华东师范大学：《植物生理学》课程资源（实验课教学课件）植物组织培养——无菌操作 Plant Physiology Experiments Tissue culture——culture and incubation

Tissue culture culture and incubation Plant Physiology Experiments

Plant Physiology Experiments Tissue culture —— culture and incubation

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

tissue culture zA technique which small tissue pieces or organs are removed from a donor plant and cultured aseptically on a nutrient medium. zBy 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. Explants

o Cultures of Organised structures MEristem cultures O Shoot tip, or shoot cultures O Node cultures of separte lateral buds ISolated root cultures O Embryo cultures o Cultures of Unorganised tissues O Callus(or tissue)cultures O Suspension (or cell)cultures O Protoplast cultures O Anther cultures Plant Tissue Culture Procedure- Background by e.f. george

z Cultures of Organised structures {Meristem cultures {Shoot tip, or shoot cultures {Node cultures of separte lateral buds {Isolated root cultures {Embryo cultures z Cultures of Unorganised tissues {Callus (or tissue) cultures {Suspension (or cell) cultures {Protoplast cultures {Anther cultures Plant Tissue Culture Procedure – Background. By E.F. George

How to Choice of Explants material correctly? ● Position o Season ●Aae Size Induction rate dedifferentiation and redifferentiation capacity

How to Choice of Explants Material correctly? zPosition zSeason zAge zSize Induction rate： ——dedifferentiation and redifferentiation capacity

The principal methods of micropropagation Meristem culture Alcr burning M tile ae cry shoot One o mom rode ct shoot Soral Ad.anne DIRECT shoots Direct Explants fern Indirect shoot STOCK PLANT someti Issua formalion INDIRECT punters MORPHOGENESIS Calus grow Cats Cavermous Indirect embryogenesis Smote embryos clue

The principal methods of micropropagation

tatt The These callus lines may also differ interaction in color and morphology I They may, for example, be compact, fab or nodular, creamy yellow or white Explant type and medium 1= Callus Induction Root-forming callus Shoot-fomlng Non-morphogenic callus Embryogenic P I callus Subculture of calluses ith diferent morphogen/c potential

Tobacco leaf Two weeks later Asparagus officinalis L nodes Seven days later

Tobacco leaf Asparagus officinalis L. nodes Seven days later Two weeks later

Asparagus officinalis L Which ne s the true callus unorganised cell masses 王伟超,沈旻,程蔚.芦笋细胞悬浮培养.KY2008-083D

Which one is the true callus ? Asparagus officinalis L. 王伟超，沈旻，程蔚. 芦笋细胞悬浮培养. KY2008-083D unorganised cell masses

Surface Sterilization Sterilants concertration Sterilization time remove effection (%) (min) ethano 70-75 easy 0.2-2 good calcium 9-10 hypochlorite easy pretty good sodium 0.5-5 easy 5-20 hypochlorite pretty good mercuric chloride 0.1-0.3 hard 2-10 excellent hydrogen 10-12 easy 5-15 good peroxide benzalkonium 0.5-5 bromide easy 20-30 good antibiotics 4-5mg/儿L) harder better Attention: Tween can improve the penetration of disinfectants and sterilization effect

Surface Sterilization Sterilants concertration (%) remove Sterilization time (min) effection ethanol 70-75 easy 0.2-2 good calcium hypochlorite 9-10 easy 5-30 pretty good sodium hypochlorite 0.5-5 easy 5-20 pretty good mercuric chloride 0.1-0.3 hard 2-10 excellent hydrogen peroxide 10-12 easy 5-15 good benzalkonium bromide 0.5-5 easy 20-30 good antibiotics 4-5(mg/L) harder 5-30 better Attention:Tween can improve the penetration of disinfectants and sterilization effect

Protocol o A short exposure in 70%ethanol, washing in water o Put into hypochlorite solution 1-5min o Washing several times in water o Absorbed water by sterilized filter paper o Transfer of the explants to the culture vesse iNcubation

Protocol zA short exposure in 70% ethanol, washing in water zPut into hypochlorite solution 1-5min zWashing several times in water zAbsorbed water by sterilized filter paper zTransfer of the explants to the culture vessel. zIncubation