Plant Physiology

Plant Physiology (Biology 327) - Dr. Stephen G. Saupe; College of St. Benedict/ St. John's University; Biology Department; Collegeville, MN 56321; (320) 363 - 2782; (320) 363 - 3202, fax; ssaupe@csbsju.edu

Plant Organ Culture - Culturing Excised Tomato Roots

Objectives: Upon completion of this laboratory, you should be able to:

use standard methods of sterile technique
surface sterilize plant tissues/organs
describe the uses for cell and tissue culture in plant physiological research
describe the process for culturing roots and the uses of root culture in plant physiology experiments

Introduction:
Various methods have been described for isolating plant parts and maintaining them in axenic culture. In general, these methods require that: (1) the plant material is in the appropriate stage (undifferentiated cells and tissues are best); (2) the plant material is surface-sterilized to kill the naturally-occurring microbial flora; and (3) the proper growth environment (i.e., appropriate nutrients, temperature, light, pH) is maintained. The appropriate conditions must usually be determined empirically for each culture and species.

Several types of plant culture can be recognized based upon the plant part cultured and the method of culture:

Organ culture. In this technique, the plant material maintains its morphological identity and has the same basic structure as the parent material. It is possible to grow several plant organs in culture including roots, shoot apices, leaves, flower parts and fruits.
Embryo Culture. Plant embryos, which normally develop from fertilized eggs in the embryo sac of the ovary, can be induced to form in culture. These somatic embryos, technically called embryoids, have been cultured from vegetative cells of many plants, from reproductive tissues other than the zygote, and from hypocotyls and cotyledons of embryos.
Tissue Culture. When small, sterile pieces of tissues containing undifferentiated cells (parenchyma cells) are placed on a suitable medium the parenchyma cells will begin to grow and divide and give rise to an undifferentiated mass of cells called a callus. Callus can be induced to differentiate and form roots and shoots by manipulating the composition of the medium.
Liquid Suspension Culture. A suspension culture consists of cells and small aggregates of cells dispersed and growing in a moving liquid medium. These can be obtained by placing a callus in a liquid medium and then agitating it on a shaker.
Pollen and Anther Culture. Pollen can be cultured by standard tissue culture techniques to form a callus. The callus can be induced to differentiate and a plantlet can be obtained. Note that the plants/cells formed are haploid.
Protoplast Culture. Protoplasts isolated from a variety of species can be placed in the appropriate medium and growth conditions. The protoplast can be induced to regenerate a cell wall and grow into a callus.

In this experiment we will study organ culture techniques. White first demonstrated that excised roots of tomatoes could be grown in culture in a defined medium. In brief, root tips are excised from young seedlings and then transferred to a liquid culture medium. After approximately 1 week, the root will have grown significantly and have many lateral roots. The tip of this root can be sub-cultured (tip culture) and the remainder of the root divided into sectors each containing 3-4 lateral roots and also sub-cultured (sector culture). The roots can be grown indefinitely in this manner if they are periodically sub-cultured into fresh medium.

Root cultures can be used in many ways including studies of carbohydrate metabolism, mineral nutrient requirements, essentiality of vitamins and other growth regulators, differentiation of the root apex and gravitropism. The advantage of using root cultures is that they grow rapidly, are relatively easy to prepare and maintain, show a low level of variability and can be easily cloned to produce a large supply of experimental tissue.

Notes On Sterile Technique: It is critical that sterile conditions be maintained throughout this experiment. The following are some general procedures for sterile technique:

Work in an area where surfaces can be cleaned and sterilized and with little air movement (i.e. not near a window). A transfer hood is ideal.
Wash hands with soap and water before starting.
Never lean over the culture; work with it out in front of you.
Whenever opening a sterile culture, etc., always hold it at an angle or keep the lid over it so that spores, dust, etc., cannot drop directly into the culture.
Disinfect the work surface before starting. This is done by wiping the surface well with either a 10% solution of bleach, a disinfectant, or alcohol (70%). It will not hurt to get a little on your hands.
All instruments that touch sterile plant materials must also be sterile. Store the instruments in 70% alcohol. Before use, the instrument is flamed in an alcohol lamp or bunsen burner. Do not hold the instrument in the flame. This procedure serves only to burn off the alcohol. Allow the instrument to cool briefly before touching the plant material. Keep all flames away from the open container of alcohol. If this solution should become inflamed, calmly cover it with a petri dish or other suitable cover.
Work quickly but precisely. Do not leave cultures open for any extended period of time.
Contaminated cultures should be autoclaved before opening.

