Plant tissue culture

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INTRODUCTION:

Plant tissue culture helps in the growth of new plants. In vitro means "in glass" but according to plant biology it means growing plants or parts of plants in glass ware. Hence because of this we use the term in "vitro plant culture" or "Plant tissue culture" this experiments are done in sterile controlled conditions. The field of plant tissue culture is based on the remarkable ability of plants. This special ability of plants allows us to separate the various parts of the plants like organs, tissues and cells and with the help of this we can grow new plants. The growth of the plant depends on the nutrient medium as well as the aseptic conditions. The culture can be sustained as a mass of undifferentiated cells for an extended period of time, or regenerated into whole plants. Plant tissue culture relies on the fact that many plant cells have the ability to regenerate a whole plant. Single cells, plant cells without cell walls, pieces of leaves, or roots can often be used to generate a new plant on culture media given the required nutrients and plant hormones.

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Haberlandt first successfully achieved plant tissue culture in 20th century. The natural plant hormone iodole acetic acid was discovered by Thimann. Hannig initiated investigation in fields of embryogenic tissue. Kotte and Robbins postulated that a true in vitro culture could be easier while using meristematic cells. (Razdan 2003).

Applications of plant tissue culture:

  • Micropropagation is widely used in forestry and in floriculture. Micropropagation can also be used to conserve rare or endangered plant species.
  • Large-scale growth of plant cells in liquid culture inside bioreactors as a source of secondary products, like recombinant proteins used as biopharmaceuticals.
  • A plant breeder may use tissue culture to screen cells rather than plants for advantageous characters, e.g. herbicide resistance/tolerance.
  • To cross-pollinate distantly related species and then tissue culture the resulting embryo this would otherwise normally die.
  • To cross distantly related species by protoplast fusion and regeneration of the novel hybrid.
  • As a tissue for transformation, followed by either short-term testing of genetic constructs or regeneration of transgenic plants.
  • For production of doubled monoploid (dihaploid) plants from haploid cultures to achieve homozygous lines more rapidly in breeding programmes, usually by treatment with colchicines which causes doubling of the chromosome number.
  • micropropagation using meristem and shoot culture to produce large numbers of identical individuals
  • Certain techniques such as meristem tip culture can be used to produce clean plant material from viruses stock, such as potatoes and many species of soft fruit.

Experiment 1:

Initiating a plant tissue from seed

Aim: To optimize the sterilization time on contamination and seed germination.

Materials: Laminar Flow Sterile Cabinet, Beakers, Petri dishes and sterile filter paper

  • 20 Seeds
  • 3 jars or Petri dishes containg Murashige and Skoog medium
  • 1 Wilson sieve in a filter jar
  • 100 mls of sterilizing solution comprising a 10%(v\v)solution of freshly prepared Domestos, a sterilizing solution
  • An empty 100 ml beaker
  • 2 sterile forceps
  • 1 pack of sterile filter paper
  • 9 McCartney bottles containing sterile distilled water (SDW)

Procedure:

  1. All the steps are performed in a Laminar Flow Air Cabinet
  2. Make up a 10% (V/V) solution of Domestos, take to this Laminar Flow Bench and pour into a Wilson Sieve jar.
  3. Label 3 jars or Petri plates with relevant species name, date, name and group name, and then write 1 as 5 min, 1 as 10 min, and 1 as 15 min.
  4. Keep 20 seeds in 10% Domestos solution is Wilson sieve jar.
  5. After 5 min remove the 5 seeds and place it in a McCartney bottle containing SDW. Pour off the water 2 other SDW water. After the third wash retain The seeds .
  6. After 10 min remove 5 seeds and follow the same washing procedure as above and retain the seeds.
  7. After 15 min remove 5 seeds wash the seeds following the procedure similar to 5 min seeds and retain the seeds.
  8. Transfer the each set of the seeds on a sterile filter paper and allow it to dry.
  9. Transfer the seeds to the respective labeled Petri dishes containing MS medium.
  10. Seal the dishes with parafilm.
  11. Keep it in a propagator for the germination for two weeks.
  12. Observe the results after two weeks.

Experiment 2:

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Micropropagation

A comparison of shoot micro propagation in in vitro plants of different species:

Introduction: micropropagation is growing plants from seeds or small pieces of tissue in sterile conditions in a laboratory in special selected medium.this are performed in careful controlled environmental. The medium that the planes grow no contain carbohydrate sources, a range of mineral salts and agar. Sometimes vitamins, amino acids, growth regulators or plant extras may be added to encourage growth. Different mediums are used for different plants.

Four basic methods can be used for micropropagation

  • Enhanced axillary shoot proliferation.
  • Node culture.
  • De novo adventitious shoot formation through organogenesis.
  • Somatic embryogenesis.
  1. Nodal stem propagation of root and tuber plants: (Agricultural applications)
  2. Aim: To compare shoot micropropagation of root and tubers crop.

    Requirements:

    • A jar of in vitro plants.
    • Forceps
    • Scissors
    • Scalpel
    • Sterile filter paper
    • Culture medium

    Procedure:

    1. Select plant which is without contamination
    2. Take out the plant material from culture vessel in the Laminar Flow Air Cabinet and keep it on a sterile filter paper
    3. Take out the larger expanded leaves in case of potato only and the apical shoot and excise the plant into nodal stem section.
    4. Place 5 nodal stem section on the freshly prepared MS medium and MI medium for plant present in a Petri dish.
    5. Seal the Petri dish with the parafilm.
    6. Write the name, date, media type, explants name and group name on the Petri dish.
    7. Observe the results after two weeks.

