English Version

Please, find herewith a summary of the Research & Development projects the Centre of Fruit Cultivars is working on:

• Classical breeding: Commercial and efficient apple breeding: durable resistance, improvement of breeding methods and valorization of homozygous plant for breeding programs.
• Rationalisation of genetic transformation of apple
• Reduced application of fungicides in apple : development of a durable system for disease control based on genetic control combined with minimal chemical applications.
• Control of post-harvest diseases in apple fruit, genetically modified with amp-genes

• The use of molecular markers in apple breeding: the study of growth characteristics and genotype identification

As apple culture in Flanders has a turnover of ca. 10 billion Belgium franks it is an important agriculture and horticulture activity. Because of the increasing importance of health care and environment a minimal use of pesticides is required. Most common pests are scab and mildew. The now available resistant cultivars are not as highly qualitative as the traditional non-resistant cultivars. Moreover, resistance has been broken down in several areas. The introduction of less susceptible and highly qualitative cultivars is crucial for the survival of the Flemish apple culture. New cultivars must be developed efficiently. The aim of this project is to improve breeding efficiency by choosing the right parent combinations, advanced selection criteria, use of molecular makers and methods to determine fruit quality.

Besides the traditional breeding between apple cultivars the potential of pyrus hybrids and possible homozygous apple cultivars will be investigated.

In summary, the aims of this project are:

A. Resistance

• Improvement of resistance level en durability by combining different resistant sources (monogenic and polygenic resistance).

• Evalulation of resistance in the fields.
• Research and development of molecular markers to identify combinations of monogenic resistance within a same genotype.

B. Improvement of breeding methods:

• Right choice of parents based on heritability of fruit quality

• Improvement of evalutation of fruit quality by simple destructive and non-destructive methods.

• Advanced and reliable pomological screening.

• Development of advanced selection methods based on correlation between seed dormancy, maturity time, juvenility, flowering time and picking time. Study on vegetative and generative growth of new selection on different rootstocks and different locations.

• Molecular characterization of progenies and parents by microsatellites and AFLP’s to study affinity.

  

  Different genotypes within one progeny

  

Non-destructive methode to determine fruit quality

C. Characterization of Pyrus hybrids and their compatibility to breed with apple and peer. 

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1. Introduction

Genetic transformation allows the introduction of a gene or a set of genes into the genome of a single cell. This single cell can then be cultured to develop into a transgenic plant. In 1989 the first transgenic apple plants were produced using Agrobacterium tumefaciens - mediated transformation. During the following years a lot of researchers – also in our lab - have contributed to develop and optimise protocols for transformation of different apple cultivars. The most important aim of transformation in apple is the production of plants resistant (or less susceptible) to different diseases (e.g. apple scab and fire blight).

In this project we aim to improve the transformation protocol for different apple cultivars. We also examine the use of an alternative selection marker based on the phosphomannose isomerase gene.

2. Improving the competence for regeneration of the explants

The quality of the explant material is an important factor that strongly influences the regeneration efficiency. In this part of the project we investigate several factors that might have an influence on the regeneration capacity :

Type of sugar, mineral salts, additives in the proliferation and regeneration medium.

Avoiding hyperhydricity by improved air-circulation in the vessels.

Type of explant (leaves, stems) and methods of wounding (scalpel, scissors,..)

Figure 1 : Influence of different macro- and micro-salts in the regeneration medium on the percentage regeneration (explants with at least 1 shoot/total number of explants).



Figure 2 : Comparison of shoot growth (elongation, vitrification, multiplication) after proliferation in media with different salt composition.

3. Use of a new selection method based on mannose

The most commonly used selection marker in apple transformation is kanamycine-resistance, encoded by the bacterial nptII gene. This selection system is based on the survival of transgenic cells on the selective agent kanamycine. They are able to inactivate the agent that is normally killing the plant cells through phosphorylation. However, the disadvantage of this selection system is that non-transgenic cells produce compounds that negatively influence the regeneration of the transgenic cells. Additionally, the ecological risks associated with the use of antibiotic resistance markers in transgenic plants is a reason to search for other selection systems.

Recently, an alternative selection system was introduced. Plant cells are transformed with the phosphomannose-isomerase gene which encodes for an enzyme that converts mannose-6-P to fructose-6-P. Mannose-6-P cannot be used as a carbon source by most plants and is used as selective agent. Transgenic cells therefor can grow and regenerate on mannose-6-P whereas non-transgenic cells will not (but they will not be killed).

Preliminary transformation experiments with the (manA)gene on a selective medium with 1 % mannose + 1 % sucrose have resulted in the production of different transgenic lines.

We will further investigate the optimal selection conditions : mannose concentration, gradual increase of selection pressure.

