In Vitro Germination and Early Vegetative Growth of Five Tomato ( Solanum lycopersicum L.) Varieties under Salt Stress Conditions

In Senegal, tomato (Solanum lycopersicum L.) cultivation is affected by salinity in many agro-ecological zones. The selection of salt tolerant varieties would be an alternative solution to enhance the production. Thus, germination and growth are studied under axenic conditions for five varieties of tomato subjected to increasing concentrations of NaCl [0, 35, 70 and 105 mM], and supplemented in an MS/2 medium for 30 days. The results reveal that salt negatively affects the evaluated parameters. The Rodeo and Lady Nema varieties have the lowest final germination rates (50%) unlike the Mongal variety (55%). These last two varieties have a decrease of 71.78% and 81.28% in the height of the stem, respectively, in the presence of NaCl at [105


Introduction
Tomato (Solanum lycopersicum L.) is one of the most important vegetable crops in the world particularly in Senegal. Globally, production and cultivated areas are constantly increasing [1], i.e. a respective increase of 2.55% and 5.08% from 1960 to the present is mentioned [2]. Despite the possibility of cultivating this species on a large scale, tomato yields in Senegal (20.05 t/ha) do not yet reach the values recorded in other countries such as the United States (96.8 t/ha), South Africa (75.5 t/ha) and China (59.4 t/ha) [2]. These insufficient yields are linked to the salinization of agricultural lands, which is one of the major constraints considerably limiting production in the world and in the Sahel in particular [3].
According to [4], 800 Mha of arable lands worldwide is affected by salinity. Indeed, among environmental stresses, salt stress is one of the main abiotic factors leading to an increase in the loss of arable lands and a drastic reduction in agricultural productivity [5]. It is, therefore, an important obstacle to food security and a major cause of land degradation, particularly, in arid and semi-arid regions [6] [7]. Added to this, the consequences of climate change are becoming more and more restrictive for the growth and development of plants, especially in semi-arid and arid areas [8]. Under these conditions of abiotic stress, the physiology of plants is disrupted [9] [10], including in tomatoes [11].
The expression of plants in response to salinity results in both water stress due to the osmotic effects induced by salt, and chemical stress, mainly due to the toxic effects of sodium. Thus, salt stress exerts at the same time osmotic, ionic and nutritional imbalances in plant species, including tomato [3]. According to [12], salinity hinders plant growth in several ways: 1) it increases the osmotic potential of the soil, making it difficult for the plant root system to access water, which provokes osmotic stress [13] [14]; 2) salinity can be associated with high concentrations of ions inside cells, which causes an inhibitory effect on plant metabolism qualified as ionic stress [15] [16]. The altered status of water leads to the reduction of initial growth and limitation of plant productivity. In most plants, growth decreases with increasing salt concentrations in the medium [17], but in others, growth is stimulated by moderate salt concentrations [18]. Salinity is an abiotic constraint that negatively influences almost all stages of the life cycle of tomato plant, leading to a reduction in its yield [19] [20]. This constraint affects the morphological parameters of the tomato as well as the relative water content of the leaves, the photosynthetic pigments, the leaves gas exchange parameters, the fluorescence of chlorophyll and the absorption of essential macronutrients [21]. According to [4], to cope with salinity, the plant implements two types of responses corresponding to the two types of stress generated by saline stress, i.e. osmotic stress and ionic stress: a rapid response resulting from an increase in external osmotic pressure [21] [22] and a slow response due to exclusion of Na + ions from the cell [23] or their compartmentalization in cells of certain organs [15] respectively.
In Senegal, land salinization particularly affects coastal areas [24] and lowlands [25]. Consequently, of the 3,800,000 ha of cultivable lands, approximately 1,700,000 ha are affected by salinization [26]. According to [27], one third of irrigated lands are affected by salinization. The salinization of senegalese lands results mainly from the different climatic phases which followed one another in the Quaternary and which provoked the invasion of the continent by marine waters [28]. In addition, there is the effect of climate changes, in particular the rainfall deficit of the 1970s and the rise in sea level (marine intrusion, floods, etc.), which have contributed to accentuating the effects of salinity downstream of the main watersheds of rivers or inlets of Senegal rivers, i.e. Senegal, Sine, Saloum and Casamance [28] [29].
Under the pressure of strong demographic growth, Senegal must imperatively find ways to increase agricultural yields in order to achieve food self-sufficiency.
Hence, it would be strategic to be able to use varieties that are more tolerant to salinity. In this context, we undertook this work, which consists of studying the in vitro behavior of five tomato varieties subjected to increasing levels of salinity (NaCl), in order to assess their behavior or even sensitivity to this abiotic stress during the germination process and the early steps of the vegetative growth.

