Demonstration of "in vitro" Morphogenesis and Totipotency of Seedling Explants

A simple exercise demonstrating plant totipotency as well as the nutritional requirements of
different plant organs employs shoot tip and root tip explants cut from aseptically germinated
seedlings. Each type of explant (excised part of the intact organism) is transferred to three simple
tissue culture media.

Background
During seed formation, the developing embryo and associated tissues tend to exclude pathogens
and foreign materials that may be in the parent plant. Contents of the seed, then, are essentially
aseptic and the resultant seedlings can be maintained in the aseptic condition if the outer surface of
the seed (seed coat) is sterilized with sodium hypochlorite (or other surface sterilant) prior to
germinating the seeds in a sterile petri dish.

Methods: Week 1
The manipulations that are required for the germination of aseptic seedlings are outlined below
and illustrated in Figure 9.1.

1. Outside the laminar flow hood: Place several (5 to 10) tomato or lettuce seeds in a small petri
dish. Fill the petri dish with a 7% chlorox solution to which a drop of wetting agent has been
added. The soapy chlorox solution is usually a good surface sterilant. Swirl the seeds
intermittently during the 10- or 15-minute chlorox treatment.

2. Preparation for aseptic transfers: Begin by washing your hands and forearms with soap,
followed by swabbing with 70% ethyl alcohol (EtOH). Sterilize the laminar flow hood by
wiping the inside (top, sides, and bottom) with EtOH. Turn on the hood; 10–15 minute
operation of the hood before use insures aseptic conditions within the work area of the hood.
Continue to swirl the seeds intermittently during the chlorox treatment. Prior to actual aseptic
transfers inside the chamber, swab hands and forearms with EtOH again; also wipe the external
surface of the petri dish before placing it inside the hood. The hood should contain the
following: a large jar which can be used as a "sink," flasks of sterile water, forceps in a beaker
of ethanol, sterile filter paper (5–7 cm diameter filter paper can be sterilized in glass petri
dishes), and sterile petri dishes which can be used as the seed germination dishes.

3. Inside the laminar flow hood: Decant chlorox and replace with sterile H2O. Rinse this way
twice. Each rinse should rest 10 minutes. Prepare the sterile germinating petri dish by
retrieving a forceps from the 70% EtOH beaker. Using the sterile forceps remove three (3)
rounds of sterile filter paper from a sterile container and place them in the germinating dish
(sterile plastic petri dish). Finally, add 5–10 ml of sterile H2O to the seeds; decant seeds and
water into the sterile germinating dish and incubate at 25°C until the next laboratory. (Both
tomato and light-insensitive lettuce seeds germinate in the light. Since shoots become green but
roots remain white under these conditions, seedling morphology is recognized more easily when
light-germinated.)

Methods: Week 2
Examine the contents of the aseptic germinating dish without opening the lid. If there is no
fungal or bacterial contamination around the seedlings, proceed; if contamination exists, request a
dish of aseptic seedlings from the instructor. The manipulation required for the transfer of seedling
explants to Mineral Salts (M) and Minimal Organic (O) growth media are outlined below and
illustrated in Figure 9.2.
1. Swab chamber, hands, and upper/lower surfaces of petri dish with 70% ethanol.
2. Place germinating dish in transfer chamber.
3. Remove scalpel or scissors from the ETOH beaker already in the hood. Slip instrument between
sheets of sterile toweling to remove ETOH (ethanol).
4. Lift one edge of lid and cut off no more than 10 mm of root tip. Excise two root tips. Lower
lid. Place scalpel back into ETOH beaker.
5. Place tubes with sterile media into the transfer chamber. (Media formulae are given in
Appendix B.) Use one tube of Minimal Organic Medium (O) and one of Mineral Salts Medium
(M). Loosen these caps.

6. Remove forceps or inoculating loop from ETOH. Slip between sterile toweling to remove
ETOH.
7. Remove excised root tip from germinating dish and transfer to the surface of the Minimal
Organic Medium (O). Transfer second root tip to the surface of the Mineral Salts Medium (M).
Caution: pick up root tip by the severed end; damage to the apical meristem disrupts mitosis!
Measure or estimate length of root tips. Record.
8. Using aseptic technique as above, prepare and transfer one shoot tip into each type of media.
Pick up the shoot tip by the severed end and insert it part way into the medium with an overall
vertical orientation of the cotyledons and shoot tip. Record size and shape of shoot tip.
9. Place the four tubes in a slant rack under lights.
10. Examine cultures each week. Record observations on the amount of growth and morphogenesis
of both root and shoot cultures.


Observations
As cultures progress it should be possible to correlate size/shape changes with the nutrient
content of the medium. A third medium, the B-deficient Medium, contains the same mineral
constituents as does the Mineral Salts Medium (M) and the same amount of sucrose as the Minimal
Organic Medium (O), but is devoid of B vitamins. Thus, this medium is referred to as B-deficient (-
B). Shoot tips and root tips have been transferred to this demonstration medium. Growth on this
medium can be evaluated and compared with growth on student experimental media M and O. The
Mineral Salts Medium (M) is the basal growth medium, supplying essential mineral nutrients for
autotrophic plant growth (Appendix C).
Predict the resultant growth in each circumstance, then monitor the growth and development of
root and shoot explants in each medium (M, O, and -B) and evaluate the following (record
observations in Table 9.1):
1. Effect of B-vitamins on:
a) shoot growth (increase in size) and morphology (change of shape), and
b) root growth and morphology.
2. Effect of organic medium containing sucrose on:
a) shoot growth and morphology, and
b) root growth and morphology.
Optional: media M, O, and -B are set up with root tip and shoot tip explants in darkness. This
set of samples can be observed along with those in the light to evaluate the effect of light as well as
media contents on the growth and development of plant organs. A row labeled "etiolation" would
be added to the bottom of Table 9.1.

Experimental observations should include the following:
1. Parameters: temperature; light quality, duration, and intensity.
2. Drawings to scale.
3. Gross measurements (length, biomass accumulation, extent of morphogenesis, totipotency,
primordia, number of branches) after 1 week and 2 weeks.
4. Net changes (Table 9.1).
5. Does the irregular orientation of the shoot explant change the growth pattern? How can these
observations be explained?