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ORCHID SEED

&
TISSUE
CULTURE
PhytoTechnology
Laboratories offers
several culture media
specifically developed
for both epiphytic and
terrestrial orchids.




PhytoTechnology Laboratories offers several media specifically developed for the in vitro culture of orchids. These media range
from basic seed sowing media to media for mericloning and stem propagation. We offer media that is composed of the basic
salts, which must be supplemented with other components before use, and media that is complete and only require the addition
of water. All of our media is manufactured according to cGMP standards in our environmentally controlled manufacturing facility
in Overland Park, KS. Each medium is first tested for specific physio-chemical specifications and then biologically tested with
two commercially significant plant cell lines. PhytoTechnology Laboratories is committed to maintaining inventory of its entire
plant tissue culture product line. Some features of our manufactured media include:

All media components meet USP or ACS quality standards
where applicable.
PhytoTechnology Laboratories has the capacity to
manufacture up to 10,000 liters of certain orchid media.
Using powder media simplifies media production and
reduces technician error when preparing media.
PhytoTechnology Laboratories offers complete orchid
media, which only require the addition of water to
prepare.
Powdered media has a shelf life of 2-3 years.
PhytoTechnology Laboratories offers secure on-line
ordering with no minimum order requirements.
Most orders are shipped within 48 hours of receipt of the
order.

Many times people question, which medium will work best for my application? We have prepared the following tables to help
with the selection process:

TERRESTRIAL ORCHID MEDIA

Seed Sowing Media

B138/ B141BM-1 Orchid Medium
M551Malmgrens Orchid Medium
T839Terrestrial Orchid Medium w/out NH4NO3
T842Terrestrial Orchid Medium w/out NH4NO3
T849Terrestrial Orchid Medium
Multiplication Medium

B142BM-2 Orchid Medium
Replate Media

T842Terrestrial Orchid Medium
PRODUCT UPDATE
EPIPHYTIC ORCHID MEDIA








Orchid seeds are very small and contain very little or no food reserves. A single seed capsule may contain 1,500 to 3,000,000 seeds. Sowing
the seed in vitro makes it possible to germinate immature seed (green pods). It is much easier to sterilize the green capsule than individual
seed after the capsule has split open. Lucke (1971) indicated that orchid seed can be sterilized when the capsule is about two-thirds ripe.
Listed below is the estimated normal ripening times of capsules for various orchid species (Lucke, 1971).

ORCHID GERNERA
TIME TO MATURITY
(MONTHS)
ORCHID GENERA
TIME TO MATURITY
(MONTHS)
Bulbophyllum 3 Laelia 9
Calanthe 4 Masdevallia 3.5
Cattleya 11 Miltonia 9
Coelogyne 13 Odontoglossum 7
Cymbidium 10 Paphiopedilum 10
Cppripedium 3.5 Phalaenopsis 6
Dendrobium 12 Stanhopea 7
Epidendrum 3.5 Vanda 20

Green Capsule Sterilization

1. Soak the green seed capsule in 100% bleach
solution for 30 minutes.
2. Dip the capsule in isopropyl alcohol or ethanol for
5-10 seconds. Remove the capsule from the
alcohol and carefully flame off the excess alcohol.
3. Under aseptic conditions, using a sterile knife or
scalpel, open the capsule and scrape out the
seed.
4. Carefully layer the seed over the surface of the
culture medium. Seal the culture vessels.
Dry Seed Sterilization

1. Collect seed and place in either a small flask ,
bottle or a shortened pipet, which has one end
sealed with cotton. Seal the other end of the pipet
with cotton once the seed have been inserted.
2. Prepare a solution containing 5-10% commercial
bleach containing a few drops of Tween 20
(Product Number P720)
3. Add the bleach solution to the flask or draw up the
solution into the pipet. Swirl the flask containing
the seed and bleach or repeatedly draw and
aspirate the bleach solution in and out of the pipet.
Replate Media

P748Orchid Replate Media
P785PhytoTech Orchid Replate Media
O156Orchid Replate Media
O139Orchid Replate Media
P668Orchid Replate/Maintenance
Medium
Seed Sowing Media

