Increasing Soybean (Glycine max L) Drought Resistance With Osmolit Sorbitol

Efforts to increase soybean production has been pursued for years in Indonesia through the process of intensification and extensification. Increased production through intensification of increasing grain yield per hectare, among others includes the improvement of cultivation system such as the use of cultivars that have superior resistance to drought. Increased soybean production has been through the expansion of planting areas utilizing available idle dry land. However, one of the constraints faced in dryland agriculture was the limited water supply due to low intensity of rainfall that leads to low crop production. In order to ensure that soybeans are cultivated on dry land remains capable of high production, it is necessary to physiologically engineered the soybean with an open stomata. The study was conducted in the greenhouse of Balai Penelitian Tanaman Serealia (BALITSEREAL) Maros, Sulawesi, Indonesia with a completely randomized block design h factorial pattern. The first factor was the water stress stadia while the second was the amount of sorbitol osmolit concentration application. Results indicated that there was an interaction between the plant height growth and number of leaves between the water clamping time and concentration of the osmolit sorbitol. The vegetative stage especially during flowering and pod formation was inhibited when the water was clamped, but by spraying osmolit sorbitol, soybean growth interms of its height and number of leaves was enhanced. This study implies that the application of osmolit sorbitol may enhance the drought resistance of soybean growth. Future research suggested that more work should be done on the application of osmolit sorbital to other agriculture crops to increase their drought resistance in the drylands.


Introduction
The main cause of low soybean production in Sulawesi, Indonesia is due to 65% of the soybean is grown in the dryland areas while the balance of 35% of the crop is grown in the cultivated and irrigated rice fields during the dry season.Part of the problems arises in dryland agriculture systems is the limited water supply which is very dependent on the amount and intensity of rainfall that leads to the relatively low crop production.For that reason, soybeans grown and cultivated in dry land must be able to withstand the drought and remain capable of producing high yield soybeans through the physiological engineered and regulated stomatal opening process.It has been well understood that drought stress crops may affect and lead to changes in molecular biochemistry, physiology and morphology of plants.Biochemical changes that occur may include the accumaulation of osmolit sorbitol applied to various crops and specific proteins involved in stress tolerance (Shinozaki & Shinozaki, 2007).Soybeans experiencing drought will close their stomata in order to reduce the process of transpiration.However, over time the stomatal closure will result in impaired growth that leads to low crop yield production.In order to prevent the stomatal closure continues over time, it is pertinent to minimize the role of abscisic acid (ABA) in the leaves by using a local compound known as osmolit sorbitol.Plants experiencing drought will trigger the abscisic acid (ABA) biosynthesis.The process occurs due to water shortage conditions in the protoplasm where oxidation process takes place continuously (Bray, 1988).Arifin (2001) further supported that the green bean plants usually experience water shortage stress during the vegetative stage and throughout their growth cycle where abscisic acid content (ABA) increased three-folds compared to plants that were not frozen.ABA is a hormone that were synthesized in the roots and translocated to the leaves, and may also be synthesized by the guard cells themselves (Assmann & Shimazaki, 1999).ABA contained in the leaves especially on guard cells normally control the stomatal closure (Turner, 1998).Therefore, the objective of this study was to assess the effects of applying osmolit sorbitol in increasing the soybean's resistance to drought through the stomatal physiological engineering.

Methods and Materials
Experiments were conducted in the Greenhouse Crops Research Institute for Maize and Cereals (BALITSEREAL) Maros, Sulawesi, Indonesia.The analysis was carried out in the Plant Crop Laboratory, Bogor Agricultural University (IPB), Bogor, Indonesia.The study was conducted in May to December 2012.The experiments used a designed randomized block design (RBD) in the two-factor factorial with three replicates.The time factor (Factor No. 1) was Water clamping (W), which consists of six standards namely, W1 = not gripped over the life of the plant, W2 = gripped throughout life (0-85 days), W3 = gripped from the beginning of growth (0-15 days), W4 = seized during active vegetative stadia (16-30 days), W5 = gripped the flowering-pod filling stadia (at the age 31-65 days), W6 = gripped the stadia seed maturity (age of 66-85 days).Meanwhile, the second factor (Factor No. 2) used a local compound concentration (osmolit sorbitol (S), which consists of four levels, namely S0 = No osmolit sorbitol sprayed, S1 = 10 ml/l, S2 = 15 ml/l, and S3 = 20 ml/l of osmolit sorbitol sprayed.Of the two factors, a total of 24 combined treatments were obtained and each was repeated three times to obtain 72 units of the experiments.The 24 combined treatments were: Determination of the amount of water provision (clamping) is based on 50% of the normal water demand and its distribution was based on the normal water requirements for the growth of soybeans in each period.The growth media was prepared with a mixture of soil and manure in the 4:1 ratio, which was filled in a polybag with a diameter of 20 cm and a height of 40 cm with an average weight of 8 kg each.Once the growth media was ready, it is filled up with water until it reached the field capacity limit.The soybean seeds were then planted as many as four points per hole.Once the soybeans reached the age of seven years old, they were then thinned untill two plants per polybag remained.With some phospohorous (P) fertilization and urea at 15 Days After Planting (DAP), the dosage of urea was given at 50 kg/ha (0.66 g urea/polybag) and SP-36 at a dose of 90 kg/ha (1.19 SP-36/polybag) at planting time.According to Fagi and Tangkuman (1995), the average amount of water requirement of soybean (variety Wilis) during the growing season was 325 mm/season.Using a water clamp control system, as much as 50% water was injected into the polybag to cater for the soybeans normal water requirement.The water clamping control time applied to the treatments are shown in Table 1.
Description: 0 = not gripped by water; X: gripped Water * = Osmolit spraying frequency adjusted with treatment Osmolit sorbitol was sprayed to the 10-85 days old soybeans at the lower leaf surface once in every five days with a concentration in accordance to the frequency of spraying treatment of 2-16 times.Osmolit sorbotil was sprayed between 1000-1200H once in every five days, particularly at 0815-0945H, 1000-1200H and 1200-1400H, despite no significant differences were observed by Ariffin (1997).The distribution and amount of water in each soybean polybags averaged at 94 ml.

