MCDB 425
Terms
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- Father of tissue culture
- Haberlandt
- Parts of plant that can be used in tissue culture without growth factors
- meristem (root tip, apical bud)
- Dedifferation requires
- growth factors (auxin, cytokinin)
- Dedifferentiated cells require (to survive/divide)
- food; can’t synthesize their own
- Medium that is used in tissue culture
- MS0 (MS zero) after Murashige and Skoog
- Auxins
- rooting hormone, signals to reform damaged parts of plants, inhibits lateral bud growth
- Cytokinins
- stimulate cell division, shoot initiation, chloroplast development, inhibits roots, stimulates lateral bud growth
- Micropropagation
- the production of whole plants from small sections of a plant called an explant (as opposed to plant tissue culture)
- Totipotency
- the ability of a single cell to express the entire genetic potential of the DNA of that cell
- Parenchyma cells
- non-meristematic cells
- (2) Forms of microprapagation
- callus production, microcuttings
- Micropropagation (4 steps)
- establish explants, including auxins and cytokinins, transfer and multiply, add auxins for rooting, then acclimatization to climate (grown in 100% humidity)
- Hardening off
- placing the plantlet from micropropagation into a soilless media with mist and placing a cover over it
- To kill viruses in tissue culture
- leave plants in growth chamber for 3-6 weeks at 100 degrees F
- Subculturing
- transferring a tissue to a new culture
- Adventitious shoot formation
- uses explants which lack meristems or buds
- used to regenerate difficult to regenerate trees (conifers) – use fascicle sheath
- Cotyledons and hypocotyls
- Colchicine
- doubles chromosome to dihaploid
- Microspore culture
- uses haploid plants to dihaploid, allows recessive traits to be expressed, fastest way to find new plant characteristics
- Somatic embryogenesis
- formation of an embryo from vegetative cells
- Somaclonal variation
- mutations arising in cells during tissue culture
- Protoplast
- plant cell that had its wall removed
- EMS
- a native plant chemical that causes SNPs
- Removal of cell wall of plants possible because of
- macerase and cellulysin
- Somatic hybridization
- fusion of two protoplasts to form one plant (used often with potatoes)
- VirA
- senses plant cell
- Vir region
- codes for vir proteins
- T-DNA
- DNA to be transferred, can be changed for use in agriculture
- VirE2
- protein that wraps all but the front of the T-DNA for transport and protection (does not enter nucleus)
- VirD2
- protein that covers the front of the T-DNA
- VirB channel
- like sex pilus, allows T-DNA through to host cell
- ATkapα
- assists nuclear transport of T-DNA by opening nuclear pore (native, normal function), binds to VirD2 only
- VIP1
- binds to VirE2, leads T-complex through cell to nucleus (native, normal function) – made to be overexpressed in plants so that they are more susceptible to genetic transformation by agrobaterium
- VirF
- regulatory protein, responsible for increasing or decreasing host range, binds to VIP1
- BiFC
- assay to detect whether two proteins interact, combine to fluoresce if they do (also yeast-two hybrid assay), can also be used with different colors to localize functions and to show the overlap or lack of overlap of proteins
- Preferred site of T-DNA integration
- Double stranded breaks (DSBs)
- KU-80
- a mutation that stabilizes agrobacterium mutations (mediates T-DNA integration)
- GUS
- a reporter gene that causes its cell to fluoresce green
- Biggest issue in biotechnology of introducing genes
- not introducing the genes, but controlling the breaks in the chromosomes such that the genes went where you wanted them to go
- IPTG
- causes constitutive expression of the lac operon
- X-Gal
- used for blue/white plating in yeast to detect presence of insertion in the lacZ gene (blue = no insertion, white = disruption of gene)
- Isoschizomers
- enzymes recognizing the same (cut) sites
- Ethidium bromide
- used as stain to detect DNA, causes it to fluoresce under UV light (in SDS-PAGE)
- Plasmids of note (4)
- 1. pSC101 2. Co1 E1 3. PBR322 4. pEGFP-C1
- pSC101
- original low copy number plasmid
- Co1 E1
- original high copy plasmid
- PBR322
- modified with amp resistance, tet resistance and replication region from pMB1 (made of three plasmids) – common until recently
- Use high copy plasmids for
- DNA replication
- Use low copy plasmids for
- protein expression (ribosome saturation)
- Amount that can be cloned into expression vector plasmids
- 5-10kb
- α-complementation
- when the LacZ gene is split between the expression vector, which has the n-terminus of the gene and is put into lacZ’M15 E. coli, which has the c terminus of the lacZ – doesn’t complement if insertion is present, still use X-Gal to detect insertion, white is still positive allows more room in the plasmid for insertion, or increases plasmid replication speed
- Three steps of PCR:
- 1. Denaturation 2. Annealing 3. Extension
- Obstacles of pEGFP-C1
- it methylates Xba1 and Bcl1 for proofreading purposes, degrades quickly without it, so customer must produce plasmid in mutant DNA without mismatch repair methlyation
- To produce DNA without methlyation
- – use dam- E. coli
- Kozak consensus site
- site at which ribosome prefers to bind to start translation – ACCAUGG
- Why use plant tissue culture (6)
- 1. Producing a clone plant 2. Expediting plant breeding 3. Propagating ‘sterile’ plants 4. Producing natural products in sterile conditions 5. Cleaning plants from pathogens 6. Producing transgenic plants
- Functions of cytokinins (6)
- 1. Stimulates cell division 2. Stimulates shoot initiation 3. Stimulates leaf expansion by cell enlargement 4. Stimulates chloroplast development 5. Stimulates lateral bud growth 6. Inhibits root development
- Advantages to somatic embryogenesis (5)
- 1. Haploid plantlets from anther culture 2. Get plantlets from genetically modified single cells 3. Get plantlets that exhibit mutations – somaclonal variation 4. Germplasm preservation 5. Make synthetic seeds
- Three stages in embryo development
- 1. Globular stage (small, round) 2. Heart stage (heart shaped) 3. Mature stage (cylindrical)
- 5 important features in recombinant E. coli strains
- 1. No recombination 2. No DNA transfer between bacteria 3. Resistance to viruses 4. Tolerant to artificial plasmids 5. No antibiotic resistance