UV Effects and Shoot Regeneration in
Tobacco Leaf Cultures
Investigation 4
Introduction:
This exercise has two primary purposes: (1) to study the interactions of representatives of two major classes plant
hormones, cytokinins and auxins, in the induction of cell division and organogenesis in tobacco leaf explants and (2)
to observe the effects of ultraviolet radiation on these processes. during the exercise, you will have a chance to learn
aseptic procedures, clonal propagation of plants, and radiation techniques.
The growth of plant cells and tissues in aseptic culture is now widespread (Evans et al., 1983) and exercises have
been developed for introductory and advanced undergraduate biology courses. This exercise builds on the
experience gained using tobacco leaf cultures in our introductory course at Barnard College and combines it with an
investigation of ultraviolet radiation effects. The effects of ultraviolet radiation have been observed on many
different organisms and UV has a long history as a mutagen for bacterial and yeast cells (cf. Jagger, 1965; Sancar
and Sancar, 1988). However, there has been little work on UV effects on isolated plant cultures because of UV’s
limited penetration through tissues. Most studies have used single cell cultures, pollen or protoplasts (cf. Negrutiu,,
1990). The development of this exercise was prompted by the use of UV for mutation induction in the selection for
herbicide resistance in tobacco cell suspension cultures by Karen Hughes at the University of Tennessee, Knoxville
(Hughes, 1983).
Induction of growth and morphogenesis in leaf explants.
When tissue is excised from the mature plant body and placed on a suitable medium, not only can it be kept alive but
some of its cells, given the proper conditions, can be prompted to undergo cell division. The medium is a solution
containing (1) the inorganic salts (macro- and micro-nutrients) that any whole plant would need to grow; (2) a
reduced carbon source, typically a carbohydrate such as sucrose; (3) a source of organic nitrogen such as ammonium
salts, amino acids or a natural mixture such as casein hydrolysate; (4) vitamins, including thiamine, nicotinic acid
and pyridoxine; and (5) the sugar alcohol, or hexitol, myo-inositol. A medium containing all five components is
basic to many plant cell and tissue cultures and is often called the basal medium. In addition, to both trigger cell
division and to control the type of growth, two types of plant growth regulators or hormones are usually needed--a
cytokinin and an auxin.
Cytokinins have as their chemical basis the purine ring adenine, one of the purines that also is found in DNA and
RNA. Cytokinins derive their name from their role in triggering cytokinesis, or cell division. In the induction of
cell division, cytokinins interact with auxins. Auxins have an indole ring as their nucleus and are typically
associated with cell and organ elongation. For example, auxin appears to play a role in the movements associated
with phototropism and gravitropism. As is typical with all plant growth regulators, auxins are involved in many
different responses. In our system, auxin is implicated more in cell division and morphogenesis than in elongation.
Cytokinin in combination with auxin, when applied to certain systems, can foster the initiation of cell division in
non-dividing and differentiated cells (a process called dedifferentiation). The combination can also foster the
formation of new organs from that population of newly and actively dividing cells (a process called redifferentiation). The relative concentrations of the two hormones are particularly important. This was reported in a
classic paper by Skoog and Miller (1957). Generally speaking (and we are always on unsteady ground when we
generalize), approximately equal amounts of cytokinin and auxin result in unorganized growth, producing a mass of
cells called a callus. If there is a higher concentration of cytokinins vis-à-vis auxin, then a callus will form and after
a period of time, shoot apices will organize from the cells and shoots will grow out of the callus tissue. If the
combination favors auxin, then callus formation will be followed by the formation of root apices and root grow will
occur. Since one leaf can be used to grow many shoots (via a callus) which can then be rooted, this is a way to
produce identical copies of a plant, or clones, and to do so in large numbers. This is cloning at the cellular level
rather than at the molecular or organ level (as with shoot cuttings; clone means twig in Greek.) For an excellent
discussion of plant development, refer to Steeves and Sussex (1989).
