PRACTICAL 2: TISSUE CULTURE

INTRODUCTION

Fresh water macrophytes play very important role in aquatic ecosystems. They provide, either directly or indirectly, food, shelter and variety of habitats for a large number of organisms, including wildfowl and economically important fish. It must be mentioned that rice, an aquatic plant, is the most important single crop species in the world. Many other aquatic plants are also of use to men as food, raw materials for industrial processes, building materials and manure in agriculture. 

Aquatic plants absorb dissolved minerals and enrich water with oxygen produced during photosynthesis. These properties are benefit to man as they assist in the maintenance of clean water and the help in the recovery of polluted water. However, in disturbs or newly constructed bodies of water rampant growth of aquatic plants may interfere with man’s use of freshwater. They may obstruct water flow, navigation or water intake. They may also create conditions favourable for pests, diseases and vector affecting humans, animals and crop plants. Although some plants lack a central stem, and plants such as mosses and ferns do not produce flowers, the anatomy of most plant can be split into four basic zones; the root stem, leaves, and flowers. All these parts play a vital role in the plant’s basic functions, including growth, reproduction, nutrient-collection and storage.

METHODOLOGY

1) PROPAGATION OF RHIZOME BUD/LEAF (GINGER)

Four gingers sized about 2 inches each were put into a beaker with an amount of dishwasher. The gauze was used to close the beaker. The beaker was let on running tap water in order to remove soil particles on the gingers. This process was run for about 10 minutes.

 

Each ginger was put into a beaker and 60% of Clorox was poured into each beaker added with 7 drops of Tween 20 for 15 minutes. Then  they were thoroughly washed once with sterilized distilled water. All beakers were shaken continuously. This process was done in laminar air flow. Then, those gingers were immersed again in 20% Clorox added with 7 drops of Tween 20 for another 10 minutes and thoroughly washed 5 times with sterilized distilled water.

The sterilized gingers were dissected to remove the outer layer under aseptic condition by using scalpel and forcep. Then, the buds of the ginger were cut for about 1mm each and it was inoculated on to the culture medium.

Final tissue culture of ginger.

2) PROPAGATION OF SEEDS

The seeds of groundnut were washed with sterile distilled water and then being shake for 3 minutes. After 3 minutes, the distilled water were decanted and 95% ethanol was added into the beaker that have washed groundnut. The mixture were shake for 1 minutes. After 1 minute, the ethanol was decant and 20% of chlorox plus 1-2 drops of Tween 20 were added into the beaker and then being shake for 10 minutes. After 10 minutes passed, the groundnut were rinsed with distilled water for 5 times and being dried in laminar flow. After the groundnut was totally dried, the seed was planted in each test tube containing Murashigae and Skoog (MS) agar.

RESULTS

Figure 1

       

Figure 2

                                                                  Figure 3

DISCUSSION

After 2 weeks, the result is out. According picture 1, explant in round jar medium showed full dark brown. Thus, according picture 2 and 3, explant in test tube medium some also turn brown neither cloudy. All the surface medium in test tube shown white mould which the explant s culture are contaiminated. This showed that produce free contamination tissue culture is difficult by M. M. Khatun et al (2016). Hence used of disinfectants agent can be one of the factor which we are using Clorox. Clorox maybe showed moderate function and cause contamination by Mol Jamsheera et al (2016). However, this experiment result is negative compare to Faridah et al (2011) where MS medium supplemented with a combination of 5.0 mg/l BAP and 2.0 mg/l IAA or 3.0 mg/l BAP and 0.5 mg/l IAA produced the highest mean number of shoots (5.6) per explant by using the same method in term of disinfectants agent and technique instead of media. Hence, Thayamini H. Seran et al (2013) claimed common problem in ginger is bacterial contamination where can be controlled by incorporation of antibiotics to initial culture medium.

REFERENCES

1.  Kanchanaree Pongchawee, Ph.D. (Aquatic Plants and Ornamental Fish Research Institute). Advanced Freshwater Aquaculture: Aquatic Plants in Aquaculture
2.  Faridah, Q. Z., Abdelmageed, A. H. A, Julia, A. A. and Nor Hafizah, R. Efficient in vitro regeneration of Zingiber zerumbet Smith (a valuable medicinal plant) plantlets from rhizome bud explants. African Journal of Biotechnology Vol. 10(46), pp. 9303-9308, 22 August, 2011. DOI: 10.5897/AJB11.1182. ISSN 1684–5315. 
3.   Jamsheera Mol, M. J, Vivas Salim, Shiji M Chemparathy, Resiya Karim and Umesh Balakrishnan T. An efficient protocol for raising contamination free micropropagation of Zingiber officinale (Ginger).  Journal of Pharmaceutical and Biological Sciences. Received: 12-07-2016 / Revised Accepted: 24-08-2016 / Published: 25-08-2016. ISSN: 2320-1924. 
4.  M. M. Khatun, T. Tanny, Abdur. M. Razzak, Firoz Alam M, Ekhlas Uddin M, Ruhul Amin and S. Yesmin.  Standardization of In Vitro Sterilization Procedures For Micropropagation of Ginger ( Zingiber officinale Rosc.). Volume-7, Issue-1, Jan-Mar-2016. ISSN: 0976-4550. 

5.  Thayamini H. Seran , 2013. In vitro Propagation of Ginger (Zingiber officinale Rosc.) through Direct Organogenesis: A Review. Pakistan Journal of Biological Sciences, 16: 1826-1835.