Plant Material
The plant material consists of seeds of five F1 hybrid tomato varieties (Solanum lycopersicum L.) supplied by the company Tropica Sem-Senegal (Technisem Novalliance Group): Ganila, Lady Nema, Mongal, Rodeo and Xewel. Their characteristics are summarized in Table 1. They were harvested and bagged in 2019,

Culture Conditions
The basic culture medium used was that of [30], the macro-elements of which were diluted by half (MS/2). To establish the salt stress, sodium chloride (NaCl) was incorporated into the culture media at final concentrations of 0, 35, 70 and 105 mM for each treatment. The pH of the culture media was adjusted to 5.7 before solidification with agar at 9 g•L −1 . The culture media corresponding to the treatments described above were distributed in culture tubes (25 × 150 mm) which were filled up with 20 mL per tube before being sterilized by autoclaving at 110˚C for 20 minutes.

Seed Disinfection and Germination Screening
The seeds of each hybrid variety were surface-disinfected with 70% alcohol, followed by soaking in a stirred batch of bleach (NaOCl) at 8˚ chlorometric sodium hypochlorite for 10 minutes.

Agro-Morphological Parameters of Growth and Biomass Determination
The trials for the in vitro growth of tomato seedlings were conducted for 30 days. After 1 month of culture, the seedlings were removed from each tube, thoroughly washed in a sterile deionized water, to remove the agarified medium from the root systems, surface-wiped with blotting paper. The following agro-morphological parameters were determined: the height of the stem, the number of leaves, the length of the taproot, the number of secondary roots and the fresh weight of the aerial and root parts. They were evaluated using a ruler for the measurements and a Sartorius precision scale (accuracy: 0.0001) for fresh

Statistical Analysis
The experiment was set up as a standard randomized design, with salt concentration chosen as a main factor variable and tomato variety as the subfactor va-

Effects of NaCl on in Vitro Germination of Five Tomato Seed Varieties
The different concentrations of NaCl (   and 96% (Rodeo).

Influence of Saline Stresses on the Average Number of Leaves
The increasing concentrations of NaCl provoked a significant decrease (P = 7.36

Impact of Salinity on the Length of the Aerial and Root Parts
The different NaCl concentrations have a significant effect (P < 2 × 10 −16 and F = 34) on the length of the aerial part (LAP) of the different varieties (  The length of the aerial part is greater than that of the root part for all varieties regardless of the applied saline stress. The importance of the development of the aerial part to the detriment of the root part is more marked in the Rodeo variety where the length of the aerial part almost triples that of the root part at different NaCl concentrations. For the other varieties, the length of the aerial part is twice or slightly longer than that of the root part at [NaCl 35 mM]. However, with the increase of salt concentrations, this difference decreases sharply to less than 17%.
This difference is less marked in the Mongal variety, with less than 1% at [NaCl 70 mM] and less than 4% at [NaCl 105 mM].

