Research Article
In vitro and in vivo Screening of Potato Cultivars Against Water Stress by Polyethylene Glycol and Potassium Humate
Agricultural and Natural Resources Research Centre of Ardabil, P.O. Box 56491-11169, Ardabil, Iran
INTRODUCTION
Water deficit is a common stress in potato production, which lead to decrease in tuber quality and yield. Because of potato susceptibility to drought (Hassanpanah et al., 2008a), preparing sufficient water is very important for increasing potato quality and quantity. It is very necessary to study about tolerance of different potato cultivars against water deficit stress and determination of potato water consumption in Ardabil. There is water deficit problem in this region. Climatic changes were occurred in Ardabil region at the recent years. These changes caused differences in precipitation dispersion, river flowing and wells water. Therefore, we have to identify agronomic characters and water need of new potato cultivars and more improvement of their quality and quantity.
Water deficit decreased number of leaves, plant water potentials (Frensch, 1997), leaf area, stem height, ground coverage, tuber number, growth and yield, canopy radiation interception and only to a lesser extent by effects on radiation use efficiency, harvest index and tuber dry matter concentration (Schittenhelma et al., 2006) and nitrate reductase (Chandra et al., 2004; Das et al., 2005; Kar et al., 2005; Xu and Guang, 2006) and increased reducing sugar content in the stem, tuber cracking and malformation, surface abrasions, hollow heart, brown center, internal brown spot, vascular discoloration or bruise, degradation of starch in the tuber stem end and total glycoalkaloids concentration (Papathanasiou et al., 1999). Reflectance indices were used to measure biomass and drought stress, changes in leaf water content (Francois and De Proft, 2005) and water stress (Bahrun et al., 2003). A set of drought tolerance genes previously found to be up-regulated in tolerant potato under drought (Schafleitner et al., 2007) was assayed for expression changes in potato under drought.
Potassium humate is an active hormone with natural origin that extracts from plants and animal remains exist in the bottom of marshes. This material is formed from N, P, K and microelements namely Mo, Cu, Zn, B, Co and Mg (Gadimov et al., 2007). Using of potassium humate increased tuber yield about 11.01 ton ha-1 under water stress condition (Hassanpanah et al., 2008a), root number (Baraldi et al., 1991) and seed numbers and weight in pea under saline stress condition and decreased nitrate amounts in leaves and roots (Gadimov et al., 2007) and in potato tubers (Hassanpanah et al., 2007) and decreased of plantlet transplantation from in vitro to greenhouse from 30 days to 13 days in MS media culture (Hassanpanah et al., 2008b).
Polyethylene glycol (PEG) is a polymer produced in a range of molecular weights. During the 1970′s and 1980′s PEG of higher molecular weight (4000 to 8000) was quite commonly used in physiological experiments to induce controlled drought stress in nutrient solution cultures. Several studies also reported theoretical or measured concentration osmotic potential relations for PEG of different molecular weights. An example for such relationship which can be roughly defined as standard calibration curves was presented by Money (1989).
Chlorophyll fluorescence is very useful to study the effects of environmental stresses on plants since photosynthesis is often reduced in plants experiencing adverse conditions, such as water deficit, temperature, nutrient deficiency, polluting agents, attack by pathogens. In condition of drought climate, shadow and deficit nutrients (the factors of decreasing of photosynthesis and protein synthesis) and the night under medium drought, but decrease in day light because of leaves plasmolysis. Certainly attraction of nitrate in condition of medium drought is more than severe drought (Scheible et al., 1997; Gadimov et al., 2007).
Crops are usually non-halophytes that tolerate only moderate salt concentrations. Nitrate reductase catalysis the first step in the nitrate-reducing pathway and is considered to be a tool to distinguish between different crop plants. Gadimov et al. (2007) were conducted in order to comparison of nitrate reductase activity in plants from different seasonal crops (barley, maize, soybean, cucumber and pea) under saline condition. They reported increase of salt level decreased nitrate reductase activity in all of organs and arrangement for tolerance of salinity pay attention to nitrate reduction in under study plants was barley, maize and soybean.