Dedifferentiation and re-differentiation results in the formation of organs and cells very different from the original
cells. In this exercise, mesophyll cells in leaf tissues will ultimately produce shoots (with stems and leaves) and
roots--entire plants. This shows that the mesophyll cells, although highly specialized, have complete sets of genetic
information or are totipotent. As the original zygote divided mitotically and became the numerous cells that
comprise the mature plant, many cells, including the mesophyll cells under study today, received and retained all the
genetic information present in that first cell. Those cells differentiated and became specialized by utilizing only
some of this “full store” of genetic information. One of the major questions facing biology is to understand how
only part of the total available information encoded by DNA is used during differentiation and how it can be reprogrammed to direct the maturation of different cells, organs, even whole plants.
Not all cells are totipotent or re-porgrammable. In some cells, the genetic information is changed during
differentiation, e.g., polyteny, gene amplification, gene movement (transposable elements), even the loss of all
nuclear material during differentiation. In other cells, the genetic information appears intact but all efforts to induce
cell division or to change the pattern of differentiation have been unsuccessful.
The ability of some plant cells to re-differentiate and form different plant organs and eve whole plants is unique to
plants; animals do not have this ability. Plants do this throughout their life. The vascular cambium results from the
re-initiation of cell division of parenchyma cells in the vascular tissue; cork cambium arises from mature epidermal
or cortical cells. Adventitious roots can form from differentiated cells in shoots (as on geranium shoot cuttings
placed in wet sand) and shoots can arise on cut roots (typical in dandelion, making it difficult to eradicate dandelion
by pulling out the shoots).
In this experiment, we are calling upon this basic ability of plants to dedifferentiate and re-differentiate new organs.
Ultraviolet Radiation
Plants absorb light energy for photosynthesis, photoperiodism and photomorphogenesis, using, for the most part,
wavelengths visible to the human eye. Other wavelengths are also absorbed and some, such as ultraviolet radiation,
can be particularly damaging to the plant or to other organisms. UV radiation is non-ionizing electromagnetic
radiation of wavelengths just shorter than those normally seen by the human eye. UV radiation is usually placed
onto three groups: UV-A, 315-400 nm; UV-B, 280-315 nm; and UV-C, shorter than 280 nm. The shorter the
wavelength, the higher the energy level, so the UV-C group is particularly effective at causing photochemical
changes and, because of this, causing extensive damage to living cells. Fortunately, the thin ozone layer in the upper
atmosphere absorbs most of the shorter UV wavelengths in sunlight (all of the UV-C and also UV-B radiation
shorter than 295 nm).
Penetration of UV radiation is relatively low through liquids, whether water in lakes and oceans or water in cells.
Lampert’s law is used to calculate the irradiance:
I/Io = e-at
I = irradiance at a particular distance into the medium or tissue
Io= initial irradiance
e = natural logarithmic base
a = absorption coefficient of medium per cm
t = distance traveled in medium (cm)
As a consequence, with multi-cellular organisms, UV radiation affects primarily superficial cells. With single cell
organisms, it will affect cells plated on agar medium or cells near the surface of liquid medium. UV will not pass
through glass or most plastics (or through opaque objects). Water, glass, and plastics, although transparent to visible
light, therefore afford some protection against UV radiation.
In cells, UV radiation is absorbed primarily by proteins and nucleic acids and the structure and function of both can
be severely affected by even short UV exposures. With UV radiation, proteins are denatured, membranes disrupted,
chromosomes break and the DNA itself can be changed. Such changes can affect the functioning of that cell as well
as any cells derived from it. If there is a permanent change to the DNA--a process called mutagenesis-- and it
occurs in a cell that produced gametes, the change or mutation can be passed on to succeeding generations. With
sufficient exposure, a cell or organism will die after UV radiation. The “germicidal” lamp emits 90% of its
irradiation at 254 nm and used in tissue culture transfer hoods ( and i other places) to kill microorganisms.
