Note: In this lab, you will learn how to make plant tissue culture media and get familiar with the procedures of micropropagating several horticultural crops. Work in a group 3-4 students throughout the semester. Turn in this lab report on April 21, 2004.
 

A. PREPARATION OF TISSUE CULTURE MEDIA (Jan 28, 2004)

1. Media Composition
Each group will prepare one or two of the following media:
 

No.	Sucrose Growth Regulators		(mg/l)y	Plants	Total No
	Mediumz	(g/l)
1.	MS	30	0.2 NAA + 1.0 BA	African violet	40 Plates
2.	MS	30	0.3 IAA + 3.0 BA	Rose, Carnation	50 Plates
3.	WPM	20	1.0 BA	Alder, aspen	40 Jars
4.	MS	30	none (solid medium)	Orchid Protocorms,Fern	50 Tubes
5.	MS	30	none (liquid medium)	Orchid Protocorms	25 Flasks

^zMedium: MS - Murashige and Skoog medium, WPM- McCown's woody plant medium.
^yGrowth regulators: naphthaleneacetic acid (NAA), benzyladenine (BA), indoleacetic acid (IAA), kinetin (KIN).

2. Procedures for Making MS Medium

a. Preparation of inorganic salts.
      You may prepare a solution of inorganic salts as shown in the attached handout. In this lab, however, the
      Murashige and Skoog (MS) salt mix which has been prepared (Sigma Chemical Co.) will be used.
      Dissolve 2.68 g of the salt mix in 500 ml of water contained in a 1-liter flask.

b. Add amino acid and inositol.

                                Glycine: 2 mg/l
                                Inositol: 100 mg/l

c. Add vitamins: Nicotinic acid: 0.5 mg/l
     Thiamin HC1: 0.1 mg/1
     Pyridoxine HC1: 0.5 mg/l

d. Add sugar or carbohydrate source
    Sucrose: 30 g/l or 20 g/l (see Table)

e. Add growth regulators.
    Growth regulator stock solutions have been prepared (most in mg/ml). Add exact amounts of growth
    regulators as specified for each medium being prepared.

f. Bring the medium volume to 1000 ml, using double distilled water and dissolve.

g. Adjust the pH of the solution.
    Using a magnetic stirrer, adjust the pH of the solution to 5.7. Use 0.1 N NaOH and 0.1 N HC1 solutions in
    small quantities. (The pH of the solution must be adjusted before adding agar: solutions containing agar tend
    to clog up the electrode of the pH meter).

h. Add agar.
    Agar (Difco Bacto-Agar) 8 g/l

i. Melt the agar in the solution.
   Heat the media on a magnetic stirrer for 30 - 40 minutes until the solution becomes clear. The media that are
    to be poured into plates should be autoclaved directly (see step l).

j. Distribution to culture tubes or jars.
   The test tubes are placed in a rack and the medium is distributed into the tubes (20 ml/tube).

k. Put caputs.
    Select the color of the caputs according to media being prepared.

l. Autoclave the media.
   Sterilize media by placing the tube racks in the autoclave for 15 minutes at 121^oC (240^oF) and at 15 PSI.
    Do not extend heating time. Take the racks out of autoclave as soon as the pressure drops to zero.

m. Harden the agar medium in slanted tubes.
     Transfer the culture tubes to a slanted tube rack while the medium is hot and cool the media in the slanted
     tubes.

n. Store media until use.
    Place the tube racks in a plastic bag and store them at 4^oC until the next week's lab.
 

    Work in a group of 3-4 students. Obtain appropriate number of tubes for each medium and initiate cultures following the procedures
    outlined below:

1. General Procedures for Tissue Culture Propagation

a. Leaf disc cultures (African violet, petunia, salpiglossis, gloxinia, chrysanthemum)

b. Shoot tip cultures (rose, ficus, spirea, birch)

c. Meristem cultures (carnation, potato, banana, gerbera, syngonium)

d. Runner tip cultures (Boston fern)

e. Protocorm cultures (cattleya, cymbidium)

2. Plant Materials to be Cultured
 
 

No.	Plant	Original Explant	Stage of Culture	Medium Number	Number per group
1.	African violet (Saintpaulia ionantha)	Leaf disc, petiole	I	1	2
2.	Rose (Rosa hybrida)	Shoot tip	I	2	2
3.	Alder (Alnus sp.)	Shoot tip	II	4	3
4.	Chrysanthemum (Dandranthema grandiflora)	Leaf disc	I	1	2
5.	Carnation  (Dianthus caryophyllus)	Shoot tip	I	2	3
6.	Aspen  (Populus canescens x grandidentata)	Shoot tip	II	4	3
7.	Carrot  (Daucus carota)	Callus	II	3	3
8.	Orchid  (Cattleya, Cymbidium)	Meristem	II	5	3
9.	Boston fern (Nephrolepis exaltata 'Bostoniensis')	Frond	II	5	3
10.	Orchid  (Cattleya, Cymbidium)	Protocorm	II	6 (liquid)	3

3. Cultural Procedures
a. Culture initiation (Stage I)
    1) Culture of leaf discs: African violet, salpiglossis, gloxinia, chrysanthemum
        Using a cork borer, take leaf discs (8-mm diam) from young leaves and sterilize them in 5% Clorox for 10 minutes.
        Then rinse the leaf discs in sterile water 3-4 times in the transfer hood. Plant the leaf discs on the agar medium, one
        disc per tube. Observe for initial swelling and callus formation from the culture.

