FasTrack Breeding for Early Flowering and Disease Resistance in Apple

Darshan M. Kadam
Scientist (Fruit Science)
Division of Fruits and Horticultural Technology
ICAR-Indian Agricultural Research Institute
New Delhi
IndianAgriculturalResearchInstitute,NewDelhi

Tree fruit industry is facing highly dynamic situations:
Climate change, reductions in available labor, increasing use
of agrichemicals, changing consumer preferences and the
spread of pathogens and insect pests.
Fruit tree breeding remains a slow, arduous process that has
changed little over the centuries. Most of the currently
dominant varieties are chance seedlings.
Breeding apple cultivar takes at least 15-20 years and costs
approximately €400 000 (Fenning and Gershenzon 2002).
A period of > 50yr is needed to obtain apple cultivar
expressing a trait originally present in a wild apple, and a fruit
quality that can compete with the world’s leading cultivars
(Schouten et al., 2006).

Limitations of conventional breeding
 Long juvenile periods
 Complex reproductive biology
 High levels of heterozygosity
 Linkage drag of undesirable traits from wild relatives
 Unsuccessful fruit setting due to abortive embryos
 Very less information available on inheritance pattern and
genomics
Gomez-Lim and Litz (2004)

Long Juvenility: Biggest Concern
Crop Duration (years)
Almond 3-4
Peach 3-4
Pistachionut 4-10
Walnut 5-9
Orange 4-6
Mandarin 4-6
Mango 3-10
Apple 6-12
Pear 6-12
Avocado 15+
Nocker and Gardiner (2014)
Length of the juvenile phase of fruit/nut crops

 Introduction of resistance genes from wild species is time-consuming
as it requires repeated pseudo-backcrossing with high quality parents
followed by selection for the desired traits within the progeny
(Baumgartner et al., 2011).
Guava Wilt Citrus PhytophthoraPomegranate Bacterial Blight
Apple Scab
Resistance Breeding: Challenging Task

Methods to manipulate fruit juvenile phase
Manipulation of photoperiod and temperature
Application of hormones or growth retardants i.e daminozide and
paclobutrazol (Yuceer et al. 2003)
Trunk ringing, bark scoring, root pruning, defoliation and
horizontal positioning (Longman et al. 1965, Tromp 1967, 1968,
Taylor et al. 1984).
Grafting on dwarfing rootstocks
Embryo rescue and seed chemical treatment
Microbudding
Early flowering mutants

Flowering Mechanism in Arabidopsis
Hanke et al 2007

IndianAgriculturalResearchInstitute,NewDelhi Expression of early flowering genes in fruit tree
Nocker SV and Gardiner S E, 2014

IndianAgriculturalResearchInstitute,NewDelhi Regulation of flowering genes in fruit crops
Lal N et al 2017

FasTrack Breeding
 FasTrack utilizes genetic engineering strategies for inducing
early flowering that produces generation cycles of one year or
less.
 Introgression of resistance genes from wild species by
hybridization with early flowering transgenic line.
 Selection of early ﬂowering seedlings carrying the trait of
interest (resistance gene) using molecular markers for further
back crossing.
 Repeated back crossing until the linkage drag is minimised and
desired parental traits are combined in a seedling population.
 The final progeny (null segregant) released for commercial use
must not carry early ﬂowering transgene.
 Technology has the potential to integrate into existing breeding
programs and addresses its limitations and vulnerabilities.

FasTrack Breeding
Hypothesis was first time proposed in Poplar
The proof of concept was first time provided in Apple using transgenic
apple plants overexpressing the BpMADS4 (Flachowsky et al. 2007).
FasTrack fruit tree breeding are currently being applied to other
perennial tree fruits such as plum and citrus (Rodriguez et al. 2014).

