In Vitro Screening of Some Tomato Commercial

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studies of tomato (Lycopersicon esculentum). Plant Cell, Tissue and Organ. Culture. 78: 1-21. [23] M. Naseem Abed Alrahman, AR. Rida, K. Ereifej, and Y. Muna.
International Journal of Biotechnology and Biochemistry. ISSN 0973-2691 Volume 7, Number 5 (2011) pp. 543-552 © Research India Publications http://www.ripublication.com/ijbb.htm

In Vitro Screening of Some Tomato Commercial Cultivars for Salinity Tolerant Magdoleen G. Osman1, Elsadig A. Elhadi2 and Mutasim M. Khalafalla1* 1

Commission for Biotechnology and Genetic Engineering, National Center for Research, P. O. Box 2404, Khartoum, Sudan. 2 Desertification Research Institute, National Center for Research, P. O. Box 728, Khartoum, Sudan.

Abstract In vitro screening of 12 tomato cultivars (Omdurman, Allakarim, Strain B., Jazera, Majd, Supermajd, Madona, Amani, Castel Rock, Rio grand, Sudan special and Peto-86) for salt tolerance at different concentrations of NaCl (0, 50, 75 and 100 mM) was investigated. To select the salt tolerant and salt sensitive cultivars, vegetative growth parameters were measured. Shoot and root growth in these tomato cultivars were decreased with increasing of NaCl concentration in the growth media. Cultivar Amani gave the longest root (6.7 cm) and heavier root fresh weight (0.28 g) at the highest level of NaCl tested while Castel Rock gave the longest shoot (8.0 cm), and heavier shoot fresh weight (1.09 g). In vitro screening of tomato through shoot tip culture is an easy and efficient method to identify salt-adapted genotypes within a limited space and time period. Keywords: Lycopersicon esculentum, NaCl, Salt tolerant.

Introduction Soil salinity is one of the most important a biotic stress that limit crop production[1, 2]. Up to 20% of the irrigated arable land in arid and semiarid regions is already salt affected and is still expanding [3]. The Soil Survey Department In Sudan, stated that the total area affected by salinity and/or sodicity was estimated at 2.5 million hectares[4]. Saline soils occur in most productive land along both bank of the Nile and its attributes[5]. The high clay content of 2:1 clay minerals makes complete reclamation of salt affected soils in Sudan impracticable. Moreover, the use of subsurface drainage system was not successful [5].

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Therefore, there is a need to test for crops tolerant to salinity. Tomato (Lycopersicon esculentum Mill.) is moderately tolerant to salinity and is typically cultivated in regions that are exposed to soil salinization [6]. In Sudan, tomato is important vegetable crop ranks second to onion among vegetable crops based on cultivated area [7]. It is grown throughout the country where irrigation water and arable land are available [8]. Its production is affected by various stresses such as disease, high temperature, draught, salinity and its vulnerability to frequent insect and pest attacks. Hence, there is a need toselect salt tolerance cultivar of tomato using modern biotechnological approaches. Salt tolerance in plants depends primarily on the genotype which determines the alteration on processes such as; exclusion of the salt, uptake and transport of salt by roots, together with metabolic and physiological events occurring at cellular level [9]. The selection of salt tolerant lines continues to challenge plant scientists, especially those working in physiology and genetics. Most crop plants, including the cultivated tomato, are sensitive to salinity, although differences between tomato cultivars have been reported [10, 11]. One strategy to reduce the deleterious effects of soil salinity on tomato production is by development of Salt -tolerant cultivars [12]. The screening of a large number of genotypes for salinity tolerance under ex vitro conditions is rather difficult since it requires a large amount of resources and space and complex interactions between the plant and different soil components. In vitro culture, on the other hand, is an ideal system for screening salt-tolerance in plants, since it can be carried out under controlled conditions with limited space and time [13]. Therefore, many attempts have been made to screen genotypes in vitro using shoot apices. Chandler et al. [14] screened genotypes of sugar beet, tobacco, Chinese cabbage and canola on media with different salt concentrations. In vitro culture of tomato has been successfully exploited for selection of tolerant cell lines for various biotic and a biotic stresses under laboratory conditions, by exploiting the genetic variability arising during in vitro culture conditions [15]. It requires comparatively less effort and fewer resources than selection under field conditions. Selection for salinity tolerance can be carried out in vitro, by culturing either explants, callus, cell suspension, protoplasts, embryos or microspores in the presence of screening agent, e.g. NaCl [11]. In vitro selection and screening for salinity tolerance have been reported in tomato by [16, 17, 18]. An in vitro shoot apex culture could be a better system for testing and selecting for salt tolerance [19]. The present investigation was focus on the utility of tomato tissue culture for in vitro selection of salt-tolerant tomato plants.

