H400, Lecture 12, 10/11/01
H400, 2001 Upcoming Due Dates
• 10/23/01 (Tuesday, not 10/21) Field Trip Reports (both trips, 1 page each)• 10/25/01 (Thursday, 1:30-3:00PM; not 10/19) Midterm Exam• 11/01/01 (not 10/31) Written Cuttage Lab Report (Experiment 1 – Even groups; Experiment 2 – Odd groups
Midterm Exam will cover:
• Lectures• Lab exercises (understand the what & why of lab exercises)• Web assignments (preparation for Geneva field trip, and for Microprop lab, see Table 2)• Textbook: (**rechk for 2001) Assigned sections of Ch 2, 9, 10, 12 & 17; see Table 2• Field Trips
UP TO and Including Lecture on Tuesday, 10/30/01
1. Mitosis / meiosis – the two types of cell division involved in plant propagation. Mitosis refers to somatic cell division involved in regeneration of new organs during asexual propagation, whereas Meiosis refers to cell division involving formation of haploid gametes and segregation of alleles, from diploid “mother” cell during the process of sexual reproduction.
Black = similarityWhite = difference 3. Mist / fog 4. Root initial / root primordium 5. In vitro / ex vitro
Midterm Exam: examples of Compare & Contrast questionsCompare and Contrast each of the following pairs (or triplet), in the context of their relevance to plant propagation. Your answer should make clear what each term refers to and the similarities and differences between them. (4 point each)
Short Essays (15 points each) (1/2 – 1 page) 1. Describe how you would determine if cuttage or micropropagation was the best method
for propagating a newly introduced ornamental selection for large-scale commercial production. Assume that you have at least a year before commercial production begins and that adaquate facilities are available for either method of propagation.
2. Briefly describe each of the stages involved in plant micropropagation via shoot culture
(as described by Murashige, 1974), including the objective(s) of each stage and the strategy used to achieve those objectives.
3. What is the effect of stock plant growth phase on rooting of cuttings and how can it be
managed to optimize rooting of difficult-to-root species? 19. Describe how light, temperature and humidity interact to influence the water potential of a
cutting and its ability to root. How are these environmental factors managed to maximize rootability in a mist system, compared to a closed case (polyethylene) moisture management system?
Also: there may be a data interpretation question
Midterm Exam: examples of Short Answer questions
Propagation via Plant Tissue Culture
PropagationCuttageGraftageLayerageDivision
Asexual PropagationCuttageGraftageLayerageDivisionMicropropagation
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Welcome to the 15 Annual conference of micropropagators
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Definitions
Plant Tissue Culture - in vitro, aseptic plant culture for any purpose including genetic transformation and other plant breeding objectives, secondary product production, pathogen elimination… or for asexual (micropropagation) or sexual propagation
Definitions
Plant Tissue Culture
Micropropagation - use of tissue culture for clonal plant propagation (including somatic embryogenesis)
In vitro seed or embryo culture - in vitro sexual propagation
Characteristics of MostMicropropagation Systems
• aseptic culture (no other organsims)• small propagule = explant• heterotrophic nutrition
History of Plant Tissue Culture Propagation at Cornell
Lewis Knudson, 1922 - 1st in vitro embryo germination (orchid) - ie the invention of in vitro embryo culture
Knudson, L, 1922, Nonsymbiotic germination of orchid seeds, Botanical Gazette LXXIII: 19
Gavino Rotor, 1949 - 1st in vitro vegetative propagation.
Rotor, G, 1949, A method of vegetative propagation of Phalaenopsis species and hybrids, AOS Bulletin (Dec 1) pp 738. - according to Arditti, (1993, Microprop of Orchids (CH 1), Wiley) Rotor (Dr. Mac Daniels student) was 1st in vitro vegetative propagation.
FC Stewart, 1959 - 1st somatic embryogenesis (carrot)
FC Stewart, MO Mapes, and K Mears, 1959, Growth and organized development of cultured cells, Amer. Journal of Botany 45:653
Explants used for micropropagation & embyo culture
• shoot meristem, tip, bud• leaf or stem (internode)• root • anther / microspore• ovule • embryo () associated seed parts
Shoot Explants
Meristem tip
Actively growing shoot tip
Dormant bud
Forcing solution (HQC, sucrose, +/- gibberellin):
allows a dormant bud to be forced under clean (indoor) conditions.
