Welcome to Scribd, the world's digital library. Read, publish, and share books and documents. See more
Download
Standard view
Full view
of .
Save to My Library
Look up keyword
Like this
55Activity
0 of .
Results for:
No results containing your search query
P. 1
Preparation of Stock Solutions for Plant Tissue Culture

Preparation of Stock Solutions for Plant Tissue Culture

Ratings:

1.0

(1)
|Views: 10,431 |Likes:
Published by Mahathir Mohmed
Murashige and Skoog (MS) medium was formulated in 1962 and the usage of this media is considerably high compared to other types of plant media.
Murashige and Skoog (MS) medium was formulated in 1962 and the usage of this media is considerably high compared to other types of plant media.

More info:

Categories:Types, Research, Science
Published by: Mahathir Mohmed on Dec 17, 2009
Copyright:Traditional Copyright: All rights reserved

Availability:

Read on Scribd mobile: iPhone, iPad and Android.
download as PDF or read online from Scribd
See more
See less

03/05/2014

 
Plant Biotechnology
1
LABORATORY EXERCISE
 Preparation of Stock Solutions for Plant Tissue Culture
Introduction
Murashige and Skoog (MS) medium was formulated in 1962 and the usage of thismedia is considerably high compared to other types of plant media. This is becauseMS medium was found to be the most suitable medium used for plant regenerationfrom tissues and calluses. MS medium was invented by plant scientistsToshioMurashigeandFolke K. Skoogduring Murashige's search for a newplant growth regulator.Initially, the media was produced based on mineral analysis of tobacco.Since the MS media contains high amount of nitrogen fixing agents, it was found outthat the MS medium was equally compatible for other plant species as well.To have a complete form of MS media, the major nutrients required were themacronutrient, micronutrient, ferum, vitamins and in most cases, presence of plantgrowth regulators or hormones gives off a better result of root and shootsformation. And in high presence of these plant growth regulators, callus can beinduced as well.Preparation of stock solutions for MS medium is a vital step in media preparation.Several nutrients must be prepared and stored in order to have a good workingmedium. Therefore, to have a working solution, the stock must be preparedbeforehand.
Objectives
1.
 
To improve the preparation of stock solutions for MS culture medium.2.
 
To improve and calculate quantities of chemicals needed for givenconcentrations and stock volumes.
Materials and Methods
Beakers (1000, 500, and 100 ml); graduated cylinder (1000, 500, and 100 ml); conicalflask (1000. 500, and 100 ml); reagent bottles (1000, 500, and 100 ml); distilledwater; magnetic stirrer; spatulas; chemical balance; weighing boat; tissue; label andpen; and chemicals for macronutrient, micronutrient, iron source, vitamin, etc(according to table)1.
 
Each group was assigned and stock solutions were prepared.2.
 
Appropriate amount of each components were weight. Different spatula andweighing boat were used for different types of chemicals.3.
 
All chemicals for each stock solution were weight and dissolved in specifiedamount of distilled water using magnetic stirrer.4.
 
Stock solutions were labelled and date of preparation was indicated.5.
 
All stock solutions were stored in a chiller at 4°C.
 