In Vitro Seed Germination
This study showed that a saline constraint has a significant and depressive effect (p < 2 × 10 −16 ) on the final germination rate of the different tomato varieties studied, alike the research carried out by [34]. Germination is the first physiological stage affected by salinity, it is thus an essential phase for early identification of varieties tolerant or not to salinity. Indeed, the germination process is very critical for plant establishment and growth specifically in the presence of adverse environmental constraints [35] [41]. The decrease in the germination percentage is due either to an increase in the external osmotic pressure [42] which affects the water absorption by the seeds [43], or to an accumulation of Na + and Clions in the cells of the embryo. The difficulty of water absorption affects the elongation of the radicle but germination can take place with low water potential thanks to certain growth regulators [44]. The seeds did not have time to put in place mechanisms allowing them to tolerate the presence of salt and thus absorb the optimal quantity of water and manage the excess of Na + and Clions.
At low concentration of NaCl, such as 35 mM, seeds of all tomato varieties germinate: a slight decrease in the final germination rate is observed globally, but the lowest rate reached 75% for the Rodeo and Xewel varieties. For a concentration of 70 mM of NaCl, the final germination rates are reduced by half.
They reached 50% for Lady Nema and Rodeo varieties. In addition to reducing the germination rate for sensitive cultivars, salt stress also delays germination and slows its speed. The decrease observed may be due to the alteration of enzymes and hormones contained in the seeds [45] or to a problem of seed hydration due to a high osmotic potential, which inhibits the emergence of the radicle off husks [46]. It would have been beneficial to introduce the increasing concentrations of salt gradually into the culture media to prevent rapid osmotic stress and ionic toxicity of the NaCl as recommended by [47].

Effects of NaCl on in Vitro Early Vegetative Growth of Vitroplants
In terms of growth and development parameters, the response of vitroplants to salt stress of the five tomato varieties varies depending on the parameter. According to the results obtained in this study, salt stress causes a delay in plant growth. The salt stress induced a reduction of more than 70% in the size of the young plants. This decrease is more marked in the Xewel variety. The authors [48] obtained a reduction in plant height of 26% in the presence of [NaCl 70 mM]. The reduction in plant growth can be explained by the fact that NaCl causes an increase in the osmotic pressure of the culture medium which prevents the absorption of water by the root system [49]. The reference [50] showed that concentrations [51]. Some varieties used in this study appeared to be sensitive to salt stress, with a reduction of more than 60% in the number of their leaves. However, [48] reported an 11% reduction in the number of leaves in tomato in the presence of [NaCl 70 mM]. In agreement with these results, previous studies had shown that tomato plants affected by salinity tend to reduce their number of leaves [52]. This reduction can be as much as 10%, which reduces photosynthesis [53]. The authors [54] explained this reduction in the number of leaves by a specific harmfulness of the Cl, ions accumulated at levels exceeding the compartmentalization capacity. According to [41], vegetative growth and, particularly leaf expansion, is severely inhibited by salt stress. The new leaves develop slowly and the senescence of the old ones accelerates. On the other hand, the toxicity of Na + ions on plants, in particular at the leaf scale can result in a significant decrease in the growth rate but also in the leaf area and the leaf elongation rate of seedlings, especially in grasses (wheat, rice, maize, etc.) and Solanaceae [55]. The reduction in growth rate is not linked specifically to the addition of NaCl but to a transient change in the plant-water relationship.
This study also revealed a decrease in the number of secondary roots. Similarly, [56] also observed a clear reduction in root volume under saline stress. These different observations are also cited by different authors as being one of the causes of the reduction in growth and vegetative productivity [57] [58], which is in accordance with our study. Thus, the presence of NaCl in the culture media causes a reduction in the fresh and dry aerial and root weights. In this experiment, the weight of the aerial parts is greater than that of the root parts; the plants favor the development of the leaves and stems to the detriment of the roots [49] [59].
The early events of plant adaptation to stress begin with the mechanisms of perception followed by signaling through the transduction of signals and messengers to activate various physiological and metabolic responses, including expression of stress response genes. The main cellular reactions developed by the plant in order to face and adapt to salt stress are inevitably preceded by a cascade of signaling and regulatory elements, which can take different pathways involving in particular that of calcium, abscisic acid (ABA), Mitogen-Activated Protein Kinases (MAPKinases), Salt Overly Sensitive" proteins (SOS) and ethylene [60].
A salt constraint provokes a depressive and significant effect on the agromorphological parameters studied. Indeed, seedlings under saline stress preferentially accumulate Na + (and Cl − ) ions in the aerial parts of rice leaves, in particular in the leaf apoplast [61]. According to their results, salt stress causes a delay in plant growth. In fact, all the varieties are affected by salt with a significant reduction of more than 70% in the length of the aerial part while the reduction in the length of the root part is more than 50%. Salt affects aerial growth more than root growth in all varieties except for the Rodeo variety in which . The reduction in the growth of vitroplants can be explained by the fact that NaCl causes an increase in the osmotic pressure of the medium, which prevents the absorption of water by the root system [49]. The authors [50] showed that salinity affects the growth of the plant at different stages of its development cycle by increasing the osmotic pressure of the soil solution, thus promoting the accumulation of certain ions in toxic concentrations in plant tissue and altering its mineral nutrition. Thus, the roots being in contact with high concentrations of NaCl lose water for the benefit of the culture medium. This leads to a decrease in volume, elongation and cell divisions. Besides, the osmotic effects of salt stress can also limit root growth and subsequently the possibilities of absorption of nutrients from the soil [15]. Indeed, the Na + and Cl − ions can interfere with transporters located on the plasma membrane of the roots, such as for example, the selective channels of K + [62].
Reduced growth may also result from increased abscisic acid concentration in the aerial part or reduced cytokinin concentrations [51]. Osmotic stress triggers the production of ABA in the roots, which is then transported to the leaves, causing the stomata to close. However, [63] and [64] explain this by a combination of the osmotic effect and the specific effect of Na + and Cl − ions. The accumulation of Na + ions in the plant limits the absorption of essential cations such as Ca 2+ and K + [16]. The K + ion, for example, is involved and required in the activation of more than 50 enzymes while Na + ions cannot provide the same function at the level of cellular metabolism. A nutritional imbalance and a reduction in plant growth occur when these essential ions (Ca 2+ and K + ) become limiting [16] [65]. A competition occurs between the Na + -K + ions for their binding to enzymes and important proteins [66]. In addition, the K + ion is vital for the synthesis of certain proteins (inhibited by the presence of Na + , the osmoregulation of plant cells, the maintenance of cell turgor, the stimulation of photosynthesis, the binding of tRNA to ribosomes (Translation affected), etc.

