PLSC368 - Plant Propagation Name________________________ Group__________
Spring Semester, 2009
Lab Exercise 3
3. ORCHID SEED GERMINATION AND PROTOCORM CULTURE
Note: This lab is designed to train students in the propagation of orchid by seed, meristem and protocorm cultures.
A. Orchid Seed Germination and Seedling Culture
1. Seed Source:
Phalaenopsis ( Phalaenopsis spp.)
Showy Pink Lady’s-Slipper Orchid ( Cypripedium reginae )
2. Germination medium: Knudson's C medium
Phytamax Medium (Sigma Co.)
Table 1. Knudson’s C medium used for orchid seed germination
Chemical mg/liter
----------------------------------------------------------------------------------------------------------------------
Calcium nitrate (Ca(NO
3
)
2
4H2O) 1,000
Potassium phosphate (KH2PO4) 250
Magnesium sulfate (MgSO
4
7H
2
O) 250
Ammonium sulfate ((NH
4
)
2
SO
4
) 500
Sucrose (C
12
H
22
O
11
) 20,000
Ferrous sulfate (FeSO
4
7H
2
O) 25
Mananese sulfate (MnSO
4
4H
2
O) 25
Agar 10,000 pH 5.0-5.2
3. Seed sterilization
Surface sterilize the seed in a 5% Clorox solution for 10 minutes and rinse in a sterile water in the transfer hood.
4. Sowing the seed
Using a metal wire loop, take out a small quantity of the surface-sterilized seed and streak them on the germination medium in the transfer hood.

5. Incubation and observation
Place the plate in the growth chamber with light and observe seed germination and protocorm development throughout the semester.
B. Protocorm Multiplication and Plant Induction
1. Protocorm Multiplication a. Plant source: Protocorms of Cymbidium orchid established in liquid culture b. Culture medium: MS medium without agar (liquid medium) c. Culture and observation
Place 2-3 protocorms in a flask containing the liquid MS medium and put the flask on a shaker.
The slow-speed gyrator culture will provide oxygen to tissues submerged in the liquid medium.
Record the number of protocorms formed per culture.
3. Plant Induction a. Plant source: Cymbidium orchid protocorms in liquid culture b. Culture medium: MS medium with agar c. Culture and observation
Using a pair of forceps, transfer protocorms from liquid medium to the agar medium (one protocorm/test tube) in the transfer hood. Incubate the culture in the light and observe shoot development from the protocorm. When shoots are about 10 cm with roots well developed, transfer the plant into a 4"-plastic pot containing orchid soil medium in the greenhouse.

PLSC 368: Plant Propagation Name_______________________ Group___________
Spring Semester, 2008
Lab Exercise 3
1. ORCHID SEED GERMINATION AND PROTOCORM CULTURE
2. Why should orchid seed be germinated on a sterile nutrient agar medium?
3. Discuss the advantages and disadvantages of propagating orchid by seed.
4. Discuss the advantages and disadvantages of orchid propagation by meristem and protocorm culture.
5. Describe the methods of orchid propagation other than the method used in this lab.
6. A brief comment on the orchid seed germination experiment you carried out.
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