25ÇÖLGEÇEN & TOKER Biol Res 41, 2008, 25-31Biol Res 41: 25-31, 2008 BRPlant regeneration of natural tetraploid Trifoliumpratense L

HATICE ÇÖLGEÇEN1 and M CIHAT TOKER2

1 Zonguldak Karaelmas University, Faculty of Arts and Science, Department of Biology, 67100, Incivez,Zonguldak, Turkey.2 Ankara University, Faculty of Science, Department of Biology, 06100 Tandogan, Ankara, Turkey.

ABSTRACT

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, apicalmeristems, epicotyl and young primary leaves were inoculated on MS and PC-L2 media containing differentconcentrations of BAP and NAA as growth regulators. The best shoot formation has been observed onexplants 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 vitroorganogenesis has been accomplished in the natural tetraploid T. pratense regenerated plants successivelytransferred 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 ofBiology, 67100 Incivez, Zonguldak, Turkey, Tel: +90 0372 257 4010-1128; Fax: +90 0372 257 41 81; E-mail:haticecolgecen@gmail.com

Received: May 9, 2007. In Revised form: March 21, 2008. Accepted: April 17, 2008

INTRODUCTION

Trifolium, a legume, makes significantcontributions to agriculture and animal feedand thus its production in the U.S. andEurope. Anatolia has been accepted as acentral origin of T. pratense (Taylor andSmith, 1979). It is utilized as an ingredientin numerous homeopathic medicines; it hasbeen used to relieve menopausal complaintsdue to its function as a phytoestrogen andalso has been used in cancer treatment dueto its anti-tumoral properties (Dixon, 2004).

Diploid forms of Trifolium species havebeen found suitable for agriculture(Gresshoff, 1980; Bhojwani, 1981; Grosserand Collins, 1984; Choo, 1988; Konieczny,1995; Kaushal et al., 2006), and plantregeneration has been successfullyaccomplished in diploid forms of T.pratense L., through various methods(Phillips and Collins, 1979; Myers et al.,1989; Radionenko et al., 1994; Carillo etal., 2004).

All three varieties of T. pratense grownin Turkey were determined as diploid,however, T. pratense collected by Elci(1982) in the Tortum vicinity of Erzurumwas determined as a natural tetraploid withlow seed setting and hard seed problems. Ithas been reported that degenerations inembryo sac might affect the rate of seedsetting as well as failure in fertilization(Algan and Bakar, 1997). The present workprimarily aimed to develop an efficient invitro regeneration system for the naturaltetraploid T. pratense.

MATERIALS AND METHODS

Plant material and culture conditions

This study examined natural tetraploid E2type, 2n = 4x = 28 chromosomes, Trifoliumpratense L. (red clover) collected from theTortum vicinity of Erzurum, Turkey, byElci (1982). The E2-type natural tetraploid

ÇÖLGEÇEN & TOKER Biol Res 41, 2008, 25-3126

T. pratense L. was grown in theexperimentation gardens of AnkaraUniversity’s Department of Biology in theFaculty of Science. Due to contaminationproblems from field samples, 15-day-oldaseptic seedlings with unifoliate primaryleaf were used as the explant source. Seedswere first sterilized in 96% ethanol for oneminute and then transferred to 10% sodiumhypochlorite solution for 10 minutes(commercial sodium hypochlorite was usedin the sterilization process). Then seedswere rinsed 3 times in autoclaved distilledwater. After being scarified with autoclavedsandpaper, seeds were germinated onhormone-free MS medium (Murashige andSkoog, 1962). Hypocotyl (0.5-1cm),cotyledon (whole and in two fragments),apical meristem (1mm), epicotyl (0.5-1cm)and young primary leaves (whole anddivided into two fragments) of asepticallygrown 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 andCollins, 1979) media were used for in vitroorganogenesis. Different concentrations ofbenzylaminopurine (BAP) and 1 mg dm-3

naphthalene acetic acid (NAA) (Table 1)were used in MS and PC-L2 media forshoot formation. All media were adjusted topH 5.8 before autoclaving. Due to thedarkening of calli after the third week, theshooted calli were subcultured onto thesame media. All the samples wereincubated at 22-24°C with a 16/8-hourphotoperiod (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 MSand PC-L2 media were transferred into PC-L2 medium containing l mg dm-3 NAA forrooting in jars (100 mm x 200 mm) underaseptic conditions and incubated at 22-24°Cwith a 16/8-hour photoperiod (irradiance of42 mmol m-2 s-1 provided by cool-whitefluorescent tubes).

The acclimatization process of therooted plantlets was carried out byremoving the jar l ids gradually with

increased durations over one week in agrowth chamber. Plantlets weretransplanted into sterilized garden soil. Thehumidity ratio of the growth chamber wasgradually decreased from 80% to 50-55%throughout. When the seedlings reached aminimum leaf number of 15, they weretransferred to the garden in late March andearly April.

Statistical analysis

The data were subjected to one-wayanalysis of variance (ANOVA) and thedifferences among means were comparedby Duncan’s multiple-range test (Duncan,1955). MS and PC-L2 media werecompared using a paired Student’s t–test.Each treatment was replicated three timesand arranged in a completely randomizeddesign. The data given in percentages weresubjected to arcsine transformation(Snedecor and Cochran, 1967) beforestatistical analysis.

