Effect of Drought on Seed Germination and Early Seedling of Tomato Genotypes using Polyethylene Glycol 6000

Tomato is a sensitive crop to a variety of environmental stresses, especially drought. Therefore, the objective of the present work was to screen for drought tolerance at seed and seedling (vegetative) levels for better understanding of drought mechanisms and identification and selection of the most tolerant tomato genotypes. Twenty four (24) tomato genotypes were screened for drought tolerance using 0%, 4% and 14% polyethylene glycol 6000 (PEG 6000). The experiment was laid in complete randomized design with three replication and three treatments. The following parameters: germination percentage, shoot length, root length, shoot and root weight and recovery date were recorded in the course of the experiment. There was no significant difference in germination percentage between control and low PEG. However, significant differences were observed between control and low concentration of PEG when compared to high PEG concentration. Water stress created by PEG 6000 at high concentration (14%) significantly reduced all the parameters measured in all the genotypes. Overall, NGB01357, L00170 and NHGB/09/113 performed better under drought conditions and could be very vital in breeding program.


Introduction
Tomato (Solanum lycopersicum L.) is an important fruit crop grown worldwide with a total world production of 159 million tons and a value of $74.1 billion on a cultivated area estimated at 5 million ha (FAO, 2011). It is also one of the most economically important crops and worth a tremendous amount of money because it gives more yields (FAOSTAST, 2013). However, the yield of tomato in West Africa particularly Nigeria is not too promising, especially when compared to that of developed countries. For instance, Nigeria production was estimated at 1,860,600 tonnes in 2010 while the United States of America estimate for the same year was 12,858,700 tonnes (FAO, 2010). Yield per hectare in Nigeria was estimated at 1/7th of that of the U.S.A (FAO, 2010). From nutritional point of view, tomato plays a vital role in providing vitamin C, carotenoids, flavonoids and phenolics for human diet (Horneburg and Myers, 2012). Tomato can be eaten as raw vegetable or can be added to other food items. Many processed products are derived from tomato such as paste, whole peeled tomato, diced products, and various forms of juice, sauces, and soups have gained significant acceptance. Tomato deserves special attention because of the role it plays in food security in West Africa. However, effort to meet food needs on a sustainable basis for a growing population is seriously reduced by biotic and abiotic stresses. Climate variability and the prevalence of extreme events, in particular drought, are harsh reality for smallholder farmers, which depend exclusively on rainfed agriculture (IPCC, 2007). Climate variability is one of the challenges faced in the world, especially in the developing countries, to maintain the food security.
Drought and flood are the two most severe environmental factors that affect crop production in West Africa. Regrettably, global climate change will increase the occurrence and severity of drought episodes (Tuberosa, 2012). Similarly, Lobell et al. (2008) reported that climate change is expected to negatively impact crop production because of variable temperatures and more frequent drought in many parts of the world by the middle of the 21st century. This makes development of varieties that can yield well under harsh environments very critical for the prevention of food shortages.
Water is of paramount importance in the production of vegetables and other crops. Worldwide, it is regarded as a restraining factor for production of horticulture and crops (Ghebremariam et al., 2013). Water is crucial for the movement of photoassimilates and nutrients, and very fundamental in all plant physiological processes (Lisar et al., 2012). Therefore, drought is the principal abiotic constraint that causes considerable yield reduction in agricultural production (Robin et al., 2003). It has been estimated that drought accounts for over 70 % of potential agriculture yield losses globally (Boyer 1982). In the same vein, Kulkarni and Deshpande (2008) indicated that crop yields are reduced by 70-80% due to drought specifically during the reproductive stage.
Nowadays, water availability for agriculture is becoming limited alongside with a projected rise in food demand for the growing world population. Therefore, developing novel cultivars with more efficient water-use and greater drought-resistance capacity is the most viable solution to ensure a sustainable agricultural production and alleviate threats to food security. Indeed, the adoption and improvement of crops suited to growth with limited water supply on drought lands is vital to ensuring food security, given the seasonal variability, population growth, and the effects of climate change. In the developing counties, the development and use of crop varieties with high water-use efficiency and high yield is particularly important for areas prone to drought, unreliable rainfall and where irrigation is unavailable or unaffordable for resource-poor farmers. Given that almost 90% of Nigeria's crops are rainfed, the huge seasonal variability associated with change in climate such as drought and flood pose a serious threat to farmers. Olaoye (2005) stated that the frequent occurrence of drought occasioned by erratic rainfall distribution and cessation of rains during the growing season is the greatest hindrance to increased production of these food and industrial crops.
Several authors have demonstrated the importance of using inducing chemicals like polyethylene glycol 6000 for the selection of drought tolerance at germination and early seedling stage (Khodarahmpour, 2011;Ghebremariam et al., 2013;Osman Basha et al. 2015;Brdar Jokanović and Zdravković, 2015). It has been reported that using PEG as a selection technique in vitro is the most trustworthy method for screening desirable genotypes and to study further the effects of water scarcity on plant germination indices (Kocheva and Georgiev, 2003;Sakthivelu et al..., 2008).
Tomato is a sensitive horticultural crop to a variety of environmental stresses, especially extreme temperature, drought, salinity and inadequate moisture stresses (Kalloo, 1993). Therefore, the objective of our work was to screen for drought tolerance at germination and seedling levels for better understanding of drought mechanisms, identification and selection of the most tolerant tomato genotypes.

MATERIALS AND METHODS Plant Materials
The material used in this study consisted of twenty four (24) tomato genotypes (Table 1). These genotypes were obtained from two different sources: twenty two (22) genotypes from the National Centre for Genetic Resources and Biotechnology (NAGRAB) Ibadan, Nigeria and 2 cultivars were bought from commercial centers of agricultural products (CCAP).