   2) Culture of shoot tips: rose, carnation, spiraea, birch, aspen
        Strip off leaves from actively growing branches and obtain shoot buds (apical, axillary), approximately 0.8-1.2 cm in
        size.Sterilize them in a 5% Clorox solution for 10 minutes and rinse them again in sterile water in the transfer hood.
        Place one explant per tube of solid medium. Observe shoot growth and proliferation.

 b. Subcultures (Stage II)
    1) Culture of shootlets: Indian laurel, birch, alder, spiraea, aspen
        Divide the shootlets from Stage II cultures of these species and subculture them on the solid media for further
        multiplication. Observe shoot multiplication on your culture; they may be divided continuously until a large number of
        plants is obtained. You also will be able to root them as they are large enough.

    2) Culture of protocorms: Cymbidium, Cattleya
        Isolate individual protocorms from the protocorm clusters of orchid and culture them separately on two solid culture
        media for plantlet generation. Place other protocorms in two different liquid media and incubate on a shaker. Observe
        shoot development from the protocorms in solid media and examine protocorm multiplication from the liquid culture.

    3) Culture of ferns: Boston fern
        Divide plantlets from the State II cultures of Boston fern and culture them on new media to increase the number of
        plants.Some rooted plantlets can be transferred to the soil medium.

    4) Culture of callus for embryogenesis: carrot
        Obtain carrot callus cultures and divide them into small pieces (2-4 mm diam.). Culture them on MS agar medium to
        induce asexual embryogenesis directly from callus tissues.

4. Incubation of Cultures
    All cultures are placed in the growth room which has light intensity of approx. 50-75 mol m^-2 sec^-2 at 22^oC. Carefully
    examine for any development of fungal or bacterial growth on the culture. All contaminated cultures must be eliminated as
    soon as possible. For lost cultures due to contamination, ask the lab assistant for additional culture material for
    replacement.
 

            Work individually or in groups. You will be responsible for further subcultures for culture multiplication, rooting, and soil
            establishment. You will be provided with various media needed for subcultures and soil establishment. Arrange for your work with
            the lab assistant in advance.

            1. Culture Multiplications (Stage II)
                If you need additional multiplication in subculture, ask the lab instructor for newly made culture media. You may take these
                multiple cultures for your own use after the semester is over.
            2. Rooting of Cultures (Stage III)
                Various culture rooting media will be prepared as needed. When your cultures are ready to be rooted, ask the lab assistant for
                appropriate media to use. The composition of rooting media varies with species. Cultures with 2-5 healthy roots can be
                transferred into soil medium.
            3. Plant Establishment in Soil (Stage IV)
                A large percentage of plantlets is lost during and after transplanting into soil, mainly due to lack of plant acclimation. Hence, be
                careful in handling in vitro grown plantlets at the Stage IV. Remove agar material carefully from the base of each culture and
                plant them gently in cell packs containing peat-lite mixes. Cover the cell pack containers with a plastic sheet for one week to
                retain high moisture conditions inside. When plants are well established, you may transplant them into larger pots.
 
 

Take notes as needed for the lab report.
 
 

PLSC 368 - Plant Propagation
Lab Exercise 2:  Questions
Spring Semester, 2004
 
 

        1. List the all ingredients of Murashige and Skoog (MS) medium in ppm concentrations.
 
 
 
 
 
 
 
 
 
 

            2. Show the concentrations of N, P, K, and Ca in the MS medium used:
 
 

                Calculations:

 
 

3. Calculate the concentrations of the following growth regulators:
 
 

Growth Regulator	Molecular Weight	mg/liter
		0.1 M	1 M	10 M	1 mM
NAA	186.2
IAA	175.2
KIN	215.2
BA	225.3

 

4. Why an agar medium containing growth regulators can not be over-sterilized in an autoclave?
 
 
 
 
 
 
 

5. Discuss the advantages of shoot tip cultures over leaf disc culture in propagation.
 
 
 
 
 
 
 
 

6. Discuss advantages of protocorm culture over seed propagation in orchid production.
 
 
 
 
 
 
 
 
 
 

7. Discuss problems that you have encountered in each stage of culture:

a.  Stage I cultures:
 
 
 

b.  Stage II cultures:
 
 
 

c.  Stage III cultures:
 
 
 

d.  Stage IV cultures:

8. Briefly describe what you have accomplished in this lab.