Uv affects nucleic acids in a number of ways, including breaking the DNA chain, forming linkages between DNA
and proteins, and forming links between different DNA strands. All these adversely affect DNA activity. The most
common effect, however, is the formation of thymine dimers, whereby two adjacent thymine bases in the DNA
chain are linked. The dimers prevent normal DNA replication and interfere with RNA and protein synthesis.
The damage can be repaired by a process called photoreactivation. It is performed by an enzyme system and driven
by UV-A and blue light (300-600 nm). If UV radiation is followed by longer wavelengths in sunlight then a high
percentage of the dimmer lesions can be repaired. The photoreactivating enzymes (photolyases) are present in
bacteria and some eukaryotic organisms but have not been found in mammalian cells (cf. Singer and Berg, 1991).
The photoreactivation ability of the cell decreases with time and typically does not last longer than 12-16 hours.
Keeping tissue that has been exposed to UV in the dark or under various safelights (usually yellow) for that time or
longer will prevent photoreactivation. There are other repair mechanisms that do not require light; so even
excluding light does not prevent some repair (Sancar and Sancar, 1988).
An appreciation of the effects of UV is particularly important at this time. Ultraviolet radiation has potent effects on
humans. It photolyzes pro-vitamin D to form vitamin D, a distinct benefit, and stimulates melanin-production in our
skin, providing a tan. Over the past 50 years, people have come to regard skin tans as attractive and desirable, a sign
of "health and vigor”. However, UV also suppresses cells of our immune systems and activates proto-oncogenes in
skin cells, leading to skin cancer. We are now witnessing a significant rise in the rate of skin cancer in the United
States that has prompted authorities to recommend limited exposure to sunlight and the use of protective sunscreens.
Gradually, we have begun to alter our attitudes towards tanning. Simultaneously, scientists have observed the
thinning of the protective ozone layer, most likely through the action in the atmosphere of nitrogen oxides produced
in exhaust emissions and chlorine resulting from the breakdown of fluorochlorohydrocarbons such as the Freon used
in refrigeration and air conditioning. We have come to realize that Uv can be dangerous at a time when our potential
exposure to UV radiation, and the exposure of other animals, plants and microbes, is increasing.
In this experiment, we will be investigating the effects of different exposures to UV radiation on cell survival, cell
division and organogenesis.
______________________
A scientific footnote: The photolytic abilities of UV are now being tapped for biological research in some
unexpected ways. For example, it is now possible to enclose ions, such as calcium, compounds such as ATP, and
hormones, such as indoleacetic acid, in "cages”. The caged ions or compounds can then be microinjected directly
into cells. The cages can be broken and their contents released by means of UV photolysis. The amount released will
depend on the length and intensity of the UV radiation. (For a recent reference see Nature 346:769-711, 1990.)
Methods and Materials
Aseptic techniques:
Since the culture medium contains sucrose and other nutrients, bacteria and fungi in the medium would grow so
rapidly that they would overrun the culture. Microorganisms, including spores, must be excluded from the culture--a
condition called asepsis must be created. This is done by (1) sterilizing everything that is involved in the process-leaf tissue, media and equipment; (2) performing any operations in a draft free and clean environment; and (3) using
proper technique.
The main instrument for sterilizing equipment and media is the autoclave, which provides steam under pressure (15
lbs/in2). The temperature under those conditions is 121 C, not the 100 C of steam at atmospheric pressure, and it is
sufficient to kill both living bacterial and fungal cells and their spores. Components of the medium that would break
down in such a high heat can be filter-sterilized and then added to the autoclaved and cooled medium. Some
implements, such a scalpels and forceps, can be autoclaved. Periodically, they can be flamed to re-sterilize.
Both benzyladenine (or benzylaminopurine, BA), the cytokinin, and naphthaleneacetic acid (NAA), the auxin, are
synthetic compounds. Both are heat and light-stable and will not degrade during autoclaving and storage.
