PLSC368 - Plant Propagation Name________________________ Group__________Spring Semester, 2009Lab 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(NO3)2 4H2O) 1,000Potassium phosphate (KH2PO4) 250Magnesium sulfate (MgSO47H2O) 250Ammonium sulfate ((NH4)2SO4) 500Sucrose (C12H22O11) 20,000Ferrous sulfate (FeSO4 7H2O) 25Mananese sulfate (MnSO4 4H2O) 25Agar 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 cultureb. Culture medium: MS medium with agarc. 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.

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PLSC 368: Plant Propagation Name_______________________ Group___________Spring Semester, 2008

Lab Exercise 3

ORCHID SEED GERMINATION AND PROTOCORM CULTURE

1 Why should orchid seed be germinated on a sterile nutrient agar medium?

2 Discuss the advantages and disadvantages of propagating orchid by seed.

3 Discuss the advantages and disadvantages of orchid propagation by meristem and protocorm culture.

4 Describe the methods of orchid propagation other than the method used in this lab.

5 A brief comment on the orchid seed germination experiment you carried out.

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