02 peil

Federal Department of Economic Affairs,
Education and Research EAER
Agroscope
The rapid cycle breeding
approach – application
and improvement
A. Peil, K. Weigl, M. Jänsch, A. Patocchi,
S. Wenzel, H. Flachowsky, M.-V, Hanke
WP2

Agroscope
The juvenile period in trees
Juvenile period in trees is long.
No flower production during this time.
Flower inducing techniques are only effective
after the plant has reached the transition
phase
Continuous flower development only in the
adult period of the tree.

Agroscope
The juvenile period in trees: apple
Hanke et al. 2007, Genes, Genomes and Genomics 1, 1-20
seedling

Agroscope
The meristem identity gene FRUITFUL (FUL) and its
homolog MADS4 gene
Floral pathway
integrator genes
SOC1, FT, LFY
Floral meristem identity genes
AP1, AP2, FUL, CAL, LFY
TFL1
Floral induction

Agroscope
Transgen expression of BpMADS4, a FRUITFUL
homolog gene of birch, in apple
25 BpMADS4 transgenic apple lines
were produced
8 lines showed solitary flowers in vitro
24 lines flowered after transfer to the
greenhouse within several weeks
Most of the flowers were
morphologically normal

Agroscope
Is it possible to obtain fruits and seeds
from these seedlings?
Greenhouse plants are cultivated under
long day conditions
Pollen of field grown apple cultivars was
harvested
Transgenic flowers were pollinated by
hand in the greenhouse
Excessive fruit drop because of the
continuous development of new flowers
The surplus of flowers was removed by
hand to avoid the fruit drop
Several fruits with fertile seeds were
harvested
Fruit of a BpMADS4 transgenic apple
plant
Seeds obtained in test crosses

Agroscope
The transgenic early flowering trait is inherited!
Transgenic BC‘1 seedling with
flowers

Agroscope
The idea behind rapid cycle breeding!
Fast and simple selection methods are needed
(tightly linked molecular markers)
Introgression of resistance genes from
wild species
Selection of resistant and transgenic
early flowering seedlings for further
crosses
Elimination of unwanted traits by
several pseudo-backcrosses
Selection of resistant seedlings which
do not carry the transgen
Utilization of seedlings for further
breeding cycles

Agroscope
Is the linkage group carrying the transgene for rapid
cycle breeding important?
x
LGagene of interest
early flowering gene
segregation in
the next
cycle

Agroscope
BpMADS4 in line T1190 is located on LG4
Resistance to Gene/QTL (Linkage group)
Powdery mildew Pl-w (LG 8), Pl-2 (LG 11), Pl-m (LG 11),
Pl-d (LG 12), Pl-1 (LG 12)
Apple scab Rvi6 (LG 1), Rvi10 (LG 1),
Rvi2 (LG 2), Rvi4 (LG 2), Rvi8 (LG 2), Rvi11 (LG 2),
Rvi15 (LG 2), VT57 (LG 2), Rvi16 (LG 3), Rvi14 (LG 6)
Rvi13 (LG 10), Rvi1 (LG 12), Rvi12 (LG 12),
Rvi5 (LG 17)
Fire Blight Mr5 (LG 3), Fiesta, Prima (LG 7), M. fusca (LG 10),
Evereste, Mf821 (LG 12)
Insects Sd-1 (LG 7), Sd-2 (LG 7), Er4 (LG 7)Er-1 (LG 8),
Er-3 (LG 8), Er-2 (LG 17)

Agroscope
Application of rapid cycle breeding to
introduce fire blight resistance (FB_E)
Pseudo-back cross (pBC) scheme
of the development of pBC’4
plants carrying the fire blight
resistance from ‘Evereste’ FB_E
– at Agroscope
Agroscope

Agroscope
Introduction of fire blight resistance from
Evereste (FB_E) – pollination results
Fb_E
+ -
BpMADS4 + 23 15
- 21 16
2013/14: 111 seeds – 75 BC’3
seedlings
Fb_E
+ -
BpMADS4 + 37 23
- 29 24
2015: 132 seeds – 113 BC’4
seedlings
No FB_E &
BpMADS4
Only
Fb_E
Only
BpMADS4
With Fb_E &
BpMADS4
Agroscope

