Schestibratov K.A., Pushin A.S., Dolgov S.V.

Artificial Climate Station "Biotron", Branch of Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry RAS. Science Avenue 6, 142290 Pushchino, Moscow region, Russia. E-mail: schestibratov@rambler.ru; dolgov@fibkh.serpukhov.su

Institute of agricultural biotechnology RAAS, Timirayzevskaya 42, Moscow, Russia

Introduction

Strawberry is an important small fruit crop throughout the world, and its production is growing steadily. Many cultivars of strawberry have already been bred, thereby enabling this crop to be cultivated under various conditions. However, there are still many factors which limit their yield significantly, one of which is the plant’s susceptibility to a variety of phytopathogenic fungi, bacteria and viruses. Gray mold or Botrytis fruit rot, caused by Botrytis cinerea, is one of the destructive diseases of strawberry responsible for substantial yield loss before and after harvest. There are very susceptible cultivars, moderately susceptible and least susceptible, no cultivar is immune (Jarvis et al., 1968; Simpson, 1991; Sutton, 1998).
The taste attributes of strawberries (Fragaria x ananassa Duch.) are regarded as significant quality factors by both consumers and the food industry. The relationship between sugars and acids of ripe fruit and sensory properties such as flavour or colour of strawberries have been studied. Stage of ripeness, age of plants, quality of ground, and genotype of variety are known to affect the quantitative variations in sugar and acids in strawberries. Other compounds also influence strawberry taste, making it complex and difficult to alter trial by breeding, and the taste of some strawberry varieties has been criticized by consumers.
The development of gene transfer techniques to crop plants has facilitated testing constitutive overexpression of different proteins involved in the plant defense system or proteins influenced on fruit taste. Plant resistance to pathogens involves the accumulation in the plant cell of proteins active in defense mechanisms such as PR proteins. The accumulation of these proteins has been shown to correlate with the development of systemic acquired resistance in plants (Van Loon, 1997; Kasprzewska, 2003). Typically, these antifungal proteins are expressed constitutively at low levels in cells and accumulate in response to fungal attack or in response to other inducers of acquired resistance. However, these mechanisms are often too weak or appear too late to be effective for protecting the plant. This strategy involves the constitutive overexpression of defense proteins that have inhibitory activity against the hyphae and/or germinating spores of the pathogen. Among these are PR proteins including glucanases (Yoshikawa et al., 1993), chitinases (Broglie et al., 1991) and thaumatin-like proteins (Liu et al., 1994). This seems to be a promising strategy also for the development of gray mold resistant strawberry.
Thaumatin II is a sweet-taste protein isolated from the fruit of the African plant katemfe (Thaumatococcus danielli). In addition to its sweet taste, this protein enhances certain flavours while masking others, binding specifically with taste receptors. On the other hand thaumatin II is a member of PR proteins (PR-5) that are induced in plants in response to infection by plant pathogens, elicitors, stress, and developmental signals. The in vitro antifungal potential of thaumatin has been reported (Vigers et al. 1991). The antifungal activity and sweet taste of thaumatin II makes it attractive candidate for use in genetic engineering to produce disease-resistant crop plants with a modified fruit taste.

Strawberry genetic transformation

Producing transgenic plants with desired properties usually encounters the problems of reproducibility of the known methods and the problems of producing plants with a low level of somaclonal variability. Genetic engineering of strawberry has been reported for the first time by James and Nehra (1990).