RESULTS

While apical meristem and hypocotylexplants induced callus in 3-4 days on PC-L2 medium, the same process took 5 dayson MS medium. On the other hand, callusinduction from the cotyledon, epicotyl, andprimary leaf explants took about 1 week onMS and PC-L2 media. Great amounts ofcalli were grown from all the explants in 3-4 weeks. In general, different-coloredwhite, yellow, and green calli were inducedon all tested media. The yellow calliobtained on both media were more friablethan the others. Among all the BAPconcentrations applied, the best callusinduction on MS medium was observedfrom apical meristem explants. Copiousamount of calli were obtained from thehypocotyl and cotyledon explants on all theBAP concentrations except the mediumcontaining 4 mg dm-3 BAP and 1 mg dm-3

NAA. The best callus induction from theepicotyl and primary leaf explants wasobserved on the medium containing 5 mgdm-3 BAP and 1 mg dm-3 NAA. On the PC-L2 medium supported with 2, 2.5, and 3 mg

27ÇÖLGEÇEN & TOKER Biol Res 41, 2008, 25-31

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ÇÖLGEÇEN & TOKER Biol Res 41, 2008, 25-3128

dm-3 BAP and 1 mg dm-3 NAA, the bestcallus was induced from apical meristemexplants. Plenty of calli were induced fromall explants on all media as well as themedium containing 4 and 5 mg dm-3 BAPand 1 mg dm-3 NAA (Table 1).

Shoot formation from white and yellowcalli of apical meristem started in thesecond and third weeks. No shootformation was observed on the calli ofhypocotyl, epicotyl, cotyledon, or primaryleaf on MS medium containing differentBAP concentrations. The highest shootformation occurred from apical meristemcallus on MS medium with 2.5 and 3 mgdm-3 BAP and 1 mg dm-3 NAA in 2-3weeks. The best number of shoot perexplants and the highest hyperhydricshoots resulted on MS medium with 2.5mg dm-3 BAP and 1 mg dm-3 NAA. Thehighest shoot formation rate was recordedin apical meristem cal lus on PC-L2medium, with 2 mg dm-3 BAP and 1 mgdm-3 NAA. Extremely low number ofshoot formation was observed on the calliof hypocotyl and epicotyl on PC-L2medium in the same concentration. Exceptthe 2 mg dm-3 BAP and 1 mg dm-3 NAAconcentration, no shoot formation wasobserved from cotyledon and primary leafcall i . The best number of shoot perexplants (Fig. 1) and the highest

hyperhydric shoots resulted on PC-L2medium with 2 mg dm-3 BAP and 1 mgdm-3 NAA. Some of the shoots producedfrom the apical meristem calli on MS andPC-L2 media established hyperhydric form(Table 2).

All the shoots induced on MS and PC-L2media have normal trifoliate leaves. Onetetrafoliate-leafed shoot formed from apicalmeristem callus on the MS with 4 mg dm-3

BAP and 1 mg dm-3 NAA (Fig. 2), and thisshoot could not be rooted. Normal apicaldevelopment occurred in some of the apicalexplants. Somatic embryogenesis did notoccur throughout the experiments.

Figure 1: Shoots on the callus of apicalmeristem on PC-L2 medium (containing 2 mgdm-3 BAP and 1 mg dm-3 NAA) (Bar = 2mm).

TABLE 2

Effect of BAP on shoot induction (%) and abnormal shoot formation (%) from calli ofapical meristems on MS and PC-L2 media.

Means ± SE. Mean followed by the same letter are not significantly different using Duncanmultiple comparison test within different concentrations of BAP.

Growth MS PC-L2regulators[mg dm-3 ] Shoot Number of Hyperhydric Shoot Number of HyperhydricBAP NAA Formation shoots [explant-1] shoot formation shoots [explant-1] shoot

2 1 40.81 ± 0.41a 0.42 ± 0.02 a b 18.95 ± 0.51 a 84.56 ± 0.00 a 1.12 ± 0.01 a 38.06 ± 0.30 a

2.5 1 45.00 ± 0.00 b 0.50 ± 0.00 a 48.02 ± 0.15 b 39.23 ± 0.00 b 0.58 ± 0.18 b 31.23 ±1.23 b

3 1 45.58 ± 0.58 b 0.38 ± 0.13 a b 33.15 ± 0.88 c 39.46 ± 0.70 b 0.61 ± 0.19 b 27.03 ± 0.47 c

4 1 32.95 ± 1.00 c 0.27 ± 0.02 b 27.13 ± 0.57 d 58.15 ± 1.86 c 0.64 ± 0.37 b 32.56 ± 0.62 b

5 1 31.33 ± 0.01 c 0.26 ± 0.01 b 32.94 ± 0.36 c 34.40 ± 1.87 d 0.22 ± 0.12 c 33.12 ± 0.85 b

29ÇÖLGEÇEN & TOKER Biol Res 41, 2008, 25-31

The shoots were rooted at the rate of94.4% on PC-L2 medium supplementedwith only 1 mg dm-3 NAA. The rooting wasobserved in a week, and the roots continuedtheir growth inside the medium (Fig. 3). Norooting occurred in the hyperhydric shootswhen taken to rooting medium and all died.

