In-vitro Screening of Tomato Genotypes for Drought Tolerance

A study was carried out to assess the drought tolerance ability of 32 tomato genotypes using PEG - 6000 induced moisture stress, on the basis of seedling parameters such as germination percentage, root length, shoot length, seedling dry weight and vigour index. In vitro screening revealed that seedling characters were reduced significantly at -0.2 MPa compared to control. Among the genotypes LE 18, LE 57, LE 27, LE 13 and LE 118 performed better under stress in terms of germination percentage, root length, shoot length, seedling dry weight and vigour index. Many of the genotypes did not germinate at -0.35 MPa. Varieties like CO 3, PKM 1, TNAU TH CO 3 and COTH 2 showed poor performance in terms of germination percentage, root and shoot length. This result can be used for pot and field level screening and manipulations in tomato cultivars for improvement of drought tolerance.

Vegetable production under drought is essential to cope up with increasing demand for vegetable crops considering increasing world population. Water is a major constraint in tomato production under rainfed condition. It is one of the important environmental factors that influence germination and plant growth. Reduced water potential in the environment can either delay or reduce the germination rate of many plant species because it interferes with the imbibition process and cell lengthening within the embryo axis (Schuab et al., 2007).
Tomato (Solanum lycopersicon) is one of the most important vegetable crops grown in India. The estimated area and production of tomato for India are about 6.34 lakh hectares and 124 lakh tons respectively and the country is the sixth largest tomato producer in the world. The average productivity of tomato in India is merely 158 q/ha while its productivity in USA is 588 q/ha. This low yield may be either due to lack of high yielding varieties, poor management practices, and abiotic and biotic stresses. High yielding variety is an important factor for maximizing the yield of tomato and to fulfill the increasing demand for this nutritionally important vegetable, tomato cultivation under drought is also advocated.
Germination of seeds under water deficit should be assessed in the laboratory under controlled temperature and humidity conditions, because, according to Lopes and Takaki (1988) the sensitivity of seeds to water stress can be influenced by different factors such as light, temperature, humidity and oxygen availability. Low water potential in the substrate can be obtained using water solutions containing saccharine, sodium chloride, manitol and *Corresponding author email :sivatnau5@gmail.com polyethylene glycol (Santos et al., 1996). However, the product used as an osmotic agent should not be absorbed by the seeds nor metabolized by the seedlings (Slavik, 1974). Polyethylene glycol 6000 can be used without restrictions as an osmotic agent (Hasegawa et al.,1984), because it does not penetrate the cells, cannot be degraded and does not cause toxicity due to its high molecular weight. Polyethylene glycol (PEG) of high molecular weight has been long used to stimulate drought stress in plants as non-penetrating osmotic agents lowering the water potential in a way similar to soil drying (Larher et al., 1993). The use of high molecular weight osmotic substances, like polyethylene glycol is one of the most popular approaches for drought induction (Landjeva et al., 2008).
Drought is among the most detrimental environmental factors limiting plant productivity through osmotic stress. Drought creates elevated osmotic pressure in the root zone and decreases the water potential, limiting plant growth. Osmotic stress leads to decrease in plant water potential, which in turn has a negative effect on water availability and almost all plant functions (Yamaguchi-Shinozaki et al., 2002). Performance of seed germination, crop growth and yield are the result of genotypic expression as modulated by continuous interaction with environment. Among the various traits which help to assess drought tolerance, seed germination percentage and root traits are more reliable on account of their high correlation with drought tolerance mechanism (Chang et al., 1982) and considerable genotypic variations for root traits existing in tomato. Hence, understanding the changes that take place in tomato roots under water deficit would help in developing cultivars better suited to the drought situation. The present study was undertaken to assess the effect of PEG-6000 induced short term moisture stress on drought tolerance of genotypes on the basis of changes in some important seedling parameters.
-(1.18 X 10-2) C -(1.18 X 10-4) C2 + (2.67 X 10-4) + (8.39 X 10-7) C2T where C = Weight of PEG (g l-1), T = Room Temperature (°C) Based on above formula, 123 and 169 g of PEG -6000 were dissolved in 1000 ml of distilled water to develop different osmoticum solutions having water potential of -0.2 and -0.35 MPa. Distilled water was used as control. Surface sterilized seeds were placed on moistened filter paper in each petridish separately. Filter paper was moistened in regular interval with respective solutions for all observations. The petridishes were kept in laboratory under room temperature. The germination percentage was recorded at every 24 h interval up to 15 days. Seeds were considered germinated when the radical was at least 2 mm long. Five seedlings were chosen randomly and seedling growth was measured by dry weight of root and shoot of the seedling. Dry weight was determined after kept in hot air oven at 80°C for 48 hrs. The length of root and shoot was measured with a ruler. The vigour index was calculated by the procedure described by Abdul Baki and Anderson (1973) as vigour index = germination percentage X (Root length + Shoot length). The data were analyzed statistically and the treatment means were compared using LSD at 5 % probability (Panse and Sukhatme, 1985).