Effects of Water Clamping Time and Osmolit Sorbitol on Soybean Growth Parameters
The effects of water clamping time and osmolit sorbitol on soybean plant height, number of leaves, leave surface area, root dry weight, leaf area, dry weight of root, relative growth rate, net assimilation rate, chlorophyll content and stomatal density can be shown in Tables 2-10, respectively.ABA is a plant hormone that acts as a natural barrier formed by itself due to the lack of water and environmental stress.Based on the analysis of clamping turning times, there was an effect of water on the ABA content in the leaves of green soybean plants, whereas spraying osmolit sorbitol provided no significant interactions as demonstrated in Tables 11-13.Results indicated that the time of water clamping had very significant effects on the weight of 100 seeds while spraying osmolit sorbitol had a no significant interaction with water clamping time as shown in Table 13.

Discussion
The results of the study clearly showed that the growth of soybean was significantly affected by the availability of water.This is because water is deemed necessary for the metabolic activities of the plant for a paired cell growth and development.Lack of water during the vegetative stadia and flowering until pod formation showed the lowest period of growth.When compared with normal plants that obtain sufficient water, lack of water at each stadia results in a decrease of crop growth.Aminah (2007) found that soybean with a water stress at 150 mm/season (below the normal requirement) showed highly significant growth differences with soybean getting water at 300 mm/season.The most sensitive and critical growth phase to drought is during the vegetative stadia, flowering and pod formation, as evidenced by the low growth measurements of their vegetative organs.Simillar results were obtained by Boyer (1996) where he observed that the growth of soybean pod formation wash and water shortages could affect the later low seed yield obtained.Aminah (2011) revealed that a strong osmotic regulation can improve the adaptability of plants during drought where the water supply is limited.Apparently, spraying osmolit sorbitol in soybeans when experiencing water shortages can increase its growth resistance to drought.This has been proven with water clamping time and spraying with osmolit sorbitol effects both in terms of plant height (for all observation time) and number of leaves (50 observations and 65 DAP).
In the process of photosynthesis, stomatal opening is an important part of the plant where the process of gas or water vapor diffusion into and out of plant tissue occurs.The process of opening and closing of stomata is one of the obstacles that occur in the diffusion process.The process of opening and closing of stomata is usually affected by stomatal guard cell turgor where it closes under the conditions of water shortage (Turner, 1998).
The results of the analysis shows that spraying osmolit sorbitol to soybeans under stress experiencing water shortages can increase its resistance to water shortages in terms of plant height and total number of leaves.Osmolit sorbitol can also control the cell's humidity where the cell turgor can be maintained with sufficient water for higher yields.

Conclusion
Soybean growth (W4) especially its plant height, leaf number, leaf area, root dry weight, content chlorophyll, and variable production is heavily reliable and sensitive to the availability of water.An osmolit sorbitol concentration of 20 ml/l sprayed on the soybeans increased its water drought resistance by controlling its physiological stomatal activity at various stadia growth and soybean yield.This study implies that the local compound osmolit sorbitol can enhance the soybean growth in drought resistance dryland areas.

Table 1 .
Water clamping time and osmolit sorbitol spraying of soybean

Table 2 .
Effect of time, water clamping and osmolit sorbital on soybean plant height growth

Table 3 .
Effect of time, water clamping and osmolit sorbital on soybean number of leaves growth

Table 4 .
Effect of time, water clamping and osmolit sorbital on soybean leaf area (cm 2 )

Table 5 .
Effect of time, water clamping and osmolit sorbital on soybean root dry weight

Table 6 .
The Effect of time, water clamping and osmolit sorbital on soybean on plant dryweight

Table 7 .
Effect of time, water clamping and osmolit sorbital on soybean relative growth rate (mg.mg -1 .days)

Table 9 .
Effect of time, water clamping and osmolit sorbital on soybean chlorophyll content (g/g)

Table 10 .
Effect of time, water clamping and osmolit sorbital on soybean stomatal density 3.2 Effects of Water Clamping Time and Osmolit Sorbitol on the Content of Abscisic Acid (ABA) and Number of Pods, Pod Weight and Seed Weight per Plant and Weight of 100 Seeds

Table 11 .
Effect of water clamping time on ABA content (ug/g)

Table 12 .
Effect of water clamping time and osmolit sorbitol on soybean total pods, pod weight and weight seeds

Table 13 .
The effects of clamping time and osmolit sorbitol on the weight of 100 seeds