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1. Introduction

Most of the fungicides used in apple growing are applied to control apple scab caused by Venturia inaequalis. By combining different sources of resistance through breeding we intend to improve the resistance level of apple trees and additionally create a durable disease resistance. Research is also focused on the reduction of fungicides by applying different types of chemicals as a result of clear assessment of biological and climatological conditions.

Fig 1 : Scab symptoms on apple leaf Fig 2 : Scab symptoms on apple

2. Quantification of plant resistance levels

This part of the research is carried out at the Royal Research Station in Gorsem. The aim is to develop a reproducible method to quantify the resistance level of varieties. This involves the definition of different levels of infection risks, based on quantitative data for inoculum density, inoculation circumstances and post infection circumstances. To quantify the resistance level, a classification system with 13 classes of scab symptoms was developed. For each plant three leaves are evaluated and classified in a class. Each class has a numerical value and with these values it is possible to calculate a TH-level for each plant.

 

3. Measurement of the natural resistance level of genotypes with distinct types of resistance

Apple cultivars can be divided into 3 classes based on disease resistance: susceptible cultivars and resistant cultivars in which the resistance is genetically controlled by one (monogenic) or by multiple genes (polygenic). To measure the resistance level we inoculate cultivars grafted on a rootstock with fungal spores and put them for 48 hours at 100% RH and 20°C. Afterwards we bring them to 70% RH and 20°C. Evaluation is done 21 days after infection. In the beginning we evaluated the plants by counting spores. Now we only do visual observations (by calculating a TH-level for each plant) because counting spores is very time consuming. By counting spores as by visual observations big differences between plants of the same cultivar can be found. This implies that it is very difficult to detect little differences in resistance level. The same differences in resistance level between polygenic, monogenic and susceptible cultivars can be found by counting spores as by calculating TH-level (based on visual observations).

Fig 1 : Results of infection tests with Venturia inaequalis on different cultivars grafted on rootstock M9. Plants were evaluated 21 days after artificial inoculation by evaluating three leaves and calculating a TH-level, first for each plant, then for all the plants of the same cultivar in each infection test. Susceptible plants have the biggest TH-levels (>30) (1/1/152, M sylvestris, IdxBR). Monogenic cultivars have a TH-level that differs from 0 (<10) because of chlorotic symptoms. Polygenic cultivars have a resistancelevel in between the monogenic and the susceptible cuktivars.

4. Improvement of the level of resistance

1. Classical breeding

We realise a more durable resistance by crossing monogenic cultivars with cultivars with polygenic resistance. The level of resistance of the progenies is evaluated by comparing the symptom expression of the progenies with the symptom expression of their ‘parents’.

2. Molecular breeding

To create a more durable resistance we transform natural resistant cultivars (monogenic or polygenic) with AMP-genes. Transformations are done by wounding the youngest leaves and infecting them with Agrobacterium . The circumstances differ for the different cultivars. Until now, we use Kanamycin as the selection marker but due to the increased critics on the use of antibiotics we are looking for another selection system.

5. Reduced use of fungicides

In an orchard different spraying schemes are compared on a polygenic cultivar to investigate if durable scab control can be achieved by a combination of minimal spraying with new cultivars with improved scab resistance. The effectiveness of four different schemes was tested: one protectant scheme and three curative schemes. Within the curative schemes one was based on biological parameters (ascospore release and infectable tissue area), one on climatological parameters and the last one on a combination of both. The results based on observations on leaves and fruit in 1999 and in 2000 show that :

• Early-season infections have to be avoided as primary infections are positively related to fruit scab. Avoiding them is necessary for an orchard free of scab with minimal spraying.
• A curative spraying scheme can control scab as good as a protectant scheme and with less fungicide applications, resulting in less fungicide residues in food and environment and in a lower production cost.

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Control of post-harvest diseases in apple fruit, genetically modified with AMP-genes

1. Introduction

A major problem in the production of apple fruit over the world, are the post-harvest losses, that are in most cases caused by fungi. An alternative for the chemical control of these fungi is the molecular breeding of apple for increased fungal resistance. At our laboratory, the apple cultivar Jonagold has been transformed with genes coding for anti-microbial peptides (AMP’s), that show in vitro a strong activity against fungi, including post-harvest fungi. In this project, the activity of the AMP’s against postharvest fungi will be studied in vivo, namely in transgenic fruits. First, the transgenic trees will be characterized molecular (Southern and expression analysis), afterwards the antifungal activity of the AMP’ will be studied by a) artificial infection tests on the transgenic fruits and b) fungal inhibition tests with extracts of transgenic fruits.

