h400, lecture 12, 10/11/01

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

.

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 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.

http://www.ladyslipper.com/compot2.html




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


• shoot organ culture (axillary or nodal)


• root organ culture


• callus culture

Http://www.cas.muohio.edu/~wilsonkg/biotech/html/greentc_.htmHttp://www.zeta.org.au/

~brianc/pcc.htm


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


somatic embryo culture

Plant Regeneration Pathways

• Organogenesis via organ culture• Direct organogenesis• Organogenesis from callus• Somatic embryogenesis


• Organogenesis via shoot organ culture

+ auxin

Organogenesis via shoot organ culture


Organogenesis via root organ culture


• Direct organogenesis

African violet leaf cultureby Michael H. Renfroe

Http://www.jmu.edu/biology/biofac/facfro/cloning/cloning.html


• 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


• Somatic embryogenesis


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

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)

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

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

Technical Considerations (How to do Microprop)

Laminar flow hood

From: Laminar Flow Hood Construction by Forester & Berger, http://envhort.ucdavis.edu/dwb/outreach.htm

http://www.osmotek.com/product.htm#lifereactorOsmotek Lifeline: Advanced products for plant tissue culture

Mechanization of Shoot Culture

*


Mechanization of Shoot 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


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