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EVALUATION OF DOUBLED
HAPLOID PRODUCTION
IN BURLEY TOBACCO
E. De Oliveira1, R. D. Miller1,2, N. Martinez1 and G. Weinberger1 1University of Kentucky
2University of Tennessee
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DOUBLING the # of CHROMOSOMES - HAPLOID cell
DOUBLED HAPLOIDS (DH) Plant breeding technique
HOMOZYGOUS diploid plants
Pollen Egg ADVANTAGES of DH Homozygous lines from heterozygous parents in a single step. 2 to 3 times faster than conventional.
Aa 100
25
AA
25
F1
F2
aa
F3 F4 F5 Fx
50
37.5 37.5
~ 0 ~50 ~50
A
x 2
a
Doubled Haploid
x 2
AA aa
Conventional
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Anther Derived Haploids (ADH): Culturing of the male gametes (anther culture)
WAYS TO GET DOUBLED HAPLOIDS
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ADH: Culturing of the male gametes (anther culture)
WAYS TO GET DOUBLED HAPLOIDS
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Maternally derived haploids (MDH): Interspecific cross N. tabacum X N. africana
WAYS TO GET DOUBLED HAPLOIDS
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OBJECTIVE What is the most effective method of generating DH
materials – ADH or MDH? HOW to develop DH lines
Part 1: Producing haploids
genotype # crosses # haploids Crosses / Haploid
TN 90LC 94 10 9.4 (GH)
GV 149 460 6 172 (GH) 29.3 (Field)
TKs 906 117 12.4 (GH) 6.6 (Field)
1460 133 ~ 11
Time: ADH: 473 days MDH: 400 days
MDH – Differential response of Genotypes
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genotype Anthers Plated
Anthers germin Hap/ ant. Germin.
Hap/ anther plated
TN 90LC 155 3 (1.9%) 21 .4
GV 149 272 39 (14.3%) 6.6 .95
TKs 2277 355 (15.6%) 7.1 1.1
2967 397 (14.7%) 7.1 .96
Efficiency of methods
ADH 0.2 flower bud/ hap 4.8 haploid/ flower bud
MDH ~ 11 flowers/ hap .09 haploid/ flower crossed
OBJECTIVE What is the most effective method of generating DH
materials – ADH or MDH? HOW to develop DH lines
Part 1: Producing haploids
ADH
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Genotype: TN 90LC 10 MDH
10 ADH
10 Inbred
EXPERIMENT
Design: Split Plot Design - 3 Locations 3 Blocks (reps) per Location 30 plants per row
Data: > Plant height - 50 D.A.T. > Plant height after topping > Leaf length > Leaf width > Number leaves/ plant > Yield
OBJECTIVE What is the most effective method of generating DH
materials – ADH or MDH? HOW to develop DH lines
Part 1: Agronomic performance of Doubled Haploids
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Plant Height at 50 D.A.T. (cm)
RESULTS
Models P value
AD x TN90 x MD <.0001
AD x TN90 <.0001
MD x TN90 0.056
Plant Height - 50th D.A.T.(cm)
Lexing Greene Woodf MEAN
ADH 91.93 100.15 152.23 114.8
TN 90 98.29 109.67 157.62 121.9
MDH 98.62 114.09 167.05 126.6
80
100
120
140
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LEXINGTON GREENEVILLE WOODF. Co.
AD TN90
TN90
MD TN90
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RESULTS Plant Height after topping (cm)
Models P value
AD x TN90 x MD 0.0004
AD x TN90 0.01
MD x TN90 0.09
Plant Height after Topping (cm)
Lexing Greene Woodf MEAN
ADH 137.96 123.31 146.64 136
TN90 142.53 129.50 146.00 139.3
MDH 144.70 131.76 148.86 141.8
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140
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LEXINGTON GREENEVILLE WOODF. Co.
AD TN90
TN90
MD TN90
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Leaf Length (cm)
RESULTS
Leaf Length (cm)
Lexing Greene Woodf MEAN
ADH 54.16 59.16 63.78 59
TN90 53.45 60.62 65.42 59.8
MDH 52.54 61.09 64.92 59.5
Models P value
AD x TN90 x MD 0.21
AD x TN90 0.07
MD x TN90 0.50
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AD TN90
TN90
MD TN90
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RESULTS Leaf Width (cm)
Leaf Width (cm)
Lexing Greene Woodf MEAN
ADH 21.17 26.03 30.04 25.75
TN90 22.13 27.13 31.05 26.77
MDH 21.12 26.97 30.37 26.15
16
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AD TN90
TN90
MD TN90
Models P value
AD x TN90 x MD 0.04
AD x TN90 0.02
MD x TN90 0.03
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RESULTS Number leaves/ Plant
Models P value
AD x TN90 x MD 0.42
AD x TN90 0.56
MD x TN90 0.08
18.0
18.5
19.0
19.5
20.0
20.5
21.0
LEXINGTON GREENEVILLE WOODF. Co.
AD TN90
TN90
MD TN90
Number of leaves/ plant
Lexing Greene Woodf MEAN
ADH 20.47 19.10 20.16 19.91
TN90 20.34 19.29 19.76 19.80
MDH 20.53 19.57 20.00 20.03
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RESULTS Yield (Kg/ha)
Yield (Kg/ha)
Lexing Greene Woodf MEAN
ADH 2631.2 2443.6 3082.9 2719.3
TN90 2604.2 2744.5 3159 2835.9
MDH 2653.8 2759.4 3260 2891.1
Models P value
AD x TN90 x MD 0.005
AD x TN90 0.04
MD x TN90 0.21
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AD TN90
TN90
MD TN90
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RESULTS
Best ADH entries
Yield (Kg/ha)
Best 20% ADH Kg/ha Yield Differ.
Mean (ADH-3 | ADH-4) 2821.8 -79.6
Mean (TN90-3 | TN90-4) 2901.6
Mean All ADH 2719.3 -116.6 Mean All TN90 2835.9
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FUTURE EXPERIMENTS
SECTION 2 - Performance of ADH and MDH lines, when used for the production of hybrid cultivars.
X TN 90LC DH
ADH and MDH on hybrids
X GV 149 DH
TKS 2002
= TN 97
= KT 204LC
msTN 90
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ACKNOWLEDGEMENTS
Beau Neal Angela Rakes
Cameron Shelton Katie Skidmore
Steven Jones Xueyi Sui
Richard Hensley Bill Pitt
ALTRIA CLIENT SERVICES
PHILIP MORRIS INTERNATIONAL
R J REYNOLDS
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