This experiment was conducted for evaluation of effect of polyethylene glycol and potassium humate on potato cultivars against water stress in in vitro and in vivo condition.
In vitro condition: This study was conducted in Biotechnology Laboratory and Greenhouse of Villkej Company in Ardabil Province, Iran in May of 2008. Plantlets produced from meristem culture of four cultivars (Agria, Savalan, Satina and Caesar) propagated by single node cuttings. Experimental design was factorial on the basis of completely randomized design with two factors in three replications. Factor A was plantlets that were produced from meristem culture of four cultivars and factor B was four treatments (One concentrations of polyethylene glycol as -1.5 bar 1L-1 MS medium, second concentrations of potassium humate (1 mL 1L MS medium) and third concentrations of PEG (-1.5 bar) with potassium humate (1 mL 1L MS medium) and other without them as control). Polyethylene glycol added by using diffusion based method (Taji et al., 2008).
Chlorophyll fluorescence of leaves measured by chlorophyll flurometer (OS-30p) since 30 days after planting. Nitrate reductase activity (NRA) measured 30 and 40 days after planting in all of organs (leaf, stem and root). Analysis of variance was done and means compared by LSD (Least significant difference) test by MSTATC software.
In vivo condition: Five plantlet from each cultivar cultured in planting beds of pitmass (Biolan) with punce (1:1 v/v) in a greenhouse. Experimental design was factorial on the basis of randomized complete block design in three replication. The plantlets were planted with 10 cm distances between rows and 10 cm between plantlets. The plantlets were irrigated after planting by normal water. Macro and micro nutrients were used to provide for nutrition of plantlets. All of practices such as irrigation and control of weeds, pests and diseases were done regularly during growth period. Control of pests and fungus diseases were done respectively by use of 250cc per ha Confidor and 400 g ha-1 Equation-Pro. Mini-tubers harvested after about two months. The traits such as minituber average weight, number and weight per plant were measured. Analysis of variance was done and means compared by LSD test, by MSTATC software. Linear correlation coefficient traits were done by SPSS software.
In vitro condition
Chlorophyll fluorescence: Analysis of variance showed significant differences between MS media culture levels on rate of Fo, Fm and Fv/m, cultivars with rate of Fv/m and MS media culture levels xcultivars on rate of Fo, Fm and Fv/m Fv/m.
The maximum leaf number, plantlet height, rate of Fo, Fm and Fv/m for MS media culture levels was under normal with potassium humate and normal toward other conditions. In order to, the rate of Fv/m increased by use of potassium humate in MS media culture under stress and normal conditions (Table 1).
The high value was found rate of Fv/m for Agria and Caesar cultivars (Table 2). The highest was belonged to Caesar under normal and normal with potassium humate; Savalan under stress conditions (Table 3).
Consequently, cultivars were ranked in order of deficit tolerance based on reductions in Fv/Fm values. Based on Fv/m ranking, Agria, Caesar and Savalan cultivars were identified as deficit tolerance.
Screening of cultivar by using chlorophyll fluorescence can provide a rapid means of gauging the freezing and chilling damage in bean, cucumber, cabbage, mandarin, grapefruit and lime (Smillie and Hetherington, 1983), freezing tolerance in potato and rice (Greaves and Wilson, 1987), effects of leaf age, irradiance and leaf water potential in potato leaves (Vos and Oyarzun, 1987), frost hardiness in black currant (Brennan and Jefferies, 1990), heat tolerance in fruit crops (Yamada et al., 1996), water stress in potato leaves induced (Basu et al., 1998), chilling susceptibility in rose (Hakam et al., 2000), photosynthetic response to elevated CO2 in high altitude potato species (Solanum curtilobum) (Olivo et al., 2002), acute disturbance in water availability (Mateja et al., 2005), fusariosis seveity in tomato plants (Wagner et al., 2006), deficit tolerance in potato (Zanandrea et al., 2006) and combined effects of selenium and drought in potato (Mateja, 2008).
