Native Deciduous Azalea Micropropagation

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Native Deciduous Azalea Micropropagation
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Presentation Overview

• What azaleas are and their significance
• The long-term objectives of this project
• This summers specific objectives
• Propagation techniques
• Explant disinfection

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Native Deciduous Azaleas
Genus Rhododendron Sub Genus Pentanthera
- Due to their highly attractive and distinctive
flowers, their diverse ecological distribution,
and their tendency to hybridize in the wild,
native deciduous azaleas represent an intriguing
system from botanical, evolutionary, and
horticultural perspectives.
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Species native to U.S.A.

• West Coast Native Azaleas
• R. occidentale

• East Coast Native Azaleas
• R. vaseyi
• R. canadense
• R. canescens
• R. austrinum
• R. flammeum
• R. periclymenoides
• R. alabamense
• R. atlanticum
• R. calendulaceum
• R. prinophyllum
• R. viscosum
• R. arborescens
• R. cumberlandense
• R. prunifolium
• R. eastmanii

• At least 17 species native to the United States.
• One species newly discovered!

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Our species
R. arborescens
R. cumberlandense
R. calendulaceum
R. viscosum
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Our Study Populations/Species

• Locations
• 1. Gregory Bald
• A. cumberlandense
• B. arborescens
• C. viscosum(?)
• 2. Copper Ridge
• A. cumberlandense
• B. arborescens
• C. viscosum(?)
• 3. Hooper Bald
• A. calendulaceum

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Unique Hybridization
Seedling 11- 1
Seedling 11- 2
Parent Gregory Bald 11 Rattlesnake Cream
Seedling 11-3
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Long Term Objectives

• The establishment of a reference plant
  population.
• Assess the correspondence between morphological,
  karyotypic and genomic parameters.
• Assess phylogenetic
• relationships amongst
• these plants.

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This Summers Objectives

• Continue production and preservation of source
  material.

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2. Refine procedures for obtaining, introducing,
and raising reference plants.
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• 3. Create a reference/preservation planting on
  Upland's Campus.

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Propagation

• Seedling
• Classic Shoot Propagation
• In Vitro or Micropropagation

1.
3.
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3.
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In Vitro Propagation
2.
1.
3.
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In-Vitro Propagation Continued
4.
5.
6.
7.
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Explant Death!

• Its normal to lose 80-90 of explants to
  contamination

Eh?
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Plant Tissue Disinfection
Standard 10 bleach 1 ml tween 20
(detergent) 10 minute wash 3x 10 minute H20
rinse
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NaDCC and Accel-TBDisinfectant Pilot Studies

• NaDCC
• 99 Dichloro-s-triazinetrione, dihydrate
• 1 others
• Chlorine-based pool sanitizer
• 55 available chlorine

• Accel-TB
• 0.5 H2O2
• 99.5 others
• BroadSpectrum disinfectant
• Fungicidal
• Tuberculocidal
• Virucidal
• Bactericidal

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NaDCC Test 1
0, 1, 1.2, 1.4, 1.6, 1.8, 2.0, and 2.2 grams/500
ml H20 1 ml tween 20 10 minute treatment 3
sterile 15 minute rinses
Contamination 67 contaminated.
Random dispersion. Browning
None Observations 1. NaDCC does not seem to be
phytotoxic at concentrations from
0-2.2g/500ml. 2. Little contamination among
control explants at 0.0gNaDCC/500 ml indicates
low levels of original explant contamination.
19-2
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NaDCC Test 2
2, 3, 4, 5, 6, 7, 9, and 11 g/500 ml H20 1 ml
tween 20 10 minute treatment 3 sterile 15 minute
rinses

Contamination None Browning 80-90 brown
death over all concentrations. Observations 1.
Phytotoxicity observed over all concentrations,
including concentrations not previously
phytotoxic to explants. 2. Still very little
contamination. 3. High levels of toxicity may be
due to smaller/younger explant pieces.
35-4
Greenhouse Source Plants
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Accel-TB Test 1
Accel-TB at strengths of 100, 90, 80, 70,
60, 50, 40, 20, and 0 100 is full
commercial strength 1 ml tween 20 10 minute
treatment 3 sterile 15 minute rinses
Contamination None Browning Complete
phytoxicity from 100-70, 50 phytotoxic from
60-0. Observations 1. Ranges 70-50 may be
effective disinfection concentrations. 2. Still
no contamination 3. Plants used in this and
NaDCC Test 2 are from greenhouse.
22-1
Greenhouse Source Plants
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NaDCC Test 3 Rinse
Outside source plants NaDCC at 3, 4, 5, 6, 7,
g/500 ml 1 ml tween 20 10 minute treatment 3
sterile 15 minute rinses
Contamination 87 contamination from
3.0g-7.0g Browning 1 or 2 deaths/concentration.
Observations 1. Contamination present in
outside plants!
8-2
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NaDCC Test 4 No Rinse
Outside source plants NaDCC at 0, 1, 2, 3, 4, 5,
7, g/500 ml 1 ml tween 20 10 minute treatment
Contaminated Brown Survival 0.0g/500ml 5 1 0 1.
0g/500ml 5 1 0 2.0g/500ml 1 5 0 3.0g/500ml 0 3
3 4.0g/500ml 2 4 0 5.0g/500ml 0 6 0 7.0g/500ml
0 6 0
5-1
browning from 10-18 days
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Accel-TB Sweet Spot Test
Outside source plants 70 - 21mins, 16mins,
13mins, 10mins, 5mins 60 - 23mins, 18mins,
14mins, 10mins, 5mins 50 - 25mins, 20mins,
15mins, 10mins, 5mins 1 ml tween 20 3 sterile 15
minute rinses
17-1
Contaminated Brown Survived 70- 83 0 17 6
0- 90 3 7 50- 83 13 4 Observations 1.
Significant contamination seen for all time
lengths and concentrations.
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Overall Observations

• Low contamination levels on greenhouse plants.
  High levels on outside plants.
• Individual plants react differently to
  disinfection techniques.
• Proposed Disinfection Tests For Next Year
• 1. NaDCC no wash 2.0g-4.0g/500ml H20 (and
  variations)
• 2. Addition of fungicide wash or soak to
  disinfection process.

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Acknowledgements

• Dr. Whipple
• Paul Lightfoot
• Earl Sommerville
• Don Hyatt
• Vivian Abney

Sunrise at Hooper Bald
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Questions?