2. Molecular analysis of the transgenic trees

B1. Southern blot analysis

By use of the southern blot technique, the number of copies of the transgene and the number of insertions can be determined. This is important because the presence of several copies can be the cause of instability, resulting in a lower expression.

B2. ELISA-analysis

Table1 : % Ah-expression in different transgenic lines

line	#ng Ah/ml	#µg prot/ml	% expression
	heated extract	rough extract
2	97,12	5542,26	0,0018
3	24,155	4090,08	0,0006
5	429,72	2711,42	0,0158
6	212,64	3540,23	0,006
7	15,818	3209,92	0,0005
8	30,495	2261	0,0013
10	10,715	2683,23	0,0004
13	64,435	1945,92	0,0033
14	38,9425	3207,91	0,0012
15	21,21	2842,05	0,0007
16	268,02	2632,24	0,0102
17	304,2464	2806,88	0,0108
19	375,5	2943,17	0,0128
24	570,8	3106,4	0,0184
25	321,752	3201,58	0,01
jona	12,64	2859,44	0,0004

3. Study of the antifungal activity of AMP’s

In the first term of the project, the resistance of the fruits to Botrytis cinerea (a fungus that penetrates by wounds) and Gloeosporium perennans (a fungus that penetrates by lenticels) will be studied by performing artificial infections at different moments of preservation. Fourteen days after infection, respectivally the number of spots and the number of infected

lenticels are counted. In the second term of the project, other fungi, namely Monilia fructigena and Nectria galligena will be used for infection tests. Figure 1 shows some transgenic apples

figure 1 : transgenic Jonagold

The in vitro antifungal activity of Rs-AFP2, Ah-AMP1 and Ace-AMP1 against different post-harvest fungi will be studied in extracts of fruit and leaf and compared to the antifungal activity of pure AMP’s (IC50-values). Fungal inhibition tests will also be executed with combinations of pure AMP’s to test the possibly synergistic effect of certain combinations of AMP’s.

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1. Study of growth characteristics : Introduction and methods

In apple breeding, there are a lot of problems which can only be dissolved by breeding improvement. However, apple breeding has a low yield because of a lot of reasons like for instance a high level of heterozygocity, a long juvenile period,… Furthermore, a lot of space is necessary to plant the seedlings and let them grow and also the high cost of their maintenance is not insignificant. An improvement of the breeding efficiency of apple is therefore of crucial importance. In this respect, molecular markers are of particular importance since molecular markers for specific characteristics allow the screening of a lot of seedlings at early stage of development. In this way, complete growth of seedlings to determine if a desired characteristic is present or not, becomes unnecessary.

However, one of the most important reasons for a low yield whiting apple breeding is the fact that little is known about the genetic control of agronomic important characteristics like for instance tree architecture. Almost no molecular markers are available for characteristics that determine tree architecture. Nevertheless, since a lot of agronomic important characteristics are polygenic, it is necessary to create genetic maps prior to determine molecular markers for growth characteristics whiting apple. Afterwards, these markers can be used to study the genetic control of the growth characteristics and to create more efficient breeding programs.

Genetic maps

To create the genetic maps, a cross between Braeburn (normal growth type) and Telamon (columnar type) was made. To search for markers to saturate the genetic maps two molecular techniques are applied : AFLP and microsatellites. These techniques are used to score polymorphism's between Braeburn and Telamon whiting the progeny. The mapping program JoinMap is used to convert the obtained data into a genetic map for Braeburn and one for Telamon. Finally, these genetic maps will be used to determine QTLs for different growth characteristics. An example of a part of an AFLP-gel is shown in fig1.

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Growth characteristics

The progeny of TelamonxBraeburn will be measured during several years for different growth characteristics like the length of the central axis, number of branches, length of branches, branching percentage, number of internodes, main length of internodes and growth speed. These characteristics will be put on the genetic maps like QTLs (Quantitative Trait Loci) and will be used to determine the molecular markers.

2. Genotype identification : introduction and methods

In classical apple breeding programs it is important to establish unique DNA profiles or fingerprints for selections in order to identify these selections unambiguously with regard to selection protection and description. Also to determine genetic relatedness and, in case of doubt, identify the true parents, these fingerprints can be used. To establish the fingerprints, 16 different microsatellites (SSR) are used. Finally, to determine whether the fingerprints are unique or not, cluster analysis is applied to the obtained data. Table 1 shows the fingerprints of some cultivars and selections which are the results of crosses between these cultivars. The 'green' alleles correspond with the alleles of the mother cultivar, the 'red' alleles correspond with the alleles of the father cultivar.

Table 1 : SSR alleles (length in basepairs) of the cultivars Arlet and James Grieves and the selections 4/3/185 and 4/2/276

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