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Bio 114 Lecture 7
Plant Biotechnology I
45
Biology
Undergraduate 1
03/06/2013

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Term
plant biotechnology is the application of laboraty based techniques for which two main reasons?
Definition

plant propagation

or

genetic improvement

Term

define tissue culture

 

Definition
the culture of plant organs, tissues, cells or protoplasts on nutrient media under sterile conditions
Term
name 4 applications of plant tissue culture
Definition

micropropagation

suspension cultures

somatic hybridisation

production of transgenic plants

Term
micropropagation. what is it important for?
Definition
it is commercially important for propagating individual plant genotypes where other methods of propagation are difficult
Term
cell suspension cultures involves the growth of small groups of cells in what type of growth medium?
Definition

liquid growth medium

 

these cell suspension cultures are commercially important for large scale synthesis of valuable plant products like antimicrobials, vitamins and food flavours

Term
a plant cell without a wall is also known as a.....
Definition

protoplast.

 

protoplast fusion is used in somatic hybridisation to produce new hybrids between sexually imcompatible plant species

Term
if i had two sexually imcompatible plant species and i wanted to create a hybrid, which application of tissue culture would i use and what method would i apply?
Definition

somatic hybridisation using protoplast fusion.

 

1. protoplasts from 2 species are mixed and induced to fuse making hybrid protoplasts

2. plants are regenerated from hybrid protoplasts by tissue culture

Term
Sharong No.3 (SR3) is a salt and drought resistant wheat. what are the 2 species used to make this hybrid?
Definition

common wheat (Triticum aestivum)

and

Tall Wheatgrass (Agropyron elongatum)

Term
most methods for making transgenic plants involve tissue culture and the regeneration of whole plants from tissue cultures. this process depends on which property possesed by many somatic plant cells?
Definition

Totipotency - this is the ability of an individual cell to divide and form all parts of the mature organism

 

totipotency depends on the capacity for dedifferentiation and redifferentiation.

Term

the first 2 steps invovled in plant tissue culture are 

 

1. isolation of a small piece of plant material (explant)  under sterile condtions

2. callus production on nutrient medium containing plant hormones


what is the final step. name the hormones promoting shoot and roots of plants

Definition

3. Organogenesis - generation of new organs from undifferentiated callus

 

root promoting hormone auxin

shoot promoting hormone cytokinin

Term
in organogenesis whole plants are produced from an undifferentiated callus. How many stages does it take to generate a whole plant?
Definition

Two Stages

 

1. Shoot regeneration (promoted by cytokinin)

2. Rooting of shoot (promoted by auxin)

Term
somatic embyrogenesis is an alternative method of plant regeneration. what does this involve and comment on the degree of ease to carry out this technique
Definition

this involes the development of embryos directly from differentiated or undifferentiated somatic cells


somatic embryogenesis is difficult in most species

Term
what term describes the phenotypic variability between individual plants derived from plant tissue culture?
Definition

somaclonal variation - this is a side effect of plant tissue culture.

 

caused by genetic changes: aneuploidy, polyploidy, chromosome structure and DNA sequence

 

Term
give an advantage and a disadvantage of somaclonal variation
Definition

advantage: additional genetic variability for plant improvement

 

disadvantage: lack of uniformity for horticulture and forestry industries

Term

name the four main steps in generating transgenic plants

 

Definition
  • Production of DNA construct
  • Transformation
  • Selection via selectable marker
  • Regenaration of whole plants from transformed cells
Term

the following are four different kinds of promoters

  • constitutive
  • tissue-specific
  • developmentally regulated
  • inducible

when is a constitutive promoter expressed?

Definition
it is on all the time throughout the plant
Term

the following are four different kinds of promoters

  • constitutive
  • tissue-specific
  • developmentally regulated
  • inducible

when is a tissue-specific promoter expressed?

Definition
in a specific part of the plant for example only expressed in root hairs
Term

the following are four different kinds of promoters

  • constitutive
  • tissue-specific
  • developmentally regulated
  • inducible

when is a developmentally regulated promoter expressed?

Definition
a developmentally regulated promoter is activated/expressed for example during fruit ripening
Term

the following are four different kinds of promoters

  • constitutive
  • tissue-specific
  • developmentally regulated
  • inducible

when is an inducible promoter expressed?

Definition
inducible promoters are only activated when a chemical treatment is applied
Term
what is a reporter gene?
Definition

 a gene attacthed to the regulatory sequence of another gene of interest


for example cell specific promoters direct the expression of Green Fluorescent Protein (GFP) a jellyfish protein in transgenic plants

Term
genes of bacterial origin that have resistant to compounds toxic to plants are known as?
Definition

selectable markers.