Plant Biotechnology
2
ResultsSTOCKMACRO1X(1 litre MS) g/L10X (g/800 mL)
NH
4
NO
3
1.6500 (1.65 x 10)(800/1000) = 13.2 gKNO
3
1.9000 (1.90 x 10)(800/1000) = 15.2 gCaCl
2
.2H
2
O 0.4400 (0.44 x 10)(800/1000) = 3.52 gMgSO
4
.7H
2
O 0.3700 (0.37 x 10)(800/1000) = 2.96 gKH
2
PO
4
0.1700 (0.17 x 10)(800/1000) = 1.36 g
MICRO1X(1 litre MS) g/L1000X (g/100 mL)
KI 0.00083(0.00083 x 1000)(100/1000)= 0.083 gH
3
BO
3
0.00620(0.00620 x 1000)(100/1000)= 0.62 gMnSO
4
.H
2
O 0.02230(0.02230 x 1000)(100/1000)= 2.23 gZnSO
4
.7H
2
O 0.00860(0.00860 x 1000)(100/1000)= 0.86 gNa
2
MoO
4
.2H
2
O 0.00025(0.00025 x 1000)(100/1000)= 0.025 gCuSO
4
.5H
2
O 0.000025(0.000025 x 1000)(100/1000)= 0.0025 gCoCl
2
.6H
2
O 0.000025(0.000025 x 1000)(100/1000)= 0.0025 g
FERUM1X(1 litre MS) g/L100X (g/500 mL)
Na
2
.EDTA 0.03730(0.03730 x 100)(500/1000)= 1.865 gFeSO
4
.7H
2
O 0.02780(0.02780 x 100)(500/1000)= 1.39 g
VITAMIN1X(1 litre MS) g/L1000X (g/100 mL)
Nicotinic acid 0.0005(0.0005 x 1000)(100/1000)= 0.05 gThiamine HCl 0.0001(0.0001 x 1000)(100/1000)= 0.01 gGlycine 0.002(0.002 x 1000)(100/1000)= 0.2 gPyridoxine HCl 0.0005(0.0005 x 1000)(100/1000)= 0.05 g
 
Plant Biotechnology
3
Plant Growth Regulator g/L g/100 mL
Benzylaminopurine(BAP)1.000 1 (100/1000) = 0.1 gNaphthaleneacetic acid(NAA)1.000 1 (100/1000) = 0.1 g
DiscussionMacronutrient
Macronutrients are normally required in millimolar (mM) quantities in most plantmedia. The macronutrient normally contains high amount of nitrogen fixing agents inthe form of ammonium (NH
4+
) and nitrate ions (NO
3-
) ions. Apart from nitrogenicsubstances, potassium, magnesium, calcium, sulphur and phosphorus are also addedas macronutrient in the form of diluted salts. Also, macronutrients serve ascomponents for structural and protoplasmic tissue.
Micronutrient
Micronutrients are needed in a very small amount. The low requirement of micronutrients can be accounted for participation of these elements in enzymaticreactions and as constituents of growth hormones. The micronutrient is the mixtureof boron, cobalt, manganese, molybdenum, copper and zinc. Excessive amountadded to the medium can cause suffocation and premature death of the explantsused.
Ferum
Ferum is required in metabolic functions such as nitrogen fixation, photosynthesisand electron carri
er during respiration’s electron transfer process. It is usually
present in the form of FeSO
4
.7H
2
O and Na
2
EDTA. In some cases, ferum is preparedtogether with micronutrient. Ferum oxidises in the presence of sunlight.Furthermore, high concentrations will cause precipitation to occur. Therefore,preparing ferum and storing the stock solution in dark environment can prolong theshelf life of ferum stock solution.
Vitamin
Vitamin is essential as it is involved in carbohydrate metabolism and the biosynthesisof some amino acids. Normally thiamine is deemed as the most important vitaminand it is introduced as thiamine hydrochloride. Other vitamins like pyridoxine,nicotinic acid are added as well. Certain plant species requires special requirementsof vitamin like biotin, riboflavin, folic acid and more. Vitamins are associated withmetabolic activity of the plant. Therefore, to have good yield, sufficient amount of vitamin should be added to the MS medium.
Plant growth regulators
Plant growth regulator (PGR), functions in initiating the root and shoot developmentof explants and embryos. They also stimulate cell division and expansion. Certainparts of plants have plant growth regulators readily available in the explants. In casesof PGR absence, PGR supplemented through medium enables growth of the

Activity (55)

You've already reviewed this. Edit your review.
1 thousand reads
1 hundred reads
Mawiyah Mahmud liked this
Mervin Hernandez added this note
Thanks for these good references.
axatpgme liked this
Nurhaizan Izan liked this
Sehalif Razak added this note
good refferences...thanks
windua liked this
Marco Cordoba liked this

You're Reading a Free Preview

Download
/*********** DO NOT ALTER ANYTHING BELOW THIS LINE ! ************/ var s_code=s.t();if(s_code)document.write(s_code)//-->