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Preperation of M&S media and sub-culturing of Arabidopsis thaliana
Cell suspension cultures
To prepare a Murashige & Skoog media and sub-culture Arabidopsis thaliana on a weekly basis
What did you set out to do? What media and method did you use – brief approx. 2 sentences
What was the results of the expirement
What impacts do the results have?
A German Scientist, (Haberlandt), at the start of the 20th century had the vision of tissue culture, when for the first time he attempted to isolate a single cell and left it in salt solution enriched with sucrose. The cells survived for one month increasing in size but failed to divide. Although he was unsuccessful he lay down the foundations for tissue culture technology. In spite of this it wasn’t until the 1970's that the technology took off through commercialization because of predicted grain shortages for future generations.
This brought about new ideas for plant growth and genetic modification of plants. One of the ways to grow plants and research was through tissue culture. This involves cells, part of/or whole plants being grown in vitrounder aseptic and nutritional controlled and environmental conditions.
The technique uses the concept of totipotency where that a single cell can divide and regenerate to produce all of the differentiated cells required to create an entire plant through cell division.
Nowadays plant tissue culture is widely used for plant multiplication through micropropagation, creating virus and disease resistant varieties of plants, including producing exact copies of plants that have desirable traits such as good flowers or fruits. Other benefits such as being able to produce mature plants quicker, reproduce plants without pollinators, production of plants from seeds that otherwise have would normally have a very low chance of germination. In addition to cleaning particular plants from viral and other infections and by quickly multiplying these plants as cleaned virus free stock forhorticultureand agriculture use.
Part of this process is cell sub culturing which involves growing plant cells in an artificial media.
(Prof. Dr. Karl-Hermann Neumann 2009) stated that most cell suspension cultures originate from callus cultures due mainly to mechanical impact in agitated liquid media.
Sub culturing is the process of repeatedly transferring some or all cellsfrom a previously created culture to a freshgrowth medium, and is used to prolong or expand the life of cells. Cells are taken under sterile conditions in a laminar flow cabinet and transferred to fresh medium which provides the necessary nutrients for the cells to survive and grow.
The medium such as Murashige & Skoog’s (M&S) “contains the correct amount of nutrients to satisfy the nutritional needs of the cells” (Gamborg, Murashige et al. 1976) and should “generally contain some or all of the following components: macronutrients, micronutrients, vitamins, amino acids or nitrogen supplements, source(s) of carbon, undefined organic supplements, growth regulators and solidifying agents” (Hussain 2012).
It is of great importance that the composition of phytohormones and plant growth regulators are correct within the media as they are the orchestrators of the development of the cells and can have a profound effect on the response of the cells.
Hormones such as auxin, cytokinin, gibberellin, ethylene, abscisic acid as well as brassinosteroids, jasmonic acid, salicylic acid all have roles in how the cells develop, and how much of each hormone in the media dictates development rate.
Arabidopsis thaliana is a small dicot plant which belongs to theBrassicaceaefamily and which is closely related to themustard plant, and for approximately the last 25/30 years since it emerged, through consensus that research should focus on a single model plant. A. thalianais now studied throughout the research community in academia, government and industry, and “significant advances in understanding plant growth and development have been made by focusing on the molecular genetics of this simple angiosperm”Meinke (1998 Oct 23) .One of the reasons that A.thaliana was chosen was the life cycle of “is short approximately 6 weeks from germination to seed maturation. Seed production is prolific and the plant is easily cultivated in restricted space” (Resource).
For the experiment everything must be aseptic. This helps to avoid cross contamination of culture samples.
Firstly the Laminar Flow Cabinet was turned on 20mins prior to cleaning as it provides the clean environment required for sub-culturing, by providing a constant positive pressure within by pushing air through filters preventing air from outside entering.
Hand washing procedures were followed ensuring all rings watches, nail polish were removed and an Ethanol solution of 70% ethanol 30% water was prepared.
The laminar flow cabinet was then cleaned from back to front by spraying liberally with the ethanol solution and wiped down with clean sterile tissue or gauze. Before spraying a second time and leaving to dry.