Figure 2: Tetrafoliate leaf on MS supplementedwith 4 mg/1 BAP and 1 mg/1 NAA.

Figure 3: The rooted shoot (young seedling) onthe PC-L2 supplemented with 1 mg/1 NAA.

After rooting, all the seedlings with aminimum of 15 leaves were transferredfrom pots to the experimentation garden bylate March and early April (Fig. 4). Theseplants continued to grow in garden. All ofthem flowered in the following floweringseason and produced fertile seeds.

DISCUSSION

The most widely used forms of T. pratenseare 2n = 14 diploid types in tissue cultureresearch and in various studies. This studyis the first tissue culture study carried outwith the natural tetraploid T. pratense L. 2n= 4x = 28 grown in Turkey. Researchersused T. pratense aseptic seedlings ofdifferent ages as explant sources (Phillipsand Collins, 1979; Bhojwani, 1981; Myerset al., 1989). In almost all the studies,apical tip and hypocotyl explants have beenpreferred and also were utilized in ourstudy. However, the ages of the seedlingsused by the researchers display variations.According to our results, 15-day-oldseedlings are the most suitable explantsources due to their totipotency properties.

Apical meristem and hypocotyl explantsinduced calli in a shorter time on PC-L2medium compared to MS medium. On theother hand, MS medium responded better ingeneral regarding callus formationcompared to PC-L2 medium. The mediumwas not found to have an impact on calluscolor; morphologies of the calli on bothmedium were similar. While white and

Figure 4: The seedlings transferred to theexperimentation garden.

ÇÖLGEÇEN & TOKER Biol Res 41, 2008, 25-3130

yellow calli were induced from the apicalmeristems in both media, the calli from theother explants were mainly yellow andgreen. Myers et al. (1989) obtained friablecalli from T. pratense on L2 (Phillips andCollins, 1979). In our study, the yellowcalli obtained from all of the explants werefound to be friable calli.

Phillips and Collins (1979) obtainedhigh numbers of shoots in 2-3 months fromthe meristem callus placed in a mediumcontaining 0.006 picloram and 0.1-10 mgdm-3 BAP concentrations, whereas, in ourstudy, apical meristem callus producedshoots in a shorter time (2-3 weeks). PC-L2medium responded better in terms of shootformation compared to MS medium. Shootnumber per explant was higher on the PC-L2 medium supplemented with 2 mg dm-3

BAP and 1 mg dm-3 NAA compared to MSmedium. Moreover, higher numbers ofhyperhydric shoots were observed in theMS medium supplemented with 5 mg dm-3

BAP and 1 mg dm-3 NAA in comparison tothe PC-L2 medium. The results of ourexperiments revealed PC-L2 to be the bestmedium for plant regeneration of thenatural tetraploid T. pratense. The bestplant growth regulator combination wasfound to be 2 mg dm-3 BAP and 1 mg dm-3

NAA.Phillips and Collins (1979) achieved

85% rooting on hormone-free MS mediumin 2-4 weeks. Researchers have rooted theshoots on different media (Gresshoff, 1980;Bhojwani, 1981; Myers et al., 1989). Interms of rooting, the same rooting mediumhas yielded optimum results for shootsobtained from both media in our study. Theobservation of rooting at the end of the firstweek and a rooting rate of 94.4 % arenoteworthy achievements. No rooting wasdone on MS medium after the achievementof such a high rate on PC-L2 medium.

Several researchers, after adjusting therooted shoots to the air medium, havetransferred them into different mixtures ofsoil and applied different rates of humidity(Phillips and Collins, 1979; Bhojwani,1981). In our study, no specific soil mixturehas been used. The soil used was sterilized,normal garden soil, and the temperaturewas set to 22-24°C with an initial humidity

rate of 80%. Researchers have mentionedthat they transferred the regenerated plantsto a greenhouse. After transferring theplantlets into individual pots containing soiland keeping them in the greenhouse briefly,we completely transferred them to externalmedium by transferring them to ourexperimentation garden. The immediateadaptation of the natural tetraploid T.pratense to garden soil can be explained bythe superior adaptation ability of ploidy-containing plants to difficult circumstances.Our plants continued growing (100%) insoil. All of them flowered in the followingflowering season and produced fertileseeds.

This study has given a method for rapidpropagation of natural tetraploid T.pratense in vitro. As previously mentioned,these diploid plants contain componentsfunctioning as phytoestrogens. Thus, theplant regeneration described in this studymay open the possibility for usingbiotechnological techniques to obtain highproduction of naturally occurring secondarymetabolites (flavonoids) in naturaltetraploid T. pratense L.

ACKNOWLEDGEMENTS

The authors wish to thank Dr. SahabettinElci and Dr. H. Nurhan Buyukkartal forproviding red clover seeds and for sharingtheir inside information pertaining to theplant.

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