PRACTICAL 1: PREPARATION OF ALGAL CULTURE MEDIUM

INTRODUCTION

Algae are simple plants that differ from the microscopic (microalgae), to large seaweeds (macroalgae), such as giant kelp. Thus, cyanobacteria, (similar to bacteria, and formerly called “blue-green algae”) as well as green, brown and red algae were example of microalgae . However, Freshwater and marine fish culture has commonly used mircroalgae as food source by De Pauw, N. and G. Persoone, 1988.

Guillards’ F/2 Medium is one the example for algae culture medium. Guillard’s (F/2) Marine Water Enrichment Solution 50 X, is a concentration that can be diluted in filtered seawater to support reconstitution of Guillard’s (F/2) Marine Enrichment medium that make up of of major nutrients, trace metals and vitamins.  Guillard’s (F/2) Marine Enrichment medium commonly use for growing of coastal marine algae (diatoms).

The objective for this experiment is to prepare different medium for algal culture.

METHODOLOGY

Preparation of marine algal medium (Guillards’ F/2 medium)

Stocks	Per litre
NaNO3	75g
NaH2PO4.2H2O	5.65g
Trace elements (chelated)	Trace elements (chelated) consist of: NA2 EDTA = 4.16g FeCl3.6H2O = 3.15g CuSO4.5H2O = 0.01g ZnSO4.7H2O = 0.022g CoCl2.6H2O = 0.01g MnCl2.6H2O = 0.18g Na2Mo4.2H2O = 0.006g
Vitamin mix	Vitamin mix consist of: Cyanocobalamin (Vitamin B12) = 0.0005g Thiamine HCl (Vitamin B1) = 0.1g Biotin = 0.0005g

Ratio to produce 1L of F/2 medium :-

1mL NaNO₃ : 1mL NaH₂PO_4.2H₂O : 1mL trace elements : 1mL vitamin mix : 1L seawater

*Add while stirring, sterilise by autoclaving for 15 minutes at 15 psi and use when cooled to room temperature.

Preparation of F/2 + Si (Guillard’s medium for diatoms)

Stocks	Per litre
NaNO3	75g
NaH2PO4.2H2O	5.65g
Trace elements (chelated)	Trace elements (chelated) consist of: NA2 EDTA = 4.16g FeCl3.6H2O = 3.15g CuSO4.5H2O = 0.01g ZnSO4.7H2O = 0.022g CoCl2.6H2O = 0.01g MnCl2.6H2O = 0.18g Na2Mo4.2H2O = 0.006g
Vitamin mix	Vitamin mix consist of: Cyanocobalamin (Vitamin B12) = 0.0005g Thiamine HCl (Vitamin B1) = 0.1g Biotin = 0.0005g
Sodium metasilicate	Sodium metasilicate consist of: Na2SiO3.9H2O = 30.0g

Ratio to produce 1L of F/2 + Si (Guillard’s medium for diatoms) :-

1mL NaNO₃ : 1mL NaH₂PO_4.2H₂O : 1mL trace elements : 1mL vitamin mix : 1L filtered seawater : 1mL sodium metasilicate

*Add while stirring, sterilise by autoclaving for 15 minutes at 15 psi and use when cooled to room temperature.

RESULTS

Figure 1 Guillards' F/2 medium

Figure 2 Guillards' F/2 + Si (diatom purpose)

DISCUSSION

Guillard’s (F/2) Marine Water Enrichment Solution is a concentrate of major nutrients, trace metals and vitamins that can be diluted in filtered seawater to support reconstitution of Guillard’s (F/2) Marine Enrichment medium. It is a widely used enriched seawater medium for growing costal marine algae (diatoms). 

The preparation of the growth medium represents a considerable part of running cost in the process of fish culture (Zhang et al., 2001). It was reported that 30-40% (max.70%) of marine hatchery operating costs can be attributed to micro-algal culture (Heasman et al., 2001). Media for the culture of marine algae generally consist of a seawater base (natural or artificial) supplemented by analytical quality nutrients (Bold & Wynne, 1985). The term ‘nutrient’ is generally applied to any element or compound necessary for algal growth. Analytical grade nutrients chemicals (macronutrients and micronutrients) represent the most expensive constituents in any culture medium (Molina et al., 2003).

Furthermore, preparation of algal culture media is labor-intensive. Media are generally prepared from premixed stock solutions. Aliquots from these stocks are measured and added to a given volume of seawater. Micronutrient stock solution, however, must be prepared by combining aliquots of pre-prepared stock solutions and adding them to a given volume of distilled water, from which aliquots are measured and added to seawater, which may complicate the preparation processes. Precipitation of one or more component of the medium, may be encountered upon inaccurate preparation or combining the stock solutions of the culture medium (Harrison & Berges, 2005).

Additionally, the nutritional value of algae produced is high and comparable to that of conventional, analytical grade fertilizers such as F/2 (Valenzuela-Espinoza et al., 1999).  

CONCLUSION

This algal culture media are easy to be prepared one of simple medium for the algal growth as they require enough nutrient. For diatom such as Chaetoceros, they need nutrient for building their outer cell wall or known as silicate.

REFERENCES

1. http://allaboutalgae.com/what-are-algae/

2. El Nabris, K. J. A. (2012). Development of cheap and simple culture medium for the microalgae Nannochloropsis sp. based on agricultural grade fertilizers available in the local market of Gaza strip (Palestine). J Al Azhar Univ Gaza (Nat Sci), 14, 61-76.