(Indoleacetic acid, the naturally occurring auxin, is heat labile.)
For the leaf tissue, neither heat nor filter sterilization can be used. Since plant (and animal) tissues are free of
organisms internally, only their surfaces need to have microorganisms removed. This can be done by a number of
agents. We will use commercial laundry bleach, which is a 5% solution of sodium hypochlorite. A brief soak in a
diluted (20% v/v) bleach solution, with a wetting agent, tween 20, will kill all surface microorganisms. Since the
leaf tissue has a waxy layer, the cuticle, the bleach does not penetrate and kill the cells. The bleach will slowly
penetrate any cut or damaged areas in the leaf. After 20 minutes, the bleach will be thoroughly removed by rinsing
with sterile water. (Any bleach remaining will continue to penetrate and eventually kill all the leaf cells.) After
rinsing, the dead tissue can then be trimmed away and the remaining healthy tissue cut up and inoculated into the
culture dishes.
All operations must be performed in a draft free space, usually a laminar flow hood, tissue culture hood, or a glove
box. The hood is cleaned with detergent and water, rinsed and dried and then either the airflow is activated or the
built in UV light turned on for a short period. A part of' the classroom can be used if windows and air vents are
closed, traffic is kept to a minimum and surfaces are thoroughly cleaned.
Once the tissue has been surface-sterilized, it must not be touched except with sterile forceps and other instruments
that have been autoclaved and/or flamed and allowed to cool. All operations must be performed with sterile
instruments. Sterile media, glassware and instruments must be carefully handled to prevent the introduction of
contaminating microorganisms. Aseptic technique is important in many investigations where bacteria, fungi or
other organisms must be excluded so that they do not interfere with the cells or reactions being studied.
Ultraviolet Radiation
In this exercise, we will investigate the effects of UV radiation (254 nm) on the processes of cell survival, growth
induction (dedifferentiation) and organogenesis (re-differentiation). Specifically, we will look at the response to
different times of exposure. The intensity of the UV radiation or irradiance is the energy received on a unit surface
in an interval of time. By using the same light bulbs and placing our explants at a fixed distance from the source, we
can vary the intensity by varying the length of exposure. With one germicidal lamp placed at 24 cm distance, the
exposure is 140 ergs/mm2/sec. We will use short exposures to investigate non-lethal effects.
Ultraviolet light does not pass through opaque objects, glass and most plastic. This makes it a particularly safe agent
in this experiment. However, because of this, we cannot irradiate the tissue with the culture dish lids in place. The
lids will be removed, the tissue will be irradiated, and then the lids will be replaced. Aseptic conditions must be
maintained during the irradiation procedure just as it was during explant preparation and inoculation.
Safety: Short-wave ultraviolet radiation can cause serious damage to skin and eyes. You will need to be very
careful. Be sure the light switch is off before opening the curtain, shade or door. Be sure to re-close the
curtain before each irradiation. Eyeglasses or safety glasses should be worn when the lamp is on. Your
instructor may also wish you to use protective gloves.
To ensure against photoreactivation, we will irradiate the tissue in dim light or under a yellow safelight. After the
lids are replaced and the culture dishes sealed with parafilm, they will be wrapped in several layers of foil and kept
in the dark for 2-3 days. At that time, the culture dishes can be safely unwrapped and placed in the lighted growth
chambers or incubators.
Replication of Data
In any experimental system, particularly biological systems, there is always the question of whether a response is
typical or not. If I ask you to run a mile and you do it in ten minutes, is this your usual time or is it somewhat fast or
slow for you? To really get some idea of you as a runner, I will need to time your running of the mile a number of
times, probably on different occasions. The average time, the range of times in which you operate (your fastest, your
slowest) and other statistics are a better indication of your running ability than one trial run.
