Deflasking of Cypripedium seedlings - Cypripedium.de

How to raise in vitro propagated Cypripedium seedlings to fully established hardy plants for the garden 

© Camiel F. de Jong, December 2004 

How to raise in vitro propagated Cypripedium 

seedlings to fully established hardy plants for the 

garden 

Camiel F. de Jong 1 

1 E-mail: camieldejong@zonnet.nl 

Introduction 

Orchids belonging to the genus Cypripedium are amongst the most attractive 

hardy plants. Previously thought to be hard to grow in one’s garden and 

impossible to propagate artificially, nowadays lab-propagated seedlings are 

readily available through the internet. Seedlings are a cheap alternative to 

mature plants. Moreover, mature plants may originate from the wild and 

consequently raising Cypripediums 

from seedlings relieves collection 

pressure from wild populations. 

However, for the beginner the 

seedlings are often easier to procure 

than to grow into a flowering plant. As 

the seedlings have been raised from 

seeds in the sterile protected 

environment of the flask, they are 

vulnerable to the hostile environment 

they face after they are removed from 

the flask. It’s difficult to piece together 

the bits of available information on 

how to successfully raise these frail 

and vulnerable seedlings into a 

vigorous and hardened plant. With this 

lack of knowledge, people start 

enthusiastically and are consequently 

discouraged by failure which is a pity 

since when a few basic rules are 

observed, flowering Cypripediums are 

not that hard to raise from seedlings. 

The most important parameters 

governing success are determined by 

proper vernalization (cold treatment) 

and choice of proper substrates to 

plant the deflasked seedlings in. In this 

Figure 1. The first seedling I raised from seed 

took 4 years after deflasking to flower and was a 

C. reginae. 

article I will discuss the important steps to grow Cypripedium seedlings 

originating from in vitro propagation to an established hardy plant. The methods 

described here work well for me but are by no means the only way. When 

specific months are mentioned one must bear in mind that these hold for the 

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climate in North-Western Europe. In these cases also the prevailing 

circumstances are mentioned so the reader is able to adjust to his or her own 

climate. 

Dormancy 

To survive the cold winter, the shoots of Cypripediums die down in autumn after 

having produced a new dormant bud. When it’s not freezing, roots continue to 

grow whereas the bud stays dormant. The dormancy of the bud is only broken 

after a three month cold period of temperatures below 5ºC ensuring the bud 

won’t develop prematurely into a shoot that will freeze to death during winter. The 

extent of the required cold period prevents the shoot from growing during warm 

spells during winter. In horticulture cold treatment is also applied and is called 

vernalization. Vernalization of Cypripedium seedlings is best carried out at 4ºC 

during a period of three months. Seedlings just out of flask have to deal with 

enough challenges already and should not be subjected to freezing. Although 

hardy plants are able to survive freezing, there’s always some damage done that 

may render the seedlings more vulnerable to attack by molds. 

If Cypripediums did not have proper vernalization, no shoot will develop. Plants 

may survive until next spring and then develop a shoot but it’s most likely they 

will perish during summer. Although some species only require a vernalization 

period of two months it’s best to subject seedlings to a vernalization period of at 

least three months to let the bud develop into a shoot in spring. 

Removal of Cypripedium Seedlings from the Flask 

When the seedlings have roots with a minimum length of around 3 cm and have 

visible dormant buds, they are ready to be removed from the flask. If not, let them 

grow on to this stage. Seedlings can be deflasked until January; after this date 

the cold period will become too short, because seedlings must be planted out 

before, or at latest in April (when only some light frost during night may occur). 

The first reason for this is that for strong shoot development the seedlings seem 

to require temperatures below 20ºC. The second reason is that for deflasked 

seedlings to develop their new bud, the growth season must be sufficiently long. 

The length of the required growth season depends on the species. The more 

Northern species such as C. guttatum, C. yatabeanum, and C. passerinum only 

require a short growing season but a relatively cold one as well. The species 

from warmer climates such as C. fasciolatum and C. kentuckiense need a longer 

growing season for proper new bud development. 

