260 1. Introduction Salinity is a widespread problem around the world, es- pecially in arid and semi-arid regions. Each year more and more land becomes non-productive due to salt accumu- lation. At least 25% of currently cultivated land through- out the world suffers from excess salinity (Bohnert and Jensen, 1996) and all major crop species are intolerant to salt (Fairbairn et al., 2000). The most economic and sus- tained way to overcome the problem of salt-stress is to develop salt-tolerant varieties (Frommer et al., 1999). In vitro culture has been widely used for the propagation and conservation of crop genetic resources in both agriculture and horticulture crops (Barakat and El-Lakany, 1992). Mutation breeding programs using gamma irradiation on apricot buds were carried out by several investigators (Legave and Garcia, 1988; Ageeva, 1989; Gulcan and Ak- soy, 1995). Legave and Garcia (1988) reported that bud sticks of five apricot cultivars were exposed to up to 70 Gy gamma rays and scored for bud survival and growth. The effect of gamma irradiation in the range 10-70 Gy on variation in the characters of apricot was also investigated (Ageeva, 1989). The varieties reacted in different ways to treatment. Induced mutations in apricot breeding were also investigated (Gulcan and Aksoy, 1995). Apricot (Prunus armeniaca) was treated with 0.3 KR gamma irradiation (source 60Co). Mutagenesis affected vigour, dry matter and vitamin C content, and the level of carotene in fruit. In vitro cultures of Japanese plum (Prunus salicina) cv. Shiro were also gamma-irradiated by Predieri and Gatti (2000). In vitro culture may offer potential for quick evaluation of germplasm against salt stress (Cano et al., 1998). Re- cently, Jain (2001) reported that tissue culture generates a wide range of genetic variation in plant species which can be incorporated into plant breeding programs. The effect of NaCl and CaCl2 in Prunus cerasifera was investigated by Lucchesini and Vitagliano (1993). The in vitro response of peach cv. Redhaven and of the peach/almond hybrid rootstock GF677 to increasing concentrations of NaCl in the medium was reported (Biricolti and Pucci, 1995). The response to increasing rates of NaCl or CaCl2 and proline on ‘Mr.S 2/5’ (Prunus cerasifera) peach rootstock cultured in vitro has also been reported (Dimassi-Theriou, 1998). Towards in vitro selection studies for salinity tolerance in Canino apricot cultivar. Effect of gamma irradiation on in vitro mutation and selection for salt-tolerance A.S. El-Sabagh*1, M.N. Barakat**, E.A-E. Genaidy*** * Department of Horticulture, Faculty of Agriculture, Damanhour branch, University of Alexandria, PO Box 22516, Damanhour, Egypt. ** Department of Crop Science, Faculty of Agriculture, Alexandria University, Egypt. *** Pomology Department National research center, Dokki, Geza, Egypt. Key words: IBA, BAP, M3 and MS3 medium, mutation gamma ray, propagation, salt tolerance. Abstract: In vitro mutation method was used to obtain salt-tolerant clone in apricot. Small propagules of Canino apricot cultivar were irradiated with gamma ray at doses of 0, 10, 25, 35, 50, 75 and 100 Gy. After 30 days from treatment, both the radio sensitivity and post-irradiation recovery were assessed as the number of proliferated shoots per explants, fresh weight of cultures, shoot length and productivity of irradiated explants. A sudden and sharp decrease in the survival percentage occurred with the dose 75 Gy, while the highest dose (100 Gy) was lethal for all propagules. A marked decline in the number of regenerated shoots per explant and fresh weight of produced cultures was associated with an increase of irradiation doses. Doses in the range of 10-75 Gy, which preserved high survival percentage of irradiated explants, seemed to be more suitable for in vitro mutation in Canino apricot cultivar. Irradiated shoots were exposed to different concentrations of NaCl which were added to the multiplication medium at the rates of 25, 50, 75, 100, 125 mM and after 30 days, vigorous shoots were selected from salinity treatments. In conclusion, apricot tissues exposed to different doses of gamma irradiation in the range of 10-75 Gy, followed by culturing the plantlets produced in a medium containing ad- ditional salts (ranging from 25 to 125 mM) can be considered a good method to identify the most tolerant mutants to salts in apricot cultivars. Adv. Hort. Sci., 2011 25(4): 260-263 1 Corresponding author: ahmed_elsabagh67@yahoo.com Received for publication 12 January 2011 Accepted for publication 12 October 2011 261 Recently, increased sodium chloride (NaCl) salinity effects on bitter almond (Amygdalus communis) (Prunus dulcis) growth, cell osmolarity and nutrient acquisition were studied in vitro (Shibli et al., 2003) and it was found that elevating salinity from 0.0 (control) to 50, 75, 100 mM NaCl resulted in reductions in shoot growth (shoot height, shoot dry weight) and rooting (rooting percentage, root number, root length). The objective of the present work was to obtain salt- tolerant clone (s) in apricot using an in vitro mutation method. 