In vitro multiplication and rooting of Camellia japonica

In this blog post I will analyse an in vitro cultivation experiment of the plant Camellia japonica L. conducted by Seglie et al.[1]

Treatments in the experiment

The scientists used a set of different treatments to examine the impact of them on the in vitro multiplication and rooting of 19 Japanese genotypes and 4 commercial cultivars of Camellia japonica.

In a first step they varied the plant hormones in the substrate, one group with 6-benzyladenine (BA) and the other with BA and gibberellic acid (GA₃). BA is a cytokinin which stimulates the cell division and inhibits apical dominance.[2] GA promotes the germinability and the elongation of plants.[3] Plants with these two substrates where then held in darkness or in a 16/8 h photoperiod (16 hours light and 8 hours darkness).

In a second experiment the plants were first multiplied on a substrate with BA and then placed on a medium with or without indole-3-butyric acid (IBA) to bring them in the rooting stage. IBA is an auxin, these plant hormones promote the rooting of plants and the apical dominance.[4] So they work as an antagonist to the cytokinins. In one group the IBA was included in a nanosponge and in the other not.

Bacterial contamination rate

In darkness the explants from auxiliary buds showed a bacterial contamination rate of 94.8%, explants from shoot tips just 54%. This is no surprise because auxiliary meristems fall in a dormancy after they are built, they only start to make a side branch when they get the signal by plant hormones.[5] In this timespan bacteria can penetrate into the meristem, in contrast the growing apical meristems let no time for the bacteria to wander to the tip of the meristem. During dormancy there is also not a high rate of metabolic activity as in the active apical meristem, so the bacteria is not much inhibited. As a conclusion I would say they took meristems from not activated auxiliary meristems and growing apical meristems, this would explain the huge difference in bacterial contamination.

Nanosponges

Nanosponges are nanocarriers, the word nano refers to materials between 1 and 100 nm, these very small carriers can be used to absorb, release and transport molecules.[6] Nanosponges are relatively new and promising, there is a wide field of applications like in medicine or in water cleaning.[7] With this small scale technique it is possible to engineer on a molecular level. As an example, toxins anywhere in human bodies can be captured and transported to the liver.[8]

In the study of Seglie et al. they used the nanosponge β-CD-NS 1:4, it is based on a β-Cyclodextrine (CD) molecule. β-CD is a ring of seven glucose molecules, it is polar on the outside and apolar on the inside, this makes it perfect to transport an apolar molecules like IBA and bring it into the plant where it gets dissolved in the polar cell water (see Image 1).[9] With this setting the IBA can be transported into the plant more efficient because the hormone does not get lost in the apolar substrate and the release of the IBA extend on a longer interval.[10]

Image 1: β-Cyclodextrine Ring (CD) with an indole-3-butyric acid (IBA) inside (assembled from
www.trc-canada.com and http://commons.wikimedia.org).

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[1] Seglie, L., Caser, M. Berruti, A., Scariot,V. (2011). Investigations on In Vitro Multiplication and Rooting in Camellia japonica L.VII International Symposium on In Vitro Culture and Horticultural Breeding.

[2] Siddiqui, W., Bhattacharjya, A., Chakraborty, I., Dhua,R.(2011). 6-Benzylaminopurine improves shelf life, organoleptic quality and healthpromotingcompounds of fresh-cut broccoli florets. Journal of Scientific & Industrial Reserch 70, S.461-465.

[3] Vandenbussche, F., Fierro, A.C., Wiedemann, G., Reski, R., Van Der Straeten, D. (2007). Evolutionary conservation of plant gibberellin signalling pathway components. BMC Plant Biology 7:65.   

[4] Campbell & Reece (2006). Biologie. München: Pearson Studium. S.971.

[5] Campbell & Reece (2006). Biologie. München: Pearson Studium. S.874 f.

[6] Cristina, B., Pacheco, I., Robbie K. (2007). Nanomaterials and nanoparticles: Sources and toxicity. Biointerphases 2:1.

[7]  Hu, C.M.J, Fang, R.H., Copp, J., Luk, B.T., Zhang, L. (2013). A biomimetic nanosponge that absorbs pore-forming toxins. Nature Nanotechnology 8, S. 336-340.

[8] Jesse Emspak (2011). Nanosponges To Suck Up Toxins. From:http://news.discovery.com/tech/nanotechnology/universal-antidote-in-a-sponge-130414.htm

[9] Seglie, L., Martina, K., Devecchi, M., Roggero, C., Trotta, F., Scariot, V. (2011). β-Cyclodextrin-based nanosponges as carriers for 1-MCP in extending the postharvest longevity of carnation cut flowers: an evaluation of different degrees of cross-linking. Plant Growth Regulation 65:3 S. 505-511.

[10] Roggero, C.M., Di Stefano, D., Tumiatti, V., Tumiatti, M., De Vecchi, M., Scariot,V., Kapila, S. (2012). Use of functionalised nanosponges for the growth, conservation, protection and disinfection of vegetable organisms. Patent WO 2013046165 A1.