Effects of Hormone Balance on Explant Growth and Morphogenesis

Background
Plant hormones, like animal hormones, are relatively small molecules that are effective at low
tissue concentrations. The two types of plant hormones used in this experiment are cytokinins and
auxins.
Cytokinins are derived from adenine and produce two immediate effects on undifferentiated
cells: the stimulation of DNA synthesis and increased cell division (Ting, 1982). Cytokinins also
produce a delayed response in undifferentiated tissue which is the formation of shoot primordia.
Both naturally occurring cytokinins, such as zeatin and synthetic analogs, such as kinetin,
demonstrate cytokinin effects (Figure 9.3). Although low tissue concentrations of cytokinins (e.g., 1
× 10-8 M zeatin) have noticeable effects, higher concentrations are found in actively dividing tissues
such as those of plant embryos and developing fruits.

Auxins are indole or indole-like compounds that stimulate cell expansion, particularly cell
elongation. Auxins also promote adventitious root development. Indoleacetic acid (IAA), a
naturally occurring auxin, and napthaleneacetic acid (NAA), a synthetic auxin, are depicted in
Figure 9.3. Only small amounts of auxin (1 × 10-6 M) are required to demonstrate an IAA response
and even smaller amounts of synthetic auxin (e.g., NAA) are required for a tissue response. The
likely reason for potency of synthetic auxins is their stability in plant tissue (i.e., the enzymes and
processes that degrade IAA do not "recognize" synthetic auxins). Synthetic auxins, then, are more
effective hormones that also last for an extended length of time. Furthermore, light influences the
physiological activity of IAA while synthetic auxins are not as light sensitive.
Plant hormones do not function in isolation within the plant body, but, instead, function in
relation to each other. Hormone balance is apparently more important than the absolute
concentration of any one hormone. Both cell division and cell expansion occur in actively dividing
tissue, therefore cytokinin and auxin balance plays a role in the overall growth of plant tissue. Since
hormone balance is presumably important to the overall effect on growth and morphological
changes, then the hormone differentials in each of the experimental media (A, B, and C) should
produce somewhat different effects on the growth and development of excised explants.

Source of Aseptic Explant Material
During seed development the embryos are formed with a placenta-like interface of intervening
tissues between parental vascular supply and the embryo proper. This circumstance depresses
passive migration of most foreign bodies and microorganisms into the developing embryo. If the
embryo which often develops aseptically is released from the seed by aseptic germination
procedures, then aseptic seedlings result. Any part of the aseptic seedling can be used as "in vitro"
experimental material. In this experiment three explant types will be used: hypocotyl (undeveloped
lower stem), epicotyl (shoot apex), and cotyledons.


Media Formulae
Media A, B, C, D, and E each contain the same complement of minerals, that is, salt base as in
Medium D (Appendix D). The effect of minerals alone on explant growth and development
constitute "basal growth rate" against which the effects of other media constituents can be
measured. Medium D, then, serves as the base-line control for endogenous growth. Medium E,
containing both essential minerals plus sucrose, constitutes the organic and inorganic control which
can be used as the base-line indicator of explant growth when both minerals and sucrose are
supplied. In addition to the substrate, sucrose, Medium E contains two organic growth factors,
inositol and thiamine, which promote sugar metabolism and general anabolic growth processes.
Medium E also contains additional phosphate thereby matching the phosphate concentrations of the
experimental media (A, B, C). The experimental media contain similar inorganic and organic
complements, but differ in hormone content.
Since cytokinins are derived from adenine, adenine sulfate has been added to each of the
experimental media (A, B, C). In addition, either kinetin or 2iP ([2-isopentenyl]-adenine), both of
which are synthetic cytokinins having immediate hormone activity, are supplemental cytokinins in
media A, B, and C. Of the three experimental media, Medium A contains the highest amount of
active cytokinin (30 mg/liter), while media B and C contain much lower amounts (2 mg/liter and 1
mg/liter, respectively).
Conversely, Medium A contains only a small amount of auxin (0.3 mg IAA/liter), while
Medium B contains a higher amount (2 mgIAA/liter). Medium C contains the lowest absolute
concentration of auxin (0.1 mg NAA/liter), but this synthetic auxin is more efficient in promoting
cell expansion and root formation than the naturally occurring auxin, IAA. Medium C, then, may
actually represent the formula with the highest physiological auxin activity.
Since cytokinin/auxin balance is reportedly important to the final overall effect on growth and
development, the results for each experimental media may be expected to differ. The balance

represented by Medium A is decidedly skewed towards a high cytokinin/low auxin ratio. Medium
B represents a more even distribution of cytokinin and auxin, while Medium C may have an
effectively higher auxin than cytokinin ratio because of the "in vivo" stability of NAA as well as its
effectiveness as an auxin.

Methods: Week 1 (Aseptic Seed Germination)
Materials:
cucumber seeds (tomato may also be used; sterilization procedure, Experiment 1)
95% ethanol
sterile jars, sterile water
25% chlorox, freshly prepared
sterile forceps or spatula
Procedure:
1. Sterilize seeds (cucumber) for 1 minute with 95% EtOH.
2. Rinse in sterile water
3. Sterilize in 25% bleach for 5 minutes and rinse three times with sterile water.
4. Transfer 10 seeds with a sterile forceps or spatula to a nutrient agar plate.
5. Incubate for 1 week (20–23°C).
Methods: Week 2
Seedling explants (approximately 1 cm in length) of aseptically germinated cucumber (or
tomato) can be cut from the seedlings as shown in Figure 9.4. Aseptic techniques including the use
of the laminar flow hood are necessary to evaluate growth experiments in which nutrient rich media
are used. An explant (hypocotyl, epicotyl, or cotyledon) should be placed on each of the following
experimental media:
A) Murashige Shoot Multiplication, Medium A,
B) Murashige Shoot Multiplication, Medium B, and
C) Murashige Shoot Multiplication, Medium C,
which contain different concentrations of growth hormones (Appendix D). For comparison, one
explant of each type should be placed on each control media:
D) Murashige and Skoog Salt Base, Medium D, and
E) Murashige Minimal Organic Medium + Sucrose with NaH2PO4⋅H20, Medium E.
Seal the petri dishes with parafilm to prevent desiccation and incubate at low light intensity until
next week. Record incubation conditions.


Discussion Questions
What effect does a hormone balance that is applied pharmacologically "in vitro" have on
seedling explant growth and morphogenesis? Which media formulations produce callus? To what
extent? On which explants? Were these results predictable? What media produce anomalous