In this study, the leaf number, plant height and rate of Fv/m were increased by use of potassium humate under stress and normal conditions. The rate of Fv/m results can be attributed to the change in the capacity of exploitation of the photochemical energy in the dark adapted state. Higher values of Fo and lower Fm can indicate a higher energy dissipation in PSII antenna system (Rohacek, 2002; Zanandrea et al., 2006).
Table 1: | Mean of measured traits under different levels of MS media culture |
Mean with the same letters in each column does not have significant difference at the 5% level of probability according to LSD. Fo: Minimal fluorescence, Fm: Maximal florescence, Fv/m: The ratio of variable fluorescence to maximal fluorescence, Normal: Without potassium humate and polyethylene glycol as control, Normal+Humate: 1 mL potassium humate in 1L MS medium, Stress: Polyethylene glycol as -1.5 bar 1L-1 MS medium, Stress+Humate: Polyethylene glycol as -1.5 bar and 1 mL potassium humate 1L-1 MS medium |
Table 2: | Mean of measured traits for potato cultivars plantlets |
Mean with the same letters in each column does not have significant difference at the 5% level of probability according to LSD |
Table 3: | Mean of measured traits for MS media culture levels on potato cultivars plantlets |
Mean with the same letters in each column does not have significant difference at the 5% level of probability according to LSD |
Tourneux et al. (2003) reported that at relative water deficits below 40% potato genotypes did not show differences in Fv/m values. The high dehydration resistance of photosystem II in potato has been reported by Tourneux and Peltier (1995) and 40 days of drought in the testing environment caused sufficient stress to lower yield, but no drop in chlorophyll fluorescence.
Nitrate reductase activity (NRA): Results of analysis of variance showed significant differences between effect of stage (S), MS media culture levels (A) and cultivars (B), AxB, SxAxB, for leaf, stem and root and SxA for stem and root, SxB for leaf and root .
The normal with potassium humate toward normal and stress with potassium humate toward stress conditions produced maximum rate of nitrate reductase activity (NRA) in all of organs (leaf, stem and root). The roots had high rate of NRA toward leaf and stem organs (Table 1). Increase of water deficit stress decreased NRA in all of organs. All of organs rate of NRA decreased as a result of water stress. Potassium humate causes to increase rate of NRA under normal and stress conditions.
Satina and Caesar had the highest rate of NRA and Savalan and Agria the lowest in all of organs (leaf, stem and root). Savalan, Caesar and Agria had high rate of NRA in root organ toward leaf and stem organs (Table 2).
The highest rate of NRA was produced since 30 days after plantlets. Since, 30 days and 40 days after plantlets had high rate of NRA in roots toward leaf and stem organs. The normal with potassium humate toward normal and stress with potassium humate toward stress conditions produced maximum rate of NRA since 30 days after plantlets. The root had high rate of NRA toward leaf and stem organs. The root had the highest rate of NRA and the leaf and stem the lowest under normal, stress with potassium humate and stress conditions since 30 days after plantlets. The root had the highest rate of NRA and the leaf and stem the lowest under normal with potassium humate since 30 and 40 days after plantlets.
Satina and Caesar had the highest rate of NRA and Savalan and Agria the lowest since 30 and 40 days after plantlets. The root had the highest rate of NRA in all of cultivars and since 30 days after plantlets. Satina and Caesar had the highest rate of NRA under normal and stress in root organ. Caesar had high rate of NRA under normal with potassium humate and stress with potassium humate (Table 3).
In this study, the rate of NRA decreased in normal with potassium humate, stress with potassium humate and stress leaves, stem and root compared with normal conditions. Increase of deficit stress decreased NRA in all of organs. This is consistent with previous observations of water-stress induced losses in maximal extractable foliar NR activity in other species (Wellburn et al., 1996). Brewitz et al. (1996) reported the rate of NRA decrease in stress leaves compare with well-water conditions in tomato crop.