Similarly, if we expose one leaf square to one minute of irradiation and note the response, we would be correct to
ask how typical that response is. For that reason, we will need to have replicates of each treatment and the minimum
number is usually three. (More replicates would be better but there are constraints of time, space and money.) We
would also like to be sure that the differences are due to changes in Uv irradiation and not other factors, such as how
much medium is in the culture dish. The experimental design calls for three explants for each of the four treatments
to be in the same culture dish and one dish for each hormonal treatment. This is the minimum number of replicates.
Good science would ask for our repeating the process. The set of four dishes for each student can be looked upon as
serving as replicates for the others, but it is not a perfect design. (Ideally, you would inoculate several dishes for
each treatment and perform the experiment on several different occasions.)
Plant Material
For this experiment, we will be using a cultivar of the common cultivated tobacco, Nicotiana tabacum, for both
historical and practical reasons. It was the plant used for the classic studies of Skoog and Miller (1957). It is also a
plant that grows well in tissue culture; it is very dependable, particularly in responding to the cytokinin-auxin media.
Tobacco has been the subject of much research, including studies of protoplast isolation and fusion and gene
transfer. This exercise could serve as a starting point for a wide range of guided research projects.
The cultivated tobacco is an amphidiplold that arose as a natural hybrid between Nicotiana sylvestris and N.
tomentosiformis. It has two sets of chromosomes from each parent and is therefore fully fertile. Having four sets of
chromosomes, not all alike, it can also be considered an allotetraploid.
Experimental Design
Each student will set up one culture dish for each of the four treatments under study:
A (high auxin medium):
B (balanced medium):
C (high cytokinin):
D (devoid of hormones):
Basal medium +0.05 μM BA +0.5 μM NAA
Basal medium +0.5 μM BA +0.5 μM NAA
Basal medium +0.5 μM BA +0.05 μM NAA
Basal medium
Each dish will be inoculated with twelve explants arranged in four rows with three explants to a row. Each row will
be exposed to Uv radiation for a different length of time. The three explants in a row will serve as replicates:
0
I
II
III
control, no irradiation
1 minute of irradiation (or shortest time assigned)
2 minutes of irradiation (or middle time assigned)
4 minutes of irradiation (or longest time assigned)
The response of the explants will be assessed at weekly intervals for up to four weeks. At each interval, the general
appearance and size of the explant and callus, and the type and number of organs (shoot and/or root) will be
observed and recorded. Qualitative and quantitative data will be presented in a formal report written in journal style.
I. Explanting
Materials:
(for each student)
Tobacco leaf: one-quarter of a fully formed but recently expanded leaf
Two 250-ml beakers
100 ml of dilute detergent solution
50 ml of 20% commercial bleach with one drop of tween 20
Four culture dishes, one each of A, B, C and D
250 ml flask with sterile doubly-distilled or ultra-pure water (capped)
Two sterile plastic petri dishes
Parafilm strips
Equipment:
Transfer or laminar flow hood
Heavy jar (e.g., Coplin jar) with alcohol (and cap)
Alcohol lamp or Bunsen burner
Two pairs of forceps
Scalpel
Ruler
Protocol:
1.
Obtain two beakers. Fill one half full with detergent solution and the other with tap water. Obtain a
piece of tobacco leaf from your instructor and immerse it in the beaker with detergent. Gently swirl the
liquid around to remove dirt and dust. Pour it off, holding the leaf segment back with your fingers. Treat
the segment very gently.
2.
Next, immerse the segment in the tap water and gently swirl again to rinse off the detergent. Gently lift
the segment out and place it on a piece of clean moist paper toweling, and cover.
3.
Thoroughly wash your hands up to the elbow. Work quickly. Do not allow the leaf to stay on the paper
too long or it will dry out and the cells will die.
4.
Quickly place the segment into a sterile petri dish under the transfer hood. Cover with a 20% bleach
and tween solution and replace the lid on the dish. Agitate the segment by swirling the petri dish around
gently about every three minutes.
