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.

Blogg_Nr2_Sven

A case study of human genome-supported changes in the understanding of diseases 


Personalized cancer-treatment

To defeat cancer there are hundreds of diffrent medicaments, which can be applied in various doses and ingestion-frequences. The aim is to identify the most effective medication with the slightest byeffect(s) to heal the patient. It is a very complex and time-consuming challange for the oncologists to find the matching treatment-strategy for the patient. Here genome-sequencing can help: Through compare the genome data of cancer-cells with the data of a healthy cell (naturally from the same patient) and the data of other patients (the DNA-data of both, healthy and cancer cell, is two terabytes big, that means three billion datapoints), oncologists can now work more efficient. Because it's impossible to regard all factors manual, geneticists (enterprise MolecularHealth) work together with computer scientists (enterprise SAP). An application should help oncologists by comparing the DNA-data with the help of a big database. "This in turn means that medics such as oncologists will have more time for their core duty – looking after their patients." 


Relevance of the complete human genome

In this case, like in the most cases, not the whole genome is important for the diagnostic / treatment. Relevant are especially the genes that differentiate in healthy and defective cells, because they are responsible for the malformation of the affected cells.


Change of survival of the patient (my opinion)

I guess the probability to improve the survival-chance of a cancer-patient through aply genome-sequencing, especially in combination with clever softwares, is really big. In short time this method shows the diffrences between the healthy and the cancer-genome. That shortens the diagnosis and the treatment-plans significant.
Once there will be a mature software with a big (DNA-)database of many cancer-patients. If then a scanned genome of a cancer-patient matches with the data of a succesfully cured former patient, a good treatment is instantly available. That doesn't mean, that it must be right the very best treatment for this patient, but it supplies the oncologists a very good initial situation and spares them a lot of time, that raises in turn the survival-probability of the patient.

http://www.sap-investor.com/en/2012/quarter-4/sap-hana/fighting-cancer-with-sap-hana.html

3.4 Personalized medicine

Clinical genome sequencing

Dangers of personalized medicine through human genome sequencing

The sequencing of one genome could predict various diseases, so that preventive measures can be adopt to avoid or at least to stem the disease. But these chances bring also disadvantages. Through the knowledge of the risk of a disease, health insurances could refuse affected people or raise their contributions.

Beside the financial reason, the ethical question is also very important. Imagine a sequencing of your genome would uncover, that you will die on cancer in 20 years. Although you could react earlier to fight against the cancer, wouldn't it be better if you would receive this information later? Wouldn't you have a better life, not counting your remaining time and knowing when you will get what sort of suffering? It could be even dangerous if there would be a number of people, who know more or less when they die (especially when they die young), because they would live more unrestrained, what could maybe increase the criminal rate.

Better managemant with the genome data

So if this proceed would be apply, the prediction should be kept back from the concerned person until the time when medical measures make an impact. But this moment ought be the latest time to inform the patients, because giving them medicaments without letting them know on what they suffer, would be even more ethical critical.

Societal concerns and lifestyle-change

The main concern of the society, like already mentioned, is the fear of an unfair treatment by the insurance companies in case of a diagnosed disease. Furthermore knowing to become diseased could change the lifestyle of a concerned person. The reactions can be very different. In some cases it could end in self-pity, in a depression, thinking often of one’s own sad destiny. This negative thinking could lead to rude or even criminal behaviour. But it's also conceivable that such a diagnose could enhance the concerned life-quality. People who react in this way want to exploit the healthy rest of their life as much more. It really depends on the character of the affected person. (Up to here, there have no references been used.)

An example how genome data has changed the diagnostic practice

Apart from all these dangers, there are certainly some positive points of the medical application of the genetic fingerprint. For example has this technology changed some cancer-diagnoses (but even more some cancer-therapy). Although the classification of cancer is mainly done through histological analysis of tissue sections or cells, there have also been used molecular markers for years for some tumour types, such as breast cancers and leukaemia. The expression level of mRNA transcript is measured through a microarray-based expression profiling. That leads to a rapid improving of the ability to classify cancers, particularly in early-stage breast cancers, colon cancers and hematologic cancers. (Dave et al., 2006; Lo et al., 2010a; O'Connel et al., 2010; Paik et al., 2004; Rosenwald et al., 2002; Wang et al., 2005). Genes included in particular cellular pathways are frequently mutated due to somatic alterations that directly conduce to the abnormal growth of the cancer cell. Detecting the presence or absence of mutations through sequencing within these genes can help predicting a patient's response to a specific targeted therapy. (Lynch et al., 2004; Paez et al., 2004)

Would this technological change improve the chance of survival of the patient? 

If a disease is referable to a genetic predisposition, it can be identified earlier with genome sequencing, so that the therapy can take place at the right moment and the progression of the disease can be observed anytime. In some cases it is only possible to detect the possibility if a disease will occur. Then the therapy can arrange preventive steps or at least the patient would be observe regularly.
In my opinion personalized medicine through genome sequencing has a big potential to improve the chance of survival of the patient for mentioned reasons. Certainly could the survival-chance of a patient be the same through conventional methods, but then it needs also luck, that the disease will be diagnosed early enough. Many diseases could be diagnosed through genome sequencing early enough, while these could keep hidden without until the patient remarks the first symptoms.