1. Plant Tissue Culture Application
I plan to go into more technical detail with tissue culture techniques than I do with some of the other molecular biology techniques. The reason is that there is much more breeder support available for molecular biology than for tissue culture techniques, and if you want to apply some of these techniques, you are much more likely to be on your own than you are if you want to apply some of the molecular biology techniques in your breeding program.

2. Development of superior cultivars
Germplasm storage
Embryo rescue
Ovule and ovary cultures
Anther and pollen cultures
Callus and protoplast culture
Protoplasmic fusion
Plant Genetic Engineering

3. Tissue Culture Applications
Micropropagation
Germplasm preservation
Somaclonal variation
Haploid & dihaploid production
In vitro hybridization – protoplast fusion
Plant genetic engineering

5. Features of Micropropagation
Clonal reproduction
Way of maintaining heterozygozity
Multiplication stage can be recycled many times to produce an unlimited number of clones
Routinely used commercially for many ornamental species, some vegetatively propagated crops
Easy to manipulate production cycles
Not limited by field seasons/environmental influences
Disease-free plants can be produced
Has been used to eliminate viruses from donor plants

6. COMPARISON OF CONVENTIONAL & MICROPROPAGATION OF VIRUS INDEXED REGISTERED RED RASPBERRIES
Duration: 6 years 2 years
Labor: Dig & replant every 2 years; Subculture every 4 weeks;
unskilled (Inexpensive) skilled (more expensive)
Space: More, but less expensive (field) Less, but more expensive (laboratory)
Required to
prevent viral Screening, fumigation, spraying None
infection:

7. Ways to eliminate viruses
Heat treatment.
Plants grow faster than viruses at high temperatures.
Meristemming.
Viruses are transported from cell to cell through plasmodesmata and through the vascular tissue. Apical meristem often free of viruses. Trade off between infection and survival.
Not all cells in the plant are infected.
Adventitious shoots formed from single cells can give virus-free shoots.

8. Elimination of viruses
Plant from the field
Pre-growth in the greenhouse
Active
growth
Heat treatment
35oC / months
Adventitious
Shoot
formation
‘Virus-free’ Plants
Virus testing
Meristem culture
Micropropagation cycle

9. Storage of Plant germplasm
In situ : Conservation in ‘normal’ habitat
rain forests, gardens, farms
Ex Situ :
Field collection, Botanical gardens
Seed collections
In vitro collection: Extension of micropropagation techniques
Normal growth (short term storage)
Slow growth (medium term storage)
Cryopreservation (long term storage
DNA Banks

10. In vitro Collection Use : Recalcitrant seeds Vegetatively propagated
Large seeds
Concern:
Security
Availability
cost

11. Ways to achieve slow growth
Use of immature zygotic embryos
(not for vegetatively propagated species)
Addition of inhibitors or retardants
Manipulating storage temperature and light
Mineral oil overlay
Reduced oxygen tension
Defoliation of shoots

12. Storage of living tissues at ultra-low temperatures (-196°C)
Cryopreservation
Storage of living tissues at ultra-low temperatures (-196°C)
Conservation of plant germplasm
Vegetatively propagated species (root and tubers, ornamental, fruit trees)
Recalcitrant seed species (Howea, coconut, coffee)
Conservation of tissue with specific characteristics
Medicinal and alcohol producing cell lines
Genetically transformed tissues
Transformation/Mutagenesis competent tissues (ECSs)
Eradication of viruses (Banana, Plum)
Conservation of plant pathogens (fungi, nematodes)

14. Cryopreservation Steps
Selection
Excision of plant tissues or organs
Culture of source material
Select healthy cultures
Apply cryoprotectants
Pregrowth treatments
Cooling/freezing
Storage
Warming & thawing
Recovery growth
Viability testing
Post-thawing

15. Cryopreservation Requirements
Preculturing
Usually a rapid growth rate to create cells with small vacuoles and low water content
Cryoprotection
Cryoprotectant (Glycerol, DMSO, PEG) 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

16. 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

18. 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 (want as much contact with selective agent as possible)
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

19. Requirements for Somaclonal Breeding
Effective screening procedure
Most mutations are deleterious
With fruit fly, the ratio is ~800:1 deleterious to beneficial
Most mutations are recessive
Must screen M2 or later generations
Consider using heterozygous plants?
But some say you should use homozygous plants to be sure effect is mutation and not natural variation
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

21. Embryo Culture Uses Rescue F1 hybrid from a wide cross
Overcome seed dormancy, usually with addition of hormone to media (GA)
To overcome immaturity in seed
To speed generations in a breeding program
To rescue a cross or self (valuable genotype) from dead or dying plant

22. Haploid Plant Production
Embryo rescue of interspecific crosses
Creation of alloploids
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)