- Paul Read et al., 1990
Leaf Explants
Seed “explant” (propgaule)
Field Trip: October 18, 2001
AnTec LaboratoryDeflasking and Compotting Slipper Orchids www.ladyslipper.com/compot2.html
Specialists in Paphiopedilum and Phramapedium (tropical ladyslipper orchids), species and hybrids.
Stem Explant: Scrophularia sp., Wansang Lim
Plant tissue culture systems used for micropropagation
• shoot organ culture (axillary or nodal)• root organ culture• callus culture• cell suspension culture• somatic embryogenesis• anther (microspore) culture
Plant tissue culture systems used for micropropagation
• shoot organ culture (axillary or nodal)
Plant tissue culture systems used for micropropagation
• root organ culture
Plant tissue culture systems used for micropropagation
• callus culture
Http://www.cas.muohio.edu/~wilsonkg/biotech/html/greentc_.htmHttp://www.zeta.org.au/
~brianc/pcc.htm
Plant tissue culture systems used for micropropagation
cell (liquid) suspension culture
Http://www.zeta.org.au/~brianc/pcc.htm
(liquid medium, agitation)
Cells and clumps of cells in suspension
http://www.bio.purdue.edu/nscort/image.html
http://mars.cropsoil.uga.edu/homesoybean/somprot.htm
Plant tissue culture systems used for micropropagation
somatic embryo culture
Plant Regeneration Pathways
• Organogenesis via organ culture• Direct organogenesis• Organogenesis from callus• Somatic embryogenesis
Plant Regeneration Pathways
• Organogenesis via shoot organ culture
+ auxin
Organogenesis via shoot organ culture
Plant Regeneration Pathways
Organogenesis via root organ culture
Organogenesis via root organ culture
Plant Regeneration Pathways
• Direct organogenesis
African violet leaf cultureby Michael H. Renfroe
Http://www.jmu.edu/biology/biofac/facfro/cloning/cloning.html
Plant Regeneration Pathways
• Organogenesis from Callus:African Violet
Use of a Protoplast Regeneration System for African Violet Improvement Traud Winkelmann, Institute for Breeding of Ornamental Species Ahrensburg, Germany
http://aggie-horticulture.tamu.edu/tisscult/proto/wink/wink.html
Plant Regeneration Pathways• Direct Organogenesis: Scrophularia sp., Wansang Lim
Plant Regeneration Pathways
• Somatic embryogenesis
http://mars.cropsoil.uga.edu/homesoybean/somprot.htm
http://mars.cropsoil.uga.edu/homesoybean/somprot.htm
Plant Regeneration Pathways• Organogenesis via organ culture• Direct organogenesis• Organogenesis from callus• Somatic embryogenesis
Revised to here 10/09/01
Micropropagation via Shoot Culture
Applications of Shoot Culture :
• propagation per se• bulking plant breeding selections• production and maintenance of disease free plants• restoration of juvenility• stock plant management
Shoot culture for propagation per se
Theoretical:Eg. Mum
Conventional prop. -- 30K plants per year
Microprop: (based on 5 fold increase every 4 wks, assuming unlimited labor, space, etc. and no losses):
1 > 5 > 25 > 125 > 625 > 3125 > 15,625 > 78,125 > 390,625 > 1,953,125 > 9,765,625 > 48,824,125 > 244,140,625
Shoot culture for propagation per se
Practical Considerations: economic niches
• French hybrid lilacs• Raspberries• Rhododendrons, Kalmia
Applications of Shoot Culture :
• propagation per se• bulking plant breeding selections• production and maintenance of disease free plants• restoration of juvenility• stock plant management
Applications of Shoot Culture :
• propagation per se• bulking plant breeding selections• production and maintenance of disease free plants• restoration of juvenility• stock plant management
Shoot culture for production and maintenance of disease free plants
• assumption: because plant tissue cultures are aspetic they are disease free
- contributed to the spread of orchid viruses
Shoot culture for production and maintenance of bacterial and fungal disease-free plants
Culture indexing: for detection of Bacteria & fungi (not virus)
Or
X
propagate
destroy
save
Shoot culture for production and maintenance of disease free plants
Virus
Effect of shoot tip (meristem) size on Stage I survival and virus elimination
Shoot culture for elimination of virus:
examples:
• Ogalvee geraniums• EMLA clonal apple rootstocks• Dasheen mosaic virus of aroids
(diffenbachia, spathophyllum, syngonium)