Previous studies on strawberry have clearly demonstrated the importance of various factors such as genotype (Liu and Sanford, 1988), type and source of explant (Nehra et al., 1989), hormonal balance and incubation conditions (Liu and Sanford, 1988; Nehra et al., 1989) on successful regeneration via adventitious organogenesis (Fig. 1). Several transformation protocols have already been developed; however, transformation frequencies are greatly influenced by the cultivar and the procedure used, e.g. 0.95% transformation frequency has been reported for the cultivar Rapella (James et al., 1990) and 6.5% for the cultivar Red Coat (Nehra et al., 1990b). High frequency of somaclonal variations in regenerated or micropropagated plants is another obstacle for successful genetic transformation of strawberry cultivars.
Agrobacterium-mediated transformation method developed by James & Nehra (1990) and improved by other researchers has been used in strawberry molecular breeding for the development of GM plants with improved properties. However, a very high frequency of somaclonal variations among transformants is a serious disadvantage of the developed method. Somaclonal variations could be revealed only by the field test evaluation of transgenic plants produced by this method.

The improved method of transgenic strawberry plant production has been developed by Schestibratov & Dolgov (2002). The present method is characterized by: 1) a low frequency of the necrosis of explants, 2) an enhanced frequency of transient expression, 3) an enhanced frequency of the transformation of transgenic tissues, an enhanced frequency of the regeneration of transgenic shoots, a higher proportion of direct transformants, a low frequency of somaclonal variations. 11 % of transformation efficiency was achieved by this protocol. It was shown that most of transformants (70-80 %) demonstrated no somaclonal variations in the field test evaluation. This protocol can be applied to transfer different genes into the plant genome. By usage of this method transgenic plants containing GUS, GUSint (Fig. 2) and thauII gene were produced.

Patent and Patent application:

Shestibratov K.A., Dolgov S.V. (2002) Method of producing transgenic plants with increased pathogen resistance. / Patent N 2002128414, 24 October 2002 (Russian).
Shestibratov K.A., Dolgov S.V. (2004) Method for producing transgenic plant with the aid of agrobacterium tumefaciens. / PCT Patent application N WO 2004/038023, 6 May 2004.

Taste modification of strawberry fruits

The sweet taste of thaumatin can be detected at threshold amounts 1600 times less than that of sucrose on weight basis (equivalent to 105 –fold less on molar basis); the threshold values are near 10-4% or 48nM, making this protein the most intensely sweet substances known. It is seems that thaumatin II gene is the most attractive one for genetic engineering of fruit crops

Sensory evaluation

Organoleptic analysis of transgenic fruits demonstrated sweetness improvement in 5 F-40-I lines accumulating thaumatin II protein (Fig. 4B). Sensory assessments on homogenized samples derived from each of S-40-I transgenic lines were performed at June 2004. The taste characteristic data of S-40-I lines are presented in Fig 4A. The fruits of S-40-I-1, 2, 3, 5 and 12 lines were characterized by higher value of sweet taste and flavour. The obtained sensory evaluation results indicated distinct differences in the sensory quality between fruits of several transformed strawberries lines and Selekta and Firework cultivars.

Sensory evaluation results indicate that the strawberry lines S-40-I-1, 2, 3, 5 and 12 used in this study had fruit better in sweetness than non-transgenic control cv. Selekta. Shape and size of berries from transgenic lines was similar as non-transgenic control cv. Selekta (Fig. 3). Other transgenic lines demonstrate taste characteristics similar as non-transgenic control. Taste panelist scores demonstrated that improved sweetness was associated with thaumatin II accumulation in tested transgenic lines. A similar property of recombinant thaumatin was noted for transgenic potato (Witty, 1992), cucumber (Szwacka et al., 2002), tomato (Bartoszewski et al., 2003). Thaumatin solution tastes sweet at concentration as low as 10-8 M. Below this taste threshold, thaumatin exhibits the property of enhancing other flavors (Higginbotham 1979).

