Tissue culture Media preparation

Transcription

1 Tissue culture Media preparation Plant Physiology Experiments

2 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 composition of the nutrient medium and other environmental parameters, the growth and development of the tissues in culture can be directed into different channels.

3 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.

4 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)

5 The Background Ⅱ Folke Karl Skoog Tsui( 崔澂 ) 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

6 The Background Ⅲ Chu C-C ( 朱至清 ) 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.

7 Tobacco A B C D (A B C D for different growth periods)

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9 Rice genetic transfomation callus induction callus co-culture the first selection rootage redifferentiation the second selection

10 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. Plant in vitro germplasm bank : 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:

11 Initiating tissue culture Explants Isolation and incubation The cultural environment Media Solidified media Liquid media Plant Tissue Culture Procedure Background. By E.F. George

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

13 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

14 The ingredients of MS medium(mg/l ) Macronutrients Micronutrients Fe 2+ Vitamin Mixture Surose Agar NH 4 NO KNO KH 2 PO MgSO 4 7H 2 O 370 CaCl 2 2H 2 O 440 MnSO 4 4H 2 O 22.3 ZnSO 4 7H 2 O 8.6 H 3 BO KI 0.83 NaMoO 4 2H 2 O 0.25 CuSO 4 5H 2 O CoCl 2 6H 2 O FeSO 4 7H 2 O 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 g 8g/L ph 5.8

15 The ingredients of N6 medium(mg/l ) (NH 4 )2SO Macronutrients Micronutrients Fe 2+ Vitamin Mixture KNO KH 2 PO MgSO 4 7H 2 O 185 CaCl 2 2H 2 O 166 MnSO 4 4H 2 O 4.4 ZnSO 4 7H 2 O 1.5 H 3 BO KI 0.83 FeSO 4 7H 2 O 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

16 Attention micronutrient should be accurate to 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

17 Processing water(100ml) macronutrients Micronutrients Vitamin Fe-EDTA Surose, Plant growth substance Adjust ph to 5.8 Autoclaving min The media should be subpackaged in several vessels, which containing agar powder

18 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.

19 Equipment

20 Journal

21 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 ) Bonga J.M., Durzan D.J Tissue culture in forestry. Netherland. Kluwer Press.( 中译本 树木组织培养 ( ) Edwin F. George, Michael A. Hall, Geert-Jan De Klerk. Plant propagation by tissue culture. 3rd edition. Springer Press. (for water purification)