PEG 6000 treatment
Tomato seeds were firstly disinfected with 3% absolute alcohol solution for 10 minutes. They were then thoroughly washed in 3 changes of distilled water in order to remove the traces of alcohol. In the course of disinfection with alcohol and rinsing with distilled water, tomato seeds were gently shaking for proper sterilization and removal of alcohol traces, respectively. With the aid of forceps twenty five (25) seeds were placed on two layers of Whatman filter papers in 9cm Petri dishes. Three treatments 0%, 4% and 14% were prepared by adding PEG 6000 weighed in scale to distilled water according to the method of Michel and Kaufman (1983) in order to obtain the osmotic potential in PEG. All the petri dishes were kept in the laboratory at a temperature of 25 0 C±2. Distilled water and prepared PEG 6000 solution were frequently added to the petri dishes as need arose.

Measurement
The first reading of seed germination was performed 48 hours after sowing, and then the counting of the germinated seeds was continued every day until the 13 th day of drought imposition. Shoot length and root length of 10 seedlings from each genotype and each replication were measured on the 13 th day. Other measurements taken was germination percentage and seed vigor index Germination index (G.I.) was computed by using the following formula: G.I.= n/d, where, n =number of seedlings emerging on day 'd' d = day after sowing.
Seed vigor index was calculated by multiplying germination (%) and seedling length (cm). The recovery rate was measured on the 21 st day. Indeed, non-germinated seeds were removed and placed in new petri dishes with moistened new filter papers and distilled water to observe their recovery rate. It should be noted that only distilled water was added when required to petri dished for a week.

Analysis of data
All data recorded were subjected to statistical analysis using -R‖ software to identify significant difference among the tomato genotypes used under the three treatments of distilled water and PEG 6000. ANOVA was performed for the assessment of the variation at 0.05 level of probability using Newman-Keuls Multiple Comparison-PostHOC test. In addition, Pearson correlation coefficient between traits measured was computed.

Germination percentage
Germination percentage is presented in

Root length
The mean root length of all tomato genotypes studied is presented in Table 5. Strong and significant differences were not found at 0% and 4% PEG in all the genotypes under the present study. At high concentration, root production was completely inhibited in NHGB/09/120 and NGB01301. In addition, significant root length reduction was observed in other genotypes treated with 14% PEG when compared with control and low concentration.

Recovery rate
To find out whether non germinated seed will show recovery and germinate, they were taken from osmotic stressed condition, the non-germinated seeds were transferred to moisten filter papers with distilled water which were placed in plastic petri dishes after 13 days and were observed for additional 7 days. After 2 days, total recovery of germination (100 %) was observed, which showed an indication of drought . Water deficit which lead to drought will definitely be exacerbated by climate change especially in rainfed zones. Therefore, there is urgent need to conduct research through screening for drought tolerance in order to develop new cultivars to be used in drought prone areas. There are several methods of screening crop genotypes but in the present study we chose to conduct the screening exercise at seed germination and seedling level using polyethylene glycols 6000 (PEG 6000) which is regarded as the most trustworthy chemical to induce water stress at the laboratory level. PEG 6000 really demonstrated through this study its ability to discriminate the most tolerant genotypes from the most susceptible ones. Table 2 shows the germination percentage of each genotype under 3 treatments i.e. 0%, 4% and 14% PEG 6000. NGB01357, L00170 and NHGB/09/113 revealed high percentage germination ranging from 28% to 40% under high concentration (14%) PEG. This indicates that they are the most drought tolerant among the 24 tomato genotypes screened. This could be explained by the fact that these drought tolerant genotypes were able to imbibe water indispensable for germination under osmotic condition created by PEG6000 solution. On the contrary, the most drought susceptible genotypes NHGB/09/120 and NGB01301 were unable to absorb water to trigger their germination and thus failed to record a single seed germination. This is followed by the susceptible genotypes which recorded low germination rate due also to the PEG inhibition. Our results are similar to previous studies on screening different crops for drought tolerance using PEG6000 (Hegarty, 1997;Turk et al..., 2004;Osman Basha et al..., 2015). It should also be noted that 14% PEG had effect on the days of germination because most of the genotypes started germinating on the 4 th day when compared to those in the control and low PEG concentration (24 hours and 2 days, respectively). Similar results were obtained by Hegarty, (1997) and Turk et al.., (2004) who reported that water stress at germination stage delayed germination or impede germination completely.

DISCUSSION
Decrease in root length was observed with increasing PEG concentration and the reduction was more pronounced in high concentration (Table 5) For the fresh weight, despite germination was recorded in some genotypes such as NG/AA/SEP/09/042, NGB01301, NG/AA/SEP/09/050, NG/MR/JAN/10/001, NGB01362, NGB01232, L00168 and NGB01250 at 14% PEG, they weighed zero gram i.e. no detectable by the weighing balance (no fresh weight). This could be explained by the fact that PEG did not permit the uptake of water and the partition of stored nutrients in the shoot and roots of the seedlings causing them to be lighter. This is a result of deleterious effect of drought on crop growth and development and biomass.

CONCLUSION
A breeding program and varietal improvement relies on sufficient genetic variability for target traits. Therefore, the traits studied under PEG stimulated drought conditions allowed us to differentiate between sensitive and tolerant genotypes. Genotypes NGB01357, L00170 and NHGB/09/113 are the most tolerant to drought while NHGB/09/120 and NGB01301 are the most susceptible to drought. PEG 6000 is a reliable chemical to induce drought condition and for rapid screening.