Transgenic strawberry with enhanced disease resistance against gray mold

Significant yield losses due to pathogen attacks occur in most of the agricultural and horticultural species. In strawberry traditional breeding programmes, resistance to pathogens is an attractive option for the management of the diseases. Many cultivars of strawberry have already been bred, thereby allowing this crop to be cultivated under various conditions. However, strawberry susceptibility to a variety of phytopathogenic fungi, bacteria and viruses is still a limiting factor for effective strawberry growing. Gray mold or Botrytis fruit rot, caused by Botrytis cinerea, is one of the most destructive diseases of strawberry, causing substantial yield loss before and after harvest (Fig. 5). Cultivars can be susceptible, moderately susceptible and minimally susceptible, but no cultivar is immune (Jarvis et al., 1968; Simpson, 1991; Sutton, 1998). Thus, gray mold resistance in strawberry plants is one of the most important traits to have been pursued in breeding programs over several decades. Due to the unavailability of breeding material with resistance against gray mold, to date disease resistance breeding has not progressed well. In this case, a transformation technique can be a useful method to produce novel breeding materials because this can make use of isolated genes from a great number of species.
The in vitro antifungal potential of thaumatin II has been reported (Vigers et al., 1991). Similar zeamatin protein only combination of thaumatin II and nikkomicyn inhibits in vitro growth of Candida albicans. Also it has been documented that thaumatin II have antifungal activity in transgenic cucumber plants (Szwacka et al., 2002). We assume that on the one hand constitutive expression of thaumatin II might be responsible for the stimulation of the plant defense system; on the other hand improved resistance may be the result of synergism with endogenous chitinases. However, the precise mechanism of tolerance in the transformed strawberry described here is not clear and further studies are necessary.
Transgenic strawberry lines with thaumatin II gene have been produced based on cv. Firework and cv. Selekta.

Disease resistance evaluation demonstrated correlation between thaumatin II expression and enhanced resistance against Botrytis cinerea. Most of tested transgenic lines expressing thaumatin II protein showed significantly smaller area of the necrotic lesions observed on the leaf disks (Fig. 6).
In conclusion, resistance to Botrytis infection can be successfully engineered into different strawberry varieties through the expression of a transgene that encodes the thaumatin II protein. Future research will show whether the use of the thaumatin II transgene will be useful in a broad range of crops for engineering disease resistance and improved sensory characteristics of fruits.

Field test evaluation

In August 2000, about 1000 m2 of transgenic strawberry plantation was established at the All-Russian Institute of Fruit Breeding, Orel city, Orel Region, Russia. 10-12 plants on each of 19 F-40-I transgenic lines and non-transgenic control of cv. Firework were used for field experiments. In vitro plantlets were transplanted to the greenhouse on February 2000. These were transferred to the field during first seven days of August 2000. 10 plants on each of S-40-I lines and cv. Selekta at the same order as F-40-I lines were transplanted in the field on April 2003. The experimental design consisted of three randomized complete blocks. The between-plant and between-row spacing were 50 cm and 1 m respectively. The plots were located such that each plot was immediately surrounded by an unplanted area (Fig. 7).
The performance of 19 strawberry transgenic lines cv. Firework and 15 lines cv. Selekta was evaluated in the field during 2000-2003 and 2003-2004 respectively. The vegetative and generative activities of field plants were evaluated according to the following main criteria: the average height of the shrub; the average number of peduncles; and the average weight of the berry. 10 out of 17 F-40-I lines with a detectable thaumatin II expression yielded as well as wild-type cv Firework and have same phenotype. 7 lines exhibited some somaclonal variation. Morphological characteristics of 12 S-40-I lines out of 14 thaumatin II accumulating lines were similar to wild-type cv. Selekta. Two lines had somewhat smaller leaves and plant heights.
To assess gene flow from cultivated to wild strawberry, we are using NPTII marker to evaluate wild F. vesca plants. Fruit samples have been picked up from F. vesca plots located at different distances from the plot of transgenic strawberry plants grown under the gauze isolation. In the first experiment F. vesca plants were located under the gauze isolation along with transgenic plants, in the second one – without isolation. To date, we have evaluated 30 plants from 6 populations. Gene escape was revealed in two populations grown under the gouze isolation near transgenic plants. In other populations NPTII marker was not present.


