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
Somaclonal variation
Embryo rescue
Ovule and ovary cultures
Anther and pollen cultures
Callus and protoplast culture
Protoplasmic fusion
In vitro screening
Multiplication

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

4. Micropropagation

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. Microcutting propagation
It involves the production of shoots from pre-existing meristems only.
Requires breaking apical dominance
This is a specialized form of organogenesis

7. Steps of Micropropagation
Stage 0 – Selection & preparation of the mother plant
sterilization of the plant tissue takes place
Stage I  - Initiation of culture
explant placed into growth media
Stage II - Multiplication
explant transferred to shoot media; shoots can be constantly divided
Stage III - Rooting
explant transferred to root media
Stage IV - Transfer to soil
explant returned to soil; hardened off

8. 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:

9. 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.

10. 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

11. Indirect Somatic Embryogenesis
Explant → Callus Embryogenic → Maturation → Germination
Callus induction
Embryogenic callus development
Maturation
Germination

12. Induction Auxins required for induction Proembryogenic masses form
2,4-D most used
NAA, dicamba also used

13. Development Auxin must be removed for embryo development
Continued use of auxin inhibits embryogenesis
Stages are similar to those of zygotic embryogenesis
Globular
Heart
Torpedo
Cotyledonary
Germination (conversion)

14. Maturation
Require complete maturation with apical meristem, radicle, and cotyledons
Often obtain repetitive embryony
Storage protein production necessary
Often require ABA for complete maturation
ABA often required for normal embryo morphology
Fasciation
Precocious germination

15. Germination May only obtain 3-5% germination
Sucrose (10%), mannitol (4%) may be required
Drying (desiccation)
ABA levels decrease
Woody plants
Final moisture content 10-40%
Chilling
Decreases ABA levels

16. Plant germplasm preservation
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

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

18. 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

19. 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)

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

22. Cryopreservation Requirements
Preculturing
Usually a rapid growth rate to create cells with small vacuoles and low water content
Cryoprotection
Cryoprotectant (Glycerol, DMSO/dimetil sulfoksida, 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

23. 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

25. Somaclonal Variation
Variation found in somatic cells dividing mitotically in culture
A general phenomenon of all plant regeneration systems that involve a callus phase
Some mechanisms:
Karyotipic alteration
Sequence variation
Variation in DNA Methylation
Two general types of Somaclonal Variation:
Heritable, genetic changes (alter the DNA)
Stable, but non-heritable changes (alter gene expression, epigenetic)
The only other source for genetic variation that I am aware of is polyploidy. As a breeder, you have to be aware of epigenetic variation because it can look like a good source to use for breeding, but since it’s not heritable, it can only be useful for vegetatively propagated crops. Sources for epigenetic variation include gene aplification, DNA methylation, and increased transposon activity. Since using somaclonal variation is the same as mutation breeding, I would like to take a look at mutation breeding in more detail.

26. Epigenetic The three main mechanisms for regulation are:
the study of gene regulation that does not involve making changes to the SEQUENCE of the DNA, but rather to the actual BASES within the nucleotides and to the HISTONES
The three main mechanisms for regulation are:
CpG island methylation (…meCGmeCGmeCGmeCGmeCGmeCGmeCGmeCG…)
acetylation and methylation of histone H3
the production of antisense RNA
CpG island methylation:
CpG islands are located in the promoter sequences of about 40% of all genes.
They are strings of ~500 bp of CG repeat. This is very specific methylation of these strings.
CpG islands are recognized by specific enzymes called methylases - they add the methyl group to the 5 position of the cytidines (all of them in the island)
This methylated DNA is recognized by methyl binding proteins, which BLOCK binding of transcription factors to the site (i.e.- it will shut down transcription of that gene!)
Modification of histones:
Previously, we talked about acetylating histones to “open up” the DNA so that transcription can occur.
In epigenetic regulation, we are actively REMOVING histones by the process of HISTONE DEACETYLASES (HDACs)
Methylation of histones can also occur - this PRECLUDES the histones from be acetylated!
Production of antisense RNA:
Antisense RNA can be transcribed and can then BIND to mRNAs and keep them from being translated (think about how the microRNAs worked!)
Production of antisense RNA can also provide steric hindrance for the production of SENSE mRNA (promoter is occluded)

27. 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

28. 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

30. Embryo Culture Uses Rescuing interspecific and intergeneric hybrids
wide hybrids often suffer from early spontaneous abortion
cause is embryo-endosperm failure
Gossypium, Brassica, Linum, Lilium
Production of monoploids
useful for obtaining "haploids" of barley, wheat, other cereals
the barley system uses Hordeum bulbosum as a pollen parent

31. 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)

32. Excision of the immature embryo:
Bulbosum technique
H. vulgare is the seed parent
zygote develops into an embryo with elimination of HB chromosomes
eventually, only HV chromosomes are left
embryo is "rescued“ to avoid abortion
Excision of the immature embryo:
Hand pollination of freshly opened flowers
Surface sterilization – EtOH on enclosing structures
Dissection – dissecting under microscope necessary
Plating on solid medium – slanted media are often used to avoid condensation

33. Culture Medium Mineral salts – K, Ca, N most important
Carbohydrate and osmotic pressure
Amino acids
Plant growth regulators

34. Culture Medium Carbohydrate and osmotic pressure amino acids
2% sucrose works well for mature embryos
8-12% for immature embryos
transfer to progressively lower levels as embryo grows
alternative to high sucrose – auxin & cyt PGRs
amino acids
reduced N is often helpful
up to 10 amino acids can be added to replace N salts, incl. glutamine, alanine, arginine, aspartic acid, etc.
requires filter-sterilizing a portion of the medium

35. Culture Medium natural plant extracts PGRs
coconut milk (liquid endosperm of coconut)
enhanced growth attributed to undefined hormonal factors and/or organic compounds
others – extracts of dates, bananas, milk, tomato juice
PGRs
globular embryos – require low conc. of auxin and cytokinin
heart-stage and later – usually none required
GA and ABA regulate "precocious germination“
GA promotes, ABA suppresses

36. “Wide” crossing of wheat and rye requires embryo rescue and chemical treatment to double the number of chromosomes.
Triticale

37. 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)

38. 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

39. 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.

40. 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

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

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

43. 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

44. 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

45. 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

46. (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

47. 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

48. Spontaneous Fusion
Protoplast fuse spontaneously during isolation process mainly due to physical contact
Intraspecific produce homokaryones
Intergeneric have no importance

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

50. 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

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

52. 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

53. 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

54. 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.

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

57. NEW SOMATIC HYBRID PLANT

58. True in vitro fertilization
A procedure that involves retrieval of eggs and sperm from the male and female and placing them together in a laboratory dish to facilitate fertilization
Using single egg and sperm cells and fusing them electrically
Fusion products were cultured individually in 'Millicell' inserts in a layer of feeder cells
The resulting embryo was cultured to produce a fertile plant

59. In Vitro Fertilization

60. 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)