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					Rozaleth Role           Tissue Culture           Assignment 12           Mar. 3, 2010

T22 – Somatic Embryogenesis from Mature Peanut Seed
Peanut is a crop that has many uses and it is also important as an economic source for countries.
However, because peanuts grow under the ground, it is very susceptible to pests and diseases. By
using tissue culture, it is aimed that the peanuts can develop a trait that would make it resistant to
those diseases and pests that affect it. With somatic embryogenesis, peanuts have been regenerated
in big numbers.

With experiments as examples, the chapter introduces the concepts on how explants can affect
embryogenesis, how auxins affect embryo induction and development, how can form of the embryo
be modified by environmental factors and how the embryos germinate and acclimatize.

General Considerations
Explant material and medium preparation.
In this chapter, the peanut cultivars used are ‘GK7’ and ‘AT127’ which has been observed to produce
many embryogenic cultures.

MS is the media used for the experiments. Different PGRs are used in different experiments and the
pH of the media is preferably at 5.8.

Exercises
Experiment 1 – in this experiment, it is aimed to observe how a particular explants tissue can affect
the development of somatic embryo. The best explants would be the actively dividing meristematic
tissues. Embryo axis may work but not as good as the previous mentioned. Mature cotyledons are
the poorest explants to be used. The PGR used is 2,4-D but with 2 different concentrations. The
whole experiment may extend to 2 weeks. The expected result would be that the explants will swell
when placed in the primary media containing 2,4 – D. When transferred to a secondary media
without 2,4 – D, the embryo will come out.

Experiment 2 – for this experiment, the effects of different concentration of auxins and the different
exposure to light will be observed. The whole experiment will be done twice. Once with 2,4-D and
exposed to light then another with 2,4-D but exposed to darkness. The cultures grown in a dark
place is expected to form embryos that are well-formed and white. The ones grown in the light. The
embryos will be green and is less succulent.
How interesting!!! The embryos grown in darkness are white and the ones in light are green!!!

Experiment 3 – 2 media will be used for this experiment. One with no auxin (primary) and another
without auxin or with low concentration of it (secondary). The ability of repetitive somatic
embryogenesis is to be observed on the secondary medium. It is expected that the somatic embryos
would start to form as it is placed on the secondary media. With a smaller concentration of 2,4-D on
the secondary media, the secondary embryos will form.

Experiment 4 – using Magenta boxes and exposure to light, germination has been observed to occur.
For this experiment, the main focus is to see the ability of the embryos to germinate and be able to
acclimatize when placed ex vitro. It is expected that roots and shoots should grow after 1 month of
being transferred to the Magenta box.
T23 – Direct Somatic Embryogenesis from Leaves and Flower Receptacles of Cineraria
Cineraria is a plant that looks like daisies. It is very notable because of the flowers it produces that
vary in color. Cineraria is considered to be a good explants because of its ability to do direct somatic
embryogenesis.

General Considerations
Growth of plants
The seeds to be used are called ‘Hansa’ mixed with ‘Cindy’. The seedlings will start to emerge from
10 days to 2 weeks, the first true leaves in 2 weeks and flowers in 26-28 weeks.

Explant preparation and basal culture media
The explant to be used is the first true leaf of cineraria, including the petiole. The media to be used
can either be MS (Murashige and Skoog) or SH (Schenk and Hilderbrandt). Among the supplements
added are sucrose, myo-inositol, thiamine-HCl, 2,4-D and BA.

Exercises
Experiment 1 – in this experiment, the 2 cultures, SH and MS, would be tested on which one works
best for the production of somatic embryos. It would take about 4 weeks to see the results. It is
expected that somatic embryogenesis will occur on both media. However, the physical
characteristics of the callus and somatic embryo would be different between the media.
This is a good compare and contrast type of test! How the salts in the medium can affect the cultures
is a good explanation as to what the plants need to survive.

Exercise 2 – with proper nutrition and PGRs present on the media, initiation of the growth of somatic
embryos occur. As initiation occurs, induction, maturation and germination of the somatic embryo is
promoted. For induction to occur, high concentrations of auxin or auxin-like PGRs are added on the
medium. For maturation to occur, the secondary medium does not require high amounts of PGRs,
salts and sugars. The whole experiment may take about 5 weeks to 11 weeks from maturation to
acclimatization.


T24 – Somatic Embryogenesis from Immature Seeds of Yellow Poplar
Yellow poplar is a tree that is very valuable because of its hard wood. It is also known because of its
desirable ornamental characteristics. Propagation of yellow poplar is done to achieve genetic
superiority. The in vitro propagation method normally used is somatic embryogenesis. The
embrogenic cultures of a yellow poplar can produce proembryonic masses which is very unique to
these trees.

PEM is the stage where the proembryos are caught on the proliferation stage. This chapter aims to
give emphasis on the techniques such as initiating the development of repetitive embryogenic
cultures from immature seeds and to produce somatic embryos from PEMs.
PEM (proembryonic masses) seems to be a non stopping event for the proembryos

Exercises
Experiment 1 – for somatic embryos to be produced, it is important that the seeds used as explants
be on the right developmental stage. It has been observed that young seeds have a higher chance of
undergoing embryogenesis than those of the mature seeds. This experiment aims to show how ages
of the seeds affect the whole embryogenesis process. The expected results are that after 2 months,
clusters of PEMs will be present.
Experiment 2 – for the second experiment, it is aimed that somatic embryos be produced from the
PEMs of the previous experiment. With a lowered concentration of auxins, the PEM gets a signal to
produce somatic embryos. The whole experiment aims to see how the absences of 2,4-D can affect
the further development of somatic embryos from the PEMs and how germination of the embryos
are affected by the presence of casein hydrosylate.


T25 – Induction of Somatic Embryogenesis in Conifers
Conifers are manufactured as pulpwood for producing papers because it has a white wood, low resin
content and has good fiber qualities. Using somatic embryogenesis, disease resistant plants can be
produced. Cytokinins and kinetins are important for somatic embryogenesis to occur. This chapter
present experiments that would teach techniques on how to establish and maintain embryogenic
cultures of spruce.
Why is the low amount of resin content on conifers preferred as wood for manufacturing paper?

Exercises
Experiment 1 – in this experiment, the induction and the maintenance of somatic embryos is aimed.
The immature seeds are preferred to be the explants used in this experiment because the frequency
of somatic embryos that can produced is high. Using BA, 2,4-D and with the presence of casein
hydrosylate, the development of somatic embryos is expected to occur.

Experiment 2 – for the second experiment, the morphology of somatic embryos are to be analyzed.
Using a microscope and staining the embryos, the structure of the embryo can be looked at. After
staining, there are important processes that can be observed in the microscope. Among these
processes is the cleavage polyembryogenesis. This is when the embryo divides into 4 separate
embryos.
Is cleavage polyembryogenesis similar to how twins can be produced in humans?


Questions:

   1. What does PEM mean?
   2. What is the pH of the MS media used for somatic embryogenesis of peanuts?
   3. What does casein hydrosylate do?
   4. Based on the experiment on cineraria, what differences in the results did the cultures
      show on both SH and MS media?
   5. What is cleavage polyembryogenesis?

				
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