In vitro Propagation of Paphiopedilum orchids

 

Among the possible techniques for propagate Paphiopedilum orchids species and hybrids a distinction is made between tissue culture- and seed germination methods.

This first technique, that confines to the mere accumlulation of clonal plants in a sterile environment (in vitro), includes amongst others: stem node culture or –allongation, axillary shoot culture, shoot apex culture method, or shoot-/root regeneration through callus induction.               
This applications basically benefits from the characteristic that totipotent cells (which can be found in coverage of wounded cut surface of plant) have the potential to form new meristematic tissue and later on can differentiate in any type of cell leading to an entire, independant plant organism [1][2].

 

Abbildung 1 : Callus induction of orchid Vanilla planifolia

Thereby samples from any region (buds, leaf, stem, root) of mother plant are cultured resulting in the formatation of undifferentiated callus and, through steered, focused initiation procedures (hormone activity) this protocorm callus provide new plant individuals.

Abbildung 2 : Regeneration of shoots and roots using tissue culture technique

Achieving good labor conditions to obtain espescially good  target tissue for good shoot- /root induction and plant growth results thus remains a main objective.

The distribution of P. orchids, based on a same method is of no market value status  yet because the problem with P. orchid species is the rarity of good basic raw material like seedlings. Especially among explants stemming from greenhouse transmitting fungal or bacterial infections is difficult to avoid and therefore not acceptable in laboratory practice. Great losses in supply related to low multiplication and plant grow were identified at a later stage due to such difficult prerequisite, but they must be dealed with in the context of the available laboratory system, technical equipement which define the conditions for propagating.
Several attempts were made to approach this question with alternative approaches  and from different angles. To remedy the situation, scientists for example investigated the role of stem cutting methodology on shoot  and root regeneration while finding out that horizontal cutting was much more efficient [3]. Another point of interest dealing with the problem of inefficient propagation is to find a protocol which allows to do the induction of roots and shooting on the same medium, whereas there were success to report [4].
Nevertheless, the P. orchids can not keep abreast of market competition adopting this methodology.

 

The second mainly practiced procedure consists of the asymbiontic seed germination method. Thereby it`s worth mentioning that Paphiopedila orchids growing naturally in nature areas live in symbiosis with funghi which provide the plant worthful nutritients. So, if the intention is to guarantee  this nutritient supply under laboratory conditions trough optimal medium composition, many others weighing  factors as pH value, light influence and temperature have to be observed and incorporated. 

 

Abbildung 3 : steps, seed germination of orchids

But the remaining difficulty is still to find the exact optimal medium compositon (proportion of minerals and organic compounds) depending in part on their role as growth retarding or -stimulating substances. Results of studies show, that, for instance B vitamins complex and other co-enzymes playing an important rule in metabolic processes  do have a positive impact on germination and plant increase in further stages, while other low-molecular-weight organics as aminoacids, proteins and nitrogen rather inhibit plant germination. But, based on this new knowledge this problem can be counteracted by adapting the laboratory conditions and the medium composition to the needs of the plant seeds [5],[6]. Overall it is important that while the in-vitro processes, the seeds as a starting material can receive water, nutricients and other organic substances added in the medium.  For this, seeds with a permeable testa are more likely to sprout. Otherwise, a low germination percentage emerge from[7].    

Approaches to solutions such as appropriate time of seed collection preparatory treatement of the seeds accelerating germination proceeding are available in different surveys [8], in this case the duration of the pretreatement is one point worthy of note. It shall be explicity mentioned here that the interaction of the different factors vary depending on the plant developement stage (seed, protocorm, plantlet,…) and the investigated Paphiopedilum species [9].

The market supply of Paphiopedilum orchids is possible thanks to this method and Paphiopedilum species and hybrids remain the only commercially-produced orchids which are not clonally propagated until now, and therefore every individual plant has a different genetic composition or « make-up ». This is essential to consider the complexity of ecolocical structures with a view to their resilience, persistence and resitance and to the preserving stocks of this endangered specie.

(Excuse for lack of figures. Illustration will be published as soon as possible.)

 

 

Literature :

 [1] Hung, L.-C. (November 2001). Paphiopedilum cloning in vitro. Abgerufen am 10. Mai 2015 von Science Direct: http://www.sciencedirect.com/science/article/pii/S0304423801002400

[2] Wen, P. J. (September 2012). Research Gate. Abgerufen am 10. Mai 2015 von Shoot multiplication of Paphiopedilum orchid through in vitro cutting methods: http://www.researchgate.net/publication/267843500_Shoot_multiplication_of_Paphiopedilum_orchid_through_in_vitro_cutting_methods

[3] Hussey, G. (1976). In vitro Release of Axillary Shoots from Apical Dominance in Monocotyledonous Plantlets. Norwich: John Innes Institute.

[4] Saleh, N. M.-Y. (2010). In vitro propagation of Paphiopedilum orchid through formation of protocorm-like bodies. Springer Science + Business Media B.V.

[5] Chen, Y. (January 2015). Asymbiotic seed germination and in vitro seedling development of Paphiopedilum spicerianum: An orchid with an extremely small population in China. Global Ecology and Conservation, Volume 3 , S. 367-378.

[6] Zeng, S. (Mai 2012). Asymbiotic seed germination, seedling development and reintroduction of Paphiopedilum wardii Sumerh., an endangered terrestrial orchid. Scientia Horticultura, Volume 138 , S. 198-209.

[7] Zeng, S. (January 2015). In vitro propagation of Paphiopedilum orchids. Critical Reviews in Biotechnology , S. 1-14.

[8] Lee, Y. (März 2011). In Vitro Culture and Germination of Terrestrial Asian Orchid Seeds. Methods in Molecular Biology, Volume 710 , S. 53-62.

[9] Long, B. (2010). In vitro propagation of four threatened Paphiopedilum species. Springer Science+Business Media B.V.