Most of the apple (Malus × domestica) growers are facing serious disease problems with apple scab which is caused by the fungus Venturia inaequalis. Developing a resistant variety in apple through classical breeding is very slow and inefficient. So, we aim at improving existing apple varieties through a new concept called “cisgenesis” which saves time and effort compared to classical breeding. Malus floribunda proved to be a good source of natural scab resistance genes. The genes HcrVf1 and HcrVf2, consisting of promoter, coding and terminator sequences in their natural configuration, were isolated from Malus floribunda and cloned into the binary vector pMF1. Apple cv. ‘Gala’ was transformed with pMF1 containing HcrVf1 and HcrVf2, individually or in combination. pMF1 can be used to obtain marker-free plants by recombinase-based excision of a fragment carrying undesired gene sequences, such as antibiotic-selection marker genes, leaving behind only the gene(s)-of-interest and one recombination site. Using this vector it is therefore possible to stack several genes by retransformation using the same selection procedure. In order to obtain durable resistance, we have the intention to combine different resistance genes from Malus either by stacking them one by one or by introducing them all together in one T-DNA. Performance of all different types of cisgenic plants will be evaluated by monitoring scab resistance levels phenotypically and by determining gene expression profiles through quantitative RT-PC

Jacobsen, E.

Joshi, S.G.

Kortstee, A.J.

Krens, F.A.

Schaart, J.G.

Schouten, H.J.

Soriano Soriano, J.M.

English

Name not available

Development of cisgenic apples with durable resistance to apple scab

Wageningen Yield 

Development of Cisgenic Apples with Durable Resistance to Apple Scab 
 
S.G. Joshi, J.M. Soriano, A. Kortstee, J.G. Schaart,  
F.A. Krens, E. Jacobsen and H.J. Schouten 
Wageningen UR Plant Breeding 
P.O. Box 16, 6700 AA Wageningen 
The Netherlands 
 
Keywords: Malus × domestica, Venturia inaequalis, marker-free plants, cisgenesis, gene 
stacking 
 
Abstract  
Most of the apple (Malus × domestica) growers are facing serious disease 
problems with apple scab which is caused by the fungus Venturia inaequalis. 
Developing a resistant variety in apple through classical breeding is very slow and 
inefficient. So, we aim at improving existing apple varieties through a new concept 
called “cisgenesis” which saves time and effort compared to classical breeding. 
Malus floribunda proved to be a good source of natural scab resistance genes. The 
genes HcrVf1 and HcrVf2, consisting of promoter, coding and terminator sequences 
in their natural configuration, were isolated from Malus floribunda and cloned into 
the binary vector pMF1. Apple cv. ‘Gala’ was transformed with pMF1 containing 
HcrVf1 and HcrVf2, individually or in combination. pMF1 can be used to obtain 
marker-free plants by recombinase-based excision of a fragment carrying undesired 
gene sequences, such as antibiotic-selection marker genes, leaving behind only the 
gene(s)-of-interest and one recombination site. Using this vector it is therefore 
possible to stack several genes by retransformation using the same selection 
procedure. In order to obtain durable resistance, we have the intention to combine 
different resistance genes from Malus either by stacking them one by one or by 
introducing them all together in one T-DNA. Performance of all different types of 
cisgenic plants will be evaluated by monitoring scab resistance levels phenotypically 
and by determining gene expression profiles through quantitative RT-PCR. 
 
