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نويسندگان : Abbas Yadollahi     تاريخ انتشار : 29.08.2013
In vitro Culture of Gisela 6 Semi-dwarf Rootstock
ABSTRACT: This research was carried out to study micropropagation of Gisela 6 rootstock in order to achieve suitable media for producing 5 thousand plants. Explants were cultured in MS and WPM media containing 2 types of cytokinins BAP and TDZ at different concentrations. The results showed that the number of shoots in MS medium containing 1.2 mg L-1 BAP was higher than the othertreatments

INTRODUCTION

Gisela 6 is a hybrid of Prunus cerasus x Prunus canescens and a semi-dwarf rootstock which is suitable for all kinds of sweet cherry. It is also satisfactory in a wide range of soils, especially heavy soils (Andersen et al. 1999). The average and slow growth of this rootstock has great value for establishment and development of modern intensive cherry orchards. Trees grafted on this rootstock don’t have suckering problem (Long and Kaiser 2010). In Iran, conventional rootstocks like Mazzard (Prunus avium) and Mahlab (P. mahlab) are mainly used as rootstocks for sweet cherry. In recent years, there has been a growing trend toward using dwarf rootstocks in Iran. Gisela 6 rootstock is one of the sweet cherry rootstocks which will become important in the Iranian fruit industry in the near future. Producing Gisela 6 rootstock in large scale by conventional methods like cutting and layering to meet the growing internal demand for high quality, disease-free and uniform planting material, seems to be difficult. The scale and speed of production of healthy plants can be enhanced by micropropagation techniques (Aka-Kacar et al. 2010). Erbenova et al. (2001) reported 50% enhancement in multiplication rate of sweet cherry rootstocks in MS medium containing 0.5 mg L-1 BAP. MS and 2MS (double macro-salts) media containing 4.4 mμ BA, 0.5 mμ NAA and 0.3 mμ GA3 were reported appropriate media for propagation of Gisela 5 (Ruzic et al. 2000). Combination of 0.5 mg L-1 BAP and 0.05 mg L -1 TDZ, and medium supplemented with 0.3 mg L-1 IBA were reported desirable for proliferation and rooting of Prunus avium, respectively (Dorkovic 2006). Tang et al. (2002) reported BAP was more effective than TDZ in shoot regeneration from leaves, and WPM was more effective than MS in caulogenesis. They also reported rooting percentage in half MS medium containing 2 mg L-1 IBA was higher than the same medium but containing 2 mg L-1 NAA. However, length and number of roots were reported greater with NAA compared to IBA. Buyukdemirci (2008) stated that MS medium containing 0.5 mg L-1 BAP, 0.01 mg L-1 IBA and 0.1 mg L-1 GA3 was the best medium for proliferation. He also reported that reducing the nitrate concentration promoted more roots from the Gisela 5 explants and the best rooting was observed in the medium supplemented with 0.5–1 mg L-1 IBA
One of the main factors in micropropagation is medium, since different media have different ingredients. Particular compound, the type of culture, the verity of plant from which explants are taken and whether the explants are taken from juvenile or mature tissues can determine the effect of cytokinins. In some plants, BAP has been reported to be more effective than TDZ )Tang et al. 2002; Ruzic and Vujovic 2008(; and in some other plants, TDZ has been shown to be more effective than BAP (Perez-Tornero et al. 2000; Bhagwat and Lane 2004; Matt and Jehle 2005; Espinosa et al. 2006; Canli and Tian 2008(. In the rooting stage, the induction of roots on explants can be crucial part in micropropagation process based on the kind of culture, the variety of plant and the age of explant (Molassiotis et al. 2003/4; Thorpe et al. 2008). The ability of plant tissues to form adventitious roots is determined through the interaction of many exogenous and endogenous factors including hormone. In most reports, exogenous auxins such as IBA, NAA or IAA were used for rooting (Ainsley et al. 2001). The above-mentioned auxins are only required at an early stage to stimulate the emergence of newly formed roots (Dobranszki and Silva 2010). Apart from hormone, other factors which can affect rooting are the chemical ingredients of the culture medium. Iron is one of the essential microelements used in micropropagation due to its essential role in many processes like chlorophyll and DNA synthesis (Dunlap and Robacker 1988). Moreover, Fe is a constituent of peroxides, which mediates IAA catabolism (Gaspar et al. 1992). Many researchers have reported the beneficial effects of Fe-EDTA replacement with Fe-EDDHA on rooting of woody plants (Maheshwari and Seth 1965; Chopra and Rashid 1969; Rashid and Street 1973; Molassiotis et al. 2003, 2004; Antonopoulou et al. 2007). The other essential components in culture medium are vitamins (Molnar et al. 2011) required in minute amounts in plant tissue culture (Torres 1989). Among four important vitamins, thiamine is more important owing to its direct involvement in the biosynthesis of some amino acids and its role as a cofactor in carbohydrate metabolism (Al-Khayri 2001). It was also reported that thiamine brought about stimulation of adventitious rooting in Taxus spp (Chee 1995). No paper has reported the effects of thiamine and Fe-EDDHA on rooting of Gisela. The objectives of this research were to study a) the effects of two types of media, two types of cytokinins at four concentrations on proliferation b) the influences of different media and rooting-hormones on rooting c) the role of thiamine and Fe-EDDHA on the rooting improvement of Gisela 6
MATERIALS AND METHODS

