Plant Physiology Experiments

Tissue culture—— Media preparation

Tissue Culture

• A technique which small tissue pieces or organs are removed from a donor plant and cultured aseptically on a nutrient medium.

• By manipulating the chemical compositionof the nutrient medium and other environmental parameters, the growth and development of the tissues in culture can be directed into different channels.

Totipotency• An ability to dedifferentiate demonstrates

that differentiated plant cells retain all the genetic information required for the development of a complete plant, a property.

The Background Ⅰ

• Its origins at the beginning of the 20th century with the work of Gottleib Haberlandt (left, plants) and Alexis Carrel (right, animals)

The Background Ⅱ

Tsui(崔澂)Folke Karl Skoog

Both organ formation and subsequent development are brought about by quantitative changes in amounts and interactions between nutrients and growth factors which are essential for growth of all cells, so that the pattern of development is determined by the relative supplies . . . of these materials at particular loci

http://www.nap.edu/readingroom.php?book=biomems&page=fskoog.html

The Background Ⅲ• Experimental results on

the establishment of the N6 medium are described on its application to anther culture of cereal crops is reviewed.

• The induction frequency of pollen plants in rice, wheat, triticale, rye and maize was higher on N6 than on Miller's or MS media.

Chu C-C （朱至清）

A B

C D

Tobacco

（A、B、C、D for different growth periods）

callus induction callus co-culture the first selection

rootage redifferentiation the second selection

Rice genetic transfomation

Why do Plant Tissue Culture?1. Fast propagation and virus removal.2. Anther and Pollen culture.3. Somaclonal variation and mutant separation.4. Protoplast culture and cell hybridation. 5. Plant artificial seed, or man-made seed.6. Give a continuous supply of young plants to

produce secondary metabolite.7. Plant ‘tissue banks*’ can be frozen, then

regenerated through tissue culture.

Chinese crop germplasm resources information system: http://icgr.caas.net.cn/Plant in vitro germplasm bank :http://www.ib.cas.cn/jgsz/zhicheng/sypt/200906/t20090603_276331.html

Initiating tissue culture

• Explants• Isolation and incubation• The cultural environment• Media• Solidified media• Liquid media

Plant Tissue Culture Procedure – Background. By E.F. George

Media

• A medium usually consists of a solution of salts supplying the major and minorelements necessary for the growth of whole plants, together with:– various vitamins (optional);– various amino acids (optional);– an energy source (usually sucrose).

relatively large amounts of some inorganic elements (the so-called major plant nutrients): ions of nitrogen (N), potassium (K), calcium (Ca), phosphorus (P), magnesium (Mg) and sulphur (S)

small quantities of other elements minor plant nutrients or trace elements): iron (Fe), nickel (Ni), chlorine (Cl), anganese (Mn), zinc (Zn), boron (B), copper (Cu), and molybdenum (Mo).

Components• Macronutrients• Micronutrients• Sugar• Plant growth substances• Vitamins• A solidifying agent• Amino acids and other nitrogen supplements• Undefined supplements such as coconuit milk

etc.• Buffers

The ingredients of MS medium（mg/L ）NH4NO3 1650

KNO3 1900

KH2PO4 170

MgSO4·7H2O 370

CaCl2·2H2O 440

MnSO4·4H2O 22.3

ZnSO4·7H2O 8.6

H3BO3 6.2

KI 0.83

NaMoO4·2H2O 0.25

CuSO4·5H2O 0.025

CoCl2·6H2O 0.025

FeSO4·7H2O 27.8

Na-EDTA 37.3

Thiamine HCl (VB1) 0.1

Nicotinic Acid 0.5

Glycine 2.0

Pyridoxine HCl (VB6) 0.5

myo-Inositol 100

Surose 30g

Agar 8g/L

pH 5.8

Vitamin Mixture

Fe2+

Micronutrients

Macronutrients

The ingredients of N6 medium（mg/L ）

（NH4）2SO3 463

KNO3 2830

KH2PO4 400

MgSO4·7H2O 185

CaCl2·2H2O 166

MnSO4·4H2O 4.4

ZnSO4·7H2O 1.5

H3BO3 1.6

KI 0.83

FeSO4·7H2O 27.8

Na-EDTA 37.3

Thiamine HCl (VB1) 1.0

Nicotinic Acid 0.5

Glycine 2.0

Pyridoxine HCl (VB6) 0.5

myo-Inositol 100

Surose 30g

Agar 8g/L

pH 5.8

Vitamin Mixture

Fe2+

Micronutrients

Macronutrients

Attention• micronutrient should be accurate to 0.0001

grams, macronutrient can be accurate to 0.01 grams.

• Macronutrient stock solution, usually 10 to 20 times, micronutrient stock solution and vitamin mixture is generally 50 ~ 100-fold.

• Stock solution preparation• Fe-EDTA preparation• Hormone preparation, storage• Water purification

Processing

water（100ml）macronutrientsMicronutrients

Vitamin Fe-EDTA

Surose,Plant growth substance

Adjust pH to 5.8

The media should be subpackagedin several vessels, which containing agar powder

Autoclaving121℃ 20min

Mission

• Sterilize water• Four dishes，including six filter papers• Four beakers• Scalpels and Nose-shaped tweezers• Two kinds of medium, including different

concentration plant growth substances, which had three bottles.

Equipment

Journal

http://www.springer.com/life+sci/plant+sciences/journal/11240http://www.plant-physiology.com/txun/index.asp

Bibliography• 卫志明. 细胞全能性和细胞工程//陈晓亚主编. 植物生理与

分子生物学（第三版） 2007. 98-133.• Bonga J.M., Durzan D.J. 1982. Tissue culture in forestry.

Netherland. Kluwer Press.(http://www.flipkart.com/tissue-culture-forestry-bonga-durzan/9024726603-mj33fycl3c#previewbook)中译本《树木组织培养》（http://hn.sslibrary.com/library.jsp?username=gphdsf ）

• Edwin F. George, Michael A. Hall, Geert-Jan De Klerk. Plant propagation by tissue culture. 3rd edition. Springer Press.

• http://www.high-tech.cn/jszx.asp?id=2 （for water purification）