5.
Agitation should continue periodically for 20 minutes.
6.
Before returning to the hood, wash your hands again. Pour off the bleach solution and rinse the
segment six times with sterile distilled water. Be sure to flame the mouth of the water flask before
you pour.
7.
After the last rinse, leave some water in the petri dish. Flame your forceps, cool by touching to the
liquid first, then transfer the leaf to a second petri dish. Pour in a little bit of water.
8.
Flame your forceps and scalpel well. Touch both to the water to cool. Trim off the bleached portions of
the leaf segment. To cut the leaf without destroying it, place the curved blade of the scalpel on the leaf
where you want to cut and then rock it back and forth. This usually works better than drawing the blade
across the leaf as if you were cutting steak. Discard the trimmed portions.
9.
Cut strips of leaf tissue about 3 mm wide. Then cut each strip into squares 3 mm on a side. You will be
placing 12 squares onto each of four dishes, so you will need at least 48 squares. You should cut about
60 to provide some spares.
actual size of 3 mm x 3 mm explant:
10. Obtain and place under the culture hood one culture dish of each of the following treatments:
A (high auxin medium):
Basal medium +0.05 μM BA +0.5 μM NAA
B (balanced medium):
Basal medium +0.5 μM BA +0.5 μM NAA
C (high cytokinin):
Basal medium + 0.5 μM BA +0.05 μM NAA
D (devoid of hormones):
Basal medium
11. Flame the forceps, touch to the liquid to cool, and transfer 12 pieces of leaf to each culture dish. Place
four pieces in a row near the top of each dish and then two additional down each row.
12. Produce a four-by-three grid, all precisely lined up and well spaced to cover the entire dish
Figure 1
13. Lay the squares on the agar surface. Do not press them into the medium. Replace the lid on each culture
dish. Carefully turn each culture dish over and on the bottom, starting at the right, label the rows with a
marking pen: 0, I, II and III. The markings indicate:
0
I
II
III
= control, no irradiation
= 1 minute irradiation or shortest time
= 2 minute irradiation or medium time
= 4 minute irradiation or longest time
i
Figure 2. The culture dish should look like this
14. The leaf squares are now ready for Uv treatment. Proceed to next section.
15. After the treatment, or if you decide to not irradiate, seal each dish with parafilm: Remove the paper
backing from the parafilm strip. Pick up a culture dish; press the lid firmly on the base; place a strip of
parafilm on the side and stretch it as you pull it around the edge.
16. Place your initials or name on the bottom of each dish. Hand in your culture dishes for your instructors
to handle or place them in their proper place in an incubator or growth chamber. They should be placed
upside down to allow vapor to condense back onto/into the medium and prevent the media from drying
out. The cultures will be incubated in light. Record the specific photoperiod and temperature regimes
used.
17. Fill in the treatments in the four dishes on the first data sheet. Use this sheet and additional sheets for
your observations.
II. UV Irradiation
Equipment: Transfer hood
Platform to hold four petri dishes and their lids in a row 24 cm from light
OR Irradiation box with Uv fixture placed 24 cm from the bench surface
Timer
Four sterile 9 x 9 cm aluminum foil squares (per student)
Protocol:
1.
When it is your turn to use the apparatus, bring your four culture dishes to the apparatus. Before
opening curtain, shade or door, check that the Uv light is off. The apparatus should have no or little
light, or a yellow safelight.
2.
Place your four culture dishes in a row on the rack under the UV light. The support has been placed so
that it is 24 cm from the bottom of the UV tubes. The dishes should be placed so that enough space is
left between each one to accommodate the lid.
3.
In turn, carefully remove the lid of a dish and place it on the rack to the left of that dish.
4.
From the supply, remove a sterile square of aluminum foil and place it the control (0) row of explants
in a dish. Repeat with the other four dishes. At this point your dishes should look like this:
5.