INTRODUCTION 
Apple is one of the important fruit crops of the world. Most of the present day 
apple cultivars are susceptible to apple scab which is caused by the fungus Venturia 
inaequalis (Cooke) G. Wint. To overcome this disease, fruit growers may spray up to 20 
times per season. There are some wild relatives within the genus of Malus which harbor 
several resistance genes for scab. One such species is Malus floribunda. Introgression of 
resistance genes from these wild species to the commercial elite susceptible cultivars 
takes a lot of time as the juvenile period of apple is very long. A second drawback of 
conventional breeding is that along with the gene(s)-of-interest other genes which code 
for undesirable traits also get introgressed due to linkage drag (Schouten and Jacobsen, 
2008). So, in order to remove as many as possible of these unwanted alleles, the breeder 
has to do repeated backcrossing and select for resistant lines with the highest quality. A 
third problem is that apple is self-incompatible. As a consequence backcrosses are not 
possible, and the original set-up of the quality cultivar cannot be attained any more after 
making a cross. Use of biotechnological tools like isolating the gene-of-interest and 
transforming it into elite, susceptible apple cultivars through Agrobacterium-mediated 
transformation (Belfanti et al., 2004) is an interesting and promising new approach in fruit 
tree breeding, for solving these three problems. However, due to strict GMO regulations 
for the transgenic crops, e.g., with respect to the presence of bacterial selectable marker 
genes aimed at antibiotic resistance and other non-plant genes, and in view of the 
acceptance of consumers, a new concept has been developed, called “Cisgenesis” 
(Schouten et al., 2006a, b). Cisgenesis can be defined as the genetic modification of a 
recipient plant with only natural gene(s) from a crossable or sexually compatible species. 
 403Proc. 1
st IS on Biotechnol. of Fruit Species
Eds.: M.-V. Hanke et al. 
Acta Hort. 839, ISHS 2009 
The gene includes its native promoter and terminator in the normal sense orientation as 
we see in the natural situation. In this case selection marker gene and other foreign genes 
present in transformants have to be removed, e.g., by a recombination-based system 
(Schaart et al., 2004) to retain only the gene(s)-of-interest and one recombination site. 
 
MATERIALS AND METHODS 
 
Gene Isolation and Vector Construction 
Malus floribunda 821 is a source of scab resistance, and of the scab resistance 
genes. Both HcrVf1 (Gene bank accession number AY397723) and HcrVf2 (Gene bank 
accession number AJ297740) were isolated with their native promoter and terminator as 
one stretch per gene from a BAC library of a scab resistant apple selection from the Plant 
Research International breeding program. Promoters for the two HcrVf genes are being 
tested in two lengths (HcrVf1 short promoter (SP HcrVf1) - 312 bp, HcrVf1 long promoter 
(LP HcrVf1) - 2 kb; HcrVf2 short promoter (SP HcrVf2) - 288 bp, HcrVf2 long promoter 
(LP HcrVf2) - 2 kb). The genes were amplified using primers that flanked the gene 
sequences with unique restriction sites. A combination of HcrVf1 and HcrVf2 with the 
long promoters was also prepared. All these fragments were cloned to pGEMT-Easy and 
the complete inserts were checked by sequencing. Subcloning was done in a binary vector 
pMF1 which is based on pRCNG (Schaart et al., 2004) and which can be used to obtain 
marker-free plants. pMF1 containing the different genes were subsequently transferred to 
Agrobacterium tumefaciens strain AGL0. 
 
Plant Transformation and Regeneration  
Agrobacterium tumefaciens strain AGL0 was used for further transformations as 
delivery system for the gene(s) of interest to the recipient plant genome. The elite, scab 
susceptible variety ‘Gala’ was used as acceptor for plant transformation according to 
Puite and Schaart (1996). Initially, explants were co-cultivated for four days after which 
they were transferred to selection medium. Explants in regeneration went through 
different stages of subculturing. First they were cultured on shoot/callus induction 
medium (SIM) in the dark until they produce shoot like structures; then they were 
transferred to SIM under the light. After sufficient shoot production they were transferred 
to shoot elongation medium (SEM). When regenerants look like complete shoots, they 
were isolated and transferred individually to shoot propagation medium (SPM) for 
maintenance and multiplication (Fig. 1). 
 
Characterization of Plant Transformants 
Putative transformants were tested for the presence of the cisgene by PCR using 
HcrVf-specific primers. The copy number and gene expression of the cisgenes will be 
studied by Southern blot and RT-qPCR. The resistance level will be determined in 
bioassays using well-characterized isolates of Venturia inaequalis. Resistance levels and 
expression levels of the genes will be linked to the individual constructs for each gene and 
to combinations of the two genes obtained directly or after gene stacking. 
 