23. Specific Examples of DH uses
Evaluate fixed progeny from an F1
Can evaluate for recessive & quantitative traits
Requires very large dihaploid population, since no prior selection
May be effective if you can screen some qualitative traits early
For creating permanent F2 family for molecular marker development
For fixing inbred lines (novel use?)
Create a few dihaploid plants from a new inbred prior to going to Foundation Seed (allows you to uncover unseen off-types)
For eliminating inbreeding depression (theoretical)
If you can select against deleterious genes in culture, and screen very large populations, you may be able to eliminate or reduce inbreeding depression
e.g.: inbreeding depression has been reduced to manageable level in maize through about 50+ years of breeding; this may reduce that time to a few years for a crop like onion or alfalfa

24. Somatic Hybridization
Development of hybrid plants through the fusion of somatic protoplasts of two different plant species/varieties
Plants bearing genes of two different species or two different genera have been produced by plant breeders since the 1930s. Scientist in the 1930s and still plant breeders today use traditional plant breeding methods to generate these transgenic plants (e.g., protoplast fusion, embryo rescue, mutagenesis, etc). These practices have not found any resistance from the public and have never been controversial outside certain scientific circles.

25. Somatic hybridization technique
1. isolation of protoplast
2. Fusion of the protoplasts of desired species/varieties
3. Identification and Selection of somatic hybrid cells
4. Culture of the hybrid cells
5. Regeneration of hybrid plants

26. Isolation of Protoplast
(Separartion of protoplasts from plant tissue)
2. Enzymatic Method
1. Mechanical Method

27. Mechanical Method Plant Tissue Cells Plasmolysis
Microscope Observation of cells
Release of protoplasm
Cutting cell wall with knife
Collection of protoplasm

28. Mechanical Method
Used for vacuolated cells like onion bulb scale, radish and beet root tissues
Low yield of protoplast
Laborious and tedious process
Low protoplast viability

29. Enzymatic Method Leaf sterlization, removal of epidermis Plasmolysed
cells
Plasmolysed
cells
Pectinase +cellulase
Pectinase
Protoplasm
released
Release of
isolated cells
Protoplasm released
cellulase
Isolated
Protoplasm

30. Enzymatic Method
Used for variety of tissues and organs including leaves, petioles, fruits, roots, coleoptiles, hypocotyls, stem, shoot apices, embryo microspores
Mesophyll tissue - most suitable source
High yield of protoplast
Easy to perform
More protoplast viability

31. (Fusion of protoplasts of two different genomes)
Protoplast Fusion
(Fusion of protoplasts of two different genomes)
1. Spontaneous Fusion
2. Induced Fusion
Intraspecific
Intergeneric
Chemofusion
Mechanical
Fusion
Electrofusion

32. Uses for Protoplast Fusion
Combine two complete genomes
Another way to create allopolyploids
In vitro fertilization
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. Spontaneous Fusion
Protoplast fuse spontaneously during isolation process mainly due to physical contact
Intraspecific produce homokaryones
Intergeneric have no importance

34. Chemofusion- fusion induced by chemicals
Induced Fusion
Chemofusion- fusion induced by chemicals
Types of fusogens
PEG
NaNo3
Ca 2+ ions
Polyvinyl alcohol

35. Induced Fusion
Mechanical Fusion- Physical fusion of protoplasts under microscope by using micromanipulator and perfusion micropipette
Electrofusion- Fusion induced by electrical stimulation
Fusion of protoplasts is induced by the application of high strength electric field (100kv m-1) for few microsecond

36. Possible Result of Fusion of Two Genetically Different Protoplasts
= chloroplast
= mitochondria
Fusion
= nucleus
heterokaryon
cybrid
hybrid
cybrid
hybrid

37. 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

38. Advantages of somatic hybridization
Production of novel interspecific and intergenic hybrid
Pomato (Hybrid of potato and tomato)
Production of fertile diploids and polypoids from sexually sterile haploids, triploids and aneuploids
Transfer gene for disease resistance, abiotic stress resistance, herbicide resistance and many other quality characters
Production of heterozygous lines in the single species which cannot be propagated by vegetative means
Studies on the fate of plasma genes
Production of unique hybrids of nucleus and cytoplasm

39. Problem and Limitation of Somatic Hybridization
Application of protoplast technology requires efficient plant regeneration system.
The lack of an efficient selection method for fused product is sometimes a major problem.
The end-product after somatic hybridization is often unbalanced.
Development of chimaeric calluses in place of hybrids.
Somatic hybridization of two diploids leads to the formation of an amphiploids which is generally unfavorable.
Regeneration products after somatic hybridization are often variable.
It is never certain that a particular characteristic will be expressed.
Genetic stability.
Sexual reproduction of somatic hybrids.
Inter generic recombination.

40. TYPICAL SUSPENSION PROTOPLAST + LEAF PROTOPLAST PEG-INDUCED FUSION

42. NEW SOMATIC HYBRID PLANT

43. Requirements for plant genetic transformation
Trait that is encoded by a single gene
A means of driving expression of the gene in plant cells (Promoters and terminators)
Means of putting the gene into a cell (Vector)
A means of selecting for transformants
Means of getting a whole plant back from the single transformed cell (Regeneration)