Plant Tissue Culture Assessment Of Aseptic Cultures Biology Essay

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It is very crucial to produce aseptic cultures when preparing tissue culture medium. This ensures that the tissue culture prepared is contaminant-free. This report shows the methodology used to obtain aseptic cultures from varies types of solution as exposed in different time period. The results also being analysed and discussed further to explain the observations that was obtained. This research is the fundamental to the techniques in plant biotechnology.

INTRODUCTION

Tissue culture is the method by producing new explants just by obtaining parts from the existing plant. It is also being used as cloning agent to produce genetically identical plants in a mass production. The parts of the plant that are highly recommended for cloning is the region where dense-mitotic division take place such as the apical and lateral meristem. For this experiment, leaves will be used as a source to generate new explants. The plant that is used in the experiment is the Crassula argentea plants. It is used because of the suitability of the plant to tolerate heat changes and ability for easy propagation. To produce good explants, the tissue culture used must be sterile to prevent any growth of contaminations such as bacteria, fungi and other microflora inside the tissue culture medium. Therefore varies treatments have been used to sterilize the tissue sample from contaminations such as by using ethanol and bleach solutions. It is crucial to kill all microorganisms on the plant tissues so that sterile tissue culture can be achieved. Different type and concentrations of medium is also being used for each plant tissue culture to observe the growth of different parts of the new explants such as formation of callus, new shoots or new roots. The tissue is then incubated for several weeks to observe any growth from the plant tissues.

MATERIALS:

No.

Materials

Quantity

Description

1.

Leaves from Crassula argentea plants

4

The leaves are used as tissues from the plant for generating new explants.

2.

70% ethanol solution

10ml

Use for the surface sterilization treatments of the plant tissue.

3.

10% commercial bleach solution

10ml

Use for the surface sterilization treatments of the plant tissue.

4.

Medium A

(MS medium + 0.1mg/l IBA + 2.0mg/l BAP)

10ml

Act as a medium for the new explants to grow.

5.

Medium B

(MS medium + 2.0mg/l IBA + 0.1mg/l BAP)

10ml

Act as a medium for the new explants to grow.

6.

Medium C

(MS medium without plant growth regulators)

10ml

Act as a medium for the new explants to grow.

METHODS:

Establishment of aseptic cultures

Healthy looking leaves are being selected and cut off from the Crassula argentea plants. Approximately 3-4 leaves are sufficient for each experiment. The leaves are then washed using running tap water to remove any dirt or contaminations from the plant.

The leaves are then cut into 9 pieces using a scalpel for surface sterilisation. Make sure that all the procedures are done in a laminar flow clean air bench.

70% ethanol and 10% commercial bleach solutions are then being poured into separate small jars. These will be used to surface sterilize the plant tissues.

Each piece of plant tissues is then inserted into different treatment solutions. 3 pieces of leaves will be placed inside the 70% ethanol solution, and the other 6 is placed inside the 10% bleach solution and the timer is started.

Each of the plant tissues are then soaked inside the treatment solution for the following time period:

70% ethanol for 2 minutes

10% commercial bleach for 20 minutes

10% commercial bleach for 30 minutes

After the period for each prescribed treatment time, the pieces of plant tissues are then washed in separate sterile purified water in small jars. This is done to remove all the traces of surface sterilant. Use 2 washes for each pieces of plant tissue and a period of 1 minute each.

The prepared pieces of leaves are then transferred into sterile petri dish to cut off its outer layers. This method is done using the sterilised instruments. The explants that were cut should not be more than 1cm cubed.

The final explants are then transferred aseptically onto medium in the culture vessels. Make sure that each 1 explant is placed at 1 medium culture vessel.

Incubation of cultures

The cultures are then sealed at the top using parafilm. This is done to prevent any contaminants from entering the cultures.

The prepared cultures then being set in the shelf and will be incubated at 25°C in the presence of light.

The cultures will be incubated for 4 weeks and growth of explants and presence of contamination is observed after that time period.

RESULTS:

Contaminations

Contaminants

Bacteria

Fungi

Yeast

Total contaminants

70% ethanol solution

17

50

5

72

10% bleach solution for 20minutes

14

18

1

33

10% bleach solution for 30minutes

6

14

0

20

Table 1: Contaminants in Tissue Cultures

Figure 1: Contaminants in Tissue Cultures

Explants growth

Medium

Number of growth in explants

Callus

Shoot

Root

Total

Medium A

(MS medium + 0.1mg/l IBA + 2.0mg/l BAP)

15

9

12

36

Medium B

(MS medium + 2.0mg/l IBA + 0.1mg/l BAP)

14

4

13

31

Medium C

(MS medium without plant growth regulators)

5

0

6

11

Table 2: Growth in Explants

Figure 2: Growth of Explants

Figure 3: Relationship between the Total Number of Contaminants and the Total Number of Explants Growth

Figure 4: Number of Explants Growth to Number of Contaminants Ratio

DISCUSSION:

Based on the data obtained, all the treatments for surface sterilization are able to provide sterile plant tissues for tissue culture method. However, the efficiency of the treatments varies from one to another.

70% ethanol for 2minutes opposed greatest contaminants to the cultures as the time period for the treatment may be too short and most the microorganisms are not fully sterile through that period of time. On the other hand, this treatment also provides the highest number of explants growth.

Treatment for 10% bleach solution for 30minutes however, provides the least amount of contaminants in the culture. But the number of growth for the treatment is also the lowest. This is due to the prolonged exposure period of sterilization that not only killed most of the contaminants but also caused the plant tissue to be damaged. Thus, resulting to a sterile non-contaminated culture but non-growth suitable tissue cultures as it killed some of the plant tissue.

Therefore, if we compared the number of explants growth to number of contaminants ratio (NE: NC) for each treatment, it is shown that the best treatment is 10% bleach solution for 20minutes because it has the highest value for NE: NC. This indicates that it is sufficient to provide sterile condition for the explants to grow as well as preventing any contaminants from intrude the process.

The growth of explants is also affected by the growth medium supplied. IBA (auxin) and BAP (cytokinin) are used at different concentration to indicate different germination forms from the explants.

Auxin hormone is a type of plant regulators which occurs naturally in plants that promote root formation. Cytokinin hormone however is another kind of plant regulator and morphogenesis that regulate cell division. This causing the formation of shoots in plant (Paciorek et. al, 2006).

Medium A with higher concentration of cytokinin compared to medium B. Therefore, medium A induced higher number of shoots formation compared to medium B.

Medium B with higher concentration of auxin compared to medium A resulting greater number of roots formation compared to medium A.

All of the medium showing a significant numbers of callus formation. This is due to the intermediate stage whereas the explants will further develop into roots or shoots. It is believed if this experiment is then further its incubation period, more distinctive forms of explants can be observed.

CONCLUSION:

Auxin hormones enhanced the formation roots in tissue culture whereas cytokinin hormones influence the shoots formation.

The best treatment for surface sterilisation for plant tissue culture is at 10% bleach solution for 20minutes.

*Word count: 1386 words

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