PPH-30306
LECTURE 1+2
Totipotent= possibility of organ regeneration. Some animals also share this capacity. Works via
dedifferentiation, zygote can differentiate in different tissues. Dedifferentiation happens in somatic cells.
Signals induce dedifferentiation:
- Abiotic stresses (wound, salt, drought)
- Biotic stresses (virus, pest)
Endogenous signals such as hormone balance. Dedifferentiation starts with genomic reconstructing and
requires chromatin 809. DNA becomes less compact, so DNA comes available. Next steps are redifferentiation
(regeneration), cell death and cell cycle (Callus).
2 types of regeneration:
- Organogenesis
o Formation of new organs (mostly root and shoot)
- Embryogenesis
o Formation of new embryo
Embryonic tissue has high generative potential (many stem cells).
Post embryonic tissue:
- Meristem
- Root pericycle
- Leaf epidermis
- Pollen
- Stem cortex
All somatic cells in vitro can regenerate due to complete control of circumstances. Meristem is virus free as
virus cannot enter the meristem.
Lecture 3+4
thin living tissues (cells) easy to see under microscope. Thick, non-transparent tissues must be treated. How to
immobilize a sample to get it sharp under the microscope?
- Biological; use binding sites like collagen
- Biochemical; use HMW
- Chemical; silane coating
- Physical; bake sections to slides on hot cells
How to remove air?
- Use low pressure (vacuum)
- Use mild soaps
- Use low surface tension solvent.
Use fitting spacers, no compression but as thin as possible.
Processing thick plant samples.
- Structural preservation
o Fixate to minimize artefacts
- Infiltrate and harden prior to sectioning
o Resin (plastic sections)
o Cryo-protectant (cryo-sections)
- Orient sample on microtome and make thin sections
- Attach sections to slides process for specific detection.
,Structural preservation
Chemical fixation
- Alcohol: fast and cheap, proteins denature. Low structural preservation; proteins precipitate.
Extraction occurs.
- Aldehydes: to crosslink proteins to preserve the structure.
- Improve accessibility of fixative. Use non -ionic detergents to lower surface tension
Cryofixation = rapid freezing in liquid propane.
Resin embedding
made of any material with minimal artefacts. Good accessibility for probes into compact tissue. Reproducible
staining’s. Time consuming.
Starch can be stained with lugol.
Fluorescence occurs when low wavelengths are absorbed, and high wavelengths are emitted. Principle:
molecule absorbs energy (gets excited), in fluorescence that electron falls back into the ground state. A photon
of lower energy is emitted (energy loss). This stokes shift allows a difference; emission visible. Jabonski diagram
explained:
- A fluorophore absorbs high energetic photon
- Electron comes temporarily in an excited vibrational energy state
- A photon of lower energy is emitted (back to ground state); fluorescence.
Fluorescence activity is sometimes depicted diagrammatically as shown in Figure 4(a) (termed a Jablonski
energy diagram). Prior to excitation, the electronic configuration of the molecule is described as in the ground
state. Upon absorbing a photon of excitation light, usually of short wavelengths, electrons may be raised to a
higher energy and vibrational excited state, a process that may only take a quadrillionth of a second (a time
period commonly referred to as a femtosecond, 10E-15 seconds). In fluorescence, during an interval of
approximately a trillionth of a second (a picosecond or 10E-12 seconds), the excited electrons may lose some
vibrational energy to the surrounding environment and return to what is called the lowest excited singlet state.
From the lowest excited singlet state, the electrons are then able to "relax" back to the ground state with
simultaneous emission of fluorescent light as illustrated in Figure 4(a). The emitted light is always of longer
wavelength than the excitation light (Stokes Law) and continues so long as the excitation illumination bathes
the fluorescent specimen. If the exciting radiation is halted, the fluorescence ceases.
, Lecture 5 + 6
Multiple peaks in flow cytometry can occur as cells divide so their DNA is doubled. Genetic skewedness due to
selection can occur in genetic mapping. Skewedness is that you have more of one parent than the other parent
in the progeny so not all recombination will be displayed. Genetic skewedness can occur as not all genes
contribute to inducement of haploid. Some genes can be lethal or be lost during inducement of haploidization.
Cybrids, generating chromosome of one parent with cytoplasmic genotype form another parent. Results in new
phenotypes of doubled haploids. To do protoplast fusion, plant must be treated with pectolytic enzymes to get
protoplasts. Enzymes can be cellulase, hemicellulose and pectinase. Hybrids are then created to be used in
combinations in breeding and bypassing sexual barriers. Challenges, ploidy level increases and somatic
incongruity, difficulties in combining plants. Incongruity exists between:
- Nucleus/nucleus; some genes of the parent cannot collaborate
- Nucleus/organelle; organelle have their own genes but also need products of genes encoded by the
nucleus.
Lecture 7+8
Clonal replication with aim for no variation. In vitro breeding goes for maximum genotypic variation.
Variant= differs from the parent in phenotype but alternations at the DNA sequence level either by molecular
or genetic means have not been proven.
Mutant= DNA sequence change has been proven.
Somaclonal variation= genetic and epigenetic variation among cell lines cultures, and plants regenerated from
such cultures.
Genetic changes are changes in DNA sequence; epigenetic changes are changes in expression of genes. Change
in gene expression often are transmitted to daughter cells. Cells can differ in morphology because gene
expression is different between cells. Mitotically transmitted differences between genetically identical plant cell
lines is called epigenetic variation. Epigenetic changes are more stable than physiological changes but less
stable than genetic changes.
The way of packaging DNA with histones affects how accessible the DNA is. As well as the methylation status.
DNA methylation is stable but may disappear (due to stress) and then leads to changes in the expression of
genes. Often in non-transcribed heterochromatic regions of chromosomes. Needed to silence and suppress
transposable elements. Mainly methylation at C next to G. Transposable elements have the potential to be
mutagenic. Use restriction enzymes to determine methylation. It cuts not on a C methylated site so check
bands. Or epigenome sequencing. DNA is treated with bisulfide, unmethylated cytosine is converted to uracil.
Methyl cytosine is not affected so you see differences in sequences.
Cytokinin habituation= growth of callus on media without cytokines. Some tissues do not require cytokinin.
Flow cytometry used to check ploidy. Transposable elements normally silenced but can be activated by DNA
methylation in tissue culture. Transposable beneficial to keep as it increases genetic variation and therefore the
adaptability to environmental changes. Mitotic cross-over (very rare) results in homozygous phenotype, loss of
heterozygosity.
Bisulphite treatment results in that unmethylated cytosine is converted to uracil. Methyl cytosine is not
affected. In a PCR, the U will be copied as T.
Genetic variation in cell cultures”
- Chromosome rearrangements
o Ploidy differences
o Inversions, translocations etc.
o DNA breaks by transportation
- Cytoplasmic changes
o Mitochondrial, chloroplast
- Nuclear encoded changes
o Somatic DNA sequence variation in plants is limited because meristems are genetically stable
However, non-meristematic (differentiated) tissues could contain aberrations. The restart of cell divisions in
previously not dividing tissue often results in chromosomal aberrations.
