505 Bulgarian Journal of Agricultural Science, 13 (2007), 505-513 National Centre for Agrarian Sciences Application of Chemical Mutagenesis to Increase the Resistance of Tomato to Orobanche ramosa L. K. KOSTOV, R. BATCHVAROVA and S. SLAVOV Agrobioinstitute, BG – 1164 Sofia, Bulgaria Abstract KOSTOV, K., R. BATCHVAROVA and S. SLAVOV, 2007. Application of chemical mutagenesis to increase the resistance of tomato to Orobanche ramosa L. Bulg. J. Agric. Sci., 13: 505-513 In this study chemical mutagenesis of tomato seeds with Ethylmethanesulfonate was used to obtain lines with increased resistance to Orobanche ramosa L. The M2 progeny of the mutant tomato plants was screened for the response to broomrape attack in large scale experiment conducted in greenhouse. Sixteen non-infested by the parasite tomato plants were selected. Their offspring was a subject of another screening for broomrape resistance using an artificial polyethylene bags system. As a result of the experiments six lines with significantly increased level of resistance to Orobanche ramosa L., were selected. Key words: Orobanche, mutagenesis, tomato, broomrape control Introduction sistant varieties, trap crops, soil solariza- tion, and biological control have their limi- Broomrape (Orobanche spp.) is wide tations. That is why the modern under- spread parasitic weed which affects crops standing for the parasite management is and a number of dicotyledonous species. the use of integrated control strategy by Broomrape causes significant losses in combining several of the known methods. tomato, tobacco and sunflower production Essential part of this strategy is the use of in many countries in the Mediterranean resistant to broomrape varieties (Sackston, region, Eastern Europe and Russia (Parker 1992; Ruso et al., 1996; Sukno et al., 1999). and Riches, 1993). Main characteristic of Development of broomrape resistance this species is the big number of seeds in the crops depends on the biology of the produced by a single plant and their long crop and the existence of natural sources period of viability. This makes the control for resistance in close related wild spe- of the parasite in agricultural areas ex- cies. The best results are reached in sun- tremely difficult. So far, known methods flower by the creation of cultivars highly for broomrape control like herbicides, re- resistant to broomrape (Pustovoit, 1976; [email protected] 506 K. Kostov, R. Batchvarova and S. Slavov Petrov, 1968; Vranceanu et al., 1980; ntara et al., 1996). A large number of mu- Parker and Riches, 1993; Melero-Vara et tations in plants and cultivars have been al., 1989). Tomato (Lycopersicon escu- achieved by the use of EMS, e.g. resis- lentum Mill.) is one of the most important tance to herbicides (Jander et al., 2003) vegetable crops in Bulgaria grown in field and male sterility (van der Veen et al., and greenhouse conditions. 1968). In tomato, EMS showed very high Tomato cultivation is a main source of efficiency by inducing different morpho- agricultural income and has vast economic logical and functional mutations (Watanabe impact in many rural regions. As it is et al., 2007). known, O. ramosa causes severe dam- The aim of this study is to create to- age to the tomato yield (Díaz et al., 2006, mato lines with increased level of resis- Parker and Riches, 1993), and is a serious tance to broomrape by the use of induced threat for the tomato production. Because chemical mutagenesis. Development of of the fast development of the agricultural tomato mutant lines resistant to broomrape sector in Bulgaria in the last few years, gives a possibility for effective control and the tomato areas are growing rapidly. The reduction in the use of chemicals which is presence of broomrape in the soil can be in accord with the contemporary tenden- a limiting condition for the tomato produc- cies for safe and environmental friendly tion or be the cause of large economic agriculture. Such lines can be used directly losses. So far there is no reliable source in the production or for donors for broom- of broomrape resistance in Solanaceae rape resistance in classical selection. family found in the nature, which makes it impossible to add this trait with close re- Materials and Methods lated or inter-specific hybridization (Jacobson, 1986). This implies the need to Plant Materials look for alternative methods to change the Tomato (Lycopersicon esculentum existing varieties so that they will have such Mill.) seeds from Bulgarian cultivar Bella resistance. were provided from IZK Maritsa. It is a Well known method used with success direct determinant cultivar suitable for field in plant breading is induced mutagenesis. production with very good qualities for the The application of induced mutagenesis has processing industry. Seeds were