General Considerations

onwardhaggardBiotechnology

Dec 12, 2012 (4 years and 9 months ago)

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Feb
28 Kim


T19 Nonzygotic embryogenesis


Gray defines an embryo as “the earliest recognizable multicellular stage of an individual
that occurs before it has developed the structures or organs characteristics of a given species.” In
many organisms, embryos are in between gametophytes and sporophyt
es. Plants are distinctive
because both zygotic and nonzygotic embryos can from.


Zygotic and nonzygotic embryos are functionally equivalent. Nonzygotic embryogenesis
has many applications, including introducing desirable characteristics and effective pro
pagation.


In vivo vs. in vitro growth conditions


Under normal circumstances,
embryos are surrounded by tissues that nourish as they
develop. Zygotic embryos get nutrition from the suspensor or endosperm. Nonzygotic embryos
develop without a coat of nutri
ents, and thus do not have a specific nutrition regiment. Usually,
the suspensor is the only source of nutrition, showing that the endosperm is not crucial for
embryogenesis and germination.


Common attributes of embryogenic culture protocols


The genotyp
e and explants chosen is the first step in embryogenesis. Age and
environmental features also play a role in success. Culturing in the dark may be needed to
prevent the effect of light (which may negatively impact cell growth). Dark culture represses
tissu
e differentiation and inhibits too fast germination.


Origin of nonzygotic embryos


Zygotic and nonzygotic embryos come from a single cell (instead of from a cell mass
through budding). This is important because genetic engineering can create a modified pl
ant,
whereas engineering in a cell in a bud would create a chimeric plant.


Obviously, nonzygotic embryos from isolated cells grow from single cells. It is harder to
tell, though, when nonzygotic embryos come from complex explants.


Initiation of embryoge
nic cells


In more developed explants, nonzygotic embryos will only from young tissues. There are
several pathways in which nonzygotic plant cells become embryos.


The instructions for embryogenesis are found in the cell and are controlled by intrinsic
fa
ctors. This fact is supported by the fact that somatic cells that have been isolated can become
embryos.


Inductive plant growth regulators


Auxins and cytokinins are needed to start embryogenesis. Auxins are thought to initiate
differential gene activati
on. Auxins are not always required when the explant is made up of
embryonic cells. This is thought to be because an induction step is not required when embryonic
cells are present.


Embryo development


The change from nonembryo to embryo may be seen when

the progenitor cell divides
unequally, creating a large vacuaolate cell and the smaller embryogenic cell. The embryogenic
cell will then continue to irregularly divide, or divide systematically to form a somatic embryo.

Feb
28 Kim


Unfortunately, nonzygotic embryos h
ave a tendency to differ from a normal growth pattern by
forming callus, germinating too early, or undergoing secondary embryogenesis.


The sequence of embryo development is: cell division, enlargement, and differentiation

(starts with embryonic vasculature development)
.


A major physical difference between nonzygotic and zygotic embryos is that zygotic
embryos are compressed and flattened due to the seed coat. Nonzygotic embryos have a
tendency for more abnormal developmen
t. They also tend to develop asynchronously, have
structural anomalies, and underdeveloped apical meristems
.




I wonder if it will be possible to replicate the exact in vitro growing environment in the lab, thus
increasing normal development.



Embryo
maturation



Another problem is that nonzygotic embryos tend not to mature properly. This problem
can be mitigated by medium conditions. High sucrose levels, times abscisic acid pulses, and
polytheylene glycol have been found to improve maturation.


Quiesc
ence and dormancy


One of the biggest differences is that nonzygotic embryos do not have a quiescent resting
phase.


Embryo germination and plant development


Obtaining plants from nonzygotic embryogenesis is quite difficult; the plant recovery is
0
-
50%,

compared to over 90% for zygotic embryogenesis. Careful thought to culture conditions
and nutrition has increased plant recovery to nearly the same levels as zygotic embryos.


Uses for embryonic cultures


Embryonic cultures have 3 main purposes: study pla
nt development, genetic engineering,
and economical propagation.


Synthetic seed technology


Synthetic seed
: an engineered somatic embryo that is meant to practically benefit
commercial plant production


This technology may be useful in areas where labor c
osts for culture and rooting are high,
such as ornamental plants.