Plant explants were provided from controlled plants kept in greenhouse of Faculty of Agriculture of Tarbiat Modares University in spring of 2011. At first, shoots were excised into 2.5 cm-long sections; then for surface disinfection, they were agitated for 5 min in a solution containing 5 drops of Tween-20 in 100 ml of water followed by washing under running water for 1 hr. For sterilization under laminar airflow chamber, firstly explants were agitated in alcohol 70% for 30 s, then in %0.01 solution of mercuric chloride for 7 min and finally they were rinsed three times with distilled water
MS (Murashige and SKoog 1962) and WPM media containing BAP and TDZ at four concentrations of 0.4, 0.8, 1.2 and 1.6 mg L-1 were investigated (Table 1)
After 30 days, at the end of the proliferation stage, the number of new shoots per explant, shoot height (cm) as well as the weight of callus (gram) were determined. Number of shoot was measured by counting newly formed shoots; and shoots height was measured by a metal ruler. For measuring the callus weight, firstly fresh cuts were made at the basal ends of the shoots and then calluses were carefully taken and weighed by scale
For the first rooting experiment, after removal of the abnormal and lower leaves, shoots with 3-5 cm length from elongated phase were separated and then individually transferred to different media supplemented with different auxins hormone (Table 2). In the second experiment of rooting, shoots with 3-5 cm length were transferred to 1/2 MS medium (macroelements half) brought about 100 percent rooting in the previous experiment, supplemented with different concentrations of thiamine and Fe-EDDHA (Table 3). After culturing shoots in different treatments, jars containing shoots were maintained in the dark for one week. After one week, all treatments were transferred to light condition. Data including percentage of rooting, length and number of roots were recorded after four weeks. Percentage of rooting, root number and root length were determined by counting rooted explants, counting the number of formed roots and measuring by using a metal ruler, respectively. Allmedia supplemented with 7 g L-1 agar (Bacterological agar, MM0317, ‘Nebotrade Ltd’), 30 g L-1 sucrose. The pH of media was adjusted to 5.75±3 with HCl 0.1N or NaOH 0.1N prior to sterilization by autoclaving at 121°C for 20 min. explants were maintained at 25±1 ºc and 16/8 hr photoperiod of cool-white light at 1250 lux for 30 days.
In the acclimation phase, peat and perlite at 3:2 (v/v) were used as medium. Plants were transferred to plastic tunnel with 24±1 ºc and 90% relative humidity. After one week, humidity was reduced.
The proliferation experiment was conducted as a factorial based on a completely randomized design (CRD) with 5 replications per treatment and two explants per replication. Statistical analysis of the data was carried out by using SAS software and difference among treatments means was compared by using Duncan's multiple range test at P≤0.05 level. For rooting, the experiments were performed as factorial based on a completely randomized design (CRD) with two factors and 5 replications per treatment and two explants per replication. Statistical analysis of the data was conducted using SAS 16 software and difference among treatments means was compared by using Duncan's multiple range test at P≤0.05 level

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