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
Midterm Exam: examples of Compare & Contrast questions
Compare 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)
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 = similarity
White = difference
3.
Mist / fog
4.
Root initial / root primordium
5.
In vitro / ex vitro
Midterm Exam: examples of Short Answer questions
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
Propagation
via Plant Tissue Culture
Propagation
Cuttage
Graftage
Layerage
Division
Asexual Propagation
Cuttage
Graftage
Layerage
Division
Micropropagation
Welcome to the
15 Annual conference
of micropropagators
. ..
.
.
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 Most
Micropropagation 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
Actively growing shoot tip
Meristem 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 Laboratory
Deflasking 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.zeta.org.au/
~brianc/pcc.htm
Http://www.cas.muohio.edu/~wilsonkg/biotech/ht
ml/greentc_.htm
Plant tissue culture systems
used for micropropagation
cell (liquid) suspension culture
(liquid medium,
agitation)
Http://www.zeta.org.au/
~brianc/pcc.htm
Cells and clumps of
cells in suspension
http://www.bio.purdue.edu/nscort/image.html
Plant tissue culture systems
used for micropropagation
somatic embryo culture
http://mars.cropsoil.uga.edu/homesoybean/somprot.htm
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 culture
by 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
propagate
Or
X
destroy
save
Culture indexing: for detection of Bacteria & fungi (not virus)
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
Restoration (?) of juvenility
% Rooting
Days to first root
Plant height
% w/ axillary bud
outgrowth
From MP Stock Plant
99.5
16
43.6 cm
74.9
From Traditional
Stock Plant
91
20
36.1 cm
69.5
from: Kristiansen, 1991, Post propagation growth of cuttings
from in vitro and in vivo propagated stock plants of
Ficus benjamina, Scientia Horticulturae 46: 315-322
Blueberry
Additional advantages associated with
Micropropagation 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
• Stage II
• Stage III
• Stage IV
Murashige, 1974
- establishment
- proliferation
- pretransplant
- transplant ex vitro
Objectives for Stage I
• asepsis
• survival
• stable growth
Briggs Nursery
Briggs Nursery
Effect of growth phase on stage I shoot
culture
from: Sanchez & Vieitez, 1991, In vitro morphogenic potential
of basal sprouts and crown branches of mature chestnut, Tree
Physiology 8:59-70.
Shoot Organ Culture
• Stage I
• Stage II
• Stage III
• Stage IV
- establishment
- proliferation
- pretransplant
- transplant ex vitro
Objectives for Stage II
• increase in number of
rootable units
Axillary shoot culture
Cytokinin
Effect of growth phase on stage I shoot
culture
from: Sanchez & Vieitez, 1991, In vitro morphogenic potential
of basal sprouts and crown branches of mature chestnut, Tree
Physiology 8:59-70.
Preformed vs. Adventitous Shoots
Rhododendron Montego with Tissue Proliferation
Shoot Organ Culture
• Stage I
• Stage II
• (Stage III
• Stage IV
- establishment
- proliferation
- pretransplant)
- transplant ex vitro
Objectives for Stage III
• +/- rooting
• +/- acclimitization ( light, RH)
Stage II – no roots
Stage III
In vitro rooting
cytokinin, auxin
Shoot Organ Culture
• Stage I
• Stage II
• Stage III
• Stage IV
- establishment
- proliferation
- pretransplant
- 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)
From: Laminar Flow Hood Construction by Forester &
Berger, http://envhort.ucdavis.edu/dwb/outreach.htm
Laminar flow hood
Technical Considerations (How to do Microprop)
Technical Considerations (How to do Microprop)
Technical Considerations (How to do Microprop)
Technical Considerations (How to do Microprop)
Mechanization of Shoot Culture
*
Osmotek Lifeline: Advanced products for plant tissue culture
http://www.osmotek.com/product.htm#lifereactor
Osmotek Lifeline: Advanced products for plant tissue culture
http://www.osmotek.com/product.htm#lifereactor
Mechanization of Shoot Culture
*
Osmotek Lifeline: Advanced products for plant tissue culture
http://www.osmotek.com/product.htm#lifereactor
Osmotek Lifeline: Advanced products for plant tissue culture
http://www.osmotek.com/product.htm#lifereactor
From TC in the home Kitchen, by Rick Walker
http://www.hpl.hp.com/botany/public_html/cp/slides/tc/tc.htm
Technical Considerations (How to do Microprop)
Murashige & Skoog basal nutrient medium
From: Phytotechnology Laboratories, http://www.phytotechlab.com/
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