Materials and methods Plant materials Twelve tomatoes (Lycopersicon esculentum Mill) varieties namely; Omdurman, Alla Kareem, Strain B., Jazera, Majd, Super majd, Madona, Amani, Castel Rock, Rio grand, Sudan special and Peto-86 were used in the present study.

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3.10.1.2 Disinfestations and seed germination Seeds were washed under continuously running tap water for 15 minutes, then washed by sterile distilled water. Seeds were disinfested under laminar flow cabinet with Clorox (0.5 % free chlorine) at concentration of 15% v/v for 15 mints then rinsed three times with sterile distilled water. 3.10.1.3 Explants preparation The disinfested seeds were germinated in bottles containing 30 ml of half –strength Murashige and Skoog (MS) medium [20] supplemented with 0.7% (w/v) agar and 3% sucrose. The cultures were incubated at 25°C±2 under cool white fluorescent light and 16 hr. photoperiod. Shoot tips of 1.5 cm length excised from 21-25 days – old in vitro produced seedling was used in this experiment.. 3.10.1.4 In vitro screening for growth under salinity stress Shoot tips explants excised from 21-25 old seedlings of each variety were cultured in the regeneration medium. The regeneration medium consisted of MS medium supplemented with 4.0 mgl-1 Kin, 7gl-1 agar, 30gl-1 sucrose and different concentrations of NaCl (0.0, 50, 75 and 100 mM).

Culture condition and data analysis All culture media were adjusted to pH 5.8 and incubated in a growth room at 25±2ºC under a photoperiod of 16 h light provided by cool white fluorescent lamps. Six replicates were used and each treatment consisted of 15 explants cultured in completely randomized design (CRD). After six weeks, the following parameters were recorded shoot length (SL), root length (RL), shoot fresh weight (SFW), root fresh weight (RFW) and number of rooted shoots (RS). Result was statistically analyzed and LSD was used for mean separation according to Steel and Torrie [21].

Results Twelve tomatoes varieties namely; Omdurman, Alla karem, Strain B., Jazera, Majd, Super majd, Madona, Amani, Castel Rock, Rio grand, Sudan special and Peto-86 were used as sources of explant in the present study. The result showed that shoot and root growth in these tomato cultivars were decreased with increasing of salinity concentration in the growth media. The highest mean shoot length (8.0 cm) was obtained by the cultivars Omdurman and Castel Rock, while Majd cultivar had the shortest one (Table 1). The mean shoot length was reduced from 7cm in the controls to 4 cm in 100 mM NaCl. Shoot length decreased significantly with the increase in NaCl concentration in the growth media. However there was no significant difference between the means shoot length of tomato plant grown in NaCl free media and those grow in 50 mM NaCl. Moreover some cultivars such as Strain B. and Amani have longer shoot length in 50 mM NaCl media than in the control. Peto-86 cultivar shoots length increased with the increased

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in NaCl concentration in the media up to 75 mM, after which decline in shoot length was observed (Table 1). On the other hand, The highest mean shoots fresh weight for cultivars was obtained for Castel Rock (1.09 g) (Table 2), followed by Allakarim and Amani (0.91g) and the difference between Castel Rock and the other cultivars was significant. . The main effect of salinity on shoot fresh weight on tomato cultivars was similar to shoot length. The highest mean shoot fresh weight was observed at 50 mM NaCl however, it was significant over the control. As for roots shoot tip of Stain B. had the highest significant number of roots (14.11) followed by Omdurman, while Majd and Supermajd had the lowest number of roots (2.7 and 2.6) respectively, (Table 3). Mean root number decreased markedly from 13.9 in the control to 0.89 at 100 mM NaCl,. The differences in root number between the control and 50 mM NaCl was not significant. At the highest salinity level (100 mM NaCl) most cultivars failed to initiated rooting except Amani, Sudan Special and Peto-86, however, Peto-86 cultivar had produced the highest root length (7.5 cm), followed by Amani (6.7 cm), while variety Majd produced the shortest roots (Table 4). Shoot tips of all cultivars on NaCl–free medium had the longest root (6.9 cm), Root length in all cultivars generally decreased significantly with the increased in NaCl concentration in the media. The mean root fresh weight generally in all cultivars decreased with the increase in NaCl level in the medium (Table 5). Strain B., Amani and Castelrock showed the highest significant root fresh weight over all other cultivars tested (0.33, 0.28, 0.27 g) respectively. Mean root fresh weight decreased significantly from 0.33 g in the control to 0.04 g at 100 mM NaCl.

