Arabidopsis thaliana

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Thale cress

Common Name: Thale cress

Scientific Name: Arabidopsis thaliana

Domain: Multicellular Eukaryote

Plant

Biosafety Level: BSL1

Benefits and applications as a model organism

Benefits :

1. Arabidopsis requires low maintenance only light, air, water and a few minerals to
   complete its life cycle. It has very limited space requirements, and is easily grown in a
   greenhouse or indoor growth chamber.
2. It has a fast life cycle, produces numerous self progeny through selfing allows for
   experiments across generations.
3. It possesses a relatively small, genetically tractable genome that can be manipulated
   through genetic engineering more easily and rapidly than any other plant. With the
   completion of the Arabidopsis genome sequencing project, we now have in hand the
   sequence of the approximately 25,500 genes in its genome. An extensive toolkit for
   manipulation has been developed over the last 20 years, including efficient mutagenesis,
   facile transformation technology, and DNA, RNA, protein, and metabolite isolation and
   detection methods.
4. Ease of crossing,high transformation efficiency, powerful reverse and forward genetics
   and the ability to do mutational screens to saturate in the laboratory have also been key
   features resulting in increased research applications of Arabidopsis thaliana. A. Thaliana
   is efficiently transformed using the Agrobacterium tumefaciens vector. Currently, a large
   number of mutant lines and genomic resources are available for the study of Arabidopsis
   thaliana.
5. Arabidopsis thaliana has become universally recognized as a model plant for such
   studies. Although it is a non-commercial member of the mustard family, it is favored
   among basic scientists because it develops, reproduces, and responds to stress and
   disease in much the same way as many crop plants.

Application

1. A. thaliana a versatile model organism for use in the biology laboratory. It is widely used
   in the fields of plant science, genetics and evolution and has helped further our
   understanding of germination and aspects of plant growth that are important in
   commercial crops. In recent years A. thaliana has even become a model organism for
   the study of the biochemical and molecular processes.

2. Arabidopsis thaliana Is a good model to understand gene isolation and function, and
   Thus, the function of many genes isolated from crop plants can be better understood via
   study of their Arabidopsis homologues. For example, Arabidopsis defense mechanisms
   against pathogens have been used directly to develop disease-resistant plants in other
   species.

3. Tissue and cell culture of Arabidopsis thaliana is also extensively used to understand
   seed germination, callus development, and for various physiological and genetic
   conditions with varying external conditions.

Maintenance of Arabidopsis thaliana

• Growth in Solid media in sterile conditions

  1. Add 4.31 g of MS basal salt mixture and 0.5 g of MES to a beaker containing 0.8 L of
     distilled water and stir to dissolve. Add distilled water to the final volume of 1 L. Check
     and adjust pH to 5.7 using 1 M OH.
  2. Divide the media into two 1 L bottles, 500 mL in each. Add 5 g of agar per bottle. Keep the lid loose.
  3. Autoclave for 20 min at 121 °C, 15 psi with a magnetic stir bar in the bottle.
  4. Place the bottles on a stir plate at low speed and allow the agar medium to cool to
     45-50 °C (until the container can be held with bare hands).
  5. Starting from this step, perform all the steps in sterile conditions in a laminar flow
     hood. Add (optional) 1-2 % sucrose and 1 mI Gamborg's Vitamin Solution, stirring to evenly dissolve
  6. Label the bottom of Petri plates with identification number or name, including the date.
  7. Pour enough media into plates to cover approximately half of the depth of the plate.
  8. Allow the plates to cool at room temperature for about an hour to allow the agar to solidify. If the plates are not to used immediately, wrap them in plastic and store at (refrigerator temperature)
  9. Surface-sterilize seeds in microcentrifuge tubes by soaking 20 min in 50 % bleach
     with the addition of 0.05 % Tween detergent.
  10. Remove all bleach residue by rinsing five to seven times in sterile distilled water.
  11. For planting of individual seeds at low density, adhere one seed to the tip of a pipette using suction, then release seed onto the agar in desired location. For planting seeds at higher densities, mix seeds in sterile distilled water (or 0.1 % cooled top agar), pour onto plate, and immediately swirl to achieve even distri-bution. Use a sterile pipette tip to adjust the distribution and remove excess water. Allow the water or top agar to dry slightly before placing the lid onto the plate.
  12. Seal with Micropore tape to prevent desiccation, while allowing slight aeration.
  13. Place the plates at 4 °C for 3 days.
  14. Transfer the plates to the growth environment. Illumination of 120-150 mol/m? s continuous light and a temperature of 22-23 °C are suitable growth conditions.