P723Orchid Seed Sowing
Medium
V895Vacin & Went Seed
Sowing Medium
K400Knudson C Medium
Multiplication/Stem
Propagation Media

O753Orchid Multiplication
Medium w/Agar
P793Orchid Multiplication
Medium
Mericloning Medium

K400 Knudson C Medium
M507Murashige Cattaleya
Orchid Medium
V891Vacin & Went Orchid
medium
ORCHID SEED GERMINATION
4. Sterilize the seed in the manner described in Step
3 for 5-10 minutes.
5. Remove the bleach solution and rinse the seed
with sterile tissue culture grade water.
6. Transfer the seed to sterile culture medium.

Replating Seedlings

1. It may take anywhere from 1 month up to 9
months for the seed to begin to germinate.
Approximately 30 to 60 days after germination
begins, it will be necessary to transfer the
seedlings to fresh media for continued growth.
2. Prepare an orchid maintenance/ replate medium
such as P748 for epiphytic orchids or T849 for
terrestrial orchids.
3. Under aseptic conditions, transfer the seedling
from the mother flask to the flask containing the
fresh medium. You should place the seedling
about apart on the medium.
4. Allow the seedlings to continue to grow and
develop. Root formation generally begins when
the plant has 2-3 leaves. Continue to transfer the
seedlings to fresh media every 30-60 days,
increasing the spacing between the plants with
each transfer. When the flask is ready for transfer
to a community pot in the greenhouse, most flasks
will have 15 to 25 plants depending upon the
species.
5. Transfer the plants into a community pot using a
finely ground orchid mix.







ORCHID STEM PROPAGATION METHODS

1. Remove any flowers that may remain on the flower stalk. Use clean, healthy, vigorous flower stalks with buds in their
nodes. Stalks on which only a few flowers have bloomed are best. Avoid old flower stalks.

2. Wash the flower stalk under running tap water for 5 minutes.

3. Prepare a 10% chlorine bleach solution and add 2-3 drops of Tween 20 (Product Number P720) to this solution.

4. Section the flower stalk into smaller section by using a clean razor blade or scalpel cutting between the nodes. Cut the
flower stalk into 50 100 mm ( ) sections leaving about 40 mm (1/2) below the node and the remainder above
the node.

5. Place the nodal section in the bleach solution (Step 3) for 15 minutes. Swirl the solution every 2-3 minutes.

6. After surface sterilizing, discard the bleach solution, and then under aseptic conditions, carefully remove the bract from
around the node.

7. Prepare a 5% chlorine bleach solution and add 2-3 drops of Tween 20 (Product Number P720) to this solution.

8. Once all of the bracts have been removed, surface sterilize the nodes in the 5% bleach solution prepared in Step 7.
Keep in this solution for 10 min, swirling the solution every 2-3 min.

9. Remove all of the bleach solution and rinse the nodes with sterile distilled water. Rinse by pouring the water over the
nodes, swirling, then pour off the water. Repeat this step three times.

10. Under sterile conditions, remove approximately 10 mm (1/8) from each end of the nodal sections using a clean, sterile
scalpel or razor. All tools should be dipped frequently in alcohol and flamed with a alcohol lamp (Product Number
B910), or heated in a glass bead sterilizer (Product Number S636) to maintain sterility.

11. Transfer the nodal section to the culture vessels containing Orchid Multiplication Medium (Product Number O753).
Insert the longer portion of the nodal section into the medium at a slight angle to just below the bud. This should result
in the emerging shoot pointing upwards.

12. Most shoots will generally appear within one month and they are ready for replating after about 60 days.

13. Many nodes exude phenolic compounds into the media which may turn the media dark brown to black. This phenolic
exudation will kill your nodes if you do not replate them to fresh media. The use of media containing charcoal will
reduce the required frequency of replating to remove phenolics as the charcoal adsorbs and binds the phenolic
compounds.

14. Replate onto Orchid Maintenance Medium (Product Number P748) and allow the plantlets to continue to develop and
root. Roots will begin to appear after 2 or 3 leaves have been produced.






