Two Types Of Plant Hormones Biology Essay

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This experiment involves plant tissue culture and two types of plant hormones, auxins and cytokinins. Plant tissue culture was done in sterile condition and in nutrient rich media. The objective of this experiment is to investigate the effects of variation of concentration and ratio of auxins and cytokinins hormones on induction of organogenesis of petunia leaves and carrots. Appropriate and specific ratio of auxins and cytokinins will regulates the growth of cells, formation of callus, shoots and roots. Higher amount of auxins will initiate roots formation and higher amount of cytokinins initiate shoots formation. Formation of callus needs both hormones. Introduction:

Plant tissue culture is compilation of techniques to grow plant cells, tissues or organs in nutrient rich culture media, control and sterile condition (Thorpe, 2007). This technique manipulates totipotency of plant which is the ability to regenerate new whole plant. Plant tissue culture is the most crucial technology in plant biotechnology. The importance of this technique is crucial tool to investigate plant physiology. Plant tissue culture plays an important role in gene engineering and technology like producing disease or pest resistance varieties which a big help in agriculture sector (Gamborg, 2002). Besides, it also helps in cloning and plant breeding.

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In tissue culture, plant growth hormones are chemicals that carry an important role in regulating formation of callus and growth of plants. Specific and suitable ratio of plant hormones is required to differentiate plant cells and form callus which is mass of undifferentiated cells (Daugherty, 2007). The callus is further differentiated to form roots and shoots and finally a whole plant. In general therere five classes of phytohormones which are auxins, cytokinins, abscisic acid, gibberellins and ethylene.The functions of auxins are initiation of roots, cell growth and encourage production of other hormones (Liu et al., 1997). Example of auxins are NAA (refer Figure 1) and indole-3-acetic acid (IAA). Cytokinins involve in formation of shoots and cell division. Combination of auxins and cytokinins regulate growth of cell and plant. Abscisic acid is an inhibitory compound that affects growth of cell. Gibberellins involve in seed germination and production of enzyme. On the other hand, ethylene affects fruit ripening and also produced during cell dividing.

Figure 1.1-Naphthaleneatic acid, NAA Figure 2. 6-Benzylaminopurine,BAP

The aim of this experiment is to investigate the effects of varying concentration and ratio of cytokinin and auxin on induction of organogenesis in petunia leaves and carrots.

Materials and Methods:

Two types of plant material were used in this experiment which is petunia leaves and carrots. This experiment was done in a laminar flow cabinet. By using sterile forceps and scalpel, the leaves were soaked in 10% (v/v) sodium hypochlorite for less than 5 minutes and the leaves were rinsed thrice with sterile water. The leaves were placed with the abaxial (lower) surface in contact with the medium in a petri dish containing MS media supplemented with a combination of high cytokinin to low auxin (refer below) and a control petri dish without addition of hormone. All the steps above were repeated using carrot slices. All of the plates were kept in room temperature. The effects of the phytohormone were recorded every week until the report due.

Series 1 = NAA: Kinetin ratios (2:0, 0.5:1, 1:0.5, 0:2), 8 petri dishes

Series 2 = NAA: BAP ((2:0, 0.5:1, 1:0.5, 0:2), 8 petri dishes Control = 2 petri dishes

Results and Discussion:

Table 1. The compiled observation of sample plates containing petunia leaves and carrots slices

Type of Plant

Treatment

Ratio

Presence of contamination

Number of the following structure

Shoot

Root

Petunia leaves

Kinetin: NAA

2:0

No

-

-

0.5:1

Yes

-

-

1:0.5

No

-

-

0:2

Yes

-

-

BAP: NAA

2:0

Yes

-

-

0.5:1

No

Yes

Yes

1:0.5

No

-

-

0:2

Yes

Yes

Yes

Control

0:0

No

Yes

Yes

Carrots

Kinetin: NAA

2:0

Yes

-

-

0.5:1

No

-

-

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1:0.5

No

-

-

0:2

No

-

1

BAP: NAA

2:0

Yes

-

-

0.5:1

No

-

-

1:0.5

No

-

-

0:2

No

1

1

Control

0:0

No

-

-

(Refer to appendices)

Petunia leaves

Based on Table 1, callus presented on all plates except 2 kinetin:0NAA and 2 BAP:0NAA plates. This followed the theory which formation of callus needs both auxins and cytokinins. Thus, callus supposed will not be form in control, 0 kinetin:2NAA and 0 BAP:2 NAA plates. Besides, formation of roots were observed on 0.5 BAP:1NAA, 0 BAP:2 NAA and control plates. The result obtained were same as the theory which auxins is the hormone that initiate formation of roots, thus, plates with higher ratio of NAA or auxins should have higher amount of roots(Liu et al., 1997).

Formation of shoots were observed on 0.5 BAP:1NAA, 0 BAP:2 NAA and control plates. The result obtained was different from the theory which plates with higher amount of cytokinins should have shoots as this hormone initiate formation of shoots (Daugherty, 2007). Other than that, contaminations of fungus were observed in four plates. Most of the tissue that was contaminated did not form both the shoot and root as the contaminant released toxin chemical that will killed the tissue culture.

Carrot slices

Based on Table 1, callus was formed on all plates. This followed the theory which formation of callus needs both auxins and cytokinins. However, callus supposed will not be form in control, 0 auxins:2cytokinins and 2 auxins:0 cytokinins plates. Besides, formation of roots were observed on 0kinetin:2NAA and 0 BAP:2 NAA plates. Theoretically, plates with higher ratio of NAA or auxins should have higher amount of roots(Liu et al., 1997). Thus, the results obtained follow the theory.

Formation of shoots were observed on 0 BAP:2NAA plate. The result obtained was different from the theory which plates with higher amount of cytokinins should have shoots as this hormone initiate formation of shoots(Liu et al., 1997). This may be due to not enough time given for the callus to develop into organ which is the shoot. Other than that, contaminations of fungus were observed in two plates with ratio of 2 cytokinins:0auxins. All of the tissue that was contaminated did not form both the shoot and root as the contaminant released toxin chemical that will killed the tissue culture.

Microbial contamination happened in plant tissue culture is common and usual contaminants are bacteria and fungus. Carrying plant tissue culture in sterile condition is the most important and hard aspect to be control. Contamination usually happened due to inefficiency of the experimenter’s skill in handling this experiment especially during the process of transferring the tissue to the nutrient rich medium. The source of contaminant may came from the dust in surrounding air or from the laboratory coat sleeve and when the experimenter talking or sneezing during the inoculation process. Thus, it is discouraged for the experimenter to talk during the experiment. This can be improved by wearing special sterile suit and wearing mask or head gear.

Other than that, there were some factors that may affect the result obtained. The factors are temperature, humidity, lights, oxygen and carbon dioxide level. As an example, carrot explants grow best at 80%-90%, but humidity below 60% lead to death of the tissue (Chawla, 2002). Too high humidity also leads to contamination as it is an optimum condition for growth of microbial organisms. As for temperature, roots formation requires low temperature while shoots formation can be induced at room temperature.

Conclusion:

In conclusion, appropriate ratio of auxins and cytokinins is a key factor in success of induction of organogenesis. Plate with higher ratio of auxins should increase formation of roots while, plate with higher ratio of cytokinins should increase formation of shoots. Formation of callus required presence of both hormones. However, not all of the results follow this theory. Besides, contamination also happened in some plates. This may be due to human errors in handling the sample which must be improved in future.

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