Conclusion


Nonzygotic embryogenesis shows cell totipotency and conserved developmental
instructions.
This lab technique allows entire plants to be grown from altered genomes.


T20
Embryogenic callus and suspension cultures from leaves of orchardgrass


The family Poacae is the most economically important plant, including corn, oats, rice,
wheat, and forage grasses. Somatic embryogenesis is the favored approach to regeneration of this

family. The experiments will use orchardgrass, a forage grass used for making hay.


General considerations

Feb
28 Kim


Growth of plants


Embryogen
-
P is the plant to use. Keep plants in pots with potting mix and fertilizer.
Plants can be split and moved to new pots be
fore becoming root bound.


Culture media


Use the Schenk and Hildebrandt basal salt mixture, with some additions (see page 192).


Exercises

Experiment 1 Culture establishment


The plant body of orchardgrass is made up of tillers. New tillers form from the base of
earlier tillers through adventitious budding. The most meristemically active areas are the base of
the inner leaves.

Procedure 20.1

1.

Remove a basal stems/leaves

2.

Separat
e leaves and keep the innermost and second most innermost leaves and cut
longitudinally. Surface disinfect.

3.

Cut into sections and place on petri dish.


Anticipated results


Normal contamination rate is 10%, though up to 50% contamination may be seen.


Exp
eriment 2 The gradient embryogenic response


Embryogenic response depends on leaf position in the tiller. There are 2 possible
responses: (1) development of embryogenic callus
(wet)
or (2) somatic embryo growth from leaf
.

Procedure 20.2

1.

Plate leaf sections

as outlines above, identify innermost and outermost leaves

2.

Culture and count # sections from each location that has embryogenic callus and direct
embryos every 8 weeks


Anticipated results


At 4
-
6 weeks, basal sections will have embryogenic callus. As l
eaf sections are more
distal, there will be a lesser callus response.


Experiment 3 Maintenance of embryogenic callus


Orchardgrass can be maintained by transferring to fresh medium every month.

Procedure 20.3

1.

Dissect callus

2.

Separate dry and wet callus an
d tissue by type and place 5 clumps/petri dish.

3.

Incubate for 4 weeks in the dark.

4.

Determine type of callus formed


Anticipated results


In transferring callus, only select areas that are making somatic embryos. There will be a
variety of callus types
produced during reculturing.


Experiment 4 Observations on monocotyledonous somatic embryos

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28 Kim



Most of the embryos produced by orchardgrass have a scutellum and coleoptiles. Some
physical details can be seen with a microscope.

Procedure 20.4

1.

Observe embryo
s and find the scutellum and coleoptiles.

2.

Observe the most common abnormal embryos.


Anticipated results


The embryos should have a scutellum and a notch. The notch is where the coleoptiles
develops from.


Experiment 5 Somatic embryo germination and plant

recovery

Procedure

20.5

1.

Tansfer somatic embryos to medium and incubate.

2.

Look for germination daily. At 2, 4, 6, and 8 days count

# shoots/roots. Find the
coleoptiles

3.

Count # shoots/callus


Anticipated results


Germination is induced by moving embryos to
medium that lack dicamba.

Most of the anticipated results in this chapter don’t really have results, they are more a further
explanation of the procedure.


Experiment 6 Initiation and manipulation of embyrogenic suspension cultures


Suspension cultures are

developed by growing callus in liquid mediu,.

Procedure 20.6

1.

Place growing callus into medium and incubate for 2 weeks.

2.

Add more medium and incubate

3.

Maintain cultures by pouring half of each culture into new flasks

4.

For embryo development, transfer half th
e culture flasks to new SH30 media and the
other half to SH30
-
C media.

5.

After 4 weeks of growth, decant and remove culture mass.

6.

Place small amounts of culture mass onto medium and incubate.


Anticipated Results


There will be proliferation but no somatic e
mbryos unless there is a nitrogen source such
as casein hydrolysate. Only the tissue from SH30
-
C media should show embryos that become
plants, showing that only media with casein hydrolysate can cause somatic embryo development.


Experiment 7 Encapsulatio
n of somatic embryos to produce synthetic seeds


Somatic embryogenesis is the most efficient way to propagate vegetative plants because
it can be scaled
-
up and has efficient plant multiplication.

Procedure 20.7

1.