Agroscope
Foreground selection results 2015
Genera-
tion
Cross
No.
Motherplants
pollinated
No.
Flowers
pollinated
No.
Fruits
harvested
No.
Seeds
No.
Seedlings
No. of seedlings per genotypic group
PLL21
Weeks
from sowing
to 1st
flowering
BpMADS4 Fb_E
Fb_E +
BpMADS4
none
BC'4
BC'3_5 x 'Modi' 1 7 3 13 11 4 5 1 1 1.1 nn
BC'3_10 0 0 0 0 0 0 0 0 0 4.8 0
BC'3_21 x 'Ladina'
1 38
1 2 2 0 0 1 1
2.2
16
BC'3_21 x 'Kanzi' 1 2 2 0 0 1 1 nn
BC'3_33 1 2 0 0 0 0 0 0 0 5.3 0
BC'3_66 1 15 0 0 0 0 0 0 0 28.9 0
BC'3_68 x 'Kanzi' 1 43 8 44* 20 4 5 8 3 11.9 nn
BC'3_69 x 'Modi'
1 20
4 8 7 3 1 3 0
5.2
0
BC'3_69 x 'Kanzi' 1 3 2 0 0 1 1 nn
BC'3_71 x 'Fuji' 1 36 3 4 4 0 2 1 1 7.0 nn
BC'3_75 1 33 0 0 0 0 0 0 0 4.0 0
BC'3_37 x ' Ladina' 1 27 2 9 8 1 3 2 2 3.4 17
BC'3_39 x 'Modi' 1 8 3 9 9 1 3 3 2 1.9 nn
BC'3_41 x 'Modi' 1 23 1 4 2 0 0 0 2 3.7 nn
BC'3_46 x 'Kanzi 1 16 6 47* 19 4 4 9 2 3.3 nn
BC'3_49 x 'Kanzi' 1 8 3 8 4 1 1 1 1 4.4 nn
BC'3_50 x 'Fuji'
1 22
1 6 5 2 1 1 1
3.0
nn
BC'3_50 x 'Modi' 3 13 12 3 3 4 2 nn
BC'3_60 x 'Modi' 1 13 2 11 6 0 1 1 4 7.0 nn
Total 15 311 42 132 113 23 29 37 24
* = only 20 seeds sown
nn = not flowering till now
Agroscope

Agroscope
Fire blight inoculation results 2014
0
20
40
60
80
100
120
5r=7
10r=4
21r=8
33r=8
66r=11
68r=9
69r=5
71r=10
75r=10
37r=11
39r=8
41r=9
46r=9
49r=11
50r=9
60r=9
Enterpriser=8
Everester=10
Galar=10
Grannyr=11
BC'3s controls
PLL21
Agroscope

Agroscope
Application of rapid cycle breeding to introduce
fire blight resistance (FB_MR5)
Resistance gene of Mr5 has been isolated and function proved in transgenic Gala
Transgenic Gala lines
Broggini et al. (2014) Engineering fire blight resistance into the apple cultivar 'Gala' using the FB_MR5 CC-
NBS-LRR resistance gene of Malus × robusta 5. Plant Biotechnology Journal accepted

Agroscope
Introduction of fire blight resistance from
M. ×robusta 5 (FB_MR5) – scheme
T1190
12120
13103 Fuji
GD
(Idared x Mr5)x
x
x
14100 Kanzix
2011
2012
2013
2014
2015 15100

Agroscope
Pollination results 2014
85 BC´3 seeds have been planted
7 BC´4 unripe seeds have been released, put on media and propagated
2 BC´4 plants carry both BpMADS4 and FB_MR5

Agroscope
Generation of transgenic seedlings of unripe seeds
without stratification
20d4d 70d
3d Water 1d gibberellic acid 3-5d D5OH 30-70 d B8

Agroscope
Improvement of the rapid cycle breeding system in apple
– development of new early flowering lines with BpMADS4
Genotype Vector construct
Transformation
efficiency (%)
Number of
transgenic
lines
Number of
lines
characterized
PNS
pHTT602-
CaMV35S::BpMADS4
1.66 25 3
Pinova p9N-CaMV35S::BpMADS4 1.47 14 2
Gala p9N-CaMV35S::BpMADS4 1.01 15 2
Mitchgla
Gala
p9N-CaMV35S::BpMADS4 0.08 1 1
Santana p9N-CaMV35S::BpMADS4 0.11 3 1