2. Materials and Methods The present work was carried out in the Biotechnology Laboratory, Crop Science Department, Faculty of Agri- culture, Alexandria University from 2001 to 2005. Small propagules of Canino apricot cultivar initiated from in vitro culture by the follwing protocol: two explants shoot tips and single node cutting; of 0.5-1 cm in length was used. Shoot tips and single node were soaked in 100 mg/l ascor- bic acid +150 mg/l citric acid for 30 min. Explants were dried for 30 min. before they were immersed in fungicide Ridomil (1 g/l solution) for 30 min. and then washed with distilled water, then soaked in Clorox (7% Sodium hy- pochlorite) for 7 min and washed with sterile distilled wa- ter three times. The explants were aseptically excised and placed in Jar containing 50-60 ml of culture medium. Each Jar contained one explant, considered as one replication. Cultures were incubated at 25±2oC under 16 hour’s illu- mination (2000 lux, day light fluorescent tubes). In order to check the phenol oxidation and to establish the explants with free from phenol, the explants were cultured on two medium (MS and M3,) supplemented with four PVP con- centrations (0.0, 40.0, 80.0, 160.0 mg/l). The effect of five medium protocols was examined: modified woody plant medium (M3) (Perez-Tornero et al., 2000), MS medium (MS1) and MS medium (Murashige and Skoog, 1962) modified by reducing KNO3 and NH4NO3 by 25%, 50% and 75% which were designated MS2 , MS3 and MS4, re- spectively. All media were supplemented with 3.0% su- crose, 4 mg/l adenine sulfate, 160 mg/l PVP, 0.4 mg/l BAP and 0.01 mg/l IBA. The pH of the media was adjusted to 5.7 by using 1.0 N HCl or 1.0 N NaOH and agar was added after adjusting the pH. The best two media (M3 and MS4) for proliferation were used to test the optimum effect of three types of cytokinins benzyladenine (BAP), Kiniten and isopentenyladenine (2iP) and their concentration on shoot tip proliferation, four different concentration of cy- tokinin 0.2, 0.4, 0.6 and 0.8 mg/l for each type using M3. Also, four different concentration of cytokinin 0.5, 1.0, 2.0 and 4.0 mg/l for each type using MS4. The proliferation was evaluated six weeks after the beginning of the experi- ment and the number of shoots, (longer than 5 mm) per explant, their length and productivity (number of shoots x the average shoot length) were recorded. Shoots of apricot derived from the shoot tips multiplication were cultured on M3 medium Perez-Tornero et al. (2000). The medium was supplement with either NAA (0.0, 0.5, 1.0, 2.0 mg/l) or IBA (0.0, 2.0, 4.0, 6.0 mg/l) were employed. In vitro mutation and selection for salt-tolerance Effect of gamma irradiation on in vitro shoot culture. Small propagules of Canino cultivar initiated from in vitro culture (Fig. 1) were irradiated in a gamma cell with a co- balt60 source at the Middle-East Regional Radioisotopes centre for Arab Countries, El-dokki, Giza with 10, 25, 35, 50, 75 and 100 Gy doses. The irradiated propagules were removed from the jar and recultured on a fresh pro- liferation medium. After six weeks incubation, impact of the irradiation was assessed by determinating the number of shoots, the fresh weight of shoot multiplication, shoot length and productivity. 0.2 mg BAP 0.4 mg BAP 0.8 mg BAP 0.6 mg BAP Fig. 1 - In vitro micro propagation of apricot cv. Canino on M3 medium derived from shoot tip explants. In vitro selection. Small propagules of the cultivar (Canino) (Fig. 1) were irradiated in a gamma cell with co- balt 60 source Gy at a dose of 10, 25, 35, 50 and 75 Gy. The propagules were subcultured five times before in vitro se- lection for salt-tolerance. Individual shoots from irradiated cultures were grown on the proliferation 0.6 mg/l BAP M3 medium supplemented with different concentrations of NaCl (25, 50, 75, 100 and 125 mM). After six weeks of incubation, the vigorous shoots were selected and trans- ferred to fresh medium free from salt. Analysis of vari- ance with SAS software (SAS Institute, 1988) was carried out. Treatment means were compared using the LSD test at 5% level probability. Data were analyzed as a factorial arrangement in Randomized Complete block design ac- cording to Steel and Torrie (1980). 