Plant Tissue Culture Aseptic Technique

Basically the techniques of plant cultures consist of taking a piece of plant such as a stem tip, meristems, seed or even an embryo and placing it in a liquid, semi-solid or solid in sterilization, nutritious medium for the tissue to growth. The common medium is gel-based such as agar or broth (Wikipedia, 2010). The formulation of medium is depends on the products whether trying to produce callus tissue, grow roots shoots (National Health Museum, 2010). After sometimes, the progenitor cells may migrate out of the tissues if there is no contamination and get enough nutrients supported by the medium (Wikipedia, 2010).

There is several thing must be considered before doing plant cultures. Firstly, selection plant source is important as there is some species disinclined to respond in tissue culture and some do not respond (National Health Museum, 2010). Thus, selects the species which is easier to grow in culture is the prior, foremost step. There are several parts of plants that can be taken directly such as the leaf in making tissue culture, but some organ of plants such as roots need to cut open to examine their structure before deciding which tissue will be selected as explants.

In this experiment, Crassula argentea has been chosen to be cultured in lab with establishment of aseptic cultures. The tissue selected for surface sterilization is larger than explants because the outer layer which has been exposed to the pathogen will be cut off later. The tissues must be cut sharply to reduce the amounts of decaying material and the possibility of being infected using sharp scalpel (Nadjeeb, 2009). The appropriate size of the piece of plant is only less or equal to 2cm because the probabilities of contamination are increase with the size.

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Next, the plant must be healthy and clean from any contamination. The plant should be wash below running tap water if it is contaminated with soil and sterilized to eliminate all microorganisms. Fungi or bacteria may grow on the plant surface and contaminate the media when they are not adequately eliminated. Surface sterilization can be done using one or combination of the following solutions ethanol, dilution of commercial bleach preparation (containing sodium hypochlorite) and aqueous solution of mercuric chloride. Mercuric chloride may be used to removal the micro-organism if the treatment by ethanol or bleach solution not successful, but it is always avoidable due to the toxicity. The treatment must be done in prescribe time because long, continuously exposure leading the chemical to kill the tissues.

Thirdly, producing a proper medium is essential to ensure transplant can develop well within it. Generally, the hormones are added before the medium is sterilized. It can be done by choosing specific hormones that promotes the growth in suitable scale. There are several types of hormones such as auxin, cytokinin and gibberelin. However, only auxins (IBA) and cytokinins (BAP) are using for plant culture in this practical. The parts of the plants which will develops are depends on the type of the hormones present in the medium as the hormones auxin helps to stimulate roots while cytokinin promote the growth of shoot (carnegiescience.edu, 2010).

Finally, the plant tissues now are ready to be culture and it is vital all the procedures take place in sterilization condition such as in laminar flow clean air bench. The tissues cannot be placed out, once it was brought into the laminar flow clean air bench to prevent contamination until all procedures are complete. Suitable site are required for growing plant tissue cultures such as in clean, warm place and have adequate light.

Tissue culture must be done in sterilization condition, placed in nutritious medium, and keep it in suitable environment for well growth. It is proving to be useful in myriad ways because it can be applied in biotechnology: to determine the plant propagation, raising and maintenance of high health status plants, and germ plasm storage (Nadjeeb, 2009). Hence, it must be done properly to obtain the good, viable tissues.

Materials and Methods

Three pieces of immature, healthy looking leaves were selected and cut for the sterilization using scalpels. Each piece was cut off in 3 parts and producing total up 9 pieces. All the pieces were brought to laminar flow clean air bench for surface sterilization treatments using 70% ethanol for 2 minutes, 10% commercial bleach solution for 20 minutes and 10% commercial bleach solution for 30 minutes. The ethanol and bleach solutions were poured into separate jar and followed by located 3 leaf pieces in the ethanol and 6 other pieces in the bleach jar. The timer was started immediately after the pieces were put into the solution. From this time onward the plant tissues has been handled aseptically in the laminar flow bench only. Each piece of plant tissue was washed separately twice in sterile purified water in 2 small bottles, McCartney bottles at least 1 minutes in each bottles after prescribe treatment time. Next, the tissues were transferred to a sterile surface (sterilized Petri dish) and the outer layers were cut off using sterilized instruments (scalpels). The final explants (less than 1 cm cubed) were transferred aseptically onto medium in the culture vessels- 1 explants per culture vessel. There are three types of media which can be seen in the table below:

Medium

Portion

Condition of explants

Aim

A

MS medium +

0.1mg/l IBA +

2.0 mg/l BAP

Absent of shoot meristem, e.g. leaf tissue and stem internode of Crassula argentea

Induce direct

organogenesis

B

MS medium +

2.0mg/l IBA +

0.1 mg/l BAP

Absent of shoot meristem, e.g. leaf tissues and stem internode

Induce callus formation

C

MS medium without plant growth regulators

Control medium

Each piece of plant tissue has been cultured on each of medium A, B and C. These materials have been handled by 25 groups to obtain reliable results as different people may handle it differently.

Finally, the tissues were incubated after all the explants were transferred to each medium. The top of the cultures were sealed with parafilm and were placed on a shelf of controlled environment room in the light at 25°C. The result of the condition of tissues have been observed 4 weeks later and recorded either there is present of contamination or the explants had growth.

Discussion

From the result obtained, it can be observed that 68% of the tissues are not growth. It is due to contamination of fungus, bacteria or yeast. The contamination may occur when the processes of sterilization on plants, culturing tool and incubator are not done well. In addition, the medium which is prepared for tissue culture also a good nutritional source for many microorganisms like fungus, bacteria and fungi. Once the contaminations are establish, they will grow rapidly, quickly and deplete the medium. They will produce toxin which affect the growth or ultimately kill the tissues (Guri et al, 1998).

Besides, there are 32% plant grow which can be observed in formation of root, shoot or callus. The tissues are capable of surviving after have been sterilized well and kept in suitable environment. There are different types of growth as the constituents of the media will vary the formation of organ. The highest percentage of formation is callus followed by root and shoot. The percentage of two or three formation such as root and callus formations are among the lowest. It happens because the nutrients only sufficient to support one type of formation which is aided by the hormones. Different types of hormones will determine different types of growth. As example, the shoot growth mostly can be seen in Medium A due to high cytokinin hormone while the root formation mostly can be seen in Medium B due to high auxin hormone. Most of callus formation also can be found in Medium B as it is a precursor of adventitious rooting (Ernst and Holtzhausen, 1987). Hence, it proves that the hormones auxin stimulates roots while cytokinin promotes shoot.

From the data, the best solution and duration of sterilization is Bleach B in 20 minutes. 20 minutes are the most suitable time because short period (10 minutes) will not sterilize all the contaminations like fungus, yeast and bacteria while longer period (20 minutes) may kill or reduce the viability of the tissues. Solutions used to sterilize explants must preserve the plant tissue but at the same time destroy any fungal or bacterial contaminants. Next, bleach is more advanced than ethanol to eliminate the contaminations. Bleach is an oxidizing agent that can destroy organism’s fold structure and leading to sterilization. For ethanol, it plays the role by dehydrating of protein and enzyme and disrupting the cell membrane to prevent bacteria keep growing. Unfortunately, the researcher found ethanol is not the best solution because some bacteria have resistant to the ethanol and uneasy to be killed (www.cte.ku.edu, 2010). Hence, the bleach is more effective than ethanol in against contamination in duration of 20 minutes.