When having reached the proper size, the seedlings are carefully removed from 

the agar and transferred to a sieve. The plants are then washed with cold water 

under the shower until all agar is removed. The seedlings are then transferred to 

a clean ziplock plastic bag with a few drops of water added to it and labeled with 

name, date of removal from the agar (and the start of vernalization!), and number 

of seedlings. At this stage I always take an analogue picture from the bag with 

the seedlings and the clearly visible label. This will later demonstrate that the 

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mature plants were raised from seeds. Of course this is not full proof but it forms 

a good indication. 

Vernalization 

Now the seedlings are ready to vernalize. The bags containing the seedlings are 

now transferred to a polystyrene-foam box. These boxes are available through 

laboratories or restaurants and are used to ship products that should be kept cool 

during transport. Make sure the box is clean. The bags with the seedlings are 

transferred to the box and the lid is closed. The box is then transferred to the 

refrigerator (4ºC). The box serves as a buffer for the fluctuations in temperature 

that occur in the refrigerator during normal operation. These temperature 

fluctuations cause condensation on the inner surface of the plastic bag and as a 

result some seedlings or parts of seedlings, dry out. The dried out parts die and 

form a good substrate for moulds when they re-hydrate again by contact with 

drops of condensation during normal storage or handling. The polystyrene box 

greatly reduces these problems. However, the seedlings should be checked 

regularly for development of mold. If this occurs, the affected seedlings must be 

removed. 

Alternatively, for larger numbers of seedlings, Bill Steele from Spangle Creek 

Labs, vernalizes his seedlings by transferring them to a polyethylene food 

storage box. Then a layer of water is added so that it’s just covering the bottom 

of the box and the box is placed in the refrigerator. Temperature changes during 

refrigeration cause condensation not only on the inner surface of the box, but 

also on the seedlings themselves, and so no desiccation occurs. 

The vernalization period should be at least three months and six months at most. 

Vernalization as described above requires the least cooled space. However, 

when one has sufficient space that can be kept frost free but below 4ºC, 

seedlings may directly be planted in the substrates and vernalized this way. This 

method is preferable as seedlings have time to settle themselves during 

vernalization, but is quite costly in terms of space. When using this method, care 

must be taken that there’s sufficient air movement to prevent molds from 

growing. However, drying out of the substrate must also be avoided. 

Substrate Mixes 

The mixes I use to plant the newly deflasked seedlings in are composed of 

Seramis, Perlite and Vulca. Seramis is a substrate that is developed for 

hydroponics. It consists of baked clay beads with a lot of pores. The material 

retains a lot of water and is also able to bind nutrients. It is not (yet?) available in 

the US but another product, Turface, seems to resemble it. This product is used 

to cover baseball fields. However, I never tested it. 

Perlite is an expanded volcanic material that retains some water but no nutrients. 

Vulca is basically pumice. Vulca itself is pumice from the Hekla volcano on 

Iceland. It makes the substrate mix somewhat drier and perhaps releases 

nutrients upon erosion. With the exception of Vulca these substrates cannot be 

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used pure but should be used in mixes. Seramis retains too much water to be 

used on its own and Perlite on the other hand dries out too fast and does not 

retain nutrients. 

A mix that works well for me contains equal parts Seramis, Perlite and Vulca. 

However, since Seramis is quite expensive it may be omitted. As Perlite gives off 

irritating dust, wear a dust mask during mixing of the substrates and wet them. 

The substrates that are used to plant newly deflasked seedlings in must be 

completely inorganic. Any organic material present in these mixes may promote 

Figure 2. Photographs of individual substrates of mixes used to deflask Cypripedium seedlings. 

The scale of the ruler is in centimeters. 

growth of molds and consequently loss of seedlings. However, there are two 

exceptions: C. acaule and C.arietinum. Seedlings of both species are planted out 

in a mix of equal parts coarse river sand and sphagnum peat. The C. arietinum 

seedlings seem to prefer more sand in the mix as compared to C. acaule 

seedlings. After planting the C. acaule seedlings MUST be watered with water to 

which vinegar 1 tablespoon (approximately 30 milliliters) of cider vinegar to 2 L of 

water is added to keep the substrate at low pH. To my knowledge, Scott Durkee 

of the Vermont Ladyslipper Company was the first to have acknowledged this. It 

is absolutely required for the survival of both C. acaule seedlings as well as 

mature plants. The plants in containers must be sheltered from rain as the rain 

washes out the acids in the substrate, yielding a pH above 4.5. In nature, C. 

acaule plants live in soils that are self sustaining acidic having a pH of around 

3.5. When the pH rises to above 4.5, water-soaked lesions develop in the leaves 

due to pathogens that are subdued at low pH but get a chance at higher pH. 