RESULTS AND DISCUSSION 
Several independent putative transformants were obtained through Agrobacterium 
mediated transformation with SP HcrVf1 and LP HcrVf1 (table. 1). Experiment with 
SP HcrVf2 and LP HcrVf2 and LP HcrVf1 + LP HcrVf2 constructs is going on. All 
independent transformants were maintained separately and are being multiplied. DNA 
was isolated from the leaflets of these individual putative transformants and analysed by 
PCR by using HcrVf1 specific primers (Fig. 2). Most of the putative transformants were 
PCR-positive and a few escapes were identified. For both SP HcrVf1 and LP HcrVf1 13 
independent PCR-positive lines were regenerated from 280 and 275 explants, 
respectively. Transformants will subsequently be used to give a dexamethasone treatment 
for induction of recombinase activity in order to remove the unwanted DNA sequences. 
 404
These sequences, including the selection marker, are located between recombination sites 
that are located on the transferred T-DNA of pMF1. 
 
CONCLUSIONS 
In this paper we presented the start of our research efforts on using cisgenesis as a 
new strategy of breeding of fruit trees. We aim to study the effect of different promoter 
lengths on expression and resistance level and the difference in performance, if any, of 
two genes combined directly or after stacking. The first, still transgenic, plants have been 
obtained, together with some cisgenic marker-free individuals that are generated thanks to 
spontaneous recombination events. All plants will be used in further characterization and 
in further research (stacking). Cisgenesis could be a better option compared to other 
methods of genetic modification such as transgenesis in dealing with GMO regulations 
and consumer acceptance. 
 
ACKNOWLEDGEMENTS 
I would like to thank TRANSFORUM, The Netherlands for the financial 
assistance of this Ph.D. project. 
 
Literature Cited 
Belfanti, E., Silfverberg-Dilworth, E., Tartarini, S., Patocchi, A., Barbieri, M., Zhu, J., 
Vinatzer, B.A., Gianfranceschi, L., Gessler, C. and Sansavini, S. 2004. The HcrVf2 
gene from a wild apple confers scab resistance to a transgenic cultivated variety. Proc 
Natl Acad Sci USA 101:886-890 
Puite, K.J. and Schaart, J.G. 1996. Genetic modification of the commercial Apple 
cultivars ‘Gala’, ‘Golden Delicious’ and ‘Elstar’ via an Agrobacterium tumefaciens-
mediated transformation method. Plant Science 119:125-133. 
Schaart, J.G., Krens, F.A., Pelgrom, K.T.B., Mendes, O. and Rouwendal, G.J.A. 2004. 
Effective production of marker-free transgenic strawberry plants using inducible site-
specific recombination and a bifunctional selectable marker gene. Plant Biotechnol. J. 
2:233-240. 
Schouten, H.J., Krens, F.A. and Jacobsen, E. 2006a. Do cisgenic plants warrant less 
stringent oversight? Nature Biotechnology 24:753. 
Schouten, H.J., Krens, F.A. and Jacobsen, E. 2006b. Cisgenic plants are similar to 
traditionally bred plants. EMBO Reports 7:750-753. 
Schouten, H.J. and Jacobsen, E. 2008. Cisgenesis and intragenesis, sisters in innovative 
plant breeding. Trends in Plant Science 13:260-261. 
 
 
Tables 
 
Table 1. Total number of putative transformants. 
 
Construct Number of 
explants used 
PCR positive 
shoots (HcrVf) 
Escapes Calli on selection 
medium 
SP HcrVf1  280 13 2  
LP HcrVf1 275 13 1  
SP HcrVf2 300   80 
LP HcrVf2 300   140 
LP HcrVf1 + LP HcrVf2 300    
 405
Figures 
 
 
 
Fig. 1. Putative HcrVf1 transformants on SPM. 
 
 
 
 
 
 
 
 
 
M  1  2   3  4   5   6  7   8   9 10 11  S  G mm M
 
Fig. 2. PCR analyses of HcrVf1 putative transformants. Amplification was obtained for 
lanes: 1,2,4,5,6,7,9. S-Santana (resistant cultivar), G- Untranformed Gala, mm- 
Master mix with out DNA, M-1 Kb+ DNA ladder 
 
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