in vitro seedlings to fully established hardy plants for the garden

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 Jong1
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E-mail: [email protected]
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 Figure 1. The first seedling I raised from seed
proper vernalization (cold treatment) took 4 years after deflasking to flower and was a
and choice of proper substrates to C. reginae.
plant the deflasked seedlings in. In this
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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How to raise in vitro propagated Cypripedium seedlings to fully established hardy plants for the garden
© Camiel F. de Jong, December 2004
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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How to raise in vitro propagated Cypripedium seedlings to fully established hardy plants for the garden
© Camiel F. de Jong, December 2004
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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How to raise in vitro propagated Cypripedium seedlings to fully established hardy plants for the garden
© Camiel F. de Jong, December 2004
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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How to raise in vitro propagated Cypripedium seedlings to fully established hardy plants for the garden
© Camiel F. de Jong, December 2004
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 11.5 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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How to raise in vitro propagated Cypripedium seedlings to fully established hardy plants for the garden
© Camiel F. de Jong, December 2004
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 Figure 4. This C. arietinum seedling flowered
weeds. One of the most detrimental two years after deflasking. However, overall
pests is liverwort. This moss tends to seedling survival was very low.
overgrow the seedlings, causing them
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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How to raise in vitro propagated Cypripedium seedlings to fully established hardy plants for the garden
© Camiel F. de Jong, December 2004
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
Figure 5. C. kentuckiense seedlings during their
deflasking although I had one C. first growing season after deflasking. This
arietinum seedling that already spectacular plant is one of the easiest to deflask.
flowered two years after it had been
removed from the flask.
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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How to raise in vitro propagated Cypripedium seedlings to fully established hardy plants for the garden
© Camiel F. de Jong, December 2004
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:[email protected]
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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