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Plant Tissue Culture

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Tang Shi

on 1 September 2013

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Transcript of Plant Tissue Culture

Plant Tissue Culture: Orchids
Method 1: Explants
The cells in the leaves, shoots, buds, roots and stem of an orchid are totipotent, even though they are differentiated.

Therefore, when transferred into a growth medium, the tissue has the potential to multiply into new orchids during micro-propagation.


A sterile work environment
Vital for micro-propagation, as microbes (bacteria, fungal spores) can easily invade tissue-growing media. Since micro-propagation is a slow process, the fast-growing bacteria or fungus will usually sap all of the nutrients from the media, starving, and eventually killing, the plant tissue.
Precautions
The person performing the experiment must wear a pair of sterile latex gloves at all times.
A laminar flow hood will be required during certain steps.
Laminar flow hood: Filters air entering, removes dust particles, microorganisms and other airborne contaminants, creating a sterile work environment. (Life Technologies)
Steps: Pollen Method
1) Under a laminar flow hood, sterile an orchid flower with 70% ethanol solution, followed by immersion for 5-30 minutes in 10% aqueous sodium hypochlorite. Rinse off the sodium hypochlorite with sterile, distilled water.

2) With a scalpel sterilized by 70% ethanol solution and heating, remove the anther of the flower. Discard the rest of the orchid.
Next, remove the filament of the anther. Discard as well.
The remaining portion of the anther contains the pollen grain.

Finishing steps:
1) Incubate at a constant temperature of 25 degrees Celsius (room temperature), with 12 hours of exposure to light (2500 lux) and 12 hours of darkness, daily. After 20 weeks, the roots of the plantlets should be developed enough to be transferred into a pot.

2) Prepare net pots, charcoal pieces and coco peat. The holes in a net pot and the porous charcoal allow water to drain away quickly, preventing the moisture-sensitive roots of an orchid from rotting. Coco peat is used as growth medium, much like the gels previously discussed.

3) Disinfect everything by washing with boiling water. Before proceeding with the potting, let it cool to room temperature.

4) Wash the plantlet in sterilized, distilled, water.

5) Fill the bottom of a net pot with a layer of charcoal. Uncap a tube containing one plantlet. Using a pair of sterile forceps, grasp the plantlet firmly by its base, and place in the plant in the pot. Fill the rest of the pot with coco peat.

6) Spray with fungicide to prevent fungus from growing and killing the orchid.

7) Grow the orchids in a greenhouse which maintains a constant temperature of 25 degrees Celsius, and a constant humidity level of 95%.
Micro-propagation
"Growing large numbers of plant cells, in-vitro, in an aseptic and closely controlled environment"
(Davidson College)
An asexual form of plant reproduction; plants produced are haploid in nature, and are genetically identical to the parent

Explant
Explant: “Living tissue transferred from an organism to an artificial medium for culture”
(Biology-Online)
Totipotent
“Having the ability to differentiate into all cell types”
(Biology-Online)
Medium
(plural: media)
“The nutrient solution in which cells or organs are grown”
(Biology-Online)

Differentiate
“A process in which cells become specialized in form and function”
(Biology-Online)

Differentiated: “Describing a cell or tissue that has undergone differentiation; with the form and function altered from being generalized to being more specific”
(Biology-Online)
Steps: (Explant Method)
1) Select an orchid plant. Under the laminar flow hood, rub the plant with 70% ethanol solution to disinfect its surface. Remove the desired amount of plant tissue by cutting it off with a scalpel that has been sterilized with 70% ethanol and heating in a flame.
2)The cut tissue is called an explant. Wash the explant in sterilized, distilled water.
3) Further sterilize the explant in 0.15% aqueous mercuric chloride for 2-10 minutes, then rinse under sterile distilled water to wash off all traces of mercuric chloride.
Alternatively, immerse in 10% aqueous sodium hypochlorite for 5-30 minutes, then rinse off with sterile distilled water.

4) With a pair of forceps place the explant in a petri dish filled with callus initiation gel. Sterilise the forceps with 70% ethanol solution and heat before it is used to pick up the explant.
The callus initiation gel is made from mixing callus initiation medium with agar. The medium contains the growth substances auxin and cytokinin, as well as sucrose and inorganic salts for nutrition to sustain growth.

