Plant Biotechnology Flashcards
(35 cards)
traditional plant breeding technique
Selective breeding
eg. by cross-pollination (transferring pollen from one plant to the STIGMA of another plant
drawbacks to traditional selective breeding techniques
long, tedious process (performed by hand) for many thousands of plants
limited by the genetic diversity of parent plants
describe mutation breeding
inducing mutations in seeds or totipotent tissue using X-rays or gammas rays
some mutations produced are not heritable
unpredictable but has produced results, especially in flower breeds
define explant
totipotent tissue (usually a bit where the plant is still growing) transferred to a nutrient medium to grow new plants
often then treated with chemical mutagens
define transgenic plant
a plant into which there has been some foreign gene inserted
Transgene may be from any source, eg. bacteria, insect etc
Major advantage of plant tissue culture
no absolute separation of germline from somatic cells
ie. can manipulate cells in culture then generate entire plant from the cultured cells
where are totipotent plant cells typically located?
in the apical meristem
plant tissues can be cultivated in solid or liquid medium. what are these two media called?
solid: callus culture
liquid: (protoplast) suspension culture
define protoplast
plant cells from which the cell wall has been removed.
usually found in liquid culture.
how is cell differentiation in explants stimulated?
by the addition of nutrients and hormones to the solid/liquid medium
epigenetic change vs genetic change
epigenetic change: affects gene expression without altering the underlying genetic sequence, hence reversible changes. may persist for a few generations but does not permanently alter the cultivar
genetic changes = mutations, permanent alterations of DNA sequences
how are transgenic plants created?
Using the Ti Plasmid (tumour-inducing) of Agrobacterium, a soil bacterium that naturally infects minor wounds in plants.
A specific segment of the Ti plasmid DNA is transferred to the plant cell nucleus via a conjugation-type event
in nature, agrobacterium is attracted to what chemical released when a plant is wounded?
minor wounds release phenolic compounds such as acetosyringone
What is T-DNA?
Transfer DNA, the specific sequence of the Ti Plasmid that is transferred to the plant cell nucleus.
in nature, bacterial T-RNA inserted into the plant chromosome causes genes expressed to produce:
Hormones –> tumour formation
Opines (sugar and amino acid derivatives)
the plant cannot utilise these, the bacteria has hijacked the plant’s metabolic system to produce its food
how have biotechnologists modified the Ti-plasmid for the genetic engineering of plants?
modified Ti plasmid:
- removed genes for hormone and opine synthesis
- removed genes not involved in transferring T-DNA so that these smaller plasmids can be used as E.coli shuttle plasmid
ALSO:
- selectable markers and inducible promotors added
- MCS
(DIAGRAM)
Agrobacterium mediated transformation of plants process
protoplasts/callus cultured with Agrobacterium with Ti plasmid containing modified T-DNA
in plate / flask
plant cells are incubated with the herbicide/antibiotic used as the selectable marker
transformed plant cells induced to produce shoot and root by altering hormone conditions
screen for transgene expression levels
Name an alternate method of getting transgenic DNA into plants
Particle bombardment
how does particle bombardment work to transfer transgenic DNA into plants?
a gun fires DNA-coated microscopic gold particles into leaf disc / callus
Two main mechanisms for the gun: pressurised gas or high voltage discharge systems
beads permeate tough cell walls, non-specific
what is chloroplast engineering? what is the major advantage compared to nuclear transformation? what is one disadvantage?
foreign genes are inserted into the chloroplast of cells, usually by a gene gun.
advantage: transgene expression levels typically very high
as each call has 50-100 chloroplasts, each with 100 copies of the chloroplast genome.
disadvantage: chloroplast derived proteins are not glycosylated
what are example of common selectable markers for DNA segments of the transgene?
npt - neomycin phosphotransferase, an antibiotic
luc gene - luciferase, emits blue light in response to a substrate
How are selectable markers removed from plants? give an example of a system.
using recombinase systems.
eg. Cre/loxP
Cre = site specific recombinase enzyme
loxP = 34 bp sequences that Cre recognises
what is the mechanism by which the Cre/loxP system operates to remove selectable markers in a transgenic plant product?
construct the transgene such that the selectable marker is flanked by loxP sites.
after transformation, Cre enzyme introduced. Recognises loxP, loops the selectable marker and excises it.
MARKED BY A loxP “SCAR”
What is CRISPR?
modern genome editing technique. Ti plasmids / gene guns rely on random insertion.
enables point mutations / insertions / deletions, all markerless