ORCHID MEDIA PREPARATION PROCEDURES

Powdered media are extremely hygroscopic and must be protected from atmospheric moisture. If possible, the entire contents
of each package should be used immediately after opening. Media stored at 2-6 C and tightly sealed should last 2-3 years.
Preparing the medium in a concentrated form is not recommended as some salts in the medium may precipitate and affect shelf
life and storage conditions. The basic steps for preparing the culture medium:

1. Measure out approximately 90% of the required final volume of tissue culture grade water, e.g. 900 ml for a final
volume of 1000 ml. Select a container twice the size of the final volume.
2. While stirring the water, add the powdered medium and stir until completely dissolved. Media containing Charcoal,
Fruit Extracts, and/or Agar will not completely dissolve.
3. Rinse the container that the medium was packaged in with a small volume of tissue culture grade water to remove
traces of the powder. Add to the solution in Step 2.
4. Add desired heat stable supplements (e.g., sucrose, gelling agent, vitamins, auxins, cytokinins, etc.) (Orchid
media B142, B141, M551, P723, P748, P785, O753, T849, and V895 are complete media and generally do not
require the addition of any other components.)
5. Add additional tissue culture grade water to bring the medium to the final volume.
6. While stirring, determine and adjust, if necessary, the medium to desired pH using NaOH, HCl, or KOH. A pH of
5.2 to 5.4 is recommended for most orchid media. For small labs or home hobbyists, pH can be adjusted by using
baking soda to raise the pH and vinegar to lower the pH of the medium.
7. If a gelling agent is used, heat until the solution is clear. Heating may be required to bring powders into solution.
8. Dispense the medium into the culture vessels before (or after) autoclaving according to your application. Add heat
labile constituents after autoclaving.
9. Sterilize the medium in a validated autoclave at 1 kg/cm
2
(15 psi), 121C, for the time period described under
Sterilization of Media.
10. Allow medium to cool prior to use.

STERILIZATON OF MEDIA

Plant tissue culture media are generally sterilized by autoclaving at 121C and 1.05 kg/cm
2
(15 psi). Media can be sterilized in
either an autoclave or pressure cooker with similar results. Recently, the use of the microwave oven has also been shown to be
successful at sterilizing media. The time required for sterilization depends upon the volume of medium in the vessel. The
minimum time required for sterilization of different volumes of medium are listed below. It is advisable to dispense medium in
small aliquots whenever possible as many media components are broken down by prolonged exposure to heat. Times for
sterilizing in a microwave are based on using a 1000-watt microwave with a turntable for more even distribution of heat. The
times required for sterilization may vary depending upon the model of the microwave, power wattage, and the number of vessels
in the microwave.
MINIMUM STERILIZATION TIME FOR PLANT TISSUE CULTURE MEDIA

Volume of Medium per Vessel
(mL)
Minimum Autoclaving Time
a

(min.)
Minimum Microwaving Time
b

(min.)
25 15-20 4-6
50 25 6-8
100 28 8-10
250 31 10-12
1000 40 NR
2000 48 NR
4000 63 NR

a
Minimum Autoclaving Time includes the time required for the liquid volume to reach the sterilizing temperature (121 C) and
15 min at 121C (Burger, 1988). Times may vary due to differences in autoclaves. Validation with your autoclave or pressure
cooker is recommended.
b
Minimum Microwaving Time includes the time require for the liquid volume to reach a temperature of 121 C and remain at
this temperature for a period of 3-4 minutes. Media used in this study contained 1.0 mL/L of PPM, Product Number P820.
Validation with your microwave is recommended.