• Growth in liquid media in sterile conditions

  Seedlings of Arabidopsis can also be grown in liquid growth media. This method
  provides large amounts of plant tissue suitable for proteomics and metabolomics or any study that requires a larger amount of starting material.Liquid culture growth is also widely used for high-throughput genomic studies. In this case, growth protocols are adapted to 96-deep-well plates with the MS media supplemented by gibberellic acid.

  1. Prepare MS media, as described in Subheading 3.1.1. Do not add agar.
  2. After the media has been autoclaved and cooled to room tem-perature, distribute 75-100-mL MS media into previously sterilized 250-mL Erlenmeyer flasks in a laminar flow hood.
  3. Add bleach- or chlorine gas-sterilized seeds to the media (add up to 10 pL of seeds to each flask, which corresponds to approximately 250 seeds).
  4. Grow seedlings under continuous light (120-150 mol/m^2 s) with gentle rotation in an orbital shaker at 120 pm for up to 2 weeks.
  5. Remove seedlings from the flask. Growth of more than 200-250 seedlings for more than 2 weeks may result in difficult removal of plant material from the flask.
  6. Remove excess media from the seedlings using filter pape. Plant material is now ready for downstream applications.
• Growth in Cell culture
  Cell suspension cultures represent a source of nearly uniform cell material for functional
  genomics and biochemical, physiological, and metabolomic studies that can be
  performed under tightly controlled environmental conditions. Several cell cultures derived
  from Arabidopsis tissue explants have been described. Among these, T87 and
  MMI/MM2d have been most widely used.
  1. Prepare 10 mg/mL thiamine stock solution by dissolving 0.1 g of thiamine in 10 mL of
     ddH,O. Filter-sterilize, aliquot 1 mL into microcentrifuge tubes, and store at -20 °C.
  2. Prepare 2,4-D stock solution by dissolving 0.2 g of 2,4-D in 100 mL of 25 % ethanol.
     Filter-sterilize, aliquot 1 mL into microcentrifuge tubes and store at -20 °C.
  3. Prepare 1 L of NI-1 media by adding 4.3 g of MS salt mixture, 30 g sucrose, 0.18 g
     KH,PO., 100 L of 10 mg/ mL thiamine stock, 220 pL of 2-mg/mI. 2,4-D stock, and 100
     mg myoino-sitol to a bottle containing 0.8 L of ddHO and stir to dissolve.
  4. Adjust the pH to 5.8 using 5 M NaOH. Add ddH2O to the final volume of 1L.
  5. Distribute 75-mL media into 250-mL Erlenmeyer flasks. Cover flasks with aluminum
     foil.
  6. Autoclave for 20 min. Let the media cool to room temperature.
  7. In a laminar flow hood, transfer 3 mL of 1-week-old I87 ce suspension culture into a
     flask containing 75 mL of NT medium .
  8. Grow the culture at 24 °C under continuous light (40 100 micromol/m^2 s) with gentle
     rotation in an orbital shaker 120 rpm.
  9. Subculture weekly by transferring cells into fresh NT-1 med.
• Growth Conditions
  The growth and development of Arabidopsis, including flowering time, is influenced by a
  number of environmental conditions in addition to the genetic background. Seeds of
  most lines germinate 3-5 days after planting under continuous light, 23 °C, adequate
  watering, and good nutrition. Plants produce their first flowers within 4-5 weeks, and
  seeds can be harvested 8-10 weeks after planting. High-quality seeds can be produced
  if watering, light, and temperature are carefully controlled. For vigorous plant growth, the
  optimum light intensity is 120-150 micromol/m^2 s and the optimum temperature is
  22-23 °C. Water requirement is strongly influenced by relative humidity. Plants tolerate
  low (20-30 %) relative humidity well, but depletion of soil moisture may occur in these
  conditions. Plant sterility may result from very high (>90 %) relative humidity, Mild
  humidity (50-60 %) is considered optimal for plant growth; however, low humidity (<50
  %) is recommended for silique maturation.