stored in brought a lot of benefits in the modern refrigerator at 4°C. agricultural production as à method for Seeds from branched broomrape (O. crops improvement and addition of new ramosa L.) were collected from infected valuable traits into the existing varieties tobacco plants in South-West of Bulgaria (Ahloowalia et al., 2001). in the region of Gotse Delchev town. Fully- Ethylmethanesulfonate (EMS) is com- matured stems of the parasite were col- monly used mutation agents in the plant lected from the tobacco fields and dried in science because of its height level of ef- shadow. The seeds were separate by sie- fectiveness. EMS belongs to the group of ves and stored at 4°C. alkylating agents which are well known mutation inducers, causing point mutations Chemical agents (like C-to-T changes) as well as loss of Ethylmethanesulfonate (EMS). EMS chromosome segments or deletions (Alca- was purchased from Sigma, USA. Application of Chemical Mutagenesis to Increase the Resistance of .... 507 EMS treatment of tomatoes for branched broomrape Tomato seeds from Bulgarian cultivar (Orobanche ramosa L.) resistance was Bella were the subject to EMS mutagen- conducted under greenhouse conditions. esis. The EMS concentration was defined Special tins (2/1 m) placed in a greenhouse based on a lethal dose in which 50% of were loaded with soil mixed with broom- the seeds lose their germination ability. rape seeds (50 mg.kg-1 soil). The soil was Before the EMS treatment the tomato taken from the field in 50 kg sacks and seeds passed through a preconditioning was autoclaved at 121°C for 30 min. Then period. They were placed in Petri dishes it was placed in the tins and broomrape on wet filter paper for 4 days at 4°C, and seeds were added. After the soil was ca- then dried for 16 hours at room tempera- refully mixed, it was distributed in 10 cm ture. After the precondition period the to- layer. The seeds were placed in rows with mato seeds were incubated in 200 ml so- 10 cm distance between the rows and 5 lution of distilled water containing 1.5% cm between the plants. In every tin there EMS in a laboratory bottle by gently shak- was one row with control plants. After the ing. Mutagenesis takes place in a fume first broomrape plants emerged above the hood. The time of treatment was 4 hours. soil, a selection of non-infested plants was After the time elapsed the laboratory bottle conducted. Tomato plans were pulled out containing the seeds was placed under and their roots were examined for the pres- water and rinsed for 15 min. The seeds ence of attached developing broomrapes. were dried on a filter paper and after this Those plants clean from broomrapes were sowed by hand in a greenhouse to obtain sown in the pots and were used for col- the seedlings. All the consumables in con- lecting M3 seeds. nection to EMS were put into 1M NaOH as a decontamination solution. At the same Artificial screening system with poly- time another 100 seeds were used as a ethylene bags (PEB) control and passed through the same pro- Polyethylene bags system for screen- cedures excluding the EMS solution treat- ing tomato plants for resistance to O. ment. ramosa was performed according to Parker and Dixon (1983), with the M3 Planting M1 progeny of M2 plants selected from the Seedlings from the EMS treated seeds greenhouse experiment. This method en- were sown in the field and were grown ables observations on every stage of until the fruit ripening period. After that 3 broomrape development which helps to fruits from each M1 plant were picked to determine the level and nature of the re- obtain the M2 seeds. The seeds were ta- sistance. Six seeds of each tomato plant ken out of the fruits, washed with water were sown in soil with perlite in small pots and left to dry at room temperature for 72 until the third real leaf emerged. Then they hours, and then placed into paper bags at were taken out and after removing the 4°C for storage. roots were placed in water for several days until new roots started to appear. Follow- Test for resistance to Orobanche ra- ing, they were carefully placed in polyeth- mosa L. ylene bags on fiberglass filter paper GF/ Experimental screening of M2 progeny A, moistened with sterile water and cov- 508 K. Kostov, R. Batchvarova and S. Slavov ered with constant thin layer of surface germinated seeds was a mark that during sterilized broomrape seeds. After the plant the EMS treatment there was an effec- was placed on the paper, the bags were tive mutagenesis process which caused closed carefully and placed in the black the dead of more then half of the seeds. box using metal hanger and covered with The effectiveness of the mutagenesis is transparent plastic box in order to keep essential for obtaining a big number of non- high moisture inside during the first days.