Table 1: Effect of NaCl on shoot length (cm) of tomato genotypes after 6 weeks of in vitro culture on MS medium supplemented with 4.0 mgl-1 Ki genotype 0.0 mM 50 mM 75 mM 100 mM means Omdurman 9.8 9.3 8.2 4.6 8.0 a Strain B. 6.4 7.6 6.6 4.4 6.3 cd Jazera 5.3 6.6 5.1 4.3 5.3 def Castel Rock 11.4 10.2 4.6 5.6 8.0 a Rio grand 5.4 5.1 4.7 4.6 4.9efg Madona 7.1 4.2 3.9 5.3 5.1 ef Majd 5.5 4.3 3.8 2.4 4.0 g Supermajd 6.3 5.7 5.6 5.5 5.8 cde Amani 7.2 10.0 6.8 6.8 7.7 ab Allakarim 9.1 8.2 6.5 2.6 6.6 c Sudan Special 7.1 7.4 6.3 6.1 6.7bc Peto-86 3.9 4.6 5.2 4.1 4.5fg Mean 7.0 a 6.96 a 5.6 b 4.7 c *Means with the same letter are not significantly different.

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Table 2: Effect of NaCl on shoot fresh weight (g) of tomato genotypes after 6 weeks of in vitro culture on MS medium supplemented with 4.0 mgl-1 Kin. genotype 0.0 mM 50 mM 75 mM 100 mM Means Omdurman 0.7 0.7 0.6 0.52 0.65 d Strain B. 1.1 0.97 0.8 0.27 0.79 bc Jazera 0.9 1.1 0.5 0.29 0.71 cd Castel Rock 1.7 1.6 0.5 0.51 1.09 a Rio grand 0.3 0.3 0.4 0.38 0.34 e Madona 0.4 0.3 0.4 0.48 0.43 e Majd 0.5 0.5 0.3 0.2 0.37 e Supermajd 0.6 0.4 0.4 0.46 0.45 e Amani 0.6 1.2 0.8 1.13 0.91 b Allakarim 1.1 1.05 1.1 0.33 0.91 b Sudan Special 0.5 0.5 0.5 0.49 0.47 e Peto-86 0.2 0.4 0.7 0.28 0.39 e Mean 0.73 a 0.76 a 0.57 b 0.45 c *Means with the same letter are not significantly different.

Table 3: Effect of NaCl on number of root of tomato genotypes after 6 weeks of in vitro culture on MS medium supplemented with 4.0 mgl-1 Kin. genotype 0.0 mM 50 mM 75 mM 100 mM Means Omdurman 22.21 18.86 10.36 0.01 12.86 a Strain B. 22.36 20.5 13.57 0.01 14.11 a Jazera 16.14 15.86 8.5 0.01 10.13 cd Castel Rock 19.5 18.21 2.02 0.01 9.95 cd Rio gran 14.46 10 5.0 0.01 7.4 e Madona 15.64 14.14 4.07 0.01 8.47 de Majd 4.86 4.71 0.86 0.01 2.6 g Super majd 5.8 2.0 3.0 0.01 2.7 g Amani 14.14 13.57 10.7 5.14 10.89 bc Allakarim 21 17.5 11 0.01 12.3 bc Sudan Special 5.43 4.7 4.5 3.9 4.6 f Peto-86 4.8 19.07 11.3 1.5 9.2 cde Mean 13.9 a 13.3 a 7.08 b 0.89c *Means with the same letter are not significantly different

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Table 4: Effect of NaCl on root length (cm) of tomato genotypes after 6 weeks of in vitro culture on MS medium supplemented with 4.0 mgl-1 Kin. genotype Omdurman Strain B. Jazera Castel Rock Rio gran Madona Majd Supermajd Amani Allakarim Sudan Special Peto-86 Mean