The roots had high rate of NRA toward leaf and stem organs. Potassium humate causes to increase rate of NRA under normal and stress conditions. NR activity decreased under water stress in strong correlation with CO2 assimilation. Therefore, a low NR activity decrease under drought could pinpoint drought tolerant genotypes. NR activity was reported to decrease under water stress (Das et al., 2005; Kar et al., 2005; Xu and Guang, 2006) and a low decrease of activity has been associated with drought tolerance (Chandra et al., 2004; Das et al., 2005). Nitrate reductase activity was significantly decreased in drought-exposed plants, but activity depletion was independent of yield or yield maintenance (Schafleitner et al., 2007).
Gadimov et al. (2007) resulted that NRA is very different related to plant type. But generally leaves are the basic portion of nitrate reduction (50-90%) and roots (6-40%) have the second place in this matter. Increase of salt level decreased NRA in all of organs. Solution of 50 mM NaCl produced maximum rate of NRA reduction observed in spring barley leaves. And solution of 100 mM NaCl caused that soybean organs show the highest sensitivity to the saline stress and decreased NRA 82% relative to control. In soybean, NRA decreased 5.2 times in leaves, 2.8 times in stems and 4.7 times in roots. This reduction was in barley and maize about 1.4-2.6 times. Arrangement for tolerance of salinity pay attention to nitrate reduction in under study plants was barley, maize and soybean.
In vivo condition: Analysis of variance showed that was significant differences between MS media culture levels on mini-tuber number and weight per plant, cultivars on mini-tuber weight average, number and weight per plant and MS media culture levelsxcultivars on mini-tuber weight per plant and average weight mini-tuber per plant.
The maximum mini-tuber weight average, number and weight per plant for was under normal with potassium humate condition (Table 1). The maximum mini-tuber weight average, number and weight per plant for Savalan cultivar (Table 2). The high value of mini-tuber number and weight per plant were found in Savalan and Caesar under normal with potassium humate condition (Table 3).
There were significant and positive correlation between leaf number with plant height; Fo with Fv/m, Fm and mini-tuber number per plant; Fm with Fv/m and mini-tuber number and weight per plant; Fv/m with mini-tuber number and weight per plant and mini-tuber number per plant with mini-tuber weight per plant and stem number with Fm and Fv/m; negative correlation (Table 4).
Table 4: | Linear correlation coefficients between different traits for MS media culture levels on potato cultivars plantlets in in vitro and in vivo conditions |
* and ** : Significant at probability levels of 5 and 1%, respectively; NRA: Nitrate Reductase Activity |
Chlorophyll fluorescence measurement is a suitable tool of study changes in the photosynthetic capacity of the plants exposed to limit water of supply. Cultivars responded differently to disturbance in water deficit. Further analysis and more measurements are needed to be able to range cultivars of potato according to their different sensitivity to acute limitation of water supply (Mateja et al., 2005). In this study, the leaf number, plant height and rate of Fv/m increased by use of potassium humate under stress and normal conditions. The rate of Fv/Fm results can be attributed to change in the capacity of exploitation of the photochemical energy in the dark adapted state. Higher values of Fo and lower Fm can indicate a higher energy dissipation in PSII antenna system (Zanandrea et al., 2006).
The cultivars were ranked in order of deficit tolerance based on reductions in rate of Fv/Fm values. Based on Fv/Fm ranking, Agria, Caesar and Savalan cultivars was identified as deficit tolerance and Satina cultivar as deficit intermediate tolerant.
Satina and Caesar had the highest rate of NRA and Savalan and Agria the lowest in all of organs (leaf, stem and root). Satina and Caesar had the highest rate of NRA under normal and stress in root organ. Caesar had high rate of NRA under normal with potassium humate and stress with potassium humate.
The maximum mini-tuber weight average, number and weight per plant for were under normal with potassium humate condition and for Savalan cultivar. The high value of mini-tuber number and weight per plant were found in Savalan and Caesar under normal with potassium humate condition.