plant physiology experimentszwslx.ecnu.edu.cn/_upload/article/files/bc/d9/4be3e9f24b...plant...
TRANSCRIPT
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)
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.
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)