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Preparation of MS Medium From Stock Solution

Preparation of MS Medium From Stock Solution

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Published by Mahathir Mohmed
Murashige and Skoog medium (MS) is the most suitable and most commonly used basic tissue culture medium for plant regeneration from tissues and callus.
Murashige and Skoog medium (MS) is the most suitable and most commonly used basic tissue culture medium for plant regeneration from tissues and callus.

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Categories:Types, Research, Science
Published by: Mahathir Mohmed on Dec 17, 2009
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12/11/2013

 
Plant Biotechnology
1
LABORATORY EXERCISE
Preparation of MS Medium from Stock Solutions
Introduction
Murashige and Skoog medium (MS) is the most suitable and most commonly used basictissue culture medium for plant regeneration from tissues and callus. It was developed byToshio Murashige and Folke K. Skoog in 1968; based primarily on the mineral analysis of 
tobacco tissue. This is a “high salt” medium due to its content of K and N salts.
Kiss
et al.
(1995) indicated shoot proliferation of pineapple
 Ananas comosus
was achieved on MSmedium with low levels of some inorganics and vitamin supplements. The shoots aftertransfers to MS medium containing NAA or IBA gave good rooting. They also reported the
invitro
axillary bud formation on the etiolated shoots and their rooting on hormone-free MSmedium. Litchi, one of the important subtropical fruit crop of Southeast Asia, has beensuccessfully regenerated
in vitro
using cotyledon, hypocotyls, and root segments on MSmedium supplemented with 0.05 mg/L NAA, 0.05 mg/L 2-Ip, 0.2 mg/L ABA, and 45 g/lsucrose (Chandra and Padaria, 1999).
Objectives
1.
 
To improve how to prepare Murashige and Skoog (MS) culture medium.
 
2.
 
To improve how to calculate quantity required from the stock solutions for a givenconcentration and volume of MS medium.
Materials and Methods
Beakers (1000, 500, and 100 ml); graduated cylinders (1000, 500, and 100 ml); conical flasks(100 ml); reagent bottles/Schott bottles (1000, 500, and 100 ml); micropipettes (1000 µl);distilled water; magnetic stirrer; spatulas; chemical balance; weighing boat; tissue; pHmeter; aluminium foil; autoclave machine; and chemicals needed: stock solutions of macronutrient; micronutrient; iron source; vitamins; plant growth regulators such as BAPand NAA; sucrose; myo-inositol; agar; and NaOH and HCl to adjust pH.
Methodology
1.
 
Five hundred millilitres of MS medium was made. The required volume of each stocksolution were calculated and obtained into 500 ml beaker on a magnetic stirrer.
 
2.
 
15 g sucrose and 0.05 g myo-inositol were added, and then stirred until fully dissolved.
 
3.
 
The volume was adjusted to about 500 millilitres with distilled water.
 
4.
 
2 ml (2000 µl) of NAA was added (to get 4 mg/L NAA) then pH was adjusted to 5.75 ±0.05 with NaOH and HCl.
 
5.
 
3.1 g of agar was added and stirred for complete mixing.
 
6.
 
The medium was heated up in microwave oven to dissolve the agar.
 
7.
 
Medium was transferred into 500 ml Schott bottle. Bottle was labelled beforeautoclaving.8.
 
The culture medium was autoclaved for 15-20 min at 121°C.
 
9.
 
After autoclaved, medium was distributed approximately 20 ml to each steriledisposable petri dishes in the laminar air flow and medium are allowed to solidifyfollowed by exposing to UV light for 20 minutes and stored for use.
 
Plant Biotechnology
2
Results
Calculation for preparation of 500 ml MS medium from stock solutionsFormula - M
1
V
1
= M
2
V
2
M
1
= Concentration of the stockV
1
= Volume will be taken from the stock (x)M
2
= Concentration of MS mediumV
2
= Volume of medium = 500 mL
Component CalculationAmount taken for500 ml MS medium
MacronutrientM
1
V
1
= M
2
V
2
(10) (x) = (1) (500 mL)x = 500/10= 50 mL from stock
50 mL
MicronutrientM
1
V
1
= M
2
V
2
(1000) (x) = (1) (500 mL)x = 500/1000= 0.5 mL from stock
0.5 mL
Ferum/iron sourceM
1
V
1
= M
2
V
2
 (100) (x) = (1) (500 mL)x = 500/100= 5 mL from stock
5 mL
VitaminM
1
V
1
= M
2
V
2
 (1000) (x) = (1) (500 mL)x = 500/1000= 0.5 mL from stock
0.5 mL
NAAM
1
V
1
=
 