Prerequisite for FasTrack Breeding
1. Early flowering transgenic protocol.
2. Knowledge of integration site and copy No. in the early
flowering transgenic line.
3. Knowledge about position of transgene and trait of interest on
the chromosome.
4. Tightly linked reliable marker for trait of interest.
5. Ensuring pollen availability and pollen vitality.
6. Assessment of morphological performance of transgenic line,
F1 and backcrossed progenies.
7. Greenhouse (biosafety level –II)

IndianAgriculturalResearchInstitute,NewDelhi FasTrack improvement in plum
Dr. Ralph Scorza
Objective 1: Developing the Fastrack System for California Dried
Plums
Objective 2: Applying the Fastrack System to California Dried Plums,
with a focus on two major desired traits: Plum Pox resistance and high-
sugar content.

IndianAgriculturalResearchInstitute,NewDelhi Six-month-old early flowering & fruiting ECF plum plants,
flowers in greenhouse, and ripe fruits

Limitations in FasTrack Breeding Scheme
In most woody fruit species, transformation and regeneration
protocol is not available.
Overexpressing or silencing of early flowering genes in plants may
lead to undesirable phenotypic effects: Plants constitutively
overexpressing BpMADS4 is often malformed and the fruit yield and
seed set is very low (Elo et al. 2007, Flachowsky et al. 2007).
USDA regulators have decided that null segregants will be outside
regulatory authority (USDA 2011, 2014).
In Europe the definition of the legal status of the null segregants is
still pending.
In India current regulations does not permit the FasTrack breeding.

Case Study -I
 Morphological evaluation of early flowering transgenic lines and
transgenic F1 seedlings
 Application of marker assisted selection to develop improved
prebreeding lines
 Pyramiding of genes from different sources using early flowering apple
tree
Objectives
Flachowsky et al 2011

Plant material: 24 BpMADS4-transgenic apple lines cv ‘Pinova’
Flachowsky et al. (2007)
Identiﬁcation of No. of T-DNA copies: Southern hybridization
Grafting experiment: T1187, T1190 grafted on NT Pinova and vice
versa
Morphological evaluation: Transgenic lines and F1 seedlings
Isolation of T DNA flanking regions: TAIL-PCR in T1190 line and
sequencing
Genetic mapping of T DNA integration sites: T1190 line
Hybridization experiment and MAS of F1 and BC1 seedlings
Material and Methodology

T1190 X Malus fusca (FB resistant)
X Regia
FB-F7, Rvi2, Rvi4
X 98/6-10
Pl1, Pl2
F1
BC1
BC2
Figure: Schematic description of the crossbred breeding scheme.

Figure: Morphological evaluation of glasshouse-grown transgenic apple lines overexpressing the BpMADS4 gene of
silver birch (Betula pendula). (a) Parthenocarpic fruits on transgenic plants without any crosspollination. (b)
Parthenocarpic fruit showing an almost normal size (60 mm in diameter). (c) Parthenocarpic transgenic fruit without
seeds. (d, e) Plants of transgenic lines T1187 (d) and T1190 (d) in comparison with an untransformed control plant of
cv ‘Pinova’. (f, g) Plants of transgenic lines T1187 and T1190 used as rootstocks and grafted with a nontransgenic
scion of ‘Pinova’ (plants on the left in both panels), or used as scion and grafted onto untransformed rootstocks of
‘Pinova’ (plants on the right in both panels).
(a) (b)
(d)
(c)
(e) (f) (g)
Results: Morphological evaluation of transgenic lines

(a)
(C)
(b)
(d)
(e)
Figure: (a) Detection of the BpMADS4 gene on genomic DNA of seven seedlings (T1–T7) by PCR
using the primers BpMADS4F and BpMADS4R. (b) Morphological evaluation of the seven seedlings
in the glasshouse. (c) Transgenic fruits grown in dense clusters. (d) Full ripe transgenic fruit of the
seedling T1 pollinated with pollen of Malus fusca. (e) Transgenic seeds of the fruit shown in (d).
Results: Morphological evaluation of F1 seedlings (T1190 X
Malus fusca)

IndianAgriculturalResearchInstitute,NewDelhi T DNA integration site in T1190 line

BpMADS4 in line T1190 is located on LG4

IndianAgriculturalResearchInstitute,NewDelhi Inheritance of molecular markers for four powdery mildew and
apple scab resistance genes and one fire blight resistance
quantitative trait locus (QTL)

Fire blight resistance assessment of the nine
transgenic F1 seedling

Inference
1. The apple line T1190 was selected for breeding purposes
and characterized with regard to the T-DNA integration
site.
2. Using line T1190, the first two generations of breeding
programme has been completed, which aims to
introgress the fire blight resistance from M. fusca.
3. Simultaneously pyramiding genes to fire blight by
application of a high-speed breeding technology and
Molecular assisted selection.