Effect of PEG on germination percentage
The cumulative germination of all the genotypes significantly decreased with increasing intensity of water deficit levels ( Table 1). Most of the genotypes had 100% seed germination except LE 20, LE 3, LE 85 and LE 100 under control condition. Among the level of stress, control recorded the highest germination percentage than other two stresses (-0.2 and -0.35 MPa). The progressive fall in the germination percentage with decreasing water potential of the environment, observed in this experiment, was caused probably by the low hydraulic conductivity of the environment, where PEG 6000 makes water unavailable to seeds, affecting the imbibition process of the seed, which is fundamental for germination (Lobato et al., 2008). Among the different genotypes, LE 18 recorded the highest germination percentage (100, 96.7 and 60.0) followed by LE 27 (100.00, 93.30 and 36.70) and LE 57 (100.00, 90.00 and 56.7 at control, -0.2 and -0.35 MPa respectively) (Fig 1).
Water stress due to drought is probably the most significant abiotic factor limiting germination and also crop growth and development (Hartmann et al., 2005). Drought stress is physiologically related, because it induces osmotic stress and affects most of the metabolic responses of plants (Djibril et al., 2005). Water deficit affects the germination of seed and the growth of seedlings negatively (Van Den Berg and Zeng, 2006).
The addition of PEG decreased the water potential and induces water stress that adversely affects the callus growth and in vitro regeneration capacity of tomato cultivars (Gopal and Iwama, 2007) . This largest reduction with PEG solution could be attributed to high viscosity, where solubility and diffusion of oxygen were reduced compared to water (Delachiave and De Pinho, 2003). The reason for decreasing germination with increasing level of stress may be due to water potential and osmotic potential as mediated by solute developed additive effect on the inhibition of seed germination (Bernstein, 1961). Water deficit conditions induced with PEG, decreased germination percentage and also delayed germination time at higher concentrations. This was due to the lower water uptake by seed resulting in decreased germination under increased concentration of PEG. Therefore, the decrease in water potential gradient between seeds and their surrounding media by the effects of PEG 6000 adversely affects seed germination (Digdem Kaydan and Mehmet Yagmur, 2008). The results showed that germination of most of the genotypes arrested completely at -0.35 MPa and genotypes varied significantly in their germination capability under reduced water potential.

Effect of PEG on root and shoot length
Increasing moisture deficit resulted in the reduction of root and shoot length in all the genotypes (Table 1). Among the different levels, control recorded the highest root length and shoot length than other two stresses (-0.2 and -0.35 MPa). The genotype, LE 18 recorded maximum root length (12.20, 8.70 and 4.30 cm) followed by LE 57 (10.50, 7.50 and 4.00 cm) LE 27 (8.80, 6.40 and 3.50 cm) at control, -0.2 and -0.35 MPa respectively. The highest shoot length was recorded in LE 18 (7.70, 5.50 and 4.20 cm) followed by LE 15 (7.60, 5.30 and 3.80 cm) and LE 27 (7.40, 5.00 and 3.40). The highest reduction in root length was observed in LE 6 (79.17 %) followed by LE 5 (71.67%) and LE 2 (69.86 %) at -0.2 MPa. The lowest reduction in shoot length was observed in LE 118 (17.65 %), LE 27 (20.37 %) and LE 18 (21.54 %) at -0.2 MPa (Fig 2).
In general, plants with longer root growth have drought resistance (Leishman and Westoby, 1994). In the present study, decreases in the external osmotic potential caused a reduction in seedling growth of tomato. The increasing concentration of PEG induced water deficit leading to decrease in root and shoot length was observed by Al-Karaki (1998). The percentage reduction in root length was higher than shoot length and this might be due to the direct contact of root with PEG solution.

Effect of PEG on root and shoot dry weight
Root dry weight and shoot dry weight significantly decreased with increasing intensity of water deficit ( Table 2). The maximum root and shoot dry weight was observed in LE 18 (10.01, 7.50 and 4.5 mg for root dry weight and 22.17, 16.60 and 4.9 mg for shoot dry weight) followed by LE 57 (9.95, 7.45 and 4.4 mg root dry weight and 22.05, 16.49 and 9.7 mg shoot dry weight) and LE 27 (9.81, 7.47 and 3.9 mg root dry weight and 21.73, 16.56 and 9.6 mg shoot dry weight) at control, -0.2 MPa and -0.35 MPa respectively. Therefore, it may be concluded that genotypes which have good seed germination and seedling growth (root/shoot length and its dry weight) under higher moisture deficit condition are superior to ensure good seedling establishment and further crop growth. The result of this study also showed that the genotypes display distinct response to drought stress. In this regard genotypic variability within species offer variable tool for studying mechanism of drought. Genotypic variations in germination and seedling growth under lower water potential were critical for quick establishment ability under water stress condition (Redona and Mackill, 1996). Seedling growth in terms of seedling dry weight, shoot and root length was also completely inhibited in most of the genotypes at -0.35 MPa. The germination percentage, shoot and root length and seedling dry weight under reduced water potential indicating their ability to tolerate drought. This was in confirmation with study of Goswami and Baruah (1994) and Jha and Singh (1997).

Effect of PEG on vigour index
Vigour index is an important parameter to assess drought tolerant capacity of crop plants. Vigour index is a combination of germination percentage with root and shoot length. PEG induced water deficit decreases the vigour index and thus might be due to decreased germination percentage and reduction of root and shoot length. Among the genotypes, the highest vigour index was recorded in LE 18 (1373.14) followed by LE 57 (1152.00) and  Table 2). The reduction in the water entry speed in the cells during the seed imbibition process (Peske and Delouche, 1985) causes reduced vigour index. The disarrangements in the cell membranes that favor faster water absorption and solute loss, can result in tissue death (Powell and Matthews, 1979).

Conclusion
Based on the present investigation, 32 tomato genotypes are grouped into three categories viz., tolerant, moderately tolerant and susceptible to the drought condition. Among the genotypes, LE 18, LE 57, LE 27, LE 14 and LE 13 are characterized as tolerant and LE 3, LE 20, LE 85, LE 100 and PKM 1 are susceptible to drought. The identified genotypes shall be taken into the field for confirmation through the physiological and biochemical analysis and including molecular approaches.