ORCHID MEDIA GUIDE
Procedure Product
Number
Product Description Comments
P 723 Orchid Seed Sowing Medium
w/ Agar
Complete Medium with Charcoal and Agar
K 400 Knudson C Orchid Medium Low salt formulation. Requires the addition of
Agar.
V 505 Vacin and Went Modified
Basal Salt Mixture
Low salt formulation. Requires the addition of
vitamins, sucrose and agar.
V 882 Vacin & Went Orchid Medium Low salt formulation. Requires the addition of
sucrose, and agar.
V 891 Vacin & Went Modified Basal
Salts
Low salt formulation. Requires the addition of
agar.
Seed Sowing
Epiphytic Orchids
V 895 Vacin and Went Modified
Basal Salt Medium
Low salt formulation. Complete Medium. Does not
contain charcoal.
B 141 BM-1 Orchid Medium Complete Medium. Does not contain charcoal.
Organic Nitrogen based medium.
M 551 Malmgrens Modified
Terrestrial Orchid Medium
Complete medium. Organic Nitrogen based media.
Seed Sowing
Terrestrial Orchids
T 849 Terrestrial (Cypripedium)
Orchid medium
Complete Medium. Does not contain charcoal.
Some species may require the addition of 1-2 mg/L
of Kinetin for best results. Inorganic Nitrogen
based media.
O 753 Orchid Multiplication Medium
w/Agar
Complete medium.
P 793 Orchid Multiplication Medium Requires the addition of a gelling agent.
Stem Props
(Phalenopsis and
other species)
M 507 Murashige Cattaleya Orchid
Multiplication Medium
Best if used at to strength. May need to be
supplemented with 1-2 mg/L of either BA or
Kinetin.
P 793 Orchid Multiplication Medium Use at to strength for best results.
M 507 Murashige Cattaleya Orchid
Multiplication Medium
Best if used at to strength. May need to be
supplemented with 1-2 mg/L of either BA or
Kinetin.
K 400 Knudson C Orchid Medium Low salt formulation. Requires the addition of
agar.
V 505 Vacin and Went Modified
Basal Salt Mixture
Low salt formulation. Requires the addition of
vitamins, agar and sucrose.
V 882 Vacin & Went Orchid Medium Low salt formulation. Requires the addition of
sucrose and agar.
Mericloning
V 891 Vacin & Went Modified Basal
Salts
Low salt formulation. Requires the addition of
agar.
P 748 Orchid Maintenance/Replate
Medium w/Banana
Complete medium. Works well with Phalenopsis,
Cattaleya, Dendrobium, and other species.
P 785 Orchid Replate Medium Complete medium. Works well with Cattaleya and
other species.
P 668 Orchid Maintenance Medium
w/Charcoal
Requires the addition of a gelling agent. Should be
supplemented with either banana powder or
coconut water. Use at to strength for best
results with some species.
O 156 Orchid Maintenance Medium
w/Banana and Charcoal
Requires the addition of a gelling agent.
O 139 Orchid Maintenance Medium
w/out Charcoal
Requires the addition of a gelling agent. Should be
supplemented with either banana powder or
coconut water
Replating
B 142 BM-2 Orchid Medium Complete Medium for Terrestrial Orchids. May
work well with Paphiopedilum and Phragmipedium
species. The addition of either banana powder,
pineapple powder or coconut water may be
beneficial.

Orchid Media Listings and Formulations: See the orchid media documents on the Product Information link on our web site.
Listed below are associated biochemicals, kits, and supplies for use with orchid culture.

Product
Number
Product Description Product Notes
Package
Size
A 296 AGAR
(Agar-Agar; Gum Agar)
Bacteriological Grade
Powder

Plant Tissue Culture Tested
CAS
Formula
MW
Storage Temp
Soluble In
9002-18-0
NA
NA
RT
WATER

100g
500 g
1 kg
5 kg
10 kg
25 kg
A 133


AGARGELLAN

A blend of agar and Gelrite.

Plant Tissue Culture Tested
CAS
Formula
MW
Storage Temp
Soluble In
NA
NA
NA
RT
WATER
500 g
1 kg
5 kg
B 852

BANANA POWDER
A mixture of Natural Banana Puree and Maltodextrin.


Plant Tissue Culture Tested
CAS
Formula
MW
Storage Temp
Soluble In
NA
NA
NA
RT
WATER
100 g
500 g
1 kg
C 325


CHARCOAL, ACTIVATED, ACID WASHED



Plant Tissue Culture Tested
CAS
Formula
MW
Storage Temp
Soluble In
64365-4-3
C
4
H
10
O
2
S
2

154.28
RT
WATER
1 kg
C 195


COCONUT WATER
Natural, No Additives


Plant Tissue Culture Tested
CAS
Formula
MW
Storage Temp
Soluble In
NA
NA
NA
2-8 C
WATER
100 mL
500 mL
1000 mL
G 469



GELRITE


Gellan Gum;
Powder

GELRITE

is a registered trademark for the Monsanto Company.

