Plant tissue culture plays important roles in agriculture field by improving the production and quality of crops using recombinant DNA technology. The mechanisms and effects of plant hormones in promoting organogenesis in different types of plants are only partially understood. Using petunia leaves and carrot, the effects of auxin and cytokinin were investigated by carrying out organogenesis. The leaves and carrots were placed in NAA, kinetin and BAP in few combinations of different ratio for 5 weeks and were observed for any growth of roots and shoots. The results showed that petunia leaves and carrot slices placed in high NAA formed roots but no shoots were formed while only callus and shoots were formed for those in high kinetin or BAP. The petunia leaves in BAP also formed shoots while no shoots were found for those placed in kinetin. This indicated that NAA promotes root formation while kinetin and BAP stimulates shoot emergence and balanced composition of auxin and cytokinin will initiate both roots and shoots formation. Also, BAP was found to be more effective in promoting shoot formation as compared to kinetin. Further research has to be carried out using different auxin and cytokinin on more plant species with longer period of time.
Keywords: Plant tissue culture, Auxin, Cytokinin, NAA, BAP, kinetin, petunia leaves, carrot, roots, shoots
Plant tissue culture is a set of laboratory procedures that involve in-vitro growing of plant cells, tissues and organs using the nutrient medium in sterile conditions (Chawla, 2009). Plant tissue culture plays important roles in crop improvement as it improves the quality of plants by exploiting the genetic information of the plants through genetic engineering (Brown & Thorpe, 1995). Advantageous traits can also be screened and inserted to the plants with this method to make the plants resistant to herbicide and pesticide and even more tolerant to the harsh environment. This in turn increases the production of crops with lesser input of cost and energy. Besides that, plant tissue culture also plays important roles in biodiversity conservation. This is because with recombinant DNA technology, endangered species of plants can be cloned and prevented from extinction. It also helps in studying the plant evolution as it allows the genetic relationship of distantly related species to be identified.
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According to Thiel et al. (2008), plant hormones are largely involved in influencing the cellular differentiation in plant tissue culture. They are organic compounds that regulate the growth and development of target cells. There are five main types of plant hormones which are the auxin, cytokinin, gibberellins, abscisic acid and ethylene. Auxin is the hormone that causes the plants to carry out cell division and elongation (Chawla, 2009). It also involves in differentiation of callus which causes the emergence of roots first meanwhile inhibiting shoot formation. It is found to inhibit the abscisic acid activities but stimulate the synthesis of ethylene. In contrast, cytokinin promotes shoot formation but suppress the formation of roots (Chawla, 2009). Gibberellins involves in the elongation or regeneration of existing meristems. Besides that, abscisic acid influences the embryogenesis but suppresses growth. In seeds, abscisic acid often plays roles in retaining the seeds in dormancy. Ethylene is a gaseous chemical produced by amino acid and diffuses in the air to promote fruits ripening and abscission (Chawla, 2009).
In this experiment, the carrot and petunia leaves were excised from the explants and were cultivated with plant tissue culture by carrying out organogenic differentiation in the MS media containing auxin (NAA) and cytokinin (kinetin or BAP). The purpose of this experiment is to investigate the effects of auxin and cytokinin in different concentrations ratios on the organogenesis of petunia leaves and carrots. The differences between kinetin and BAP on the growth of petunia leaves and carrots were also studied and compared.
Materials and methods:
In this experiment, two different types of plants were used which are the petunia leaves and carrots. For the petunia leaves, 18 robust and healthy leaves were excised using the sterilized forceps and scalpel. These leaves were soaked in the 10% (v/v) sodium hypochlorite for not more than 5 minutes and were rinsed thrice with sterile water in a laminar flow cabinet. Meanwhile, 8 petri dishes containing the Murashige-Skoog (MS) media for tissue culture supplemented with different combination of cytokinin and auxin concentration which were the NAA: Kinetin and NAA: BAP with ratio 2:0, 0.5:1, 1:0.5 and 0:2 respectively were labeled. Besides that, a control petri dish with MS media without supplement of any plant hormones was also set up. For each of these 9 petri dishes, 2 petunia leaves were placed with the abaxial surface in contact with the MS media.
The procedure was repeated with carrots slices which were cut from the carrot with sterilized forceps and scalpel. 3 carrot slices were placed on each of the petri dishes. The effects of the plant hormones on the petunia leaves and carrot slices were checked every three days for 5 weeks and the results were recorded. Any leaves or carrot slices contaminated with fungi were eliminated by transferring the uncontaminated leaves to the new petri dish MS media during the experiment.
From the result, it was shown that all the petunia leaves and carrot slices in all petri dish with NAA, kinetin or BAP formed callus. As proposed by Che et al. (2006), when the explants are transferred to culture media, plant hormones such as auxin and cytokinin will initiate callus formation. This shows that the NAA, kinetin and BAP have caused the leaves or carrot slices to differentiate into callus. As for the control petri dish without auxin and cytokinin, there should not have any callus, roots or shoots formed as plant hormones are needed in order to have callus formed from cell differentiation. The petunia leaves have no callus and roots but shoots were formed while the carrot slices form callus but not roots and shoots. This may due to the contamination of plants hormones during the excision and transferring of leaves to the MS media as the forceps and scalpel may not be sterilized properly.