M
2
V
2
(1000) (x) = (4) (500mL)x = 2000/1000= 2 mL from stock
2 mLDiscussionComparing MS medium with other culture medium
MS medium - one of the most successful media - was formulated by analyzing the inorganiccomponents in tobacco plants and then adding them to medium in amounts similar to those
found in the plants. It has comparatively high salt levels compared to White’s
medium(White, 1963) but lower than B5 medium (Gamborg
et al.
, 1968). The type of tissue culturemedium selected depends on the species to be cultured. According to Andreu and Marin(2005), WPM was the medium that promoted a better establishment of the
Prunus insititia
 cultures, but MS supported higher multiplication rates. In contrast, MS was better thanWPM for both explant establishment and multiplication of chokecherry
Prunus virginiana
 (Zhang
et al 
., 2000), and mature wild cherry (Hammatt and Grant, 1997)
Carbon sources
In tissue culture, plants lose their ability to synthesize their own food, thus they depend onexternal factors to make energy. Sucrose as a carbon source is known to be the best. This isbecause it is cheap and easily available. Added to that, sucrose can be autoclaved along with
 
Plant Biotechnology
3the media, which is preferred over filter sterilization. Sucrose hydrolyses during autoclavinginto more efficiently utilized energy source, such as fructose and glucose. Sucrose isessential for various metabolic activities. It is needed for differentiation of xylem andphloem in cultured cells. Sucrose is cheaper than glucose and has a more positive waterpotential.
 
However sucrose hydrolysis during autoclaving is dependant on pH factors, and it does notoccur at pH settings of 6.0 (George
et al.,
2007). Sucrose is less effective for monocot plants(Bhojwani and Razdan, 1996). Sucrose can also pose as a limiting factor to growth in certaincultures. The presence of sucrose in the media specifically inhibits chlorophyll formation andphotosynthesis, thus it endures a less feasible autotrophic condition. Sucrose may notpromote organogenesis as good as glucose.Alternative types of carbon source available; respectively in decreasing order are glucose,maltose and raffinose. Fructose is less effective and mannose and lactose were the leastsuitable. Other types of carbon source are galactose, cellobiose, melibiose, and trehalose,but these carbon source show inferior results as compared to sucrose.
Agar
Agar is the most commonly used gelling agent. When agar is mixed with liquid, it forms a gelthat melts at about 100°C and solidifies at about 45°C. It is highly stable, clear, non-toxic andresistance to metabolism during culture (Henderson and Kinnersley, 1988; Sahay, 1999). Allagars contain impurities, such as inorganic salts, organic compounds, phenolics, and longchain fatty acids: amounts and types vary depending on the manufacturer. Interactionswere found between agar performance and plant species and regeneration processes. Agarquality could affect, in principle, all developmental processes, where the regeneration of adventitious shoots and roots being the most sensitive (Scholten and Pierik, 1998).The concentration of agar may be critical to plant response in culture. Stoltz (1971)described the effects of concentrations of agar on the growth of mature
Iris
embryos.Somatic embryos of 
Picea
(Tremblay and Tremblay, 1991), shoot apical meristems of 
Picea
 (Romberger and Tabor, 1971), protoplasts of red cabbage (Koda
et al.
, 1988), and anthercultures of tobacco (Kohlenbach and Wernicke, 1978) have been shown to be sensitive toagar quality. The agar concentrations commonly used in plant culture media range between0.5% and 1%.Scholten and Pierik (1998) reported that no relationship could be found between the priceand quality of the agars. The performance of agars can be improved by washing agarsbefore use (Shillito
et al.
, 1983)When greater purity is needed, agarose may be used. Because of the additional purification(Johansson, 1988), agarose is considerably more expensive than agar (Kao, 1981). Mediumsolidified with Gelrite has the advantage of being clear, which agar-solidified medium is not.Consequently contamination is more easily detected at an early stage (Anonymous, 2006a).Gelrite requires more stirring than agar when being added to media. Phytagel is an agarsubstitute produced from a bacterial substrate but results in clumping. Therefore, to

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