Murashige and Skoog medium (M&S) is the most commonly used medium for plant propagation in vitro.
To make the M&S medium for culturing
- 1L of Deionised water (DIW) was measured out using a graduated cylinder and poured into a Duran bottle.
- 20ml of the DIW was removed and a mark placed on the bottle where the meniscus lay.
- A magnetic stirrer was placed in the Duran, and the Duran was placed on to a stirring table with the heat turned off.
- Using the analytical scales 4.3g of Basel salt was weighed then added to the DIW and left to dissolve.
- 30g of sucrose was then weighed and added.
- The pH of the media is also important as it affects both the growth of plants and activity of plant growth regulators. It is adjusted to the value between 5.4 - 5.8.
Calibration of a pH meter
- A Two-Point Calibration Procedure was used
- The electrode was rinsed with distilled water.
- The electrode was placed in a pH = 7 buffer solution, until it stabilized and read approx. 7 and then calibrated button was pressed.
- The electrode was removed from the solution and rinsed with distilled water.
- The electrode was then placed in the second buffer solution (pH = 4.00)
- Remove the electrode from the second buffer solution and rinse it with distilled water.
- Set calibration.
- Re-place the electrode in the pH = 7.00 buffer solution, for storage between tests.
Once the pH meter had been calibrated1M potaasium hydroxide was added using a dropper pipette until the concentration read 5.8 on the pH meter. (Reading off 6.06)
This study required a final working concentration 500 μg/l NAA and 50 μg/l Kinetin.
To achieve this 0.5ml (500μl) of NAA and 0.05ml (50μl) was measured from a stock solution and added using precision pipettes.
The mark on the bottle was then re-measured and was seen to be level with the original mark and pH re-checked – (6.06)
The solution was then divided between 10 Erlenmeter flasks, and sealed with tin foil lids before being labelled with the following information : - M&S, Date, and Initials of group.
The flasks were then removed and placed in the autoclave for 20mins at a temperature of 121°C. The autoclave is a Sterilizationunit (Pressure Cooker) that uses heat to kill all spores, such asfungi,bacteria,and viruses that may have been on the surface, or within the culture media. The flasks were removed from the autoclave before being left to cool at room temperature. (Autoclaved flasks can be stored for up to 2 months in a room at temp of 4°C)
The procedure to establish a Kinetin stock solution incluing calulations
An explanation of how Autoclave induces sterility
Sub-culturing A.thaliana in this media
Growth conditions of the cultures over the week
Staining with FDA (if Used)
Making Wet-mount Slides of Plant Cells
Some samples can be placed directly under the microscope. However, many samples look better when placed in a drop of water on the microscope slide. This is known as a "wet mount." The water helps support the sample and it fills the space between the cover slip and the slide allowing light to pass easily through the slide, the sample, and the cover slip.
This was done by taking a clean, scratch free glass slide. Use a pipette to take a drop from the sample to be viewed.
Place 1 - 2 drops of the culture in the middle of the slide If too much water is added, the cover slip will float creating a water layer that is too thick. If too little water is added, the specimen may be crushed or dry out too quickly.
Lower a clean cover slip over the drop as though it were hinged at one side to avoid bubbles.
Examine the preparation under microscope first under 4 x followed by 40 x and 100x magnification
Discard the cover slip.
Viewing through the microscope, Brownian motionmovement was seen, this is a vibrational random movement caused by particlessuspended in thefluidcolliding. If the movement was seenas directional and motile it could indicate bacteria being present.
Using 90 ml of medium per 250 ml flask. Sub-culture on a weekly interval, even if the tissue growth rate appears slow.
Hussain, A. Q., I. A. Nazir H. and Ullah, I. (2012). "Plant Tissue Culture: Current Status and Opportunities, Recent Advances in Plant in vitro Culture,." from http://www.intechopen.com/books/recent-advances-in-plant-in-vitro-culture/plant-tissue-culture-current-status-and-opportunities.