 

 

Term
the E. coli gene 'nptII' has antibiotic resistance and is used as the slectable marker in some constructs for plant transformation. What specifically does this gene confer resistance to?
Definition
nptII codes for neomycin phosphotransferase. This inactivates a range of aminoglycoside antibiotics such as kanamycin and neomycin
Term
the herbicide resistance Streptomyces hygroscopicus bar gene is also used as a seleactable marker. which herbicide does this inactivate?
Definition
phosphinothricin (glufosinate) - active ingredient of Basta™ herbicide
Term

getting DNA into plants can be done via two methods : Naked DNA delivery systems and Natural delivery systems.

name the 2 methods used to execute the naked DNA delivery system

Definition

Particle bombardment AKA Biolistics

Electroporation 

 

Naked DNA delivery methods are suitable for dicots (tomatoes, potatoes) and monocots (cereals)

Term

getting DNA into plants can be done via two methods : Naked DNA delivery systems and Natural delivery systems.

 

in which type of plant transformation are Natural delivery systems used and which bacteria is commonly implicated?

Definition

Natural delivery mainly used to transform Dicots but has been used successfuly with some rice (monocot) genotypes

 

the bacteria commonly used is 

Agrobacterium tumefaciens

Term

in naked dna delivery by particle bombardment what are the particles coated in?

how fast are these particles fired at the plant cell?

Definition

particles coated in gold

 

fired through a particle gun at 600m/sec

these particles penetrate cell walls and membranes - dna released and incorporated into the plant chromosomess

Term
in naked DNA delivery the particles are fired at the plant callus, leaf, epidermal strips at high velocity (600m/sec) what helps to accelerate the DNA coated particles?
Definition

gas acceleration tube contains high pressure helium gas 

OR

by electrical discharge

 

the target cell may be callus, whole leaves or epidermal strips

Term

electroporation is usually used with plant p..........s

It involves the use of a short high voltage electrical discharge making PM permeable to DNA and other polar molecules temporarily. the membrane reseas leaving the membrane intact

Definition
protoplasts
Term
Agrobacterium tumefaciens is a plant pathogen that produces tumours in many plant species. name the disease it typically causes in plants
Definition
Crown Gall Disease
Term
A tumefaciens is a natural genetic engineer  because it can transfer a small segment of its DNA into the plant genome. What is the name of this section of DNA and what does it carry?
Definition

the section transferred is the T-DNA.

 

this carries genes that induce uncontrolled cell division

and

direct the synthesis of opines. - these are amino acids the bacterium can use as nutrients but the plant cannot 

Term
where is the T-DNA found?
Definition
on the Ti plasmid (tumour-inducing plasmid) this is ~200kb
Term

T-DNA is bounded by ... regions called the l... and r...... borders (TL and TR)

both borders are flanked by a .....-bp direct repeat sequence

 

the b........ and r........ are the only sequences necessary for ............. into the plant DNA

Definition

two

left

right

25

borders

repeats

intergration

Term
when using T-DNA to transfer genes to a plant where would we insert the genes we wish to transfer to the target plant?
Definition
between the Tl and Tr borders. the regions in between these can be removed and we can insert the genes we wish to transfer to the plant
Term
what is floral dipping? does this require tissue culture?
Definition

in this method flowers are immersed in a suspension of Agrobacterium.

This does NOT require any tissue culture

This is the method of choice for Arabidopis thaliana but not widely aplicable to other species. Transformation of germ line occurs at <0.5%

Term
the generation of new organs from an explant or callus
Definition
organogenesis
Term
the development of embryos directly from somatic cells
Definition
somatic embryogenesis
Term
variability between plants derived from tissue culture
Definition
somaclonal variation
Term
name the 4 types of promoters used in DNA constructs
Definition

constitutive promoter

inducible promoter

tissue-specific promoter

developmentally regulated promoter

Term
a type of gene used to study the activity of a promoter in transgenic plants
Definition
reporter genes
Term
a type of gene used to select for transformed cells or plants
Definition
selectable marker
Term
name two techniques used for delivering naked DNA to cells
Definition
biolistics and electroporation
Term
Agrobacterium tumefaciens causes?
Definition
Crown Gall Disease
Term
the region of the Ti plasmid that is transferred to the plant is the ?
Definition
T-DNA
Term
A simple method for transforming some plants without tissue culture is?
Definition
Floral Dipping
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