Agroscope
Development new early flowering lines with BpMADS4
T-Line Genotype Number of T-
DNA copies
In vitro early
flowering
T-Line Genotype Number of T-
DNA copies
In vitro early
flowering
T915 Pinova 1 yes T935 Gala 1 yes
T918 Pinova 2 no T936 Gala 6 no
T919 Pinova 2 yes T937 Gala 3 no
T920 Pinova 2 no T938 Gala 2 yes
T928 Pinova 1 yes T954 Gala n.d. no
T934 Pinova 2 no T962 Mitchgla Gala 1 yes
T950 Pinova 2 no T963 Santana 3 no
T951 Pinova 2 no T971 Santana 2 yes
T913 Gala 1 yes T972 Santana 1 yes
T916 Gala 2 yes

Agroscope
Determination of the linkage group where BpMADS4
was integrated
T-Line
Amplicon Fragment corresponding to plant genome Fragment corresponding to T-DNA
(bp) Size (bp) LG Contigs Identity
(%)
Size (bp) Identity
(%)
Border
motif
Deletions
(bp)
T938† 1,715 1,398 15 MDC042808.9 99 317 100 RB 24
828 182 15 MDC042808.9 99 55 100 LB 16
T962 739 400 2 MDC009026.62 96 329 100 RB 23
612 161 2 MDC009026.62 99 451 100 LB 211
T972 1,287 969 9 MDC021272.342 97 318 100 RB 34
2,734 2302 9 MDC021272.342 98 432 100 LB 230

Agroscope
Early flowering BpMADS4 lines established
T-Line Genotype T-DNA insertions
Number LG
T1190a PNS 1 4
T1189 PNS 1 8
T1236 PNS 1 16
T1240 PNS 1 14
T915 Pinova 1 10
T920 Pinova 2 7
T935 Gala 1 5
T938 Gala 2 15
T962 Mitchgla Gala 1 2
T972 Santana 1 9

Agroscope
Test crosses: pollination with Regia
T-Line
Number of
flowers
Number of Number of Number of seedlings
pollinated ripe fruits seeds Overall Flowered in the first
year
T1189 PNS 27 1 8 6 2
T1236 PNS 20 4 34 27 9
T1240 PNS 18 2 8 4 0
T915 Pinova 75 17 115 29 2
T913 Gala 22 1 1 0 0
T935 Gala 26 2 9 3 n.d.
T938 Gala 16 2 12 5 n.d.
T972 Santana 1 0 n.d. n.d. n.d.

Agroscope
Phenotype of early flowering BpMADS4 lines
Disadvantage of the constitutive flowering gene expression
T913 Gala Mitchgla
Gala
T962 T915 PinovaT928
reduced growth, high fruit drop
<80°— spreading 80–90°— drooping>90°— weeping
Gala T962 GM T913 Gala

Agroscope
Improvement of the rapid cycle breeding system in
apple - using TFL1 (repress the repressor)
Floral pathway
integrator genes
SOC1, FT, LFY
Floral meristem identity genes
AP1, AP2, FUL, CAL, LFY
TFL1
Floral induction
The shoot meristem identity
gene TERMINAL FLOWER 1
(TFL1) represses flowering

Agroscope
Morphological deformations of flower organs
Fruit with normal seeds
Plants are not suitable for breeding
RNAi mediated down regulation of MdTFL1-1 in
apple reduces vegetative growth and generation time

Agroscope
Use of an inducible promoter in rapid cycle breeding
to promote flowering and normal plant growth
42°C, 2h
4 weeks
Silencing of MdTFL1 expression
using the heat-inducible promoter
Gmhsp17.5-E from soybean
Development of 5 transgenic
lines of the apple cultivars
Pinova and Gala
Heat shock treatment to induce
silencing

Agroscope
Heat shock silencing of MdTFL1 gene expression
Pinova T967
Successful activation of the
promoter gene construct via heat
No weeping plant habit, no
marginally plant habit differences
But: no flower production on in
vitro and glasshouse plants after
heat induction
MdTFL1 genes were down-
regulated by heat in transgenic
and non-transgenic controls

Agroscope
Conclusions
A BpMADS4 early flowering line can be successfully used to
introduce fire blight resistance in a short generation time (around
one cycle/year)
A set of early flowering lines from Pinova, Gala, Santana and
Mitchgla carrying BpMADS4 on different linkage groups has been
developed
The approach to repress MdTFL1 by the heat induction of RNAi to
promote flowering was not successful

Agroscope
Thanks for your attention
Agroscope

Agroscope
Seven seedlings of F1 generation.
Transgenic seedlings are marked by arrow
Transgenic seedlings flower within several weeks
BpMADS4 is expressed in transgenic seedlings
SM
T1165
T1187
T1190
Blank
Seedling1
Seedling2
Seedling3
Seedling4
Seedling5
Seedling6
Seedling7
BpMADS4 Expression
Is the transgenic early flowering trait inhereted?