• Maintenance in Growth Chambers

  1. Clean down the entire growth chamber using sterilization agents and fungal
     spore eliminating agents.

  2. Height temperature(40-45 C) treatment of the room without ventilation.

  3. The recommended growth temperature is 21-23°C. Night temperature should be
     2-4 °C lower than daytime temperature.Use continuous light or a long-day
     photoperiod if you wish to accelerate the reproductive cycle. Short days (less
     than 12 h) favor growth of vegetative tissue and delay flowering.

• Seed handling storage and preservation
  Seed storage is important as when seeds deteriorate, they lose vigor and eventually the
  ability to germinate. The rate of this "aging" is determined by interactions of the
  temperature and moisture content at which seeds are stored, and unknown cellular
  factors that affect the propensity for damage.With the increase of storage temperature
  and seed moisture content, the life span of the seeds decreases. Seeds left at room
  temperature and ambient relative humidity lose viability within approximately 2 years.

  Seed stored dry at 4°C or -20°C should last decades.

  1. For active collections which are accessed often, store seeds at 4 °C and 20-30 %
     relative humidity. Control of humidity is typically achieved by a dehumidification system
     in the cold room. Note that the control of relative humidity provides a safety factor in
     case seed containers are not sealed properly.
  2. For long-term or archival storage, the recommended temperature is sub zero, preferably -20 °C and also preferably 20 % relative humidity.
  3. For open containers such as envelopes, seeds can be stored at 15-16 °C, with a
     relative humidity maintained very carefully at 15 %. Under this controlled environment,
     seeds will maintain suitable low moisture content. Storing seeds at relative humidity < 15% will not increase shelf life and may actually accelerate deterioration.
• Genetic crosses
  Some species of Arabidopsis, particularly A. thaliana, are mostly self-pollinating, especially in a growth chamber or greenhouse setting where insect populations are minimized. It should be noted that the pollen of Arabidopsis does not disperse through the air. Therefore, crossing Arabidopsis is mainly conducted through manual emasculation of flowers just prior to flower opening, followed by hand transfer of pollen from the desired male parent to the stigma of the emasculated flower.
  1. Select the appropriate parent plants. Choose young plants at early stages of
     flowering. Avoid using the first flowers in the inflorescence, which are usually less fertile,
     and the smaller flowers produced by mature plants.
  2. Prepare the female parent:
     (a) Select a stem with two to three flower buds, in which the tips of the petals are barely
     visible and before the anthers begin to deposit pollen on the stigma.
     (b) Remove siliques, leaves, and any open flowers above and below the selected buds
     on the chosen stem with a small pair of scissors; avoid damaging the stem.
     (c) Remove the sepals, petals, and all six stamens from the selected flower buds using
     the precision clamping tweezers, leaving the pistil intact.
  3. Prepare the male parent: Select a newly opened flower with anthers that are
     dehiscent. These flowers will contain fresh pollen that will contribute to the success of
     the cross. Remove the flower by squeezing near the pedicel with tweezers.
  4. Pollinate the female parent by taking the fully open flower from the male parent and
     brushing the anthers over the bare stigma of the female parent. Visually confirm that
     pollen has been deposited on the stigma.
  5. Label the crosses, placing tape on the stem of the female plant, noting the male and
     female parent and the date of the cross.
  6. Inspect developing siliques over the next several days. Successful crosses are visible
     after 3 days when the siliques start elongating. Siliques are ready for harvest once they
     turn brown, but before they shatter.
  7. Harvest siliques by cutting them with scissors and placing them into a microcentrifuge
     tube or a small paper envelope.
  8. Air-dry seeds at room temperature, preferably at 20-30 % relative humidity, for 1-3
     weeks. Thresh seeds if necessary.
• Floral Dip Transformation with Agrobacterium tumefaciens

Genome

https://www.ncbi.nlm.nih.gov/datasets/taxonomy/3702/