Auxin: “A group of plant growth substances responsible for raising the pH around cells, making the cell wall less rigid and allowing elongation.” – Encourages root formation
(Biology-Online)

Cytokinin: “Class of plant hormones active in promoting cell division.” – Encourages shoot formation
(Biology-Online)

5) To seal in moisture, cover the petri dish in plastic wrap. Incubate at a constant temperature of 25 degrees Celsius (room temperature), with 12 hours of exposure to light (2500 lux) and 12 hours of darkness, daily, for 16-20 weeks.
The light conditions mimic the natural daylight cycle. Moisture and light are required for photosynthesis and respiration in the plant tissue cells- two processes necessary for survival in plants.
6) The plant tissue will develop into a callus. There should be signs of shoot and root growth present in the callus. Multiple shoots will usually grow from the same explant and form a callus together.
Callus: “undifferentiated plant tissue produced at wound edge; can be grown in-vitro”
(Biology-Online)
3) Uncap an autoclaved conical flask containing liquid basal medium. Briefly heat the mouth of the flask with flame. Using a pair of forceps sterilized in the same way as the scalpel, transfer the anther into the flask. Cover with plastic wrap.


Basal medium: “An unsupplemented medium which promotes the growth of many types of microorganisms which do not require any special nutrient supplements.”
(Biology-Online)
4) To promote callus formation, incubate the flask at a temperature of 23-27 degrees Celsius, in complete darkness, for 10 days.

5) For 3-8 weeks, proceed to expose the callus to 16 hours of light at 3500 lux and 8 hour of darkness, daily, at 23-27 degrees Celsius.
Place on top of a vibrating shaker that will regularly shake the flask. This prevents the newly forming plantlets from clumping together into one large lump, which would reduce the number of plantlets (from multiple to only one), and by extension, plants, that can be grown from the callus.

6) There will be signs of shoot and root growth present in the callus

7) Under a laminar flow hood, split the callus into its separate plantlets.

8) Sterilize the surface of the conical flask with 70% alcohol solution. Remove the plastic wrap and sterilize the mouth of the flask.
Sterilise the surface of an autoclaved tube of rooting medium with 70% ethanol solution. Uncap the tube. Briefly heat the mouth of a tube over flame to kill microbes.
With a sterilized (70% ethanol solution, heat) pair of forceps, transfer one plantlet into a tube of rooting medium.

9) Disinfect a new cap over by heating it over flame. Use the new cap to cover the tube, pressing firmly to ensure than the fit is secure.

10)Repeat for all plantlets present in the conical flask.

7) Under a laminar flow hood, split the callus into its separate shoots.

8) Sterilize the surface of an autoclaved tube of rooting medium with 70% ethanol solution. Uncap the tube. Briefly heat the mouth of a tube over flame to kill microbes.
With a sterilized (70% ethanol solution, heat) pair of forceps, transfer each shoot section into its own tube.

9) Disinfect a new cap over by heating it over flame. Use the new cap to cover the tube, pressing firmly to ensure than the fit is secure.

Bibliography
All documents and websites were accessed on 30 August, 2013
Davidson College. Plant Tissue Culture. Retrieved from www.bio.davidson.edu/people/kabernd/seminar/2002/method/amy/aj.htm
Sigma Aldritch, Explant Sterilization, Retrieved from www.sigmaaldrich.com/life-science/molecular-biology/plant-biotechnology/tissue-culture-protocols/explant-sterilization.html#explant
Plant Tissue Culture, Case Study 3, Demostration of tissue culture for teaching, Retrieved from www.liv.ac.uk/~sd21/tisscult/case_study_3.htm
WizQiz, Micropropagation through Plant tissue culture : Detailed methodolog, Retrieved from www.wiziq.com/tutorial/41707-Micropropagation-through-Plant-tissue-culture-Detailed-methodolog
Venamy Orchids, Orchid Culture Guide, Retrieved from
www.orchidsusa.com/1Introduction.htm#1.6Taxonomynomenclature
Life Technologies, Laminar Flow Hood, Retrieved from www.lifetechnologies.com/sg/en/home/references/gibco-cell-culture-basics/cell-culture-equipment/laminar-flow-hood.html
Biology-Online, Dictionary, Retrieved from www.biology-online.com/dictionary
My Journey Towards Conservation: Propagating Orchids from tissue culture/flasks, Srinivas Garuduchar
Orchid Micropropagation: Regeneration Competence of Anther Culture, Vishal Sharma, Govt. P.G College for Girls, Sector 11, Chandigarh (Panjab University), India

Cell and Tissue culture Technique (Anther Culture), Md. Shafikur Rahman, Department of Biotechnology, Faculty of Agriculture,
Patuakhali Science and Technology University, Bangladesh

Plant Tissue Culture, APS Education Center

Micropropagation of an orchid Dendrobium strongylanthum Rchb.f., Kong, Q.1, Yuan, S.Y.2, Végvári, Gy.3, 1Department of Biology, Honghe University, Mengzi 661100, China 2Department of Agronomy, Honghe University, Mengzi 661100, China 3Department of Fruit Science, Corvinus University of Budapest, H-1118 Budapest, Hungary, publish 2007, International Journal of Horticultural Science
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