Close the curtains or door and turn on the Uv light. After one minute, turn off the light. At this point, I,
II and III have all received a one minute dose.
6.
Move each aluminum square to the right to cover the second row as well (now 0 and I are covered).
7.
Close the curtains or door and turn on the Uv light. Irradiate for one minute and turn off the light. At
this point, II and III have received two minutes total of irradiation.
8.
Open the curtains or door, move each aluminum square to the right to cover the third row as well (0, I
and II are now covered).
9.
Close the curtains or door and irradiate for two minutes. Turn off the light. The total irradiation for III
is now four minutes.
10. Open the curtains, carefully remove the foil squares, and replace each lid.
11. Seal the culture dishes with parafilm (see the last step of the explanting procedure.)
During and after the irradiation, be careful to work under very dim light to prevent
photo-induced DNA repair.
12. Wrap all four culture dishes in two layers of aluminum foil. The packet can now be moved out of the
transfer hood.
13. Place in the incubator or hand in to your instructors.
14. Measure the size, in millimeters, of the remaining, uninoculated explants. If your explants are square,
then the length of one side should do. Record your data. Calculate the average explant size (1ength or
area) at the start of the treatment.
15. After three days, the culture dishes should be removed from the aluminum foil and placed, upside
down, in the incubator.
III. Observations
1.
At weekly intervals, for up to four weeks, examine the culture dishes. Do not remove the parafilm or
lift the lid. Observe the cultures through the lid and, after inverting the dish, through the agar. Feel free
to place under a dissecting microscope.
2.
Observe and record your observations. Record on the data sheets or construct your own table for
recording data. Several are provided. You may photocopy these to provide as many as you need. You
might decide to use several sheets for each week's observations--some for qualitative observations,
some for actual measurements.
3.
Observe and record the general appearance of the explants, including their color. Record the
appearance of each explant in a treatment (A media, 0 time; A media, I time, and so forth). Summarize
the data for the three replicates of each treatment.
4.
Determine the size (e.g., length) of the explants, record your data and calculate the average size and
standard deviation for the three replicates in each irradiation regime for each treatment.
5.
Look for any signs of cell growth, usually at the cut edges of the explants. Record your observations.
You may wish to measure the extent of callus formation (in millimeters) or create an arbitrary and
relative scale (e.g., 0 = no growth; 1 = some growth; 2 = moderate growth; 3 = maximum growth).
6.
Carefully observe and record the number and type of shoots or roots that form on any explant and
record. With some genotypes, organogenesis may be visible as early as two weeks after inoculation; in
others, it may require four weeks growth. Some treatments may give both roots and shoots on the same
explants; others only one or the other; some none. Calculate the average number of shoots and roots for
the replicates in a treatment and regime.
IV. Report
Write a report on your results in journal style. Use these instructions as the Introduction and Methods and
Materials sections. You will need to supply the three other parts:
Results. Your reporting of what you found including any data whether included in the narrative or supplied
as tables or charts.
Discussion. An analysis of your results, relating them to what you set out to investigate and to the broader
questions of morphogenesis and Uv radiation effects. You may wish to consider some of the questions
posed below in framing your discussion.
Abstract. Summarize what you have done in one paragraph of not more than 150 words.
Questions for consideration:
Did the behavior of the leaf tissue agree with what was expected on the different media? How might you
explain any differences?
Did both shoots and roots appear on same explant and how might you explain such a response?
What were the responses to Uv radiation vis-à-vis tissue survival, cell grow and morphogenesis?
Were the responses dose-dependent? How might you explain this phenomenon?
Did the responses to Uv change as the tissues grew? How might you explain such changes?
How might you explain the low level or absence of visible mutations in regenerated shoots in terms of the
nature of mutations?
Given that the cultivated tobacco is an amphidiploid (or allotetraploid), how might you explain the low
level or absence of visible mutations in regenerated shoots?
Were any chimeras observed and how might you explain their appearance?
What do your results reveal about UV effects?