T18 - Shoot organogenesis from petunia leaves

stubbornnessuglyBiotechnology

Dec 12, 2012 (4 years and 11 months ago)

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Cowles
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Shoot organogenesis from cotyledon explants of watermelon


Watermelon is an important crop, especially in southern states. However, it is susceptible to several
diseases so chemicals and used and the fields are rotated.
This makes it a good
candidate

for genetic
engineering.

Watermelon can be grown from shoots induced on cotyledon explants. They mature in 6
-
10 weeks.

this process has been used to make genetically engineered and polyploid plants.

the first experiment examines differences in sh
oot formation in different areas of the cotyledon.

Not all
areas of an explant have equal regeneration capabilities.

in vitro

germinated seedlings have th
e best
potential in watermelons.

First seeds are soaked for 4
-
15 hours. The outer coat is removed and
the seedling inside is taken out.
Several of these are surface disinfested

and plated on germination media for 5
-
7 days
. The cotyledons
are then cut off

without including the shoot tip as it will inhibit shoot formation.

They are then cut into
smaller piec
es and cultured bottom side

(abaxial)

down.

Afte
r

4 weeks the number of shoots from each
area of the original cotyledon are counted.

The most should come from the base of the cotyledon.

Shoots are then placed on shoot elongation medium for 10
-
21 days. When

they root they can be put
into soil and acclimatized.

I wonder what it is about the base of the cotyledons that make them better at shoot formation.

I really
can't think of anything that would do that. There is so much that we don't understand about
devel
opment.

The genotype of a plant is critical for determining competence for organogenesis.

Thus it is import
ant to
test different cultivars.

The seedli
ngs

if different cultivars

should be prepared as above and cotyledon
bases should be
placed

on media.

After 9
-
12 days they should develop buds along cut edges.

After 4
weeks record the number of explants with shoots or buds.

This data can then be analyzed with an
ANOVA to determine which cultivars were better.

Any shoots can then be removed, cultured on
e
longati
on media and then acclimatized.

Dark pretreatments improve shoot regeneration in several plants including beans, tomato
es, pea
rs and
potatoes. The role of darkness is not understood but it may preserve light sensitive PGRs and also results
in thinne
r cell walls which may allow for more PGR movement.

That's interesting that darkness would have an influence. I guess that seedlings would normally begin
growth underground where i
t's dark, so it makes sense.

I wonder what the advantage of thinner walls
wo
uld be when you're underground. Maybe it's just a mechanism to minimize energy expenditure since

they can't photosynthesize underground.

Seedlings are pre
pared as described above, the only difference being that half of them are cultivated in
the light and
half in the dark.

Basal cotyledon explants are then grown on me
dia, elongated and
acclimatized. Explants from seedlings that were grown in the dark should produce more buds that those
cultured in the light.

Cowles
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Direct and indirect shoot organogenesis fro
m leaves of
Torenia fournieri

Torenia fournieri

is commonly called wishbone flower, bluewings or torenia. It's anthers fuse into a
wishbone shape
. It has been used to study somaclonal variation.

In this exercise we will induce direct
and indirect shoot formation.

Young leaves should be removed from the 2nd and 3rd nodes and disinfested. They should be cut into
pieces and cult
ured abaxial side on the medium.

They can then be cultured

and 21
-
23C un
der 50
-
60
umol/m2*s light

with periodic checking to see how many shoots they are producing.

For direct shoot formation the above procedure should be used and the explants should
be cultured on
direct organogenesis medium.

After 4
-
6 weeks of growth the numb
er of shoots should be counted
. The
shoots can then be placed on MS medium without PGRs for further growth. After 3
-
5 weeks the
plantlets can be transplanted and then acclimatized over 2
-
3 weeks.

Torenia can also produce organogenic callus which has merist
ematically active cells.

The ploidy of these
cells varies dramatically due to a fast division rate, leading to genetic instability which is the
disadvantage of callus.

I didn't know why callus produced so much genetic variation. It makes sense that extreme
ly rapid division
would lead to more errors and thus more variation.

I would be interested to know what processes make
division less accurate when it is performed rapidly, and what phases of mitosis are effected the most.

For indirect shoot formation the p
rocess is the same as above with the explants being cultured on callus
induction medium.

Fresh weight should be calculated on a weekly basis to measure callus formation.

The explants should be
subcultured

on callus induction medium after 3
-
4 weeks.

After a

total of 5
-
6
weeks the callus can be removed and cultured on indirect organogenesis medium.

After an additional 4
-
6 weeks the number of shoots formed can be counted.

Multiple shoots should be produced on each
callus.

T18
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Shoot organogenesis from petunia

leaves

Garden variety petunias have a complex history and are hybrids from several different petunia species.

It is considered a cultigen since it did not originate in the wild.

Petunia is usually propagated by seeds,
but some hybrids have to be propagate
d via cuttings.

I wondered what the difference between and cultivar and a cultigen was. A cultivar is a variety of plant
that is selected for its superior characteristics

and propagated in a manner that preserves them.

A
cultigen is a plant that has been d
eliberately altered by humans via artificial selection
.

Petunia is related to tobacco. It is easy to culture in the lab
, which makes it good for study and lab
exercises.

These exercises will explore the effects of auxin and cytokinin on shoot, root and callus
formation.

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Seedlings should be grown to be used in the lab experiments.
Red flowering varieties should be avoided
since they don't respond well in vitro.

Flies shoul
d be killed since they may introduce pathogens.

The first exercise will examine the effects of PGRs.

While only 1 types of PGR will be added to each
treatment, there will be a low concentration of other endogenous PGRs in the explant.

Thus adding a
PGR jus
t changes the ratio of auxin/cytokinin.

Leaves should be removed from plants and disinfested.
The edges of the leaf should be removed and it should be cut horizontally so each explant contains part
of the central vein.

Thes
e should be placed

abaxial side d
own

on media with different PGRs
.

they should
be cultured in cool fluorescent light

and

should then be monitored for shoot, root and callus formation.

Roots should form on media with auxin, while shoots will form on cytokinin media.

The most callus will
fo
rm on media with both cytokinin and auxin.

It's interesting to me that media with both cytokinin and auxin produce callus. I wonder why this doesn't
happen in the plant since both auxin and cytokinin are present. Perhaps it requires higher level of PGRs
th
an are normally found
in vivo.

The next experiment compares the performance of plants resulting from direct and indirect
organogenesis.

Explants are prepared as above and placed on medium with
cytokinin.

The same is done
for callus.

After 4 weeks any shoot
s formed are removed and cultured on MS medium.

After the shoots
have rooted they are transplanted and acclimatized. Data collected on the number of shoots formed
and the size and appearance of the resulting plants is compared between direct and indirect
o
rganogenesis
.

Much more variability should be observed among the plants obtained via indirect
organogenesis.

Questions

1.

What are 3 factors that influence watermelon cotyledon shoot formation?

2.

What causes the ploidy variation observed in callus?

3.

What is
organogenic callus?

4.

Compare and contrast cultivars and cultigens.

5.

T/F measureable variation should result in plants created via indirect organogenesis.