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Planting the Seedlings 

It is best to plant seedlings in 

rectangular containers that are around 

18-20 cm deep and are 30 by 50 cm, 

depending on the amount of seedlings. 

Drill some holes in the bottom to avoid 

water accumulation. I prefer the large 

containers as they are least subject to 

fluctuations in moisture content. Fill 

the containers with the substrate mix 

until 5 cm below the rim. Flatten the 

surface and put the seedlings on with 

the bud facing upwards. Cover the 

seedlings with additional substrate 

until only the tip of the bud is just 

visible. Put in a label with name and 

date etc, and spray with water until it 

leaks out at the bottom. Use a fine 

spray to ensure all substrate is wetted. 

The substrate is so well draining that 

when a normal spurt of water is used it 

directly drains to the bottom without 

wetting adjacent substrate. The 

seedlings are now ready to be put in 

the cold for vernalization or in case 

they have already been vernalized in 

bags they can be put outside when 

severe frosts are out of the air. Place 

them in a spot without direct sunlight 

during the late morning and afternoon. 

When the plants are outside, I apply 1- 

1.5 gram of the slow release fertilizer 

Osmocote® to the substrate surface. 

The release of nutrients from the 

Osmocote® granules is temperaturedependent 

and lasts 6 months for the 

variety I use. This variety contains also 

trace elements and has a N-P-K-Mg 

ratio of 15, 9, 15, 2%, respectively. It 

ensures a constant availability of low 

levels of nutrients. When conventional 

fertilizer is used, nutrient levels rise 

dramatically at time of application and 

drop sharply after watering or raining. 

The high levels of nutrients during 

application of conventional fertilizer 

Figure 3. C. acaule seedlings at various stages 

in their development. The upper panel 

represents acaule seedlings just after 

deflasking. The middle panel shows the same 

seedlings that emerged in their first year after 

deflasking, and the lower panel shows the 

seedlings in their third growing season. At this 

stage they have to be repotted to ensure good 

growth during next season. To the original mix 

of 50/50 peat and coarse sand 10% larch 

needles are added to the mix. Seedlings are 

repotted when the new growth has matured in 

summer 

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also promote pathogen growth. The constant low levels of nutrients supplied by 

slow release fertilizer ensure a constant uptake during the growing season and 

the overall levels of nutrient uptake during the year are much higher. Again, the 

secret is to avoid fluctuations as much as possible. Do NOT fertilize C. acaule 

and C. arietinum. 

It’s important to keep the growing area clean and well ventilated in order to avoid 

the development of molds. Also, remove any dead seedlings or dead parts. In the 

event molds develop, some fungicides may be used. When gray mold (Botrytis 

cinerea) appears, it can be controlled with products containing chlorothalonil. 

However, gray mold is often the result of an untidy growing area and is more a 

question of prevention instead of spraying chemicals. 

Another problem that may occur is damping off. This is caused by Pythium 

species and may be controlled by Previcur® N (active agent: Propamocarb HCl). 

However, in most countries this agent is only available for professional use. 

I am still in the process of identifying pathogens that attack Cypripediums and 

this will perhaps be the subject of a coming article. 

When seedlings had proper vernalization and are subjected to temperatures 

conducive to growth, the buds will start stretching within two to three weeks. At 

this point, aphids may often attack the 

newly developing shoots resulting in 

malformation or even complete death. 

To prevent this problem, I spray the 

seedlings with Admire® (Active agent: 

Imidacloprid). This is a systemic 

insecticide that has the additional 

advantage that snails seem to have 

less appetite for treated seedlings. 

Most of the time spraying once a 

season is sufficient but when aphids 

reappear again an additional spray 

should be applied. 

When the seedlings are grown 

outside, weeds must be controlled. 