Plant Tissue Culture Tested
CAS
Formula
MW
Storage Temp
Soluble In
71010-52-1
NA
NA
RT
WATER
100 g
250 g
500 g
1 kg
2.5 kg
5 kg
10 kg
G 386


D-GLUCOSE, ANHYDROUS
ACS Reagent


Plant Tissue Culture Tested
CAS
Formula
MW
Storage Temp
Soluble In
492-62-6
CH
2
OH(CHOH)
4
CHO
180.16
RT
WATER
500 g
1 kg
5 kg
25 kg
K 424 KEIKI PASTE


Plant Tissue Culture Tested
CAS
Formula
MW
Storage Temp
8006-54-0
NA
NA
RT
50 mL
K 483


KINETIN SOLUTION (1mg/mL)
(6-Furfuryalaminopurine)

Plant Tissue Culture Tested
CAS
Formula
MW
Storage Temp
525-79-1
C
10
H
9
N
5
O
215.2
-20 to -0 C
100 mL
M 825


MES
2-(N-Morpholino)ethanesulfonic Acid
Free Acid; PKa=6.1 @ 25C;
Useful pH range 5.5-6.7
Plant Tissue Culture Tested
CAS
Formula
MW
Storage Temp
Soluble In
4432-31-9
C
6
H
13
NO
4
S
195.2
RT
WATER
100 g
500 g
1 kg
N 605


-NAPHTHALENEACETIC ACID SOLUTION (1mg/mL)
(NAA; 1-Naphthaleneacetic Acid)

Plant Tissue Culture Tested
CAS
Formula
MW
Storage Temp
86-87-3
C
12
H
10
O
2

182.6
0-5 C
100 mL
P 775

PEPTONE
Type 1
Enzymatic Digest From Meat

Plant Tissue Culture Tested
CAS
Formula
MW
Storage Temp
Soluble In
73049-73-7
NA
NA
RT
WATER
100 g
500 g
1 kg
P 780 PEPTONE, GLYSATE
From Gelatin

Plant Tissue Culture Tested
CAS
Formula
MW
Storage Temp
Soluble In
73049-73-7
NA
NA
RT
WATER
500 g
1 kg
Product
Number
Product Description Product Notes
P 862


PINEAPPLE POWDER
Dried Pineapple Puree w/Maltodextrin
Use at 5-30 g/L

Plant Tissue Culture Tested
CAS
Formula
MW
Storage Temp
Soluble In
NA
NA
NA
RT
WATER
P 820



PPM-PLANT PRESERVATIVE MIXTURE



Plant Tissue Culture Tested
CAS
Formula
MW
Storage Temp
Soluble In
NA
NA
NA
2-8 C
WATER
S 391


SUCROSE
(a-D-Glucopyranosyl-B -D-fructofuranoside; Saccharose)


Plant Tissue Culture Tested
CAS
Formula
MW
Storage Temp
Soluble In
57-50-1
C
12
H
22
O
11

342.3
RT
WATER
T 872

TOMATO POWDER
Dried Tomato Puree w/Maltodextrin


Plant Tissue Culture Tested
CAS
Formula
MW
Storage Temp
Soluble In
NA
NA
NA
RT
WATER
O 799 ORCHID SEED SOWING KIT, EPIPHYTIC ORCHIDS

P723--Orchid Seed Sowing Medium
P748--Orchid Replate Medium
P785Orchid Replate Medium
C913--Culture Vessels
F951--Forceps
P820PPM
P958--pH Indicator Strips
S963--Scalpel
S970--Scalpel Blades
Instruction Manual
O 788 ORCHID SEED SOWING KIT, TERRESTRIAL ORCHIDS