Agroscope
Are ther transgenic lines with one early flowering
gene integration?
Nine single copy lines were detected
which could be used as parental
lines
The line T1190 was chosen for a
further breeding programme
The segregation of the early
flowering transgene was studied on
600 seedlings (1:1)
Isolation of the integration site in
line T1190
SM
T1165
T1166
T1167
T1168
T1185
T1186
T1187
T1188
T1189
T1190
T1236
T1237
T1238
Pinova
Plasmid
Southern hybridization of BamHI restricted DNA
using a BpMADS4 specific probe

Agroscope
The juvenile period in trees: ApplePlant
species
Gene Homolog
to
Type of
expression
Early flowering Reference
Pyrus communis
La France ACO1 Antisense yes (in vitro) Gao et al. 2007
Spadona,
Conference
MdTFL1.1 TFL1.1 RNAi yes Flaishman et al.
unpubl
Malus domestica
Orin MdTFL1 TFL1 Antisense yes Kotoda et al. 2003,
2006
Holsteiner
Cox, Pinova,
Galaxy
MdTFL1 TFL1 RNAi yes Szankowski et al.
2009; Flachowsky et
al. unpubl
Orin MdFT FT overexpr yes (in vitro) Kotoda et al. 2010
Pinova MdFT FT overexpr no /yes (in vitro) Hättasch et al 2009;
Tränkner et al. 2010
Orin AFL1 / AFL
2
LFY overexpr no Kotoda et al. 2003
Rubinola MdSOC1/
MdFT1
SOC1/FT overexpr not yet Zhu et al. 2010

Agroscope
Ectopical expression of flowering genes
in fruit trees
Gene Source Expression in Early
flowering
Reference
FT Arabidopsis Citrus sinensis x Poncirus
trifoliata
yes Pena et al. pers. Comm.
LFY Arabidopsis Citrus sinensis x Poncirus
trifoliata
Citrus sinensis
Malus domestica Pinova
Malus domestica Gala
yes
yes
no
no
Pena et al. 2001
Pena et al. pers. comm.
Flachowsky et al. 2010
Schaart et al. unpublished
AP1 Arabidopsis Citrus sinensis x Poncirus
trifoliata
Citrus sinensis
Malus domestica M26
Fortunella crassifolia
yes
yes
no
yes
Pena et al. 2001
Pena et al. pers. Comm
Zhu et al. 2009
Duan et al. 2010
BpMADS4 Betula pendula Malus domestica Pinova yes Flachowsky et al., 2007
CiFT Citrus unshiu Pyrus communis
Poncirus trifoliata
yes
yes
Matsuda et al. 2009
Endo et al. 2005
MtFT Medicago
trunculata
Olea europaea yes Cerezo et al. 2010

YOUR LOGO
Fruits 2014
BC’2_43(2012) x Fuji BC’3_46 x Kanzi
weight [g] heighth [mm] width Ø [mm]
minimum 33 42 39
maximum 118 58 63
average 65 48 51
weight [g] heighth [mm] width Ø [mm]
minimum 35 41 41
maximum 58 48 49
average 46 45 44

Agroscope
RNAi mediated down regulation of MdTFL1-1 in
apple reduces vegetative growth and generation time
Kotoda et al. (2006):
Antisense down regulation was not very effective
Flowering 8 months after grafting
RNAi based strategy in four apple cvs using
(pHELLSGATE::MdTFL1)
Precocious flowering in vitro of 20 lines
Flowering of these lines in the greenhouse
within a few weeks followed by death or strong
inhibited vegetative growth
MdTFL1 expression was reduced
Transgenic plants showed a reduced growth
Flachowsky et al. 2012, Tree Physiology 32, 1288-1301

Agroscope
The Fast-track breeding approach can be extended
to other cultivars and linkage groups

Agroscope
Julius Kühn- Institute (JKI) Germany:
Henryk Flachowsky
Stephanie Wenzel
Kathleen Barthel
Andreas Peil

02 peil

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02 peil