Plant Tissue Culture - Workforce 3 One

kissimmeemisologistBiotechnology

Dec 14, 2012 (4 years and 8 months ago)

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Plant Tissue Culture

Laboratory Exercise


Seed
germination of Bean and Corn

What is asexual plant propagation?



Identical offspring are produced by a single
parent


Continuous sexual reproduction


risk
producing plants that have lost desirable traits
and gained unwanted ones


Introduction to Plant Propagation



Asexual reproduction
by
Cuttings



Cuttings are pieces of stems, leaves or roots
placed in appropriate media


vermiculite,
sand, perlite, potting soil or water



Existing cells in growing tissue
will develop
into missing roots, stems or leaves



Introduction to Plant Propagation


Asexual reproduction
by
Tissue Culture



One or few cells


excised from parent
plant


placed in a media
containing nutrients



Cells will divide to
produce


Callus


Callus


produces stems,
roots and leaves


P
lant tissues of interest to
Biotechnologists


Meristematic

tissue


Are regions of cell
division


dividing and
differentiating


Tissue culture uses
parts


meristematic

tissues


W/o


no plant tissues
or organs can be
produced


Where are meristems present?


Found in growing tips of
plant



Root tips, Shoot tips,
branch tips, flower and
leaf buds



Vascular cambium


Width of plant

Cloning of Plant Tissue Culture


Few cells or piece of plant are grown in sterile
media


agar



Sugar, vitamins and hormones



Optimum growing
conditions


Differentiation

into shoots, leaves and roots
of a new plant

Cloning of Plant Tissue Culture


Shoots and roots are
growing


transplanted
into soil or another
medium



Transplanted PTC


humidity must be high
-

to avoid dehydration

Hormone function in plants


Hormones/Plant growth regulators


regulate
seed germination + growth of shoots, leaves,
roots and flowers



Produced in root and shoot meristems



Two groups


auxins

and
cytokinins

Hormone function in plants


Auxin



produced in shoot
tips

IAA



Causes
cell elongation,
leaf development and
root tip elongation



Synthetic
auxins



1
-
naphthaleneacetamide,
2,4
-
D herbicide (death of
plants)



Hormone function in plants


Cytokinins


cause cell division
-

Zeatin


Commercial version


Kinetin



Ratio of auxin to cytokinin important in both
stem and root elongation



Ethylene


controls fruit ripening and leaf
development

Hormone function in plants


Gibberellin



seed, leaf bud germination, stem
elongation and leaf development



Abscisic acid (ABA)


regulates bud
development and seed dormancy



Phytochrome



pigment controls flowering

Advantages of PTC


More

plantlets in
less

time



Clones

of parents with
no unwanted

variant
offspring



Plants
difficult to be grown by seed
germination or stem or leaf cuttings


Example


orchids

takes five years

Factors to consider in PTC


The
species

and
variety

of plant material



The
media

and
media ingredients



Preparation of
plant samples, media and
equipment

(
sterility and temperatures
)



Done in laminar flow hoods

Plant Genetic Engineering


Agrobacterium
tumefaciens


crown
gall disease

in plants


Ti plasmid (tumor
-
inducing) has t
-
DNA
(transfer DNA)


Tumor inducing genes
and growth hormones
on tDNA


Plant Genetic Engineering


Ti crown gall inducing plasmid is 200 Kb in
length


Bacterial plasmid


5
-
8 Kb


Scientists remove oncogenes and replace
with useful genes


genetic engineering


Virulence genes (vir) controls tranformation

-

Directs transfer of t
-
DNA from plasmid into
plant chromosomal DNA


Genetically engineered Ti plasmid


pBI121


Reporter
genes


GUS gene codes for ß
-
glucuronidase


Breaks down
carbohydrates


5
-
bromo
-
4
-
chloro
-
3
-
indolyl
-
beta
-
D
-
glucuronic acid (X
-
Gluc
)
-

blue precipitate

Agrobacterium tumefaciens


In plant transformation


bacteria do not
enter into target cell


Stay outside and insert Ti plasmid



Incorporated into chromosomal DNA



Transcription and Translation


This project is funded by a grant awarded under the President’s Community Based Job Training Grant as implemented by the
U.S. Department of Labor’s Employment and Training Administration (CB
-
15
-
162
-
06
-
60). NCC is an equal opportunity
employer and does not discriminate on the following basis:


against any individual in the United States, on the basis of race, color, religion, sex, national origin, age disability, pol
iti
cal
affiliation or belief; and


against any beneficiary of programs financially assisted under Title I of the Workforce Investment Act of 1998 (WIA), on the
basis of the beneficiary’s citizenship/status as a lawfully admitted immigrant authorized to work in the United States, or hi
s
or her participation in any WIA Title I
-
financially assisted program or activity.


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