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Tissue Culture Techniques

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.

The manipulations required for the germination of aseptic seedlings

Method

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.)

4. 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 manipulations required for the germination of aseptic seedlings

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.

The manipulations required for the transfer of seedling explants to Mineral Salts (M) and Minimal Organic (O) growth media.