  3. Organogenesis form leaf explants :( Horticulture applications)
  4. Aim: To induce organogenesis in horticultural ornamentals.

    Requirements:

    • A jar of Begonia plant
    • Forceps
    • Scissors
    • Scalpel
    • Sterile filter paper
    • Culture medium
    • Parafilm

    Procedure:

    1. Select culture of Begonia plant without contamination.
    2. Remove a plant from the Petri dish in the Laminar Flow Air Cabinet and place it onto a sterile filter paper.
    3. Cut the leaves of Begonia and place it in Petri dish containing AV medium.
    4. Place 5 leaves on the medium facing the adaxial surface towards the medium in one Petri dish and adaxial surface facing towards the medium in another Petri dish.
    5. Seal the Petri dish with the parafilm
    6. Write the name, date, media type, explants name and group name on the Petri dish.
    7. Observe the results after two weeks.

  5. Shoot micropropagation of fruit and nut species: (Horticulture applications)
  6. Aim: To induce and compare the shoot micropropagation of two different fruit and nut species.

    Requirements:

    • An explants from hazel (Corylus avellana) and currants (Ribes spp).
    • Forceps
    • Scissors
    • Scalpel
    • Sterile filter paper
    • One 9 cm Petri dishes and one jar containing sterile freshly prepared DKW medium
    • Parafilm

    Procedure:

    1. Select culture of Hazel nut and Currants plant without contamination.
    2. Remove a plant from the Petri dish in the Laminar Flow Air Cabinet and place it onto a sterile filter paper.
    3. Cut away the shoots from the clump and remove plant part showing senescence and count the number of shoots.
    4. Cut again the explants until it contains 2-4 leaves attached to the base of a stem on case of Ribes spp.
    5. In case of hazel nut sub culturing is done using nodal stem cutting i.e. Cut the stem showing nodal part and remove leaves and apical meristem .
    6. Place 3-5 plantlets onto a Petri dish in case of Ribes spp. and onto the jar containing DKW medium in case of hazelnut.
    7. Seal the Petri dish and plastic bottle with the parafilm.
    8. Write the name, date, media type, explants name and group name on the Petri dish.
    9. Observe the results after two weeks.

  7. Nodal propagation of forest tree species; (forestry)
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    Aim: To induce micropropagation using nodal stem propagation in forest tree species.

    Requirements:

    • An explant from parica (Schizolobium amazonicum)
    • Forceps
    • Scissors
    • Scalpel
    • Sterile filter paper
    • One jar containing sterile freshly prepared DKW medium
    • Parafilm

    Procedure:

    1. Select culture of parica plant without contamination.
    2. Remove a plant from the Petri dish in the Laminar Flow Air Cabinet and place it onto a sterile filter paper.
    3. Cut away the shoots and remove plant part showing senescence.
    4. Subculture is done using nodal stem cutting i.e. cut the stem showing nodal part and remove leaves and apical meristem.
    5. Place nodal cuttings onto the bottle containing DKW medium.
    6. Seal the plastic bottle with the parafilm.
    7. Write the name, date, media type, explant name and group name on the Petri dish.
    8. Observe the results after two weeks.

Experiment 3:

Practical studies of somatic embryogenesis (Morphological observations)

Aim: To observe different stages of embryos .

Requirements:

  • a binocular microscope
  • Somatic embryo culture of Daucus carrota

Procedure:

  1. Observe the somatic embryo culture using binocular microscope for the different stages.
  2. Jot down the stages observed.

Experiment 4:

Method for encapsulation of somatic embryos to produce artificial seeds

Aim: To produce artificial seeds of Daucus carrota from somatic embryos.

Requirements:

  • somatic embryo culture of carrot (Daucus carrota)
  • Forceps
  • Scalpel
  • Sterile filter paper
  • MS medium dispensed into 9 cm Petri dish
  • A solution of 3% (W/V) Alginate
  • A solution of 100mM CaCl2
  • A 100 ml beaker
  • A sterile 9 cm Petri dish
  • SDW

Procedure:

  1. Transfer the embryos to the alginate solution carefully agitate it gently taking care of no bubbles form, until they are completely submerged into the liquid.
  2. Suck up the embryos together with alginate solution using Pasteur pipette. Wipe out the excess of alginate solution outside of the pipette.
  3. Now drop the alginate solution with the somatic embryos carefully into the calcium solution, shaking simultaneously ultmately results in the formation of artificial seeds. Ca++ on contact with alginate polymerizes encapsulating the embryos.
  4. Drain the excess of the medium away using Wilson sieve in 100 ml of empty beaker.
  5. Wash the arificial seeds with SDW.
  6. Capture the encapsulated embryos; place at least 5 artificial seeds on MS medium in a 9 cm Petri plate.
  7. Seal the Petri dish with the parafilm.
  8. Write the name, date, media type, explant name and group name on the Petri dish.
  9. Observe the results after two weeks.

Referencing:

  • Razdan, M. K. 2003. Introduction to Plant Tissue Culture. p.375. [Online] Available from: http://books.google.co.uk/books?id=oe_liIY_tVsC&printsec=frontcover