They should be removed as soon as 

they appear since when they are 

allowed to grow bigger the seedlings 

are also uprooted upon removal of the 

weeds. One of the most detrimental 

pests is liverwort. This moss tends to 

overgrow the seedlings, causing them 

Figure 4. This C. arietinum seedling flowered 

two years after deflasking. However, overall 

seedling survival was very low. 

to rot during winter, and if they survive at all, it prevents the new shoot from 

emerging. When liverwort appears, it should be removed immediately. I don’t 

know of any treatment available against liverwort that does not harm the 

seedlings as well. Manual removal is the only option so far. 

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Additional Care 

In fall the seedlings have developed their new dormant bud and may be removed 

from the inorganic substrates when they look sufficiently hardy and placed in the 

substrates used for mature plants. If they are still frail they may be left in the 

inorganic substrate for another year. Most of the seedlings may be left outside 

now. However, C. fasciolatum and C. plectrochilon seedlings must be protected 

against wet winters with alternating warm and cold spells as these will effectively 

kill them. Care must also be taken that seedlings do not “freeze up”, caused by 

continuous freezing and thawing. The freeze/thaw cycles not only cause damage 

to the plants themselves but also result in continuous expansion and 

recompression of especially the inorganic substrates. This causes seedlings and 

small plants to “float” to the surface 

exposing their rhizome and roots to 

dry winter air. The roots or the whole 

seedling consequently dries out killing 

the seedling. Also when only parts are 

dried out and re-hydrate during rain, 

they will present a good substrate for 

molds that will eventually kill the whole 

seedling. This may be a problem only 

in sea climates with alternating warm 

and cold spells depending on wind 

direction. In climates where winters 

are cold and plants are covered with 

snow this will be less of a problem. 

However, when it occurs the plants 

should be covered with fresh 

substrate. 

In spring the cycle starts again, and in 

general the first seedlings will start to 

flower four to five years after 

deflasking although I had one C. 

arietinum seedling that already 

flowered two years after it had been 

removed from the flask. 

Figure 5. C. kentuckiense seedlings during their 

first growing season after deflasking. This 

spectacular plant is one of the easiest to deflask. 

Concluding Remarks 

In this article I have attempted to describe how to successfully deflask 

Cypripedium seedlings. It is a description of what kind of techniques work well for 

me and is the outcome of six years experience of sowing and deflasking 

Cypripedium seedlings. It is intended as a base for growers who start deflasking 

Cypripedium seedlings and to push them in the right direction and encourage 

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people to raise Cypripediums from seedlings themselves; the sight of a flowering 

Cypripedium raised from a seedling by yourself is very rewarding. 

For beginners I would recommend C. reginae, C. kentuckiense, C. californicum 

and C. parviflorum ssp pubescens to start with. C. reginae seedlings are by far 

the cheapest and easiest to procure. C. kentuckiense is one of the most striking 

species of the genus and is also one of the easiest to deflask. However, 

seedlings of this species are more difficult to find and are more expensive. Below 

the reader will find some addresses for obtaining Cypripedium seedlings. The 

author also has a varying offer of Cypripedium seedlings. 

Acknowledgements: 

I wish to thank Bill Steele, (Spangle Creek Labs, http://www.uslink.net/~scl/) for useful 

comments and critically reading the manuscript. 

Copyrights 

This article may be distributed freely for private means in its entire form. For 

reproduction of individual parts or figures ask permission of the author. It is 

prohibited to distribute this article or its parts for commercial purposes without 

explicit permission of the author. 

Sources of Cypripedium seedlings 

For Europe: The author has a varying offer of Cypripedium seedlings and may be 

contacted via: 

mailto:camieldejong@zonnet.nl 

For North America: Bill Steele from Spangle Creek Labs offers a variety of 

Cypripedium seedlings. See his excellent website at: 

http://www.uslink.net/~scl/ 

Additional addresses are available through the Cypripedium Forum of Michael 

Weinert: 

http://www.cypripedium.de/forum/messages/574.html 

Also available on the web: 

“Artificial pollination of Cypripedium species” by the same author at 

http://www.cypripedium.de/forum/pollination.pdf 

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Deflasking of Cypripedium seedlings - Cypripedium.de

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