Acclimatization of Tissue Culture Plants
Content Definitions: plant tissue culture, micropropagation Stages of micropropagation Methods of micropropagation, production system, plants propagated by TC
Acclimatization definition, need for acclimatization Characteristics of TC plants & acclimatization process Planting, stages of acclimatization Acclimatization facilities, acclimatization equipment & planting tools Soil, plant protection, characteristics of successful acclimatization
Potting, shipping, troubleshooting
Plant Tissue Culture: Definitions ďƒ˜ Plant
tissue culture: aseptic culture of plant protoplasts, cells, tissues or organs under conditions which lead to cell multiplication or regeneration of organs or whole plants
ďƒ˜ Micropropagation: Vegetative Propagation of Plants by Tissue Culture
Stages of Micropropagation
1
2
1 - Avihai 2 - www.fsagx.ac.be/pc/images/phas_accl_re.jpg 3 - www.fsagx.ac.be/pc/images/phas_accl_re.jpg
3
Picture: Dubon
Methods of Micropropagation ďƒ˜ Organogenesis: most plants ďƒ˜ Embryogenesis: Coffee, date palms Coffee embryogenesis (photos from Jimma, Ethiopia)
Micropropagation Production System Tissue Culture Laboratory Acclimatization Greenhouse Potting Nurseries
Plants Produced by Micropropagation ďƒ˜ Mass propagation: banana, sugar cane, pineapple, apple rootstocks, peach-almond rootstocks, ginger, coffee, date palms, vanilla, paulownia, eucalyptus, bamboo, house plants from Araceae family (Philodendron, etc.), orchids, ferns. ďƒ˜ Virus & disease free mother plants: potato, sweet potato, carnation, gypsophylla, strawberry
Acclimatization (Definition): A process by which physiologically and anatomically adjust from in vitro to ex vitro conditions.
Why acclimatization is required? A tissue culture plant is characterized by: Poor cuticle development: epi-cuticular wax needs to be formed.
Abnormal stomatal development and function. Either are not properly depressed. Or do not open and close properly. Non- or poorly functional roots. Poorly developed photosynthetic system.
Cuticle: Waxy layer water proofing upper leaves.
Abnormal stomatal development
Photosynthetic System of TC Plants. Poorly differentiated leaf structure. Poorly developed chloroplasts. Supplied carbohydrate source to independent carbon fixation. 1. DNA
4. Grana
2. Ribosomes 5. Stroma 3. & 6. Outer Membrane
7. Starch Grain
Characteristics of Plant Readiness for Acclimatization Dark green leaves, no signs of vitrification (hyper-hydration). Leaves of small size Short roots on all the plants in the TC vessel, hairy roots are preferred
Preferably, no microbial contamination
Characteristics of Acclimatization Process Time: Acclimatization is relatively slow process, may take weeks. Accordingly, plant’s starch reserves are important. Humidity: The plant is transferred from high relative humidity to lower relative humidity Plant protection: Transfer from clean environment to presence of pathogens. Light: Transfer from conditions of low light intensity to high light intensity. Photosynthesis: plants should transform from low photosynthetic competence (heterotrophic nutrition) to photosynthetic competence.
Planting ďƒ˜The culture vessels brought from the lab should be stored at shade and avoid extreme (high or low) temperatures ďƒ˜Opening the vessels for several days before the planting is not recommended: may create contamination or drying ďƒ˜The plant should be separated, avoid cutting
Planting (cont.)
Agar should be removed by gentle shaking and absorb by towel paper. Sorting* before inserting to soil cells. Planting site: shady place, with high humidity the planting trays should be positioned on table Following the planting, the planted trays should be taken to the acclimatization table
Sorting of Plants TC plants should be sorted before being inserted to the soil Sorting to the following categories: big, medium, small, clumps Sorting allow uniformity of plants in acclimatization trays, efficient work, synchronized transfer from Acc-1 to Acc-2 Picture: www.ncsu.edu/jcraulstonarboretum/photography.../
Planting conditions Detached soil – planted celled trays are put on acclimatization tables at insect proof greenhouse/tunnel The soil should be previously wetted, do not water the trays following the planting Increased humidity (90% or more) and reduced light intensity (~10KLux)
Stages of Acclimatization STAGE
DAYS
Planting of plantlets from TC.
1 day.
Primary acclimatization – the plants are mostly dependent Secondary acclimatization: plants become independent
7- 14 days 14-28 days
Photo: Rahan Meristem
Acclimatization facilities Wet cover (for example: cheesecloth) Humidity tent. Overhead mist. Fog system.
Acclimatization Equipment Steam generator / soil mix solar sterilization. Soil mixing device Nursery trays Planting tools Soil cover:
Cheesecloth
Planting tools Long forceps Planting stick (pencil) Hole making pattern.
Labels & pen. Planting forms.
Planting Form Date: _______ Var: ________ # plants: _____ Used vessels:___
GRAN NAIN
Soil Mix Functions Soil mix should provide: Physical support Nutrition – minerals water Root aeration
Soil Mix Components Nutrition / organic matter: peat moss, coco peat, Coffee husks, compost, decomposed FYM, rice shells, etc. Water holding: vermiculite, volcanic gravel (tuff), etc.
Aeration/drainage – Perlite, Volcanic gravel, wood charcoal, etc. Question: Why tuff can give both water holding and drainage?
Crop Protection Sources of plant pests & diseases: Plant born: arrive with the TC plant
Soil born: pests and diseases from local soil or artificial soils Air born: pests carried by the air Facility & tools born Vector transmitted: pests carried by other organisms, e.g. virus carried by insect
Factors Affecting Successful Acclimatization Source plants: activated photosynthetic system, protecting wax layers, root system Balanced soil mix
Adequate planting Humidity and shade Pest and disease control
The Following Step: Potting In many crops the acclimatized plants are too small to stand harsh field planting conditions: hot & dry weather, floods, etc. For these crops, potting is required as intermediate step between acclimatization and field planting. Examples: Banana, Pineapple, coffee
Direct Field Planting of Acclimatized Plants Paulownia: Acclimatized plants can be planted directly in the field
Production of Mother Plants by Tissue Culture
TC
Gen.0
Gen.1
Gen.2, 3
Shipping Acclimatized Plants
Source: www.galiltec.com/eng/Tech_Microp.html
Troubleshooting Potential acclimatization problems may stem from:
TC plants provided (physiology, disease, genetics) Acclimatization facility Soil mix preparation Delicate crops
Pests and diseases
Simple Acclimatization Facilities Simple adequate solution: wooden table covered with PE sheet
Simple inadequate solution: tunnel on the floor subjected to soil-born pests and diseases
Troubleshooting: Soil Mix Preparation Using the right proportions of local materials: For organic matter: compost, coffee husks, rice shells
For drainage: volcanic gravel (tuff), coarse sand For water holding – local soil (small amount) Good drainage should be the top priority
Troubleshooting: Delicate crops Delicate crops: vitrified, embryos, etc The approach: adding intermediate stage
between TC in lab and acclimatization in GH Solutions: growth on autoclaved soil mix in transparent
box at growth room
Troubleshooting: Mutations SCV Heliconia A – Chlorophyl (leaves)
B – Low stature C- Pseudostem+petiole D- Normal
Dwarf soma-clonal banana variant www.fao.org/ag/agp/agps/pgr/Pic5.jpg