B141BM-1Orchid Seed Sowing
Medium
B142BM-2 Orchid Medium
M551Malmgrens Modified Terrestrial
Orchid Medium
T849Terrestrial Orchid Medium
C913 Culture Vessels
F951--Forceps
P820PPM
P958--pH Indicator Strips
S963--Scalpel
S970--Scalpel Blades
Instruction Manual
B 939 BEAKERS, GRADUATED, GRIFFIN
PMP, AUTOCLAVABLE
2000 mL
B 960

BEAKERS, GRADUATED, GRIFFIN
PMP, AUTOCLAVABLE
4000 mL

C 913

CULTURE VESSEL,
Clear Polypropylene, w/ Lid
4-5/8D x 2-7/8H,
Autoclavable
C 932 CULTURE VESSEL,
Clear Polypropylene, w/ Lid
4-5/8D x 5 H,
Autoclavable

C 032 CULTURE BOTTLE, GLASS, 32OZ (964 mL);
Milk bottle; w/logo & graduation marks



Product
Number
Product Description Product Notes Package Size
C 962

CYLINDERS, GRADUATED
PMP, AUTOCLAVABLE
10 mL
1 ea

C 964

CYLINDERS, GRADUATED
PMP, AUTOCLAVABLE
100 mL
1 ea

C 968 CYLINDERS, GRADUATED
PMP, AUTOCLAVABLE
1000 mL
1 ea

C 972

CYLINDERS, GRADUATED
PMP, AUTOCLAVABLE
2000 mL

1 ea

D 940 PETRI DISH
Disposable, Sterile
20 ea
500 ea
F 934 FLASKS, ERLENMEYER, 125 mL, WIDE MOUTH
Kimax
12/pkg
48/case
F 979 FLASKS, ERLENMEYER, 250 mL, WIDE MOUTH
Kimax
12/pkg
48/case
F 980 FLASKS, ERLENMEYER, 500 mL, WIDE MOUTH
Kimax
6/pkg
24/case
F 985

FLASK, ERLENMEYER
1000 mL, Wide Mouth
1 ea
6 ea
F 986

FLASK, ERLENMEYER
2000 mL, Wide Mouth

1 ea
4 ea
S 981

STOPPER, RUBBER, No. 6- 1/2, ONE-HOLE 25 ea
100 ea
S 983 STOPPER, RUBBER, No. 8, ONE-HOLE 25 ea
100 ea
S 984 STOPPER, RUBBER, No. 10, ONE-HOLE 6 ea
25 ea
50 ea
F 950

FORCEPS, DRESSING, 6
2.4 cm
1-9 ea
10+
F 951

FORCEPS, DRESSING, 8
3.1 cm
1-9 ea
10+
F 952

FORCEPS, DRESSING, 10
3.9 cm
1-9 ea
10+
F 953

FORCEPS, DRESSING, 12
4.7 cm
1-9 ea
10+
F 955

FORCEPS, CURVED, 6
2.4 cm
1-9 ea
10+
F 956 FORCEPS, CURVED, 8
3.1 cm
1-9 ea
10+
F 957 FORCEPS, BAYONET
8.25 Length (21 cm)
1-9 ea
10+
P 958 pH INDICATOR STRIPS
4.0-7.0 pH Range
100 ea
P 959 pH INDICATOR STRIPS
0-14 pH Range
100 ea
S 970

SCALPEL BLADE, NO. 10
Sterile Carbon Steel; Individually Wrapped
100/box
S 971

SCALPEL BLADE, NO. 11
Sterile Carbon Steel; Individually Wrapped

100/box
S 963

SCALPEL HANDLE, NO 3
5 Length (2.0 cm)
1-9 ea
10+
S 973

SCALPEL HANDLE, NO 3L
8 Length (3.1 cm)

1-9 ea
10+
PhytoTechnology Laboratories, LLC
SHIPPING ADDRESS: 7895 MASTIN OVERLAND PARK, KS 66204
MAILING ADDRESS: P.O. BOX 13481 SHAWNEE MISSION, KS 66282
PHONE: 1-888-749-8682 913-341-5343 FAX: 1-888-449-8682 913-341-5442
VISIT OUR WEB SITE AT: WWW.PHYTOTECHLAB.COM