Harvest somatic embryos and select mature e
mbryos

2.

Treat embryos with alginic acid. For a control, place some on medium without treatment.

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28 Kim


3.

Pipette one embryo at a time into a CaCl2 solution. Transfer the resulting calcium
alginate containing embryo beads to a liquid SH0 solution. Place beads on soli
dified SH0
medium and incubate.


Anticipated Results


After dropping the embryo into CaCl2, a bead should surround the embryo. The alginic
acid is converted to calcium alginate, creating a synthetic seed coat to offer protection.


T21 Somatic embryogenesis from seeds of melon


Melons have hundreds of cultivars. They are at risk for a variety of diseases, and native
resistance is not enough. Genetic engineering is thought to be a solution.


General Considerations

Seed sources


Purchase a pound of seeds outlined on page 206.

Preparation of explants


Imbibe seeds before dissection.
Then remove seed coat and remove embryos. Surface
disinfect embryos.


Culture media


The medium used is “Murashige and Skoog Basal Medium with Sucrose
and Agar,”
which can be ordered. Embryo initiation (EI) and embryo development (ED) media are also
needed.



Exercises

Experiment 1 Effect of explant type on somatic embryogenesis


Embryogenesis only occurs from certain areas of the melon. This experimen
t will
demonstrate the optimal site for embryogenically competent cells.

Procedure 21.1

1.

Dissect zygotic embryos. Place on petri dish of EI medium. Maintain in darkness.

2.

Check for contamination.

3.

After 2 weeks, transfer to ED medium.

4.

After 3 and 5 weeks on E
D medium, count # explant type that produced a somatic
embryo.


Anticipated results


On EI medium, the explants will grow to several times their staring size. If there is a
change in color to green, the needed dark conditions were not met.


On ED medium,

explants will continue to grow and become green. Callus will start to
develop and somatic embryos will be seen around 4 weeks.



Experiment 2 Effect of genotype on somatic embryogenesis


This experiment studies 3 different genotypes to understand effect

of genotype on
embryogenesis.

Procedure 21.2

1.

Dissect embryos and culture only basal cotyledon explants.

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28 Kim


2.

Place on EI medium and incubate in dark.

3.

At 3 and 5 weeks, find the difference in embryogenesis among the genotypes.


Anticipated results


There will be big differences, with clear differences in embryogenesis.


Experiment 3 Embryogenic culture maintenance

Procedure 21.3

1.

Place basal cotyledon explants, callus, or somatic embryos onto petri dishes of EI

and ED

2.

Look at differences in growth
and embryogenesis at week 2. Transfer to fresh medium.
Repeat once more.

3.

Compare embryogenic response


Anticipated results


Cultures on ED medium produce a few plants or dies. Cultures on EI medium grow as
callus, but number of embryos decreases. There wil
l also be abnormal changes in morphology.
The underlying reasons for this are unknown.


Experiment 4 Observation and categorization of embryonic stages


The melon has a rapid embryogenic response, making it a good say to study embryo
stages.

Procedure 21.
4

1.

Choose cultures from previous experiments that have been responsive.

2.

After transferring to ED medium, observe 2x/week. Focus on explants with the most
somatic embryos.

3.

Sketch the morphology and location of embryos on the explants. Every week, redraw.

4.

Ide
ntify the embryonic stages (globular, heart
-
shaped, torpedo, and cotyledonary
embryonic).

5.

See which embryonic stage has the most plant development.



Anticipated results


After finding responsive explants, you will see normal and abnormal embryogeneis. Al
l
the stages of embryogenesis can be seen.


Questions

1.

How do zygotic and nonzygotic embryos differ in growth conditions?

a.

Zygotic: surrounded by nutritive tissues; nonzygotic: develops without the coat,
usually the suspensor is the only nutrition source

2.

The

instructi
ons for embryogenesis is contained and controlled by external factors. T or
F?

a.

F

3.

Name 3 differences in development between nonzygotic and zygotic embryos.

a.

Nonzygot
ic lack a resting phase and tend not to mature properly zygotes are
compressed and
flattened because of their seed coat

4.

Zygotic and nonzygotic embryos are functionally equivalent, T or F?

a.

T

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28 Kim


5.

What is the range of plant recovery from nonzygotic embryogenesis?

a.

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