Case Study- II
Objective
 Development of null sergeants of the fifth generations (BC’4)
carrying the Fb_E fire blight resistance gene within shorter
span of time

Foreground selection of seedlings: SSR markers ChFbE01, ChFbE09,
ChFbE02 and ChFbE06 for Fb_E locus
Estimation of the percentage of ”Evereste” genome in null
segregants: Six linkage group 12 SSR markers CH05d04, CH04g04, CH01f02, CH03c02
and Hi07f01
Fire blight resistance assessment: 15 genotypes of the last generation (null
segregants) were assessed by shoot Inoculation using the E. amylovora strain.
Verification of the presence of Rvi6 and Fb_F7 in the 5th
generation: Fb_F7 using SCAR markers AE10-375 and GE-8019 and Rvi6 using the
SSR marker CH-Vf1 and SCAR marker AL07
Material and methods

19/62 9/25 12/72 28/113
Detailed breeding scheme

Figure: Four seedlings derived from the cross of BC’2_2 (Fb_E/BpMADS4) and ‘Granny Smith’,
representing the four groups of seedlings (see picture) that are generated in each generation (N.B.
Fb_E and BpMADS4 are independently inherited). BpMADS4 genotypes show the typical slender
habitus. The seedlings without BpMADS4 recover the habitus of a non-transgenic apple seedling
No Fb_E &
BpMADS4 Only Fb_E
Only
BpMADS4
With Fb_E
& BpMADS4

Figure: The 18 seedlings of the BC’3_2014/BC’4 generation carrying Fb_E and
lacking BpMADS4. (a) 15 BC’3_2014/BC’4 seedlings showing a regular habitus for
an apple seedling. (b) Three BC’3_2014/BC’4 seedlings showing a compact
habitus we called ‘ananas’ (i.e., pineapple). (c) Seedling BC’3_2014_47

Figure: Pictures of transgenic flowers and fruit with abnormalities. a
Flower without pistil. b, c Flowers with more than the expected five petals.
d Apple fruit with seven instead of the expected five seed chambers

BC’3_2014_47

GS BC’2_2 none BpMADS4 BpMADS4/ Fb_E Evereste Gala Galaxy
r=8 r=9 n=4, r=70 n=4, r=28 Fb_E n=9, r=84 r=7 r=10
n=7, r=58
Figure: Fire blight resistance levels of BC’3 plants derived from the cross BC’2_2
(Fb_E/BpMADS4) and Granny Smith’ subdivided into four groups depending on
the inheritance of Fb_E and BpMADS4.

Inference
 Advanced selections with genes of “wild” origin, purified
from genetic drag, can be developed 4–5 times faster than
by classical breeding.
 Fifth generation (BC’4) segregant carrying the Fb_E fire
blight resistance gene within 7 years
 USDA regulators have decided that the final products of the
early flowering system will be outside regulatory authority,
as long as these genotypes have been tested for
phenotype and molecularly shown to not contain
transgenes or pieces of transgenes (USDA 2011, 2014;
Mcgary et al 2017)

Conclusion & Path Ahead
 FasTrack utilizes genetic engineering strategies, but the product
released for commercial use is not a genetically modified plant. Thus,
the produce might escape regulatory hurdles and may be more
acceptable to the public.
 FasTrack breeding has the potential to hasten the existing fruit
breeding programs for incorporating genes of “wild” origin, purified
from genetic drag.
 This technology has the potential to revive and expand tree breeding
in general.

FasTrack Breeding for Early Flowering and Disease Resistance in Apple

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