Plant regeneration of natural tetraploid Trifolium pratense L

The regeneration of natural tetraploid T. pratense, originated from Erzurum-Turkey, is reported in this study. This plant has low seed setting and hard seed problems due to polyploidy. Hypocotyl, cotyledon, apical meristems, epicotyl and young primary leaves were inoculated on MS and PC-L2 media containing different concentrations of BAP and NAA as growth regulators. The best shoot formation has been observed on explants initiated from apical meristem placed on PC-L2 medium that includes 2 mg dm-3 BAP and 1 mg dm-3 NAA. 94.4% of the shoots originated from calli were rooted on PC-L2 medium with 1 mg dm-3 NAA. In vitro organogenesis has been accomplished in the natural tetraploid T. pratense regenerated plants successively transferred to the field. Key terms: red clover (Trifolium pratense L.), Fabaceae, organogenesis, plant regeneration. Corresponding Author: Hatice Çölgeçen, Zonguldak Karaelmas University, Faculty of Arts and Sciences, Department of Biology, 67100 Incivez, Zonguldak, Turkey, Tel: +90 0372 257 4010-1128; Fax: +90 0372 257 41 81; E-mail: [email protected] Received: May 9, 2007. In Revised form: March 21, 2008. Accepted: April 17, 2008 INTRODUCTION Trifolium, a legume, makes significant contributions to agriculture and animal feed and thus its production in the U.S. and Europe. Anatolia has been accepted as a central origin of T. pratense (Taylor and Smith, 1979). It is utilized as an ingredient in numerous homeopathic medicines; it has been used to relieve menopausal complaints due to its function as a phytoestrogen and also has been used in cancer treatment due to its anti-tumoral properties (Dixon, 2004). Diploid forms of Trifolium species have been found suitable for agriculture (Gresshoff, 1980; Bhojwani, 1981; Grosser and Collins, 1984; Choo, 1988; Konieczny, 1995; Kaushal et al., 2006), and plant regeneration has been successfully accomplished in diploid forms of T. pratense L., through various methods (Phillips and Collins, 1979; Myers et al., 1989; Radionenko et al., 1994; Carillo et al., 2004). All three varieties of T. pratense grown in Turkey were determined as diploid, however, T. pratense collected by Elci (1982) in the Tortum vicinity of Erzurum was determined as a natural tetraploid with low seed setting and hard seed problems. It has been reported that degenerations in embryo sac might affect the rate of seed setting as well as failure in fertilization (Algan and Bakar, 1997). The present work primarily aimed to develop an efficient in vitro regeneration system for the natural tetraploid T. pratense. MATERIALS AND METHODS Plant material and culture conditions This study examined natural tetraploid E2 type, 2n = 4x = 28 chromosomes, Trifolium pratense L. (red clover) collected from the Tortum vicinity of Erzurum, Turkey, by Elci (1982). The E2-type natural tetraploid ÇÖLGEÇEN & TOKER Biol Res 41, 2008, 25-31 26 T. pratense L. was grown in the experimentation gardens of Ankara University’s Department of Biology in the Faculty of Science. Due to contamination problems from field samples, 15-day-old aseptic seedlings with unifoliate primary leaf were used as the explant source. Seeds were first sterilized in 96% ethanol for one minute and then transferred to 10% sodium hypochlorite solution for 10 minutes (commercial sodium hypochlorite was used in the sterilization process). Then seeds were rinsed 3 times in autoclaved distilled water. After being scarified with autoclaved sandpaper, seeds were germinated on hormone-free MS medium (Murashige and Skoog, 1962). Hypocotyl (0.5-1cm), cotyledon (whole and in two fragments), apical meristem (1mm), epicotyl (0.5-1cm) and young primary leaves (whole and divided into two fragments) of aseptically grown seedlings provided explant tissues. The explants were cultured in petri dishes (100 mm x 15 mm). MS (2% sucrose, 0.8% agar) and PC-L2 (2.5 % sucrose, 0.8 % agar, Phillips and Collins, 1979) media were used for in vitro organogenesis. Different concentrations of benzylaminopurine (BAP) and 1 mg dm-3 naphthalene acetic acid (NAA) (Table 1) were used in MS and PC-L2 media for shoot formation. All media were adjusted to pH 5.8 before autoclaving. Due to the darkening of calli after the third week, the shooted calli were subcultured onto the same media. All the samples were incubated at 22-24°C with a 16/8-hour photoperiod (irradiance of 42 μmol m-2 s-1 provided by cool-white fluorescent tubes). Rooting and ex vitro acclimatization The shoots (1-1.5 cm) obtained from MS and PC-L2 media were transferred into PCL2 medium containing l mg dm-3 NAA for rooting in jars (100 mm x 200 mm) under aseptic conditions and incubated at 22-24°C with a 16/8-hour photoperiod (irradiance of 42 mmol m-2 s-1 provided by cool-white fluorescent tubes). The acclimatization process of the rooted plantlets was carried out by removing the jar l ids gradually with increased durations over one week in a growth chamber. Plantlets were transplanted into sterilized garden soil. The humidity ratio of the growth chamber was gradually decreased from 80% to 50-55% throughout. When the seedlings reached a minimum leaf number of 15, they were transferred to the garden in late March and early April. Statistical analysis The data were subjected to one-way analysis of variance (ANOVA) and the differences among means were compared by Duncan’s multiple-range test (Duncan, 1955). MS and PC-L2 media were compared using a paired Student’s t–test. Each treatment was replicated three times and arranged in a completely randomized design. The data given in percentages were subjected to arcsine transformation (Snedecor and Cochran, 1967) before statistical analysis.