Roots were formed on the petunia leaves in 2:0 NAA and kinetin and 2:0 NAA and BAP. This appeared to be the same for the carrot slices in 2:0 NAA and kinetin and 2:0 NAA and BAP as there were also roots formed. This is due to the high level of auxin which stimulates the transcription of auxin-responsive gene to promote root formation from the callus (Moubayidin et al., 2009). However, there was no shoot formed on all these leaves and carrot slices. This may due to the reason that the formation of shoot requires cytokinin and this hormone was absent. Hence, no shoots were formed. Also, the high level of auxin will suppress the shoot formation of the callus (Shimizu-sato et al., 2009).
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The petunia leaves in 1:0.5 NAA and BAP formed shoots and roots. The auxin induces the formation of roots while the cytokinin induces the formation of shoots. However, the petunia leaves in 1:0.5 NAA and kinetin did not form shoots and roots. This is because BAP is more effective than kinetin in initiating shoot formation as it is the most active form of cytokinin (Khandel et al., 2011). Similarly, the carrot slices in 1:0.5 NAA and kinetin and 1:0.5 NAA and BAP did not form shoot and roots. According to Moubayidin et al. (2009), only balanced composition of auxin and cytokinin will causes the explants to form roots and shoots. Since the ratio of auxin and cytokinin used was 1:0.5 at which the concentration of cytokinin was lesser, the absence of shoots may due to low concentration of cytokinin which is insufficient to induce the formation of shoot on the leaves. Also, the presence of cytokinin which suppresses the differentiation in root meristematic cells may also inhibit the root from emerging (Moubayidin et al., 2009). The time taken for the experiment is also one of the reasons as the time may not be enough to see the shoot and root formation.
As for the 0.5:1 NAA and kinetin, roots were found to form on the petunia leaves and carrot leaves in respectively as auxin which induces root formation was present. However, the leaves and carrot slices in 0.5:1 NAA and BAP did not have roots formation. Besides that, shoots were also formed on the petunia leaves placed on 0.5:1 NAA and BAP but this did not appear to be the same for leaves in 0.5:1 NAA and kinetin. As stated by Chawla (2009), cytokinin promotes the cell division and elongation of shoots. As compared to the leaves in 0.5:1 NAA and kinetin at which no shoots are formed, it can be seen that BAP than the kinetin is better in inducing shoot formation (Buah et al., 2010). The carrot slices in 0.5:1 NAA and kinetin or BAP did not form shoot. One possible reason for the absence of roots and shoot on petunia leaves and carrot slices is that there was not sufficient time for the shoot and root formation to be observed and longer time is required.
It can also be seen that only the petunia leaves in 0:2 NAA and BAP showed the emergence of shoot. This indicated that cytokinin in the form of BAP acts in stimulating the cell division and differentiation to form shoot. The absence of shoots for the petunia leaves in 0:2 NAA and kinetin showed that kinetin is weaker in initiating shoot formation as compared to BAP. The rest of the petunia leaves and carrot slices in 2:0 NAA and kinetin and 2:0 NAA and BAP did not have shoots. This is because the time taken for the observation of the leaves and carrot was too short and the shoots were yet to emerge. All the leaves and carrot slices in 0:2 auxin to cytokinin did not have roots as NAA was not added to the media to promote root initiation and elongation.
In this experiment, carrot slices and petunia leaves were used to study the effects of auxin and cytokinin. This was to allow comparisons of effectiveness of tissue culture using different types of explants to be done. As stated by Akin-Idowu et al. (2009), carrot is the root of a plant with actively-dividing meristematic cells and it provides advantages to successful tissue culture with higher mass of differentiated cells. Petunia leaves on the other hand contain less actively-dividing cells. Thus, the callus, root and shoot formation of carrot slices should be more as compared to that of petunia leaves.
One of the leaves was removed in petri dish with 1:0.5 and 0:2 NAA and kinetin respectively. This is because the leaves were found to be infected with micro-organisms such as fungi due to contamination. This may cause the petunia leaves to die off, leading to unnoticeable effects of the kinetin and NAA on the growth of the leaves. According to Odutayo et al. (2007), the presence of microbes in the plant tissue culture retards shoots and roots formation. Thus, in order to get accurate results, the contaminated leaves were removed to prevent the spreading of the microbes to the other leaf on the petri dishes. The contamination of the tissues culture may due to insufficient sterilization of the forceps and scalpels used. Also, the petri dishes containing the MS media with the leaves may also not fully enclose which creates passage for the microbes to enter.
In conclusion, high amount of NAA promotes root formation while inhibiting shoot formation. On the other hand, kinetin or BAP in high concentration initiates shoot formation but suppressing root formation. The absence of NAA causes no roots to be formed while the absence of kinetin or BAP causes no formation of shoots. The absence of roots on leaves or carrot slices with NAA or absence of shoot on leaves and carrot slices with kinetin or BAP may due to the reason that insufficient time was given to observe the plants and the roots and shoots were yet to emerge. BAP is more effective in stimulating the emergence of shoot as compared to kinetin as it is the most active cytokinin. Also, carrot is more effective than petunia leaves in tissue culture as it contains actively dicing meristematic cells.
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