cryopreservation Requirements
Storage
Usually in liquid nitrogen (-196oC) to avoid changes in ice crystals that occur above -100oC
Thawing
Usually rapid thawing to avoid damage from ice crystal growth
Recovery
Thawed cells must be washed of cryo-protectants and nursed back to normal growth
Avoid callus production to maintain genetic stability

49. Synthetic Seeds