Publications

1. Dolgov S.V. et al. (1999). Expression of thaumatinII gene in horticultural crops. In: Developments in plant breeding. Vol. 8 "Genetics and Breeding for Crop Quality and Resistance" ed. S. Mugnozza, E. Porceddu and M. Pagnotta. Kluwer Ac. Publ. Dordrecht,, Boston, London, pp 165-172.
2. Dolgov S.V., Lebedev V.G., Anisimova S.S., Lavrova N., Serdobinskiy L.A., Tjukavin G.B., Shadenkov S.A., Lunin V.G. (1999) Phytopathogene resistance improvement of horticultural crops by plant-defensin gene introduction. In: Developments in plant breeding. Vol. 6 "Genetics and Breeding for Crop Quality and Resistance" ed. S. Mugnozza, E. Porceddu and M. Pagnotta. Kluwer Ac. Publ. Dordrecht,, Boston, London, pp 111-118.
3. Firsov A. P., Dolgov S.V. (1999). Agrobacterial transformation and transfer of the antifreeze protein gene of winter flounder to the strawberry, Acta Horticulture 484, pp 581-586.
4. Schestibratov K and Dolgov S (2001) Genetic engineering of strawberry cv. Firework. / in Abstracts of 43rd ETCS Congress, Granada, Spain, September 30-October 3, 2001.
5. Schestibratov K and Dolgov S (2002) Molecular breeding of strawberry cv Firework for enhanced disease resistance and taste improvement by introduction of thauII and rs-afp3 genes./ in Abstracts of Agricultural Biotechnology International Conference, Saskatoon, Saskatchewan, Canada, 15-18 September, 2002.
6. Schestibratov K and Dolgov S (2002) Molecular breeding of strawberry cv Firework for enhanced disease resistance and taste improvement by introduction of thauII and rs-afp3 genes. / in Abstracts of 1st EPSO Conference: Networks in Plant Biology, Brunnen, Switzerland, 27-31 October, 2002.
7. Shestibratov K.A., Dolgov S.V. (2002) Method of producing transgenic plants with increased pathogen resistance. / Patent N 2002128414, 24 October 2002 (Russian).
8. Shestibratov K.A., Lebedev V.G., Lavrova N.V., Korneeva I.V., Kharchenko P.N., Dolgov S.V. (2003) Fruit taste modification of horticultural crops by thaumatin II gene introduction. / 7th International congress of plant molecular biology, Barselona, Spain, June 23-28, 2003.
9. Shestibratov K.A., Dolgov S.V. (2004) Method for producing transgenic plant with the aid of agrobacterium tumefaciens. / PCT Patent application N WO 2004/038023, 6 May 2004.
10. Shestibratov K.A., Dolgov S.V. (2004) Genetic engineering of strawberry cv Firework and Selekta for taste improvement and enhanced disease resistance by introduction of thauII gene. / in Abstracts of 5th International Strawberry Symposium, Coolum, Australia, 5-10 September, 2004.
11. Shestibratov K.A., Lebedev V.G., Korneeva I.V., Lavrova N.V., Dolgov S.V., Kharchenko P.N. (2004) Improving fruit quality and pathogen resistance by genetic engineering methods. / Bulgarian-Russian seminar “The application of molecular diagnostic in breeding, variety testing and analysis of transgenic plants” Varna, Bulgaria, 3-7 July, 2004.
12. Dolgov S.V., Shestibratov K.A. (2004) Enhanced disease resistance and taste improvement of apple and strawberry by introduction of PR-protein genes. / in Abstracts of XV International Plant Protection Congress, Beijing, China, May 11-16, 2004.
13. Shestibratov K.A., Dolgov S.V. (2004) Method for producing transgenic plant with the aid of agrobacterium tumefaciens. / PCT Patent application N WO 2004/038023, 6 May 2004.
14. Shestibratov K.A., Dolgov S.V. (2005) Transgenic strawberry plants expressing a thaumatin II gene demonstrate enhanced resistance to Botrytis cinerea. Sci. Hort. (in press).