3. Results and Discussion Effect of gamma irradiation on in vitro apricot culture The basic requirement for effective use of mutation in- duction in plant breeding programs is the analysis of ra- dio sensitivity of the explant material (Walther and Sauer, 262 1986). Predieri (2001) reported that one of the first steps in mutagenic treatment is the estimation of the most ap- propriate dose to apply. The aim of the present work was to determine the radio-sensitivity of in vitro apricot cul- ture, as assessed by the number of regenerated shoots, the fresh weight of shoot multiplication, the shoot length and productivity in order to select the suitable dose of gamma irradiation to conduct in vitro mutation for improvement. The collected data, reported in Table 1, indicate that a clear decrease in in vitro traits occurred with increas- ing irradiation dose. Complete lethality (100% death) was observed with an irradiation dose higher than 75 GY (Fig. 2). Several other studies have been conducted on the ra- dio-sensitivity of in vitro cultures of fruits, such as Pru- nus avium (Walther and Sauer, 1985), kiwifruits (Shen et al., 1990), grapevine (Lima da Silva and Doazan, 1995; Charbaji and Nabulsi, 1999) and Prunus salicina (Predieri and Gatti, 2000). Previously, Laneri et al. (1990), working with Gerbera jamesonii, stated that in a mutation breed- ing experiment, the dose chosen for the main experiment should result in the highest survival of irradiated explants and that a low inhibition of the rate of production of new shoots gives the highest efficiency in recovering useful mutants. In light of these studies, the results obtained in the present investigation suggest that doses of 10 Gy to 75 Gy seem to be the most suitable for inducing mutation for apricot improvement. In vitro mutation and selection for salt-tolerance in Cani- no apricot cultivar Mutation breeding can be employed as a promising technique that allows diversification of apricot. Induced mutations change only one or a few specific traits of an elite cultivar without undesired additional variations (Predieri, 2001). In fact Predieri concluded that the most suitable method may be mutation treatment and propaga- tion of in vitro axillary shoots without passage through undifferentiated growth, and it can contribute to fruit im- provements without upsetting the requirements of the fruit industry nor the consumers. Through in vitro selection, mutation with a useful agronomic trait, e.g. salt or drought tolerance or disease resistance, can be isolated in a short time (Jain, 2001). The present work was conducted to obtain salt-tolerant clone(s) in apricot cv. Canino using in vitro shoot muta- tion. Small propagules were irradiated with 0, 10, 25, 35, 50, or 75 Gy and explants were multiplied for five sub- cultures. The generated irradiated shoots were subjected to a salt (NaCl) which was added to the medium with the concentrations 25, 50, 75, 100, or 125 mM. The number of vigorous shoots of cv. Canino showed marked differences in their in vitro salinity tolerance (Table 2). It is clear that the number of vigorous shoots decreased rapidly with in- creasing salinity. The highest number of vigorous shoots was obtained in medium supplemented with 25 mM selec- tive agent of salinity when the propagules were exposed to 25 and 50 Gy, respectively (Table 2). From these results, it can be concluded that apricot cv. Canino tissues exposed to different doses of gamma irradiation in the range 10-75 Gy, followed by culturing in medium containing a higher concentration of additional salts (ranging from 25 to 100 mM) can be considered a good method to identify mu- tants in apricot cv. Canino which are the most tolerant to salts. FAO/IAEA (1997) reported that plant biotechnology in combination with mutation induction and conventional breeding might open new frontiers for obtaining salt-toler- ance rice varieties. The application of mutation techniques in breeding has increased constantly over the past years. These techniques must be rapid to keep pace with the large quantity of breeding materials generated after mu- tagenesis. Screening under field conditions is difficult due Table 1 - Effect of gamma irradiation six weeks after the treatment on in vitro apricot shoot Gamma irradiation (gy) doses Weight Number of shoots Shoot length Productivity Control 5.85 a 23.40 a 6.15 a 147.36 a 10 3.58 b 14.30 b 2.99 b 42.27 b 25 3.45 b 13.80 b 3.14 b 42.52 b 35 2.40 bc 9.60 bc 3.63 b 41.68 b 50 1.50 cd 6.00 cd 0.77 c 11.05 c 75 0.68 d 2.70 d 0.48 c 4.18 c Means within a column or a row followed by the same letter(s) are not significantly different at the 0.05 level of probability. Table 2 - Effect of irradiation doses on the number of vigorous shoots of apricot cv. 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