Experiment 1 and 2 Plant tissue culture

Experiment 1: preparation of plant tissue culture medium; Experiment 2: explants preparation preparation and callus formation Objectives:

1. To prepare the stock solution of macronutrient, micronutrient and vitamins of the basal MS medium. 2. To prepare the stock solution solu tion of phytohormones. 3. To prepare the MS medium with or without the supplementation of phytohormones. 4. To gain experience in disinfection of explants and aseptic techniques. 5. To establish cultures for callus induction experiments. Introduction:

In general, a nutritive medium contains macronutrients, micronutrients, vitamins, sugars and sugar alcohols. A medium may also include amino acids, nucleic acid bases, or other organic molecules. The medium without any phytohormones is known as basal medium. The addition of phytohormones to basal medium is to stimulate the growth and development of the explants in a particular fashion. Auxins and cytokinins are most commonly employed in tissue culture media. Once a medium formulation has been selected and the solution is prepared, the pH of the medium must be adjusted. The pH for many plant culture media is set from 5.7 to 5.8 as this pH range keeps most of the ions in charge state where they are available for absorption by the plant cells. Subsequently, the medium in a culture vessel will be sterilized or autoclaved. Before placing explants in culture on the nutrient agar, surface disinfection of explants by using diluted commercial chlorine bleach is required. The concentration of chlorine bleach and the length of time the tissue is in it can vary. Improperly sterilized tissue will result contamination which is arise from the explants. Contamination by fungi appears as fuzzy growth whereas bacterial contamination appears as a halo around the explants. A callus culture is an unorganized plant tissue growing on a solidified medium. New growth will form on the outside of the transferred callus as callus tissue is a heterogeneous accumulation of old and young cells.

Materials:

Sterilized Distilled water, 4 pieces beaker (250ml), measuring cylinder (100ml), glass petri dishes, stainless steel blade, forceps and scalpels.

Non-sterilized 5 macronutrients solution (2ml), micronutrients (2ml), FeNaEDTA (2ml), vitamins (2ml), 2,4-D (2ml), sucrose(6g), aluminium foil, culture vials, agar (1.6g), pH meter, microwave, parafilm, Clorox bleach, muslin cloth, ethanol, Tween-20, rubber band and plant leaves.

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Procedures (flowchart): Preparation of medium:

Culturing of the explants:

2ml of each materials + 6g of

Wash explants under running tap

sucrose (5macronutrients,

water (30 mins)

micronutrients, FeNaEDTA, vitamins) Prepare 100 ml 10% Clorox in sterile water and add 2-3 drops of

Dissolve in 100ml dH2O

Tween-20.

Adjust to pH 5.7 ± 0.1 Wash explants in Clorox solution Make up to 200ml

and shake it (10 mins).

Adding 1.6g agar

Wash explants in sterile water for 5, 10 and 15 minutes.

Put into microwave (2-4 mins) to dissolve the agar

Cut the explants into 5mm x5mm in glass petri dish.

Dispense the hot solution into culture vials and cover

Inoculate the explants pieces to

them with aluminium foils.

petri dish with agar.

Label the culture vials and

Seal the petri dish with parafilm

autoclave at 121˚C, 15psi (15

and make observation for few

mins)

weeks.

Callus induction (in vitro plantlets are provided): Flame the top 20 mm of

Cut the in vitro leaf explants

Place the explants with the

in vitro plantlet’s vial

into 5mm x5mm squares.

adaxial side up on the MS medium and MS supplemented with 1.0 mg/l 2.4-D.

Label all the culture vials

Repeat step 3 to 4 by

Flame the culture vials and

and incubate in the culture

placing the explants with

aluminium foil cap and seal it

room. Make observation

abaxial side up.

with parafilm.





Results:

Culturing explant Week 1

Type of contamination: fungus; Percentage of contamination: about 40% (4 of 10 explants contaminated); Morphology of contaminant: fuzzy, white.

Callus induction Medium without 2,4-D

Medium with 2.4-D

Week 1

The explants are remained the same.   

Shape and color of explants: regular. Degree of callus formation: none Morphology of callus formed: none

The explants are remained the same.   






Week 2

Callus formation 

 

Shape and color of explants: small brownish lump around the explant explant Degree of callus formation: small Morphology of callus formed: assembly of cell lump, brown.

Callus formation 

 

Shape and color of explants: small brownish lump around the the explant Degree of callus formation: moderate Morphology of callus formed: assembly assembl y of cell lump and some thread-like structure -

Remained the same.   

-


Explants die. 

 

Shape and color of explants: smaller;brown/ white. Degree of callus formation: none Morphology of callus formed: none

Explants die. 

