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 1 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 1 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 2 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 3 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. 4 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 5 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. 6 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 7 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 8
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