Practical 7: Aquatic Macrophyte Tissue Culture

Introduction

Our ecosystems are being polluted day by day and there are less people out there care about it, it is the main cause that our sensitive plants died because they are unable to cope with the change. Aquatic systems continue to be among the world’s most threatened ecosystems (Zedler and Kercher, 2005). The threats will lead the aquatic biota become increasingly stressed, resulting in reduced growth and reproduction (Sim et al., 2006) and ultimately death, leading to a decline in species richness (Hart et al., 1990). So, in order to preserve them as well as wanting to grow faster, tissue culture was introduced.

Objective
To successfully culture Anubias nana sample using only leaf cuttings on agar media.

To observe the growth of leaf sample on MS media.

Materials and Method

Our explant are Anubias nana.

In the laminar flow, the bottle are sterilized and mouth of the medium bottle before pouring. 

 The Murashigae and Skoog(MS) agar are poured into sample bottle and let it cooled down.

 Explant are cutted 1cm each, 4 pieces of leaves are cutted.

 The explants are washed with distilled water and 2 to 3 drops of tween 20 (Soap like)

 Explants are shaken for 3 minutes without stopping and the bubble are presented.

 After that. rinsed with distilled water for 3 minutes (shake continuously).

Here is our model of the shaking mechanisms.

Washed the explants with 70%, 50%, 30% and 10% of chlorox for 1 minute for each treatment and rinsed with distilled water for 3 minutes ( with continuous shaking) 

 Explants that are sterilized are kept in the bottle to prevent contamination before use.

 In the laminar flow,  explants are washed with 70% ethanol ( shake for 3 minutes), then rinsed with distilled water (shake for 3 minutes as well).

 The explants are then transferred to on top of tissue paper to cut the dead parts.

The dead parts which are in yellow colors.

 

The explants are planted on the surface of the agar.

4 of the explants are successfully planted.

Results

After 2 weeks, It was observed that most sample has a growth of fungus and bacteria which was due to contamination during handling which can be seen in sample TP1, TP2 and TP3 with a high growth of contamination on TP2. TP1 on the other hand had minimal contamination but no difference was observed. However, TP4 was successful with no contamination and developing red edges on the area it was cut which will soon develop into a callus.

Image : TP2 with great degree of contamination.

Image : Shows the various contamination which occur on TP1, TP2 and TP3 with TP1 having least contamination.

Image : TP4 free from any contamination and red edges shows the future development of callus.

Discussion

Aquatic plants can take up excessive nutrients and also play a crucial role in creating a favourable environment for a variety of chemical, biological and physical processes that contribute to the nutrient removal and degradation of organic compounds (Chong et al, 2004). Tissue culture are widely use might because harvesting of aquatic plants from their natural habitat will become a threat to the species richness (Lauzer, 2004). Besides, tissue culture have advantage such as good quality of planting materials which disease and virus free at a competitive price while conserving aquatic plants in their natural habitat. Large scale plant production also can be programmed and preservation of plant species in vitro is also possible (Yapabandara and Ranasinghe, 2006), in addition, Many tissue cultured water plant species show a more bushy growth with more adventitious shoots, qualities that many will appreciate (Christensen, 1996). However tissue culture must be culture in a sterile environment as you can see from TP1, TP2 and TP3 which have bacteria contamination, it might be cause by infection during preparation or the seal are not tight enough, we are lucky to have TP4 uncontaminated and the red edges shows that the explants are growing. 

Conclusion

Tissue culture can be hard to culture sometimes due to the cleanliness of the air in the environment and even laminar flow could not prevent it, if there are no contaminations, high chances are the explants going to grow.

Reference

Chong, Y. X., Hu, H.Y. and Qian, Y. (2004). Advances in utilization of macrophytes in water pollution control, Tech. Equip. Env. Pol. Contr. 4 : 36–40.

Christensen, C. (1996). Tropical aquarium plants Denmark. Aquaphyte online: University of Florida. http://www.tropica.dk. Cited 10 January 2008. 

Hart, B. T., Bailey, P., Edwards, R., Hortle, K., James, K., McMahon, A., Meredith, C. and Swadling, K. (1990). Effects of salinity on river, stream and wetland ecosystems in Victoria, Australia. Water Res. 24, 1103–1117. 

Lauzer, D. (2004). In vitro embryo culture of Scirpus acutus. Plant Cell, Tissue and Organ Culture 76: 91-95.

Sim, L. L., Chambers, J. M. and Davis, J. A. (2006). Ecological regime shifts in salinised wetland systems. I. Salinity thresholds for the loss of submerged macrophytes. Hydrobiologia 573: 89–107.

Yapabandara, Y. M. H. B. and Ranasinghe, P. (2006). Tissue culture for mass production of aquatic plant species. 

Zedler, J. B. and Kercher, S. (2005). Wetland resources: status, trends, ecosystems services, and restorability. Annu. Rev. Environ. Resour. 30: 39–74.