Applications of Shoot Culture :
• propagation per se• bulking plant breeding selections• production and maintenance of disease free plants• restoration of juvenility• stock plant management
From MP Stock PlantFrom TraditionalStock Plant
% Rooting 99.5 91Days to first root 16 20Plant height 43.6 cm 36.1 cm% w/ axillary budoutgrowth 74.9 69.5
Restoration (?) of juvenility
from: Kristiansen, 1991, Post propagation growth of cuttingsfrom in vitro and in vivo propagated stock plants of Ficus benjamina, Scientia Horticulturae 46: 315-322
Blueberry
Additional advantages associated withMicropropagation via shoot culture
• Stock plant management- Bamboo Nurseries (philodendron) - before MP 75% of gh space devoted to stock plants
•Enhanced field performance - related to “rejuvenation”
-Red Rasp / C&W noted vigor and suckering: MP > Conventional-thornless blackberry (Sqartz, et al, 1983)-Strawberry - Zimmerman, 1986-Aster, mum, and hosta (mum: 100 divisions vs. 10) - lasts 1 season (Simart in Zimmerman)
• Extend propagation season / especially combined with cold storage of shoot and / or other types of culture
• Facilitate international export
Stages of Shoot Organ Culture• Stage I - establishment• Stage II - proliferation• Stage III - pretransplant• Stage IV - transplant ex vitro
Resume 10/18/99
Murashige, 1974
Objectives for Stage I
• asepsis• survival• stable growth
Briggs Nursery
Briggs Nursery
from: Sanchez & Vieitez, 1991, In vitro morphogenic potential of basal sprouts and crown branches of mature chestnut, TreePhysiology 8:59-70.
Effect of growth phase on stage I shoot culture
Shoot Organ Culture• Stage I - establishment• Stage II - proliferation• Stage III - pretransplant• Stage IV - transplant ex vitro
Objectives for Stage II
• increase in number of rootable units
Axillary shoot culture
Cytokinin
from: Sanchez & Vieitez, 1991, In vitro morphogenic potential of basal sprouts and crown branches of mature chestnut, TreePhysiology 8:59-70.
Effect of growth phase on stage I shoot culture
Preformed vs. Adventitous Shoots
Rhododendron Montego with Tissue Proliferation
Shoot Organ Culture• Stage I - establishment• Stage II - proliferation• (Stage III - pretransplant)• Stage IV - transplant ex vitro
Objectives for Stage III
• +/- rooting• +/- acclimitization ( light, RH)
Stage II – no roots
In vitro rooting cytokinin, auxin
Stage III
Shoot Organ Culture• Stage I - establishment• Stage II - proliferation• Stage III - pretransplant• Stage IV - ex vitro establishment
Stage IV – fog or fine mist at Briggs Nursery
Later stage acclimitization (Stage IV) at Briggs Nursery
Stage IV fog tunnel at KPL
Stage IV – Rhododendron at Prides Corner Nursery
Stage IV
Stage IV losses
Stage IV losses
Technical Considerations (How to do Microprop)
Laminar flow hood
From: Laminar Flow Hood Construction by Forester & Berger, http://envhort.ucdavis.edu/dwb/outreach.htm
Technical Considerations (How to do Microprop)
Technical Considerations (How to do Microprop)
Technical Considerations (How to do Microprop)
Technical Considerations (How to do Microprop)
http://www.osmotek.com/product.htm#lifereactorOsmotek Lifeline: Advanced products for plant tissue culture
Mechanization of Shoot Culture
*
http://www.osmotek.com/product.htm#lifereactorOsmotek Lifeline: Advanced products for plant tissue culture
http://www.osmotek.com/product.htm#lifereactorOsmotek Lifeline: Advanced products for plant tissue culture
Mechanization of Shoot Culture
*
http://www.osmotek.com/product.htm#lifereactorOsmotek Lifeline: Advanced products for plant tissue culture
From TC in the home Kitchen, by Rick Walkerhttp://www.hpl.hp.com/botany/public_html/cp/slides/tc/tc.htm
From: Phytotechnology Laboratories, http://www.phytotechlab.com/
Murashige & Skoog basal nutrient medium
Technical Considerations (How to do Microprop)
Plant Tissue Culture Media / Additional Components
• vitamins: thiamine (vitamine B1), pyradoxine, etc.• Phytohormnes (auxin / cytokinins)
Effect of auxin and cytokinin / auxin on organogenesis in vitro
Cytokinin >
Auxin
Make new slide:
[Structure of cytokinin (BAP) here]
Cytokinins used in plant tissue culture• (full spelling) BAP• (full spelling) 2iP• zeatin• thiadiazuron• kinetin