Biomass Determination
Plants are not equal when it comes to salt stress. Some are sensitive while others are tolerant to salinity. This determines their biomass production in the presence of salt and governs their classification into four categories: True halophytes (Salicornea sp., the mangrove, Spartina sp., Cakile maritima), Facultative halophytes (Plantago maritima), Resistant non halophytes (Hordeum sp.), Glycophytes or halophobes (Phaseolus vulgaris, Glycine max, Solanum sp.) [67]. In all plant species, both halophytes and glycophytes, the salinity of the environment leads, above a certain threshold, to a reduction in the produced biomass. However, the degree of inhibition of growth depends on the genus, species, variety as  [62]. However, the depressive effect of NaCl at high concentration is in general more marked in the aerial organs than in the root organs. This difference in sensitivity between absorption organs and photosynthetic organs is described as characteristic of glycophytes [69]. Indeed, the inhibition of root growth is generally less marked than that of the aerial parts for which an accumulation of Na + and Cl − ions can inhibit growth and become toxic to the plant [70]. This would be a strategy developed by the plant to accumulate resources and energy that allow it to fight against salt stress [71].
Salinity tolerance in plants has several aspects: detoxification, regulation and restoration of homeostasis, and growth control. Among the mechanisms of plant salt tolerance is the exclusion and compartmentalization of Na + . This is the most effective strategy for avoiding the toxicity of Na + at metabolic sites in the cytoplasm. Thus, in barley, tomato and tulip, it has been shown that Na + /H + activity at the root level increases in response to the presence of Na + ions [72] [73] [74]. In addition, overexpression of the NHX1 gene promoter improves tolerance to salinity in Arabidopsis [75], tomato [76] and rice [77].

Conclusion
The reaction of plants to salt stress is through adaptive, morphological, anatom- • a first group formed by the vigorous and fairly tolerant Lady Nema and Mongal varieties; • a second group made up of varieties Ganila and Xewel, slightly less vigorous, and therefore moderately sensitive; • and, a third group composed by the Rodeo variety, which is not very vigorous and is sensitive to salinity.
Finally, these results will be of an important contribution to a better management of the cultivation of tomato varieties in semi-arid or arid zones where the quality of agro-ecological soils becomes unfavorable to this agronomic speculation because of the natural or the water irrigation-related salinization of lands.