0.0 mM 8.2 8.1 3.9 5.5 8.4 8.4 4.5 4.1 7.3 5.4 7.4 11.6 6.9 a

50 mM 4.7 6.1 7.4 8.3 9.5 9.5 2.5 3.1 8.7 4.6 5.6 11.5 6.7 b

75 mM 0.8 2.9 4.0 1.1 0.4 0.4 0.01 2.9 5.6 2.5 4.9 6 2.6 c

100 mM 0.01 0.01 0.01 1.57 0.01 0.01 0.01 0.01 5.2 0.01 3.21 1 0.9 d

Means 3.4 efg 4.2 cde 3.8 def 4.1 def 4.5 b 4.5 b 1.7 h 2.5 gh 6.7 a 3.1 fg 5.3 bc 7.5 a

*Means with the same letter are not significantly different.

Table 5: Effect of NaCl on root fresh weight (g) of tomato genotypes after 6 weeks of in vitro culture on MS medium supplemented with 4.0 mgl-1 Kin. genotype 0.0 mM 50 mM 75 mM 100 mM Means Omdurman 0.45 0.06 0.025 0.01 0.14 c Strain B. 0.6 0.41 0.19 0.07 0.33 a Jazera 0.15 0.25 0.01 0.01 0.11 cd Castel Rock 0.43 0.43 0.2 0.04 0.27 a Rio grand 0.32 0.21 0.01 0.01 0.14 c Madona 0.31 0.05 0.15 0.01 0.13 c Majd 0.26 0.09 0.11 0.01 0.12 c Supermajd 0.15 0.04 0.03 0.01 0.06 d Amani 0.31 0.5 0.23 0.1 0.28 a Allakarim 0.49 0.2 0.1 0.01 0.21 b Sudan Special 0.32 0.3 0.13 0.09 0.21 b Peto-86 0.09 0.4 0.2 0.1 0.20 b Mean 0.33 a 0.24 b 0.12 c 0.04 d *Means with the same letter are not significantly different.

Discussion In vitro plant tissue culture is useful and quick tool to evaluate plant tolerance to salt

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stress. Many studies were carried out through using different tissue culture methods [22]. The result shows that shoot and root growth in these tomato cultivars were decreased with increasing NaCl concentration in the growth media in general agreement with Naseem et al., [23]. Reduction in growth with increasing salinity in growth media may be attributed to water deficit or ion toxicity associated with excessive ion uptake particularly of Na+ and Cl- [24]. Nutrients imbalance as a result of depressed uptake, shoot transport and impaired internal distribution of minerals especially K+ and Ca+2 may also explained the reduction in plant growth [25]. At 50 mM NaCl in the growth media shoot growth was not significantly affected by the presence of salt in the growth media. However, root length and root dry weight were significantly decreased with increase in NaCl concentration. Root growth was more adversely affected by increasing NaCl concentration in the growth media than shoot growth [26, 27, 28]. Root growth is suggested by Cano, et al. [17] as better characteristic for evaluating salt tolerance of tomato species through in vitro shoot apex culture than shoot growth Moreover, Cano et al. [17] suggest that shoot apex culture may be useful for rapid evaluation and screening of tomato segregate populations in a breeding program. However, Chandler et al. [14] concluded that at least for the species studied, shoot-apex culture was not suitable for selecting for salt tolerance. Amani gave best performance at high level of NaCl and good characteristics in root and shoot length and root fresh weight. Also Castel Rock gave the bet result for shoot length, shoot fresh weight and root fresh weight followed by Omdurman and strain B. giving the best shoot length, number of root and root fresh weight. Cultivated tomato is generally classified as being moderately salt-sensitive. Different genotypes of tomato displayed widely different degrees of salinity tolerance [29, 30, 13]. These result were in agreement with the previous findings concerning the physiological responses of tomato cultures to salt treatments. Marked differences in the behavior of both susceptible and tolerant tomato genotypes were evident [32, 33, 34, 35, 11, 36]. Yet, an understanding of the mechanisms that plants use to cope with high salinity is necessary to select and develop tomato plants that are more tolerant to salinity. Rus et al. [36] also found that adaptation capacity to salinity varies with the genotype’s degree of tolerance. Pérez-Alfocea et al. [37] detected different salt stress responses among several tomato cultivars, from an halophytic behavior,

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