 

Shape and color of explants: smaller; brown Degree of callus formation: none Morphology of callus formed: none





Discussion:

The culture media used for in vitro cultivation of explants are composed of complex mixture of salts, vitamins, carbon source, solidifying agents, plant growth regulators and antibiotics. The complex mixture of salts is divided into three groups: macroelements, microelements and iron source. The examples of macroelements are N, K, P, Ca, S and Mg and they are building blocks of amino acid, proteins, nucleic acid, cell wall and enzyme cofactors. However, the micronutrients like zinc, copper and cobalt are usually part of certain enzymes. The iron is specified into a new group because it is usually added in form of FeEDTA. EDTA forms complex with the iron in order to slow down and continuous release iron into the medium. The vitamins usually added to promote plant’s growth whereas the carbon source is needed as the explants have insufficient chlorophyll or maturity to produce food by itself. The carbon source that commonly used is sucrose which is readily assimilated and relatively stable. The solidifying agent in this experiment is agar. It plays plays supporter role for cultures and it has many advantages such as it does not react with media constituents, it does not digested by plant enzymes and it can remain stable at all incubation feasible temperature. The plant growth regulator involved in this experiment is 2, 4-D which is a synthetic auxin. It is absorbed through the leaves and is translocated to the meristems of the plants. Auxins promote cell division and cell growth, cause cell elongation and swelling of the tissues. Specifically, it induces formation of callus and adventitious roots in explants. The antibiotics that prevent growth of microbes are not used because it may inhibit cell growth. From the results above, the culturing explants in petri dishes are contaminated with fungus from the first week but the explants in culture vials (callus induction) are not contaminated with any fungus or bacteria. The reason is due to the different source of explants. The source of former is from outside plantlet while the latter is from the in vitro plantlet. Hence, although the outside plantlet’s leaf was washed and sterilized with Clorox bleach and Tween-20, there may be some dusts or spores hidden inside the cracks of plant leaf. In contrast, the in vitro plantlet was already in aseptic condition, therefore there will be no any contaminants inside the vial. For the callus induction part, the explants at the first week have no changes because the explants cells may prepare the materials like proteins for dedifferentiation or growth. In week 2, only 1 of explants without 2,4-D forms callus, another two remain the same and the remaining explants were dead. The reason for the no changes in two explants culture is because the absence abs ence of 2,4-D 2 ,4-D and they may ma y require longer time to form callus. cal lus. On the other hand, there are 2 explants with 2,4-D forming callus and another three were dead. There are more samples with 2,4-D forming callus than the without 2,4-D one as the presence of 2,4-D (auxins). Besides that, the samples with 2,4-D also have root formation which fits the desired result. Apart from the PGR factor, other factors such as genotypes, physical growth factors, original position of explants within the plants and growth factors also influences the callus





There is several precaution steps needed to be carried out during experiment. The FeEDTA compound is light-sensitive, so it must be always covered with aluminium foil. Next, vitamins and hormones should be stored in fridge after being used as they readily readil y to be oxidized when exposure to atmosphere. The aluminium foil caps for culture vials should not be tightly packed during autoclaving because the difference of air pressure may cause the flow out of medium from culture vials. Moreover, the working areas and equipments should be sterilized with alcohol before performing the tissue culture.

Questions:

1. If a culture requires 4.5% (w/v) sucrose, how much sucrose would you add to make the following amounts of medium (a) 2L; (b) 500ml; (c) 250ml; (d) 10ml? 4.5% (w/v) =4.5g/100ml as 1% (w/v) =1g/100ml, a) 4.5g/100ml = x g/2000ml x = 90g c) 4.5g/100ml = z g/250ml z = 11.25g

b) 4.5g/100ml = y g/500ml g/500ml y = 22.5g d) 4.5g/100ml = w g/10ml w = 0.45g

2. From which regions of the freshly isolated explants does the callus originate? The callus is originated at the cut surface and within veins of the explants. 3. What is the function of Tween-20 in the process of surface sterilization? It functions as a surfactant, or wetting wetti ng agent, to enhance the ability of the bleach to penetrate cracks in the plant sample to more fully sterilize the sample. 4. If u need 2.5mg/l 2,4-D in 1 liter of medium and the stock solution of 2,4-D contains 10mg/100ml, how much stock solution do you need? M1V1 = M2V2 (2.5mg/L)(1L) = (10mg/0.1L) V 2 V2 = 0.025L 5. If you have a 15mg/100ml stock solution soluti on of BAP and you need 1mg BAP in 25ml, how much stock solution would you add to 125ml of medium? M1V1 = M2V2 (15mg/100ml) V 1 = (125ml) (0.04mg/ml) V1 = 33.33 ml 6. There are 2150mg kinetin (MW=215) in 1L of medium. Express this in terms of molarity.







Conclusion:

In a nut shell, different techniques in plant tissue culture provide certain advantages such as the production of multiple plants in the absence of seeds or necessary pollinators to produce seeds. In this experiment, the object for f or study is callus and it is very useful as genes can be inserted into it and it will regenerate into whole plant that will perform the beneficial or desirable traits. This technique is significant to many biological fields especially botany, agriculture and forestry.

References:

1) 2) 3) 4) 5) 6) 7) 8) 9)

http://www.sarracenia.com/faq/faq6010i.html http://www.ehow.com/list_6368801_types-tissue-culture-contamination.html http://www.planttissueculturemedia.com/ http://answers.yahoo.com/question/index?qid=20080725174318AAiXG5e http://www.tissue-cell-culture.com/docs/libary/tc_trouble.html http://www.fungin.com/overview.htm http://www.academicjournals.org/ajar/pdf/pdf2007/mar/odutayo%20et%20al.pdf http://www.sarracenia.com/faq/faq6010i.html http://www.sarracenia.com/faq/faq6010i.html

Experiment 1 and 2 Plant tissue culture

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