Preparing Sterile Tomato Roots:

Wipe down the working surface with disinfectant and allow it to evaporate.
Obtain a package of sterile paper towels. Unfold some towels on the bench top as a work surface.
Obtain a ripe, unblemished tomato with an unbroken skin and gently wash it in tap water and then carefully dry.
Surface sterilize a tomato by dipping it in 70% alcohol. Then, soak in 10% bleach for 15 minutes. Using a scalpel that has been dipped into 70% alcohol and then flamed, cut the skin of the tomato along four quadrants starting from the stem end. Tear the fruit into four sections. Be careful not to touch the inner part of the fruit with your fingers, the scalpel (which is no longer sterile) or any other unsterile object.
Using flame-sterilized forceps or the end of the scalpel, remove about 10 seeds and place them in a sterile petri dish. Remove as much pulp from the seeds as possible without jeopardizing the sterility of the seeds.
Transfer 5-10 seeds to each of 2 sterile petri dishes containing a layer of filter paper. Add just enough sterile water to each dish until the seeds just barely fail to float.
Seal the dishes with parafilm, wrap in aluminum foil and place in an incubator at 25 C to germinate. The seeds should germinate within a few days.

Culturing Sterile Tomato Root Tips:

Using a flame sterilized scalpel, excise approximately 10 mm of the root tip of 5 seedlings and then transfer one tip to each of five vessels containing White's Medium (see recipe). [NOTES: Only open the dish partially so that the lid still covers the contents of the dish. Holding the flask with one hand, remove the lid of the flask with the pinky of your other hand. While holding the flask at a 45 angle, transfer the root tips to the container. Flame the mouth of the flask before inserting the stopper. Avoid touching the sides of the flask with the instruments, root tips, etc.; Instruments should be sterilized after each operation by dipping them in alcohol and then igniting the alcohol with a flame. Select only healthy, uncontaminated seedlings.]
Repeat step 1, transferring one tip to each of five vessels containing White�s medium minus sucrose.
After approximately one week, measure the length of the primary root in the tip cultures and count the number of lateral roots on each root. Record these data.
The roots can be sub-cultured, if desired, by removing 10 mm tips (tip cultures) or using sections containing at least one lateral root (sector cultures).

References:

Machlis, L and J.G. Torrey. 1956. Plants in Action. Freeman.
Dodds, J.H. and L.W.Roberts. 1983. Experiments in Plant Tissue Culture. Cambridge University Press.
Stribling, J.M. 1983. Pioneers in Plant tissue culture. Carolina Tips 46:33-35.
Street, H.E. 1967. Excised Root Culture. In: Methods in Developmental Biology. Wilt and Wessels.

Data:

Table 1. Organism
scientific name
common name

Table 2. Seed germination data
date seeds planted
date tip culture started
date root culture analyzed

Table 3: Growth of tomato roots in White's medium with and without 2% sucrose
Sample	White's + 0% sucrose		White's + 2% sucrose
	Length (mm)	# lateral roots	Length (mm)	# lateral roots
1
2
3
4
5
6
7
8
9
10
mean

Data Analysis and Conclusions:

Which treatment did you predict (hypothesize) will have longer roots? Explain.
Which treatment did you predict (hypothesize) will have the greater number of lateral roots? Explain.
Calculate the mean primary root length (mm) and mean # lateral roots for each treatment.
Do your data support your predictions? Perform a suitable statistical test (i.e., unpaired t-test).
Write your null hypothesis. What is the p value? What does it mean? Did the treatment have a statistically significant impact on root growth and branching?
Prepare an abstract describing this experiment.