Blog nr3 ‒ In vitro propagation of Paphiopedilum orchids

In vitro propagation of Paphiopedilum orchids (P.o.)

In vivo Paphiopedilum orchid

The focus of this blog is on the asymbiotic in vitro seed germination of P.o. with its pitfalls. To understand this, it is essential to know the in vivo process of symbiotic germination. Other processes are only described briefly, otherwise it would break the mold.

Under natural conditions P.o. are propagated vegetative (asexual; division of the rhizome) or generative (sexual; propagation through seed) (Sprunger, 2012).

Generative in vivo propagation of P.o.

Seed capsula of an orchid
with thousands of seeds

Orchid seeds don’t have reserve-substances (endosperm) to feed the germinating embryo, which is about 30 cells big. Under in vivo conditions they get nutrients from  mycorrhizal fungi. Normally mycorrhizal fungi form symbioses (the symbiosis it self is called mycorrhiza) with other plants, but in the case of in vivo orchid germination, only the orchid profits until the plant can operate photosynthesis. Orchid seeds release attractants which make mycorhizal hyphae grow towards those seeds with the aim to digest them. The hyphae are capable to break through the hard, germinate-inhibiting and waterproof shell (carapace; according to current knowledge it is in fact still not clarified if the carapace is germinate-inhibiting due to its waterproofness or to germinate-inhibiting substances or both) and penetrate in the outer cells of the embryo. This activates the germinating-process of the orchid seed. The embryo now turns the table and digests the hyphae. As long as the orchid has no leaves in order to conduct photosynthesis, it acts as a fungal parasite. Later on, when the orchid carries leaves, it forms a mycorrhizal symbiosis. This germinating-process is called symbiotic seed germination, although it isn’t really a symbiosis. (Bernert, 2006 - 2015)

Seed of Cephalanthera longifolia
with anemochoric extensions,
in the center the dark carapace

Symbiotic seed germination

In vitro propagation of P.o.

Besides the adoption of the mechanisms of in vivo propagation in the laboratory, there are possibilities to circumvent the natural principles, for example the replacement of the carapace-breaking mycorrhizal fungus. The methods can be divided in two main techniques:

1)      Vegetative propagation:

a.      Propagation through the division of axillary buds is a conventional but very inefficient and time consuming method (Zeng, et al., 2015).

b.      Propagation through rhizome division.

When the plant has at least six vegetation-points after flowering, the rhizomes can be split up into fragments, which should content at least three bulbs and at least two bunches of leaves. The connected roots are divided carefully, dead root-parts are removed and treated with active carbon to protect the cut surface.

(Sprunger, 2012);  (Violeta, kein Datum); (Unger, 2013)

2)      Generative propagation:

a.      Symbiotic seed germination (with mycorrhizal fungus)

b.      Asymbiotic seed germination (without mycorrhizal fungus)
(Bernert, 2006 - 2015)

For symbiotic in vitro seed germination of P.o., the appropriate mycorrhizal fungus is isolated and cultivated separately on agar. After all the required nutrition are added (macro- and micronutrients, which the plant would get out of soil under in vivo conditions), the P.o.-seeds are  placed on this breeding ground. The interaction with the mycorrhizal fungus will ensure the germination (Sprunger, 2012). Some steps in asymbiotic seed germination are analogue to the symbiotic seed germination, for example the mixture of agar, and can be taken from the text below. 

Asymbiotic in vitro seed germination

In asymbiotic seed germination under in vitro conditions, there is no mycorrhizal fungus used. So how the carapace is vanquished? How is the seedling fed until its autotrophic nutrition through photosynthesis?

Orchid seeds on Agar

The American botanist Lewis Knudson found in 1921 an asymbiotic method for in vitro germinating of orchid seeds. It was the first practical procedure for in vitro propagation of any plant under axenic conditions (Zeng, et al., 2015). As breeding ground he used agar, a polysaccharide out of algae that isn’t degraded from plants, mixed with  water (about 6g agar / L water), so that the seedling doesn’t drown and is grounded, but is still capable to penetrate the substrate. It is important to take distilled respectively deionised water, otherwise it would be impossible to compose the optimal nutrition-solution for the orchid. To ensure sterile conditions, that is required for in vitro cultures, and to break through the carapace, the seeds are put in a bleaching agent, for example 0,5% Natriumhypochlorit (NaOCl) (Bernert, 2006 - 2015). Now the embryo needs all the nutrition for building the plant up to

In vitro orchid

the stadium of leaf-building. Then, with photosynthesis, the synthesized assimilates will replace the energy-delivering nutrition, which P.o. takes from mycorrhiza under in vivo conditions (most other plants take it from the endosperm). The exacter the amounts of the ingredients, the bigger the chance for surviving. As replacement for the endosperm, sucrose is taken. Glucose could also be taken, but there are clues that during sterilisation, glucose has a plant-toxic impact. Beside macro- and microelements, the orchid needs also vitamins of the b-complex, phytohormones, myo-inositol (plays an important role as the structural basis for a number of secondary messengers) and active carbon (binds toxic products like phenols in his giant, inner surface). Indeed active carbon binds a lot of other substances, what gives the impression of taking away important molecules from the plant, like the extremely rare phytohormones. According to Dr. Claus Rüdiger Bernert, active carbon releases substances that the plant has used up, because there is an equilibrium between adsorption and desorption. (Bernert, 2006 - 2015)

If you want to imitate in vitro propagation of P.o. or other orchids as described above, have a look at the website www.orchideen-im-garten.de (chapter Gastbeiträge), where Dr. Claus Rüdiger Bernet reports of his experiences. Indeed it is in German, but you should be able to read it. Besides of a list with the exact amount of the required substances, there are also very good advice for private citizens on how to replace for private citizens unavailable substances (for example with pineapple- or potato-juice). These istructions are highly practical and based on theoretical background.

References

Bernert, C. R. (2006 - 2015). Orchideen im Garten. Von http://www.orchideen-im-garten.de/gast_saat1.php abgerufen

Bernert, C. R. (2006 - 2015). Orchideen im Garten. Von http://www.orchideen-im-garten.de/gast_saat7.php abgerufen

Sprunger, S. (2012). In-vitro Kultur einheimischer Orchideen. Basel: Schweizerische Orchideenstiftung am Herbarium Jany Renz - Botanisches Institut der Universität Basel.

Unger, M. (2013). Pagewizz. Von http://pagewizz.com/orchideen-tipps-fuer-die-vegetative-vermehrung/ abgerufen

Violeta, R. (kein Datum). Pflanzenfreunde. Von http://www.pflanzenfreunde.com/paphiopedilum.htm abgerufen

Zeng, S., Huang, W., Wu, K., Zhang, J., Teixeira da Silva, J., & Duan, J. (2015). In vitro propagation of Paphipedilum orchids. USA: informa healthcare - Critical Reviews in Biotechnology.